Bioreactor screening platform for modeling human system biology and screening for agents acting on muscle force of heart
阅读说明：本技术 用于模拟人系统生物学和关于药剂对心脏的肌力作用进行筛选的生物反应器筛选平台 (Bioreactor screening platform for modeling human system biology and screening for agents acting on muscle force of heart ) 是由 D·D·特兰 K·D·科斯塔 李登伟 于 2018-11-29 设计创作，主要内容包括：公开了针对肌力作用快速筛选化合物的两级或两层系统和方法。该系统包含在第一层中的工程化心脏组织条(CTS),该CTS包含包埋于生物相容性凝胶中的心肌细胞,例如人心室心肌细胞,其中凝胶包含至少两个生物相容性结构支撑物,例如用于升高凝胶的聚二甲基硅氧烷柱。该系统还包含在第二层中的设备,该设备包含至少一个类器官模块,其包含至少一个类器官盒和镜布置,其中每个类器官盒包含类器官。该系统还包含至少一个检测装置,例如高速照相机,用于检测该系统第一层中CTS凝胶的挠曲和/或用于检测该系统第二层的一个或多个类器官盒中组织或类器官的行为。该方法包含将化合物施加至心脏组织条并检测凝胶响应于该化合物的施加而发生的任何挠曲,以检测表现出可能的肌力作用的化合物,并将该化合物引入类器官模块,其中该类器官模块中心脏组织或类器官的收缩性改变鉴定出具有肌力作用的化合物。还提供了一种制造心脏组织条的方法。第二层系统也可用于离体制造和监测、表征、操纵或测试一种或多种类器官(例如人的类器官)的方法中。该系统、方法、设备和组合物可用于多种情况,包括评估潜在治疗剂的功效和/或毒性。(A two-stage or two-layer system and method for rapid screening of compounds for muscle strength effects is disclosed. The system comprises an engineered Cardiac Tissue Strip (CTS) in a first layer, the CTS comprising cardiomyocytes, such as human ventricular cardiomyocytes, embedded in a biocompatible gel, wherein the gel comprises at least two biocompatible structural supports, such as polydimethylsiloxane columns for elevating the gel. The system also includes a device in the second layer, the device comprising at least one organoid module comprising at least one organoid cassette and a mirror arrangement, wherein each organoid cassette comprises an organoid. The system also includes at least one detection device, such as a high speed camera, for detecting the flexing of the CTS gel in the first layer of the system and/or for detecting the behavior of tissue or organoids in one or more organoid cassettes in the second layer of the system. The method comprises applying a compound to a strip of cardiac tissue and detecting any deflection of the gel in response to the application of the compound to detect the compound exhibiting a possible inotropic effect, and introducing the compound into an organoid module, wherein a change in contractility of the cardiac tissue or organoid in the organoid module identifies the compound having the inotropic effect. A method of manufacturing a strip of cardiac tissue is also provided. The second layer system may also be used in methods of manufacturing and monitoring, characterizing, manipulating or testing one or more organoids ex vivo (e.g., human organoids). The systems, methods, devices, and compositions can be used in a variety of contexts, including assessing the efficacy and/or toxicity of potential therapeutic agents.)
1.A system for screening compounds for changes in muscle contractility, comprising:
(a) a first stage screening apparatus comprising:
(i) a biocompatible gel comprising a plurality of cardiomyocytes;
(ii) a biocompatible support device for suspending the biocompatible gel, wherein the biocompatible gel and the biocompatible support device form a cardiac tissue strip;
(iii) a detection device for detecting movement of the biocompatible gel; and
(iv) a power source for applying an electrical pacing stimulus to the biocompatible gel; and
(b) a secondary screening apparatus comprising:
(v) at least one organoid module comprising at least one organoid cassette, wherein the organoid cassette comprises a medium inlet, a medium outlet, and at least one wall compatible with an external detection device, wherein each organoid cassette comprises a biomaterial comprising at least one human cell that is a human embryonic stem cell, a human adult stem cell, a human induced pluripotent stem cell, a cell derived from human tissue, or a progenitor cell of human tissue, and wherein at least one organoid cassette comprises cardiac biomaterial;
(vi) a mirror arrangement for simultaneously monitoring any biological development of the biological material in each organoid cassette; and
(vii) a detection device for observing the biological development of the biological material monitored in each organoid cassette.
2. The system of claim 1, wherein the cardiomyocytes are human cardiomyocytes.
3. The system of claim 2, wherein the human cardiac muscle cell is a human ventricular cardiac muscle cell.
4. The system of claim 2, wherein the human cardiomyocytes are derived from at least one human pluripotent stem cell.
5. The system of claim 1, wherein the cardiomyocytes are at least 106Individual cells/ml are present.
6. The system of claim 1, wherein the biocompatible gel comprises matrigel.
7. The system of claim 6, wherein the matrigel is present at a concentration of at least 0.5 mg/ml.
8. The system of claim 6, wherein the biocompatible gel further comprises collagen.
9. The system of claim 8, wherein the collagen is human collagen type I.
10. The system of claim 8, wherein the collagen is present at a concentration of at least 1 mg/ml.
11. The system of claim 1, wherein the support apparatus is at least two vertical support members.
12. The system of claim 11, wherein the vertical support member is made of polydimethylsiloxane.
13. The system of claim 11, wherein there are two vertical support members.
14. The system of claim 13, wherein the two vertical support members are approximately circular in cross-section, having a diameter of about 0.5 mm.
15. The system of claim 1, wherein the strip of cardiac tissue is about 26.5mm in length by about 16mm in width by about 6mm in height.
16. The system of claim 1, wherein the detection device is a high-speed camera.
17. The system of claim 1, wherein the mirror arrangement of the second stage screening apparatus comprises at least one angled cone mirror.
18. The system of claim 1, wherein the biological material is at least one tissue or at least one organoid.
19. The system of claim 18, further comprising another organoid, the other organoid being a heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gallbladder, lung, small intestine, colon, bladder, prostate, uterus, tumor, eye, skin, blood, or vascular organoid.
20. The system of claim 19, wherein the other organoid is a cardiac organoid.
21. The system of claim 1, wherein the second stage screening apparatus further comprises an electrode in adjustable relationship to a tissue or organoid in at least one organoid cassette.
22. The system of claim 1, wherein the second stage screening device further comprises a temperature control element, a light source, a module access port, or any combination thereof.
23. The system of claim 1, further comprising a data processor in electronic communication with the detection device, temperature control element, light source, module access port, or any combination thereof.
24. The system of claim 23, wherein the detection device is a digital camera, at least one pressure sensor, or a combination of a digital camera and at least one pressure sensor.
25. The system of claim 1, further comprising a tissue having at least one human cell.
26. The system of claim 1, further comprising a monitor.
27. The system of claim 1, comprising a plurality of organoid modules.
28. The system of claim 1, further comprising an interconnected fluid-switching network, wherein the network comprises a plurality of fluid lines, a plurality of valves, at least one pump, and at least one fluid tank.
29. The system of claim 28, further comprising a port for introducing a compound.
30. The system of claim 28, wherein said interconnected fluid-switched network comprises fluid communication between at least two organoid cassettes.
31. The system of claim 28, wherein the fluid is a culture medium.
32. The system of claim 28, wherein the fluid exchange network provides automated media exchange.
33. The system of claim 1, further comprising a pneumatic pressure controller.
34. The system of claim 33, wherein the pneumatic controller controls 0 in at least one module or in one or more organoid cassettes2And C02Of the concentration of at least one of the above.
35. The system of claim 1, further comprising a drug perfusion apparatus for delivering a compound to the cell, tissue, or organoid.
36. A method of manufacturing a strip of cardiac tissue, comprising:
(a) providing a biocompatible mold having a length of about 26.5mm by a width of about 16mm by a height of about 6 mm;
(b) forming a biocompatible gel conforming to the mold, wherein the biocompatible gel comprises matrigel, collagen, and a plurality of cardiomyocytes; and
(c) securing at least two vertical support members to the biocompatible gel, thereby forming a strip of cardiac tissue.
37. The method of claim 36, wherein the vertical support member is secured to the biocompatible gel by embedding the vertical support member in the biocompatible gel formulation prior to gelation.
38. The method of claim 36, wherein the vertical support member is affixed to the biocompatible gel by adhesion or by mechanical attachment.
39. A method of screening for a compound having a muscle-strength effect, comprising:
(a) pacing the cardiac tissue strip of claim 1 with an electrical stimulus at a pacing rate of 0.5Hz, 1.0Hz, 1.5Hz, or 2.0Hz, in the presence or absence of a candidate muscle force compound;
(b) detecting any movement of the paced cardiac tissue strip in the presence or absence of the candidate muscle force compound;
(c) comparing motion of a cardiac tissue strip paced in the presence of the candidate muscle force compound to motion of a cardiac tissue strip paced in the absence of the candidate muscle force compound;
(d) determining that the candidate muscle force compound is a potential muscle force compound when the motion of the cardiac tissue strip paced in the presence of the compound is different from the motion of the cardiac tissue strip paced in the absence of the compound; and
(e) administering the potential inotropic compound to a tissue or organoid in the secondary screening device of claim 1 and monitoring the tissue or organoid's response to the compound, wherein an alteration in contractility identifies the potential inotropic compound as an inotropic compound.
40. The method of claim 39, wherein the pacing rate is 1.0 Hz.
41. The method of claim 39, wherein the muscle force compound is identified as a potentially negative muscle force compound when motion of the cardiac tissue strip paced in the presence of the muscle force compound is less than motion in the absence of the muscle force compound.
42. The method of claim 39, wherein the muscle force compound is identified as a negative muscle force compound when:
(a) the motion of the paced cardiac tissue strip in the presence of the inode is less than the motion in the absence of the inode; and
(b) the tissue or organoid in the secondary screening device exhibits reduced contractility in the presence of the myogenic compound as compared to the absence of the myogenic compound.
43. The method of claim 39, wherein the myodynamia compound is identified as a potential positive myodynamia compound when the motion of the paced cardiac tissue strips is greater in the presence of the myodynamia compound than in the absence of the myodynamia compound.
44. The method of claim 39, wherein the myodynamia compound is identified as a positive myodynamia compound when:
(a) the motion of the paced cardiac tissue strip in the presence of the inode is greater than the motion in the absence of the inode; and
(b) the tissue or organoid in the secondary screening device exhibits increased contractility in the presence of the myogenic compound as compared to the absence of the myogenic compound.
45. The method of claim 39, wherein the compound is a drug, a viral vector, conditioned media, extracellular vesicles, other cells, or any combination thereof.
46. A tissue monitoring system comprising
(a) At least one organoid module comprising a plurality of organoid cassettes, wherein each organoid cassette comprises a culture medium inlet, a culture medium outlet, and at least one wall compatible with an external detection device, wherein a plurality of said organoid cassettes each comprise a biomaterial comprising at least one human cell, wherein said cell is a human embryonic stem cell, a human adult stem cell, a human induced pluripotent stem cell, a cell derived from human tissue, or a progenitor cell of human tissue;
(b) a mirror arrangement for simultaneously monitoring any biological development of the biological material in each of at least two organoid boxes; and
(c) a detection device for observing biological development of the biological material monitored in each of at least two organoid cassettes.
