Culture medium for culturing primary hepatocytes in duck embryonic period and application of culture medium

Abstract

The invention discloses a culture medium for culturing primary hepatocytes in a duck embryonic period and application of the culture medium. The invention provides a culture medium. The concentration of FSK is 20 to 30 [mu] M, the concentration of SB431542 is 10 to 15 [mu] M, the concentration of DAPT is 1 to 1.5 [mu] M, the concentration of IWP2 is 0.5 to 0.75 [mu] M, the concentration of LDN193189 is 0.1 to 0.15 [mu] M, the volume percentage content of a mycillin solution is 0.5 to 2%, the volume percentage content of a B27 additive is 1 to 3%, the volume percentage content of a GlutaMAX additive is 0.5 to 1.5%, the concentration of HGF is 10 to 20 ng / mL, the concentration of TNF-alpha is 10 to 20 ng / mL, and the concentration of IL-6 is 10 to 20 ng / mL. The volume percentage of the fetal calf serum is 1-3%, and the balance is the basic culture medium. By adopting the culture medium provided by the invention, obvious in-vitro proliferation of the duck embryo hepatocytes is realized, and the purity and in-vitro maintenance time of the hepatocytes are greatly improved.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a culture medium for culturing duck embryonic primary hepatocytes and application thereof. Background Art

[0002] The liver is a complex and important organ that plays an important role in metabolism and detoxification. Since it is difficult to study liver metabolism, viral action, and drug action mechanisms in vivo, it is necessary to establish a primary hepatocyte model. Duck primary hepatocytes can be used as a cell model for studying viral infection mechanisms, bacterial infection mechanisms, drug screening, etc.

[0003] In the prior art, duck hepatocytes are difficult to proliferate in vitro. Conventional culture media cannot promote the proliferation of hepatocytes, but greatly promote the proliferation of non-parenchymal cells. Isolated duck primary hepatocytes usually begin to mesenchymalize or disintegrate after 2-3 days of culture. Therefore, the development of a culture medium for duck primary hepatocyte culture has important scientific research value and market value. Summary of the invention

[0004] The purpose of the invention is to provide a culture medium for culturing duck embryonic primary hepatocytes and application thereof.

[0005] The present invention first provides a composition, comprising the following components: FSK, SB431542, DAPT, IWP2, LDN193189, B27 additive, GlutaMAX additive, HGF, TNF-α and IL-6.

[0006] Specifically, the composition consists of the following components: FSK, SB431542, DAPT, IWP2, LDN193189, B27 additive, GlutaMAX additive, HGF, TNF-α and IL-6.

[0007] In the composition, the ratio of each component is as follows: 20-30 μmol FSK: 10-15 μmol SB431542: 1-1.5 μmol DAPT: 0.5-0.75 μmol IWP2: 0.1-0.15 μmol LDN193189: 10-30 mL B27 additive: 5-15 mL GlutaMAX additive: 10-20 μg HGF: 10-20 μg TNF-α: 10-20 μg IL-6.

[0008] Specifically, in the composition, the ratio of each component is as follows: 20 μmol FSK: 10 μmol SB431542: 1 μmol DAPT: 0.5 μmol IWP2: 0.1 μmol LDN193189: 20 mL B27 additive: 10 mL GlutaMAX additive: 20 μg HGF: 20 μg TNF-α: 20 μg IL-6.

[0009] The composition may also include penicillin and streptomycin and / or fetal bovine serum.

[0010] Specifically, the composition consists of the following components: FSK, SB431542, DAPT, IWP2, LDN193189, B27 additive, GlutaMAX additive, HGF, TNF-α, IL-6, penicillin and streptomycin.

[0011] In the composition, the ratio of each component is as follows: 20-30 μmol FSK: 10-15 μmol SB431542: 1-1.5 μmol DAPT: 0.5-0.75 μmol IWP2: 0.1-0.15 μmol LDN193189: 50000-200000 U penicillin: 50-200 mg streptomycin: 10-30 mL B27 additive: 5-15 mL GlutaMAX additive: 10-20 μg HGF: 10-20 μg TNF-α: 10-20 μg IL-6.

[0012] Specifically, in the composition, the ratio of each component is as follows: 20 μmol FSK: 10 μmol SB431542: 1 μmol DAPT: 0.5 μmol IWP2: 0.1 μmol LDN193189: 150000U penicillin: 150 mg streptomycin: 20 mL B27 additive: 10 mL GlutaMAX additive: 20 μg HGF: 20 μg TNF-α: 20 μg IL-6.

[0013] Specifically, the composition consists of the following components: FSK, SB431542, DAPT, IWP2, LDN193189, B27 additive, GlutaMAX additive, HGF, TNF-α, IL-6, penicillin, streptomycin and fetal bovine serum.

