In-vivo organized cell preparation as well as preparation method and application thereof

Abstract

The invention relates to a preparation method and application of an in-vivo organized cell preparation, in particular to a cell therapy technology for forming functional liver tissues in the spleen. According to the technology, hepatocyte-like cells are obtained by extracting skin fibroblasts and utilizing a chemical reprogramming technology, and matrix conditions suitable for hepatocyte colonization are constructed in the spleen, so that functional regeneration of healthy liver tissues in the spleen is realized, and the liver-like cells are used for treating various diseases; comprising but not limited to single-gene hereditary liver diseases such as familial hypercholesterolemia and phenylketonuria.

Description

Background Art

[0002] Live cell drugs are the third drug revolution after small molecule chemical drugs and biological macromolecule drugs. The current live cell drugs are mainly functionalized blood system cells, such as hematopoietic stem cells, lymphocytes, etc., and their indications are limited to blood system diseases and limited types of tumor immunotherapy. Functional cells other than blood system cells are mainly various epithelial cells. Using functionalized epithelial cells as drugs can treat more diseases, such as liver disease, neurodegenerative diseases, endocrine system diseases, musculoskeletal system diseases, etc. However, epithelial cells cannot play a role through direct infusion like blood system cells. For example, direct infusion of liver cells into the patient's body cannot be used for effective liver disease treatment. Most epithelial cells need to form structurally specific tissues and organs in the body to function. Therefore, the in vivo therapeutic application of epithelial cells requires an in vivo "organization" process, and it is urgent to develop new technologies to promote therapeutic epithelial cells to form "therapeutic organs" in the body to exert their biological therapeutic effects. To solve this key technical problem, it is necessary to construct a place with a wide space and rich blood vessels in the body to support the development of functional epithelial cells in the body to form tissue structures similar to natural tissues and organs.

[0003] Chinese patent document "CN112481192B Chemical small molecule composition and method for direct reprogramming of fibroblasts into hepatocytes by chemical induction in vitro and in vivo" and Chinese patent document "CN104830906B A method for obtaining functional human liver parenchymal cells through reprogramming" provide methods for obtaining human liver parenchymal cells with mature functions from outside the human body by using in vitro chemical induction or in vitro reprogramming. The above-mentioned hepatocytes can still maintain key functions after a large number of amplifications, and can be used in applications such as in vitro drug development, bioartificial liver support systems, and the construction of humanized liver mice. However, the above-mentioned technology can only achieve the regeneration of hepatocytes in vitro, and cannot achieve the "in vivo organization" process. Summary of the invention

[0004] The present invention provides an in vivo organized cell preparation, a preparation method and an application thereof, which realizes the regeneration of functional liver tissue in the spleen through gene editing, chemical reprogramming and organ matrix remodeling technology, providing a new approach for the treatment of various diseases.

[0005] In a first aspect, the present invention provides an in vivo organoid cell preparation.

[0006] Further, the in vivo organogenic cell preparation includes functional epithelial cells and organ matrix remodeling agents;

[0007] Furthermore, the functional epithelial cells are one or more of human or non-human hepatocytes, pancreatic islet cells, thyroid cells and neuronal cells, and the cell sources include but are not limited to mice, rats, rabbits, monkeys, pigs, dogs and humans;

[0008] Furthermore, the functional epithelial cells are hepatocytes, and the hepatocytes are hepatocytes induced by pluripotent stem cells, hepatocytes induced by reprogramming, primary extracted epithelial cells or genetically modified hepatocytes;

[0009] Furthermore, the genetically modified hepatocytes are hepatocytes modified by TALEN, ZFN, CRISPR / Cas9 or their derivative technologies, and the derivative technologies include but are not limited to base editing technology, lead editing technology and double base editing technology;

[0010] In some embodiments, the hepatocytes are hepatocytes induced by chemical reprogramming;

[0011] Furthermore, the organ matrix reforming agent includes fibroblasts, and the fibroblasts are fibroblasts derived from skin, heart, liver, lung or kidney;

[0012] Furthermore, the fibroblasts are fibroblasts that have undergone directed differentiation;

[0013] Furthermore, the directed differentiation method is: digesting the fibroblasts and plating them on a cell culture dish, adding 10 ng / mL TGF-α, 10 ng / mL bFGF and 10 μmol / L TGF-β1 signaling pathway inhibitor SB431542, and stimulating continuously for 7 days.