47. The system of claim 46, wherein the mirror arrangement includes at least one angled cone mirror.
48. The system of claim 46, wherein the biological material is at least one tissue or at least one organoid.
49. The system of claim 48, wherein the organoid is a heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gall bladder, lung, small intestine, colon, bladder, prostate, uterus, tumor, eye, skin, blood, or vascular organoid.
50. The system of claim 49, wherein the organoid is a cardiac organoid.
51. The system of claim 46, further comprising an electrode in adjustable relationship to the cells, tissue or organoids in at least one organoid cassette.
52. The system of claim 46, wherein the detection device is a recording device.
53. The system of claim 46, further comprising a temperature control element, a light source, a module access port, or any combination thereof.
54. The system of claim 46, further comprising a data processor in electronic communication with the detection device, temperature control element, light source, module access port, or any combination thereof.
55. The system of claim 52, wherein the recording device is a digital camera, at least one pressure sensor, or a combination of a digital camera and at least one pressure sensor.
56. The system of claim 46, further comprising a tissue having at least one human cell.
57. The system of claim 46, further comprising a monitor.
58. The system of claim 46, comprising a plurality of organoid modules.
59. The system of claim 1, further comprising a media mixer.
60. The system of claim 59, wherein the media mixer is a magnetic stirring device or a rotating disk.
61. The system of claim 46, further comprising an interconnected fluid-switching network, wherein the network comprises a plurality of fluid lines, a plurality of valves, at least one pump, and at least one fluid tank.
62. The system of claim 61, further comprising a port for introducing a compound.
63. The system of claim 62, wherein the compound is a candidate therapeutic agent, a drug, a viral vector, conditioned media, extracellular vesicles, other cells, or any combination thereof.
64. The system of claim 61, wherein said interconnected fluid-switched network comprises fluid communication between at least two organoid cassettes.
65. The system of claim 61, wherein the interconnected fluid-switching network provides a partially common fluid delivery path for at least two organoid cassettes, a partially common fluid exhaust path for at least two organoid cassettes, or a partially common fluid delivery path and a partially common fluid exhaust path for at least two organoid cassettes.
66. The system of claim 61, wherein the fluid is a culture medium.
67. The system of claim 61, wherein said fluid exchange network provides automated media exchange.
68. The system of claim 46, further comprising a pneumatic pressure controller.
69. The system of claim 68, wherein the pneumatic controller controls 0 in at least one module or in one or more organoid cassettes2And C02Of the concentration of at least one of the above.
70. The system of claim 46, further comprising a plurality of module access ports.
71. The system of claim 46, further comprising a drug perfusion apparatus for delivering a therapeutic agent to the cell, tissue, or organoid.
72. A method for determining the biological activity of a compound, comprising administering a compound to a cell, tissue or organoid in the system of claim 46, and monitoring the response of the cell, tissue or organoid to the compound.
73. The method of claim 72, wherein the compound is a drug, a viral vector, conditioned media, extracellular vesicles, other cells, or any combination thereof.
74. The method of claim 72, wherein said biological activity is modulation of the function of said cell, tissue or organoid, thereby identifying said compound as a therapeutic agent for treating an organ homology disorder of said cell, tissue or organoid.
75. The method of claim 72, wherein the assay measures the toxicity of the compound.
76. A method for determining the biological activity of a compound, comprising administering a compound to a plurality of cells, tissues or organoids in the system of claim 61 through an interconnected fluid-exchange network, and monitoring the response of the cells, tissues or organoids to the compound.
77. The method of claim 76, wherein the compound is a drug, a viral vector, conditioned media, extracellular vesicles, other cells, or any combination thereof.
78. The method of claim 76, wherein said biological activity is modulation of the function of said cell, tissue or organoid, thereby identifying said compound as a therapeutic agent for treating an organ homology disorder of said cell, tissue or organoid.
79. The method of claim 76, wherein the assay measures the toxicity of the compound.
80. An organoid manufactured using the system of claim 46, wherein said organoid comprises a plurality of differentiated cells.
Technical Field
The present disclosure relates generally to the field of medical health and cardiac physiology, and more particularly to the field of medical devices that provide a versatile bioreactor platform for monitoring the function of human tissue engineered organoids, and to the field of medical treatments that provide methods and screening for bioactive compounds, such as compounds or agents that have a myogenic effect on the heart.
Background
The traditional discovery and development of new drugs and therapeutics for heart disease has been an inefficient and expensive process. Due to the lack of a suitable human model, cardiotoxicity becomes a common leading cause of drug withdrawal, even for non-cardiovascular (e.g., cancer) drugs. Although traditional animal models such as rodents, dogs and pigs can be used, there are major species differences in both anatomy and function. Human pluripotent stem cells (hpscs) have been proposed to fill this gap, but conventional two-dimensional culture and experiments with single cells or disordered clusters do not adequately reproduce the phenotype of the human heart. Therefore, previous hPSC-based drug screening models focused on patterns such as single cell viability and electrophysiological effects, making them useful tools for cardiotoxicity screening. Newer two-dimensional models are also focused primarily on electrophysiology and proarrhythmia to assess cardiotoxicity. To date, only a few hPSC-based drug screening systems have been developed to study the effect of drugs on cardiac contractility, but alternative indicators of contractility have been developed for quantifying contractility at the single cell level. These hPSC-based drug screening systems include strain-embedded 2D muscle membranes, cardiac micro-tissue, cardiac micro-wire or bio-wire (biowire), mini-myocardium (μ HM) and force-producing engineered cardiac tissue (EHT), which, although not as physiologically functional as natural auditory tissue, is a limitation common to all engineered tissue systems developed to date.
The process of developing and approving the sale of therapeutic agents useful for treating diseases or conditions in humans or other animals is a lengthy, expensive and uncertain process. Despite the delay in introducing new therapeutic agents into a clinical or veterinary setting for treating individuals with disease, the public recognizes the importance of ensuring that new therapeutic agents are effective with minimal adverse side effects. At present, preclinical drug testing relies on experimental animals to determine the safety and efficacy of new drugs. Animal drug testing is a slow, expensive and unreliable indicator of human patients taking drugs safely. This is evident when one recognizes that adverse effects are a major cause of drug failure in clinical trials of candidate therapeutics for treating diseases and conditions. Therefore, there is an urgent need to develop complementary methods for preclinical drug testing to assess the safety and efficacy of experimental therapeutics.
Human pluripotent stem cell (hPSC) technology offers an exciting new opportunity for an in vitro therapeutic screening platform. These cells have many advantages over animal models, including human origin, suitability for culture, and the ability to generate patient-specific cell lines for genetic disorders (e.g., long QT syndrome). Differentiated cells derived from hpscs (e.g., heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gall bladder, lung, small intestine, colon, bladder, prostate, uterus, blood vessels, tumors, eye or skin) have been used with tissue engineering methods to reproduce various aspects of the three-dimensional environment of natural tissues in order to better mimic human functions. The major bottleneck in adopting these new predictive platforms for therapeutic screening is the lack of tools capable of culturing and monitoring the function of such tissue models.
To further enhance the predictive power of in vitro therapeutic agent screening, tools are needed to support modular system biology methods to predict multi-organ or complete "body" responses. One disadvantage of current in vitro screening platforms is that they typically assess the response of individual organs. In contrast, the body is a complex and dynamic system composed of multiple organs with complex interactions. For example, certain drugs are more active as metabolite derivatives (e.g., doxorubicin vs. adriamycin), and simple screening platforms may not be able to replicate the clinical pharmacological pathway correctly. Some studies have begun to link different organ testing platforms (e.g., organ chips), but only at the microscopic level (e.g., microfluidic devices), not applicable to systematic biological methods for large tissue engineered organoids.
Therefore, there remains an urgent need to develop new therapeutic testing platforms that are able to better predict the effects of candidate therapeutic agents on tissue engineered organoids in vitro, and to develop new tools to allow "organ" to "organ" interactions to analyze the overall functional response (i.e., systemic response). In addition, there is a need for accurate and efficient screening of compounds or agents that have a desired effect on organs effectively mimicked using in vitro tissue engineered organoids or tissues.
Despite the efforts expended to advance cardiac care, there remains a need in the art for more rapid and reliable identification of cardiac drugs with muscle strength effects. Positive inotropic agents are agents that enhance contractility of the heart, and are generally prescribed for patients with congestive heart failure or cardiomyopathy, or in some cases for patients who have had a recent heart attack. The negative inotropic agent can weaken cardiac contraction, and can be used for treating hypertension and angina pectoris. These muscle force effects may be effective when applied to the appropriate condition, but may also be toxic to other patients. Thus, the ability to sensitively detect these effects is invaluable for any cardiac efficacy/toxicity screening.
Disclosure of Invention
The present disclosure provides a three-tier system and related methods to facilitate drug discovery/screening using engineered human ventricular cardiac tissue strips (hvCTS) in a first tier of screening, which can be used to rapidly assay compounds with myodynamia effects and is suitable for large throughput screening formats. This first screening is typically combined with a second screening of compounds using the human ventricular organoid chamber (hvCOC) disclosed herein. Two-layer screening of compounds with myodynamic effects allows for a hierarchical focus on a compound of interest, while obtaining data regarding the effect of the compound on cells (e.g., cardiomyocytes, such as human ventricular cardiomyocytes) obtained in the first layer of screening using hvCTS, and combining this data with the effect of the compound on higher biological structures of related organoids (e.g., cardiac organoids) to confirm the results of the first layer of screening and reveal the effect at any organoid or organ level that is not apparent from the cells used in the first layer of screening. This method not only allows a rapid, progressive focus on compounds with myogenic effects, but also evaluates the effect of compounds in two different but related environments, namely a cell-based environment and an organoid or organ-based environment. Using this method, results are better than using any single-stage screening (e.g., currently used compound screening systems and methods)Method) to obtain more accurate and more reliable results in terms of reproducibility. The present disclosure further provides the possibility of optimal combination of third layer screening with first and second layer screening. In the third screen, the hvCOC organoid screening was extended to multiple organoids using the hvCOC system for multiple organs. The third level of screening produces data that is even more reliable than the data obtained from the two level of screening. Furthermore, the multi-class organ hvCOC format allows for the use of multiple organoids of the same type in the screening, e.g., cardiac organoids, and/or the use of different organoids simultaneously in a third tier of screening, using the universal multi-class organ hvCOC system disclosed herein. (As used herein, "organoid" generally refers to organ-like biological material, but the term may also refer to tissue that may be considered organ-like biological material. the meaning of terms used herein will be apparent from the context of their use.) this technique is general in that it is applicable to identifying compounds with positive muscle force effects and compounds with negative muscle force effects. In some embodiments, the human ventricular cardiac tissue comprises hPSC-derived Ventricular Cardiomyocytes (VCM). Single-cell characteristics of such cells, e.g. electrophysiology (action potential, Ca)2+Treatment), transcriptome, proteome, etc. have been widely characterized. As disclosed herein, various cells, such as human ventricular cardiomyocytes, can be used to develop organoids for the second tier hvCOC system and for the third tier multi-class organ hvCOC system.
