Primary pig hepatocyte proliferation medium and primary pig hepatocyte proliferation culture method

Abstract

The present invention provides a primary pig hepatocyte proliferation culture medium and a proliferation culture method of primary pig hepatocytes, and relates to the field of biotechnology. The primary pig hepatocyte proliferation culture medium provided by the present invention is composed of a liquid basal culture medium, a cell culture nutrient additive, a growth factor, a transforming growth factor-β pathway inhibitor, a WNT signaling pathway activator, a reactive oxygen scavenger, taurine, galactose, an antibiotic and serum. Through the synergistic effect between the various components, not only can long-term in vitro culture of pig hepatocytes be achieved, but also the normal morphology of hepatocytes can be maintained, and some physiological characteristics and biological functions of hepatocytes can be maintained, which is helpful to promote the application of pig hepatocytes in bioartificial livers.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a primary pig hepatocyte proliferation culture medium and a primary pig hepatocyte proliferation culture method. Background Art

[0002] The treatment of liver failure is still a global problem. Bioartificial liver (BAL) is one of the main effective means of treating liver failure and a research hotspot. The selection of appropriate hepatocytes is the core issue of bioartificial liver (BAL). Currently, the hepatocytes used in bioartificial liver mainly include human hepatocytes, liver tumor cell lines and xenogeneic animal hepatocytes. Due to the difficulty in obtaining human hepatocytes and the potential tumorigenicity of liver tumor cell lines, xenogeneic animal hepatocytes are widely used in the research of bioartificial liver. Among them, pig hepatocytes have become the most widely used xenogeneic animal hepatocytes in bioartificial liver due to their easy large-scale acquisition, similar size and structure to human hepatocytes, and physiological and metabolic functions that are closest to human hepatocytes. However, primary pig hepatocytes have a short survival time in vitro and are not easy to be cultured for a long time in vitro, which limits their application in bioartificial liver. Therefore, a stable and effective method and culture medium for long-term in vitro culture of primary pig hepatocytes is crucial.

[0003] The Chinese invention patent application with publication number CN1869205A discloses that the SV40 large T antigen gene and human telomerase reverse transcriptase are introduced into primary pig hepatocytes by using a retroviral vector method, thereby achieving long-term in vitro culture of primary pig hepatocytes. However, this method introduces the entire viral genome into cells, which has potential tumorigenicity, and is therefore limited in clinical application.

[0004] The Chinese invention patent application with publication number CN109022348A discloses a simple and efficient method for isolating and culturing primary pig hepatocytes, but the method only focuses on isolating and obtaining primary pig hepatocytes with large numbers and high activity. The culture medium used is H-DMEM culture medium, containing 10% fetal bovine serum, 100u / ml penicillin, 100u / ml streptomycin double antibody, and 5ug / ml insulin, which cannot allow primary pig hepatocytes to be cultured in vitro for a long time. The Chinese invention patent application with publication number CN107043738A discloses a serum-free culture medium for culturing primary pig hepatocytes, which can maintain the biological characteristics and biological functions of primary pig hepatocytes, but cannot allow primary pig hepatocytes to be cultured in vitro for a long time.

[0005] At present, there is no patent reporting a culture medium for long-term in vitro culture of primary pig hepatocytes.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The first object of the present invention is to provide a primary porcine hepatocyte proliferation culture medium, which can achieve long-term in vitro culture of hepatocytes to solve the above-mentioned problems.

[0008] The second object of the present invention is to provide a method for proliferating and culturing primary pig hepatocytes.

[0009] In a first aspect, the present invention provides a primary porcine hepatocyte proliferation medium, comprising the following components:

[0010] Liquid basal culture medium, cell culture nutrient supplements, growth factors, transforming growth factor-β pathway inhibitors, WNT signaling pathway activators, reactive oxygen species scavengers, taurine, galactose, antibiotics and serum.

