Scaffold-free 3D hepatocyte micro-tissue as well as preparation method and application thereof

Abstract

The invention provides a scaffold-free 3D hepatocyte micro-tissue as well as a preparation method and application of the scaffold-free 3D hepatocyte micro-tissue. According to the method, through a one-step transplantation strategy, hepatocytes and interstitial cells are mixed and then subjected to 3D co-culture, and the scaffold-free 3D hepatocyte micro-tissue with a tissue structure similar to that of a natural liver is prepared. According to the method, the step of repeated injection in the traditional technology is simplified, the clinical operability is improved, and the prepared 3D hepatocyte micro-tissue can be widely applied to drug screening, toxicity testing, establishment of a liver disease model, liver tissue engineering and cell transplantation and treatment of liver diseases.

Description

Background Art

[0002] As an organ with a vast space and abundant blood supply, the spleen does provide an ideal environment for liver cell regeneration. Its rich vascular network helps to provide sufficient nutrients and oxygen to the regenerated liver cells, which is essential for cell growth and survival. However, the natural structural characteristics of the spleen - the scarce matrix content and loose structure - have become the main obstacles to the growth and functionalization of liver cells on it. Due to the lack of sufficient matrix support, it is difficult for liver cells to stably adhere to the spleen, and thus it is difficult to achieve effective regeneration and functional construction.

[0003] Therefore, the prior art for constructing functional liver tissue in the spleen requires that the spleen matrix structure be modified to establish a microenvironment suitable for liver cell regeneration, and then liver cells are transplanted into the modified spleen to achieve functional construction of liver tissue. For example, Chinese patent document CN118161530A, a preparation for modifying organ matrix and its application, discloses a preparation for modifying organ matrix, especially the modification of spleen matrix, which can increase the matrix collagen content of the spleen and provide a good regenerative microenvironment for the growth of epithelial functional cells to support the growth and functionalization of liver cells; the technical strategy of this patent requires two injections of the spleen, which limits its clinical transformation and application. Summary of the invention

[0004] To solve the above problems, the present invention provides a scaffold-free 3D hepatocyte microtissue, a preparation method and its application. The method prepares 3D hepatocyte composite microspheres based on 3D culture technology through a one-step transplantation strategy, and only needs to be injected into the spleen once to establish a tissue structure similar to that of a natural liver in the spleen. The 3D hepatocyte microspheres of the present invention have a tissue structure similar to that of a natural liver and can achieve functional regeneration of liver tissue to the greatest extent. The 3D hepatocyte microspheres of the present invention can be used for drug screening, toxicity testing, establishment of liver disease models, liver tissue engineering and cell transplantation, and treatment of liver diseases, and have broad application prospects.

[0005] The first aspect of the present invention provides a scaffold-free 3D hepatocyte microtissue.

[0006] Further, the scaffold-free 3D hepatocyte microtissue is composed of hepatocytes and mesenchymal cells;

[0007] Furthermore, the hepatocytes and the mesenchymal cells are mixed in vitro at a ratio of (5-15):1, and then the scaffold-free 3D hepatocyte microtissue is formed through a 3D co-culture process;

[0008] Preferably, the hepatocytes and the mesenchymal cells are mixed in vitro at a ratio of 10:1, and then the scaffold-free 3D hepatocyte microtissue is formed through a 3D co-culture process;

[0009] Further, a hepatocyte culture medium was used for 3D co-culture, wherein the hepatocyte culture medium was: 10% fetal bovine serum, 0.1 μM dexamethasone, 10 μg / L EGF, 20 μg / L HGF, 20 μg / L TGF-α, 4.2 mg / L insulin, 5 μg / L sodium selenite and 3.8 mg / L transferrin were added to DMEM / F12 culture medium;

[0010] Furthermore, the hepatocytes are one or more of primary hepatocytes, hepatocytes induced by pluripotent stem cells and hepatocytes induced by direct reprogramming, and the cell sources include but are not limited to mice, rats, rabbits, monkeys, pigs, dogs and humans;

[0011] Furthermore, the interstitial cells are one or more of fibroblasts, adipocytes, smooth muscle cells, endothelial cells, perivascular cells and immune interstitial cells derived from the heart, liver, lung or kidney, and the cell sources include but are not limited to mice, rats, rabbits, monkeys, pigs, dogs and humans;

[0012] Furthermore, the scaffold-free 3D hepatocyte microtissue is spherical, and the diameter of the spherical scaffold-free 3D hepatocyte microtissue ranges from 50 microns to 500 microns.

