阅读说明：本技术 肝前体细胞和内皮细胞共培养在治疗急性肝损伤中的应用 (Application of co-culture of liver precursor cells and endothelial cells in treatment of acute liver injury ) 是由 鄢和新 刘文明 于 2020-12-08 设计创作，主要内容包括：本发明涉及医药技术领域,具体是一种人肝前体细胞与内皮细胞共培养体系,以及共培养后的肝细胞球在制备急性肝损伤治疗药物中的应用。本发明采用无基质胶、低血清共培养体系,发现共培养后,可以有效增加促进肝细胞功能的提升,操作简单、花费低、培养周期短,海藻酸盐微囊包被后可以明显提高急性肝损伤小鼠的生存率,高达90％以上,远高于对照组的16.7％。与多潜能干细胞诱导得到的类肝细胞和原代肝细胞相比,肝前体细胞将更具有临床实用价值,为干细胞移植治疗肝损伤提供了新的思路和替代治疗方案。(The invention relates to the technical field of medicines, in particular to a co-culture system of human liver precursor cells and endothelial cells and application of co-cultured hepatocyte balls in preparation of a medicine for treating acute liver injury. The invention adopts a co-culture system without matrigel and with low serum, and finds that after co-culture, the improvement of the function of the liver cells can be effectively improved, the operation is simple, the cost is low, the culture period is short, and the survival rate of mice with acute liver injury can be obviously improved after the alginate microcapsules are coated, and is more than 90 percent and far higher than 16.7 percent of that of a control group. Compared with the hepatic cell like cells and primary hepatic cells obtained by the induction of the pluripotent stem cells, the hepatic precursor cells have more clinical practical value, and provide a new thought and alternative treatment scheme for treating liver injury by stem cell transplantation.)

1. A co-culture medium of human liver precursor cells and endothelial cells is characterized in that Advance DMEM/F12 is used as a basal medium, 1 XB 27 supplement, 1 XL-glutamine reagent, 1 XP pH regulator, 1 XP primary cell antibiotic and 1 XP penicillin-streptomycin are added, 1-20mM nicotinamide, 1-10mM N-acetylcysteine, small molecular compound 1-50ng/ml HGF, 1-100ng/ml EGF, 1-10 mu M CHIR-99021, 0.1-20 mu M Y27632, 300 and 700nM TGF-beta inhibitor A8301, 1-100mg/ml vitamin C and 1-10ng/ml OSM are added simultaneously; additionally, 1-20ng/ml VEGF, 1-20ng/ml FGF10, 0.1-10% FBS were added.

2. A co-culture method of human liver precursor cells and endothelial cells is characterized by comprising the following steps: the hepatic precursor cells and endothelial cells grown to logarithmic phase were counted separately by Tryple digestion at 1: 1 by 2 x 10⁵Inoculating the cells into a low-adhesion 6-well plate at a density, and adding 3ml of fresh human liver precursor cells and endothelial cells co-culture medium according to claim 1; in the culture process, the cells gradually gather to form hepatocyte spheroids, and then the hepatocyte spheroids are converted into a low proliferation state for 5 days.

3. A hepatocyte cell pellet co-cultured by the method of claim 2.

4. Use of the co-cultured hepatocyte cell of claim 3 in the preparation of a medicament for the treatment of acute liver injury.

5. The use of claim 4, wherein the acute liver injury treatment is a cell therapy drug.

6. A microencapsulated formulation characterized in that the hepatocyte cells obtained by co-culture according to claim 3 are coated with alginate microcapsules.

7. A microencapsulated formulation as defined by claim 6 wherein the process for preparing the microencapsulated formulation comprises: collecting the co-cultured hepatocyte pellet of claim 3 into a centrifuge tube, and discarding 2 x 10 of the supernatant⁶The solution/ml is re-suspended in the sodium alginate solution, so that bubbles are avoided to the greatest extent; loading the cell-sodium alginate mixed solution into a syringe pump, starting an electrostatic droplet generator, adjusting the pump speed, preparing sodium alginate gel beads, and dripping the sodium alginate gel beads into a beaker containing 100 mmol/of calcium chloride solution for full calcification for 20 minutes; transferring the calcium alginate gel beads to a 50ml centrifuge tube, adding a PLL solution with the volume 5 times that of the gel beads, and oscillating at room temperature to form a film for 5-8 minutes; fully washing the microcapsule for 3 times by using 0.9 percent sodium chloride, and then adding sodium citrate solution with the volume about 8 times of the microcapsule volume to liquefy the gel beads for 2 to 5 minutes; after the liquefied rubber beads are washed, the liquefied rubber beads need to be treated by 0.15% sodium alginate solution for about 10 minutes, and after being fully washed, the liquefied rubber beads can be directly transplanted in vivo.

8. Use of a microencapsulated formulation as defined in claim 6 in the preparation of a medicament for the treatment of acute liver injury.

Technical Field

The invention relates to the technical field of medicines, in particular to a co-culture system of matrigel-free low-serum liver precursor-like cells (hepLPCs) and endothelial cells (HUVEC), which can obviously improve the liver functions of the liver precursor cells (hepLPCs), and after being coated by alginate microcapsules, the survival rate of mice with acute liver injury can be obviously improved, thereby providing a new thought and an alternative treatment scheme for treating liver injury by stem cell transplantation.