The hvCOC system used in the second and third layers of the multi-layer systems and methods of the present disclosure is the first platform that simultaneously characterizes multiple in vitro tissue engineered tissues or organoids, including a mirror arrangement and a single detection device. Fluid-switching networks are provided on which organoids are interconnected to create a "mini-human" system that mimics the systemic drug response of human patients and can be used as a default in vivo model in place of animal testing. The semi-automated platform includes a number of features to facilitate the investigation of functional responses to delivered drugs, such as environmental control (e.g., temperature and C0)2) High speed camera, synchronized pressure-volume recording, interconnected fluid exchange system, drug perfusion, intra-organoidPressure control, mechanical stimulation and electrical stimulation. These features are designed to improve the culturing procedure, allow for examination of long-term drug exposure of tissues or organoids, and allow for simultaneous multi-tissue and/or multi-organ drug responses. Current in vitro therapeutic agent screens typically only assess acute responses of a single tissue or organoid, which expands the challenge and is costly. By using a single camera with a mirror arrangement for multiple types of organ imaging, this system is more scalable than currently available designs.
To develop the next generation in vitro mannequins, the bioreactor platform includes modular organoid cassette systems and fluid switching networks, enabling flexible system biology approaches. "plug and play" organoid cassettes expedite the process of imaging various tissues and/or organoid combinations of interest within a bioreactor. In addition, the circulation and exchange of culture medium between tissues or organoids may reproduce the circulatory system of a human. Signaling factors and metabolites may be freely exchanged between tissues or organoids and may affect the drug response of one or more tissues or organoids. For example, the metabolite adriamycin alcohol is known to be more cardiotoxic than the chemotherapeutic agent doxorubicin itself. This "body-in-a-jar" technique facilitates drug discovery and medical research with precision or personalization and is superior to organ-chip techniques that often fail to fully reproduce organ function due to the lack of three-dimensional tissue.
New molecular entities are characterized using clinically relevant endpoints (e.g., ejection fraction in heart tissue, permeability in lung tissue) and then classified using automated computer algorithms (e.g., machine learning) trained to detect biological activity and toxicity patterns. The multi-class organ imaging platforms disclosed herein increase throughput and can be used for higher content screening. By increasing throughput, the system becomes easier to screen for preclinical drugs. The platform is also used to probe basic biology in tissue engineered human constructs.
In addition to a layer of hvCTS systems and methods, the present disclosure provides a universal bioreactor platform for developing engineered organoid tissues that more closely mimics the corresponding humanThe in vivo structure and function of body organs. The bioreactor platform allows for control of various environmental variables and allows for real-time or end-point monitoring of tissue or organoid function using various probes and monitors. In addition, the present disclosure provides improved environmental control to achieve temperature control and various gases such as C02And control of oxygen. A well-controlled environment allows for stable culture of cells, and thus can be harvested for long periods of time for drug screening. Using a combination of a high-speed camera and a pressure sensor, the present disclosure provides a method of combining spatiotemporal motion of a displaced tissue or organoid (e.g., a contracting cardiac organoid) with pressure recording to measure pressure-volume relationships. In addition, the present disclosure provides a complex system for fluid exchange within a bioreactor platform by the synergistic use of fluid pumps, three-way valves, and fluid tanks, as opposed to some systems that use simple hydrostatic pressure systems. The fluid exchange system also provides for the connection of any number of organoids within the bioreactor. The fluid exchange system of the present disclosure provides the additional functionality of controlling the delivery of media for feeding, aspirating the media, mixing bioactive components (i.e., therapeutic agents), and injecting bioactive agents on an acute schedule (e.g., bolus injection) or a chronic schedule (e.g., perfusion). Biologically active components may include, but are not limited to, pharmaceutical compounds, viral vectors, conditioned media, progenitor cells, and extracellular vesicles. The fluid exchange system also provides for cleaning, rinsing or flushing of the fluid lines. In addition, the present disclosure provides mechanical stimulation of developing tissues or organoids by applying methods for mechanical stretching to the tissues or organoids having a cavity. By applying mechanical tension, the present disclosure provides a means for manipulating a tissue or organoid so long as the mechanical tension can act as a mechanical transduction signal. In contrast to electrical pacing of human cardiac organoids via field stimulation, the present disclosure provides point stimulation of such organoids, resulting in more accurate and refined stimulation of organoid tissue. With point stimulation, conductivity measurements within a tissue or organoid (e.g., brain, heart) can be accomplished, for example, by using optical mapping techniques. Further, in accordance with the present disclosure, machine learning principles are used in tissue or organoid behavior analysisThe application of (a) is expected to improve the assessment of the outcome of treatment response by fully analyzing and understanding the high dimensional parameter space. The present disclosure provides all of the benefits in an integrated package, which represents a significant advancement in the field of therapeutic screening, including new approaches, i.e., experimental assays that are expected to improve the prevention, treatment, and/or amelioration of symptoms of various diseases and conditions.
In one aspect, the present disclosure provides a system for screening compounds for changes in muscle contractility, comprising: (a) first stage screening apparatus comprising: (i) a biocompatible gel comprising a plurality of cardiomyocytes; (ii) a biocompatible support device for suspending a biocompatible gel, wherein the biocompatible gel and the biocompatible support device form a cardiac tissue strip; (iii) a detection device for detecting movement of the biocompatible gel; and (iv) a power source for applying an electrical pacing stimulus to the biocompatible gel; and (b) a second stage screening apparatus comprising: (v) at least one organoid module comprising at least one organoid cassette, wherein the organoid cassette comprises a medium inlet, a medium outlet, and at least one wall compatible with an external detection device, wherein each organoid cassette comprises a biomaterial comprising at least one human cell that is a human embryonic stem cell, a human adult stem cell, a human induced pluripotent stem cell, a cell derived from human tissue, or a progenitor cell of human tissue, wherein the at least one organoid cassette comprises cardiac biomaterial; (vi) a mirror arrangement for simultaneously monitoring any biological development of the biological material in each organoid cassette; and (vii) a detection device for observing the monitored biological development of the biological material in each organoid cartridge. In some embodiments, the cardiomyocyte is a human cardiomyocyte, such as a human ventricular cardiomyocyte. In some embodiments, the human cardiac muscle cell is derived from at least one human pluripotent stem cell. In some embodiments, the cardiomyocytes are present in an amount of at least 106Individual cells/ml are present. In some embodiments, the biocompatible gel comprises matrigel, e.g., a biocompatible gel in which matrigel is present at a concentration of at least 0.5mg/ml or 1 mg/ml. Matrigel may be obtained from stock solutions containing, for example, at least 5mg/ml or at least 10mg/ml matrigel. In some implementationsIn one embodiment, the biocompatible gel further comprises collagen. The present disclosure also provides embodiments wherein the collagen is type I human collagen. Some embodiments comprise a biocompatible gel, wherein the collagen is present at a concentration of at least 1mg/ml, for example, about 2 mg/ml. In some embodiments, the same biocompatible gel composition is used to form cardiac tissue strips in the first screening and cardiac organoids in the second screening. In some embodiments, the support apparatus is at least two vertical support members (e.g., embodiments in which there are two vertical support members), and those vertical support members may be made of polydimethylsiloxane. In some embodiments, the vertical support members (e.g., two vertical support members) are approximately circular in cross-section, with a diameter of about 0.5 mm. In some embodiments, the strip of cardiac tissue is a strip of cardiac tissue that is about 26.5mm in length by about 16mm in width by about 6mm in height, such as 26.5mm in length by 16mm in width by 6mm in height. In some embodiments, the detection device is a high-speed camera.
In some embodiments of the system, the mirror arrangement of the second stage screening apparatus comprises at least one angled cone mirror. The present disclosure also contemplates embodiments wherein the biological material is at least one cardiac tissue or at least one cardiac organoid. In some embodiments, the heart organoids are cultured in the same system as the other organoids. Thus, some embodiments also encompass another type of organ that is a heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gall bladder, lung, small intestine, colon, bladder, prostate, uterus, tumor, eye, skin, blood, or vascular organoid, for example where the other type of organ is a heart organoid. In some embodiments, the second stage screening apparatus further comprises an electrode in adjustable relationship to the tissue or organoid in the at least one organoid cassette. In some embodiments, the secondary screening apparatus further comprises a temperature control element, a light source, a module access port, or any combination thereof. In some embodiments, the system further comprises a data processor in electronic communication with the detection device, the temperature control element, the light source, the module access port, or any combination thereof. It is also understood that wherein the detecting means is a digital camera, at leastEmbodiments of a pressure sensor or a combination of a digital camera and at least one pressure sensor. In some embodiments, the system further comprises a tissue having at least one human cell. In some embodiments, the system further comprises a monitor. Embodiments of the system also include a plurality of organoid modules. In some embodiments, the system further comprises an interconnected fluid-switching network, wherein the network comprises a plurality of fluid lines, a plurality of valves, at least one pump, and at least one fluid tank. Embodiments are also contemplated in which the system further comprises a port for introducing the compound. In some embodiments, the interconnected fluid-exchange network comprises fluid communication between at least two organoid cassettes. In some embodiments, the fluid is a culture medium. In some embodiments, the fluid exchange network provides automated media exchange. Some embodiments of the system further comprise a pneumatic controller. In some embodiments, the pneumatic controller controls 0 in at least one module or in one or more organoid cassettes2And C02Of the concentration of at least one of the above. In some embodiments, the system further comprises a drug perfusion apparatus for delivering the compound to the cell, tissue, or organoid.
Another aspect of the present disclosure relates to a method of manufacturing a cardiac tissue strip, comprising: (a) providing a biocompatible mold having a length of about 26.5mm by a width of about 16mm by a height of about 6 mm; (b) forming a biocompatible gel conforming to the mold, wherein the biocompatible gel comprises matrigel, collagen, and a plurality of cardiomyocytes; and (c) securing at least two vertical support members to the biocompatible gel, thereby forming a strip of cardiac tissue. In some embodiments of the method, the vertical support member is secured to the biocompatible gel by embedding the vertical support member in a biocompatible gel formulation prior to gelation. In some embodiments, the vertical support member is affixed to the biocompatible gel by adhesion or by mechanical attachment.
Another aspect of the present disclosure relates to a method of screening for a compound having a myodynamic effect, comprising: (a) applying a test compound to a strip of cardiac tissue at a specific concentration (or equivalent volume of vehicle); (b) measuring spontaneous contractions of the cardiac tissue strip without electrical pacing; (c) pacing a cardiac tissue strip disclosed herein with an electrical stimulus at a pacing rate of 0.5Hz, 1.0Hz, 1.5Hz, or 2.0Hz, and measuring contractions of the cardiac tissue strip; (d) repeating the above steps with increasing concentrations of the test compound; (e) data obtained from cardiac tissue strips with low contractility (e.g. 0.01mN) were eliminated without treatment; (f) dose-response curves (contractile force versus dose) were plotted and dose-response curves of compound and vehicle treatment were compared to determine whether myodynamia activity was present (by distinguishing true pharmacological effects of test compounds from artifact effects of vehicle treatment); and (g) administering the potential inotropic compound to the tissue or organoid in the second stage screening device disclosed herein and monitoring the cardiac tissue or organoid for response to the compound, wherein a change in contractility (e.g., developing stress, stroke volume, and work in stroke in the cardiac organoid) identifies the potential inotropic compound as the inotropic compound. The present disclosure also provides a method of screening for a compound having a myodynamia effect, comprising: (a) pacing the cardiac tissue strip of
The examples disclosed below describe examples of each of the screening methods described above. In some embodiments, the pacing rate is 1.0 Hz. In some embodiments, the muscle force compound is identified as a potentially negative muscle force compound when motion of the paced cardiac tissue strip is less in the presence of the muscle force compound than in the absence of the muscle force compound. In some embodiments, when (a) the motion of the cardiac tissue strip paced in the presence of the myodynamia compound is less than the motion in the absence of the myodynamia compound; and (b) identifying the myodynamia compound as a negative myodynamia compound when the tissue or organoid in the secondary screening device exhibits decreased contractility in the presence of the myodynamia compound as compared to the absence of the myodynamia compound. In some embodiments, a myodynamia compound is identified as a potential positive myodynamia compound when the motion of the paced cardiac tissue strip is greater in the presence of the myodynamia compound than in the absence of the myodynamia compound. In some embodiments, when (a) the motion of the cardiac tissue strip paced in the presence of the myodynamia compound is more than the motion in the absence of the myodynamia compound; and (b) identifying the myodynamia compound as a positive myodynamia compound when the tissue or organoid in the secondary screening device exhibits increased contractility in the presence of the myodynamia compound as compared to the absence of the myodynamia compound. In some embodiments of this aspect of the disclosure, the compound is a drug, a viral vector, conditioned medium, extracellular vesicles, other cells, or any combination thereof. In some embodiments, the cardiac tissue or organoid is connected to (i.e., in fluid communication with) other organoids, such as the liver, to detect system-level responses to the test compound.
