阅读说明：本技术 用于吸引和破坏特定生物元件的微流控芯片 (Microfluidic chip for attracting and destroying specific biological elements ) 是由 贝诺特·查洛特 让·玛丽·拉米雷斯 塞巴斯蒂安·梅恩斯 泽维尔·加里奇 科林·皮涅斯 伊莎 于 2020-03-12 设计创作，主要内容包括：本发明涉及一种用于吸引和破坏特定生物元件的微流控芯片,所述芯片包括：-贮存器(1),其由包含能够吸引生物元件的化学引诱物化合物的基质组成；-至少一个微通道网络(2),其布置在贮存器(1)和芯片的外部环境(3)之间,并且允许化学引诱物化合物传递到该环境,并允许存在于该环境中的生物元件传递到贮存器(1)；-至少一个电极(4),其布置在贮存器(1)和微通道网络(2)之间或布置在与微通道网络(2)相同的位置处,该电极(4)能够产生电场,使得该生物元件在向贮存器(1)传递的过程中被破坏。(The present invention relates to a microfluidic chip for attracting and destroying specific biological elements, said chip comprising: -a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting biological elements; -at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to this environment and the biological elements present in this environment to pass to the reservoir (1); -at least one electrode (4) arranged between the reservoir (1) and the microchannel network (2) or at the same location as the microchannel network (2), the electrode (4) being capable of generating an electric field such that the biological element is destroyed during transfer to the reservoir (1).)

1. A microfluidic chip for attracting and destroying specific biological elements, the chip comprising:

-a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting said biological element;

-at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to said environment and the biological elements present in said environment to pass to the reservoir (1);

-at least one electrode (4) arranged between the reservoir (1) and the microchannel network (2) or at the same location as the microchannel network (2), the electrode being capable of generating an electric field such that the biological element is destroyed during transfer to the reservoir (1).

2. Microfluidic chip according to claim 1, comprising a lower part (5) and an upper part (6), said lower part (5) comprising a part of said reservoir (1), said network of microchannels (2) and said electrodes (4), said upper part (6) comprising a part of said reservoir (1) and being adapted to be arranged above said lower part (5), said upper part (5) and lower part (6) being fixed together.

3. Microfluidic chip according to any of claims 1 to 2, wherein the reservoir (1), the network of microchannels (2) and the electrode (4) are all ring-shaped and wherein the reservoir is located in the center of the chip.

4. The microfluidic chip according to any of claims 2 to 3, wherein the upper portion (6) and/or the lower portion (5) comprise a ring cavity i) (8) and a ring cavity ii) (10), the ring cavity i) (8) being capable of receiving the substrate comprising the chemoattractant compound through one or more openings (9) communicating with the external environment (3) and opening into the ring cavity i) (8); the annular cavity ii) (10) is arranged between the reservoir (1) and the network of microchannels (2) and is adapted to receive a liquid into which the chemoattractant compound can diffuse through one or more openings (11) communicating with the external environment (3) and opening into the annular cavity ii) (10).

5. Microfluidic chip according to anyone of claims 2 to 4, wherein the upper portion (6) and the lower portion (5) are made of biocompatible material.

6. Microfluidic chip according to any one of the preceding claims, wherein the matrix comprising the chemoattractant compound is composed of a biocompatible material, preferably a cross-linked polymer.

7. Microfluidic chip according to any one of the preceding claims, wherein the mass percentage of chemoattractant compound to substrate in the reservoir (1) is between 0.1% and 20%, preferably between 0.5% and 10%, and more preferably between 0.5% and 5%.

8. Microfluidic chip according to anyone of the preceding claims, wherein the chemoattractant compound contained in the reservoir (1) is selected from at least one of the following compounds: CXCL12, CCL5, CCL2, CCL3, CCL7, CCL19, CCL21, CCL22, CCL25, CXCL1, CXCL5, CXCL6, CXCL8, CX3CL1, TGF α, TGF β, FGF, PDGF, EGF, VEGF.

9. Microfluidic chip according to any of the preceding claims, wherein each microchannel comprised in the microchannel network (2) has a height of 1 to 40 μm, a width of 1 to 40 μm and a length of 30 to 250 μm.

10. Microfluidic chip according to anyone of the preceding claims, wherein the electrodes (4) are interdigitated electrodes.

11. Microfluidic chip according to any one of the preceding claims, wherein the electrode (4) is a wireless electrode, the chip comprising an antenna activatable by a second antenna.

12. Microfluidic chip according to anyone of the preceding claims, wherein the electric field generated by the electrodes (4) is capable of destroying the biological element by irreversible electroporation.

13. Microfluidic chip according to any one of the preceding claims, wherein the electric field generated by the electrode (4) is a pulsed electric field of 1000 to 6500V/cm, preferably 3000 to 5000V/cm, at a frequency of 1Hz and for a duration of 100 to 200 μ s.

Technical Field

The present application relates to the field of microfluidic devices capable of attracting and destroying specific biological elements. More specifically, the present application relates to a microfluidic chip capable of attracting and destroying specific biological elements (e.g., prokaryotic or eukaryotic cells) in vivo (in vivo).

Background

In france, approximately 400000 new cancer cases were diagnosed annually, of which approximately 150000 deaths occurred.

Different types of cancer exist, depending on the tissue in which the cancer occurs. Solid tumors characterized by local clustering of cancer cells are distinct from blood cell tumors, which spread by the circulation of cancer cells in the bone marrow or blood. Blood cell tumors, also known as hematopoietic cancers, are cancers that affect the blood or lymphoid organs, such as leukemias and lymphomas.

With respect to solid tumors, this cancer is different from adenocarcinoma, which is a cancer produced by epithelial tissues. Sarcomas, which differ from solid tumors, are cancer cells that occur in so-called supporting tissues, such as osteosarcomas on bone, liposarcomas in fat, and myosarcomas in muscle.

Solid cancers account for 90% of the total number of cancers in both men and women, with prostate, lung and colorectal cancers being the three most common cancers in men and breast, colorectal and lung cancers being the three most common cancers in women. Although the cancer mortality rate in men decreases by 1.5% per year and in women by 1% per year (normalized ratio) between 1980 and 2012, there is still a need for effective solutions to treat and prevent these solid tumors and their complications.

To date, surgery is one of the main treatment modalities for solid tumors, involving resection of the entire tumor, where possible, while possibly also resecting the tissue surrounding the tumor, called resection margin. Surgery can be a single treatment when the tumor is a highly localized tumor, particularly when the tumor is in an early stage, but it is also often combined with medical treatments such as radiation therapy (which is also a local treatment) and/or chemotherapy (which is a systemic treatment that potentially acts on all cancer cells in the body) or other treatments.

The advantage of treating solid tumors by local surgery is the ability to remove the entire tumor where possible, and to preserve organs and anatomical structures that are not affected by cancer cells. It also avoids the side effects caused by radiotherapy, such as the occurrence of burns or radiotherapy-induced cancers, and chemotherapy-induced side effects, such as skin reactions, nausea, vomiting, diarrhea, muscle pain, fatigue, hair loss, and chemotherapy-induced cancers.

To reduce the recurrence rate, the resection area includes the area of healthy tissue surrounding the tumor, i.e., the resection margin. At this stage, several situations must be considered. In the first case, the tumor cells of the primary tumor are included in the resection area and will recur. In the second case, some of the tumour cells are located outside the resection area, possibly in situ or at a distance from the resection area, and therefore may recur in situ or at a distance from the resection area, forming metastases, i.e. secondary colonies of cancer cells that spread to a distance from the organ affected by the initial tumour, which are the source of so-called "metastatic" cancer in an organ different from the organ in which the solid tumour is located.

Therefore, it is necessary to prevent local recurrence and the risk of metastatic cancer as much as possible after resection of a solid tumor.

Chemotherapy may be used after local surgical removal of a tumor, which is referred to as "adjuvant chemotherapy," to prevent recurrence and/or formation of metastatic cancer. However, as mentioned above, many side effects are associated with this drug treatment. Among the most significant side effects associated with the use of chemotherapy, reference may be made in particular to chemotherapy-induced cancers, which are by definition new tumors resulting from the treatment of patients with cytotoxic drugs directed against first malignant tumors; reference may also be made to the risk of developing resistance of cancer cells to chemotherapy, both of which strongly limit the possibility of eliminating said cells.

Thus, to date, no effective solution has been found to effectively eliminate the remaining cancer cells after solid tumor resection to prevent and/or reduce the risk of local recurrence and/or development of metastatic cancer without the side effects of short and medium-term drugs.

In response to these needs, the present invention proposes a microfluidic chip for attracting and destroying specific biological elements, in particular eukaryotic cells, such as cancer cells. The chip is particularly suitable for implantation in a solid tumor resection area in the human body to attract and destroy remaining cancer cells in the body.