Background

The liver is the largest organ of the human body and plays an important role in the human body. It regulates carbohydrate metabolism, urea and lipid metabolism, storage of essential nutrients, production of plasma proteins, secretion of bile acids and liver, etc.; meanwhile, the liver is used as a substance energy conversion center of the human body, and sufficient substrates and cytochrome enzymes directly/indirectly participate in the biotransformation of the human body to various foreign substances. China is a high-incidence country of hepatitis infection, and thus the incidence of severe liver disease is also very high in China. Liver transplantation remains the most effective means of treating severe liver disease. The liver cell is the most abundant parenchymal cell in the liver, is the 'gold standard' of the research in the field of the liver, and plays a significant role in vitro culture, drug toxicity screening and the research in the aspect of bioartificial liver.

Three strategies are mainly adopted for in vitro hepatocyte culture: firstly, the direct culture of liver cells in vitro, secondly, the directional differentiation culture of liver cells in vitro by stem cells, and thirdly, the culture of hepatic cell-like cells by inducing the transdifferentiation of fiber progenitor cells. In vitro differentiation techniques using human Embryonic Stem Cells (hESCs) and Induced pluripotent Stem Cells (hipSCs) can induce differentiation of Stem Cells into "hepatoid Cells" with hepatocyte markers and partial hepatocyte function [ Cells J, 2016,165(7):1586 1597 ]. Fibroblast transdifferentiation into hepatocytes with partial hepatocyte function can be induced by in vitro chemical induction or transfection of hepatocyte-specific transcription factors. However, recent studies have shown that such in vitro reprogramming may lead to genetic and epigenetic changes, inducing genomic instability and genetic mutations, and that transplantation of transgenic biomaterials into humans faces major ethical problems, and that these deficiencies will lead to enormous risks for hESC and hipSC-derived "hepatocyte-like" cell transplantation [ Dudley S C, J.Beware of cell bearing targets: cell replacement therapy and arrhythmic risk [ J ]. Circ Res,2005,97(2):99-101 ]. Hepatocytes are therefore considered an ideal cell source for cell transplantation and therapy. In 2017, the group of inventors found that mature hepatocytes could be induced to be transformed into pro-hepatic-like cells with proliferative capacity by small molecular combination in vitro and have the potential to differentiate into mature hepatocytes [ Wu H, Zhou X, Fu G B, He Z Y, Wu H P, You P, Ashton C, Wang X, Wang H Y, Yan H X. reversible transformation between liver cells and liver promoters for in vitro hepato expansion [ J ]. Cell Res,2017,27(5):709 and 712 ]. The precursor cells generated by the reprogramming related signal channels of the liver cells are efficiently activated in vitro through small molecule combination, the safety and the uniformity are high, and the method has a certain clinical application value in the treatment of acute liver injury. Meanwhile, an HPV E6/E7 system is introduced to obtain immortalized liver precursor cells, so that a large number of liver precursor cells can be obtained, and a large number of cell sources are provided for cell therapy [ Xiao J, Wang X, Wu Y, ZHao Q, Liu X, Zhang G, ZHao Z, Ning Y, Wang K, Tan Y, Du B.synergistic effect of recalcrol and HSV-TK/GCV therapy on hormone cells [ J ]. Cancer Biol Ther,2019,20(2):183-191 ]. In view of cellular safety, we have also introduced the HSV-TK suicide system for ease of clearance. The gene-transduced cells express functional HSV-TK, which can induce the conversion of nontoxic Ganciclovir (GCV) into toxic GCV-triphosphate to inhibit DNA synthesis, thereby causing cell death [ Jianyonong Xiao, Xialoan Wang, Yingya Wu, Qing Zhao, Xiaodong Liu, Guangxian Zhuang, Zengqiiang Zhao, Yizohen Ning, Kun Wang, Yuhui Tan, Biaoyan Du.synergistic effect of resveratrol and HSV-TK/GCV therapy on multiple cells.cancer Biol Ther.2019; 20(2):183-191.].

In recent years, for the complexity of in vitro simulator tubes, the cell culture mode is also evolved from traditional 2D (2Dimension) culture to Co-culture (Co-culture) to Organoid (Organoid), although organoids are becoming the focus of research more and more in recent years, the drawbacks thereof cannot be evaded, i.e. firstly, the cell survival in the center of organoids is affected due to the passive diffusion of organoids and oxygen, so the size of organoids is limited to a few millimeters; (II) more and more organoids are reported to be immature even after prolonged culture, similar to embryonic and fetal tissues, but not adult tissues, with restricted developmental understanding [ Lancaster M A, Renner M, Martin C A, Wenzel D, Bicknell L S, Hurles M E, Homfay T, Penninger J M, Jackson AP, Knoblich J A. Cerebral organic models human branin depletion and microcephaly [ J. Nature,2013,501(7467): 373. Pharma 379 ] ]; and thirdly, the operation is complex and the culture cost is high. Endothelial Cells (EC) may play a key role in overcoming the above limitations [ Garcia M D, Larina I V.vascular definition and hemodynamic purpose in the mouse yolk sac [ J ]. Front physiology, 2014,5(308) Dye B R, Dedhia P H, Miller AJ, Nagy M S, White E S, Shell L D, Spence J R.A bioengineered amino proteins in vivo development and distribution of multiple stem cell derived human lung ligands [ J.Elife, 2016,5 ]. Therefore, a set of controllable new technology system for culturing liver cells is urgently needed to be established, and normal mature liver cells with better functions are cultured in vitro.

