CN112029708A - Stem cell culture system - Google Patents
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- 210000000130 stem cell Anatomy 0.000 title claims abstract description 31
- 238000004113 cell culture Methods 0.000 title claims abstract description 18
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 66
- 239000000243 solution Substances 0.000 claims abstract description 57
- 239000007853 buffer solution Substances 0.000 claims abstract description 33
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 26
- 238000005406 washing Methods 0.000 claims abstract description 23
- 238000003756 stirring Methods 0.000 claims abstract description 22
- 239000012475 sodium chloride buffer Substances 0.000 claims abstract description 21
- 239000011159 matrix material Substances 0.000 claims abstract description 19
- 238000001914 filtration Methods 0.000 claims abstract description 17
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 16
- 239000004202 carbamide Substances 0.000 claims abstract description 16
- 239000006228 supernatant Substances 0.000 claims abstract description 12
- 238000005303 weighing Methods 0.000 claims abstract description 12
- 238000000502 dialysis Methods 0.000 claims abstract description 11
- 239000001963 growth medium Substances 0.000 claims abstract description 11
- 230000007774 longterm Effects 0.000 claims abstract description 9
- 238000004140 cleaning Methods 0.000 claims abstract description 6
- 238000005520 cutting process Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 210000002826 placenta Anatomy 0.000 claims abstract description 6
- 210000001778 pluripotent stem cell Anatomy 0.000 claims abstract description 6
- 239000002244 precipitate Substances 0.000 claims abstract description 6
- 239000000126 substance Substances 0.000 claims abstract description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 40
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 30
- 239000012498 ultrapure water Substances 0.000 claims description 30
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 25
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 20
- 239000003513 alkali Substances 0.000 claims description 15
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 10
- 102000057297 Pepsin A Human genes 0.000 claims description 10
- 108090000284 Pepsin A Proteins 0.000 claims description 10
- 229940111202 pepsin Drugs 0.000 claims description 10
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 7
- 102000004190 Enzymes Human genes 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 5
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 claims description 5
- 229940088598 enzyme Drugs 0.000 claims description 5
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 claims description 5
- 229940049954 penicillin Drugs 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 229960005322 streptomycin Drugs 0.000 claims description 5
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
- 235000020776 essential amino acid Nutrition 0.000 claims description 2
- 239000003797 essential amino acid Substances 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 15
- 230000000694 effects Effects 0.000 abstract description 7
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000032677 cell aging Effects 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
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Abstract
The invention provides a stem cell culture system, in particular to the technical field of cells, which comprises a humanized matrix and a humanized culture medium, wherein the humanized matrix and the humanized culture medium support long-term culture of human pluripotent stem cells; the humanized matrix was prepared as follows: s1, cutting the fresh placenta into blocks, cleaning twice, and filtering; s2, washing with cold 1M sodium chloride buffer solution, wherein the time for replacing clear water is 6-10 hours for one time, and washing is 6 times; s3, washing with cold 0.3M acetic acid solution for 3 days, changing the solution every 6 to 10 hours, and weighing after filtering when changing the 0.3M acetic acid solution for the ninth time; s4, centrifuging the weighed substances at the temperature of 8-12 ℃ for 1 hour, removing the supernatant, collecting the precipitate, weighing, adding 1.8M urea buffer solution according to the mass-volume ratio of 1: 1, and stirring at the temperature of 8-12 ℃ for 24 hours; s5, centrifuging for 30min at the temperature of 8-12 ℃, putting the supernatant into a dialysis bag with the temperature of 30KD, and dialyzing the liquid to obtain the humanized matrix. The invention can ensure the stability and activity of the cell state.
Description
Technical Field
The invention belongs to the technical field of cells, and particularly relates to a stem cell culture system.