[0014] In the composition, the ratio of each component is as follows: 20-30 μmol FSK: 10-15 μmol SB431542: 1-1.5 μmol DAPT: 0.5-0.75 μmol IWP2: 0.1-0.15 μmol LDN193189: 50000-200000U penicillin: 50-200mg streptomycin: 10-30mL B27 additive: 5-15mL GlutaMAX additive: 10-20μg HGF: 10-20μg TNF-α: 10-20μg IL-6: 10-30ml fetal bovine serum.

[0015] Specifically, in the composition, the ratio of each component is as follows: 20 μmol FSK: 10 μmol SB431542: 1 μmol DAPT: 0.5 μmol IWP2: 0.1 μmol LDN193189: 150000U penicillin: 150 mg streptomycin: 20 mL B27 additive: 10 mL GlutaMAX additive: 20 μg HGF: 20 μg TNF-α: 20 μg IL-6: 20 ml fetal bovine serum.

[0016] The present invention also provides a culture medium, named as culture medium A, which comprises: FSK concentration of 20-30 μM, SB431542 concentration of 10-15 μM, DAPT concentration of 1-1.5 μM, IWP2 concentration of 0.5-0.75 μM, LDN193189 concentration of 0.1-0.15 μM, penicillin-streptomycin solution volume percentage of 0.5%-2%, B27 additive volume percentage of 1-3%, GlutaMAX additive volume percentage of 0.5-1.5%, HGF concentration of 10-20 ng / mL, TNF-α concentration of 10-20 ng / mL, IL-6 concentration of 10-20 ng / mL, and the remainder being basal culture medium.

[0017] Specifically, the composition of the culture medium A is as follows: the concentration of FSK is 20 μM, the concentration of SB431542 is 10 μM, the concentration of DAPT is 1 μM, the concentration of IWP2 is 0.5 μM, the concentration of LDN193189 is 0.1 μM, the volume percentage of penicillin-streptomycin solution is 1.5%, the volume percentage of B27 additive is 2%, the volume percentage of GlutaMAX additive is 1%, the concentration of HGF is 20 ng / mL, the concentration of TNF-α is 20 ng / mL, the concentration of IL-6 is 20 ng / mL, and the remainder is basal culture medium.

[0018] The present invention also provides a culture medium, named as culture medium B, which consists of the culture medium A and fetal bovine serum.

[0019] In the medium B, the volume percentage of the fetal bovine serum is 1-3%

[0020] In the culture medium B, the volume percentage of the fetal bovine serum is 2%.

[0021] Specifically, any of the above basic culture media is William's E culture medium.

[0022] FSK (Forskolin), CAS No. is 66575-29-9.

[0023] SB431542 (SB-431542, CAS No. is 301836-41-9).

[0024] DAPT, CAS No. is 208255-80-5.

[0025] IWP2, CAS No. is 686770-61-6.

[0026] LDN193189 (LDN-193189), CAS No. is 1435934-00-1.

[0027] The product forms of FSK, SB431542, DAPT, IWP2 and LDN193189 are all powders, which are prepared into 5 mM stock solutions with DMSO and added in the form of stock solutions when used.

[0028] Specifically, the penicillin-streptomycin solution is a 100× penicillin-streptomycin solution.

[0029] Specifically, the penicillin-streptomycin solution contains 10000 U / mL penicillin and 10 mg / mL streptomycin.

[0030] Specifically, the B27 additive is a 50× B27 additive.

[0031] Specifically, the GlutaMAX additive is a 100× GlutaMAX additive.

[0032] Specifically, the GlutaMAX additive contains 200 mM L-alanyl-L-glutamine dipeptide.

[0033] Specifically, the HGF is recombinant human HGF.

[0034] Specifically, the HGF is Recombinant Human Hepatocyte growth factor.

[0035] Specifically, the IL-6 is recombinant human IL-6.

[0036] Specifically, the IL-6 is Recombinant Human IL-6 Protein.

[0037] Specifically, the TNF-α is recombinant human TNF-α.

[0038] Specifically, the TNF-α is Recombinant Human TNF-alpha Protein.

[0039] Specifically, the composition of the culture medium A is: FSK storage solution, SB431542 storage solution, DAPT storage solution, IWP2 storage solution, LDN193189 storage solution, penicillin-streptomycin solution, B27 additive, GlutaMAX additive, HGF, TNF-α, IL-6 and William's E culture medium.

[0040] Specifically, the composition of the culture medium B is: FSK storage solution, SB431542 storage solution, DAPT storage solution, IWP2 storage solution, LDN193189 storage solution, penicillin-streptomycin solution, B27 additive, GlutaMAX additive, HGF, TNF-α, IL-6, fetal bovine serum and William's E culture medium.