[0013] The second aspect of the present invention provides a method for preparing the above-mentioned scaffold-free 3D hepatocyte microtissue.

[0014] Furthermore, the method comprises the following steps:

[0015] S1. Using a two-step in situ collagenase perfusion method to separate and extract hepatocytes, resuspending the separated hepatocytes in a hepatocyte culture medium to prepare a hepatocyte suspension;

[0016] S2. Using directed differentiation induction factors to induce the mesenchymal cells to differentiate into specific mesenchymal cells suitable for liver tissue regeneration, and resuspending the induced mesenchymal cells in a hepatocyte culture medium to prepare a mesenchymal cell suspension;

[0017] S3, mixing the hepatocyte suspension obtained in S1 and the interstitial cell suspension obtained in S2, and performing 3D culture with a hepatocyte culture medium, wherein the hepatocytes and interstitial cells initiate aggregation and self-assembly into spherical cell microtissues through cell-cell adhesion molecules (such as E-cadherin) and cell-matrix interactions, thereby obtaining the scaffold-free 3D hepatocyte microtissue;

[0018] Further, in step S1, the hepatocytes are one or more of primary hepatocytes, hepatocytes induced by pluripotent stem cells, hepatocytes induced by chemical reprogramming, and hepatocytes edited by genes, and the concentration of the prepared hepatocyte suspension is 10 2 -10 5 / mL, preferably 10 3 / mL;

[0019] Further, in step S2, the interstitial cells are one or more of fibroblasts, adipocytes, smooth muscle cells, endothelial cells, perivascular cells and immune interstitial cells;

[0020] Further, in step S2, the directed differentiation inducing factors are at least two of growth factors, cytokines, chemokines and chemical small molecules that regulate the adhesion and proliferation of mesenchymal cells;

[0021] In some embodiments, the directed differentiation inducing factors are growth factors and cytokines;

[0022] In some embodiments, the directed differentiation inducing factors are growth factors and chemokines;

[0023] In some embodiments, the directed differentiation inducing factors are growth factors and chemical small molecules;

[0024] In some embodiments, the directed differentiation inducing factors are growth factors, cytokines and chemokines;

[0025] In some embodiments, the directed differentiation inducing factors are growth factors, chemokines and small chemical molecules;

[0026] In some embodiments, the directed differentiation inducing factors are cytokines and chemokines;

[0027] In some embodiments, the directed differentiation inducing factors are cytokines and chemical small molecules;

[0028] In some embodiments, the directed differentiation inducing factors are cytokines, chemokines and chemical small molecules;

[0029] In some embodiments, the directed differentiation inducing factors are chemokines and small chemical molecules;

[0030] In some embodiments, the directed differentiation inducing factors are growth factors, cytokines, chemokines and chemical small molecules;

[0031] Further, the growth factors include but are not limited to EGF, HGF, FGF, IGF, VEGF, TGF-β or their equivalent analogs, pharmaceutical preparations, precursors and / or combinations thereof;

[0032] Further, the cytokines include but are not limited to IFN-γ, CSFs, ILs, TNF-α, or their equivalent analogs, pharmaceutical preparations, precursors or combinations thereof;

[0033] Further, the chemokines include but are not limited to CXCL8, CXCL6, CXCL3, CXCL9, CXCL13 or their equivalent analogs, pharmaceutical preparations, precursors and / or combinations thereof;

[0034] Further, the chemical small molecules include but are not limited to CHIR99021, AM580, EPZ004777, 616452, or their equivalent analogs, pharmaceutical preparations, precursors and / or their combinations;

[0035] Further, in step S2, the concentration of the growth factors is 5-40 μg / L EGF, 10-20 μg / L HGF, 5-40 μg / L FGF, 2-20 μg / L IGF, 10-40 μg / L VEGF, 10-20 μg / L TGF-β;

[0036] Further, in step S2, the concentration of the cytokines is 1-40 μg / L IFN-γ, 5-25 μg / L CSFs, 1-20 μg / L ILs, 5-25 μg / L TNF-α;

[0037] Further, in step S2, the concentration of the chemokines is 5-15 μg / L CXCL8, 10-25 μg / L CXCL6, 5-15 μg / L CXCL3, 10-20 μg / L CXCL9, 10-20 μg / L CXCL13;