[0014] Furthermore, the cell number ratio of the functional epithelial cells to the fibroblasts is 1:(0.5-2), preferably 2:1.

[0015] The second aspect of the present invention provides the induction process of chemically reprogramming hepatocytes.

[0016] Furthermore, the induction process includes:

[0017] 1. Prepare hepatocyte culture medium:

[0018] DMEM / F12 medium was supplemented with 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;

[0019] 2. Prepare chemical reprogramming medium A: add chemical small molecules 20 μmol / L CHIR99021, 10 μmol / L RepSox (E-616452), 50 μmol / L Forskolin, 0.05 μmol / L AM580 and 5 μmol / L EPZ004777 to the hepatocyte culture medium prepared in step 1;

[0020] 3. Fibroblast culture:

[0021] 1) First, observe the cells under a microscope to ensure that the cells are well attached, free of contamination, and in a healthy state;

[0022] 2) Aspirate the culture medium, gently rinse the cells twice with PBS, add an appropriate amount of trypsin to digest the cells, and when the cells become round and detach from the bottom, add an equal volume of complete culture medium to neutralize the trypsin;

[0023] 3) Transfer the cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells with an appropriate amount of fresh complete medium. Pipette thoroughly to disperse the cells into a single cell suspension. 2 Density of planking;

[0024] 4) Place the culture plate in an incubator at 37°C and 5% CO concentration for 24 hours;

[0025] 4. On the second day after the skin fibroblasts (MSF) were plated, after the cell state was stable, the chemical reprogramming medium prepared in step 2 was used to induce the culture of the plated skin fibroblasts (MSF), and the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 4 days;

[0026] 5. Prepare chemical reprogramming medium B: Add chemical small molecules 20 μmol / L CHIR99021, 10 μmol / L RepSox (E-616452) and 50 μmol / L Forskolin to the hepatocyte culture medium prepared in step 1. Use the obtained chemical reprogramming medium B to further culture the skin fibroblasts cultured in step 4 for 4 days;

[0027] 6. Prepare chemical reprogramming medium C: add chemical small molecules 3 μmol / L CHIR99021, 2 μmol / L RepSox (E-616452) and 2 μmol / L Forskolin to the hepatocyte culture medium prepared in step 1, and further culture the skin fibroblasts obtained in step 5 with the obtained chemical reprogramming medium C for 4 days. During the induction process, replace the chemical reprogramming medium C once every 4 days. Continue induction for 30 days to obtain induced hepatocytes (CiHeps).

[0028] The third aspect of the present invention provides a method for cell organogenesis using the in vivo organogenesis cell preparation.

[0029] Further, the method comprises:

[0030] (1) using an organ matrix remodeling agent to reshape the matrix structure of a target organ, wherein the target organ is the spleen, the peritoneal cavity or the renal capsule;

[0031] Further, the target organ is the spleen;

[0032] Furthermore, the organ matrix reforming agent is fibroblasts;

[0033] Furthermore, the process of using fibroblasts to reshape the spleen matrix structure includes: extracting skin fibroblasts, digesting and plating the second to third generation fibroblasts on a cell culture dish, adding 10 ng / mL TGF-α, 10 ng / mL bFGF and 10 μmol / L TGF-β1 signaling pathway inhibitor SB431542, and stimulating continuously for 7 days; then using a 31G syringe to inject 5*10 6 A directed induced fibroblast (i-MSF) was used to transform the spleen;

[0034] (2) Transplanting the functional cells into the target organ described in (1) above to achieve functional regeneration of the functional cells in the target organ, wherein the functional cells are one or more of hepatocytes, pancreatic islet cells, thyroid cells and neuronal cells, preferably hepatocytes.