In other aspects, the present disclosure provides a system and method involving hvCOC of the second or third layer of the multilayer system and method described herein. One of these aspects relates to a tissue monitoring system comprising (a) at least one organoid module comprising a plurality of organoid cassettes, wherein each organoid cassette comprises a fluid (e.g., culture medium) inlet, a fluid (e.g., culture medium) outlet, and at least one wall compatible with an external detection device, wherein each of the plurality of organoid cassettes comprises a biomaterial comprising at least one human cell, wherein the cell is a human embryonic stem cell, a human adult stem cell, a human induced pluripotent stem cell, a cell derived from human tissue, or a progenitor cell of human tissue; (b) a mirror arrangement for simultaneously monitoring any biological development of the biological material in each of the at least two organoid boxes; and (c) a detection device for observing biological development of the biological material monitored in each of the at least two organoid cassettes. The wall compatible with the external detection device is a substantially flat, substantially vertical physical barrier at the periphery of the organoid box that effectively prevents loss of fluid (e.g., culture medium) due to leakage from the organoid box, while effectively allowing passage of electromagnetic radiation of any wavelength detectable by the external detection device (e.g., a digital camera). Exemplary walls compatible with the external detection device are glass or any effectively transparent thermoset or thermoplastic. Biological material is any cell or group of similar or different cells, tissues, organoids, organ systems, and is typically of human origin. Biological development of a biomaterial refers to any increase in the number or size, structure (e.g., size, shape, color, topology), or function/behavior (e.g., rhythmic or arrhythmic contractions) of the biomaterial relative to its state at a previous point in time. The tissue monitoring system disclosed herein provides a system comprising a plurality of biological cells of a higher order, such as found in a tissue or organ. Thus, a tissue monitoring system includes a system for monitoring a tissue, organoid, or organ. In some embodiments, the system comprises a tissue engineered organoid, e.g., a three-dimensional tissue engineered organoid, or a tissue, e.g., an ex vivo tissue. Organoids comprise the cellular and tissue characteristics of an organ such that the organoid exhibits at least one biological function of the corresponding organ. The mirror arrangement may be any arrangement of one or more mirrors that provides for monitoring of biological material in the form of one or more cells, tissues, organoids or organs in a plurality of organoid cassettes using fewer detection devices than organoid cassettes to improve monitoring efficiency and reduce monitoring costs. The system for tissue monitoring described in this paragraph can also be used as the hvCOC system described herein as the second and third layers of the multi-layered system that can be used to screen compounds for muscle strength activity.
In some embodiments, the mirror arrangement comprises at least one angled cone mirror. In some embodiments, the biological material is at least one tissue or at least one organoid, for example, embodiments in which the organoid is a heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gall bladder, lung, small intestine, colon, bladder, prostate, uterus, tumor, eye, skin, blood, or vascular organoid. In some embodiments, the organoid is a cardiac organoid. In some embodiments, the system further comprises an electrode in adjustable relationship to the cells, tissue or organoid in the at least one organoid cassette, e.g., for point stimulation (e.g., point electrical stimulation) and/or point monitoring of the behavior of at least one cell of the tissue or organoid. In some embodiments, the detection device is a recording device, such as a camera. In some embodiments, the recording device is a digital camera, at least one pressure sensor, or a combination of a digital camera and at least one pressure sensor. In typical embodiments where the recording device is at least one pressure sensor, there is a 1:1 correspondence between pressure sensors and organoid cassettes, i.e., one pressure sensor per organoid cassette containing the cells, tissues or organoids being monitored. In some embodiments, the system further comprises a temperature control element (e.g., a heater), a light source (e.g., an LED light or lamp), a module access port, or any combination thereof. In some embodiments, the system further comprises a data processor in electronic communication with the detection device, the temperature control element, the light source, the module access port, or any combination thereof. The electronic processor provides software and/or hardware-based control of the elements of the system and apparatus to control the environment, e.g., control fluid (e.g., media) flow by controlling pumps and valves in the interconnected fluid-switching network, and/or control heaters, lights, detection devices (e.g., cameras), and processes the results to generate a table that can be used for operator analysis (fig. 16).
The hvCOC system disclosed herein can also comprise a tissue having at least one human cell. In some embodiments, the system further comprises a monitor. In some embodiments, the system comprises a plurality of organoid modules. In some embodiments, the system further comprises a media mixer, such as a magnetic stirring device or a rotating disk.
In some embodiments, the hvCOC systems disclosed herein further comprise an interconnected fluid-switching network, wherein the network comprises a plurality of fluid lines, a plurality of valves, at least one pump, and at least one fluid tank. The interconnected fluid-switching network comprises any combination of fluid lines, valves, pumps, tanks, and/or organoid cassettes as disclosed herein, which are capable of collectively moving fluid through more than one path, and preferably between or among multiple organoid cassettes. Typical embodiments of hvCOC systems comprising interconnected fluid-switching networks control fluid flow using a data processor to control at least one valve and at least one pump. In some embodiments, the hvCOC system further comprises a port for introducing a compound, e.g., a therapeutic agent, a candidate therapeutic agent, a drug, or a candidate drug. In some embodiments, the interconnected fluid-switched network comprises fluid communication between at least two organoid cassettes, and in typical embodiments, the fluid communication is controlled by a data processor controlling at least one valve and at least one pump. In some embodiments, the interconnected fluid-exchange network provides a partially common fluid delivery path for the at least two organoid cassettes, a partially common fluid exhaust path for the at least two organoid cassettes, or a partially common fluid delivery path and a partially common fluid exhaust path for the at least two organoid cassettes. In typical embodiments, the fluid is a culture medium. In some embodiments, the fluid exchange network provides automated media exchange, for example by controlling fluid (e.g., media) exchange using a data processor to control at least one valve and at least one pump. In some embodiments, the hvCOC system further comprises a pneumatic controller, e.g. controlling 0 in at least one module or in one or more organoid cassettes2And C02A gas pressure controller of the concentration of at least one of (1). In some embodiments, the hvCOC system also includes a plurality of module access ports. Also contemplated are embodiments of the hvCOC systems disclosed herein, which further comprise a delivery system for delivering a therapeutic agent to a cell, tissue or organoidThe medicament infusion device of (1). In typical embodiments, a drug perfusion apparatus comprises a port for introducing a compound, at least one valve, and at least one pump, the port being coupled to at least one fluid line that extends into a organoid cassette containing a tissue, organoid, or organ to be exposed to the compound. Optionally, the drug perfusion apparatus further comprises a fluid line, and optionally, at least one valve and at least one pump, to remove fluid, such as culture medium, from the organoid cassette.
Another aspect of the present disclosure relates to a method for determining the biological activity of a compound, comprising administering the compound to a cell, tissue or organoid in the hvCOC system disclosed herein, and monitoring the response of the cell, tissue or organoid to the compound. In some embodiments, the compound is a drug, a viral vector, a conditioned medium, an extracellular vesicle, other cells, or any combination thereof. In several embodiments, the compound is delivered with a chemical or biological agent known to have an effect on the biological material in the organoid box providing the compound. In such embodiments, the effect of the compound on changes caused by a chemical or biological agent is monitored. In these embodiments, the compound and the chemical or biological agent may be co-administered or the administration may be offset in time. In some embodiments, the biological activity is modulation of the function of a cell, tissue, or organoid, thereby identifying the compound as a therapeutic agent for treating an organ-homologous disorder of the cell, tissue, or organoid. In some embodiments, the assay measures the toxicity of the compound.
A related aspect of the disclosure relates to a method for determining the biological activity of a compound, comprising administering the compound to a plurality of cells, tissues or organoids in the hvCOC system disclosed herein through an interconnected fluid-exchange network, and monitoring the response of the cells, tissues or organoids to the compound. In some embodiments, the compound is a drug, a viral vector, a conditioned medium, an extracellular vesicle, other cells, or any combination thereof. The compound may be delivered with or without chemical or biological agents having known effects on the biological material in the organoid box receiving the compound. In some embodiments, the biological activity is modulation of the function of a cell, tissue, or organoid, thereby identifying the compound as a therapeutic agent for treating an organ-homologous disorder of the cell, tissue, or organoid. In some embodiments, the assay measures the toxicity of the compound.
Another aspect of the present disclosure is an apparatus for monitoring organoid function comprising at least one organoid module comprising (a) a plurality of organoid cassettes, wherein each organoid cassette comprises a fluid (e.g., culture medium) inlet, a fluid (e.g., culture medium) outlet, and at least one wall compatible with an external detection device; (b) a scope arrangement for simultaneously monitoring at least two organoid cassettes; and (c) a detection device. In some embodiments, the detection device is a recording device. In some embodiments, the hvCOC system also includes a temperature control element, a light source, a module access port, or any combination thereof. In some embodiments, the hvCOC system also includes a data processor in electronic communication with the detection device, the temperature control element, the light source, the module access port, or any combination thereof.
Another aspect of the disclosure relates to organoids produced using the hvCOC system disclosed herein, wherein the organoids comprise a plurality of differentiated cells. In some embodiments, the organoid is a heart, brain, nerve, liver, kidney, adrenal gland, stomach, pancreas, gallbladder, lung, small intestine, colon, bladder, prostate, uterus, blood, tumor, eye, or skin organoid.