[0041] The present invention also protects the use of any of the above compositions in preparing a culture medium;

[0042] The purpose of the culture medium is as follows (a1) or (a2) or (a3):

[0043] (a1) Cultivation of duck primary hepatocytes;

[0044] (a2) Promote the proliferation of duck primary hepatocytes;

[0045] (a3) Enriching and / or purifying duck primary hepatocytes.

[0046] Specifically, the duck primary hepatocytes are isolated from duck embryos.

[0047] Specifically, the duck embryo is a duck embryo of embryonic age 8 or 14.

[0048] The present invention also protects the use of any of the above culture media, which is as follows (a1) or (a2) or (a3):

[0049] (a1) Cultivation of duck primary hepatocytes;

[0050] (a2) Promote the proliferation of duck primary hepatocytes;

[0051] (a3) Enriching and / or purifying duck primary hepatocytes.

[0052] Specifically, the duck primary hepatocytes are isolated from duck embryos.

[0053] Specifically, the duck embryo is a duck embryo of embryonic age 8 or 14.

[0054] The present invention also provides a method for culturing duck primary hepatocytes, comprising the following steps: using any of the above culture media to culture duck primary hepatocytes.

[0055] Specifically, the duck primary hepatocytes are isolated from duck embryos.

[0056] Specifically, the duck embryo is a duck embryo of embryonic age 8 or 14.

[0057] The present invention also protects the application of the method, which is as follows (b1) or (b2):

[0058] (b1) Promote the proliferation of duck primary hepatocytes;

[0059] (b2) Enriching and / or purifying duck primary hepatocytes.

[0060] Specifically, the duck primary hepatocytes are isolated from duck embryos.

[0061] Specifically, the duck embryo is a duck embryo of embryonic age 8 or 14.

[0062] Specifically, any of the ducks mentioned above is a Beijing duck.

[0063] Specifically, any of the above-mentioned ducks is a Z-type Beijing duck.

[0064] The present invention uses a simplified hepatocyte separation method to compare the number and functional indicators of hepatocyte separation throughout the duck embryonic period, and comprehensively determines the most suitable embryonic age for hepatocyte separation. On this basis, the in vitro culture conditions of human hepatocytes are tested and other chemical small molecules that promote hepatocyte growth are added, and finally an optimized culture medium is obtained. The culture medium provided by the present invention realizes significant in vitro proliferation of duck embryonic hepatocytes, greatly improving the purity of hepatocytes and the in vitro maintenance time. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 This is a photograph of cells stained by the trypan blue exclusion method in Example 1.

[0066] Figure 2 This is a photograph of cells after glycogen staining in Example 1.

[0067] Figure 3 This is a photograph of cells after immunofluorescence staining in Example 1.

[0068] Figure 4 The positive rate results are statistically analyzed based on the photos after immunofluorescence staining in Example 1.

[0069] Figure 5 It is the albumin content in the cell culture supernatant at each sampling time point in Example 2.

[0070] Figure 6 It is the aspartate aminotransferase content in the cell culture supernatant at each sampling time point in Example 2.

[0071] Figure 7 It is the lactate dehydrogenase content in the cell culture supernatant at each sampling time point in Example 2.

[0072] Figure 8 The figure is a comparison of the aspartate aminotransferase content in the culture supernatant obtained using various test culture media at the same culture time in Example 2.

[0073] Fig. 9 The figure is a comparison of the lactate dehydrogenase content in the culture supernatant obtained using various test culture media at the same culture time in Example 2.

[0074] Fig.10 This is a photograph of cells after glycogen staining in Example 2.

[0075] Fig.11 This is a photograph of cells after immunofluorescence staining in Example 2 (48 hours).

[0076] Fig.12 This is a photograph of cells after immunofluorescence staining in Example 2 (96 hours).

[0077] Fig.13 This is a photograph of cells after immunofluorescence staining in Example 2 (144 hours).

[0078] Fig.14 This is the hepatocyte growth curve in Example 2.

[0079] Fig.15 These are morphological observation photographs of the above steps in Example 3 using culture medium 1-0, culture medium 1-2%, culture medium 1-4% or culture medium 1-6%.

[0080] Fig.16 These are morphological observation photographs of the above steps in Example 3 using culture medium 9-0, culture medium 9-2%, culture medium 9-4% or culture medium 9-6%.

