CN117165514A - Animal-source-free supplement, culture medium and application thereof in inducing differentiation of directional endoderm cells - Google Patents

Animal-source-free supplement, culture medium and application thereof in inducing differentiation of directional endoderm cells Download PDF

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CN117165514A
CN117165514A CN202311093054.2A CN202311093054A CN117165514A CN 117165514 A CN117165514 A CN 117165514A CN 202311093054 A CN202311093054 A CN 202311093054A CN 117165514 A CN117165514 A CN 117165514A
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mug
animal
directional
medium
endoderm
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王小丰
鲁素芳
吴双双
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Hangzhou Ruipu Chenchuang Technology Co ltd
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Hangzhou Ruipu Chenchuang Technology Co ltd
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Abstract

The invention discloses an animal-source-free supplement, an animal-source-free culture medium comprising an induced differentiation-oriented endoderm cell without the animal-source supplement, and application of the animal-source-free culture medium in the induced differentiation-oriented endoderm cell. The animal-free supplement comprises the following components: biotin, DL alpha-tocopheryl acetate, human recombinant insulin, human transferrin, corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, l-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, and triiodothyronine. The invention provides a serum-free and animal-derived supplement and a culture medium for the first time. The supplement and the culture medium do not contain serum and animal source components, and can efficiently induce the directional endoderm differentiation of the pluripotent stem cells by being combined with small molecules such as Activin A, CHIR99021 and the like, and the differentiation efficiency is up to 95%. The invention overcomes the potential safety hazard of biological products caused by factors such as undefined components, animal sources and the like of the traditional supplement.

Description

Animal-source-free supplement, culture medium and application thereof in inducing differentiation of directional endoderm cells
Technical Field
The invention relates to an animal-source-free supplement, a culture medium and application thereof in inducing differentiation of directional endoderm cells.
Background
The formation of directional endoderm (Definitive endoderm, DE) is a key stage in embryo development during human embryo development, which is a precursor for the formation of internal organs such as liver, lung, pancreas, etc.
Multipotent stem cells hipscs not only have unlimited proliferation potential, but also have no associated ethical and ethical issues that make hipscs an ideal choice for the study and treatment of many diseases. Researchers have been struggling to develop efficient and reliable differentiation protocols that induce the differentiation of multipotent stem cells hipscs into directional endodermal fate.
The directional endoderm cells induced by the technical means can further guide them to differentiate along a specific pathway, obtaining various endoderm derived functional cells such as liver cells, islet cells, alveolar cells, and the like. Efficient induction of directional endodermal differentiation has the potential to revolutionarily alter regenerative medicine and to bring new therapeutic possibilities for patients with organ failure, genetic diseases and other afflicting diseases.
In the prior art, the culture medium for inducing and defining the endoderm cells is generally prepared by adding various supplements to a basic culture medium, wherein the supplements generally comprise various active small molecules such as growth factors and the like, but most of the supplements contain serum and animal-derived components. The clinical products require that the cell preparation cannot contain animal-derived components because animal nucleic acids, or infected bacterial and viral nucleic acids, etc., and proteins that may cause immune effects on the recipient may remain in the animal-derived components. Although animal-derived components can be detected at the time of subsequent quality detection, the use of materials containing animal-derived components is still not allowed for safety at the time of drug approval, even if only materials as auxiliary supporting properties. Therefore, there is a need to develop supplements and media for inducing oriented endoderm cells without animal sources.
Disclosure of Invention
The invention aims to provide an animal-source-free supplement and a culture medium for inducing differentiation of directional endoderm cells, which have simple components, do not contain animal-source components such as serum and the like, can avoid detection of the animal-source components before clinical application, and are more suitable for clinical application.
The technical scheme adopted by the invention is as follows:
an animal-free supplement, the supplement comprising the following components:
biotin, DL alpha-tocopheryl acetate, human recombinant insulin, human transferrin, corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, l-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, and triiodothyronine.