Disclosure of Invention

The invention aims to establish a co-culture system of liver precursor cells and endothelial cells, which does not need matrigel, has low serum and no differentiation, has short culture period, improves the liver function of the liver precursor cells through the interaction between cells, thereby improving the survival rate of mice with acute liver injury and further providing a new thought and an alternative treatment scheme for treating the liver injury by stem cell transplantation.

Co-culture of hepatocytes with endothelial cells was reported as early as 2013, and Takanori Takebe found that iPSC was co-cultured with endothelial cells (HUVEC) and Mesenchymal Stem Cells (MSC) to form Liver Bud, and that specific hepatocytes (immature endoderm cells for tracking the fate of hepatocytes) were self-assembled into three-dimensional iPSC-LB by interaction with endothelial cells and Mesenchymal cells, and were vascularized, and researchers used mixed culture media of multipotent stem cells, endothelial cells and Mesenchymal stem cells, respectively, in culture, but the authors were still unable to circumvent the preclinical potential risks that may be brought by the use of multipotent stem cells [ Takebe T, Sekine K, Enomura M, Koike H, Kimura M, Ogaieri T, Zhang R, Ueno, Zhening Y W, Kokine N, Azeyas, Adchia H, Tariffia H. varied human plasma J. upright et al. [ Natural Manual J. upright Bud. SC, 2013,499(7459):481-484.]. Despite this, the Takebe study highlights the enormous potential of organoid embryos grown in vitro to treat organ failure. The invention establishes a low serum culture condition which can co-culture the liver precursor cells and the endothelial cells on the basis of predecessor, can obviously improve the functions of the liver cells without a differentiation process, and has the advantages of low culture cost, short period and simple operation.

Similar to our invention, recent studies found that mouse liver blood sinus endothelial cells (LSEC) can promote LPCs to improve hepatocyte function [ Kim Y, Rajagopalan P.3D liver cells multiple main liver cells and liver single end biliary cells phenotypes [ J ]. PLoS One,2010,5(11): e15456 ], and the mechanism of production of liver and gall organs by a liver-specific duct system has not been explored [ Yap K, Gerrand Y W, Dingle AM, Yooh G C, Morrison W A, Mitchell G M.Liver single end biliary cells process reaction and differentiation of liver tissues [ 120251, 091J ], (091J.) ]. In combination with Hepatocytes, Endothelial Cells and Kuffer Cells may mimic the in Vitro progression of NAFLD [ Suurmond C E, Lasli S, Van Den Donder F W, Ung A, Kim H J, Bandaru P, Lee K, Cho H J, Ahadian S, Ashamakhi N, Dokmeci M R, Lee J, Khadenossi A. in Vitro Human Liver Model of Nonalco Stemonic Stemonatic Stemonatitis by Cocurating hepatotes, Endothelil Cells, and Kupffer cell [ J ]. Adv Healthc Mater,2019,8(24): E1379 ], further disclosing broad prospects for co-culture [ Kostalin R, ess F, applied G D, Westing L, filtration T, Westing T A.A, tissue of tissue, tissue of filtration J, tissue of filtration of tissue of the culture of the Human tissue of the genus of the species of the genus of NAFLEXCEL, biemel K M, Darnell M, Hoffmann S A, Knospel F, Wonne E C, Knobeloc D, Nussler A K, Gerlach J C, Andersson T B, Zeilinger K.Serum-free culture of primary human hepatocytes in a minor lipid-fiber biorator for pharmaceutical in vitro students [ J ]. J Tissue Eng Regen Med,2015,9(9): 1017. 1026 ]. It is well recognized that co-cultures with endothelial cells can maintain good differentiation potential and function relatively stably with hepatocytes, thereby enhancing specific liver function. However, the specific mechanism is unknown. There is no mechanism for hepatic differentiation in the human 3D co-culture system. Based on this, we obtained from hepLPCs or hepLPCs + HUVEC 1: 1 (named vhepLPCs) 3D culture systems were set up without matrigel and low FBS. We will elucidate the mechanism of intercellular interactions to understand the key mechanisms of hepatocyte maturation. Lays a foundation for the engineering application of the hepatic precursor cells.