[0081] Fig.17 This is the hepatocyte growth curve in Example 4. DETAILED DESCRIPTION

[0082] The present invention is further described in detail below in conjunction with specific embodiments. The examples given are only for illustrating the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation on the present invention in any way. The experimental methods in the following examples, unless otherwise specified, are all conventional methods, carried out in accordance with the techniques or conditions described in the literature in the art or in accordance with the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are all repeated three times, and the results are averaged. Unless otherwise specified, the cell culture conditions are all: 37°C, 5% CO2. Glycogen PAS staining kit: Solebao Company, product catalog number G1360. The embryonic age corresponds to the number of days of incubation, for example, a duck embryo of 14 embryonic age refers to the embryo in the breeder duck egg that was incubated to the 14th day. The duck embryo breeds in the examples are all Z-type Beijing ducks. References to Z-type Beijing duck: Progress in breeding of Z-type Beijing duck, Zhang Yunsheng, Huang Wei, Hou Shuisheng. Progress in breeding of Z-type Beijing duck [C] / / Poultry Branch of China Animal Husbandry Association, Jiangxi Provincial Animal Husbandry and Veterinary Bureau. Proceedings of the 4th China Waterfowl Development Conference. National Waterfowl Industry Technology Research and Development Center, Beijing Animal Husbandry and Veterinary Research Institute, Chinese Academy of Agricultural Sciences;, 2011: 5. PBS buffer (7.0-7.3): contains 2.68mM KCl, 1.47mM KH2PO4, 137mM NaCl, 8.0mM Na2HPO4, and the balance is water.

[0083] Example 1: Effect of embryonic age on the isolation of primary hepatocytes

[0084] 1. Isolation of primary hepatocytes from embryos of different embryonic ages

[0085] Embryos were taken from breeder duck eggs on the 6th, 8th, 10th, 12th, 14th, 16th, 18th, 20th and 22nd days of incubation, and primary cells were prepared.

[0086] The method is the same as step 1 of Example 2 to obtain primary cells.

[0087] 2. Comparative Performance

[0088] 1. Take the primary cells prepared in step 1, resuspend them in DMEM medium containing 10% fetal bovine serum, and then stain them using the trypan blue exclusion method. Count the total cell number and the number of live cells of the primary cells prepared from the liver of each embryo, and calculate the cell survival rate (i.e., the percentage of the number of live cells to the total number of cells, referred to as the survival rate).

[0089] Results Figure 1 .

[0090] 2. Take the primary cells prepared in step 1 and add 5×10 5 The cells were inoculated into 6-well plates at a density of 100 / ml and cultured for 24 hours. The culture medium was discarded and PBS buffer was added for washing. Then, the glycogen PAS staining kit was used according to the instructions. The results of glycogen staining are shown in Figure 2 (Scale bar: 100 μm). Statistics Figure 2 The total cell area in each field of view and the total area of ​​the positively stained area in the cells were used to obtain the percentage of the total area of ​​the positively stained area to the total cell area (recorded as the glycogen content) to measure the glycogen storage.

[0091] 3. Take the primary cells prepared in step 1 and add 5×10 5 The cells were inoculated at a density of 100 / ml into a 6-well plate, and after culturing for 24 hours, immunofluorescence staining targeting albumin was performed (see step 4, 2 of Example 2 for the method).

[0092] The results of immunofluorescence staining are shown in Figure 3 (Scale bar: 50 μm). Statistics Figure 3 The total number of cells and the number of positive cells (immunofluorescence positive cells are positive hepatocytes) in each field of view were calculated, and the purity (also known as the positive rate, that is, the percentage of immunofluorescence positive cells in the total cells) was calculated. The results are shown in Figure 4 .

[0093] The three indicators (viable cell number result, glycogen content result and positive hepatocyte number result) obtained in the above steps were graded and assigned values ​​according to Table 1, and the three indicators accounted for 1 / 3 respectively. The total score was calculated by multiplying the product of the indicator and the graded score by 100%, which was the scoring result. The total score is shown in Table 2. Primary hepatocytes isolated from embryos of 8 or 14 embryonic ages had the best performance.

[0094] Table 1

[0095]

[0096] Table 2

[0097]

[0098]

[0099] Example 2: Optimization of culture medium for duck embryonic primary hepatocyte culture

[0100] 1. Preparation of primary cells

[0101] 1. Take a culture dish with a diameter of 90 mm and add 20 ml of sterile saline containing 1% sodium heparin, which is culture dish 1; take two culture dishes with a diameter of 90 mm and add PBS buffer, which are culture dishes 2 and 3; place culture dishes 1, 2 and 3 on ice for precooling.

[0102] 2. Take 14-year-old duck eggs, clean the shells with saline, and disinfect with 75% ethanol aqueous solution, then remove the duck embryo and separate the liver.

[0103] 3. Take the liver obtained in step 2 and place it in culture dish 1 first, shave off the obvious fibrous membrane and gallbladder on the liver tissue, then use bent forceps to gently squeeze the liver to promote the release of blood in the liver tissue, and then place the liver in culture dish 2 and culture dish 3 in turn for washing.