Microfluidic devices for use in the field of cancer therapy have been described in the prior art. Reference may be made in particular to WO2018/089989a1, which describes an extracorporeal cancer treatment apparatus that treats cancer by subjecting a biological fluid, such as blood, to electromagnetic radiation specific to the type of cancer cell targeted and capable of destroying that type of cancer cell.

Reference is also made to document US2018111124a1, which describes a microfluidic device comprising one or more microfluidic channels and one or more wireless bipolar electrode arrays for achieving high-flux capture of tumor cells circulating in a solution of conductive ions by applying an alternating electric field of 40kHz to a biological sample. The cells thus captured can be used to diagnose cancer or assess the efficacy of a cancer treatment.

No prior art documents describe or suggest such microfluidic chips: it is used to attract and destroy cancer cells, preferably in vivo, after resection of a solid tumor, thereby preventing and/or reducing the risk of local recurrence and/or development of metastatic cancer.

More generally, there is no prior art document describing or suggesting a microfluidic chip for attracting and destroying specific biological elements, preferably in vivo, comprising: a reservoir containing a matrix comprising a chemoattractant compound capable of attracting biological elements; at least one microchannel network, the microchannel network communicating the reservoir with an environment external to the chip; and at least one electrode capable of generating an electric field that disrupts the biological element.

Disclosure of Invention

The present invention relates to a microfluidic chip for attracting and destroying specific biological elements, said chip comprising:

-a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting said biological element;

-at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to said environment and the biological elements present in said environment to pass to the reservoir (1);

-at least one electrode (4) arranged between the reservoir (1) and the microchannel network (2) or at the same location as the microchannel network (2), the electrode (4) being capable of generating an electric field such that the biological element is destroyed during transfer to the reservoir (1).

Drawings

Fig. 1 is a cross-sectional view of a chip according to the present invention.

Fig. 2 shows the upper part (6) of the chip as seen from above.

Fig. 3 shows the lower part (5) of the chip as seen from above.

Fig. 4 shows an exploded view of the bottom of a chip according to the invention.

Fig. 5 shows an exploded view of the top of a chip according to the invention.

Fig. 6 shows a cross-sectional view of a chip according to the invention at a scale of 5:0.5 cm.

Fig. 7 shows a cross-sectional view of the chip at a 5:1cm scale.

FIG. 8 is an inverted fluorescence micrograph under a 5X objective at the microchannel level showing breast cancer tumor cells (MDA-MB-231) without gradient (14).

Figure 9 is an inverted fluorescence micrograph under a 5X objective at the microchannel level showing breast cancer tumor cells (MDA-MB-231) (14) being drawn into the chip under a fetal bovine serum gradient generated from 1% fetal bovine serum outside the chip and 10 μ Ι pure fetal bovine serum within the reservoir.

FIG. 10 is an inverted fluorescence micrograph under a 10X objective at the microchannel level showing breast cancer tumor cells (MDA-MB-231) without gradient (14).

FIG. 11 is an inverted fluorescence micrograph under a 5X objective at the central reservoir level of the chip showing breast cancer tumor cells (MDA-MB-231) without gradient (14).

Figure 12 is an inverted fluorescence micrograph under a 10X objective at the microchannel level showing breast cancer tumor cells (MDA-MB-231) (14) being drawn into the chip under a fetal bovine serum gradient generated from 1% fetal bovine serum outside the chip and 10 μ Ι pure fetal bovine serum within the reservoir.

FIG. 13 is an inverted fluorescence microscope image under a 5X objective at the level of the central reservoir of the chip, showing breast cancer tumor cells (MDA-MB-231) (14) being drawn into the chip under a fetal bovine serum gradient generated from 1% fetal bovine serum outside the chip and 10 μ l pure fetal bovine serum within the reservoir.

FIG. 14 is an inverted fluorescence micrograph under a 10 Xobjective at the microchannel level, showing breast cancer tumor cells (MDA-MB-231) (14) being drawn into the chip under a chemisorbent SDF-1 gradient (1 μ g of chemisorbent SDF-1 in the central reservoir of the chip).

FIG. 15 is an inverted fluorescence micrograph under a 5X objective at the level of the center reservoir of the chip showing breast cancer tumor cells (MDA-MB-231) (14) being drawn into the chip under a chemisorbent SDF-1 gradient (1 μ g of chemisorbent SDF-1 present in the center reservoir of the chip).

FIG. 16 is a graph showing the concentration of CO at 5%₂And 95% humidity for 48 hours, inverted fluorescence microscopy images of breast cancer tumor cells (MDA-MB-231) (14) on slides containing interdigitated electrodes without electroporation.

FIG. 17 is an inverted fluorescence microscope image of breast cancer tumor cells (MDA-MB-231) on a slide containing interdigitated electrodes after destruction by electroporation with a 1Hz, 5V electric field applied for 100 μ s at time d0 and d0+3 hours with interdigitated electrodes.

FIG. 18 is an inverted fluorescence micrograph at the microchannel level showing breast cancer tumor cells (MDA-MB-231) without gradient and pulsed electric fields.

FIG. 19 is an inverted fluorescence micrograph at the microchannel level showing breast cancer tumor cells (MDA-MB-231) are drawn into the chip under an SDF-1 gradient (1 μ g of chemisorption SDF-1 in the central reservoir of the chip) in the absence of a pulsed electric field.

FIG. 20 is an inverted fluorescence micrograph at the microchannel level showing breast cancer tumor cells (MDA-MB-231) being drawn into the chip with an SDF-1 gradient (1 μ g of chemisorption SDF-1 in the center reservoir of the chip) and a 1Hz, 5V pulsed electric field of 100 ms.

FIG. 21 is a graph showing the release of BSA-FITC (simulated SDF-1) from an alginate matrix contained in a microfluidic chip according to the present invention over a period of 50 days.

FIG. 22 is a graph showing the release of SDF-1 from an alginate matrix over a 50 day period.

Detailed Description

Applicants have developed a microfluidic chip to attract and destroy biological elements, such as prokaryotic or eukaryotic cells. The microfluidic chip is particularly advantageous for attracting and destroying cancer cells in vivo after resection of a solid tumor to prevent and/or reduce the risk of local recurrence and/or development of metastatic cancer.

Definition of

A "microfluidic chip" is a device comprising a network of microchannels, i.e. micron-sized channels, obtained by etching or moulding a material, connected to each other, and connecting the chip interior to the chip exterior by drilling through the inlets and outlets of the chip in order to perform the desired function. The microfluidic chip may be obtained by a specific process such as deposition and electrodeposition, etching, bonding, injection molding, stamping, soft lithography, anodic bonding or any other technique. These manufacturing processes are known to those skilled in the art. In the context of the present invention, the desired function of a microfluidic chip is to be able to attract a specific biological element, preferably a eukaryotic cell, such as a cancer cell, in vivo and to destroy it in vivo.

A "network of microchannels" corresponds to a plurality of channels connected to the outside of the chip by inlets and outlets drilled through the chip. The microchannels may for example be moulded or manufactured directly in the material of the microfluidic chip. The number of microchannels varies depending on the diameter of the chip, the width of the microchannels, or the spacing between the microchannels. For example, for a chip with a diameter of 1 centimeter and a channel width of 10 μm, the number of microchannels may be approximately 1500.

The height of the channel corresponding to each microchannel constituting the microchannel network may be from several micrometers to several hundred micrometers, and the length may be from several hundred micrometers up to several millimeters. The cross-section of the microchannels may in principle have any two-dimensional shape, such as square, rectangular, circular or a combination thereof. The microchannels may be straight or curved.

The width of the microchannel is the horizontal distance of the two points that are farthest apart on opposite edges of the cross-section. The height of the microchannel is the perpendicular distance from the farthest point on the opposite edge of the cross-section. The length of a microchannel is the distance between the two ends of the channel, and the length of the microchannel corresponds to the largest dimension. The two shorter dimensions generally define the aforementioned cross-section.

In the context of the present invention, "electrode" refers to any element capable of conducting an electric current and comprises two conductors separated by an air gap having a pitch of 1 μm to 1 mm. For clarity, the invention will refer to the electrodes in the singular, but those skilled in the art will understand that an electrode is made up of two conductors separated by an air gap that can conduct current.

The microfluidic chip is preferably "in vivo implantable", i.e. the microfluidic chip is intended to be implanted in a living body and can be implanted in a living body, preferably a mammal, in particular a human. More specifically, this means that the chip can be implanted in a living body without disturbing or degrading the tissue with which it is in contact, and that the chip can function in vivo, i.e., attract and destroy a specific biological element, preferably a eukaryotic cell such as a cancer cell, in vivo, and destroy it in vivo.