Other features and advantages of the disclosed subject matter will become apparent from the following detailed description, including the drawings. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Drawings
FIG. 1 is a screening platform for the myodynamia effects of agents on human hearts. In a method of screening for negative and/or positive inotropic agents, a three-layer flow diagram of a system comprising a screening platform is used. The first stage involves hvCTS (human ventricular cardiac tissue strip) screening. Initially, compounds were tested in hvCTS systems using a range of doses and different pacing rates (e.g., spontaneous, 0.5, 1, 1.5, and 2Hz) (n ≧ 5). Next, IC50 was determined for compounds identified as negative inotropic agents, while a second tier of screening was performed for compounds identified as positive inotropic agents or compounds that did not exhibit inotropic effects. The second layer of screening involved hvCOC (human ventricular cardiac organoid chamber) screening. Initially, compounds (n ≧ 4) were tested using hvCOC at the dose range reported for the first-layer screen and the pacing frequency reported for the first-layer screen. The dose-response relationship was analyzed for developing pressure, stroke work and cardiac output. In this second screen, the hvCOC screen was used to confirm compounds identified as positive inotropic agents in the first screen. EC50 was determined for compounds identified as inotropic agents. A third level of screening may also be used. The third screen was a hvCOC screen with multiple organoids. In this third screen, compounds (n ≧ 4) were tested using multiple classes of organ hvcocs at the dose range reported for the first screen and the pacing rate reported for the first screen. The dose-response relationship was analyzed for developing pressure, stroke work and cardiac output. The myodynamic effect exhibited by a given compound is thus confirmed in the presence of other organoids, indicating the behavior of the compound in one or more organs or organ systems. Consistent with the flow chart, human ventricular cardiac tissue strip (hvCTS) screening exemplifies the results of testing compounds in the hvCTS system at dose ranges and different pacing rates (e.g., spontaneous, 0.5, 1, 1.5, and 2Hz) (n > 5). The compound was determined to be a negative inotropic agent based on hvCTS results and IC50 determined from the fitted dose-response curve. Compounds were identified as positive inotropic agents based on hvCTS results and a second screening with human ventricular cardiac organoids screening, as described in USSN 62/592,083, which is incorporated herein by reference. Compounds that showed inotropic effects in the first screening with hvCTS can be identified by the second screening with human ventricular cardiac organoids. Compounds subjected to the second tier human cardiac organoid screening were analyzed at the dose range and pacing rate taught by the first tier hvCTS screening disclosed herein. The dose-response relationship was analyzed for developing pressure, stroke work and cardiac output. The results can be used to confirm that a compound is a positive inotropic agent, and then EC50 assays were performed on such compounds.
FIG. 2 engineered human ventricular cardiac tissue strips (hvCTS) and force measurements. A. Engineered human ventricular cardiac tissue strips (hvCTS) and force measurements. Schematic of hvCTS construction in custom designed PDMS bioreactor molds. One million hPSC-CM per tissue was mixed with ice-cold bovine type I collagen and Matrigel and polymerized to produce self-assembled hvCTS, which was immobilized between flexible end posts 0.5mm in diameter. After 7 days of culture, hvCTS was kept in the original mold for test compound screening, where twitch force was measured by tracking end post deflection in real time as the contractile tissue was field stimulated and the dose of test compound was escalated. Using hvCTS held in the original mold and by applying the beam bending equation based on elastic theory (F { (3 pi ER)4)1\2a2(3L-a)]Where F is the tissue contractile force; E. r and L represent Young's modulus, radius and length of PDMS columns, respectively;ais the height of the tissue above the post; is the measured tip deflection) to calculate the force to obtain a force measurement. B. Representative traces show the contraction profiles of nifedipine (nifedipine), isoproterenol, and tocainide (tocainide) at 0Hz to 2Hz electrical pacing. C. Dose-response curves for each drug were fitted for different pacing rates. Nifedipine, a negative inotropic agent, showed a decrease in EC50 from 2.27 μ M to 0.544 μ M as the pacing rate was increased from 0.5Hz to 2 Hz. The positive inotropic agent isoproterenol showed consistent EC50 with increasing pacing rates in the range of 0.031pM to 0.059 pM. At all frequencies tested, tocainide had no dose-dependent effect on contractile force. D. Force frequency analysis showed that nifedipine, at all pacing rates, caused a decrease in contractile force as the concentration increased. At all tested pacing rates, isoproterenol caused an increase in contractile force with increasing concentration. No relevant dose-dependent effects were observed with tocainide.
Figure 3 force-frequency analysis, normalized to the force generated at 0.5Hz (30bpm) at drug-free baseline conditions, shows that known myotonics cause a change in contractile force with increasing dose at all pacing frequencies within the effective concentration range. Negative inotropic agents including 2 of the calcium channel blockers and 4 class I antiarrhythmic drugs showed a decrease in contractility with increasing compound concentration at all 4 frequencies. Positive inotropic agents showed an increase in contractility with increasing drug concentration at all 4 frequencies. Data show mean + SEM for n-4-8.
Figure 4 force-frequency analysis, normalized against the force generated at 0.5Hz (30bpm) under drug-free baseline conditions, shows that contractile force responds to dose escalating 17 unknown compounds at all pacing frequencies over the effective concentration range. Unknown compounds were classified as positive inotropic, negative inotropic, and inotropic compounds according to force-frequency analysis. Of the 17 compounds, two (2) were misidentified. Data show mean + SEM for n-3-8.
Figure 5 screening of known myotonics with hvCTS. A. Representative traces show contraction profiles of known positive inotropic agents, negative inotropic agents, and drugs without known inotropic effects. B. The fitted drug dose-response curves at 1Hz pacing rate showed positive, negative and inotropic effects. C. IC50 for drugs exhibiting negative inotropic effects calculated at 1Hz pacing rate was compared to EC50 for drugs exhibiting positive inotropic effects. Left, from left to right: amitriptyline (amitriptyline), nifedipine, Quinidine (Quinidine), Lidocaine (Lidocaine), and Flecainide (Flecainide). Middle drawing, from left to right: lisinopril (Lisinopril), glibenclamide (Gilbenclamide), Norepinephrine (Norepinephrine), Dobutamine (Dobutamine), Caffeine (Caffeine), Milrinone (Milrinone), and Digoxin (Digoxin). D. Comparison of EC50/IC50 determined by blind and non-blind screening for myotonics.
Figure 6. representative hvCTS twitch force traces of 17 compounds screened in a blinded study. Unknown compounds were classified as positive inotropic, negative inotropic, and inotropic compounds according to force-frequency analysis. Of the 17 compounds, two (2) were misidentified.
FIG. 7 Blind screening of compounds with hvCTS. A. Representative traces show contraction profiles of drugs classified as negative, positive and no effect after data analysis. B. The fitted drug dose-response curves at 1Hz pacing rate showed positive, negative and inotropic effects. C. IC50 for drugs exhibiting negative inotropic effects calculated at 1Hz pacing rate was compared to EC50 for drugs exhibiting positive inotropic effects. Left, from left to right: amitriptyline, nifedipine, quinidine, lidocaine, and flecainide. Middle drawing, from left to right: lisinopril, glyburide, norepinephrine, dobutamine, caffeine, milrinone, and digoxin. D. Comparison of EC50/IC50 determined by blind and non-blind screening for myotonics.
FIG. 8 comparison of drug action in hvCTS and hvCOC. The effect of isoproterenol on human ventricular heart organoid chamber (hvCOC, a 3D heart organoid) function shows an increase in stroke work, cardiac output and pressure volume ring. A more robust increase in stress in the hvCOC heart organoids was observed relative to the increase in contractility in response to isoproterenol observed in hvCTS.
Fig. 9.a) a schematic view of a bioreactor system comprising an
Figure 10. three-dimensional rendering of an
Fig. 11.a) schematic view of a fluid exchange system for organoid cassettes comprising fluid lines, pumps, valves, pressure sensors and fluid tanks. Depending on the function, fresh medium is added to the medium tank using a particular arrangement of valves and pumps, such as B) suction or C). A detailed description of the illustrated embodiment of the bioreactor system is provided in example 6.
Figure 12.a) a graphical representation of a fluid exchange system consisting of fluid lines, pumps and valves directing culture medium between multiple organoid cassettes within a module. Multiple organoid types can be connected to simulate a "body in tank". B) A heart organoid with sufficient pumping capacity can be used as the only biological pump to form a self-powered "body-in-tank". Example 6 provides additional description of these embodiments of the bioreactor system.
Fig. 13.a) shows an embodiment of a bioreactor system showing inlet and outlet paths for medium exchange through a valve controlled organoid 1 (left pane: a cardiac organoid; and a right pane: liver organoids). B) Schematic of a mechanical stimulation system in which a reversible fluid pump is connected to the
Figure 14, for operation of the bioreactor platform or system LabVIEW front panel schematic. A) A plurality of organoids acquisition preview window. B) An environmental control panel. C) Electrical stimulation parameters. The user may choose to control the voltage power, alter the frequency, select the chamber to stimulate and decide to send a continuous stimulus or a single pulse. D) Real-time pressure and volume data of four different organs. Pressure is represented by the grey line and organoid volume is represented by the black line. E) The parameters are recorded.
FIG. 15 MATLAB analysis to generate average P-V loops from the collection. A) The average volume contraction curve of the tissue is calculated. Each volume contraction of the beat is plotted as a scatter plot, with the maximum contraction time set to t-0 seconds. The average curve is shown as a red solid line. B) The mean P-V loop plot for multiple contractions is summarized. The red circles represent values at the sampling time points.
Figure 16 is a flow diagram of LabVIEW software for monitoring cells, tissues and organoids in the systems and devices disclosed herein. The flow chart schematically illustrates the software-based control of environmental variables, such as temperature and C02Horizontal, and software-based control of features of the system and apparatus, such as lens control, illumination control, and electrical stimulation control of cells, tissues, and/or organoids contained in the system or device.
FIG. 17 screening of known myotonics with hvCTS. A. Representative traces show contraction profiles of known positive inotropic agents, negative inotropic agents, and drugs without known inotropic effects. B. Dose-response curves for positive and negative inotropic agents fitted at a pacing rate of 1 Hz. The dose-response relationship of drugs without known muscle action is irrelevant. C. IC of negative inotropic agent calculated at 1Hz pacing rate50EC for comparative and positive inotropic agents50And (6) comparing.
Detailed Description
The present disclosure provides multi-layer systems and related methods for screening compounds for muscle force effects, and may also be used to assess the effects, e.g., toxicity, of other compounds on biological cells, tissues, and/or organs. The systems and methods effectively identify and characterize positive and/or negative inotropic agents. Typical configurations involve a two-layer system and related methods, where the first layer is designed to provide accurate and rapid, versatile, and cost-effective initial screening of compound myodynamia. The first layer system comprises a Cardiac Tissue Strip (CTS), such as a human ventricular cardiac tissue strip (hvCTS) supported in a manner that allows significant flexibility in gel movement, and associated detection (e.g., recording) means to capture gel movement in the presence or absence of a test compound. CTS is easy to prepare and is used in a simple method to screen compounds for their ability to induce gel movement of embedded cells. The first layer system and method is applicable to high throughput formats as well as conventional formats. The second layer screening systems and methods involve tissues and organoids that are developed and maintained in organoid cassettes typically located in organoid modules, as described herein. The second layer of screening involves exposing the tissue or organoid to a compound, preferably a compound exhibiting myodynamic effects, in a cassette placed in an environment where the detection (e.g., recording) device can monitor organoid behavior. The environment also typically provides for the delivery and removal of fluids, such as culture media and compound-containing fluids, under controlled conditions, requiring various controls to maintain an environment compatible with tissue or organoid viability. The two-layer system is used in a two-layer process that reveals compounds having muscle strength effects at the cellular, tissue and/or organoid or organ level, thereby improving the accuracy and reliability of results obtained in screening compounds having muscle strength effects. In addition, the present disclosure provides three-tier systems and methods that relate to the two-tier systems and methods described above and complement the third-tier systems and methods that relate to multi-class organ (or multi-tissue or mixed tissue and organoid) modular systems and related methods. In this third level, multiple tissues and/or organoids are developed and maintained in different organoid cassettes that can be conveniently located in a single organoid module (it is understood that organoid cassette organoid modules can contain tissues or organoids) which can be of the same type (e.g., heart) or of different types (e.g., heart and liver). This typical arrangement conveniently allows a single mirror system (e.g. a pyramid system) to be used in conjunction with a single detection (e.g. recording) device. Information is obtained regarding the myogenic effect of the compound on the cells and the effect of the compound on one or more homologous tissues, organoids or organs, or a plurality of different tissues, organoids or organs, including the myogenic effect, when the compound is subjected to the three-layer system and method. The triple-tier screening system and method further enhances the data obtained in terms of accuracy, reliability and repeatability, and adds manageable costs in terms of money and time.
