CN113383766A - Freezing storage liquid for stem cell freezing storage and preparation method thereof - Google Patents
Freezing storage liquid for stem cell freezing storage and preparation method thereof Download PDFInfo
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- CN113383766A CN113383766A CN202010171833.XA CN202010171833A CN113383766A CN 113383766 A CN113383766 A CN 113383766A CN 202010171833 A CN202010171833 A CN 202010171833A CN 113383766 A CN113383766 A CN 113383766A
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- cryopreservation
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- 210000000130 stem cell Anatomy 0.000 title claims abstract description 24
- 239000007788 liquid Substances 0.000 title claims abstract description 23
- 238000007710 freezing Methods 0.000 title claims abstract description 21
- 230000008014 freezing Effects 0.000 title claims abstract description 21
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 238000003860 storage Methods 0.000 title abstract description 6
- 238000005138 cryopreservation Methods 0.000 claims abstract description 85
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 75
- 150000001413 amino acids Chemical class 0.000 claims abstract description 63
- 239000000243 solution Substances 0.000 claims abstract description 61
- 239000004372 Polyvinyl alcohol Substances 0.000 claims abstract description 36
- 229920002451 polyvinyl alcohol Polymers 0.000 claims abstract description 36
- 239000000463 material Substances 0.000 claims abstract description 32
- 239000011664 nicotinic acid Substances 0.000 claims abstract description 29
- 210000002966 serum Anatomy 0.000 claims abstract description 23
- 239000007853 buffer solution Substances 0.000 claims abstract description 9
- 239000000203 mixture Substances 0.000 claims abstract description 3
- 229940024606 amino acid Drugs 0.000 claims description 50
- 235000001014 amino acid Nutrition 0.000 claims description 50
- 230000003993 interaction Effects 0.000 claims description 16
- 108090000765 processed proteins & peptides Chemical class 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 claims description 13
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 claims description 13
- 150000001875 compounds Chemical class 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 12
- 239000002253 acid Substances 0.000 claims description 11
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 claims description 10
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims description 9
- 229960002429 proline Drugs 0.000 claims description 9
- 238000006116 polymerization reaction Methods 0.000 claims description 8
- 229920000724 poly(L-arginine) polymer Polymers 0.000 claims description 7
- 239000004475 Arginine Substances 0.000 claims description 6
- PHOQVHQSTUBQQK-SQOUGZDYSA-N D-glucono-1,5-lactone Chemical compound OC[C@H]1OC(=O)[C@H](O)[C@@H](O)[C@@H]1O PHOQVHQSTUBQQK-SQOUGZDYSA-N 0.000 claims description 6
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 claims description 6
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 claims description 6
- 239000004473 Threonine Substances 0.000 claims description 6
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 6
- 229960003121 arginine Drugs 0.000 claims description 6
- DQJCDTNMLBYVAY-ZXXIYAEKSA-N (2S,5R,10R,13R)-16-{[(2R,3S,4R,5R)-3-{[(2S,3R,4R,5S,6R)-3-acetamido-4,5-dihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy}-5-(ethylamino)-6-hydroxy-2-(hydroxymethyl)oxan-4-yl]oxy}-5-(4-aminobutyl)-10-carbamoyl-2,13-dimethyl-4,7,12,15-tetraoxo-3,6,11,14-tetraazaheptadecan-1-oic acid Chemical class NCCCC[C@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)CC[C@H](C(N)=O)NC(=O)[C@@H](C)NC(=O)C(C)O[C@@H]1[C@@H](NCC)C(O)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 DQJCDTNMLBYVAY-ZXXIYAEKSA-N 0.000 claims description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims description 5
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 claims description 5
- 150000001720 carbohydrates Chemical class 0.000 claims description 5
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 5
- -1 GDL-L-Thr Chemical class 0.000 claims description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 4
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 claims description 4
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims description 4
- 235000003704 aspartic acid Nutrition 0.000 claims description 4
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 claims description 4
- 230000009881 electrostatic interaction Effects 0.000 claims description 4
- 239000012091 fetal bovine serum Substances 0.000 claims description 4
- 125000000524 functional group Chemical group 0.000 claims description 4
- 229910052739 hydrogen Inorganic materials 0.000 claims description 4
- 239000001257 hydrogen Substances 0.000 claims description 4
- 230000007062 hydrolysis Effects 0.000 claims description 4
- 238000006460 hydrolysis reaction Methods 0.000 claims description 4
- 230000002209 hydrophobic effect Effects 0.000 claims description 4
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 4
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- HYLXOQURIOCKIH-VQVTYTSYSA-N Thr-Arg Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CCCNC(N)=N HYLXOQURIOCKIH-VQVTYTSYSA-N 0.000 claims description 3