In the sense of the present invention, "biological element" refers to any element that contains genetic information in the form of RNA or DNA and that may be found within a living organism, i.e. in vivo, such as prokaryotic cells, eukaryotic cells and microorganisms. Among the biological elements that are conceivable, reference may be made to prokaryotic cells, such as bacteria; and eukaryotic cells, such as animal cells.

"specific biological element" or "target biological element" refers to a biological element of interest in the context of use of the chip, i.e., a biological element that is intended to be attracted and destroyed in vivo. In the case of a microfluidic chip, the chemoattractant compound present in the reservoir is selected to attract the biological element of interest.

By "at the same location as the microchannel network" is meant that the electrodes are arranged at the level of the microchannel network, more precisely above or below the microchannel network. Preferably, the electrodes are arranged above the network of microchannels.

Preferably, in the context of the present invention, the biological element of interest is a prokaryotic or eukaryotic cell, more preferably, the biological element of interest is a eukaryotic cell. More preferably, the eukaryotic cell is a cancer cell, preferably a metastatic cancer cell.

A "cancer cell" is a cell that: one or more major DNA lesions have occurred in the cell, transforming normal cells into cancer cells capable of proliferating to form a population of the same transformed cells (i.e., a tumor). In particular, when the cell in question has a plurality of characteristics, such as: unaffected by cell growth regulatory signals, the ability to escape the programmed cell death process and the ability to divide indefinitely.

More specifically, in the context of the present invention, "cancer cells" refer to cells from a so-called primary or primary solid tumor, present at or near the area of resection of the solid tumor.

Preferably, in the context of the present invention, "cancer cells" also refers to "metastatic cancer cells", i.e. cancer cells which are capable of or have migrated from the original tumor to the whole body through the blood or lymphatic vessels and which are capable of or have proliferated in large numbers in one or more tissues in the vicinity of or at a distance from said tumor, thereby forming metastases at the origin of the "metastatic cancer" or "metastatic tumor". In this context, cancer cells are cells derived from so-called secondary or tertiary solid tumors, which correspond to metastases in a second or third tissue or organ, unlike the original tumor.

For the sake of clarity, reference is made in the singular to "a specific biological element", "a prokaryotic cell", "a eukaryotic cell", "a cancer cell", "a metastatic cell", it being understood that the microfluidic chip according to the invention is capable of attracting and destroying several of said elements and cells in vivo. It should also be noted that the microfluidic chip according to the invention is capable of attracting and destroying in vivo several specific biological elements, which may have different properties, which are attracted to the chip by a specific selection of one or more chemoattractant compounds present in the reservoir.

In the context of the present invention, the term "preventing" refers to reducing the risk of having a particular disease or condition, reducing or slowing the onset of the symptoms of the disease. For example, in the context of the present invention, the term "prevention" may correspond to a reduction of the risk of spread of infection (when the biological element is a prokaryotic cell); or reducing the risk of local recurrence of cancer and/or the risk of the appearance of metastases (particularly metastatic cancer) (when the biological element is a cancer-type eukaryotic cell).

In the context of the present invention, the term "treatment" refers to the amelioration or reversal of a given disease or disorder or at least one discernible symptom. The term "treating" may also refer to reducing or slowing the progression of a disease or disorder, or the onset of symptoms of a disease or disorder. For example, in the context of the present invention, the term "treatment" may correspond to a reduction or slowing of the progression of the infection when the biological element is a prokaryotic cell, or to a reduction or slowing of the appearance of metastases, more particularly metastatic cancers, when the biological element is a cancer-type eukaryotic cell.

In the sense of the present invention, the microfluidic chip is preferably intended to be implanted in the body of a subject.

The subject in the context of the present invention is a living body, preferably a mammal, more particularly a human, child, man or woman.

"solid cancer" or "solid tumor" refers to an individualized mass of cancer cells in the skin, mucosa, bone, etc. or any other tissue present in an organ, i.e., cancers derived from epithelial cells (such as skin, mucosa, glands, etc.) and sarcomas derived from cells of connective and supporting tissues (such as bone, cartilage, etc.).

Preferably, in the context of the present invention, "solid cancer" or "solid tumor" refers to a cancer such as in breast, lung, prostate, bladder, salivary gland, skin, small intestine, colorectal, thyroid, cervix, endometrium and ovary, lip-mouth-larynx, kidney, liver, brain, testis, pancreas, preferably breast cancer. These examples of solid tumors are non-limiting.

By "chemoattractant compound" is meant any compound capable of attracting a biological element, preferably cells expressing at their surface the particular membrane receptor of the compound, by chemotaxis, the movement of which depends on the concentration gradient of the chemoattractant compound. In the context of the present invention, a chemoattractant compound is capable of inducing a shift in one or more specific biological elements, depending on the concentration gradient of the compound in positive chemotaxis, wherein the biological elements move towards the region where the concentration of chemoattractant compound is highest.

In particular, in the context of the present invention, a chemoattractant compound is considered "suitable for attracting" a particular biological element when the chemoattractant compound allows the particular biological element to move within the microfluidic chip, in particular when the chemoattractant compound allows the particular biological element to move towards the chemoattractant compound reservoir where the concentration of chemoattractant compound is highest. The skilled person will know how to characterize a particular biological element of interest in order to select a chemoattractant compound suitable for attracting that element within the chip.

In the context of the present invention, chemoattractant compounds are selected based on the particular biological element of interest.

Where the biological element is a eukaryotic cell, the chemoattractant compound is selected according to the type of membrane receptor expressed by the cell.

More precisely, when the biological element is a eukaryotic cell, the chemoattractant compound may be a cytokine, i.e. a polypeptide or soluble protein synthesized by the cell and which acts remotely on other cells via membrane receptors to modulate their activity and function, said chemoattractant compound being selected from the group consisting of chemokines, granulocyte and macrophage colony-stimulating factors, such as M-CSF, G-CSF, CSF-1; growth factors and transforming growth factors, such as TGF α, TGF β, EGF, betacellulin (betacellulin), amphiregulin (ampheregulin), heregulin (heregulin), HBEGF, FGF, VEGF; tumor necrosis factors such as NGF, TNF α, TNF β; interferons, such as IFN α, IFN β, IFN γ, IFN λ; and interleukins, such as IL-1 to IL-38.

Preferably, when the biological element is a eukaryotic cell (such as a cancer cell), the chemoattractant compound is selected from the group consisting of a chemokine, a growth factor and a transforming growth factor.

Chemokines are small proteins from 8 to 14 kilodaltons, characterized by the presence of four conserved cysteine residues, allowing their three-dimensional structure to form. Chemokines can be divided into four subfamilies, called the CXC or alpha family (i.e., the first two cysteines are separated by any amino acid), the CC or beta family, the CX3C or delta family, and the C or gamma family, depending on the spacing between the two cysteines at the N-terminal position. For example, a chemokine in the context of the present invention may be selected from the following chemokines: CXCL12 (also referred to as stromal cell derived factor 1(SDF-1)), CCL5, CCL2, CCL3, CCL7, CCL19, CCL21, CCL22, CCL25, CXCL1, CXCL5, CXCL6, CXCL8, CX3CL 1.

Growth factor is a low molecular weight protein (less than 30 kilodaltons) that stimulates cell proliferation and is recognized by specific membrane receptors, usually tyrosine kinases. The growth factor in the context of the present invention may for example be selected from TGF α or TGF β (transforming growth factor α or β), FGF (fibroblast growth factor α), EGF (epidermal growth factor), betacellulin, amphiregulin, heregulin, HBEGF, VEGF (vascular endothelial growth factor), PDGF (platelet-derived growth factor).

When the biological element is a prokaryotic cell, such as a bacterium, the chemoattractant compound may be a peptide containing a formylated N group, such as N-formylmethionyl-leucyl-phenylalanine (FMLP), or a carbohydrate molecule, such as glucose.

In the context of the present invention, the chemoattractant compound is comprised in a matrix consisting of a biocompatible material as defined in the present invention. The biocompatible material of the matrix may be specifically selected according to the chemoattractant compound, the desired release profile, and the use scenario of the microfluidic chip.

In the context of the present invention, "external environment" means the tissue located around the microfluidic chip, more precisely the tissue in direct contact with the chip, within 300mm, preferably within 150mm, more preferably within 100mm around the chip when the microfluidic chip is implanted in vivo.

By "resecting a solid tumor" is meant removing, ablating, or resecting the solid tumor, for example, by surgery.

Accordingly, the present invention relates to a microfluidic chip for attracting and destroying specific biological elements in vivo.

Reference numerals used in the context of the present detailed description refer to the numbers used in this application to describe the invention, but are not limited thereto.

A first subject of the invention relates to a microfluidic chip for attracting and destroying specific biological elements, said chip comprising:

-a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting biological elements;

-at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to said environment and the biological elements present in said environment to pass to the reservoir (1);

-at least one electrode (4) arranged between the reservoir (1) and the microchannel network (2) or at the same location as the microchannel network (2), the electrode being capable of generating an electric field such that the biological element is destroyed during transfer to the reservoir (1).