[0011] As a further technical solution, the liquid basal culture medium includes at least one of DMEM, DMEM / F12, William's E and RPMI1640;

[0012] Preferably, the volume proportion of the serum is 1%-20%;

[0013] Preferably, the concentration of taurine is 0.1-10 mM;

[0014] Preferably, the concentration of galactose is 0.1-1 g / L.

[0015] As a further technical solution, the antibiotic includes at least one of a penicillin-streptomycin solution, a penicillin-streptomycin-amphotericin B mixed solution and a penicillin-streptomycin-gentamicin mixed solution, and the volume proportion of the antibiotic is 1%-3%.

[0016] As a further technical solution, the cell culture nutrient additive includes at least one of L-alanyl-glutamine, N-2 serum-free additive and B-27 serum-free additive;

[0017] The volume proportion of the L-alanyl-glutamine is 0.1%-2%;

[0018] The volume proportion of the N-2 serum-free additive is 0.1%-3%;

[0019] The volume proportion of the B-27 serum-free additive is 0.1%-3%.

[0020] As a further technical solution, the growth factor includes at least one of epidermal growth factor, hepatocyte growth factor and platelet-derived growth factor;

[0021] The concentration of the epidermal growth factor is 1-50ng / ml;

[0022] The concentration of the hepatocyte growth factor is 1-50ng / ml;

[0023] The concentration of the platelet-derived growth factor is 1-100 ng / ml;

[0024] As a further technical solution, the transforming growth factor-β pathway inhibitor includes at least one of SB431542, LY364947, K02288 and A-83-01;

[0025] The concentration of SB431542 is 0.01-10uM;

[0026] The concentration of LY364947 is 0.01-5uM;

[0027] The concentration of K02288 is 0.01-10uM;

[0028] The concentration of A-83-01 is 0.01-5uM.

[0029] As a further technical solution, the WNT signaling pathway activator includes at least one of a recombinant Wnt3a protein and a glycogen synthase kinase 3β inhibitor;

[0030] The glycogen synthase kinase 3β inhibitor includes at least one of CHIR99021 and TWS119;

[0031] The concentration of the recombinant Wnt3a protein is 1-50 ng / ml;

[0032] The concentration of CHIR99021 is 1-10uM;

[0033] The concentration of TWS119 is 1-50uM.

[0034] As a further technical solution, the active oxygen scavenger includes at least one of sodium pyruvate, N-acetyl-L-cysteine, DPI and vitamin C;

[0035] The concentration of sodium pyruvate is 0.01-1uM;

[0036] The concentration of N-acetyl-L-cysteine ​​is 0.01-2uM;

[0037] The concentration of the DPI is 0.01-5uM;

[0038] The concentration of the vitamin C is 1-20ug / ml.

[0039] As a further technical solution, the primary porcine hepatocyte proliferation medium is composed of the following components:

[0040] William's E liquid basal medium, N-2 serum-free supplement 1.5vol%, B-27 serum-free supplement 2vol%, L-alanyl-glutamine 1.5vol%, epidermal growth factor 15ng / ml, hepatocyte growth factor 20ng / ml, A-83-01 2uM, LY364947 1uM, CHIR99021 5uM, recombinant Wnt3a protein 2ng / ml, sodium pyruvate 0.1uM, N-acetyl-L-cysteine ​​0.2uM, vitamin C 10ug / ml, taurine 1mM, galactose 0.1g / L, penicillin-streptomycin 2vol% and serum 10vol%.