- QOLYAJSZHIJCTO-VQVTYTSYSA-N Thr-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(O)=O QOLYAJSZHIJCTO-VQVTYTSYSA-N 0.000 claims description 3
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 claims description 3
- 238000003760 magnetic stirring Methods 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- 239000004471 Glycine Substances 0.000 claims description 2
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 2
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 2
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 claims description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 claims description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 claims description 2
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 2
- 239000004472 Lysine Substances 0.000 claims description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 2
- 235000004279 alanine Nutrition 0.000 claims description 2
- 229960003767 alanine Drugs 0.000 claims description 2
- 125000003368 amide group Chemical group 0.000 claims description 2
- 229940098773 bovine serum albumin Drugs 0.000 claims description 2
- 235000014633 carbohydrates Nutrition 0.000 claims description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 2
- 125000002843 carboxylic acid group Chemical group 0.000 claims description 2
- 238000004113 cell culture Methods 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 2
- 229940124280 l-arginine Drugs 0.000 claims description 2
- 238000010907 mechanical stirring Methods 0.000 claims description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 claims description 2
- 125000000719 pyrrolidinyl group Chemical group 0.000 claims description 2
- 235000009697 arginine Nutrition 0.000 claims 3
- 235000008521 threonine Nutrition 0.000 claims 3
- 235000013930 proline Nutrition 0.000 claims 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims 1
- 125000003277 amino group Chemical group 0.000 claims 1
- 230000003592 biomimetic effect Effects 0.000 claims 1
- 235000013922 glutamic acid Nutrition 0.000 claims 1
- 239000004220 glutamic acid Substances 0.000 claims 1
- 235000014304 histidine Nutrition 0.000 claims 1
- 235000018977 lysine Nutrition 0.000 claims 1
- 229920001308 poly(aminoacid) Chemical class 0.000 claims 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 claims 1
- 210000004027 cell Anatomy 0.000 abstract description 11
- 230000004083 survival effect Effects 0.000 abstract description 8
- 210000000056 organ Anatomy 0.000 abstract description 4
- 210000001519 tissue Anatomy 0.000 abstract description 3
- 239000002994 raw material Substances 0.000 abstract description 2
- 231100000053 low toxicity Toxicity 0.000 abstract 1
- 238000000034 method Methods 0.000 description 11
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 9
- 210000003954 umbilical cord Anatomy 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000052 comparative effect Effects 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 235000012209 glucono delta-lactone Nutrition 0.000 description 3
- 239000003761 preservation solution Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- GVUVRRPYYDHHGK-VQVTYTSYSA-N Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 GVUVRRPYYDHHGK-VQVTYTSYSA-N 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 229960003681 gluconolactone Drugs 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 238000004017 vitrification Methods 0.000 description 2
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 230000032823 cell division Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 208000037887 cell injury Diseases 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 239000012595 freezing medium Substances 0.000 description 1
- 238000001415 gene therapy Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/125—Freeze protecting agents, e.g. cryoprotectants or osmolarity regulators
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/10—Preservation of living parts
- A01N1/12—Chemical aspects of preservation
- A01N1/122—Preservation or perfusion media
- A01N1/126—Physiologically active agents, e.g. antioxidants or nutrients
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dentistry (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a freezing storage solution for stem cell freezing storage and a preparation method thereof, wherein each 100mL of the solution contains: 0.1-50g of bionic ice control material, 0.1-15mL of DMSO, 0.1-30mL of serum and the balance of buffer solution. The cryopreservation liquid takes bionic ice control materials such as amino acids or polyvinyl alcohol and the like as main components, has low DMSO content, low toxicity and definite components, and can achieve the same or even higher cell survival rate as the existing cryopreservation liquid. The cryopreservation liquid has simple composition, convenient raw material source and low cost, and can be widely applied to cryopreservation of various cells, tissues or organs.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and particularly relates to a freezing medium for stem cell freezing preservation and a preparation method thereof.