[0035] In some embodiments, the functional cells are hepatocyte-like cells (iHeps) obtained using reprogramming technology or hepatocytes obtained using gene editing technology, and the gene editing technology is CRISPR / Cas9 gene editing technology.

[0036] The fourth aspect of the present invention provides the use of the in vivo organized cell preparation in tissue engineering.

[0037] Furthermore, the in vivo organized cell preparation can be used to treat various diseases, including but not limited to familial hypercholesterolemia, phenylketonuria and other monogenic hereditary liver diseases.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention achieves the regeneration of functional liver tissue in the spleen through gene editing, chemical reprogramming technology and organ matrix remodeling technology, thereby providing a new approach for the treatment of various diseases, especially for those monogenic hereditary liver diseases that are difficult to be effectively treated with traditional treatments, such as familial hypercholesterolemia. Therefore, it has broad application prospects and is expected to bring revolutionary breakthroughs in the medical field.

[0040] (2) The in vivo organized cell preparation provided by the present invention greatly expands the scope of indications for cell therapy, allowing therapeutic epithelial cells to form "therapeutic organs" in vivo. The preparation can not only be used to treat liver system diseases, but can also be expanded to the treatment of various diseases such as diabetes and neurodegenerative diseases, exerting its biological therapeutic efficacy, thereby bringing revolutionary breakthroughs to the medical field and developing unprecedented new living cell drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 . Schematic diagram of the process of cell “organization” in the spleen

[0042] Figure 2 .Bright field observation of morphological differences between MSF and CiHeps

[0043] Figure 3 .Results of determination of type I collagen content in spleen

[0044] Figure 4 . Diagram of the process of liver cell organogenesis

[0045] Figure 5 .CRISPR / Cas9 gene editing system corrects CiHeps-LDLR gene defect

[0046] Figure 6 . Schematic diagram of detection of LDL-C content in serum DETAILED DESCRIPTION

[0047] 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 intended to limit the scope of the present invention.

[0048] 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.

[0049] In order to further understand the present invention, the present invention is further described in detail below in conjunction with the best embodiment.

[0050] Example 1

[0051] This example summarizes the overall operation process of cell organogenesis. Figure 1 The process includes the following steps:

[0052] (1) extracting skin fibroblasts;

[0053] (2) Using reprogramming technology to obtain hepatocytes (iHeps), the gene editing of hepatocytes can also be further performed according to the characteristics of the disease;

[0054] (3) Directed activation of skin fibroblasts to create matrix conditions in the spleen suitable for hepatocyte colonization;

[0055] (4) Transplanting hepatocyte-like cells or modified hepatocytes into the spleen to achieve functional regeneration of healthy liver tissue in the spleen, ultimately achieving the treatment of multiple diseases.

[0056] Example 2

[0057] This embodiment provides a method for obtaining chemically reprogrammed induced hepatocytes (CiHeps) using chemical reprogramming technology, comprising:

[0058] 1. Prepare a hepatocyte culture medium, the ingredients of which include: adding 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 to DMEM / F12 culture medium.

[0059] 2. Plate and culture skin fibroblasts:

[0060] 1) First, observe the cells under a microscope to ensure that the cells are well attached, free of contamination, and in good health.

[0061] 2) Aspirate the culture medium, gently rinse the cells twice with PBS, add an appropriate amount of trypsin to digest the cells, and when the cells become round and detach from the bottom, add an equal volume of complete culture medium to neutralize the trypsin.

[0062] 3) Transfer the cell suspension into a 15 mL centrifuge tube and centrifuge at 1000 rpm for 5 minutes. Discard the supernatant and resuspend the cells with an appropriate amount of fresh complete medium. Pipette thoroughly to disperse the cells into a single cell suspension. 2 Density of planking.

[0063] 4) The culture plate was placed in an incubator at 37°C and 5% CO for 24 hours.

[0064] 3. Prepare chemical reprogramming medium A: Add chemical small molecules 20 μmol / L CHIR99021, 10 μmol / L RepSox (E-616452), 50 μmol / L Forskolin, 0.05 μmol / L AM580 and 5 μmol / L EPZ004777 to the hepatocyte culture medium prepared in step 1.