[0038] Further, in step S2, the concentration of the chemical small molecules is 1-20 μmol / L CHIR99021, 2-10 μmol / L AM580, 1-15 μmol / L EPZ004777, 5-20 μmol / L 616452;

[0039] Further, in step S2, the concentration of the prepared interstitial cell suspension is 10 1 -10 4 / mL, preferably 10 2 / mL;

[0040] Further, in step S3, the mixing volume ratio of the hepatocyte suspension to the mesenchymal cell suspension is 10:1-100:1, preferably 20:1, more preferably 10:1;

[0041] Further, in step S3, the hepatocyte culture medium used is: 10% fetal bovine serum, 0.1 μM dexamethasone, 10 μg / L EGF, 20 μg / L HGF, 20 μg / L TGF-α, 4.2 mg / L insulin, 5 μg / L sodium selenite and 3.8 mg / L transferrin are added to DMEM / F12 culture medium.

[0042] The third aspect of the present invention provides a hepatocyte culture medium for the above-mentioned scaffold-free 3D hepatocyte microtissue.

[0043] Furthermore, the formula of the hepatocyte culture medium is: 10% fetal bovine serum, 0.1 μM dexamethasone, 10 μg / L EGF, 20 μg / L HGF, 20 μg / L TGF-α, 4.2 mg / L insulin, 5 μg / L sodium selenite and 3.8 mg / L transferrin are added to DMEM / F12 culture medium.

[0044] Since the survival rate of hepatocytes is low, a higher survival rate of hepatocytes can be obtained by resuspending hepatocytes and interstitial cells using the above-mentioned hepatocyte culture medium.

[0045] A fourth aspect of the present invention provides the use of the above-mentioned scaffold-free 3D hepatocyte microtissue in disease treatment and tissue engineering.

[0046] Furthermore, the scaffold-free 3D hepatocyte microtissue can be used for drug screening, toxicity testing, establishment of liver disease models, liver tissue engineering and cell transplantation, and treatment of liver system diseases.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] (1) The scaffold-free 3D hepatocyte microtissue of the present invention is closer to the physiological microenvironment of the human body, that is, the scaffold-free technology can rely on the adhesion and interaction of cells themselves to form a 3D structure without relying on artificial scaffold materials. This method is closer to the natural microenvironment of cells in the body.

[0049] (2) Traditional 3D culture often uses natural or synthetic scaffold materials (such as collagen, gelatin, polycaprolactone, etc.), which have residual chemical components or degradation products that are toxic to cells or the surrounding environment and can induce immune responses during in vivo experiments or transplantation. The scaffold-free technology of the present invention avoids the potential toxicity and immune response of scaffold materials, thus eliminating the above risks, improving the safety and biocompatibility of the culture system, and can be widely used in clinical research.

[0050] (3) The scaffold-free 3D hepatocyte microtissue of the present invention has better flexibility and wider practicality: by regulating cell density, culture medium components, etc., cell clusters of different sizes can be flexibly constructed, which is more suitable for forming large-scale cell clusters or tissue structures in dynamic culture systems (such as suspension culture, bioreactors).

[0051] (4) The one-step transplantation strategy of the present invention can improve clinical operability and reduce clinical application costs: that is, 3D hepatocyte composite microspheres are prepared based on 3D culture technology, and only one injection into the spleen is required to establish a tissue structure in the spleen similar to that of a natural liver.

[0052] (5) The scaffold-free 3D hepatocyte microspheres prepared by the present invention can better reflect the characteristics of the in vivo cellular microenvironment and its tissue structure. At the same time, compared with monodispersed cells, the cell microspheres are easier to colonize in the spleen and integrate with the surrounding host tissues, thereby maximizing the functional regeneration of liver tissue, thereby using the regenerated liver tissue to treat liver diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 A schematic diagram of a technical process is provided for an embodiment of the present invention;

[0054] Figure 2 The gene expression of fibroblasts induced by the combination of factors;

[0055] Figure 3 Schematic diagram of the formation process of 3D hepatocyte microspheres observed by bright field;

[0056] Figure 4 The cell distribution of 3D hepatocyte microspheres was determined by immunofluorescence staining.

[0057] Figure 5 This is the situation of functional liver tissue regeneration in the spleen formed after 3D hepatocyte microspheres were transplanted into the spleen. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical solution and advantages of the present invention more clear, the present invention is further described in detail below. However, it should be understood that the description herein is only used to explain the present invention and is not used to limit the scope of the present invention.