More precisely, the invention relates to an in vivo implantable microfluidic chip for attracting and destroying specific biological elements (preferably eukaryotic cells), said chip comprising:

-a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting biological elements in vivo;

-at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to said environment and the biological elements present in said environment to pass to said reservoir (1);

-at least one electrode (4) arranged between the reservoir (1) and the microchannel network (2) or at the same location as the microchannel network (2), the electrode being capable of generating an electric field such that the biological element is destroyed during in vivo transfer to the reservoir (1).

According to a preferred aspect, the present invention relates to an in vivo implantable microfluidic chip for attracting and destroying eukaryotic cells, preferably cancer cells, said chip comprising:

-a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting said cells in vivo;

-at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to said environment and the cells present in said environment to pass to the reservoir (1);

-at least one electrode (4) arranged between the reservoir (1) and the microchannel network (2) or at the same location as the microchannel network (2), the electrode (4) being capable of generating an electric field such that the cells are destroyed during in vivo transfer to the reservoir (1).

According to a first particular embodiment, the present invention relates to an in vivo implantable microfluidic chip for attracting and destroying specific biological elements (preferably eukaryotic cells, more preferably cancer cells), said chip comprising:

-a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting said cells in vivo;

-at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to said environment and the cells present in said environment to pass to the reservoir (1);

-at least one electrode (4) arranged between the reservoir (1) and the network of microchannels (2), said electrode (4) being capable of generating an electric field such that the cells are destroyed during in vivo transfer to the reservoir (1).

According to a second particular embodiment, the present invention relates to an in vivo implantable microfluidic chip for attracting and destroying specific biological elements (preferably eukaryotic cells, more preferably cancer cells), said chip comprising:

-a reservoir (1) consisting of a matrix comprising a chemoattractant compound capable of attracting said cells in vivo;

-at least one microchannel network (2) arranged between the reservoir (1) and an environment (3) external to the chip and allowing the chemoattractant compound to pass to said environment and the cells present in said environment to pass to the reservoir (1);

-at least one electrode (4) arranged at the same location as the network of microchannels (2), said electrode (4) being capable of generating an electric field such that the cells are destroyed during in vivo transfer to the reservoir (1).

Preferably, in this embodiment, the electrodes are arranged above or below the microchannel network, more preferably, the electrodes are arranged above the microchannel network.

The matrix of the reservoir (1) containing the chemoattractant compound is formed from a biocompatible material.

The chemoattractant compound contained in the matrix of the reservoir (1) attracts in vivo by positive chemotaxis certain biological elements within the chip, which migrate to the reservoir (1) where the concentration of chemoattractant compound is highest. The network of microchannels (2) allows diffusion of the chemoattractant compound from the reservoir (1) to the external environment (3) of the chip and migration of the specific biological element from the external environment (3) of the chip to the interior of the chip, reaching the reservoir (1). The electrodes (4) are arranged between the reservoir (1) and the network of microchannels (2) such that when a particular biological element enters the chip from the external environment (3) through the network of microchannels (2) and reaches the reservoir (1), it is destroyed by an electric field generated by said electrodes (4) during in vivo transfer to the reservoir (1).

In the context of the present invention, the biological element is preferably a cancer cell, in particular a cancer cell derived from a cancer or a solid tumor. The migration of the cells inside the chip is performed by adhesion to a support on which the electrodes (4) are arranged, the electric field generated by the electrodes then being able to destroy the cells during their transfer in vivo to the reservoir (1).

The microchannel network (2) of the microfluidic chip according to the invention is preferably located on the outer edge of the chip, i.e. in contact with the external environment (3), so as to ensure communication between said environment and the internal space of the chip.

The chip according to the invention may comprise several microchannel networks (2), for example two microchannel networks. According to a preferred aspect, the microfluidic chip according to the invention comprises a single electrode (4). According to another aspect, the microfluidic chip according to the invention comprises several electrodes (4).

According to a particular embodiment of the invention, the electrodes may be arranged more specifically as:

-on both sides of at least one microchannel network;

-within at least one microchannel network;

-between at least two microchannel networks; or

Adjacent to at least one network of microchannels.

The microfluidic chip according to the first subject of the invention comprises a lower part (5) and an upper part (6), a reservoir (1) containing a chemoattractant compound matrix, a network of microchannels (2) and electrodes (4) being able to be contained in the upper part (6) and/or lower part (5) of the chip independently of each other.

The upper part (6) of the microfluidic chip is adapted to be arranged above the lower part (5) to form a cover, said upper (6) and lower (5) parts being fixed together.

According to a particular aspect, the microfluidic chip according to the invention comprises a lower part (5) and an upper part (6), the lower part (5) comprising a portion of the reservoir (1), the network of microchannels (2) and the electrodes (4), the upper part (6) comprising a portion of the reservoir (1) and being adapted to be arranged above the lower part (5), said upper and lower parts being fixed together.

According to another particular aspect, the microfluidic chip according to the invention comprises a lower part (5) and an upper part (6), the lower part (5) comprising a portion of the reservoir (1) and the electrode (4), the upper part (6) comprising a portion of the reservoir (1) and the network of microchannels (2), the upper part being adapted to be arranged above the lower part (5), the upper part and the lower part being fixed together.

With respect to the electrode (4), "contained in" means that the electrode is deposited and/or integrated on the lower and/or upper part of the chip. Preferably, the electrodes are integrated on the lower part of the chip.

The reservoirs (1) contained in the lower part (5) and the upper part (6) are single reservoirs, a part of which is located in the upper part of the chip and another part is located in the lower part of the chip.

In the context of the present invention, the expression upper portion (6) "adapted to be arranged above the lower portion (5) to form a cover" means that the shape of the upper portion (6) is such that it adapts to the shape of the lower portion (5) on which it is placed, without interfering with the function of each element constituting the lower portion (5), and allowing the formation of a cover closing the chip.

Advantageously and preferably, the upper part (6) and the lower part (5) of the microfluidic chip are circular, so that the chip can be implanted in the body without damaging the tissue.

Preferably, the chip is circular, e.g. the upper and lower parts have a semi-elliptical or hemispherical shape, such that when the upper part is arranged on the lower part, the microfluidic chip is elliptical or spherical, respectively.

The microfluidic chip according to the first subject matter has a size suitable for implantation in the body and is of the order of a few centimeters, preferably 0.5cm to 5cm, more preferably 1cm to 3cm, still more preferably 1 cm.

More specifically, the invention relates to a microfluidic chip wherein the reservoir (1), the microchannel network (2) and the electrodes (4) are all annular and wherein the reservoir is located in the center of the chip.

As the name implies, a ring is the shape of a ring. Since the reservoir (1) is in a central position and the network of microchannels (2) has an annular shape, this particular configuration ensures that the chemoattractant compound contained in the reservoir (1) diffuses radially to the external environment (3) and that the biological element is attracted uniformly from said environment to the reservoir (1). Likewise, the annular shape of the electrodes (4) arranged between the reservoir (1) and the network of microchannels (2) advantageously ensures an effective destruction of each biological element penetrating the chip from any direction.

The lower part (5) and the upper part (6) of the microfluidic chip may be fixed to each other by any physical or chemical means (12) suitable for in vivo use. As examples of physical or chemical fixing means, reference may be made to screws or adhesives suitable for in vivo use of the chip, or to hollow elements present in the lower and upper parts, respectively, connecting them together and projecting elements arranged opposite thereto.

Preferably, in the context of the present invention, the upper part (6) comprises one or more openings through which one or more screws can be inserted, and the lower part (5) comprises one or more nuts adapted to receive said one or more screws.

More preferably, the upper part (6) of the microfluidic chip according to the invention comprises a central opening through which a screw can be inserted, and the lower part (5) comprises a central nut capable of receiving said screw. In this preferred embodiment, the annular reservoir (1) is arranged around a central opening of the upper chip part (6) and a central nut of the lower chip part (5).

In particular, the microfluidic chip comprises one or more seals (7) capable of sealing the chip, said seal (7) being arranged between the lower part (5) and the upper part (6) above the network of microchannels (2).

By "seal capable of sealing the chip" is meant a seal having the property of not allowing fluids, such as blood, that may be present in the external environment (3) to enter the chip, and not allowing liquids and substances present inside the chip to leave the chip through any other location than the network of microchannels (2). One or more seals (7) are above the microchannel network (2) which do not impede diffusion of the chemoattractant compound to the external environment (3) nor the passage of specific cells present in said environment through the chip to the reservoir (1). Advantageously, the seal forms a support region between the upper and lower portions of the chip.