The invention is based on the prophase [ Zhenyu Wang, Weijian Li, Hongshu jin, Ming Ding, Gongbo Fu, Tianjie Yuan, Weijian Huang, Mengjun Dai, Dan Tang, Min Zeng, Yi Chen, Hongdan Zhuang, Xuejing Zhu, Yuan Pen, Qigen Li, Wei-Feng Yu, He-Xin Yan, Bo Zhu. 6690-6705.Wei-Jian Li, Xue-Ju Zhu, Tian-Jie Yuan, Zhen-Yu Wang, Zheng-Qian Bian, Hong-Shu Jug, Xiao Shi, Cai-Yang Chen, Gong-Bo Fu, Wei-Jian Huang, Yao-Ping Shi, Qian Liu, Min Zeng, Hong-Dan Zhang, Hong-Ping Wu, Wei-Feng Yu, Bo Zhai, He-Xin Yan et al and an exogenous polypeptide biological instrumentation tongue juice embedded with a hand 3D-layerd human promoter-cell juice extract juice front, Ju-8; 12(551) eaba5146 ], establishing a co-culture system of human liver precursor cells and endothelial cells to form vhepLPCs spheres, and finding that the liver function of the vhepLPCs after co-culture is obviously improved and verified from multiple aspects of gene level, protein level and functional experiments; meanwhile, the vhepLPCs can obviously improve the survival curve of mice with acute liver injury, and the survival rate of the mice coated with the alginate microcapsules can reach over 90 percent. Conventional cell transplantation methods include spleen transplantation and portal vein transplantation, and inevitably cause bleeding risk due to surgery. Alginate is a biologically inert material that protects hepatocytes from the host. After the alginate microcapsule is adopted for coating, on one hand, immune rejection is avoided, and on the other hand, cells can freely pass through a substrate and protein, so that a new strategy is provided for cell treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect of the present invention, there is provided a co-culture medium for human hepatoblasts and endothelial cells, comprising Advance DMEM/F12 as a basal medium, and 1 XB 27 supplement (Gibco), a 1 XL-glutamine reagent (e.g., GlutaMAX-I), a 1 XP regulator (HEPES), a 1 XP primary cell antibiotic (e.g., 0.1mg/ml primocin), and 1 XPcillin-streptomycin, simultaneously adding 1-20mM nicotinamide, 1-10mM N-acetylcysteine, 1-50ng/ml HGF of small molecular compound, 1-100ng/ml EGF, 1-10 mu M CHIR-99021, 0.1-20 mu M Y27632, 300-700nM TGF-beta inhibitor A8301, 1-100mg/ml vitamin C and 1-10ng/ml OSM; additionally, 1-20ng/ml VEGF, 1-20ng/ml FGF10, 0.1-10% FBS (Corning) were added.

Further preferably, the co-culture medium of human hepatoprogenitor cells and endothelial cells is prepared by adding 1 XB 27 supplement (Gibco), 1 XL-glutamine reagent (such as GlutaMAX-I), 1 XP pH regulator (HEPES), 1 XP cell antibiotic (such as 0.1mg/ml primycin) and 1 XP streptomycin to Advance DMEM/F12 as a basal medium, and simultaneously adding 10mM nicotinamide, 1.56mM N-acetylcysteine, small molecule compound 25ng/ml HGF, 50ng/ml EGF, 3. mu.M R-02199, 10. mu. M Y27632, 500nM TGF-beta inhibitor A8301, 50mg/ml vitamin C, 2ng/ml OSM; 10ng/ml VEGF, 10ng/ml FGF10, 1% FBS (Corning) were additionally added.

Further, the basic culture medium advanced DMEM/F12 is a commercial culture medium: invitrogen, 12634-.

In a second aspect of the present invention, there is provided a method for co-culturing human hepatic precursor cells and endothelial cells, comprising the steps of:

the hepatic precursor cells and endothelial cells grown to logarithmic phase were counted separately by Tryple digestion at 1: 1 by 2 x 10⁵The cells were seeded into a low-adhesion 6-well plate at a density, and 3ml of a fresh co-culture medium (modified co-culture medium) for human hepatic precursor cells and endothelial cells as described above was added; the cells gradually conglomerate during the culture process to form hepatocyte spheroids (sphenoids), and further change to a low proliferation state, and the cells are cultured for 5 days。

In a third aspect of the present invention, there is provided a hepatocyte pellet co-cultured by the co-culture method as described above.

In a fourth aspect, the invention provides a use of the hepatocyte spheroids obtained by co-culture as described above in the preparation of a medicament for treating acute liver injury.

Furthermore, the acute liver injury treatment drug is a cell therapy drug.

In the fifth aspect of the invention, a microencapsulated preparation is provided, wherein the microencapsulated preparation is a hepatocyte spheroid obtained by co-culturing human hepatic precursor cells and endothelial cells and coated by alginate microcapsules.

Further, the preparation method of the microencapsulated preparation comprises the following steps: the hepatocyte spheroids co-cultured as described above were collected in a centrifuge tube, discarded and resuspended in an appropriate volume of sodium alginate solution (2 x 10)⁶Ml), avoiding the generation of bubbles as much as possible; loading the cell-sodium alginate mixed solution into a syringe pump, starting an electrostatic droplet generator, adjusting the pump speed, preparing sodium alginate gel beads, and dripping the sodium alginate gel beads into a beaker containing 100 mmol/of calcium chloride solution for full calcification for 20 minutes; transferring the calcium alginate gel beads to a 50ml centrifuge tube, adding a PLL solution with the volume 5 times that of the gel beads, and oscillating at room temperature to form a film for 5-8 minutes; fully washing the microcapsule for 3 times by using 0.9 percent sodium chloride, and then adding sodium citrate solution with the volume about 8 times of the microcapsule volume to liquefy the gel beads for 2 to 5 minutes; after the liquefied rubber beads are washed, the liquefied rubber beads need to be treated by 0.15% sodium alginate solution for about 10 minutes, and after being fully washed, the liquefied rubber beads can be directly transplanted in vivo.