[0059] Unless otherwise defined, all technical terms and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention, and the terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The reagents and instruments used herein are all commercially available, and the characterization means involved can refer to the relevant descriptions in the prior art, which will not be repeated herein.

[0060] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

[0061] Example 1

[0062] This example provides a preparation process of scaffold-free 3D hepatocyte microspheres, as shown in the attached Figure 1 The construction process includes:

[0063] The mechanism of 3D hepatocyte microsphere formation is as follows: The present invention uses ultra-low adsorption cell culture plates to prepare 3D hepatocyte microspheres. In the absence of other external constraints, cells adsorb to each other through adhesion molecules (such as E-cadherin) and freely aggregate in three-dimensional space to form a tightly aggregated structure. The sphere is an efficient structure that encapsulates the maximum volume under the minimum surface area, thereby minimizing free energy. Therefore, spheroidization becomes the result of energy optimization.

[0064] 1. Hepatocytes (or hepatocytes modified by CRISPR / Cas9) and fibroblasts (or other mesenchymal cells) were mixed at different ratios (cell number ratio) of 5:1, 7:1, 10:1, 15:1 and 20:1, and spheroid 3D culture was performed using ultra-low attachment culture plates. The technical parameters of the ultra-low attachment culture plates are as follows (taking 96-well ultra-low attachment culture plates as an example):

[0065] Table 1 Ultra-low attachment cell culture plate parameters and specific parameter values / characteristics

[0066]

[0067] 2. Implant the prepared 200-micron 3D hepatocyte microspheres into the spleen to detect the colonization effect and functional regeneration of the 3D hepatocyte microspheres in the spleen.

[0068] The following table shows the 3D hepatocyte microspheres obtained by mixing at different ratios.

[0069] Table 2 Spheroidization rate and cell survival rate of 3D hepatocyte microspheres with different ratios

[0070]

[0071] The ratios in the above table show that the optimal ratio of hepatocytes to fibroblasts is 10:1.

[0072] Example 2

[0073] This embodiment provides a method for extracting primary hepatocytes, the method comprising:

[0074] (1) Prepare the required Percoll purification solution in advance: Each mouse needs to prepare the purification solution according to the ratio of 3.3mL Percoll separation solution, 4mL DMEM culture medium, and 10μL 1mol / L hydrochloric acid. Preheat 100CDU / mL type IV collagenase and HBSS in a 37℃ water bath. Prepare the hepatocyte culture medium in advance: add 10% FBS, 1% glutamine, 1% sodium pyruvate, 1% penicillin-streptomycin solution, 1% insulin-transferrin-selenium additive, 1μmol / L dexamethasone, 10ng / mL EGF, 20ng / mL HGF, and 20ng / mL TGF-α to the DMEM / F12 culture medium.

[0075] (2) Mice were anesthetized by intraperitoneal injection of 1.25% ready-to-use tribromoethanol solution.

[0076] (3) Fix the mouse on the workbench with its abdomen facing upward and secure its limbs with tape.

[0077] (4) Thoroughly clean the chest and abdominal areas with 75% alcohol, then use forceps and ophthalmic scissors to make an incision in the lower abdomen and cut open the epidermis and peritoneum.

[0078] (5) The laparotomy is continued upward until the liver, PV, and inferior venous cava (IVC) are fully exposed.

[0079] (6) Insert the perfusion needle into the PV and quickly flush it with HBSS. The liver will turn white quickly.

[0080] (7) Cut the IVC, and after the liver is drained of blood, push type IV collagenase at a constant speed of 5 mL / min for digestion.

[0081] (8) During the digestion process, the liver gradually swells and softens. After digestion, the gallbladder is removed and the liver is placed in a culture dish filled with pre-cooled culture medium. The liver is quickly torn into pieces with flat-blade forceps, and the hepatocytes pour out of the liver capsule.

[0082] (9) The obtained cell suspension was screened through a 70 μm mesh, transferred to a centrifuge tube, and centrifuged at 4°C, 1000 rpm, for 1 min.

[0083] (10) After discarding the supernatant, the adherent hepatocytes were selected using Percoll purification solution and centrifuged at 4°C, 1000 rpm for 10 min.

[0084] (11) After discarding the supernatant, rinse the cells with pre-chilled HBSS and centrifuge at 1000 rpm for 3 min at 4°C.