In particular, the invention relates to a microfluidic chip wherein the upper part (6) and/or the lower part (5) comprises a ring cavity i) (8) and a ring cavity ii) (10), the ring cavity i) (8) being capable of receiving a substrate comprising a chemoattractant compound, the ring cavity ii) (10) being arranged between the reservoir (1) and the microchannel network (2) being capable of receiving a liquid into which the chemoattractant compound is capable of diffusing.

More specifically, the invention relates to a microfluidic chip wherein the upper part (6) and/or the lower part (5) comprises an annular cavity i) (8) and an annular cavity ii) (10), the annular cavity i) (8) being able to receive a matrix comprising a chemoattractant compound through one or more openings (9) communicating with the external environment (3) and opening into the annular cavity i) (8); the annular cavity ii) (10) is arranged between the reservoir (1) and the network of microchannels (2) and is adapted to receive a liquid into which the chemoattractant compound can diffuse through one or more openings (11) communicating with the external environment (3) and opening into the annular cavity ii) (10).

More particularly, the invention relates to a microfluidic chip wherein the upper part (6) and the lower part (5) comprise an annular cavity i) (8) and an annular cavity ii) (10), the annular cavity i) (8) being able to receive a matrix comprising a chemoattractant compound through one or more openings (9) communicating with the external environment (3) and opening into the annular cavity i) (8); the annular cavity ii) (10) is arranged between the reservoir (1) and the network of microchannels (2) and is adapted to receive a liquid into which the chemoattractant compound can diffuse through one or more openings (11) communicating with the external environment (3) and opening into the annular cavity ii) (10).

Preferably, the chemoattractant compound has been added to the matrix of the reservoir (1) prior to addition to the annular cavity i) (8). Alternatively, the chemoattractant compound may have been added to the annular chamber i) (8) either before or after addition to the chamber in which the reservoir (1) matrix is located.

Preferably, after the upper part (6) has been placed over the lower part (5), a matrix is added to the annular cavities i) (8) and ii) (10) through the openings (9, 11), respectively, the matrix comprising the chemoattractant compound and the liquid into which the chemoattractant compound can diffuse.

The liquid into which the chemoattractant compound can diffuse is preferably an aqueous solution, such as saline or a physiological buffered solution, such as an aqueous solution containing Phosphate Buffered Saline (PBS).

The impermeability of each chamber after said addition operation is ensured by closing the openings with a seal (for example made of PDMS or any other suitable material) located at the outlet of these openings (9, 11) and communicating with the external environment (3).

Preferably, an annular cavity i) (8) is included in the upper (6) and lower (5) parts of the chip, one or more openings (9) to the cavity being located in the lower part (5) of the chip, and an annular cavity ii) (10) and one or more openings (11) to the cavity are included in the upper part (6) of the chip.

The chip according to the invention is made of a biocompatible material. In particular, the invention relates to a microfluidic chip wherein the upper part (6) and the lower part (5) are made of biocompatible material.

More precisely, such upper (6) and lower (5) parts and the elements they comprise are made of biocompatible material. More precisely, the upper part (6), the lower part (5), the reservoir (1), in particular the matrix containing the chemoattractant compound, the network of microchannels (2), the electrodes (4) and the seal (7) are all made of biocompatible materials.

By "biocompatible material" or "biomaterial" is meant a material that does not interfere with or degrade the biological environment in which it is used, even in direct or indirect, transient or sustained contact with internal tissues and fluids of the human or animal body. As examples of biocompatible materials that can be used in the context of the present invention, reference may be made, without being exhaustive, to glass, ceramics (such as alumina, zirconia, hydroxyapatite), metals and metal alloys (such as titanium, platinum), polymers of natural origin (such as collagen, agarose, chitosan, carrageenan, xanthan and alginates), or degradable synthetic polymers (such as polyesters and polyanhydrides) or non-degradable polymers (such as polyurethanes, celluloses and their derivatives, vinyl polymers, etc.

Preferably, the upper part (6), the lower part (5) and the microchannel network (2) are made of a polymer of synthetic origin, such as Polydimethylsiloxane (PDMS) or Polyetheretherketone (PEEK), independently of one another. Even more preferably, the upper part (6) and the lower part (5) are made of Polyetheretherketone (PEEK).

Preferably, the microchannel network (2) is made of Polyetheretherketone (PEEK).

Preferably, the electrode (4) is made of titanium and/or platinum, more preferably titanium and platinum.

Preferably, the reservoir (1), in particular the matrix comprising the chemoattractant compound, is made of collagen and/or alginate, more preferably, said reservoir (1), in particular said matrix, is made of alginate.

In a particular and preferred way, the upper part (6), the lower part (5) and the network of microchannels (2) of the chip according to the invention are made of Polyetheretherketone (PEEK), the reservoir (1) of chemoattractant compound is made of alginate, and the electrodes (4) are made of titanium and platinum.

According to a particular aspect, the invention relates to a microfluidic chip wherein the matrix comprising the chemoattractant compound is made of a biocompatible material, preferably a cross-linked polymer. Preferably, the cross-linked polymer constituting the matrix is a polymer of natural origin, in particular collagen and/or alginate, preferably alginate.

Crosslinking of a polymer corresponds to the process of forming one or more three-dimensional networks from a leading or branched polymer by chemical and/or physical means. The person skilled in the art knows how to induce crosslinking of the polymer according to the polymer in question, which may be carried out by heating and/or by using a crosslinking agent, as an example. A "crosslinked" polymer is one in which some of the chains are linked together by strong or weak bonds.

For example, crosslinking of collagen can be performed at room temperature using a crosslinking agent such as ammonia gas, oxidized sugar, or aldehyde, and crosslinking of alginate can be performed at room temperature in a calcium chloride bath.

Even more preferably, the biocompatible material constituting the matrix comprising the chemoattractant compound is an alginate which is cross-linked in a calcium chloride bath, preferably at room temperature.

The cross-linking of the biocompatible material constituting the matrix comprising the chemoattractant compound advantageously allows a sustained release of the compound. By "sustained release" is meant the kinetic presentation of controlled and continuous release of the chemoattractant compound over a period of time. Preferably, in the context of the present invention, the release of the chemoattractant compound takes place for 3 days to 6 months, preferably for 15 days to 3 months.

According to a particular aspect, the invention relates to a microfluidic chip wherein the mass percentage of chemoattractant compound to substrate in the reservoir (1) is between 0.1% and 20%, preferably between 0.5% and 10%, more preferably between 0.5% and 5%.

One skilled in the art will know how to determine the amount of chemoattractant compound in the reservoir based on the context of use of the chip, the particular biological element of interest, the desired release time of the chemoattractant compound, and the biological material comprising the reservoir matrix.

When the specific biological element is a eukaryotic cell, preferably a cancer cell, the chemoattractant compound contained in the reservoirs of the microfluidic chip and in particular in the reservoir matrix is preferably selected from chemokines, such as CXCL12, also known as stromal cell derived factor 1(SDF-1), CCL5, CCL2, CCL3, CCL7, CCL19, CCL21, CCL22, CCL25, CXCL1, CXCL5, CXCL6, CXCL8, CX3CL1, growth factors and transforming growth factors, such as TGF α or β (transforming growth factor α or β), FGF (fibroblast growth factor α), EGF (epidermal growth factor α), β -cell, amphiregulin, heregulin, HBEGF, PDGF (platelet derived growth factor), VEGF (vascular endothelial growth factor).

More specifically, the invention relates to a microfluidic chip wherein the chemoattractant compound contained in the reservoir (1) of the chip is selected from at least one of the following compounds: CXCL12, CCL5, CCL2, CCL3, CCL7, CCL19, CCL21, CCL22, CCL25, CXCL1, CXCL5, CXCL6, CXCL8, CX3CL1, TGF α, TGF β, FGF, PDGF, EGF, VEGF.

According to another aspect, when the specific biological element is a prokaryotic cell, preferably a bacterium, the chemoattractant comprised in the reservoir (1) of the microfluidic chip, in particular the chemoattractant in the matrix of the reservoir (1), is preferably selected from carbohydrate molecules.

The chemoattractant compounds can be used alone or in combination with one or more of the other chemoattractant compounds and/or other compounds described above, which are capable of improving, directly or indirectly, the ability of the chemoattractant compounds to attract a particular biological element, such as a eukaryotic cell, particularly a cancer cell; such as carbohydrate (glucose) and/or lipid (fatty acid) molecules capable of providing the necessary energy (in the form of ATP after degradation of glucose or fatty acid) for the survival of biological elements, in particular eukaryotic cells, further in particular cancer cells. Other molecules can also be used in conjunction with the chemoattractant, such as oxygen. Oxygen can be transported through hemoglobin or synthetic hemoglobin. Oxygen is an important molecule for the survival and proliferation of cells, especially cancer cells. Preferably, the chemoattractant compound is used in combination with one or more carbohydrate (glucose) and/or lipid (fatty acid) molecules.