Background
Stem cells are a class of cells that have unlimited or immortal self-renewal capacity, capable of producing at least one type of highly differentiated progeny cells. The definition of stem cells has been revised and defined from different levels over the years. Most biologists and physicians now believe that stem cells are a class of cells derived from embryos, fetuses, or adults that have the ability to self-renew and proliferate and differentiate without restriction under certain conditions, and that are capable of producing daughter cells with the same phenotype and genotype as themselves, and also capable of producing specialized cells that make up body tissues and organs, and also capable of differentiating into progenitor cells. Stem cell culture can promote the long-term growth of human stem cells, and can also maintain the multidirectional differentiation potential. The stem cell culture medium and the serum-free nutrient additive of the stem cells are used together, so that the cell adherence and proliferation effects are better; however, the existing stem cell culture system is easy to cause the phenomena of unstable state and cell aging of cells;
disclosure of Invention
The invention aims to provide a stem cell culture system which can ensure the stable state and activity of cells.
The invention provides the following technical scheme:
a stem cell culture system comprises a humanized matrix and a humanized culture medium which support the long-term culture of human pluripotent stem cells; the humanized matrix was prepared as follows:
s1, cutting fresh placenta into blocks, adding water, stirring for 6-10 hours at the temperature of 8-12 ℃, then screening, washing with clear water at the temperature of 8-12 ℃, replacing the clear water for 6-10 hours for one time and for two times, filtering for the second time, suspending the intercepted tissues with cold 1.5M sodium chloride buffer solution, and stirring at the temperature of 8-12 ℃;
s2, washing with cold 1M sodium chloride buffer solution, replacing the clear water for 6-10 hours once, washing for 6 times, filtering when replacing the sodium chloride buffer solution for 6 times, suspending the intercepted tissue with cold 0.3M acetic acid solution, and stirring at the temperature of 8-12 ℃;
s3, washing with cold 0.3M acetic acid solution for 3 days, changing the solution every 6 to 10 hours, and weighing after filtering when changing the 0.3M acetic acid solution for the ninth time;
s4, centrifuging the weighed substances at the temperature of 8-12 ℃ for 1 hour, removing the supernatant, collecting the precipitate, weighing, adding 1.8M urea buffer solution according to the mass-volume ratio of 1: 1, and stirring at the temperature of 8-12 ℃ for 24 hours;
s5, centrifuging for 30min at the temperature of 8-12 ℃, putting the supernatant into a dialysis bag with 30KD, dialyzing for 2 h at the temperature of 8-12 ℃ by taking triethanolamine buffer solution and 0.5% chloroform as surrounding solution, then thoroughly cleaning the outer surfaces of a beaker and the dialysis bag, continuing dialyzing at the temperature of 8-12 ℃ by taking cold triethanolamine buffer solution as surrounding solution, changing the triethanolamine buffer solution once every 2 h for 3 times, and finally dialyzing for 3 times at the temperature of 8-12 ℃ overnight to obtain dialyzed liquid, namely the humanized matrix;
preferably, the humanised medium contains the sodium chloride precipitated fraction of human plasma, 100U/m litre penicillin, 100. mu.g/m litre streptomycin, 10mM non-essential amino acid NEAA, 10mM mercaptoethanol.
Preferably, in step S1, the sodium chloride buffer is prepared as follows: 40 g of sodium chloride and 15 ml of tris (hydroxymethyl) aminomethane buffer are dissolved in Mi l of iQ water, p hours is adjusted to 7.0, and the volume is adjusted to 500 ml.
Preferably, in step S1, the buffer solution of tris (hydroxymethyl) aminomethane is prepared as follows: dissolving 200 g of tris (hydroxymethyl) aminomethane alkali in 600 ml of ultrapure water, adjusting p hours to 7.0, and adding ultrapure water to a constant volume of 1L.
Preferably, in step S3, pepsin is prepared as follows: pepsin was added at a tissue mass ratio of 1: 300, 200 g/l of the enzyme was suspended with the tissue mass in 0.2M acetic acid solution and stirred at 10 ℃ for 8 hours.
Preferably, in step S4, the urea buffer is prepared as follows: 240.0 g of urea, 12.1 g of tris (hydroxymethyl) aminomethane alkali and 18.0 g of sodium chloride are dissolved in 1.8L of ultrapure water, the concentration is carried out for C L, the p hour is adjusted to 7.4, and the volume of the ultrapure water is adjusted to 2L.