[0041] In a second aspect, the present invention provides a method for proliferation and culture of primary pig hepatocytes, comprising: inoculating primary pig hepatocytes onto a culture support pre-coated with extracellular matrix protein, and then culturing and amplifying the primary pig hepatocytes in the primary pig hepatocyte proliferation culture medium.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] The primary porcine hepatocyte proliferation medium provided by the present invention is composed of a liquid basal medium, cell culture nutrient additives, growth factors, transforming growth factor-β pathway inhibitors, WNT signaling pathway activators, reactive oxygen scavengers, taurine, galactose, antibiotics and serum. Through the synergistic effect of the various components, it can not only achieve long-term culture of hepatocytes (more than 100 days), but also maintain the normal morphology of cells, express normal hepatocyte markers, such as ALB and HNF4α, etc., and maintain certain liver function gene expressions, such as ALB, UGT1A1, CYP3A4, etc. The medium is helpful to promote the application of porcine hepatocytes in bioartificial livers. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0045] Figure 1 These are freshly isolated primary porcine hepatocytes;

[0046] Figure 2 These are porcine hepatocytes cultured in vitro for 9 days;

[0047] Figure 3 To culture the 35th generation of porcine hepatocytes in vitro;

[0048] Figure 4 The immunofluorescence results of hepatocyte markers (HNF4α, ALB) of pig hepatocytes cultured in vitro at the 35th passage;

[0049] Figure 5 These are bright field photos of pig hepatocytes cultured in the culture media provided in Example 1, Example 2, and Example 3 on the 30th day;

[0050] Figure 6 These are bright field photos of pig hepatocytes cultured in the culture medium provided in Comparative Examples 1-5 on the 10th day. DETAILED DESCRIPTION

[0051] The embodiments of the present invention will be described in detail below in conjunction with the embodiments and examples, but it will be appreciated by those skilled in the art that the following embodiments and examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of the present invention. If specific conditions are not specified, proceed according to normal conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0052] The term "primary cells" generally refers specifically to the P0 cells that have just been separated. Cells that have been cultured and cells that have changed their characteristics are not called primary cells.

[0053] The term "primary cell culture" generally refers to cell culture immediately after cells, tissues and organs are taken directly from the body. More strictly speaking, it refers to the culture before successful subculture, when the cells retain the basic properties of the original cells.

[0054] In a first aspect, the present invention provides a primary porcine hepatocyte proliferation medium, comprising the following components:

[0055] Liquid basal culture medium, cell culture nutrient supplements, growth factors, transforming growth factor-β pathway inhibitors, WNT signaling pathway activators, reactive oxygen species scavengers, taurine, galactose, antibiotics and serum.

[0056] The culture medium provided by the present invention, through the synergistic effect of the above components, can not only realize long-term in vitro culture of hepatocytes, but also maintain the normal morphology of hepatocytes, maintain some physiological characteristics and biological functions of hepatocytes, and help promote the application of porcine hepatocytes in bioartificial livers.

[0057] It should be noted that the culture medium provided by the present invention is suitable for culturing primary cells of animal liver, and the animal liver is pig liver tissue.

[0058] In some preferred embodiments, the liquid basal culture medium includes, but is not limited to, DMEM, DMEM / F12, William's E and RPMI1640, or other liquid culture medium that can be used for animal cell culture and is well known to those skilled in the art.

[0059] The volume percentage of the serum may be, for example, but not limited to, 1%, 2%, 4%, 6%, 10% or 20%.

[0060] In some preferred embodiments, antibiotics are used to inhibit the growth of bacteria other than hepatocytes. In the present invention, the antibiotics include at least one of a penicillin-streptomycin solution (i.e., a mixed solution of penicillin and streptomycin, and the same applies below. For example, the reagent can be selected from Shanghai Yuanpei Biotechnology Co., Ltd., with a product number of S110JV), a penicillin-streptomycin-amphotericin B mixed solution (the reagent can be selected from Beijing Solebow Technology Co., Ltd., with a product number of P7630), and a penicillin-streptomycin-gentamicin mixed solution (the reagent can be selected from Beijing Solebow Technology Co., Ltd., with a product number of P1410). The volume proportion of the antibiotic can be, for example, but not limited to, 1%, 2% or 3%;

[0061] In some preferred embodiments, the cell culture nutrient supplement includes at least one of L-alanyl-glutamine, N-2 serum-free supplement, and B-27 serum-free supplement;

[0062] The volume percentage of the L-alanyl-glutamine may be, for example, but not limited to, 0.1%, 0.5%, 1%, 1.5% or 2%;

[0063] The volume percentage of the N-2 serum-free additive may be, for example, but not limited to, 0.1%, 0.5%, 1%, 2% or 3%;

[0064] The volume percentage of the B-27 serum-free additive may be, for example, but not limited to, 0.1%, 0.5%, 1%, 2% or 3%.