[0065] 4. On the second day after the skin fibroblasts (MSF) were plated, after the cell state was stable, the chemical reprogramming medium A prepared in step 3 was used to induce the culture of the plated skin fibroblasts (MSF), and the cells were placed in a cell culture incubator at 37°C and 5% CO2 for 4 days.

[0066] 5. Prepare chemical reprogramming medium B: Add chemical small molecules 20 μmol / L CHIR99021, 10 μmol / L RepSox (E-616452) and 50 μmol / L Forskolin to the hepatocyte medium prepared in step 1. The skin fibroblasts obtained in step 4 were further cultured with the obtained chemical reprogramming medium B in a cell culture incubator at 37°C and 5% CO2 for 4 days.

[0067] 6. Prepare chemical reprogramming medium C: add chemical small molecules 3 μmol / L CHIR99021, 2 μmol / L RepSox (E-616452) and 2 μmol / L Forskolin to the hepatocyte culture medium prepared in step 1, and further culture the skin fibroblasts obtained in step 5 with the obtained chemical reprogramming medium C in a cell culture incubator at 37°C and 5% CO2. During the culture process, replace the chemical reprogramming medium C once every 4 days. Continue induction for 30 days to obtain induced hepatocytes (CiHeps).

[0068] The results are as follows Figure 2 As shown in the figure, after 30 days of induction of skin fibroblasts by different combinations of chemical small molecules CHIR99021, RepSox (E-616452), Forskolin, AM580, EPZ004777, CH55 and UNC0638, the induced hepatocytes and MSF had obvious morphological differences: MSF was long spindle-shaped, while CiHeps had the typical morphology of hepatocyte-like epithelial cells, with enlarged and rounded nuclei and marble-like spreading growth.

[0069] Example 3

[0070] This embodiment provides a method for remodeling spleen matrix structure by fibroblasts, comprising:

[0071] (1) Extract skin fibroblasts, digest the second to third generation fibroblasts and plate them on a polystyrene cell culture dish, add 50 ng / mL bFGF and culture for 24 hours.

[0072] (2) Use a 31G syringe to inject 5*10 6 A directed induced fibroblast (i-MSF) is used to transform the spleen. The method of directed induction is shown in Example 2.

[0073] (3) Seven days after transplantation, spleen samples were collected and the type I collagen content in the spleen was detected using an ELISA kit.

[0074] The results are attached Figure 3 As shown, the graph demonstrates that the type I collagen content of the engineered spleen is significantly increased.

[0075] Example 4

[0076] In this example, the morphology and distribution of liver cells in the spleen were detected by hematoxylin and eosin (H&E) stains (hereinafter referred to as HE staining), proving that the process of organization of cells in the spleen to form functional liver tissue has been achieved. The HE staining and detection process is as follows:

[0077] (1) Dewaxing: Soak the sections in xylene (25 min) → anhydrous ethanol (3 min) → 95% ethanol (3 min) → 90% ethanol (3 min) → 80% ethanol (3 min) → 70% ethanol (3 min) → 60% ethanol (3 min) → 50% ethanol (3 min) → 30% ethanol (3 min) → double distilled water (3 min).

[0078] (2) Hematoxylin staining: Immerse the sections in hematoxylin stain for 45 seconds and then wash with tap water until there is no excess floating color.

[0079] (3) Eosin staining: The sections were sequentially treated with 50% ethanol (3 minutes) → 60% ethanol (3 minutes) → 70% ethanol (3 minutes) → 80% ethanol (3 minutes) → 90% ethanol (3 minutes) → 95% ethanol (3 minutes), and then stained with eosin dye for 15 seconds.

[0080] (4) Dehydration and transparentization: The sections were sequentially subjected to 95% ethanol (3 minutes) → anhydrous ethanol (3 minutes) → xylene (10 minutes).

[0081] (5) After sealing with neutral resin, the required tissue images can be collected under a bright field microscope.