One skilled in the art will carefully select one or more chemoattractant compounds depending on the particular biological element targeted.

To this end, when the biological element is a eukaryotic cell, the membrane receptors expressed by the targeted cell must be analyzed beforehand to ensure the specificity of the chemoattractant compound or compounds selected.

For example, when the particular biological element is a cancer cell expressing the transmembrane receptor CXCR4 (e.g., a human breast cancer cell), the chemoattractant compound of choice is stromal cell derived factor 1 (SDF-1).

Similarly, when the particular biological element is a cancer cell of lung cancer, the chemoattractant compound selected is Epidermal Growth Factor (EGF) or transforming growth factor alpha (TGF α).

The microchannels constituting the microchannel network (2) of the microfluidic chip according to the invention may be parallelepipeds, cylinders, scales, truncated cones or a mixture of these shapes.

Each of the microchannels comprised in the microchannel network (2) of the microfluidic chip according to the invention may have a height of 1 μm to 500 μm, preferably 50 μm to 150 μm, a width of 1 μm to 500 μm, preferably 50 μm to 150 μm, and a length of 30 μm to 1mm, preferably 30 μm to 500 μm.

Specifically, in the context of the present invention, each microchannel comprised in the microchannel network (2) has a height of 1 μm to 40 μm, a width of 1 μm to 40 μm, and a length of 30 μm to 250 μm.

Preferably, in the context of the present invention, each microchannel comprised in the microchannel network (2) has a height of from 5 μm to 20 μm, a width of from 5 μm to 20 μm, and a length of from 100 μm to 200 μm, preferably 200 μm.

Each microchannel included in the microchannel network may have its own dimension that is independent of the dimensions of the other microchannels included in the microchannel network.

Similarly, when a chip includes several networks of microchannels, each microchannel of the same network, as well as each microchannel included in a different network of microchannels, may have their respective sizes and shapes independently of each other.

Preferably, in the context of the present invention, all microchannels comprised in the network of microchannels have the same shape and the same dimensions.

According to a particular aspect, the invention relates to a microfluidic chip wherein the electrodes (4) are interdigitated electrodes.

The "interdigital electrodes" correspond to electrodes (4) in which each conductor is multi-dentate. Preferably, each conductor has a diameter of 200 μm to 400 μm, preferably about 315 μm, and is spaced from the other conductor by an air gap of 10 μm to 50 μm, preferably about 15 μm.

Even more preferably, the electrode (4) is a wireless electrode remotely powered by an antenna (13).

For example, the remote power supply may operate by wireless communication using the ISM (industrial, scientific and medical) band at a frequency range of 13.553-13.567 MHz.

More specifically, the invention relates to a microfluidic chip in which the electrode (4) is a wireless electrode, said chip comprising an antenna that can be activated by a second antenna. Preferably, when the microfluidic chip is implantable in vivo, the second antenna is preferably placed outside the body.

"antenna" refers to an electronic component made of conductive material that forms one or more coils. A magnetic field is generated when a current passes through the antenna. The magnetic field may be discharged as electrical energy by the antenna. Thus, the antenna transmits current through the tissue through the inductance. Preferably, in the context of the present invention, the antenna is an inductor.

The antenna (13) allows remote powering of the electrode (4).

The antenna may also allow information to be transmitted remotely.

The second antenna to be positioned outside the body has a transmitter function, and the antenna included in the wireless electrode has a receiver function.

One skilled in the art will carefully adapt the resonant frequency, distance, and alignment between the transmitter and receiver depending on the efficiency of the wireless power to be transmitted.

The antenna included in the chip and the second antenna are made of a biocompatible material, preferably the same material as used for the preparation of the electrode (4), preferably titanium.

More specifically, when the chip is implanted in vivo, the diameter of the antenna is sufficient to transmit power through the tissue by inductance, preferably the diameter of the antenna is of the same order of magnitude as the thickness of the tissue to be passed through, preferably the diameter of the antenna is 5mm to 100mm, even more preferably 10mm to 80 mm.

According to a preferred aspect, the second antenna is integrated into a patch for external application to the skin, said patch comprising a physiologically acceptable external carrier, i.e. an external carrier compatible with the skin, mucous membranes and appendages of the skin.

The second antenna may also be located in an external box.

According to a particular aspect, the invention relates to a microfluidic chip in which the electric field generated by the electrodes (4) is capable of destroying the biological element by irreversible electroporation.

Preferably, the electric field generated by the electrodes is a pulsed electric field, which corresponds to a selective non-thermal treatment of short duration (typically a few microseconds to a few milliseconds).

The application of a pulsed electric field over a particular cell results in the accumulation of charge on the membrane surface and an increase in the transmembrane potential of the cell membrane. The attractive forces between the oppositely signed charges accumulated on both sides of the cell membrane cause the cell membrane to be compressed, and the elastic forces tend to oppose this compression. When the applied pulsed electric field exceeds a critical value, the voltage compression force is greater than the elastic force, and holes appear on the cell membrane. When the strength of the pulsed electric field is high and/or the duration of the treatment is long, the permeability of the cell membrane is increased and the destruction of the cell membrane becomes irreversible.

In the context of the present invention, the pulsed electric field generated by the electrodes on a particular biological element induces irreversible electroporation of said biological element. More precisely, when the biological element is a prokaryotic or eukaryotic cell, the pulsed electric field generated by the electrodes generates pores, in particular nanopores, in the cell membrane, inducing an irreversible deregulation of the homeostasis of the cell, leading to cell death by apoptosis or necrosis.

More specifically, the electric field generated by the electrode (4) is a pulsed electric field with a frequency of 1Hz, a voltage of 1000V/cm to 6500V/cm for a duration of 100. mu.s to 200. mu.s, preferably a pulsed electric field of 3000V/cm to 5000V/cm.

Preferably, the pulsed electric field of duration between 100 μ s and 200 μ s is generated by the electrodes several times a day, preferably at least 3 times a day, for a period of time suitable for the use scenario of the chip.

According to a particular embodiment of the invention, the microfluidic chip comprises means for measuring the number of specific biological elements entering the microfluidic chip and/or the number of biological elements destroyed by the electrodes.

In a conceivable approach, reference may be made in particular to an electrical impedance measurement system via electrodes generating an electric field that destroys biological elements, or to a measurement system by other electrodes dedicated to the measurement method, and the measurement system may be included in the upper and/or lower part of the microfluidic chip.

Electrical impedance is a measure of the opposition of a system to the movement of charge when a potential difference is applied to the system. In other words, it is equivalent to the ratio of the voltage applied to the system to the current generated. In the context of the present invention, the measurement serves to quantify the specific biological element entering the microfluidic chip, which adheres to the support, inducing a change in electrical impedance, and to quantify the specific biological element destroyed by the electrodes, which is separated from the electrodes, thus inducing a change in electrical impedance.

Preferably, the means for measuring the number of biological elements entering the microfluidic chip and/or the number of biological elements destroyed by the electrodes is an electrical impedance measurement method. In a particular embodiment of the invention, the information may be collected and transmitted by wireless communication techniques, for example, by the ISM (industrial, scientific and medical) band with a frequency range of 13.553MHz to 13.567 MHz.

A second subject of the invention relates to the use of a microfluidic chip according to the first subject of the invention for attracting and destroying specific biological elements.

More specifically, the invention relates to the use of a microfluidic chip according to the first subject of the invention for attracting and destroying specific biological elements in vivo, or to a method for attracting and destroying specific biological elements in vivo, said chip being implanted in vivo.

More precisely, the present invention relates to the use of a microfluidic chip according to the first subject of the invention for treating or preventing the proliferation and dissemination of specific biological elements (e.g. prokaryotic or eukaryotic cells) in a subject, or to a method for treating or preventing the proliferation and dissemination of specific biological elements (e.g. prokaryotic or eukaryotic cells) in a subject, wherein said chip is implanted in the body of the subject.

According to one aspect, the present invention relates to the use of a microfluidic chip according to the first subject of the invention for treating or preventing infections caused by prokaryotic cells (e.g. bacteria), or a method for treating or preventing infections caused by prokaryotic cells (e.g. bacteria), wherein said chip is implanted in the body of a subject.

According to a preferred aspect, the present invention relates to the use of a microfluidic chip according to the first subject of the present invention for treating or preventing proliferation and dissemination of eukaryotic cells, in particular cancer cells, or a method for treating or preventing proliferation and dissemination of eukaryotic cells, in particular cancer cells, preferably after resection of a solid tumor of a subject, wherein said chip is implanted in the body of the subject.

More precisely, the present invention relates to the use of a microfluidic chip according to the first subject of the present invention for preventing the risk of local cancer recurrence and/or metastatic carcinogenesis in a subject, or a method for preventing the risk of local cancer recurrence and/or metastatic carcinogenesis in a subject, wherein said chip is implanted in the body of the subject.