[0065] In some preferred embodiments, the growth factors include, but are not limited to, epidermal growth factor, hepatocyte growth factor, and platelet-derived growth factor;

[0066] The concentration of the epidermal growth factor may be, for example, but not limited to, 5 ng / ml, 10 ng / ml, 15 ng / ml, 20 ng / ml, 40 ng / ml or 50 ng / ml;

[0067] The concentration of the hepatocyte growth factor may be, for example, but not limited to, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 40 ng / ml or 50 ng / ml;

[0068] The concentration of the platelet-derived growth factor may be, for example, but not limited to, 1 ng / ml, 5 ng / ml, 10 ng / ml, 20 ng / ml, 40 ng / ml, 60 ng / ml, 80 ng / ml or 100 ng / ml;

[0069] In some preferred embodiments, the transforming growth factor-β pathway inhibitor includes at least one of SB431542, LY364947, K02288, and A-83-01;

[0070] The concentration of SB431542 may be, for example, but not limited to, 0.01uM, 0.1uM, 1uM, 5uM, 8uM or 10uM;

[0071] The concentration of LY364947 may be, for example, but not limited to, 0.01uM, 0.1uM, 1uM, 2uM or 5uM;

[0072] The concentration of K02288 may be, for example, but not limited to, 0.01uM, 0.1uM, 1uM, 5uM, 8uM or 10uM;

[0073] The concentration of A-83-01 may be, for example, but not limited to, 0.01uM, 0.1uM, 1uM, 2uM or 5uM.

[0074] In some preferred embodiments, the WNT signaling pathway activator comprises at least one of a recombinant Wnt3a protein and a glycogen synthase kinase 3β inhibitor;

[0075] The glycogen synthase kinase 3β inhibitor includes at least one of CHIR99021 and TWS119;

[0076] The concentration of the recombinant Wnt3a protein may be, for example, but not limited to, 1 ng / ml, 2 ng / ml, 5 ng / ml, 10 ng / ml, 25 ng / ml or 50 ng / ml;

[0077] The concentration of CHIR99021 may be, for example, but not limited to, 1uM, 5uM, 8uM or 10uM;

[0078] The concentration of TWS119 may be, for example, but not limited to, 1 uM, 5 uM, 10 uM, 20 uM, 40 uM or 50 uM.

[0079] In some preferred embodiments, the active oxygen scavenger includes at least one of sodium pyruvate, N-acetyl-L-cysteine, DPI and vitamin C;

[0080] The concentration of sodium pyruvate may be, for example, but not limited to, 0.01uM, 0.1uM, 0.5uM or 1uM;

[0081] The concentration of the N-acetyl-L-cysteine ​​may be, for example, but not limited to, 0.01uM, 0.1uM, 0.5uM, 1uM or 2uM;

[0082] The concentration of the DPI may be, for example, but not limited to, 0.01uM, 0.1uM, 0.5uM, 1uM, 2uM or 5uM;

[0083] The concentration of the vitamin C may be, for example, but not limited to, 1 ug / ml, 5 ug / ml, 10 ug / ml, 15 ug / ml or 20 ug / ml.

[0084] In some preferred embodiments, the concentration of taurine may be, for example, but not limited to, 0.1 mM, 0.5 mM, 1 mM, 5 mM or 10 mM.

[0085] In some preferred embodiments, the concentration of galactose may be, for example, but not limited to, 0.1 g / L, 0.2 g / L, 0.5 g / L or 1 g / L.