[0082] The reprogrammed hepatocytes were transplanted into the transformed spleen, and histological analysis was performed to detect the organogenesis of hepatocytes in the spleen at different time points. Figure 4 As shown in the figure, hepatocytes can colonize and grow well in the spleen, and the hepatocytes have clear edges and a large nuclear-to-cytoplasmic ratio, showing a good cell state. And over time, the hepatocytes in the spleen form liver-specific microstructures, indicating that the hepatocytes undergo an organization process in the spleen.

[0083] Example 5

[0084] This example uses CRISPR / Cas9 gene editing technology and the cell organization method provided by the present invention to correct the low-density lipoprotein receptor (LDLR) gene defect in familial hypercholesterolemia.

[0085] Among them, mice with LDLR gene defects are selected, the skin fibroblasts of the mice are extracted, and the skin fibroblasts are converted into hepatocytes through the above-mentioned chemical reprogramming technology, that is, hepatocytes with LDLR gene defects are obtained.

[0086] Familial hypercholesterolemia (FH) is an autosomal dominant hereditary disease, mainly due to genetic abnormalities that cause cholesterol metabolism to be different from that of ordinary people, resulting in significantly increased low-density lipoprotein cholesterol (LDL-C) blood. This disease can lead to premature atherosclerotic cardiovascular disease (ASCVD) in patients. Common pathogenic genes include mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (ApoB), proprotein convertase subtilisin / kexin type 9 (PCSK9) and LDL receptor adaptor protein 1 (LDLRAP1), among which LDLR gene mutations are the most common.

[0087] The specific operation process of this embodiment is as follows:

[0088] (1) According to the location of the gene to be inserted, design the sgRNA sequence complementary to the target site and ensure that there is a PAM sequence (5'-NGG-3') downstream of the target site.

[0089] (2) Use bioinformatics tools (CRISPOR) to assess the off-target risk of sgRNA and select sgRNA with high specificity.

[0090]

[0091]

[0092] (3) Insert the designed sgRNA sequence into the plasmid expression vector (containing the Cas9 nuclease gene and the sgRNA expression cassette).

[0093] (4) Constructing a donor plasmid template (including upstream and downstream homology arms and LDLR gene cDNA fragment). Specifically, according to the insertion position of the LDLR gene, confirm the gene sequence on both sides of the target site, design the upstream and downstream homology arms, and connect the upstream and downstream homology arms to both sides of the LDLR gene cDNA fragment respectively.

[0094] (5) Use viral vectors to simultaneously deliver the Cas9 / sgRNA expression vector and donor DNA into LDLR gene-deficient hepatocytes.

[0095] (6) Screening and verification of edited hepatocytes.

[0096] The results are as follows Figure 5 As shown, the CRISPR / Cas9 gene editing system can correct the CiHeps-LDLR gene defect, and the LDLR gene-corrected transdifferentiated hepatocytes CiHeps can express LDLR protein.

[0097] Example 6

[0098] This example treats monogenic hereditary liver disease by regenerating liver tissue in the spleen. Specifically, the transdifferentiated liver cells that have been corrected by gene are implanted into the modified spleen to treat familial hypercholesterolemia by utilizing the hepatocyte organization process in the spleen. The efficacy of the cell therapy product of regenerating liver tissue in the spleen is evaluated by detecting the content of low-density lipoprotein (LDL-C) in serum.

[0099] Method for detecting LDL-C content in serum: HDL, CM, VLDL and other lipoproteins (except LDL) first change their structure and dissociate under the action of surfactants, and the released micronized cholesterol molecules react with cholesterol enzyme reagents. The generated hydrogen peroxide is consumed in the absence of coupling agents and does not show color. At this time, the LDL particles are still intact. Adding reagents containing coupling agents can dissociate LDL particles and release cholesterol. Cholesterol produces hydrogen peroxide under the catalysis of cholesterol esterase and cholesterol oxidase. In the presence of 4-aminoantipyrene, hydrogen peroxide is catalyzed by oxides to react to form red quinone compounds of benzoquinone imine phenazone. Because the cholesterol molecules of other lipoproteins have been removed, the color depth is proportional to the amount of LDL-C.