[0104] 4. After completing step 3, place the liver in a pre-cooled 4 mL centrifuge tube (containing PBS buffer containing 1‰ BSA), then discard the liquid in the centrifuge tube and cut the liver in the centrifuge tube into 1 mm 3 Following the tissue blocks, gently wash the tissue blocks 2-3 times with pre-chilled PBS buffer until the supernatant is clear.

[0105] 5. After completing step 4, transfer the tissue block to a 50 mL centrifuge tube, add 10 mL of 37°C preheated digestion solution and mix by pipetting, digest in a 37°C water bath for 20 minutes (oscillate every 5 minutes), then add 20 ml of DMEM medium containing 10% fetal bovine serum to the centrifuge tube to terminate the digestion, gently pipette, then filter with a 200-mesh cell sieve and collect the filtrate.

[0106] Digestion solution: contains 1 g / L DNase I (Roche, product catalog number is 11284932001), 2 g / L type II collagenase (Gibco, product catalog number is 17101015), and the balance is PBS buffer.

[0107] 6. Take the filtrate obtained in step 5, centrifuge at 1000 rpm for 5 min, discard the supernatant, add DMEM medium to resuspend the precipitate, then centrifuge at 50 g / min for 5 min and discard the supernatant. The remaining precipitate is the primary cells.

[0108] 2. Preparation of 15 Culture Media

[0109] FSK (Forskolin; CAS No. is 66575-29-9): MCE Company, product catalog number is HY-15371. SB431542 (SB-431542; CAS No. is 301836-41-9): MCE Company, product catalog number is HY-10431. DAPT (CAS No. is 208255-80-5): MCE Company, product catalog number is HY-13027. IWP2 (CAS No. is 686770-61-6): MCE Company, product catalog number is HY-13912. LDN193189 (LDN-193189; CAS No. is 1435934-00-1): MCE Company, product catalog number is HY-12071B. The product forms of FSK, SB431542, DAPT, IWP2 and LDN193189 are all powders, which are prepared into 5 mM stock solutions with DMSO and added in the form of stock solutions when used.

[0110] Penicillin-streptomycin solution (100×) (Specification: containing 10000U / mL penicillin and 10mg / mL streptomycin): Servicebio, product catalog number G4003-100ML. B27 additive (50×): Gibco, product catalog number 17504044. GlutaMAX TM Additive (100×) (Specification: containing 200 mM L-alanyl-L-glutamine dipeptide): Gibco Company, product catalog number is 35050061.

[0111] HGF (Recombinant Human Hepatocyte growth factor): ABclonal, product catalog number RP01602. IL-6 (Recombinant Human IL-6 Protein): ABclonal, product catalog number RP01688. TNF-α (Recombinant Human TNF-alpha Protein): ABclonal, product catalog number RP00993. ITS-G (Insulin-Transferrin-Selenium Supplement) (100×): icellbioscience, product catalog number iCell-4507.

[0112] William's E medium: Gibco, product catalog number is 12551032.

[0113] The composition of the basic culture medium is: FSK storage solution, SB431542 storage solution, DAPT storage solution, IWP2 storage solution, LDN193189 storage solution, penicillin-streptomycin solution, B27 additive, GlutaMAX TM Supplements and William's E medium. In the basal medium, the concentration of FSK is 20μM, the concentration of SB431542 is 10μM, the concentration of DAPT is 1μM, the concentration of IWP2 is 0.5μM, the concentration of LDN193189 is 0.1μM, the concentration of penicillin-streptomycin solution is 1.5% (volume percentage), the concentration of B27 additive is 2% (volume percentage), and the concentration of GlutaMAX TM The concentration of the additive is 1% (volume percentage).

[0114] Culture medium 1 to culture medium 15 are all composed of additives and basal culture medium (all of which are natural pH). The additives are shown in Table 3 (1-15 in the first row represent culture medium 1 to culture medium 15, and √ indicates that the additive is present). The concentration of HGF in the culture medium is 20 ng / mL. The concentration of TNF-α in the culture medium is 20 ng / mL. The concentration of IL-6 in the culture medium is 20 ng / mL. The concentration of ITS-G in the culture medium is 1% (volume percentage).

[0115] Table 3

[0116] Additives 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 HGF √ √ √ √ √ √ √ √ TNF-α √ √ √ √ √ √ √ √ ITS-G √ √ √ √ √ √ √ √ IL-6 √ √ √ √ √ √ √ √

[0117] 3. Preliminary screening of culture medium by testing biochemical indicators

[0118] The test culture media are the 15 culture media prepared in step 2.