Three-dimensional (3D) engineered tissues have advantages over two-dimensional (2D) preparations in drug screening methods because 3D exhibits greater physiological relevance, including gene expression and electrophysiology, when compared to 2D monolayers. Consistent with this view, the following facts are demonstrated herein: higher order 3D tissues are functionally more mature and more accurately display contractile responses when exposed to known agents.
One advantage of the hvCTS in the first layer of the screening system disclosed herein is that the shrinkage characteristics are measured intact in the PDMS mold without the need to transfer the hvCTS to another container, thereby achieving a higher degree of standardization and higher throughput while retaining the possibility of long-term experiments. It has been recognized that there may be variations between batches of tissue, and that approximately 10-fold variations may result. Without wishing to be bound by theory, this variation may be due to variations in the geometry and elasticity of the artificially fabricated PDMS columns, leading to variations in force calculations. Inclusion and exclusion criteria have been introduced based on about 200 hvCTS, which have been analyzed to objectively and systematically identify outliers that are less than 2% of the hvCTS made for the study. In particular, those hvCTS that developed a co. olmn (< 10% percentile) force upon pacing at 1Hz under baseline, drug-free conditions were excluded from the study.
The hvCTS system comprises a biocompatible gel. The gel may be prepared from(e.g., Cultrex)) Or any biocompatible compound or mixture capable of forming a gel and providing an environment in which the cells contained in the gel are capable of functioning in a manner consistent with their natural physiological function. Biocompatible gels can be formed from agarose, gelatin, and other compounds and compound mixtures known in the art, wherein
Embodiments of the hvCTS system include biocompatible gels that include cardiomyocytes, such as human cardiomyocytes (e.g., human ventricular cardiomyocytes). The cardiomyocytes may be derived from cardiac tissue, cardiac organs, or may be derived from human pluripotent stem cells. Cardiomyocytes can be present in a range of concentrations in a biocompatible gel, e.g., 104-109Individual cells/ml, e.g. 106Concentration of individual cells/ml biocompatible gel. The primary focus of the disclosed systems and methods is cardiomyocyte behavior and cardiac physiology, producing "hvCTS" in the name of the disclosed systems, which contemplate biocompatible gels containing myocytes (e.g., skeletal and smooth myocytes) and other cells suitable for physiological monitoring using the disclosed systems. The same biocompatible gel is also suitable for preparing the heart organoids used in the second layer screening, allowing direct comparison of the results obtained in the two layer screening.
In embodiments of the hvCTS system that include cardiomyocytes, the biocompatibility of the gel allows the cardiomyocytes to interact in a manner that results in a collective ability to contract and relax under conditions that induce contraction and relaxation behavior in vivo. In particular, the cardiomyocytes contained in the biocompatible gel co-contract under electrical stimulation at the pacing rates disclosed herein, and the contractile force can be altered by known inotropic agents that increase the contractile force induced by a given electrical pulse and known negative inotropic agents that decrease the contractile force induced by a given electrical pulse. The electrical pulses may be provided by any suitable power source capable of delivering electrical pulses of 10V for a duration of 5 milliseconds at a delivery or pacing rate of 0.1-10Hz, for example by providing a pacing rate of 0.5, 1.0, 1.5, and/or 2.0 Hz.
Formation of the biocompatible gel is achieved using a mold of any suitable set of dimensions and is made of any material compatible with gelation of the biocompatible gel material, e.g. containing human ventricular cardiomyocytesIn some embodiments, the mold is made ofGelling and cardiomyocyte compatible polydimethylsiloxane formation. A typical shape of the mold cavity is a rectangular prism (i.e., a three-dimensional rectangle) whose depth (i.e., height or thickness) is not less than the intended thickness of the biocompatible gel to be formed in the mold. As disclosed in example 1, a typical mold cavity may have dimensions of about or exactly 26.5mm x 16mm x 6mm, but it will be appreciated that the dimensions of the biocompatible gel and the mold used for its formation may be scaled up or down and may vary greatly from the exemplary dimensions disclosed.
The hvCTS system disclosed herein also provides a biocompatible support device for biocompatible gels. The biocompatible support device allows for placement of the biocompatible gel in air, away from any solid surface support other than the biocompatible support device. The biocompatible support device allows the biocompatible gel to have a greater freedom of movement, particularly in the vertical plane. As a result of the greater freedom of movement, any movement of the biocompatible gel due to contraction can be detected while reducing or minimizing interference from the support structure. Thus, the biocompatible support structure for the biocompatible gel is at least two vertical support members, and typically two vertical support members (e.g., legs or posts). The vertical support member may be constructed of any biocompatible material capable of stably supporting the weight of the biocompatible gel. An exemplary material for supporting a device (e.g., a vertical support member) is Polydimethylsiloxane (PDMS). Any shape compatible with the function of the support device in stably supporting the biocompatible gel is contemplated, exemplary shapes being at least one strut having a cross-sectional shape in the form of a circle, oval, rectangle, triangle, or at least a pentagonal geometric plane. In embodiments comprising at least two vertical support members as the biocompatible support device, the vertical support members may be the same or different in shape. An exemplary biocompatible support device is two approximately circular posts made of PDMS with a diameter of about 0.5 mm. Typically, the height of the vertical support members is similar or the same, resulting in the biocompatible gel being substantially horizontal, including an appearance that is level to the naked eye. The support device, e.g., the at least two vertical support members of some embodiments, is affixed to the biocompatible gel by adhesion or mechanical attachment or surface tension, or the support device may be integrated into the biocompatible gel by embedding the support device in a biocompatible gel formulation (i.e., a biocompatible gel material) during gel formation. The biocompatible gel and the biocompatible support device are combined to form a tissue strip, and when the cells contained in the biocompatible gel are cardiomyocytes, the tissue strip is a cardiac tissue strip.
The hvCTS system also includes a detection device, which is any camera, camcorder, film recorder or digital recording device capable of detecting transient movements of the biocompatible gel. An exemplary recording device is a high speed camera, as described in example 1, wherein the camera can record about 100 frames per second. In some embodiments, the detection device is in electronic communication with a source of computing power (e.g., a computer) that can record and optionally analyze any movement detected by the device. The software that can confer the recording and/or analyzing function, for example on a computer, comprises motion detection/analysis software that is robust enough to process the motion data detected by the detection means. An exemplary software is LabView software.
Book of JapaneseA method of manufacturing a cardiac tissue strip by providing a biocompatible mold (e.g., PDMS mold) having a cavity of appropriate shape and size to form a desired biocompatible gel is also provided. Introducing a biocompatible gel material (e.g.And type I human collagen) with a plurality of cells (e.g., human ventricular cardiomyocytes) and adding the mixture to the mold cavity under conditions in which gelation of the gel material will occur. A sufficient amount of gel material is added to the mold to cover the bottom of the cavity and fill the cavity to the height required for the biocompatible gel. For containing
The hvCTS system can also be used to screen for inotropic agents or compounds that alter cardiac contractility. The system can be used to screen positive inotropic agents, i.e., compounds that induce an increase in contractility, and to screen negative inotropic agents that decrease contractility. In practice, the hvCTS system with a biocompatible gel containing cells of interest (e.g., human ventricular cardiomyocytes) is exposed to a lead supplying a power source at a pacing rate of 0.1-10Hz (e.g., 0.5Hz, 1.0Hz, 1.5Hz, or 2.0Hz) for a duration of 5 milliseconds at 10V. A pulse is applied to the biocompatible gel, with or without the biocompatible gel contacting the candidate myogenic agent, and the detection device is operable. In typical embodiments, any movement of the biocompatible gel is detected and analyzed using a computational power source (e.g., a computer) in electronic communication with a detection device, which is typically a high speed camera, but may also be a camcorder, a film recorder, or a digital detection and optionally recording device capable of detecting movement of the biocompatible gel. A compound is identified as a myogenic agent if the movement of the compound causes a change in the biocompatible gel relative to the movement of the gel in the absence of the compound. A compound is identified as a negative inotropic agent if exercise is less in the presence of the compound relative to absence; and a compound is identified as a positive inotropic agent if the biocompatible gel movement is greater in the presence of the compound relative to in the absence. Screening methods using the disclosed hvCTS system provide a rapid initial screen for biologically active compounds capable of modulating cardiac contractility. The disclosed methods are suitable for high throughput screening of compounds and provide rapid, cost-effective and accurate preliminary assessment of potential therapeutic agents. It is expected that the methods and systems disclosed herein will be used as a first level of multi-level development work to identify cardiac therapeutic agents, for example, by performing a second-order screen for compounds identified as positive or negative inotropic agents in the disclosed methods using, for example, cardiac organoids that will be found in cardiac organoid chambers in systems engineered for screening for bioactive compounds.
To confirm the effectiveness and reliability of hvCTS and hvCOC systems as in vitro screening systems for identifying contractile responses to a variety of pharmacological compounds, pharmacological compounds of the major drug classes were screened in blind and non-blind environments. In the blind screening disclosed in the examples contained herein, all negative inotropic agents pass an EC in the reported range of human ventricular tissue preparations50The estimate is correctly identified. Furthermore, the disclosed hvCTS system is sensitive enough to accurately discern differences in potency between the same class of drugs, as demonstrated in the case of L-type calcium channel blockers. As with other engineered cardiac tissues previously reported, the hvCTS system responds poorly to positive inotropic agents (including isoproterenol and digoxin) [3, 4 ]]. Analysis of drug responses at increasing concentrations over the pacing frequency range enables us to accurately identify those pharmacological compounds with the reported positive inotropic effects. Although most positive inotropic agents had little effect on hvCTS when successfully measured, the EC50 measurement was found to be comparable to that knownLiterature values are consistent and only 1 of the 8 tested compounds was found to be out of range. Thus, the experimental results reported herein demonstrate that a simple and rapid hvCTS screening system reduces false positives and false negatives. The predictive power of the hvCTS screening system was further evaluated in a blind screen and the accuracy of negative, positive and negative muscle action was found to be 100%, 86% and 80%, respectively. Interestingly, the hvCOC screening system disclosed herein, which is suitable for the second layer of a two-or three-layer system, is a screening system with an early maturing environment that generates physiologically complex parameters that exhibit enhanced positive inotropic force. Based on these results, the two-tier screening system is particularly useful for avoiding false positives and false negatives. The use of such a screening system will facilitate drug discovery by leading to better overall success, closing the long-term gap between inaccurate animal models and human patients. Thus, it is expected that the screening system disclosed herein will lead to better overall screening success and revival of drug development pipelines.
Most of the EC50 values reported for various cardiac active agents were obtained from in vitro isolated myocardial bars, and the disclosed hvCTS system was comparable to such isolated myocardial bars. Isolated mouse or rat hearts have also been studied to measure cardiac function of various drugs with cardiovascular effects. However, even for these animal models, it is rarely reported to have EC50Estimated dose range. The experimental results disclosed herein provide the first EC50 value for a variety of pharmacological compounds in a human heart tissue-based assay system.