[0086] In some preferred embodiments, the primary porcine hepatocyte proliferation medium consists of the following components:

[0087] William's E liquid basal medium, N-2 serum-free supplement 1.5vol%, B-27 serum-free supplement 2vol%, L-alanyl-glutamine 1.5vol%, epidermal growth factor 15ng / ml, hepatocyte growth factor 20ng / ml, A-83-01 2uM, LY364947 1uM, CHIR99021 5uM, recombinant Wnt3a protein 2ng / ml, sodium pyruvate 0.1uM, N-acetyl-L-cysteine ​​0.2uM, vitamin C 10ug / ml, taurine 1mM, galactose 0.1g / L, penicillin-streptomycin 2vol% and serum 10vol%.

[0088] By further optimizing and adjusting the various components in the primary porcine hepatocyte proliferation culture medium, the culture time of hepatocytes can be extended while maintaining the normal morphology of hepatocytes and maintaining some physiological characteristics and biological functions of hepatocytes.

[0089] In a second aspect, the present invention provides a method for proliferation and culture of primary pig hepatocytes, comprising: inoculating primary pig hepatocytes onto a culture support pre-coated with extracellular matrix protein, and then culturing and amplifying the primary pig hepatocytes in the primary pig hepatocyte proliferation culture medium.

[0090] The present invention does not impose any specific restrictions on the detailed steps of the proliferation culture method. The proliferation culture method may be, for example, as follows:

[0091] ① Pre-coating the culture support with extracellular matrix protein;

[0092] The extracellular matrix protein can be collagen I, Matrigel and other extracellular matrix proteins.

[0093] Preferably, the coating concentration of collagen I is 0.1-10ug / cm 2 .

[0094] Preferably, the coating concentration of the Matrigel matrix gel is 0.1%-10%.

[0095] The culture support refers to a culture bottle or a culture well plate, etc.

[0096] ② Prepare primary cell suspension with common complete culture medium, inoculate the cell suspension into the culture support pre-coated in step ①, and culture in a 5% CO2, 37°C incubator to allow it to adhere to the wall;

[0097] The common complete culture medium is DMEM containing 10%-15% serum and 1%-3% secondary antibody (penicillin and streptomycin).

[0098] The primary cells are primary pig hepatocytes separated by collagenase perfusion method.

[0099] ③ After a few hours, change the medium to remove the non-adherent hepatocytes and replace it with the aforementioned porcine hepatocyte proliferation medium;

[0100] The several hours are preferably 2-4 hours.

[0101] ④ Place in a 5% CO2, 37℃ incubator for culture.

[0102] ⑤ Change the medium every 2-3 days. The culture medium used is the aforementioned pig hepatocyte proliferation medium.

[0103] ⑥ After the cells grow to cover the culture support, they are subcultured. The subculture steps are as follows: pre-coat the new culture support for more than half an hour as in step ①; discard the old culture medium, wash twice with sterile PBS, add an appropriate amount of trypsin for digestion; place in a 5% CO2, 37°C incubator for digestion for 2-3 minutes; add an equal amount of ordinary DMEM full culture to terminate digestion; transfer all liquids to a centrifuge tube, centrifuge at 1400RPM for 3 minutes; discard the supernatant, add an appropriate amount of the aforementioned porcine hepatocyte proliferation medium to prepare a cell suspension, transfer the cell suspension to a new culture dish, and shake it evenly by the cross method.

[0104] ⑦Continue to culture in a 5% CO2, 37℃ incubator.

[0105] The culture method provided by the present invention is simple, can realize long-term in vitro culture of pig hepatocytes, and can maintain the normal morphology of the hepatocytes and maintain some physiological characteristics and biological functions of the hepatocytes.