Background
Cryopreservation refers to keeping the biological material at ultralow temperature to slow down or stop cell metabolism and division, and to continue to develop once normal physiological temperature is restored. Since the advent, this technique has become one of indispensable research methods in the field of natural science, and has been widely adopted. Stem cells are widely used in clinical treatment and regenerative medicine due to their functions of repairing tissue cell damage, replacing damaged cells, and stimulating the regeneration of body's own cells. For example, the human umbilical cord mesenchymal stem cells of the newborn fetus have wide clinical application prospect due to low immunogenicity, high cell content, strong proliferation capacity and the like; the cryopreservation of the human umbilical cord mesenchymal stem cells provides possibility for wide clinical application of the human umbilical cord mesenchymal stem cells. In addition, as the world population ages, the need for cryopreservation of donated human cells, tissues or organs available for regenerative medicine and organ transplantation is also increasing dramatically. Therefore, how to efficiently store precious cells, tissues and organ resources in a freezing way becomes a scientific and technical problem to be solved urgently.
The most common cryopreservation method currently used is vitrification freezing. The vitrification freezing technology can make the liquid inside and outside the cell become glass state directly in the fast freezing process, so as to avoid the damage caused by the formation of ice crystal in the freezing process. However, prior art cryopreservation reagents are not effective in controlling the growth of ice crystals during the rewarming process, thereby damaging the cells. Stem cells, such as mesenchymal stem cells, have the potential for self-renewal and multipotentiality and can be widely used in stem cell transplantation, autoimmune diseases or gene therapy vectors and the like. The prior cryopreservation reagent for stem cells generally comprises components such as a cryoprotectant, a stem cell growth medium and the like, and achieves the aims of supporting cells and reducing the formation of ice crystals. However, the growth medium easily causes the stem cells to be differentiated blindly, and the directional differentiation potential of the stem cells after recovery is influenced.
Disclosure of Invention
The present invention provides a cryopreservation solution, each 100mL of the solution containing: 0.1-50g of bionic ice control material, 0.1-15mL of DMSO, 0.1-30mL of serum and the balance of buffer solution.
According to the present invention, the cryopreservation liquid does not contain polyols and sugars.
According to the invention, the bionic ice control material is selected from at least one of polyvinyl alcohol or amino acid bionic ice control materials.
According to the present invention, the amount of polyvinyl alcohol contained in the cryopreservation solution per 100mL is preferably 0.1 to 6 g.
According to the invention, the PVA is chosen from isotactic PVA, syndiotactic PVA and atactic PVA in one or a combination of two or more thereof, for example the PVA has a syndiotacticity of from 15% to 65%, in particular for example from 40% to 60%, from 53% to 55%. Atactic PVA is preferred, for example PVA with a syndiotacticity of 45% to 65%.
According to the present invention, the PVA may be selected from the group consisting of PVA having a molecular weight of 10-500kDa or higher, for example, 10-30kDa, 30-50kDa, 80-90kDa, 200-500 kDa.
According to the invention, the PVA may be chosen from those having a degree of hydrolysis greater than 80%, for example a degree of hydrolysis of 80% to 99%, 82% to 87%, 87% to 89%, 89% to 99%, 98% to 99%.
According to the invention, the amino acid bionic ice control material is selected from one or more than two of amino acid, polyamino acid or peptide compounds.
As an embodiment of the invention, the amino acid bionic ice control material can be an amino acid containing an ice-philic group and a hydrophilic group, or a polyamino acid or peptide compound consisting of the amino acid containing the ice-philic group and the amino acid containing the hydrophilic group.
The hydrophilic group is a functional group capable of forming a non-covalent interaction with a water molecule, such as a hydrogen bond, van der waals interaction, electrostatic interaction, hydrophobic interaction, or pi-pi interaction with water; can be selected from hydroxyl (-OH), amino (-NH)2) Carboxylic acid group (-COOH), or amide group (-CONH)2) And the like;
the ice-philic group is a functional group that can form a non-covalent interaction with ice, such as a hydrogen bond with ice, van der waals interactions, electrostatic interactions, hydrophobic interactions, or pi-pi interactions; illustratively, the oxophilic group may be selected from the group consisting of hydroxyl (-OH), amino (-NH)2) Phenyl (-C)6H5) Or pyrrolidinyl (-C)4H8N).
Illustratively, the amino acid bionic ice control material is selected from one or two of arginine, threonine, proline, lysine, histidine, glutamine, aspartic acid, glycine and the like, or polyamino acid consisting of the amino acids; for example, the amino acid bionic ice control material is a combination of arginine and threonine.
In one embodiment of the invention, the amino acid bionic ice control material is polyamino acid (the polymerization degree is not less than 2), preferably the polymerization degree is 2-40 (such as the polymerization degree is 6, 8, 15, 20 and the like), and the polyamino acid is an amino acid homopolymer, such as one or a combination of more than two of poly-L-proline, poly-L-arginine and the like.