In the context of these uses and methods, the microfluidic chip is implanted in the subject at a location 0.1cm to 20cm, preferably 1cm to 10cm, more preferably 5cm, from the area of resection of a solid tumor or a focus of bacterial infection. Preferably, the microfluidic chip is implanted as soon as possible at the level of the resection area, once the solid tumor has been resected, preferably immediately after said tumor has been resected.

In the context of these uses and methods, preferably, the pulsed electric field is generated several times per day, preferably at least 3 times per day, more preferably 8 times per day, with the electrodes, the duration of the pulsed electric field being adapted to the use scenario of the chip.

"context of use of the chip" refers to the type of specific biological element targeted, i.e., when the element is a bacterium, the type of bacterial infection; or, when the element is a cancer cell, the type of cancer cell targeted, in particular the type of solid tumor removed, and the stage of the tumor.

Depending on the scenario, the pulsed electric field may be generated over a period of 3 days to 6 months, preferably 2 weeks to 4 months, to treat in a cyclically repeating fashion, with or without a rest period.

For example, if the resected solid tumor is in an advanced stage, the electrodes generate pulsed electric fields at least 3 times per day, preferably 8 times per day, over a period of 8 to 16 weeks. For example, if the resected solid tumor is in an advanced stage, the electrodes generate at least 8 pulsed electric fields per day over a period of 8 to 16 weeks.

In the context of the above-mentioned use, when the specific biological element is a cancer cell, the microfluidic chip may be used alone, or in combination, simultaneously or sequentially, with other pharmaceutical compounds, such as anti-cancer compounds, including chemotherapeutic agents and/or hormones and/or immunotherapeutic agents and/or targeted therapeutic compounds and/or radiotherapy.

Finally, the invention also relates to a microfluidic chip according to the first subject for attracting and destroying specific biological elements in vivo, said chip being implanted in vivo according to the preceding implementation conditions.

More precisely, the invention relates to a microfluidic chip according to the first subject for treating or preventing the proliferation and dissemination of specific biological elements (for example prokaryotic or eukaryotic cells) in a subject, wherein said chip is implanted in the body of the subject according to the aforesaid implementation conditions.

According to one aspect, the present invention relates to a microfluidic chip according to the first subject for the treatment or prevention of infections caused by prokaryotic cells (e.g. bacteria), wherein said chip is implanted in the body of a subject.

According to a preferred aspect, the present invention relates to a microfluidic chip according to the first subject for treating or preventing the proliferation and dissemination of eukaryotic cells, in particular after the resection of a solid tumor in a subject, wherein said chip is implanted in the body of the subject according to the preceding implementation conditions.

Even more preferably, the present invention relates to a microfluidic chip according to the first subject for preventing the risk of local cancer recurrence and/or metastatic cancer development in a subject, wherein said chip is implanted in the body of the subject according to the preceding implementation conditions.

In the context of these uses and methods, when the specific biological element is a eukaryotic cell, in particular a cancer cell, the cell is preferably derived from a cancer of the breast, lung, prostate, bladder, salivary gland, skin, small intestine, colorectal, thyroid, cervix, endometrium and ovary, lips, throat, kidney, liver, brain, testis, pancreas, preferably a breast cancer. Examples of such solid tumors are non-limiting.

The invention will be further illustrated by the following figures and examples. However, these examples and drawings should not be construed as limiting the scope of the present invention.

Examples

Example 1: manufacture of microfluidic chips for in vitro concept studies

For in vitro conceptual validation, a reservoir containing a chemoattractant was placed at the center of the microfluidic chip.

a) Integration of microelectrodes

In a so-called "piranha" solution (H)₂SO₄:H₂O₂3:1) to clean and increase the hydrophilic character of the glass substrate, and then dehumidified on a hot plate at 150 ℃ for 15 minutes to promote a negative-type photosensitive resin (from micro chemicals)nLOF 2020).

The resin was deposited on the substrate by spin coating (3000rpm, 30s) to obtain a thickness of 2 μm. The solvent was evaporated by annealing on a hot plate at 110 ℃ for one minute. To initiate crosslinking of some portions of the photoresist, the sample was subjected to UV exposure (70 mJ/cm)²). This activation will change the local properties of the resin, which after baking will become soluble or insoluble in the solvent. If the resin is negative, a portion exposed to UV rays during development will become solid, while another portion will dissolve. To continue the crosslinking reaction initiated by UV exposure, the plate was then subjected to a post-exposure bake (PEB) to provide the energy required for 1 minute of treatment at 110 ℃. Finally, this development step will dilute the non-crosslinked portion of the resin in a tetramethylammonium hydroxide based solvent (TMAH, from micro chemicals)726 MIF).

Then, a titanium/platinum (20/200nm) deposit was sputtered, followed by so-called "lift-off" (acetone and NANO)^TMREMOVER PG from micro chemicals) to remove resin residues. At the end of this step, electrodes are formed on the glass plate.

Using silicon nitride (Si)₃N₄) To ensure electrical insulation of the parts that should not be electrically conductive. To this end, the above-described photolithography process is repeated and additionalAnd (4) an alignment step. Obtaining 200nm Si in PECVD (plasma enhanced chemical vapor deposition) frame₃N₄And (4) depositing. The regions not protected by the resin are then etched by a reactive ion etching process (ICP RIE). At the end of the process, the device was cleaned using REMOVER PG (from MicroChemicals) and air plasma.

b) Mold fabrication Using MicroChemicals SU-8 photosensitive epoxy

Silicon plates (diameter 76.2mm) were prepared in a "piranha" solution and then dehumidified at 15 to 150 ℃. The SU-8 resin was deposited on the silicon plate by spin coating (3000rpm, 30s) to obtain the desired thickness (10 μm or 100 μm depending on whether SU-82010 or SU-82100 is used). The solvent evaporation is carried out by minimizing the mechanical stress in the resin (5 ℃/min) by very slow heating and very gentle cooling ramp. For a sample having a thickness of 10 μm, the sample was subjected to 125mJ/cm²And for a sample thickness of 100 μm, subjecting the sample to 250mJ/cm²365nm UV exposure. The plate was then subjected to a post-exposure bake (PEB) (10 μm thick sample, baked at 95 ℃ for 4 minutes; 100 μm thick sample, baked at 95 ℃ for 30 minutes). Finally, through development, the uncrosslinked portions of the SU-8 resin can be diluted in a solvent ("SU-8 developer solution" consisting mainly of Propylene Glycol Monomethyl Ether Acetate (PGMEA)). At the end of this step, it will allow the structuring of the microchannel pattern that remains on the silicon plate.

c) Structuring of micro-channels of a chip and final assembly of the chip

After the mold is made, it is cured by mixing it with its curing agent (Sylgard from Dow)^TM184 silicone elastomer kit, ratio 1:10) to prepare a Polydimethylsiloxane (PDMS) silicone polymer. Air bubbles formed during mixing were removed using a dryer and a vacuum pump. Once the PDMS was degassed, it was poured onto a SU-8 mold placed in a petri dish and placed in an oven at 80 ℃ for at least 2 hours to complete the crosslinking process. After cooling downThe PDMS was peeled off and cut with a circular blade to obtain a cylindrical shaped device. The resulting PDMS was then pierced with a punch to form a central hole to contain the chemoattractant. The final step involves bonding PDMS on a glass substrate to encapsulate the channels. The process is carried out by using O₂Or activating the surface of PDMS by air plasma generator to remove Si-CH of PDMS₃Conversion of the functional group to Si-OH. In contact with glass (SiO)₂) After contact, a permanent Si-O-Si covalent bond will be able to be created.

d) Electrical detection instrument

The chip is equipped with interdigitated electrodes (distance: 15 μm) which apply an electric field (2000V/cm to 5000V/cm) strong enough to cause irreversible electroporation of specific cells, thus inducing apoptosis or necrosis of the cells which have penetrated the system. During on-chip applications, square-wave signals (voltage/frequency/high-voltage time: 3V to 7.5V/1 Hz/100. mu.s) were output from a function generator (TG2511A TTi) and displayed by an oscilloscope (TBS1032B Tektronix). The second channel of the oscilloscope was connected to a high precision shunt resistor (0.1W, LVR01R1000FE70 from Vishay) to produce an image of the current flowing through the electrode (4). To automate the experiment, the instrument was connected to a computer (Raspberry Pi 3 Model B +, operating System: Linux Raspbian 9(Stretch)) via a USB connection. The "virtual instrument software architecture" (VISA) communication is ensured by the "open source" PyVISA library and Python scripts are executed periodically by the crontab program of the operating system.

e) Manufacture of release matrices

A3% (w/v) alginate solution in water was deposited in a porous mold having the shape of a reservoir of the chip. The molds were soaked in a calcium chloride cross-linking bath for 24 hours, after 3 washes with Milli-Q water, the matrix was frozen at-20 ℃, and subsequently lyophilized.