Preferably, in step S5, the triethanolamine buffer is prepared as follows: 15 g of tris (hydroxymethyl) aminomethane alkali and 20 g of sodium chloride are dissolved in 1.5L of ultrapure water, the concentration is carried out for C l, the p h is adjusted to 7.0, and the volume of the ultrapure water is adjusted to 2L.
The invention has the beneficial effects that:
the invention can ensure the stable state and activity of cells, promote the long-term growth of human stem cells and simultaneously maintain the multidirectional differentiation potential. The stem cell culture medium and the serum-free nutrient additive of the stem cells are used together, so that the cell adherence and proliferation effects are better.
Detailed Description
Example 1:
a stem cell culture system comprises a humanized matrix and a humanized culture medium which support the long-term culture of human pluripotent stem cells; humanized culture medium contains sodium chloride precipitated fraction of human plasma, 100U/m L penicillin, 100 μ g/m L streptomycin, 10mM nonessential amino acid NEAA, 10mM mercaptoethanol; the humanized matrix was prepared as follows:
s1, cutting fresh placenta into blocks, adding water, stirring at 8 ℃ for 6 hours, screening, washing with clear water at 8 ℃, replacing the clear water for 6 hours for one time and two times, filtering for the second time, suspending the intercepted tissue with cold 1.5M sodium chloride buffer solution, and stirring at 8 ℃; the preparation process of the sodium chloride buffer solution is as follows: dissolving 40 g of sodium chloride and 15m L of tris (hydroxymethyl) aminomethane buffer solution in Mi L of iQ water, adjusting p hours to 7.0, and fixing the volume to 500 ml; the buffer solution of tris (hydroxymethyl) aminomethane is prepared as follows: dissolving 200 g of tris (hydroxymethyl) aminomethane alkali in 600 ml of ultrapure water, adjusting p hours to 7.0, and adding ultrapure water to a constant volume of 1L;
s2, washing with cold 1M sodium chloride buffer solution, wherein the time for replacing clear water is 6 hours, washing is 6 times, filtering is carried out when the sodium chloride buffer solution is replaced for the 6 th time, and the intercepted tissue is suspended by cold 0.3M acetic acid solution and stirred at the temperature of 8 ℃;
s3, washing with cold 0.3M acetic acid solution for 3 days, changing the solution every 6 hours, and weighing after filtering when changing the 0.3M acetic acid solution for the ninth time; the preparation with pepsin was as follows: adding pepsin into the tissue at a mass ratio of 1: 300, suspending 200 g/L enzyme and tissue blocks in 0.2M acetic acid solution, and stirring at the temperature of 8 ℃ for 6 hours;
s4, centrifuging the weighed substances at 8 ℃ for 1 hour, removing supernatant, collecting precipitate, weighing, adding 1.8M urea buffer solution according to the mass-volume ratio of 1: 1, and stirring at 8 ℃ for 24 hours; the urea buffer was prepared as follows: 240.0 g of urea, 12.1 g of tris (hydroxymethyl) aminomethane alkali and 18.0 g of sodium chloride are dissolved in 1.8L of ultrapure water, the concentration is carried out for C L, the p hour is adjusted to 7.4, and the volume of the ultrapure water is constant to 2L
S5, centrifuging for 30min at the temperature of 8 ℃, putting the supernatant into a dialysis bag with 30KD, dialyzing for 2 hours at the temperature of 8 ℃ by taking triethanolamine buffer solution and 0.5% chloroform as surrounding solution, then thoroughly cleaning the outer surfaces of a beaker and the dialysis bag, dialyzing at the temperature of 8 ℃ continuously by taking cold triethanolamine buffer solution as surrounding solution, changing the triethanolamine buffer solution once every 2 hours for 3 times, and finally dialyzing at the temperature of 8 ℃ overnight to obtain dialyzed liquid, namely the humanized matrix; the triethanolamine buffer solution is prepared as follows: dissolving 15 g of tris (hydroxymethyl) aminomethane alkali and 20 g of sodium chloride in 1.5L of ultrapure water, adjusting the volume for p hours to 7.0 when the volume is concentrated for C liters, and fixing the volume to 2 liters by using the ultrapure water;