In an embodiment of the invention, the peptidic compound consists of an ice-philic amino acid, such as: threonine (L-Thr), glutamine (L-Gln), aspartic acid (L-Asn), etc., with other hydrophilic amino acids selected from arginine, proline, alanine, etc., or Gluconolactone (GDL) or other sugars.
The peptide compound is a peptide formed by more than two amino acid units, such as: 2-8 amino acid units, specifically 2, 3, 4, 5 amino acid units; each amino acid unit is different. The arrangement of the hydrophilic amino acid and the other hydrophilic amino acid in the peptide compound is not particularly limited, and the peptide compound can be connected by amino acid connecting groups or chemical bonds known in the art, for example, the hydrophilic amino acid and the hydrophilic amino acid can be arranged in a single interval, or a plurality of hydrophilic amino acids can be connected to form a hydrophilic amino acid fragment or a hydrophilic amino acid fragment, and then the hydrophilic amino acid fragment or the hydrophilic amino acid fragment is respectively connected with the hydrophilic amino acid (or fragment) and the hydrophilic amino acid (or fragment).
In an embodiment of the invention, the peptidic compound is a polypeptide, such as at least one of L-Thr-L-Arg (TR), L-Thr-L-Pro (TP), L-Arg-L-Thr (RT), L-Pro-L-Thr (PT), L-Thr-L-Arg-L-Thr (TRT), L-Thr-L-Pro-L-Thr (TPT), L-Ala-L-Ala-L-Thr (AAT), L-Thr-L-Cys-L-Thr (TCT). The polypeptides may be prepared by methods known in the art for polypeptide synthesis, for example by solid phase synthesis.
In another embodiment, the peptidic compound is a glycopeptide derivative, such as a gluconolactone or other glycopeptide derivative synthesized from carbohydrates and amino acids, such as GDL-L-Thr, GDL-L-Ser, and GDL-L-Val. The glycopeptide derivatives may be prepared according to art-known methods for the reaction of saccharides and amino acids, such as solid phase synthesis or by reacting saccharides and amino acids in an organic solvent.
According to the invention, the content of the amino acid bionic ice control material in each 100mL of the cryopreservation solution is 0.5-50g, preferably 1.0-35g, for example, when the amino acid bionic ice control material is amino acid, the content of the amino acid bionic ice control material can be 5.0-35g, preferably 15-25 g; when the amino acid bionic ice control material is polyamino acid, the content of the amino acid bionic ice control material can be 0.5-9.0g, and preferably 1.0-5.0 g.
According to the present invention, the buffer may be selected from at least one of DPBS or hepes-buffered HTF buffers or other cell culture buffers known in the art.
According to the invention, the serum can be selected from human serum albumin or a substitute thereof, such as sodium dodecyl sulfate, for human cryopreservation subjects, and fetal bovine serum or bovine serum albumin for non-human cryopreservation subjects.
According to the invention, the DMSO content is between 0.1 and 15mL, preferably between 1.0 and 10mL, more preferably between 1 and 7.5mL, for example between 1.5 and 5mL, per 100mL of cryopreservation solution.
According to the invention, the serum content is 0.1-30mL, such as 5.0-20mL, 10-15mL, per 100mL of cryopreservation solution.
According to the invention, the pH of the cryopreservation solution is from 6.5 to 7.6, for example from 6.9 to 7.2.
In one embodiment of the present invention, the cryopreservation solution comprises the following components per 100 mL:
0.5-50g amino acid
DMSO 0.1-10mL
0.1-30mL of serum
The balance of the buffer solution.
Preferably: the cryopreservation liquid comprises the following components in each 100 mL:
L-Arg 2.0-20g
L-Thr 1.0-10g
DMSO 0.1-10mL
serum 5.0-20mL
DPBS margin.
As one embodiment of the present invention, the cryopreservation solution contains the following components per 100mL volume:
0.5-9.0g of polyamino acid
DMSO 0.1-10mL
Serum 5.0-20mL
The balance of the buffer solution.
Preferably: the cryopreservation liquid comprises the following components in volume per 100 mL:
poly-L-proline or poly-L-arginine 1.0-8.0g
DMSO 0.1-10mL
Serum 5.0-20mL
DPBS margin.
As one embodiment of the present invention, the cryopreservation liquid comprises the following components per 100mL volume:
PVA 1.0-5.0g
DMSO 0.1-10mL
0.1-20mL of serum
The balance of the buffer solution.
Preferably: the cryopreservation liquid comprises the following components in volume per 100 mL:
PVA 1.0-4.0g
DMSO 4-10mL
10-20mL of serum
DPBS margin.