[0106] The present invention is further described below by means of specific examples and comparative examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0107] Example 1

[0108] A primary porcine hepatocyte proliferation culture medium, comprising the following components:

[0109] William's E liquid basal medium, N-2 serum-free supplement 1.5vol%, B-27 serum-free supplement 2vol%, L-alanyl-glutamine 1.5vol%, epidermal growth factor 15ng / ml, hepatocyte growth factor 20ng / ml, A-83-01 2uM, LY364947 1uM, CHIR99021 5uM, recombinant Wnt3a protein 2ng / ml, sodium pyruvate 0.1uM, N-acetyl-L-cysteine ​​0.2uM, vitamin C 10ug / ml, taurine 1mM, galactose 0.1g / L, penicillin-streptomycin 2vol% and serum 10vol%.

[0110] Example 2

[0111] A primary porcine hepatocyte proliferation culture medium, comprising the following components:

[0112] DMEM liquid basal culture medium, N-2 serum-free supplement 1 vol%, B-27 serum-free supplement 1 vol%, L-alanyl-glutamine 0.5 vol%, epidermal growth factor 10 ng / ml, hepatocyte growth factor 10 ng / ml, A-83-01 1uM, LY364947 1uM, CHIR99021 5uM, sodium pyruvate 0.5uM, N-acetyl-L-cysteine ​​0.5uM, vitamin C 5ug / ml, taurine 5mM, galactose 0.2g / L, penicillin-streptomycin 2 vol% and serum 20 vol%.

[0113] Example 3

[0114] A primary porcine hepatocyte proliferation culture medium, comprising the following components:

[0115] RPMI1640 liquid basal medium, N-2 serum-free supplement 0.5vol%, B-27 serum-free supplement 0.5vol%, L-alanyl-glutamine 1vol%, epidermal growth factor 10ng / ml, hepatocyte growth factor 10ng / ml, A-83-01 2uM, LY364947 1uM, CHIR99021 5uM, recombinant Wnt3a protein 2ng / ml, sodium pyruvate 0.1uM, N-acetyl-L-cysteine ​​0.1uM, vitamin C 20ug / ml, taurine 2mM, galactose 0.5g / L, penicillin-streptomycin 2vol% and serum 10vol%.

[0116] Comparative Example 1

[0117] A culture medium, which differs from the culture medium in Example 1 in that it does not contain epidermal growth factor and hepatocyte growth factor.

[0118] Comparative Example 2

[0119] A culture medium, which differs from the culture medium in that it does not contain sodium pyruvate, N-acetyl-L-cysteine, and vitamin C.

[0120] Comparative Example 3

[0121] A culture medium, which differs from Example 1 in that it does not contain A-83-01 and LY364947.

[0122] Comparative Example 4

[0123] A culture medium, which differs from that in Example 1 in that it does not contain recombinant Wnt3a protein and CHIR99021.

[0124] Comparative Example 5

[0125] A culture medium, which differs from that in Example 1 in that it does not contain taurine and galactose.

[0126] Test Example 1

[0127] The culture medium provided in Examples 1-3 and Comparative Examples 1-7 was used to culture hepatocytes in the following manner:

[0128] 1. Use 2% Matrigel to coat the culture flask, preferably covering the bottom of the culture flask, and place it in a 37°C incubator for half an hour before use.

[0129] 2. Primary porcine hepatocytes were isolated by collagenase perfusion. Figure 1 shown.

[0130] 3. Prepare primary porcine hepatocyte suspension with DMEM containing 10%-15% serum and 1%-3% secondary antibody, inoculate it into coated culture flasks, and culture it in a 5% CO2, 37°C incubator.

[0131] After 4.2 hours, the medium was changed to remove the primary porcine hepatocytes that were not attached to the wall, and the medium was replaced with the medium of Examples 1-3 and Comparative Examples 1-5, and cultured in a 5% CO2, 37°C incubator.

[0132] 5. Change the medium every 2-3 days. The medium used is the aforementioned pig hepatocyte proliferation medium.