example 2:
a stem cell culture system comprises a humanized matrix and a humanized culture medium which support the long-term culture of human pluripotent stem cells; humanized culture medium contains sodium chloride precipitated fraction of human plasma, 100U/m L penicillin, 100 μ g/m L streptomycin, 10mM nonessential amino acid NEAA, 10mM mercaptoethanol; the humanized matrix was prepared as follows:
s1, cutting fresh placenta into blocks, adding water, stirring for 8 hours at 10 ℃, screening, washing with clear water at 10 ℃, replacing the clear water for 8 hours for one time and two times, filtering for the second time, suspending the intercepted tissue with cold 1.5M sodium chloride buffer solution, and stirring at 10 ℃; the preparation process of the sodium chloride buffer solution is as follows: dissolving 40 g of sodium chloride and 15m L of tris (hydroxymethyl) aminomethane buffer solution in Mi L of iQ water, adjusting p hours to 7.0, and fixing the volume to 500 ml; the buffer solution of tris (hydroxymethyl) aminomethane is prepared as follows: dissolving 200 g of tris (hydroxymethyl) aminomethane alkali in 600 ml of ultrapure water, adjusting p hours to 7.0, and adding ultrapure water to a constant volume of 1L;
s2, washing with cold 1M sodium chloride buffer solution, wherein the time for replacing clear water is 8 hours, washing is 6 times, filtering is carried out when the sodium chloride buffer solution is replaced for the 6 th time, and the intercepted tissue is suspended by cold 0.3M acetic acid solution and stirred at the temperature of 10 ℃;
s3, washing with cold 0.3M acetic acid solution for 3 days, changing the solution every 8 hours, and weighing after filtering when changing the 0.3M acetic acid solution for the ninth time; the preparation with pepsin was as follows: adding pepsin into the tissue at a mass ratio of 1: 300, suspending 200 g/L enzyme and tissue mass in 0.2M acetic acid solution, and stirring at 10 deg.C for 8 hr
S4, centrifuging the weighed substances at 10 ℃ for 1 hour, removing the supernatant, collecting the precipitate, weighing, adding 1.8M urea buffer solution according to the mass-volume ratio of 1: 1, and stirring at 10 ℃ for 24 hours; the urea buffer was prepared as follows: 240.0 g of urea, 12.1 g of tris (hydroxymethyl) aminomethane alkali and 18.0 g of sodium chloride are dissolved in 1.8L of ultrapure water, the concentration is carried out for C L, the p hour is adjusted to 7.4, and the volume of the ultrapure water is constant to 2L
S5, centrifuging for 30min at the temperature of 10 ℃, putting the supernatant into a dialysis bag with 30KD, dialyzing for 2 hours at the temperature of 10 ℃ by taking triethanolamine buffer solution and 0.5% chloroform as surrounding solution, then thoroughly cleaning the outer surfaces of a beaker and the dialysis bag, dialyzing at the temperature of 10 ℃ by taking cold triethanolamine buffer solution as surrounding solution, changing the triethanolamine buffer solution once every 2 hours for 3 times, and finally dialyzing at the temperature of 10 ℃ overnight to obtain dialyzed liquid, namely the humanized matrix; the triethanolamine buffer solution is prepared as follows: 15 g of tris (hydroxymethyl) aminomethane alkali and 20 g of sodium chloride are dissolved in 1.5L of ultrapure water, the concentration is carried out for C l, the p h is adjusted to 7.0, and the volume of the ultrapure water is adjusted to 2L.