[0100] The LDL-C detection kit was purchased from Elabscience, with the catalog number: E-BC-K205-M. The specific steps are as follows:

[0101] (1) Blank well: Take 5 μL of double distilled water and add it to the corresponding well of the ELISA plate;

[0102] Standard wells: Take 5 μL of the low-density lipoprotein cholesterol standard working solution of the kit and add it to the corresponding wells of the ELISA plate;

[0103] Sample well: Take 5 μL of the sample to be tested and add it to the corresponding well of the ELISA plate;

[0104] (2) Add 180 μL of the enzyme working solution 1 of the kit to each well in step ①;

[0105] (3) Mix well and incubate at 37°C for 5 min;

[0106] (4) Measure the OD value of each well using a 546 nm ELISA reader and record it as A1;

[0107] (5) Add 60 μL of the enzyme working solution 2 of the kit to each well in step ④;

[0108] (6) Mix well and incubate at 37°C for 5 min;

[0109] (7) Measure the OD value of each well using a 546 nm ELISA reader and record it as A2.

[0110] (8) Calculation of results:

[0111] The calculation formula of serum (plasma) low-density lipoprotein cholesterol content is:

[0112]

[0113] annotation:

[0114] ΔA determination: ΔA value of the test well - ΔA value of the blank, ΔA: A2-A1

[0115] ΔA standard: standard well ΔA value - blank ΔA value, ΔA: A2-A1

[0116] c: Concentration of the standard

[0117] f: dilution factor of the sample before adding it to the detection system

[0118] The results are as follows Figure 6 As shown, this product has excellent therapeutic effects, which is more than 60% higher than the intrahepatic transplantation scheme currently used in clinical practice, demonstrating the huge application potential of "organized" hepatocyte therapy products in clinical practice.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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. An in vivo organoid cell preparation, characterized in that: The cell organogenesis preparation includes functional epithelial cells and organ matrix remodeling agents, wherein the functional epithelial cells are one or more of liver cells, pancreatic islet cells, thyroid cells and neuronal cells, and the organ matrix remodeling agent is fibroblasts, and the cell number ratio of the functional epithelial cells to the fibroblasts is 1:(0.5-2).

2. The in vivo organoid cell preparation according to claim 1, characterized in that: The functional epithelial cells are hepatocytes, which are hepatocytes induced by pluripotent stem cells, chemically reprogrammed hepatocytes, primary extracted hepatocytes or genetically modified hepatocytes. The genetically modified hepatocytes include hepatocytes obtained by TALEN, ZFN, CRISPR / Cas9 or their derivative technologies, and the derivative technologies include but are not limited to base editing technology, lead editing technology and double base editing technology.

3. The in vivo organoid cell preparation according to claim 2, characterized in that: The hepatocytes are chemically induced hepatocytes, and the induction process of the chemical reprogramming includes: (1) Preparation of hepatocyte culture medium: Add fetal bovine serum, dexamethasone, EGF, HGF, 20 μg / L TGF-α, 4.2 mg / L insulin, 5 μg / L sodium selenite and 3.8 mg / L transferrin to DMEM / F12 culture medium; (2) preparing chemical reprogramming medium A; (3) The skin fibroblasts are plated and cultured. On the second day of the plate culture, the skin fibroblasts are statically cultured in a cell culture incubator at 37° C. and 5% CO 2 for 4 days using the chemical reprogramming medium A prepared in (2). (4) preparing a chemical reprogramming medium B, and using the obtained chemical reprogramming medium B to culture the skin fibroblasts obtained by culturing in step (3) for 4 days. (5) preparing a chemical reprogramming medium C, and culturing the fibroblasts obtained in (4) with the obtained chemical reprogramming medium C. During the culturing process, the chemical reprogramming medium C was replaced once every 4 days, and the culturing was continued for 30 days to obtain induced hepatocytes (CiHeps).