[0119] Take the primary cells prepared in step 1 and resuspend them in the test medium to make the cell concentration 5×10 5 / ml, then inoculated into a 6-well plate (2ml per well) and cultured for 6 days. The supernatant was sampled after 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours and 144 hours, and the albumin (ALB) content, aspartate aminotransferase (AST) content and lactate dehydrogenase (LDH) content in the supernatant were detected using a fully automatic biochemical analyzer. The higher the albumin content, the better the liver cell function. The lower the aspartate aminotransferase and lactate dehydrogenase content, the lower the degree of cell damage.

[0120] The albumin content in the cell culture supernatant at each sampling time point is shown in Figure 5 The content of aspartate aminotransferase in the cell culture supernatant at each sampling time point is shown in Figure 6 The lactate dehydrogenase content in the cell culture supernatant at each sampling time point is shown in Figure 7Comparison of the aspartate aminotransferase content in the culture supernatant obtained using various test culture media at the same culture time Figure 8 Comparison of lactate dehydrogenase content in the culture supernatant obtained by using various test culture media at the same culture time Fig. 9 .

[0121] Based on the results of albumin content, medium 1 was screened out. Based on the results of aspartate aminotransferase content and lactate dehydrogenase content, medium 3, medium 7, medium 9 and medium 11 were screened out. Therefore, based on this step, the following 5 culture media were preliminarily screened out from 15 culture media: medium 1, medium 3, medium 7, medium 9 and medium 11.

[0122] 4. Further screening of culture medium

[0123] The test culture media are the following five culture media prepared in step 2: culture medium 1, culture medium 3, culture medium 7, culture medium 9 and culture medium 11.

[0124] 1. Detection of glycogen by glycogen staining

[0125] Glycogen is a substance formed by the polymerization of a large number of glucose molecules. It is stored in the liver and is an important energy storage substance of the liver. Glycogen is detected by periodic acid Schiff staining (PAS). A positive reaction of glycogen shows red or purple-red color, and the cell nucleus is blue.

[0126] ① Take the primary cells prepared in step 1 and resuspend them in the test medium to make the cell concentration 5×10 5 The cells were then inoculated into 6-well plates (2 ml per well) and cultured for 48, 96 or 144 hours.

[0127] ②After completing step ①, discard the liquid in the well, add PBS buffer for washing, and then use the glycogen PAS staining kit and operate according to the instructions. The results of glycogen staining are shown in Fig.10 (Scale bar: 100 μm). Statistics Fig.10 The total cell area in each field of view and the total area of ​​the positive staining region in the cell were used to obtain the percentage of the total area of ​​the positive staining region to the total cell area (recorded as glycogen content) to measure the glycogen storage. The results of glycogen content are shown in Table 4. Medium 3 had the worst effect, and medium 9 had the best effect.

[0128] Table 4

[0129]

[0130]

[0131] 2. Detection of albumin by immunofluorescence

[0132] ① Take the primary cells prepared in step 1 and resuspend them in the test medium to make the cell concentration 5×10 5 The cells were then inoculated into 6-well plates (2 ml per well) and cultured for 48, 96 or 144 hours.

[0133] ②After completing step ①, discard the liquid in the wells, add PBS buffer for washing, and then perform immunofluorescence detection. The primary antibody used is Albumin Rabbit pAb (ABclonal, product catalog number A0353, used after 1:100 dilution), the secondary antibody used is FITC-labeled donkey anti-rabbit IgG (Servicebio, product catalog number GB22403, used after 1:200 dilution), and DAPI is used to stain the cell nucleus.

[0134] The results of immunofluorescence staining are shown in Figures 11 to 13 ( Fig.11 Corresponding to 48 hours of culture, Fig.12 Corresponding to 96 hours of culture, Fig.13 Corresponding to 144 hours of culture; 100x scale bar: 100 μm; 400x scale bar: 50 μm). Statistics Fig.11 The total number of cells and the number of positive cells (immunofluorescence positive cells, i.e. positive hepatocytes) in each field of view were used to calculate the purity (also known as the positive rate, i.e. the percentage of immunofluorescence positive cells in the total cells). The results are shown in Table 5.

[0135] Table 5

[0136] 48 hours 96 hours 144 hours Medium 1 96.60％ 98.45％ 98.20％ Medium 3 96.65％ 98.16％ 97.14％ Medium 7 96.54％ 98.35％ 98.50％ Medium 9 98.08％ 98.43％ 98.40％ Medium 11 96.75％ 98.18％ 98.15％

[0137] 3. Create a hepatocyte growth curve

[0138] Take the primary cells prepared in step 1 and resuspend them in the test medium to make the cell concentration 5×10 5 / ml, and then inoculated into a 12-well plate (1 ml per well), cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days and 9 days, and then the cell number was counted using a cell counter.

[0139] Results Fig.14 The best results were obtained with medium 1 and medium 9.