[0133] 6. After the cells have grown to cover the entire culture dish, they are subcultured. The subculture steps are as follows: pre-coat a new culture dish for more than half an hour as in step ①; discard the old culture medium, wash twice with sterile PBS, and add an appropriate amount of trypsin for digestion; place in a 5% CO2, 37°C incubator for digestion for 2-3 minutes; add an equal amount of ordinary DMEM full culture medium to terminate digestion; transfer all liquids to a centrifuge tube and centrifuge at 1400RPM for 3 minutes; discard the supernatant, add an appropriate amount of the aforementioned porcine hepatocyte proliferation medium to prepare a cell suspension, transfer the cell suspension to a new culture dish, and shake well using the cross method.

[0134] 7. Continue to culture in a 5% CO2, 37°C incubator.

[0135] The porcine hepatocytes were cultured in vitro for 9 days using the culture medium provided in Example 1. Figure 2 As shown, pig hepatocytes cultured to passage 35 were Figure 3 The immunofluorescence results are shown in Figure 4 As shown, the immunofluorescence results showed that when pig hepatocytes were cultured in vitro to the 35th generation, the hepatocyte markers HNF4α and ALB showed a high level of expression.

[0136] The bright field photographs of pig hepatocytes cultured in the culture medium provided in Example 1, Example 2, and Example 3 until the 30th day are as follows: Figure 5 As shown, the results show that pig hepatocytes can be cultured for a long time (more than 30 days) and maintain good proliferation ability by culturing the culture medium in Examples 1-3.

[0137] The bright field photograph of pig liver cells cultured on the 10th day using the culture medium provided in Comparative Example 1-5 is as follows Figure 6 As shown, it can be seen that the cells cultured in the culture medium with reduced components have poor growth and proliferation, shortened survival time, and poor cell morphology and state. When pig liver cells were cultured in the culture medium in Comparative Examples 1-5, the liver cells survived for a maximum of 10 days and lost their normal cell morphology.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primary porcine hepatocyte proliferation medium, characterized in that: It is composed of the following components: William's E liquid basal medium, N-2 serum-free supplement 1.5 vol%, B-27 serum-free supplement 2 vol%, L-alanyl-glutamine 1.5 vol%, epidermal growth factor 15 ng / ml, hepatocyte growth factor 20 ng / ml, A-83-01 2uM, LY364947 1uM, CHIR99021 5uM, recombinant Wnt3a protein 2ng / ml, sodium pyruvate 0.1uM, N-acetyl-L-cysteine ​​0.2uM, vitamin C 10ug / ml, taurine 1mM, galactose 0.1g / L, penicillin-streptomycin 2vol% and serum 10vol%; Or it may be composed of the following components: DMEM liquid basal medium, N-2 serum-free supplement 1 vol%, B-27 serum-free supplement 1 vol%, L-alanyl-glutamine 0.5 vol%, epidermal growth factor 10 ng / ml, hepatocyte growth factor 10 ng / ml, A-83-01 1 uM, LY364947 1 uM, CHIR99021 5 uM, sodium pyruvate 0.5 uM, N-acetyl-L-cysteine ​​0.5 uM, vitamin C 5 ug / ml, taurine 5 mM, galactose 0.2 g / L, penicillin-streptomycin 2 vol% and serum 20 vol%; Or it may be composed of the following components: RPMI1640 liquid basal medium, N-2 serum-free supplement 0.5vol%, B-27 serum-free supplement 0.5vol%, L-alanyl-glutamine 1vol%, epidermal growth factor 10ng / ml, hepatocyte growth factor 10ng / ml, A-83-012uM, LY364947 1uM, CHIR99021 5uM, recombinant Wnt3a protein 2ng / ml, sodium pyruvate 0.1uM, N-acetyl-L-cysteine ​​0.1uM, vitamin C 20ug / ml, taurine 2mM, galactose 0.5g / L, penicillin-streptomycin 2vol% and serum 10vol%.

2. A method for the proliferation and cultivation of primary pig hepatocytes, characterized in that: include: The primary porcine hepatocytes are inoculated onto a culture support pre-coated with an extracellular matrix protein, and then cultured and expanded in the primary porcine hepatocyte proliferation medium of claim 1.

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