The invention further provides an animal-free concentrated supplement which is prepared from the animal-free concentrated supplement, wherein the animal-free concentrated supplement is 100 x concentrated solution, and the animal-free concentrated supplement is prepared by taking MCDB131 culture medium as a solvent, and comprises the following components in concentration:
200-350 mug/mL biotin, 80-150 mug/mL DL alpha-tocopherol acetate, 350-500 mug/mL human recombinant insulin, 400-600 mug/mL human transferrin, 1-5 mug/mL corticosterone, 1000-2000 mug/mLD-galactose, 80-150 mug/mL ethanolamine hydrochloride, 80-150 mug/mL reduced glutathione, 150-300 mug/mL L-carnitine hydrochloride, 80-150 mug/mL linoleic acid, 80-150 mug/mL linolenic acid, 0.5-0.8 mug/mL progesterone, 1500-2000 mug/mL putrescine, 1-2 mug/mL sodium selenite and 0.1-0.5 mug/mL triiodothyronine.
Preferably, the animal-source-free concentrated supplement takes MCDB131 culture medium as a solvent, and comprises the following components in concentration:
250 μg/mL biotin, 100 μg/mL DL alpha-tocopherol acetate, 400 μg/mL human recombinant insulin, 500 μg/mL human transferrin, 2 μg/mL corticosterone, 1500 μg/mLD-galactose, 100 μg/mL ethanolamine hydrochloride, 100 μg/mL reduced glutathione, 200 μg/mL left-handed carnitine hydrochloride, 100 μg/mL linoleic acid, 100 μg/mL linolenic acid, 0.63 μg/mL progesterone, 1610 μg/mL putrescine, 1.44 μg/mL sodium selenite, and 0.20 μg/mL triiodothyronine.
Further, the present invention also provides an animal-free medium for inducing differentiated directional endoderm cells, the medium comprising the animal-free supplement described above, in particular, the animal-free medium for inducing differentiated directional endoderm cells comprises directional endoderm medium 1 and directional endoderm medium 2, the directional endoderm medium 1 being a MCDB131 medium-based medium, comprising the following components:
2 to 3.5 mug/mL biotin, 0.8 to 1.5 mug/mL DL alpha-tocopherol acetate, 3.5 to 5 mug/mL human recombinant insulin, 4 to 6 mug/mL human transferrin, 0.01 to 0.05 mug/mL corticosterone, 10 to 20 mug/mLD-galactose, 0.8 to 1.5 mug/mL ethanolamine hydrochloride, 0.8 to 1.5 mug/mL reduced glutathione, 1.5 to 3 mug/mL L-carnitine hydrochloride, 0.8 to 1.5 mug/mL linoleic acid, 0.8 to 1.5 mug/mL linolenic acid, 0.005 to 0.008 mug/mL progesterone, 15 to 20 mug/mL putrescine, 0.01 to 0.02 mug/mL sodium selenite and 0.001 to 0.005 mug/mL tri-iodothyronine;
1-2 mM glutamine, 50-100U/mL penicillin, 50-100 mug/mL streptomycin, 3-5 mM glucose, 80-120 ng/mL TGF-beta activator, 0.2-0.3 mM vitamin C, 5-8 mu M Wnt signal pathway activator, 40-60 nM PI3K/mTOR inhibitor and 8-15 mu M ROCK inhibitor.
Further, preferably, the TGF-beta activator is Activin A; the Wnt signal path activator is CHIR-99021; the PI3K/mTOR inhibitor is PI-103; the ROCK inhibitor is Y27632.
Preferably, the composition of the directional endoderm medium 1 is: a basal medium based on MCDB131 medium; the following final concentrations of ingredients:
2.5. Mu.g/mL biotin, 1. Mu.g/mL DL alpha-tocopherol acetate, 4. Mu.g/mL human recombinant insulin, 5. Mu.g/mL human transferrin, 0.02. Mu.g/mL corticosterone, 15. Mu.g/mLD-galactose, 1. Mu.g/mL ethanolamine hydrochloride, 1. Mu.g/mL reduced glutathione, 2. Mu.g/mL left-handed carnitine hydrochloride, 1. Mu.g/mL linoleic acid, 1. Mu.g/mL linolenic acid, 0.0063. Mu.g/mL progesterone, 16.1. Mu.g/mL putrescine, 0.0144. Mu.g/mL sodium selenite, and 0.002. Mu.g/mL triiodothyronine;
2mM glutamine, 100U/mL penicillin, 100. Mu.g/mL streptomycin, 4.5mM glucose, 100ng/mL Activin A, 0.25mM vitamin C, 6. Mu.M CHIR-99021, 50nM PI-103 and 10. Mu. M Y27632.