[0085] (12) The cells that settle at the bottom of the tube are primary hepatocytes. After cell counting, the extracted primary hepatocytes are plated in a culture dish coated with type I rat tail collagen and placed at 37°C and 5% CO. 2 cultured in a cell culture incubator.

[0086] Example 3

[0087] This embodiment provides a method for extracting and directional inducing primary skin fibroblasts, the method comprising:

[0088] (1) Prepare the culture medium for primary mouse skin fibroblasts (MSF) in advance: add 10% fetal bovine serum (FBS), 1% penicillin-streptomycin solution, 10 ng / mL bFGF, and 10 ng / mL EGF to DMEM / F12 culture medium.

[0089] (2) Mice were anesthetized by intraperitoneal injection of 1.25% ready-to-use tribromoethanol solution.

[0090] (3) Use a shaver to shave the hair on the back of the mouse, and then use a depilatory cream to further remove hair until the skin is smooth and hairless.

[0091] (4) Wipe loose hair with 75% alcohol and disinfect the mice, then transfer them to a clean bench for subsequent operations.

[0092] (5) Cut off the hair-free skin on the back of the mouse, remove excess subcutaneous fat, and rinse with Hank's balanced salt solution (HBSS) until the HBSS is clear and no longer turbid.

[0093] (6) Prepare a digestion solution with dispase and 0.25% trypsin in a ratio of 2:1. Cut the cleaned skin into large pieces and place them in the digestion solution. Transfer to a 37°C constant temperature incubator, 90 rpm, and digest for 1 hour.

[0094] (7) After digestion is complete, use forceps and ophthalmic scissors to separate the epidermis from the dermis and remove excess adipose tissue.

[0095] (8) Cut the dermis into small pieces and transfer to 0.25% collagenase type I for further digestion. Digestion was continued in a constant temperature incubator at 37°C and 90 rpm for 2 hours.

[0096] (9) Add serum-containing culture medium to terminate digestion and filter the digestion solution through a 70 μm mesh to obtain a single-cell suspension.

[0097] (10) Place the obtained cell suspension in a centrifuge tube and centrifuge at 1000 rpm for 5 minutes.

[0098] (11) Wash once with HBSS and centrifuge again at 1000 rpm for 3 min.

[0099] (12) After cell counting, the extracted MSF was plated at an appropriate cell density and cultured in a cell culture incubator at 37° C. and 5% CO 2 .

[0100] (13) When the cell confluence reaches 90%, the cells are passaged.

[0101] (14) The second to third generation fibroblasts were digested and plated on a cell culture dish, and 10 ng / mL TGF-α, 10 ng / mL bFGF, and 10 μM TGF-β1 signaling pathway inhibitor SB431542 were added for continuous stimulation for 7 days.

[0102] The results are as follows Figure 2 As shown, in fibroblasts differentiated by TGF-α, bFGF and SB431542 (the combination is abbreviated as 3C), the mRNA expression levels of factors Egf, Hgf, Vegfd and Bmp6 that are beneficial to hepatocyte growth were significantly increased, while the expression level of Ctgf gene that induces hepatocyte apoptosis was significantly decreased.

[0103] Example 4

[0104] This embodiment provides a 3D co-culture method of primary hepatocytes and induced fibroblasts, the method comprising:

[0105] (1) Primary hepatocytes and induced fibroblasts were prepared into hepatocyte suspensions and interstitial cell suspensions in different ratios using hepatocyte culture medium, and the extracted hepatocytes and induced fibroblasts were mixed at a ratio of 10:1 (cell number ratio).

[0106] (2) 10,000 cells were added to each well of a 3D 96-well plate and cultured in hepatocyte culture medium at 37°C and 5% CO. 2 Culture the cells in a cell culture incubator for 5-7 days.

[0107] (3) The formula of hepatocyte culture medium is as follows: DMEM / F12 containing 10% fetal bovine serum, 0.1 μM dexamethasone, 10 μg / L EGF, 20 μg / L HGF, 20 μg / L TGF-α, 4.2 mg / L insulin, 5 μg / L sodium selenite, and 3.8 mg / L transferrin.

[0108] Example 5

[0109] In this embodiment, the formation process of 3D hepatocyte microspheres was observed by bright field microscopy. Bright field microscopy is a compound optical microscope that uses light to illuminate samples to generate images.