[0140] Based on the above results, the most suitable culture media were determined to be culture medium 1 and culture medium 9. Both culture medium 1 and culture medium 9 can significantly promote the proliferation of duck hepatocytes in vitro and increase the in vitro culture maintenance time, while ensuring the purity of hepatocytes and inhibiting the growth of non-parenchymal cells.

[0141] Example 3: Further Optimization of Culture Medium

[0142] The test culture media were the following 8 culture media:

[0143] Medium 1-0: Medium 1;

[0144] Culture medium 1-2%: composed of 2% (volume percentage) fetal bovine serum and 98% (volume percentage) culture medium 1;

[0145] Medium 1-4%: composed of 4% (volume percentage) fetal bovine serum and 96% (volume percentage) medium 1;

[0146] Medium 1-6%: composed of 6% (volume percentage) fetal bovine serum and 94% (volume percentage) medium 1;

[0147] Medium 9-0: Medium 9;

[0148] Medium 9-2%: composed of 2% (volume percentage) fetal bovine serum and 98% (volume percentage) medium 9;

[0149] Culture medium 9-4%: composed of 4% (volume percentage) fetal bovine serum and 96% (volume percentage) culture medium 9;

[0150] Culture medium 9-6%: composed of 6% (volume percentage) fetal bovine serum and 94% (volume percentage) culture medium 9;

[0151] Culture medium 1 is culture medium 1 prepared in step 2 of Example 2; culture medium 9 is culture medium 9 prepared in step 2 of Example 2.

[0152] The primary cells prepared in step 1 of Example 2 were resuspended in the test medium to a cell concentration of 5×10 5 The cells were then inoculated into 6-well plates (2 ml per well), cultured for 48, 96 or 144 hours, and then morphological observations were performed.

[0153] The morphological observation photos of the above steps using medium 1-0, medium 1-2%, medium 1-4% or medium 1-6% are shown in Fig.15 . Fig.15In the figure, the first row is a high-magnification photo (100 times, scale bar 100μm), the second row is a high-magnification photo (100 times), the third row is a high-magnification photo (100 times), and the fourth row is a low-magnification photo (50 times, scale bar 500μm). The results of the series of culture media using culture medium 1 showed that the addition of serum can significantly promote the growth rate of cells, including hepatocytes and non-parenchymal cells (mainly evaluated by the size of the liver mass area); during the culture process, under the comprehensive 4% serum condition, hepatocytes proliferated fastest and could quickly fill the bottom of the culture well, while non-parenchymal cells were relatively few, followed by 2%, and 6% hepatocytes were relatively few, among which non-parenchymal cells were densely present; the addition of serum accelerated the mesenchymalization of hepatocytes (transformation of hepatocytes into non-parenchymal cells), and the time of mesenchymalization was also advanced as the serum concentration increased (mesenchymalization in the blue box).

[0154] The morphological observation photos of the above steps using medium 9-0, medium 9-2%, medium 9-4% or medium 9-6% are shown in Fig.16 . Fig.16 In the figure, the first row is a high-magnification photo (100 times, scale bar 100 μm), the second row is a high-magnification photo (100 times), the third row is a high-magnification photo (100 times), and the fourth row is a low-magnification photo (50 times, scale bar 500 μm). The results of the series of culture media using culture medium 9 show that the three serum concentrations have similar efficiency in promoting the proliferation of hepatocytes through the size of the hepatocyte clusters, while the 4% and 6% serum concentrations will promote the mesenchymalization of hepatocytes (mesenchymalization in the blue box), and the 2% serum condition can not only promote the proliferation of hepatocytes, but also no mesenchymalization of hepatocytes occurs. Serum has a significant proliferation effect on hepatocytes, but because serum also has a significant promoting effect on non-parenchymal cells, long-term use will significantly increase the mesenchymalization of hepatocytes. Therefore, the use of low-concentration serum in combination with culture medium 9 can make hepatocytes proliferate rapidly in the early stage and have no obvious mesenchymalization in the later stage.

[0155] Example 4: Verification of the effect of culture medium

[0156] The test culture media were the following 7 culture media:

[0157] ① Culture medium 9-2%: composed of 2% (volume percentage) fetal bovine serum and 98% (volume percentage) culture medium 9;

[0158] ② Medium 9: Medium 9;

[0159] ③ Culture medium 1-2%: composed of 2% (volume percentage) fetal bovine serum and 98% (volume percentage) culture medium 1;

[0160] ④ Medium 1: Medium 1;

[0161] ⑤Basic culture medium - 2%: composed of 2% (volume percentage) fetal bovine serum and 98% (volume percentage) basic culture medium;

[0162] ⑥Basic culture medium;

[0163] ⑦1.5 times basal medium: the contents of FSK, SB431542, DAPT, IWP2, and LDN193189 are all increased to 1.5 times, and the others are the same as the basal medium;

[0164] Culture medium 1 is culture medium 1 prepared in step 2 of Example 2; culture medium 9 is culture medium 9 prepared in step 2 of Example 2, and basal culture medium is basal culture medium prepared in step 2 of Example 2.