The directional endoderm culture medium 2 is based on MCDB131 culture medium and comprises the following components:
2 to 3.5 mug/mL biotin, 0.8 to 1.5 mug/mL alpha-tocopherol acetate, 3.5 to 5 mug/mL human recombinant insulin, 4 to 6 mug/mL human transferrin, 0.01 to 0.05 mug/mL corticosterone, 10 to 20 mug/mLD-galactose, 0.8 to 1.5 mug/mL ethanolamine hydrochloride, 0.8 to 1.5 mug/mL reduced glutathione, 1.5 to 3 mug/mL L-carnitine hydrochloride, 0.8 to 1.5 mug/mL linoleic acid, 0.8 to 1.5 mug/mL linolenic acid, 0.005 to 0.008 mug/mL progesterone, 15 to 20 mug/mL putrescine, 0.01 to 0.02 mug/mL sodium selenite and 0.001 to 0.005 mug/mL tri-iodothyronine;
1-2 mM glutamine, 50-100U/mL penicillin, 50-100 mug/mL streptomycin, 3-5 mM glucose, 40-60 ng/mL TGF-beta activator, 0.2-0.3 mM vitamin C.
Preferably, the composition of the directional endoderm medium 2 is: a basal medium based on MCDB131 medium; the following final concentrations of ingredients:
2.5. Mu.g/mL biotin, 1. Mu.g/mL DL alpha-tocopherol acetate, 4. Mu.g/mL human recombinant insulin, 5. Mu.g/mL human transferrin, 0.02. Mu.g/mL corticosterone, 15. Mu.g/mLD-galactose, 1. Mu.g/mL ethanolamine hydrochloride, 1. Mu.g/mL reduced glutathione, 2. Mu.g/mL left-handed carnitine hydrochloride, 1. Mu.g/mL linoleic acid, 1. Mu.g/mL linolenic acid, 0.0063. Mu.g/mL progesterone, 16.1. Mu.g/mL putrescine, 0.0144. Mu.g/mL sodium selenite, and 0.002. Mu.g/mL triiodothyronine;
2mM glutamine, 100U/mL penicillin, 100 μg/mL streptomycin, 4.5mM glucose, 50ng/mL Activin A, 0.25mM vitamin C.
Further, the directional endoderm medium 1 and the directional endoderm medium 2 can be prepared from a concentrated supplement without animal source, the directional endoderm medium 1 having a composition of:
the MCDB131 culture medium is taken as a basic culture medium, and comprises the following components:
100 Xanimal-derived concentrate supplement, 1-2 mM glutamine, 50-100U/mL penicillin, 50-100 μg/mL streptomycin, 3-5 mM glucose, 80-120 ng/mL TGF-beta activator, 0.2-0.3 mM vitamin C, 5-8 μM Wnt signaling pathway activator, 40-60 nM P3K/mTOR inhibitor, and 8-15 μM ROCK inhibitor.
The composition of the directional endoderm culture medium 1 is:
the MCDB131 culture medium is taken as a basic culture medium, and comprises the following components:
100 Xanimal source-free concentrated supplement, 1-2 mM glutamine, 50-100U/mL penicillin, 50-100 mug/mL streptomycin, 3-5 mM glucose, 40-60 ng/mL TGF-beta activator, 0.2-0.3 mM vitamin C.
The invention also provides application of the animal-source-free culture medium in inducing differentiation of directional endoderm cells.
The invention further provides a culture method for inducing and differentiating the pluripotent stem cells into the directional endoderm cells, which comprises the steps of culturing the pluripotent stem cells by adopting the animal source-free culture medium for inducing and differentiating the directional endoderm cells, and differentiating the pluripotent stem cells into the directional endoderm cells.
In the present invention, the pluripotent stem cells are human induced pluripotent stem cells.
Further, the method is that the pluripotent stem cells are cultured for 1 day by using the directional endoderm culture medium 1, then are cultured for 3 days by changing to the directional endoderm culture medium 2, and the directional endoderm cells are obtained by inducing differentiation.
Further, the culture is carried out in an incubator under the condition of 37 ℃ and 5% CO 2 。
Further, when cultured with directional endoderm medium 2 for 3 days, the medium was changed every day.