In a sixth aspect, the invention provides an application of the microencapsulated preparation in preparing a drug for treating acute liver injury.

The invention has the advantages that:

according to the invention, a matrigel-free and low-serum co-culture mode is established for the first time to culture the hepatic precursor cells and the endothelial cells, and the finding shows that the function of the hepatic cells can be obviously improved after co-culture, and the alginic acid microcapsule coated vheppLPCs spheres can obviously improve the survival time of mice with acute hepatic injury, so that a new strategy is provided for promoting cell therapy.

Drawings

FIG. 1: safety identification of liver precursor cells hepLPCs; a: the hepLPCs are transfected into an HSV-TK/GCV suicide system through lentivirus mediation, and the GCV treatment can obviously inhibit cell activity for 48 hours to form dose dependence; b: after GCV treatment for 48 hours, q-PCR detects that the expression of apoptosis-related markers CASP3 and BCL-2 is obviously increased compared with a control group; c: the liver precursor cells transfected with GFP are treated by GCV to obviously inhibit the cell activity; d: the high-density hepatic precursor cells hepLPCs are inoculated subcutaneously to NSG mice, 50mg/kg GCV is injected into the abdominal cavity of an experimental group, the materials are obtained after 5 days, embedding is carried out, and the change of cell activity is observed under a mirror, so that the proportion of living cells after GCV treatment is obviously reduced compared with a control group, physiological saline is used as the control, and each group of mice is more than 3.

FIG. 2: establishing a co-culture system of liver precursor cells and endothelial cells without matrigel and with low serum; a: 2D monolayer culture of hepLPCs, 3D hepLPCs sphenoids alone, 3D hepLPCs coculture with HUVEC of vhepLPCs sphenoids, 2D monolayer differentiation of 14 days hepLPCs 4 states of cell morphology change, bars: 100 μm; b: q-PCR detects the expression of liver function related markers ALB and HNF4A, and the expression of ALB and HNF4A in the co-cultured vhepLPCs is obviously higher than that of the other three groups; c: glycogen staining of hepLPCs and vhepLPCs spheres was performed to identify the ability of cultured cells to accumulate glycogen, bars: 20 μm; d: hepLPCs and vhepLPCs spheres were oil red stained to identify the capacity of the cells for lipid metabolism, bars: 20 μm; e: hepLPCs and vhepLPCs could inhale indocyanine violet, suggesting that sphenoids have good liver function, bars: 20 μm.

FIG. 3: co-culture can promote hepatocyte maturation; a: firstly, determining that endothelial cells can survive under a low serum condition under a co-culture condition, and detecting the expression condition of an endothelial cell marker VEGFA through q-PCR (polymerase chain reaction), wherein the VEGFA expression is obviously increased after co-culture; b: WB detected the expression of hepLPCs and vhepLPCs spheres CD31, and it was found that the expression of CD31 was significantly increased after co-culture; c: IF further verified that ALB was also partially expressed in sphenoids after co-culture, indicating that two cells interacted in this culture system, bars: 100 μm; d: q-PCR (polymerase chain reaction) is used for detecting the expression conditions of liver function markers ALB and CYP3A4 of hepLPCs and vhepLPCs spheres, and the liver function marker expression is obviously increased after co-culture; e: further verification of protein level; f: immunofluorescence results showed that hepLPCs sphenoids alone partially expressed hepatocyte markers, whereas almost all sphenoids after co-culture expressed Albumin, CYP3a4, HNF4A, bars: 100 μm.

FIG. 4: the microencapsulated vhepLPCs sphenoids can obviously improve the survival rate of mice with acute liver injury; a: the hepLPCs/vhepLPCs spheres with better activity are wrapped in alginate microcapsules according to a certain density, the wrapping efficiency is more than 50% in bright field, and the bars: 100 μm; b: the activity of the coated alginate microcapsule is detected, and the Calcein-AM/EthD-1 staining result shows that the cell activity is more than 90 percent; c: the CCL4 treatment of NSG mice induced acute hepatic injury, transplantation of microencapsulated sphenoids for treatment the next day, daily weighing within 7 days, orbital vein blood collection, recording of mouse survival conditions, and plotting of survival curves according to mouse survival time show that the survival rate of vhepLPCs sphenoids at 7 days is 90.9%, which is obviously higher than that of hepLPCs sphenoids by 70% and that of a control group by 16.7%; d: the next day after the operation, the abdominal cavity of the mouse is cut open, and the microcapsules in the experimental group are all enriched around the liver; e: ALT and AST detection shows that the ALT and the AST are in lower levels before transplantation (-1), the mice are in obvious injury state on the transplantation day (0), the ALT and the AST are obviously reduced in a treatment group compared with a control on the first day (1) after operation, and no obvious difference exists among three groups after 3 days; f: histological staining results show that the control group had significant liver damage, while the treated mice had intact liver morphology.

Detailed Description

The following examples are provided to illustrate specific embodiments of the present invention.