Example 3:
a stem cell culture system comprises a humanized matrix and a humanized culture medium which support the long-term culture of human pluripotent stem cells; humanized culture medium contains sodium chloride precipitated fraction of human plasma, 100U/m L penicillin, 100 μ g/m L streptomycin, 10mM nonessential amino acid NEAA, 10mM mercaptoethanol; the humanized matrix was prepared as follows:
s1, cutting fresh placenta into blocks, adding water, stirring for 10 hours at the temperature of 12 ℃, then screening, washing with clear water at the temperature of 12 ℃, replacing the clear water for 10 hours for one time and for two times, filtering for the second time, suspending the intercepted tissues with cold 1.5M sodium chloride buffer solution, and stirring at the temperature of 12 ℃; the preparation process of the sodium chloride buffer solution is as follows: dissolving 40 g of sodium chloride and 15m L of tris (hydroxymethyl) aminomethane buffer solution in Mi L of iQ water, adjusting p hours to 7.0, and fixing the volume to 500 ml; the buffer solution of tris (hydroxymethyl) aminomethane is prepared as follows: dissolving 200 g of tris (hydroxymethyl) aminomethane alkali in 600 ml of ultrapure water, adjusting p hours to 7.0, and adding ultrapure water to a constant volume of 1L;
s2, washing with cold 1M sodium chloride buffer solution, wherein the time for changing clear water is 10 hours, washing is 6 times, filtering is carried out when the sodium chloride buffer solution is changed for the 6 th time, the intercepted tissue is suspended by cold 0.3M acetic acid solution, and stirring is carried out at the temperature of 12 ℃;
s3, washing with cold 0.3M acetic acid solution for 3 days, changing the solution every 10 hours, and weighing after filtering when changing the 0.3M acetic acid solution for the ninth time; the preparation with pepsin was as follows: adding pepsin into the tissue at a mass ratio of 1: 300, suspending 200 g/L enzyme and tissue blocks in 0.2M acetic acid solution, and stirring at 12 ℃ for 10 hours;
s4, centrifuging the weighed substances at the temperature of 12 ℃ for 1 hour, removing supernatant, collecting precipitates, weighing, adding 1.8M urea buffer solution according to the mass-volume ratio of 1: 1, and stirring at the temperature of 12 ℃ for 24 hours; the urea buffer was prepared as follows: 240.0 g of urea, 12.1 g of tris (hydroxymethyl) aminomethane alkali and 18.0 g of sodium chloride are dissolved in 1.8L of ultrapure water, the concentration is carried out for C L, the p hour is adjusted to 7.4, and the volume of the ultrapure water is constant to 2L
S5, centrifuging for 30min at the temperature of 12 ℃, putting the supernatant into a dialysis bag with 30KD, dialyzing for 2 hours at the temperature of 12 ℃ by taking triethanolamine buffer solution and 0.5% chloroform as surrounding solution, then thoroughly cleaning the outer surfaces of the beaker and the dialysis bag, dialyzing at the temperature of 12 ℃ by taking cold triethanolamine buffer solution as surrounding solution, changing the triethanolamine buffer solution once every 2 hours for 3 times, and finally dialyzing for 3 times at the temperature of 12 ℃ overnight, wherein the dialyzed liquid is the humanized matrix; the triethanolamine buffer solution is prepared as follows: dissolving 15 g of tris (hydroxymethyl) aminomethane alkali and 20 g of sodium chloride in 1.5L of ultrapure water, adjusting the volume for p hours to 7.0 when the volume is concentrated for C liters, and fixing the volume to 2 liters by using the ultrapure water;
the invention can ensure the stable state and activity of cells, promote the long-term growth of human stem cells and simultaneously maintain the multidirectional differentiation potential. The stem cell culture medium and the serum-free nutrient additive of the stem cells are used together, so that the cell adherence and proliferation effects are better.
Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will understand that various changes, modifications and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A stem cell culture system, comprising: the culture medium comprises a humanized matrix and a humanized culture medium which support the long-term culture of the human pluripotent stem cells; a humanised matrix prepared by the steps of:
s1, cutting fresh placenta into blocks, adding water, stirring for 6-10 hours at the temperature of 8-12 ℃, then screening, washing with clear water at the temperature of 8-12 ℃, replacing the clear water for 6-10 hours for one time and for two times, filtering for the second time, suspending the intercepted tissues with cold 1.5M sodium chloride buffer solution, and stirring at the temperature of 8-12 ℃;
s2, washing with cold 1M sodium chloride buffer solution, replacing the clear water for 6-10 hours once, washing for 6 times, filtering when replacing the sodium chloride buffer solution for 6 times, suspending the intercepted tissue with cold 0.3M acetic acid solution, and stirring at the temperature of 8-12 ℃;
s3, washing with cold 0.3M acetic acid solution for 3 days, changing the solution every 6 to 10 hours, and weighing after filtering when changing the 0.3M acetic acid solution for the ninth time;
s4, centrifuging the weighed substances at the temperature of 8-12 ℃ for 1 hour, removing the supernatant, collecting the precipitate, weighing, adding 1.8M urea buffer solution according to the mass-volume ratio of 1: 1, and stirring at the temperature of 8-12 ℃ for 24 hours;
s5, centrifuging for 30min at the temperature of 8-12 ℃, putting the supernatant into a dialysis bag with 30KD, dialyzing for 2 h at the temperature of 8-12 ℃ by taking triethanolamine buffer solution and 0.5% chloroform as surrounding solution, then thoroughly cleaning the outer surfaces of the beaker and the dialysis bag, continuing dialyzing at the temperature of 8-12 ℃ by taking cold triethanolamine buffer solution as surrounding solution, changing the triethanolamine buffer solution once every 2 h for 3 times, and finally dialyzing for 3 times at the temperature of 8-12 ℃ overnight to obtain dialyzed liquid, namely the humanized matrix.
2. A stem cell culture system according to claim 1, wherein: the humanised medium contained the sodium chloride precipitated fraction of human plasma, 100U/m litre penicillin, 100. mu.g/m litre streptomycin, 10mM non-essential amino acid NEAA, 10mM mercaptoethanol.
3. A stem cell culture system according to claim 2, wherein: in step S1, the process of preparing the sodium chloride buffer is as follows: 40 g of sodium chloride and 15 ml of tris (hydroxymethyl) aminomethane buffer are dissolved in Mi l of iQ water, p hours is adjusted to 7.0, and the volume is adjusted to 500 ml.
4. A stem cell culture system according to claim 3, wherein: the buffer solution of tris (hydroxymethyl) aminomethane is prepared as follows: dissolving 200 g of tris (hydroxymethyl) aminomethane alkali in 600 ml of ultrapure water, adjusting p hours to 7.0, and adding ultrapure water to a constant volume of 1L.
5. A stem cell culture system according to claim 4, wherein: the preparation with pepsin was as follows: pepsin was added at a tissue mass ratio of 1: 300, 200 g/l enzyme and tissue mass were suspended in 0.2M acetic acid solution and stirred at 8 to 12 ℃ for 6 to 10 hours.
6. A stem cell culture system according to claim 5, wherein: the urea buffer was prepared as follows: 240.0 g of urea, 12.1 g of tris (hydroxymethyl) aminomethane alkali and 18.0 g of sodium chloride are dissolved in 1.8L of ultrapure water, the concentration is carried out for C L, the p hour is adjusted to 7.4, and the volume of the ultrapure water is adjusted to 2L.
7. A stem cell culture system according to claim 5, wherein: the triethanolamine buffer solution is prepared as follows: 15 g of tris (hydroxymethyl) aminomethane alkali and 20 g of sodium chloride are dissolved in 1.5L of ultrapure water, the concentration is carried out for C l, the p h is adjusted to 7.0, and the volume of the ultrapure water is adjusted to 2L.
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| CN101044235A (en) * | 2004-09-08 | 2007-09-26 | 威斯康星校友研究基金会 | Culturing human embryonic stem cells |
| CN102586176A (en) * | 2012-01-11 | 2012-07-18 | 中国科学院生物物理研究所 | Novel animal source-free and feed layer-free human pluripotent stem cell culture system |
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| CN101044235A (en) * | 2004-09-08 | 2007-09-26 | 威斯康星校友研究基金会 | Culturing human embryonic stem cells |
| CN102586176A (en) * | 2012-01-11 | 2012-07-18 | 中国科学院生物物理研究所 | Novel animal source-free and feed layer-free human pluripotent stem cell culture system |
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