Further, in the method, the pluripotent stem cells are obtained by amplification culture according to the following method:
the pluripotent stem cells were cultured with mTESR1 at 37℃until the cell fusion degree reached 80-90%, digested into single cells, resuspended in mTESR1 stem cell culture medium containing 10 mu M Y27632, and inoculated into a vector, and induced to differentiate after one day.
The carrier is a culture plate.
The invention has the beneficial effects that:
the invention provides a serum-free protein and animal-free supplement and a culture medium comprising the animal-free supplement for the first time, wherein the supplement and the culture medium do not contain serum and animal-free components, and can efficiently induce the directional endoderm differentiation of pluripotent stem cells by being combined with small molecules such as Activin A, CHIR99021 and the like, and the differentiation efficiency is as high as 95%. The invention overcomes the potential safety hazard of biological products caused by the factors of undefined components, animal sources and the like of the traditional supplement, and can be used for clinical products. The animal-source-free supplement has the advantages of simple formula, easy preparation, low cost, stable quality and the like, has a wide application prospect in clinical research, and is favorable for commercialization and mass production application of products.
Drawings
FIG. 1A morphology of multipotent stem cell hiPSC clones.
FIG. 2A morphology of pluripotent stem cell hiPSC single cell plating.
FIG. 3 is a morphology of the oriented endoderm cells obtained four days after induced differentiation of the oriented endoderm.
FIG. 4 is a graph showing the results of measuring the differentiation efficiency of directional endoderm by a flow meter.
Detailed Description
The following describes the technical scheme of the present invention with specific examples, but the scope of the present invention is not limited thereto.
Example 1
1. SM15 supplement 100 x formulation:
15 small molecules were dissolved in MCDB131 basal medium to make 100 x concentrate: biotin 250. Mu.g/mL, DL-alpha-tocopherol acetate 100. Mu.g/mL, human recombinant insulin 400. Mu.g/mL, human transferrin 500. Mu.g/mL, corticosterone 2. Mu.g/mL, D-galactose 1500. Mu.g/mL, ethanolamine hydrochloride 100. Mu.g/mL, glutathione (reduced) 100. Mu.g/mL, L-carnitine hydrochloride 200. Mu.g/mL, linoleic acid 100. Mu.g/mL, linolenic acid 100. Mu.g/mL, progesterone 0.63. Mu.g/mL, putrescine 1610. Mu.g/mL, sodium selenite 1.44. Mu.g/mL, and triiodothyronine 0.20. Mu.g/mL, to prepare the SM15 supplement. The final concentrate is diluted by 100 times for use, and the supplement is packaged after preparation, and stored at-20deg.C.
The SM15 supplement small molecule sources used in the examples are shown in table 1 below:
TABLE 1
2. Multipotent stem cell hiPSC passaging:
step 1: multipotent Stem cells hipscs were digested with slesr (Stem Cell, cat# 05872) preheated at 37 ℃ for 2min, and large clones were digested into small clones.
Step 2: the small clones were collected into centrifuge tubes, after centrifugation at 300g for 3min, the slesr waste liquid supernatant was aspirated away.
Step 3: the tube was charged with mTESR1 (Stem Cell, cat# 85850) Stem Cell medium containing 5. Mu. M Y27632 (Selleck, cat# S1049) small molecules and resuspended as a uniform small clone suspension.
Step 4: the small clone suspension was added to a Matrigel (BD BioSciences, cat# 356231) coated 6-well plate and gently swirled to disperse the small clones uniformly in the wells.
Step 5: after 24h Kong Zhongshang clear was aspirated, 2mL fresh mTeSR1 (Stem Cell, cat# 85850) Stem Cell medium was exchanged.
Step 6: and after the confluence of cloning reaches about 80-90%, the cloning is digested into single cell plates to induce differentiation.
3. Multipotent stem cell single cell hiPSC plating:
step 1: the growth confluence of multipotent stem cells hiPSC reaches about 80-90%, the culture supernatant is aspirated, and 1mL of Actutase (EMD Millipore, cat#SCR 005) preheated at 37 ℃ is added for digestion for 3-5min.
Step 2: after cloning and digesting into single cells, collecting single cells into a centrifuge tube, centrifuging for 3min at 300g, and sucking away the supernatant of the digestion solution.
Step 3: the mTeSR1 stem cell culture medium containing 10 μ M Y27632 small molecules was added to the centrifuge tube and resuspended to a uniform single cell suspension.