The matrix was then gently placed in the chip.

Example 2: study of cytokine Release Profile

(A) To protect the chemoattractant compound SDF-1 from enzymatic proteolysis in vitro and in vivo, the chemoattractant compound SDF-1 is complexed with BSA, usually in a ratio of 1.51 molecules of Bovine Serum Albumin (BSA) and 10 molecules of SDF-1. BSA is a 6kDa large fetal bovine serum albumin, while SDF-1 is an 8kDa small cytokine. Since the release of SDF-1 from the matrix is by diffusion, the molecule with the largest hydrodynamic radius will have the largest effect on the diffusion of the complex: i.e., BSA. Thus, as a first step, the model was used to study the release profile of SDF-1 compound from microfluidic chips fabricated according to example 1. To maximize detection of nanoscale concentrations, we chose to detect Fluorescein Isothiocyanate (FITC) conjugated BSA by High Performance Liquid Chromatography (HPLC) coupled fluorescence detector.

Sample preparation:

-depositing 3.2 μ g of BSA-FITC on a matrix of lyophilized alginate contained in a chip;

incubation at 37 ℃ for 30 minutes.

The study was started:

in a gas-tight box, submerge the chip in 1.5ml PBS.

Incubation with shaking at 37 ℃ for the duration of the study, i.e. 50 days.

At different times, 750. mu.l of the release solution were sampled and the samples were analyzed by HPLC (water/acetonitrile).

The chromatographic column used is a chromatographic column designed for the detection of proteins such as BSA-FITC.

Add 750 μ Ι of fresh PBS to the release medium to compensate the previous sample.

As a result:

this study confirmed that BSA-FITC was released continuously from the alginate matrix contained in the microfluidic chip according to the present invention for 50 days, and 99% release was achieved after 50 days (fig. 21).

(B) In the second step, the release of SDF-1 from the alginate matrix over a period of 50 days was quantified using an ELISA assay.

Sample preparation:

-complexing of SDF-1 with BSA (0.1%);

-depositing 1 μ g of SDF-1 on a freeze-dried alginate matrix;

incubation at 37 ℃ for 30 minutes.

The study was started:

-immersing the substrate in 1.5ml PBS in an airtight box;

incubation with shaking at 37 ℃ for the duration of the study, i.e. 50 days.

750. mu.l samples of the release solution were taken at different times and analyzed by means of an ELISA kit dedicated to the detection of SDF-1, according to the supplier's instructions.

To the release solution, 750 μ l of fresh PBS was added to compensate the previous sample.

As a result:

this study demonstrated sustained release of SDF-1 from the alginate matrix over a period of only 23 days, while 40% release was achieved after 23 days (fig. 22).

Example 3: implementation of in vitro microfluidic chip

A) Attraction to cells in the chip

Microfluidic chips were implemented in vitro to test their ability to attract MDA-MB-231 breast cancer cells, which are epithelial cells of breast tumors. The compound "stromal cell derived factor" SDF-1 is a chemoattractant compound capable of attracting MDA-MB-231 cells.

1) MDA-MB-231 cells stably transfected with Green Fluorescent Protein (GFP) were trypsinized at D0. Then, 20000 cells were seeded in a 35mm petri dish with a microfluidic chip placed in the center. Cells were cultured in Dulbecco's modified Eagle's Medium/nutrient mixture F-12(DMEM F12) + Glutamine + 1% fetal bovine serum + 1% antibiotics (streptomycin, penicillin).

The central reservoir of the microfluidic chip was loaded with 33 μ l of DMEM F12+ glutamine + 1% fetal bovine serum + 1% antibiotics (streptomycin, penicillin) (fig. 8), or 33 μ l of DMEM F12+ glutamine +10 μ l pure fetal bovine serum + 1% antibiotics (streptomycin, penicillin) (fig. 9). At 5% CO₂And 95% humidity for 7 days, photographs were taken using an inverted fluorescence microscope.

It was found that the cells moved towards the reservoir, and also specifically into the reservoir, under both test conditions, with the number of cells being greater with 10 μ l of pure fetal bovine serum present within the reservoir.

2) MDA-MB-231 cells stably transfected with Green Fluorescent Protein (GFP) were trypsinized at D0. Then, 100000 cells were seeded in a 50mm diameter petri dish with a microfluidic chip placed in the center. Cells were cultured in Dulbecco's modified Eagle's Medium/nutrient mixture F-12(DMEM F12) + Glutamine + 1% fetal bovine serum + 1% antibiotics (streptomycin, penicillin).

The central reservoir of the microfluidic chip was loaded with 50 μ l of DMEM F12+ glutamine + 1% fetal bovine serum + 1% antibiotics (streptomycin, penicillin) (fig. 10 and 11), or 50 μ l of DMEM F12+ glutamine +10 μ l fetal bovine serum + 1% antibiotics (streptomycin, penicillin) (fig. 12 and 13), or 50 μ l of DMEM F12+ glutamine +1 μ g SDF-1+ 1% antibiotics (streptomycin, penicillin) (fig. 14 and 15).

At 5% CO₂And 95% humidity for 8 days, photographs were taken using an inverted fluorescence microscope.

Under all test conditions, in particular in the presence of a fetal bovine serum gradient (fig. 12 and 13), even more significantly in the presence of a compound SDF-1 gradient (fig. 14 and 15), it was found that the cells moved towards the reservoir, and also specifically into the reservoir.

B) Ability of interdigitated electrodes to disrupt cells

1) The ability of interdigitated electrodes to generate an electric field to destroy MDA-MB-231 cells was tested. For this purpose, MDA-MB-231 cells stably transfected with Green Fluorescent Protein (GFP) were trypsinized at D0. 60000 cells were then seeded into PDMS wells fixed on a glass slide, in which circular interdigitated electrodes were placed. The media used were: DMEM F12+ glutamine + 10% fetal bovine serum + 1% antibiotics (streptomycin, penicillin).

At 5% CO₂And 95% humidity for 48 hours, a monolayer of cells covering the entire slide (which contained interdigitated electrodes) was obtained (FIG. 16), then passed over the slideAn electric field of 5V 100. mu.s 1Hz was applied to the plate to electroporate the cells, and a photograph was taken 3 hours later with an inverted fluorescence microscope (FIG. 17).

About 70% of the cells were observed to be destroyed after 3 hours after electroporation.

2) The microfluidic chip according to the present invention was tested for its ability to attract MDA-MB-231 cells and to destroy the cells by electroporation.

For this purpose, MDA-MB-231 cells stably transfected with Green Fluorescent Protein (GFP) were trypsinized at D0. 50000 cells were then seeded in a petri dish of 50mm diameter, and the microfluidic chip comprised interdigitated electrodes and two networks of microchannels placed in the centre of the petri dish.

Cells were cultured in Dulbecco's modified Eagle's Medium/nutrient mixture F-12(DMEM F12) + Glutamine + 1% fetal bovine serum + 1% antibiotics (streptomycin, penicillin).

Three conditions were tested:

loading the central reservoir of the chip with 50 μ l of DMEM F12+ glutamine + 1% fetal bovine serum + 1% antibiotics (streptomycin, penicillin) without gradient and without pulsed electric field (fig. 18).

In the absence of pulsed electric field, the central reservoir of the chip was loaded with 50 μ l of DMEM F12+ glutamine + SDF-11 μ g + 1% antibiotics (streptomycin, penicillin) (FIG. 19).

In the presence of a pulsed electric field, the central reservoir of the chip was loaded with 50. mu.l of DMEM F12+ glutamine + SDF-11. mu.g + 1% antibiotics (streptomycin, penicillin) (FIG. 20).

After 3 days of culture at 5% CO₂And 95% humidity for 7 days, during which electroporation was applied under C conditions (5V 100ms 1Hz) every 3 hours. Photographs were taken using an inverted fluorescence microscope.

This study demonstrated that in the presence of a chemoattractant SDF-1 gradient and in the absence of an electric field, MDA-MB-231 cells were attracted to the central reservoir and retained in the space between the two microchannel networks.

In the presence of chemoattractant SDF-1 and a pulsed electric field applied every 3 hours (5V 100 μ s 1Hz), the cells pass through the first network of microchannels and are electroporated.

In all cases, the cells cannot span both microchannel networks.

These different experiments have demonstrated the ability of microfluidic chips to attract and destroy biological elements, and in particular, the ability of the chips to attract breast cancer tumor cells (MDA-MB-231) under a gradient of fetal bovine serum, more notably under a gradient of chemoattractant SDF-1, and to destroy these kinds of tumor cells by pulsed electric fields.