Example (b):

first, experimental material

The B27 serum-free additive used in the invention is purchased from Yuanbei GmbH; N-acetyl-L-cysteine (N-acetyl cysteine), Nicotinamide (Nicotinamide), vitamin C was purchased from Sigma-Aldrich; recombinant proteins Hepatocyte Growth Factor (HGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF) and epithelial growth factor (FGF10) were purchased from Peprotech; small molecule compounds Rock inhibitors (Y-27632), TGF-beta inhibitors (A-83-01) were purchased from ceramic biochemistry; inhibitors of oncostatin m (osm), GSK3 beta (CHIR99021) were purchased from TargetMol; the used hepatic precursor cells were derived from an immortalized cell line established before the subject group (Zhenyu Wang, Weijian Li, Hongshu Jing, Ming Ding, Gongbo Fu, Tianjie Yuan, Weijian Huang, Mengjun Dai, Dan Tang, Min Zeng, Yi Chen, Hongdan Zhuang Zhang, Xuejing Zhu, Yuan Pen, Qiagen Li, Wei-Feng Yu, He-Xin Yan, Bo Zhui. Generation of hepatic spiders using human-derived vertical promoter-cells for detecting liver toxicity diagnosis reagent, 2019: 90-Jiang 6705, Jiang-Yang Shen-Huang, Jiang-Huang-Huang Yang, Jian-Huang Shu-Huang Shu, Weijie Yang Huang-Huang Zhen Huang, Weijie-Huang Zhen Huang-Huang Shu Huang Shu, Weijie-Huang Zhen Huang Dan, Weijie-Huang Dan-Huang, Wei Huang Dan-Huang, Wei Huang Dan-Huang, Wei Dan-Huang Dan, Wei Dan, Wei Dan, Wei-Feng Yu, Bo Zhai, He-Xin Yan. An extracorporeal biochemical liver embedded with 3D-layred human liver promoter-like cells extracts aid liver failure in pixels. Sci Transl Med.2020Jul 8; 12 (551.) eaba5146.) and cultured using a specific cell culture medium (i.e., TEM propagation medium) (Zhenyu Wang, Weijian Li, Hongshu Jing, Ming Ding, Gongbo Fu, Tianjie Yuan, Weijian Huang, Mengjun Dai, Dan Tang, Min Zeng, Yi Chen, Hongdan Zhang, Xuejing Zhu, Yuan Pen, Qi Li, Wei-Feng Yu, He-Xin Yan, Bo Zai. Generation of pharmaceutical man lipids using a human-derived promoter-cells for pharmaceutical delivery diagnosis, pharmaceutical delivery diagnosis, Sep 20118; 6690-6705.Wei-Jian Li, Xue-Ju Zhu, Tian-Jie Yuan, Zhen-Yu Wang, Zheng-Qian Bian, Hong-Shu Jug, Xiao Shi, Cai-Yang Chen, Gong-Bo Fu, Wei-Jian Huang, Yao-Ping Shi, Qian Liu, Min Zeng, Hong-Dan Zhang, Hong-Ping Wu, Wei-Feng Yu, Bo Zhai, He-Xin Yan et al and an exogenous polypeptide biological instrumentation tongue juice embedded with a hand 3D-layerd human promoter-cell juice extract juice front, Ju-8; 12(551) eaba 5146.); GCV is available from TargetMol; advanced DMEM medium purchased from Gibco; penicillin and streptomycin dual antibiotic was purchased from borui biomedical corporation; the primary cell antibiotic, primocin, was purchased from Invivogen; hepes solution and GlutMax solution were purchased from shanghai culture bio; PBS Buffer (Phosphate Buffer Solution) and Tryple for washing and resuspension of cells were purchased from Gibco; the medium used for the 2D hepatoprecursor cell experiments, Dulbecco's Modified Eagle Medium (DMEM), was purchased from Gibco; fetal Bovine Serum (FBS) was purchased from Corning; low adhesion well plates for cell culture were purchased from Corning; the qi ma device was purchased from Yuyan biology.

The device comprises an electrostatic liquid drop generator (YD-06), a syringe pump (razel a99), a universal shaking table, a lifting table, a beaker, an iron wire ring, a syringe, a metal long needle head and a metal short needle head; 1.5% purified sodium alginate, 0.15% purified sodium alginate, 100mmol/L calcium chloride, 0.9% sodium chloride, 0.05% (W/V) Polylysine (PLL), 55mmol/L sodium citrate.

Second, Experimental methods

The invention uses Graphpad7 biometric software to analyze the corresponding experimental data, and uses two-tailed unpaired t test and one-way ANOVA analysis of variance to compare the difference between two or more groups of different treatments. All statistical data, using at least three independent samples or replicates, are expressed as means ± s.e.m., where ns is insignificant and P <0.05 is considered statistically different, where P, P <0.01, P < 0.0001.

1. Establishment of a serum-free 3D culture system of hepatic precursor cells:

the hepatic precursor cells grown to log phase were counted by Tryple digestion at 2 x 10⁵Each well is inoculated into a low-adhesion 6-well plate, 3ml of fresh modified medium is added, the specific components are Advance DMEM/F12 as a basic culture medium, 1 XB 27 supplement (Gibco), 1 XL-glutamine reagent (such as GlutaMAX-I), 1 XP regulator (HEPES), 1 XP primary cell antibiotic (such as 0.1mg/ml primocin) and 1 XP-streptomycin are added, 10mM nicotinamide, 1.56mM N-acetylcysteine, small molecule compound 25ng/ml HGF, 50ng/ml EGF, 3 uM CHIR-99021, 10 mu M Y27632, 500nM TGF-beta inhibitor A8301, 50mg/ml vitamin A8301Plain C, 2ng/ml OSM. During the culture process, the cells gradually gather to form sphenoids, and then the cells are converted into a low proliferation state. After 5 days of culture, samples can be taken for relevant cytological experiments.