Step 4: the single cell suspension was added to Matrigel coated 12 well plates, each well was seeded with approximately 50-60 ten thousand cells, and gently swirled to uniformly disperse single cells in the well.
Step 5: after 24h, the culture supernatant in the wells was aspirated and the initiation of the induction of differentiation directed endoderm was initiated.
4. Inducing differentiation of directional endoderm: stage1 (4 days)
Preparation of Directional endoderm Medium 1Stage1:day1 (S1D 1) Medium based on MCDB131 Medium, containing the following final concentrations of the components:
100 XSM 15, 2mM glutamine, 100U/mL penicillin, 100. Mu.g/mL streptomycin, 4.5mM glucose, 100ng/mL Activin A, 0.25mM vitamin C, 6. Mu.M CHIR-99021, 50nM PI-103 and 10. Mu. M Y27632.
Preparing a directional endoderm culture medium 2Stage1:day2-day4 (S1D 2-S1D 4) culture medium, wherein the culture medium is based on MCDB131 culture medium and contains the following components in final concentration: 100 XSM 15, 2mM glutamine, 100U/mL penicillin, 100 μg/mL streptomycin, 4.5mM glucose, 50ng/mL Activin A, 0.25mM vitamin C.
Step 1: on the first day, after the S1D1 culture medium is fully preheated in a water bath pot at 37 ℃, the waste liquid supernatant in 12 holes is sucked, and 1.5mL of fresh and preheated S1D1 culture medium is replaced.
Step 2: the next day, after the S1D2-S1D4 culture medium is fully preheated in a water bath pot at 37 ℃, the waste liquid supernatant in 12 holes is sucked, and 2mL of fresh and preheated S1D2-S1D4 culture medium is replaced.
Step 3: on the third day, after the S1D2-S1D4 culture medium is fully preheated in a water bath pot at 37 ℃, the waste liquid supernatant in 12 holes is sucked, and 2mL of fresh and preheated S1D2-S1D4 culture medium is replaced.
Step 4: on the fourth day, after the S1D2-S1D4 culture medium is fully preheated in a water bath pot at 37 ℃, the waste liquid supernatant in 12 holes is sucked, and 2mL of fresh and preheated S1D2-S1D4 culture medium is replaced.
Step 5: on the fifth day, induced differentiation was completed, the supernatant of the waste liquid in the 12 wells was aspirated, and 1mL of Actutase (EMD Millipore, cat#SCR 005) was added to digest for 3-5min.
Step 6: after cells in the well plate were digested into single cells, the single cells were collected in an EP tube, centrifuged at 300g for 3min, the supernatant of the digested solution was aspirated, 200. Mu.L of a fixative (BD Biosciences, cat #51-2090 KZ) was added, and incubated at 4℃for 30min in a refrigerator.
Step 7: after incubation for 30min, 300g of the centrifuge was centrifuged for 3min and the supernatant of the fixative was aspirated.
Step 8: one wash pass, add 200. Mu.L of wash buffer (BD Biosciences, cat#51-2091 KZ) to resuspend single cells.
Step 9: after centrifugation at 300g for 3min, the supernatant was aspirated and 200. Mu.L of wash buffer was added to resuspend single cells.
Step 10: according to the following steps of 1:200 antibodies PE anti-human FOXA2 (BD Biosciences, cat # 561589) and APC anti-human SOX17 (BD Biosciences, cat # 562594) were added and incubated in a refrigerator at 4℃for 2h.
Step 11: after incubation for 2h in a refrigerator at 4 ℃, washing for 2 times by a wash buffer (repeating the steps 8-9), and loading the solution into a BD FACS Calibur flow cytometer flow meter to detect the induction and targeting efficiency of the differentiation of the endoderm by the combination of small molecules such as SM15 supplement without animal sources, active A, CHIR-99021 and the like.
5. Experimental data results:
the culture status of hiPSC clones before plating is shown in fig. 1, the hiPSC clones are very compact, smooth in edge and clear in nuclear to cytoplasmic ratio.
hiPSC clones were plated in 12-well plates at 50-60 ten thousand cells per well after single cell digestion with Ackutase, and recorded after 24h by photographing, as shown in FIG. 2.
Multipotent stem cells hiPSC plated single cells induced differentiation of directional endoderm, recorded 4days after initiation of differentiation, as shown in fig. 3, it was seen that cells had grown to 100% confluence, and cells in the well plate were very compact, spread flat and free of heterogeneity.