[0110] (1) Place the ultra-low attachment 3D cell culture plate on the microscope sample stage and secure it using a dedicated clamp or adapter.

[0111] (2) Turn on the light source and shine the light evenly on the bottom of the orifice plate.

[0112] (3) Adjust the coarse adjustment knob to roughly focus the cell area, and then use the fine adjustment knob to precisely adjust the focus.

[0113] (4) Adjust the microscope light source brightness and aperture size to optimize contrast.

[0114] (5) Take an image and save it.

[0115] Observation results such as Figure 3 As shown in the figure, the formation process of 3D hepatocyte microspheres was collected by bright field microscopy. It can be seen from the figure that with the increase of culture time, the external morphology of 3D hepatocyte microspheres presents a relatively regular circle with a diameter of 200 microns.

[0116] Example 6

[0117] In this example, the cell distribution of 3D hepatocyte microspheres was detected by immunofluorescence staining technology.

[0118] (1) The 3D hepatocyte microspheres were fixed with 4% paraformaldehyde at room temperature for 10 minutes and then rinsed with PBS three times.

[0119] (2) Add 5% bovine serum albumin (BSA) containing 0.5% Triton X-100 and block the membrane at room temperature for 45 minutes.

[0120] (3) Discard the blocking solution and rinse with PBS three times. Add the prepared primary antibody to the cell microspheres and incubate at 4°C overnight.

[0121] (4) Wash off the primary antibody with PBS, add the prepared secondary antibody to the cell microspheres, strictly avoid light, and incubate at room temperature for 45 minutes.

[0122] (5) Wash off the secondary antibody with PBS, add the prepared DAPI staining solution to the cell microspheres, strictly avoid light, and incubate at room temperature for 5 minutes.

[0123] (6) Wash the tissue with PBS and double distilled water to remove the stain. Add anti-fluorescence quenching mounting medium to the tissue for mounting.

[0124] (7) Collect images under a fluorescence microscope.

[0125] The results are as follows Figure 4 As shown, the 3D hepatocyte microspheres expressed the hepatocyte-specific protein CYP1A2, and fibroblasts were evenly distributed in the cell microspheres.

[0126] Example 7

[0127] This embodiment provides a process for transplanting 3D hepatocyte microspheres into the spleen, which process includes the following steps:

[0128] (1) Seven days after the spleen translocation surgery, the mice were anesthetized, and the excess hair at the spleen position of the mice was trimmed with curved scissors, and the skin at the spleen position was moistened with 70% ethanol.

[0129] (2) Place the mouse on its side with its left side facing up on a heating blanket. It is recommended to place a glass rod with a diameter of about 5 mm under the mouse's body to make the spleen protrude more prominently and facilitate intrasplenic injection.

[0130] (3) Intrasplenic injection of 3D hepatocyte microspheres (200 microspheres, 2*10 6 During the injection, the syringe is at a 45-degree angle to the spleen, and 50 μL of 3D hepatocyte microsphere suspension is injected slowly and evenly at a distance of about 4 mm from the tail of the spleen. During the injection, the spleen should be observed to rise slowly.

[0131] (4) About 1 minute after the injection is completed, slowly withdraw the needle. To prevent leakage of the injection solution, press the injection site with a sterile cotton ball for more than 5 minutes.

[0132] (5) Clean the mouse skin with 70% ethanol until the anesthesia disappears and place the mouse back into a clean cage.

[0133] By transplanting 3D hepatocyte microspheres into the spleen, the results are as follows Figure 5 As shown, 3D hepatocyte microspheres can colonize well in the spleen and express hepatocyte-specific proteins ALB and CYP1A2, indicating that liver tissue can be constructed in the spleen with just one transplant.

[0134] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working process and related instructions of the system described above can refer to the corresponding process in the aforementioned method embodiment, and will not be repeated here.

[0135] Those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working process and related instructions of the storage device and processing device described above can refer to the corresponding process in the aforementioned method embodiment and will not be repeated here.

[0136] The term "comprise" or any other similar term is intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus / device that includes a list of elements includes not only those elements but also other elements not expressly listed, or also includes elements inherent to such process, method, article, or apparatus / device.

[0137] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A scaffold-free 3D hepatocyte microtissue, characterized in that: The scaffold-free 3D hepatocyte microtissue is composed of hepatocytes and interstitial cells. The hepatocytes and interstitial cells are mixed in vitro at a ratio of (5-15):1, and then the scaffold-free 3D hepatocyte microtissue is formed through a 3D co-culture process.