2. Establishment of a co-culture system of liver precursor cells and endothelial cells:

the hepatic precursor cells and endothelial cells grown to logarithmic phase were counted separately by Tryple digestion at 1: 1 by 2 x 10⁵The cells were densely seeded in a low-adhesion 6-well plate, and 3ml of fresh modified co-culture medium was added, specifically composed of Advance DMEM/F12 as a basal medium, 1 XB 27 supplement (Gibco), 1 XL-glutamine reagent (e.g., GlutaMAX-I), 1 XH pH regulator (HEPES), 1 XD primary cell antibiotic (e.g., 0.1mg/ml primocin), and 1 XPS-streptomycin, together with 10mM nicotinamide, 1.56mM N-acetylcysteine, small molecule compound 25ng/ml HGF, 50ng/ml EGF, 3. mu.M CHIR-99021, 10. mu. M Y27632, 500nM TGF-beta inhibitor A8301, 50mg/ml vitamin C, 2ng/ml OSM. 10ng/ml VEGF, 10ng/ml FGF10, 1% FBS (Corning) were added additionally taking into account the endothelial cell growth conditions. During the culture process, the cells gradually gather to form sphenoids, and then the cells are converted into a low proliferation state. After 5 days of culture, samples can be taken for relevant cytological experiments.

3. Liver function experimental verification:

adding the collected hepLPCs/vhepLPCs spheres into Trizol to extract total RNA, and performing related transcriptomics identification; after the hepLPCs/vhepLPCs sphenoids are added into the protein lysate, the liver function related markers can be detected, such as Albumin and CYP3A 4; the functional aspects can be oil red dyeing, PAS dyeing, ICG inhalation, immunofluorescence dyeing and the like.

4. Acute liver injury mouse model

Male wild type NSG mice weighing approximately 18-20g at 6-8 weeks used in the experiment were purchased from the Nanjing model animal research center and tested in the southern model animal house. The using amount of CCL4 is 10ml/kg, and the CCL4 and olive oil are mixed according to the proportion of 1:9 and placed in a dark place at normal temperature for standby. Mice were randomly divided into 3 groups of 11 mice each, and were intraperitoneally injected with CCL 4200. mu.l, and cell transplantation was performed the next day.

Microencapsulation and transplantation of hepLPCs/vhepLPCs sphenoids

The hepLPCs/vhepLPCs spheres were collected in a centrifuge tube, discarded and resuspended in the appropriate volume (2 x 10)⁶Ml) of sodium alginate solution (Shandong Mingyue), avoiding the generation of bubbles as much as possible. And loading the cell-Alginate mixed solution to a syringe pump, starting an electrostatic drop generator, adjusting the pump speed, preparing sodium Alginate gel beads, and dripping the sodium Alginate gel beads into a beaker containing 100 mmol/of calcium chloride solution for full calcification for 20 minutes. Transferring the calcium alginate gel beads to a 50ml centrifuge tube, adding a PLL solution with the volume 5 times that of the gel beads, and shaking the solution to form a film for 5 to 8 minutes at room temperature. The microcapsules were washed thoroughly 3 times with 0.9% sodium chloride and the gel beads were liquefied by adding about 8 volumes of sodium citrate solution for 2-5 minutes. Because the subsequent in vivo transplantation test is required, the liquefied rubber beads need to be treated by 0.15 percent sodium alginate solution for about 10 minutes after being washed, and can be directly used for in vivo transplantation after being fully washed. The injection is performed by intraperitoneal injection, and each mouse is 100 mu l. Empty microcapsules served as control.

6. Immunohistochemistry:

after the immune tissue slices are placed at 60 ℃ and baked for 60 minutes, the xylene I, II and III are dewaxed for 5 minutes each time, and are dehydrated by 100 percent, 95 percent and 75 percent ethanol for 5 minutes each time, and then the slices are placed in double distilled water for soaking for 5 minutes after the dehydration. Then taking out tissue sections for antigen retrieval: boiling in 0.01M citric acid buffer for 15 min; naturally cooling to room temperature. Post-blocking endogenous peroxidase Activity, 3% H₂O₂And 15 min. Soaking and cleaning with double distilled water for 3 times, and sealing with Goat serum (5% coat serum in PBS) for 1h to improve antibody specificity. The antibody was then diluted according to the antibody instructions, carefully added dropwise to the sections to complete coverage, and incubated overnight at 4 ℃. The following day tissue sections were removed from the 4 ℃ freezer and, to prevent rapid temperature flaking, PBST was washed 3 times after approximately 20-30 minutes at room temperature. A secondary antibody, 1:200 (2% BSA in PBS), was added dropwise to the cells from the same species as the primary antibody, and incubated at room temperature for 30min (wet-box tending). After PBST washing, DAB color development for 1-10min, use now first. The reaction was terminated when the tissue portion appeared brick red. Note that the time should not exceed 10 minutes to prevent false positives. Further washing with double distilled water for 2 times, and placing the slices intoStaining in hematoxylin solution. Washing off excessive hematoxylin with running water after 2 minutes before adding ethanol hydrochloride differentiation solution, taking out the slices after the slices are placed in the differentiation solution for about 1 to 2 seconds, returning the running water to blue for 5 minutes through the running water, re-dehydrating, sealing the slices with neutral resin, and observing under a mirror after air drying.