[0165] The primary cells prepared in step 1 of Example 2 were resuspended in the test medium to a cell concentration of 5×10 5 / ml, and then inoculated into a 12-well plate (1 ml per well), cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days or 7 days, and then the cell number was counted using a cell counter.

[0166] The results of cell number are shown in Fig.17 .

[0167] The basal medium was used as the reference medium. Relative cell number = (the number of cells in a certain medium at a certain time - the number of cells in the reference medium at the same culture time) ÷ the number of cells in the reference medium at the same culture time × 100%. The results of relative cell number are shown in Table 6. Serum has a very significant promoting effect on the proliferation rate of in vitro cultured hepatocytes. The best effect was achieved when the medium was 9-2%.

[0168] Table 6

[0169]

[0170]

[0171] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including those that depart from the disclosed scope of this application. Changes made using conventional techniques known in the art may be applied to some of the basic features within the scope of the following claims.

Claims

1. A composition comprising the following components: FSK, SB431542, DAPT, IWP2, LDN193189, B27 additive, GlutaMAX additive, HGF, TNF-α and IL-6; In the composition, the ratio of each component is as follows: 20-30 μmol FSK: 10-15 μmol SB431542: 1-1.5 μmol DAPT: 0.5-0.75 μmol IWP2: 0.1-0.15 μmol LDN193189: 10-30 mL B27 additive: 5-15 mL GlutaMAX additive: 10-20 μg HGF: 10-20 μg TNF-α: 10-20 μg IL-6.

2. The composition according to claim 1, characterized in that: In the composition, the ratio of each component is as follows: 20 μmol FSK: 10 μmol SB431542: 1 μmol DAPT: 0.5 μmol IWP2: 0.1 μmol LDN193189: 20 mL B27 additive: 10 mL GlutaMAX additive: 20 μg HGF: 20 μg TNF-α: 20 μg IL-6.

3. The composition according to claim 1, characterized in that: The composition further comprises penicillin and / or streptomycin and / or fetal bovine serum.

4. A culture medium, named as culture medium A, comprising: FSK at a concentration of 20-30 μM, SB431542 at a concentration of 10-15 μM, DAPT at a concentration of 1-1.5 μM, IWP2 at a concentration of 0.5-0.75 μM, LDN193189 at a concentration of 0.1-0.15 μM, penicillin-streptomycin solution at a volume percentage of 0.5%-2%, B27 additive at a volume percentage of 1-3%, GlutaMAX additive at a volume percentage of 0.5-1.5%, HGF at a concentration of 10-20 ng / mL, TNF-α at a concentration of 10-20 ng / mL, IL-6 at a concentration of 10-20 ng / mL, and the remainder being basal culture medium.

5. The culture medium according to claim 4, characterized in that: The composition of the culture medium is as follows: FSK concentration is 20 μM, SB431542 concentration is 10 μM, DAPT concentration is 1 μM, IWP2 concentration is 0.5 μM, LDN193189 concentration is 0.1 μM, penicillin-streptomycin solution volume percentage is 1.5%, B27 additive volume percentage is 2%, GlutaMAX additive volume percentage is 1%, HGF concentration is 20 ng / mL, TNF-α concentration is 20 ng / mL, IL-6 concentration is 20 ng / mL, and the remainder is basal culture medium.

6. A culture medium, named culture medium B, consisting of culture medium A and fetal bovine serum; the culture medium A is as described in claim 4 or 5.

7. Use of the composition according to any one of claims 1 to 3 in preparing a culture medium; The purpose of the culture medium is as follows (a1) or (a2) or (a3): (a1) Cultivation of duck primary hepatocytes; (a2) Promote the proliferation of duck primary hepatocytes; (a3) Enriching and / or purifying duck primary hepatocytes.

8. The use of the culture medium according to any one of claims 4 to 6, which is as follows (a1) or (a2) or (a3): (a1) Cultivation of duck primary hepatocytes; (a2) Promote the proliferation of duck primary hepatocytes; (a3) Enriching and / or purifying duck primary hepatocytes.

9. A method for culturing duck primary hepatocytes, comprising the following steps: culturing duck primary hepatocytes using the culture medium described in any one of claims 4 to 6.

10. The use of the method according to claim 9, which is as follows (b1) or (b2): (b1) Promote the proliferation of duck primary hepatocytes; (b2) Enriching and / or purifying duck primary hepatocytes.

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