4. The in vivo organoid cell preparation according to claim 3, characterized in that: In step (1), the hepatocyte culture medium comprises 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 added to DMEM / F12 culture medium.

5. The in vivo organoid cell preparation according to claim 3, characterized in that: In step (2), the method for preparing the chemical reprogramming medium A is to add chemical small molecules 20 μmol / L CHIR99021, 10 μmol / L RepSox (E-616452), 50 μmol / L Forskolin, 0.05 μmol / L AM580 and 5 μmol / L EPZ004777 to the hepatocyte culture medium; in step (4), the method for preparing the chemical reprogramming medium B is to add chemical small molecules 20 μmol / L CHIR99021, 10 μmol / L RepSox (E-616452) and 50 μmol / L Forskolin to the hepatocyte culture medium; in step (5), the method for preparing the chemical reprogramming medium C is to add 3 μmol / L CHIR99021, 2 μmol / L RepSox (E-616452) and 2 μmol / L Forskolin to the hepatocyte culture medium.

6. The in vivo organogenic cell preparation according to claim 1, characterized in that: The fibroblasts are directed differentiated fibroblasts derived from skin, heart, liver, lung or kidney. The directed differentiation method comprises digesting the fibroblasts and plating them on a cell culture dish, adding TGF-α, L bFGF and TGF-β1 signaling pathway inhibitor SB431542, and continuously stimulating for 5-10 days.

7. A method for regenerating liver tissue in the spleen using the in vivo organogenetic cell preparation according to any one of claims 1 to 6, characterized in that: The method comprises: 1) remodeling the matrix structure of the spleen using an organ matrix remodeling agent, wherein the organ matrix remodeling agent includes fibroblasts and a bioactive material; 2) Transplanting the hepatocytes into the spleen modified in 1) above to achieve functional regeneration of the functional cells in the target organ, wherein the hepatocytes are chemically induced hepatocytes, hepatocyte-like cells obtained by reprogramming technology, or hepatocytes obtained by gene editing technology, and the gene editing technology is CRISPR / Cas9 gene editing technology.

8. The method according to claim 7, characterized in that In step 1), the process of remodeling the spleen matrix structure using the organ matrix remodeling agent includes: 1) Extract skin fibroblasts, digest the extracted fibroblasts and plate them on a cell culture dish, add TGF-α, bFGF and TGF-β1 signaling pathway inhibitor SB431542, and stimulate them continuously for 7 days; 2) Use a 31G syringe to inject 5*10 6 In this study, we used a directed induced fibroblast (i-MSF) to transform the spleen matrix.

9. The method according to claim 7, characterized in that: In step 2), the hepatocytes are hepatocytes obtained by CRISPR / Cas9 gene editing technology to correct the CiHeps-LDLR gene defect, and the CRISPR / Cas9 gene editing process includes: 1) According to the location of the gene to be inserted, design the sgRNA sequence complementary to the target site and ensure that there is a PAM sequence (5'-NGG-3') downstream of the target site; 2) Use bioinformatics tools (CRISPOR) to assess the off-target risk of sgRNA and select sgRNA with high specificity; 3) Insert the designed sgRNA sequence into the expression vector (containing the Cas9 nuclease gene and sgRNA expression cassette); 4) Constructing a donor plasmid template (including upstream and downstream homology arms and LDLR gene cDNA fragment). Specifically, according to the insertion position of the LDLR gene, confirm the gene sequence on both sides of the target site, design the upstream and downstream homology arms, and connect the upstream and downstream homology arms to both sides of the LDLR gene cDNA fragment respectively; 5) Using viral vectors to simultaneously deliver Cas9 / sgRNA expression vectors and donor DNA into LDLR gene-deficient hepatocytes; 6) Screening and validation of edited hepatocytes.

10. Use of the in vivo organized cell preparation according to any one of claims 1 to 6 in tissue engineering, characterized in that: The in vivo organoid cell preparation can be used to treat various diseases, including but not limited to familial hypercholesterolemia, phenylketonuria and other monogenic inherited liver diseases.

Images