Thirdly, experimental results:

1. stability characterization of liver precursor cells

In the invention, the pre-hepatic cells of our team are constructed into immortalized hepLPCs through a lentivirus-mediated HPV E6/E7 system. Meanwhile, we introduce the HSV-TK/GCV suicide system into hepLPCs. To verify its safety, we treated with gancyclovir (gcv) and found that the viability of hepLPCs was significantly inhibited 48h after in vitro treatment (fig. 1A). Gene expression showed a significant increase in expression of the apoptosis marker CASP3, BCL-2 (fig. 1B). The lentivirus fluorescent labeling system is adopted to transfect HepLPCs, so that the HepLPCs are provided with GFP fluorescent proteins (GFP-HepLPCs), the in-vivo change is favorably traced, and meanwhile, GFP-HepLPCs cell lines are established by amplification after GFP positive cells are sorted in a flow mode. Significant cell death was also evident upon GCV treatment (fig. 1C). Mixing 5 x 10⁶The cells were transplanted subcutaneously into NSG immunodeficient mice, GCV was intraperitoneally injected at 50mg/kg, and after 5 days, the cells were sampled, and the experimental group of GFP-positive cells was found to show a significant decrease in illumination under the microscope (FIG. 1D).

2. Establishing co-culture system of liver precursor cells and endothelial cells

In order to obtain culture conditions with relatively complex structure and good hepatocyte function, the invention firstly compares the differences among the traditional 2D culture, 3D suspension culture, 3D co-culture of the hepatic precursor cells and the endothelial cells, and the cell morphology after 14 days of 2D differentiation, finds that the cells all present good states, and under the condition of 3D low serum, the co-culture of the hepatic precursor cells and the endothelial cells still maintains good cell viability (fig. 2A). Taking out a proper amount of cells, extracting total RNA, carrying out reverse transcription to obtain cDNA, carrying out function gene detection on mature hepatocytes by qPCR, and finding that the 3D co-culture has higher expression than the other three groups of liver function markers ALB and HNF4A (figure 2B). Oil red staining, glycogen staining, indigo violet uptake experiments all showed that vhepLPCs sphenoids had good cellular function (fig. 2C, D, E).

3. Promoting hepatocyte maturation following co-culture

In the culture process, we find that the co-culture of vhepLPCs can promote the maturation of liver cells. First we confirmed that endothelial cells were viable in the matrigel-free, low serum co-culture system, verified by gene expression and protein levels (fig. 3A, B), and immunofluorescence further corroborated the experimental results (fig. 3C). Then, the expression level of the liver-related marker ALB and CYP3A4 of vhepLPCs is obviously increased through transcript level detection (figure 3D), and the expression level of Albumin and CYP3A4 is obviously increased after co-culture (figure 3E) through further verification of three different batches of proteins. Immunofluorescence results were consistent with protein levels, and hepLPCs sphenoids alone partially expressed hepatocyte markers, and the expression uniformity of Albumin, CYP3a4, and HNF4A was significantly better after co-culture (fig. 3F).

4. After co-culture, the survival rate of mice with acute liver injury can be obviously improved

In order to deeply evaluate the function of the vhepLPCs spherioids, the present invention included the hepLPCs spherioids and vhepLPCs spherioids in alginate microcapsules, which are a biologically inert material for protecting hepatocytes from the host, considering that portal vein transplantation and spleen transplantation cause blood coagulation risk. Protecting cells from immune rejection by the host is freely shuttled between the substrate and the protein. Microencapsulation efficiency was greater than 50% (fig. 4A). Cell viability assay was performed after coating and Calcein-AM/EthD-1 staining showed good cell viability (FIG. 4B). In order to verify the in vivo curative effect, the CCL 4-induced acute liver injury mouse is constructed, and the next day is injected with 1 x 10 of the abdominal cavity⁶Cells, daily weighing after operation, orbital vein blood sampling and mouse survival status recording. The survival curves are plotted as shown in fig. 4C, and it is found that the 7-day survival rate of the mice in the vhepplcs group reaches 90.9%, which is significantly higher than 70% of the hepLPCs alone and 16.7% of the control group, wherein the three-day survival rate of the mice in the vhepplcs group is even higher than 100%, which is significantly different from the control group. When the abdominal cavity of the mice was cut open, the microcapsules in the experimental group were significantly concentrated around the liver, while the control group was relatively diffuse (fig. 4D). Blood index detection shows that the experimental group is on the first post-operationALT, AST were significantly lower than the control group a day (fig. 4E). Histological staining results showed significant tissue necrosis in the control group and relatively intact tissue morphology in the experimental group (fig. 4F). In conclusion, the alginate microcapsule-coated vhepLPCs spheres can significantly improve the survival rate of mice with acute liver injury, and provide a new idea and an alternative treatment scheme for treating liver injury by stem cell transplantation.

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full range of equivalents.