Blind studies have shown that the first-level screening has limitations in detecting positive inotropic effects of compounds, with false positive (lisinopril) and false negative (dopamine) results. As heart organoids become more mature, it is expected that positive inotropic effects will depend on more sophisticated adrenergic signaling and calcium processing and thus can be detected more accurately and sensitively. The secondary screening device provides a platform for simultaneous monitoring of one or more human organs (including at least one cardiac organoid) and provides for automated culture, regulation, signaling between multiple organoids, and evaluation of functional human substitutes for high-content, species-specific in vitro screening of potential myogenic compounds to enhance preclinical testing of efficacy and toxicity of new therapeutics as a preferred alternative to animal testing. The use of a secondary screening system is expected to accurately identify myogenic compounds in a reliable manner using a rapid and cost-effective screening method. Simultaneous recording of data from multiple organoids is expected to greatly improve consistency and throughput, particularly for experiments analyzing long-term drug responses (i.e., over 24 hours and up to several weeks). Gaps exist in the current in vitro therapeutic agent screening methods, and rare adverse reactions cannot be explained. Therapeutic screening is usually performed in an acute fashion (i.e., within 5-30 minutes after drug exposure, with a short data acquisition time) and may not accurately predict long-term effects or miss rare events. The secondary platform also provides many of the features required for the screening system, such as rapid, high-throughput initial screening at
Human stem cells and their derivatives provide exciting opportunities for the development of in vitro therapeutic screening platforms because they are of human origin and function similarly to the natural human cells after differentiation. However, standard two-dimensional (2D) cell cultures or three-dimensional (3D) embryoid bodies cannot replicate the essential features of actual human organ tissues. Researchers have reproduced the characteristics of human tissue when using tissue engineering techniques to engineer predictive organoid models. Combining rapid initial screening of cells contained in a biocompatible gel with multiple organoids that can be used for secondary screening in a single drug screening platform can and is expected to further improve the reconstitution of human function, thus increasing our ability to predict therapeutic effects in humans, providing a more efficient and elegant alternative to animal testing.
The present disclosure provides a modular imaging platform in which a data processor (e.g., a computer) simultaneously controls at least one or more recording devices (e.g., a camera), wherein each image may record the behavior of a gel containing cardiomyocytes or may record the behavior of multiple organoids (fig. 9). Figure 16 shows a software diagram for implementing such control. The organoid module of the secondary device is depicted as a single bioreactor housing for multiple organoid cassettes, which features facilitate therapeutic agent screening (fig. 10). These features include (a) a mirror arrangement (e.g., a keratoscope) for multi-class organ acquisition using a single recording device, (b) a fluid exchange system for automated medium exchange (fig. 11) and (c) controlled exchange of medium between organoids (fig. 12). Other features include (d) a mixer (e.g., a magnetic stirrer) for efficient mixing of the culture medium solution; (e) temperature control element and C02A control element for controlling physiological environmental conditions for long term culture and collection; (f) pressure control for mechanical stimulation during tissue formation and culture (fig. 13B); (g) electrical point stimulation for controlling the rate of beating; (h) perfusion for automated delivery of a therapeutic agent; and (i) a valve for directing fluid through the organoid (fig. 13A).
Examples of the invention
Example 1
In this example, a method for conducting the study disclosed in the following example is described.
Cardiomyocytes were derived from human pluripotent stem cells. All experiments used cells derived from the human embryonic stem cell line (hESC line) hES2(ES02, Wicell, Madison, WI, ETSA) propagated in feeder-free culture in mTeSR1 medium (stem cell Technologies) on Matrigel. Using prior developments[1]And incorporated herein by reference, using a small molecule Wnt inhibitor IWR-1 to effect directed cardiac differentiation. After
Generation of hvCTS constructs. On day 14 of differentiation, the cell clusters were lysed into single cells using a trypsin/EDTA solution (0.025% trypsin/EDTA; Invitrogen), held for 20 minutes, and then resuspended in differentiation medium. After 48 hours, cells were harvested by centrifugation (300g, 5 min) at
Drug screening was performed with hvCTS. First, a pharmacological compound to be used for screening is prepared. Compounds for non-blind "open label" screening were purchased from Sigma and stock solutions were prepared at the highest possible concentration according to product information. Working concentrations in the 5-log range were prepared for each drug.
In one blind screening described herein (see, e.g., example 4), powders of 17 compounds were reconstituted in DMSO and prepared according to the information provided by the supplier. Working concentrations in the 5-log range were prepared for each drug according to the dose range recommendations provided by the supplier.
Dose response and force measurements. The medium containing neonatal calf serum was replaced with DMEM high glucose medium containing HEPES but no phenol red for optical measurement of the forces generated in hvCTS. The optimal volume for these experiments was determined to be 5 mL. Using an integrated flexible PDMS column as a force sensor, the deflection of the column was captured in real time by a high speed camera (100 frames/sec) and LabView software (National Instruments, Austin, TX, USA), and the twitch force was calculated using the beam bending equation derived from elastic theory as previously describedAs described and incorporated herein by reference. These measurements were obtained by maintaining hvCTS in the original mold from which it was produced and soaking hvCTS in 37 ℃ medium. For each drug, the force generated in response to the 9 doses was measured by adding the drug to hvCTS in half-log increments in a cumulative manner. For each dose, the forces generated using the field electrodes at 0Hz, 0.5Hz, 1Hz, 1.5Hz and 2Hz electrical pacing were measured.
And (6) analyzing the data. Data were calculated and analyzed by a custom MatLab program. The force frequency curve for each drug was generated by plotting the forces generated at different concentrations at 0.5Hz, 1Hz, 1.5Hz and 2 Hz. All forces were normalized to the force generated at 0.5Hz (100%) at no drug baseline conditions.
Dose-response curves were constructed by plotting the force generated at 1Hz as a function of the logarithm of the drug concentration. The force generated under the drug-free baseline condition was defined as 100%. The force generated at each drug concentration was then normalized to the force generated at the relevant no-drug baseline condition. The EC50 for each test drug was estimated by fitting to a four parameter dose-response equation as shown below using commercial software (GraphPad software inc.): y ═ Bottom + (Top-Bottom)/(l + 1O)(LogEC50-X) Hill slope). X is the logarithm of the drug concentration; y is the normalized force generated at a pacing rate of 1 Hz; top and Bottom are the Y values of the Top and Bottom flat areas of the curve itself; the baseline is the Y value defining 0% (maximal inhibition by the standard drug). Hill Slope (Hill Slope) quantifies the steepness of the dose-response curve (the higher the Hill Slope, the steeper the curve).
Production of human ventricular cardiac organoid chambers (hvcocs). Initial step of hvCOC manufacture, from dissociation of cell clusters to preparation with
To create the chamber, we follow the procedure disclosed in and incorporated by reference herein29A similar procedure. Briefly, the top of a 3 × 3 × 6cm polystyrene bioreactor was modified to allow a
The balloon was filled with distilled water to the desired chamber size and placed concentrically in a 2% agarose mold in phosphate buffered salineProduced in water (PBS) and made by inserting a 13mm diameter test tube (VWR) into the center of the agarose. The agarose mold forms the outer mold boundary and the catheter balloon forms the inner boundary. The catheter was pulled gently towards the hypotube and clamped over the hypotube with a nylon screw compressor clamp (Fisher Scientific) to ensure that the balloon did not move within the mold. 1mL of ice-cold sterile tissue mixture was transferred to the mold to ensure complete coverage of the porous polyethylene ring. The whole apparatus was brought to 37 ℃ and 5% C02The gel polymerization was initiated by incubation for 2 hours and then immersed in NCS medium. After 24 hours, the top of the bioreactor with the hypodermic tubing, catheters and tissues was removed from the agarose and transferred to another polystyrene bioreactor containing 20mL of NCS medium. In this environment, at 37 ℃ and 5% C02The tissue was then maintained for 10 days, with half of the medium replaced daily, during which time the tissue was compacted around the balloon nucleus to form an artificially engineered heart organoid.
Characterization of the contractile function of hvCOC. After 10-12 days of culture, the silastic balloon was deflated and carefully removed from the organoid interior. A high sensitivity differential pressure sensor (Millar) was inserted into the lumen of the chamber through a closed loop fluid system connected to a large media reservoir. A digital camera (PixelLINK) mounted outside the bioreactor allows direct tissue monitoring with images acquired at 43 frames/second. Chamber pressure and digital video were acquired simultaneously using a custom acquisition program built into LAB VIEW (National Instruments) and chamber cross-sectional area was extracted from the video using a custom script in matlab (mathworks). The chamber volume is estimated by assuming an equivalent sphere of the same cross-sectional area. Functional pump analysis was performed using a GRASS S88x stimulator (Astro-Med) at the specified frequency with field stimulation of 63 mV/mm. The stimulation pulse length is 50 milliseconds. Both the pressure signal and the video signal were passed through a digital low pass filter with a 13Hz cutoff frequency in MATLAB prior to data analysis.
Example 2
Drug screening protocols based on hvCTS were developed. To validate hvCTS as a drug screening model, hvCTS was electrically paced at 0Hz, 0.5Hz, 1Hz, 1.5Hz, and 2 Hz. The results show a flat to positive force-frequency relationship from 0.5Hz to 1Hz and a negative force-frequency relationship from 1Hz to 2Hz, with decreasing contractile force produced as pacing rate increases (fig. 2 and 3). When nifedipine, a negative inotropic agent, was administered to hvCTS, a dose-dependent decrease in contractile force production was observed at all tested pacing rates (fig. 2C, 2D and 3). The IC50 determined at 0.5Hz, 1Hz, 1.5Hz, and 2Hz was 2.3. mu.M, 0.62. mu.M, 0.61pM, and 0.54pM, respectively. When the positive inotropic agent isoproterenol was administered to hvCTS, a dose-dependent increase in contractile force production was observed at all tested pacing rates (fig. 2C and 2D). EC50 determined at 0.5Hz, 1Hz, 1.5Hz, and 2Hz was 0.039pM, 0.059pM, 0.054pM, and 0.031pM, respectively. No consistent change in the resultant contractile force was observed at the different frequencies tested for tocainide. Since there were no significant differences in measured EC/IC50 at different pacing rates, and a positive force-frequency relationship was observed only at 0.5Hz to 1Hz, EC/IC50 was determined for subsequent screening at a pacing rate of 1Hz after analyzing the force-frequency relationship for all concentrations of the drug test to determine the dose-dependent effect on contractile force produced in the tested hvCTS.
Example 3
Screening for agents known to affect cardiac contractility. As a next validation step, drugs with known effects on cardiac contractility were tested. A total of 13 drugs known to have a positive, negative or no effect on cardiac contractility were tested. The hPSC-CM used to make the hvCTS used in the study had an average purity of 80. + -. 2.2% cTnT + cells. At baseline, spontaneous contraction occurred in 65.7% (n ═ 70) hvCTS. All hvCTS produce contractile force under electrical stimulation. When paced electrically at 1Hz, the average contractile force generated was 0.076+0.025mN (n ═ 70). Those hvCTS (see table 1) that produced less than o.olmn (less than 10% percentile) force when paced at 1Hz under baseline drug-free conditions were excluded from the study.