2. The scaffold-free 3D hepatocyte microtissue according to claim 1, characterized in that: The hepatocytes are one or more of primary hepatocytes, hepatocytes induced by pluripotent stem cells and hepatocytes induced by direct reprogramming, and the cell sources include but are not limited to mice, rats, rabbits, monkeys, pigs, dogs and humans; the interstitial cells are one or more of fibroblasts, adipocytes, smooth muscle cells, endothelial cells, perivascular cells and immune interstitial cells derived from the heart, liver, lung or kidney, and the cell sources include but are not limited to mice, rats, rabbits, monkeys, pigs, dogs and humans.

3. The scaffold-free 3D hepatocyte microtissue according to claim 1, characterized in that: The scaffold-free 3D hepatocyte microtissue is spherical, and the diameter of the spherical scaffold-free 3D hepatocyte microtissue ranges from 50 microns to 500 microns.

4. A method for preparing a scaffold-free 3D hepatocyte microtissue according to any one of claims 1 to 3, characterized in that: The method comprises the following steps: S1. extracting liver cells and preparing liver cell suspension; S2. After inducing the mesenchymal cells to differentiate into specific mesenchymal cells suitable for liver tissue regeneration using directed differentiation induction factors, a mesenchymal cell suspension is prepared; S3. After mixing the hepatocyte suspension obtained in S1 and the interstitial cell suspension obtained in S2, 3D culture is performed using a hepatocyte culture medium to allow the hepatocytes and interstitial cells to self-assemble into spherical cell microtissues, thereby obtaining the scaffold-free 3D hepatocyte microtissue.

5. The preparation method according to claim 4, characterized in that: In step S1, the hepatocytes are one or more of primary hepatocytes, hepatocytes induced by pluripotent stem cells, hepatocytes induced by chemical reprogramming, and hepatocytes edited by genes, and the concentration of the prepared hepatocyte suspension is 10 2 -10 5 / mL, preferably 10 3 / mL.

6. The preparation method according to claim 4, characterized in that: In step S2, the interstitial cells are one or more of fibroblasts, adipocytes, smooth muscle cells, endothelial cells, perivascular cells and immune interstitial cells, and the directional differentiation inducing factors are at least two of growth factors, cytokines, chemokines and chemical small molecules that regulate the adhesion and proliferation of interstitial cells. The concentration of the prepared interstitial cell suspension is 10 1 -10 4 / mL, preferably 10 2 / mL.

7. The preparation method according to claim 6, characterized in that: The growth factors include but are not limited to EGF, HGF, FGF, IGF, VEGF, TGF-β or their equivalent analogs, pharmaceutical preparations, precursors and / or their compositions; the cytokines include but are not limited to IFN-γ, CSFs, ILs, TNF-α, or their equivalent analogs, pharmaceutical preparations, precursors or their compositions; the chemokines include but are not limited to CXCL8, CXCL6, CXCL3, CXCL9, CXCL13 or their equivalent analogs, pharmaceutical preparations, precursors and / or their compositions; the chemical small molecules include but are not limited to CHIR99021, AM580, EPZ004777, 616452, or their equivalent analogs, pharmaceutical preparations, precursors and / or their compositions.

8. The preparation method according to claim 4, characterized in that: In step S3, the mixing volume ratio of the hepatocyte suspension to the mesenchymal cell suspension is 1:1-100:1, preferably 20:1, and more preferably 10:

1.

9. The preparation method according to claim 4, characterized in that: In step S3, the hepatocyte culture medium used is: 10% fetal bovine serum, 0.1 μM dexamethasone, 10 μg / L EGF, 20 μg / L HGF, 20 μg / L TGF-α, 4.2 mg / L insulin, 5 μg / L sodium selenite and 3.8 mg / L transferrin are added to DMEM / F12 culture medium.

10. Use of the scaffold-free 3D hepatocyte microtissue according to any one of claims 1 to 3, or the scaffold-free 3D hepatocyte microtissue obtained by the preparation method according to any one of claims 4 to 9 in disease treatment and tissue engineering, characterized in that: The scaffold-free 3D hepatocyte microtissue can be used for drug screening, toxicity testing, establishment of liver disease models, liver tissue engineering and cell transplantation, and treatment of liver system diseases.

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