After 4days of induced differentiation, actuase is digested into single cells, and the directional endoderm differentiation efficiency Marker is detected by a flow meter, and the obtained result is shown in figure 4, and the double positive efficiency of FOXA2/SOX17 is as high as 95%.

Claims (10)

1. An animal-free supplement characterized in that the animal-free supplement comprises the following components:
biotin, DL alpha-tocopheryl acetate, human recombinant insulin, human transferrin, corticosterone, D-galactose, ethanolamine hydrochloride, reduced glutathione, l-carnitine hydrochloride, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, and triiodothyronine.
2. An animal-free concentrate supplement made from the animal-free supplement of claim 1, which is a 100 x concentrate in MCDB131 medium as solvent, comprising the following concentrations of components:
200-350 mug/mL biotin, 80-150 mug/mL DL alpha-tocopherol acetate, 350-500 mug/mL human recombinant insulin, 400-600 mug/mL human transferrin, 1-5 mug/mL corticosterone, 1000-2000 mug/mLD-galactose, 80-150 mug/mL ethanolamine hydrochloride, 80-150 mug/mL reduced glutathione, 150-300 mug/mL L-carnitine hydrochloride, 80-150 mug/mL linoleic acid, 80-150 mug/mL linolenic acid, 0.5-0.8 mug/mL progesterone, 1500-2000 mug/mL putrescine, 1-2 mug/mL sodium selenite and 0.1-0.5 mug/mL triiodothyronine.
3. The animal-free concentrated supplement of claim 2, wherein the animal-free concentrated supplement is in MCDB131 medium as a solvent and comprises the following components in the following concentrations:
250 μg/mL biotin, 100 μg/mL DL alpha-tocopherol acetate, 400 μg/mL human recombinant insulin, 500 μg/mL human transferrin, 2 μg/mL corticosterone, 1500 μg/mLD-galactose, 100 μg/mL ethanolamine hydrochloride, 100 μg/mL reduced glutathione, 200 μg/mL left-handed carnitine hydrochloride, 100 μg/mL linoleic acid, 100 μg/mL linolenic acid, 0.63 μg/mL progesterone, 1610 μg/mL putrescine, 1.44 μg/mL sodium selenite, and 0.20 μg/mL triiodothyronine.
4. An animal-free medium for inducing differentiation of directional endoderm cells, characterized in that the animal-free medium comprises the animal-free supplement of claim 1.
5. The animal-derived free medium for inducing differentiated directional endoderm cells according to claim 4, wherein the animal-derived free medium for inducing differentiated directional endoderm cells comprises directional endoderm medium 1 and directional endoderm medium 2, and the directional endoderm medium 1 is a basal medium of MCDB131 medium, comprising the following components:
2 to 3.5 mug/mL biotin, 0.8 to 1.5 mug/mL DL alpha-tocopherol acetate, 3.5 to 5 mug/mL human recombinant insulin, 4 to 6 mug/mL human transferrin, 0.01 to 0.05 mug/mL corticosterone, 10 to 20 mug/mLD-galactose, 0.8 to 1.5 mug/mL ethanolamine hydrochloride, 0.8 to 1.5 mug/mL reduced glutathione, 1.5 to 3 mug/mL L-carnitine hydrochloride, 0.8 to 1.5 mug/mL linoleic acid, 0.8 to 1.5 mug/mL linolenic acid, 0.005 to 0.008 mug/mL progesterone, 15 to 20 mug/mL putrescine, 0.01 to 0.02 mug/mL sodium selenite and 0.001 to 0.005 mug/mL tri-iodothyronine;
1-2 mM glutamine, 50-100U/mL penicillin, 50-100 mu g/mL streptomycin, 3-5 mM glucose, 80-120 ng/mL TGF-beta activator, 0.2-0.3 mM vitamin C, 5-8 mu M Wnt signal pathway activator, 40-60 nM PI3K/mTOR inhibitor and 8-15 mu M ROCK inhibitor;
the directional endoderm culture medium 2 is based on MCDB131 culture medium and comprises the following components:
2 to 3.5 mug/mL biotin, 0.8 to 1.5 mug/mL DL alpha-tocopherol acetate, 3.5 to 5 mug/mL human recombinant insulin, 4 to 6 mug/mL human transferrin, 0.01 to 0.05 mug/mL corticosterone, 10 to 20 mug/mLD-galactose, 0.8 to 1.5 mug/mL ethanolamine hydrochloride, 0.8 to 1.5 mug/mL reduced glutathione, 1.5 to 3 mug/mL L-carnitine hydrochloride, 0.8 to 1.5 mug/mL linoleic acid, 0.8 to 1.5 mug/mL linolenic acid, 0.005 to 0.008 mug/mL progesterone, 15 to 20 mug/mL putrescine, 0.01 to 0.02 mug/mL sodium selenite and 0.001 to 0.005 mug/mL tri-iodothyronine;
1-2 mM glutamine, 50-100U/mL penicillin, 50-100 mug/mL streptomycin, 3-5 mM glucose, 40-60 ng/mL TGF-beta activator, 0.2-0.3 mM vitamin C.