Calcium channel blockers, including verapamil (verapamil), nifedipine, bepridil (bepridil) and mibefradil (mibefradil), all showed a dose-dependent decrease in contractility produced in the hvCTS tested, with calculated IC50 of 0.078 μ M, 4.2pM, 3.3pM and 38pM, respectively, exposed to the calcium channel blocker at a pacing rate of 1Hz (fig. 3, 17B and 17C). Two class I antiarrhythmics, namely propiram (disopyramide) and flecainide (flecainide), also showed a dose-dependent reduction in contractility produced, with a significantly higher EC50 of 80pM and 75pM, respectively (fig. 3, 17B and 17C), while the other two class I antiarrhythmics, procainamide (procainamide) and tocainide, did not show any effect on contractility produced at all concentrations and pacing frequencies tested (fig. 17B). Beta agonists, including isoproterenol and dobutamine, are known positive inotropic agents that show a small but constant dose-dependent increase in contractile force produced at all tested pacing rates, with EC50 of 0.15pM and 0.015pM, respectively, where contractile force was measured using a 1Hz pacing rate (fig. 17B and 17C). Other known inotropic agents, including the calcium sensitizers levosimendan, the KATP channel inhibitor glibenclamide, and the phosphodiesterase inhibitor milrinone, also showed dose-dependent increases in contractile force produced at all pacing frequencies, with EC50 in the submicromolar range (levosimendan 0.15 pM; glibenclamide 0.93 pM; milrinone 0.41pM) (fig. 17B and 17C).
Table 1: comparison of EC50/IC50 values for test compounds measured with the hvCTS assay and reported as literature.
Table 2 summarizes the analysis of baseline data obtained from open label, non-blind drug screening assays.
Table 2: baseline data for viability, spontaneous frequency and purity of hPSC-CM used in hvCTS for open-label non-blind drug screening assays
Example 4
The pharmacological compounds affecting cardiac contractility were screened blindly. Blind screening of drugs was performed to study the effect of drugs on cardiac contractility. A total of 17 drugs with positive, negative or no effect on cardiac contractility but unknown identity at the time of testing were tested using the hvCTS screening protocol as described above. The average contractile force produced when electrically paced at 1Hz was 0.060+0.005mN (n-128). Of the 17 drugs screened, amitriptyline, nifedipine, quinidine, lidocaine and flecainide were found to exhibit a dose-dependent decrease in contractile force at all tested pacing rates and were therefore correctly identified as having a negative myodynamic effect when the drug identity was unknown (fig. 7B; see also fig. 4, 5 and 6). The IC50 values determined at 1Hz were 0.45mM, 0.15. mu.M, 0.20mM, 0.44. mu.M and 15pM, respectively (FIGS. 5C and 7C). Based on force-frequency analysis at all concentrations, glibenclamide, norepinephrine, dobutamine, caffeine, milrinone, and digoxin were correctly identified as having positive inotropic effects on cardiac contraction (fig. 4, 5B, 6, and 7B). EC determined at 1Hz pacing rate50The values were 3.2. mu.M, 0.35pM, 0.25pM, 0.29pM, 2.8pM and 3.7pM, respectively (FIGS. 5C and 7C). Dopamine, a catecholamine, is also a beta-agonist and therefore has a positive inotropic effect, with no significant effect on the contractility generated in our hvCTS system. The other four drugs, including aspirin, pravastatin, tolbutamide, and ramipril, did not have known myogenic effects and did not show any dose-dependent effect on contractility in the hvCTS tested (figures 4, 6, and 7B). Interestingly, lisinopril, which had no known effect on contractility, showed a slight increase in contractility produced with increasing concentration, and was therefore classified as having a positive inotropic effect. In general, blind screening using the hvCTS screening protocols and systems disclosed herein yields overall predictionsThe capacity was 0.76, the sensitivity was 0.78 and the specificity was 0.76.
Table 3 summarizes the analysis of baseline data obtained from blind drug screening assays.
Table 3: baseline data for viability and purity of hPSC-CM used in hvCTS for blinded drug screening assays
Development power at 1HZ
% CTNT + (by batch)
Number of values
128
30
Minimum value
0.0020
38.60
25% percentile
0.01869
61.30
Median value
0.0445
73.45
75% percentile
0.0875
85.83
Maximum value
0.2700
92.20
10% percentile
0.007918
42.19
90% percentile
0.1338
87.88
Mean value of
0.05951
71.46
Standard deviation of
0.05326
16.68
Standard error of mean
0.004707
3.045
Example 5
Second tier drug screening protocol using hvCOC. Since the increase in contractile force produced by hvCTS in response to compounds known to have positive inotropic effects is small, the effect of these positive inotropic agents was studied in higher order three dimensional (3D) constructs (i.e., hvcocs) that are significantly larger in size (e.g., 1 million versus 1 million cardiomyocytes) than the hvCTS disclosed herein and have a pump-like configuration as opposed to the trabecular-like configuration of hvCTS. The hvCOC construct, which received isoproterenol treatment, caused a concentration-dependent increase in stroke volume, cardiac output and developed pressure (fig. 8). When compared to the maximum effect obtained at 10pM isoproterenol, hvCOC caused 132% of baseline pressure compared to hvCTS caused 113% of baseline pressure (fig. 8).
Example 6
A bioreactor. The present disclosure provides custom bioreactors for culturing at least one, and in some cases, a variety of tissue engineered human organs. In some embodiments, a bioreactor is used as the second layer screening apparatus, as disclosed herein. The device is designed to allow interconnection and simultaneous measurement of multiple organoids, and has features that enhance the reproducibility and efficiency of organoid functional testing by enabling an operator to perform subsequent characterization in the same bioreactor with minimal manipulation or intervention.
FIG. 9 provides a high level schematic illustrating the versatility of the disclosed bioreactor system. Figure 9A shows an
Figure 10 shows a perspective view of the
Figure 11 illustrates elements of one embodiment of a bioreactor system that relate to fluid movement, such as media flow, particularly fluid movement related to the addition or supply of fresh media and the removal or suction of spent or spent media. Figure 11A shows the entire fluid exchange system for a
Turning now to the features of FIG. 11B in relation to one embodiment of the bioreactor system for pumping media, media 93 is in contact with cassette media-adapter D tubing 86, which is attached to adapter D valve 67. Organoid-fitting D tubing 85 is also attached to fitting D valve 67. Further, the joint D valve 67 is attached to a joint D-pump C pipe 87, which is attached to the pump C72. Pump C72 is attached to pump C-fitting C tubing 88, which is attached to fitting C valve 66. The junction C valve 66 is attached to a junction C-mix/recycle tank conduit 89, which in turn is attached to the mix/recycle tank 73. In some embodiments, the media 93 is recirculated and directed to the mixing/recirculation tank 73. The joint C valve 66 is also attached to a joint C-waste conduit 90 leading from the joint C valve 66 to waste.
In operation, the supply of cells in the organoid involves the assembly highlighted in fig. 11C, including
Additional attachment assemblies are described that provide fluid communication within the system and allow additional functions including, but not limited to, therapeutic additive dilution, perfusion of therapeutic agents, therapeutic washing of organoids, flushing of fluid lines, and the like.
Fig. 12 shows a high-level schematic of the fluid exchange within
Figure 13 illustrates a method of moving fluid into and out of the
As will be apparent to those skilled in the art, some features of the bioreactor are optional, and most features are present in various embodiments. In some embodiments, the cells may be derived from any mammalian species or
At least one
The remaining components of the system include tanks such as
In some embodiments, the organoid model has fluid pathways for inflow and outflow (fig. 13A). Valves (e.g., check valves, solenoid valves) control the direction in which fluid moves into and out of the organoid with the chamber. In some embodiments, a single shaft or tube for inflow and outflow is contemplated (fig. 13B). The fluid pump controls the rate of fluid flow into and out of the organoid. In some embodiments, unequal inflow and outflow fluid rates are used to control the amount of fluid within the organoid. Adjusting the volume within the organoid cavity results in mechanical stretching of the flexible organoid. In some embodiments, the stretching is applied as a step function (passive stretching) or a sigmoid function (cyclic stretching). In many organoid types, mechanical stretching is considered to be a mechanically transduced signal. In some embodiments, the combination of mechanical and electrical stimulation provides a more robust response for therapy screening.
The fluid exchange system automates routine media changes, regulates intraluminal pressure, perfuses therapeutic agents during screening and exchanges media between organoids (fig. 11 and 12). The fluidic system consists of a series of microfluidic pumps, a three-way valve controlled by a digital output plate, and a culture medium reservoir. Altering the valve configuration changes the direction of travel of the media. In some embodiments, fluid may be added to or removed from a hollow vertically mounted shaft to which an organoid is attached to adjust hydrostatic pressure. Pressure transducer 95 and signal conditioners (e.g., OPP-M and LifeSens) sense the average pressure within the organoids and communicate with the pump via Lab VIEW to adjust the desired intra-cavity pressure. Additionally, fluid exchange systems are used to mix and perfuse compounds to organoids. The solution was pumped out of the additive tank and mixed with the circulating medium. The compound is then perfused into the
Example 7
A bioreactor control. Custom LabVIEW code automatically performs a large part of the process, including hardware and software. Each
Example 8
And (4) data capture. Pressure and volume data from the bioreactor are simultaneously recorded to generate a pressure-volume curve in the relevant contracted organoid. High speed digital cameras (Allied Vision) acquire images up to 100 frames/second. Organoid volumes are estimated by assuming equivalent spheres with the same cross-sectional area. A single acquisition typically involves multiple contractions. To characterize the average contraction characteristics of organoids, MATLAB code first divides the curve into discrete contractions. The data for each contraction is then aligned and averaged (fig. 15A). The average pressure curve and the average volume curve can then be plotted as an average P-V ring (fig. 15B).
The recorded high-speed bright-field video (e.g., optical flow) is analyzed to characterize the motion pattern of the contracting organoids. Changes in the contraction profile are analyzed to confirm the therapeutic effect on organoid contractility. To handle the large volume of multi-dimensional data acquisition, machine learning algorithms determine key parameters related to treatment response and ultimately classify unknown therapeutic agents into classes of interest. Additionally, machine learning can be performed concurrently with long-term data collection to identify rare anomalous events and minimize data storage. For example, the long-term data acquisition may be divided into a series of consecutive acquisitions. As further acquisition continues, completed acquisition is sent to a buffer for analysis. Machine learning (e.g., binary support vector machines) will evaluate any anomalies in the function, such as rare anomalous events, from the data in the buffer. If an anomaly is detected, the relevant data is permanently stored and the normal functional data is discarded.
Example 9
An organoid module. In some embodiments of the bioreactor disclosed herein, a housing (about 25x25x15 cm) made of sterilizable material and detection/recording device 2 (e.g., a camera) and temperature control element 4 (e.g., a heating unit) is attached to the top of
Since micro-tissues lack key features of larger organs (e.g., diffusion limitation of thicker tissues), they are not ideal choices for simulating human organ responses. Bioreactors, which allow fluid exchange between multiple macroscopic organoids, recapitulate key physiological and pharmacological features of the human body. The ability to measure multiple functional properties in a simplified human biomimetic model provides a new approach to bridge the long-term gap between traditional cell culture systems, in vivo animal models and clinical trials. In combination with the induced somatic reprogramming of hpscs, the "human in can" system is expected to become a universal platform for next generation drug discovery, cardiotoxicity screening, disease modeling, and other ethnic, gender, and patient-specific applications.
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It will be apparent from the context of the citation that each reference cited herein is incorporated herein in its entirety or by reference in relevant part.
It is to be understood that while the claimed subject matter has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the claimed subject matter, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the spirit and scope of the disclosed subject matter.