6. An animal-derived free medium for inducing differentiation of directional endoderm cells according to claim 5, wherein said TGF- β activator is Activin a; the Wnt signal path activator is CHIR-99021; the PI3K/mTOR inhibitor is PI-103; the ROCK inhibitor is Y27632.
7. The animal-derived free medium for inducing differentiation of directional endoderm cells of claim 5, wherein the composition of directional endoderm medium 1 is: a basal medium based on MCDB131 medium; the following final concentrations of ingredients:
2.5. Mu.g/mL biotin, 1. Mu.g/mL DL alpha-tocopherol acetate, 4. Mu.g/mL human recombinant insulin, 5. Mu.g/mL human transferrin, 0.02. Mu.g/mL corticosterone, 15. Mu.g/mLD-galactose, 1. Mu.g/mL ethanolamine hydrochloride, 1. Mu.g/mL reduced glutathione, 2. Mu.g/mL left-handed carnitine hydrochloride, 1. Mu.g/mL linoleic acid, 1. Mu.g/mL linolenic acid, 0.0063. Mu.g/mL progesterone, 16.1. Mu.g/mL putrescine, 0.0144. Mu.g/mL sodium selenite, and 0.002. Mu.g/mL triiodothyronine;
2mM glutamine, 100U/mL penicillin, 100 μg/mL streptomycin, 4.5mM glucose, 100ng/mL Activin A, 0.25mM vitamin C, 6 μM CHIR-99021, 50nM PI-103 and 10 μMY27632;
the composition of the directional endoderm culture medium 2 is: a basal medium based on MCDB131 medium; the following final concentrations of ingredients:
2.5. Mu.g/mL biotin, 1. Mu.g/mL DL alpha-tocopherol acetate, 4. Mu.g/mL human recombinant insulin, 5. Mu.g/mL human transferrin, 0.02. Mu.g/mL corticosterone, 15. Mu.g/mLD-galactose, 1. Mu.g/mL ethanolamine hydrochloride, 1. Mu.g/mL reduced glutathione, 2. Mu.g/mL left-handed carnitine hydrochloride, 1. Mu.g/mL linoleic acid, 1. Mu.g/mL linolenic acid, 0.0063. Mu.g/mL progesterone, 16.1. Mu.g/mL putrescine, 0.0144. Mu.g/mL sodium selenite, and 0.002. Mu.g/mL triiodothyronine;
2mM glutamine, 100U/mL penicillin, 100 μg/mL streptomycin, 4.5mM glucose, 50ng/mL Activin A, 0.25mM vitamin C.
8. Use of the animal-derived supplement of claim 1 or the animal-derived medium of any one of claims 4 to 7 for inducing differentiation of directional endoderm cells.
9. A method for culturing pluripotent stem cells into directional endoderm cells by induction, which comprises culturing pluripotent stem cells into directional endoderm cells using the animal-derived medium for inducing differentiation of directional endoderm cells according to any one of claims 5 to 7.
10. The method according to claim 9, characterized in that the method is: the pluripotent stem cells are cultured for 1 day by using directional endoderm culture medium 1, then are cultured for 3 days by changing to directional endoderm culture medium 2, and are induced to differentiate to obtain the directional endoderm cells.
CN202311093054.2A 2023-08-29 2023-08-29 Animal-source-free supplement, culture medium and application thereof in inducing differentiation of directional endoderm cells Pending CN117165514A (en)

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