The invention also provides a preparation method of the cryopreservation liquid, which comprises the following steps:
(1) dissolving the bionic ice control material in a part of buffer solution, and adjusting the pH value to form a solution 1;
(2) dissolving DMSO in a part of buffer solution to prepare a solution 2;
(3) and (3) mixing the solution 1 and the solution 2 after cooling to room temperature, adjusting the pH value, and fixing the volume to a preset volume by using a buffer solution to obtain the frozen preservation solution.
According to the production method of the present invention, the serum is added when the cryopreservation solution is used.
According to the preparation method, when the bionic ice control material is polyvinyl alcohol, water bath heating is adopted and stirring is carried out; for example, the water bath temperature is 65-85 deg.C, 70-80 deg.C; the stirring is mechanical stirring such as magnetic stirring.
The invention also provides application of the cryopreservation liquid in stem cell cryopreservation. The stem cells can be cryopreserved using any stem cell freezing method known in the art, and in one embodiment, comprises adding a freezing fluid with stem cells into a cryopreservation tube, placing the cryopreservation tube in an isopropanol cryopreservation box, and freezing. In another embodiment, the cryopreservation solution described above is used for cryopreservation of stem cells using the microdroplet method.
Advantageous effects
The cryopreservation solution provided by the invention takes a polyvinyl alcohol or amino acid bionic ice control material as a main ice control component, the source is wide, the biocompatibility is good, the prepared cryopreservation reagent can be free of a cell culture medium, the components are determined, the composition is simple, and the higher cell survival rate can be maintained. The cryopreservation liquid provided by the invention is convenient in raw material source and low in cost, and can be widely applied to cryopreservation of various stem cells.
Detailed Description
The preparation method of the present invention will be described in further detail with reference to specific examples. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
The polymerization degree of the poly-L-proline adopted in the embodiment of the invention is 8 or 15, and the molecular weight is 795 or 1475; the polymerization degree of the poly-L-arginine is 8 or 15, and the molecular weight of the poly-L-arginine is 1267 or 2361.
In the embodiment of the invention, the survival rate is the average survival rate of 2-3 repeated experiments.
Examples
1. Preparing a cryopreservation solution: the following formula is adopted to prepare the cryopreservation liquid
The total volume of the cryopreservation solution A is 100mL, 2.0g of poly-L-arginine is ultrasonically dissolved in 25mL of DPBS, and the pH value is adjusted to 7.0 to obtain a solution 1; dissolving 10mL of DMSO in 20mL of DPBS to obtain solution 2, mixing the two solutions when the solution 1 and the solution 2 return to room temperature, adjusting the pH to 7.0, adding DPBS to a constant volume to make up the balance to 85% of the total volume, storing 15mL of serum separately, and adding the solution before use.
Cryopreservation liquid B: the total volume is 100mL, 2.0g of PVA is heated in a water bath at 80 ℃ and is dissolved in 25mL of DPBS by magnetic stirring, the pH value is adjusted to 7.0 after the PVA is completely dissolved and is cooled to the room temperature, 10mL of DMSO is dissolved in 25mL of DPBS to obtain a solution 2, the two solutions are mixed when the solution 1 and the solution 2 return to the room temperature, the pH value is adjusted, the constant volume is supplemented to 85% of the total volume, and 15mL of serum is separately stored and is added when a preservation solution is used.
Comparative example:
frozen preservation solution 1 #: each 1mL of the culture medium contained 10% (v/v) DMSO, 15% (v/v) fetal bovine serum, and the balance of alpha-MEM.
Application example:
cryopreservation of human umbilical cord mesenchymal stem cells was performed using the cryopreservation liquids of the above examples and comparative examples according to the protocol in table 1.
And (3) freezing and storing the human umbilical cord mesenchymal stem cells in a freezing and storing tube of 300 mu L:
the cryopreservation method of the human umbilical cord stem cells used by the invention specifically comprises the following steps: digesting human umbilical cord mesenchymal stem cells on a culture dish with 25% pancreatin for 2-3 minutes, then putting the cells into an equal volume of culture solution (10% FBS + a-MEM culture medium), gently blowing and beating until the stem cells are completely shed, centrifuging for 5 minutes at 1000r, discarding supernatant, adding 300 mu L of freezing solution into a centrifuge tube, gently blowing and beating to disperse stem cell clusters, adding the freezing solution with the stem cells into a 2ml cryopreservation tube, putting the cryopreservation tube into an isopropanol cryopreservation box, putting the cryopreservation tube into a refrigerator at-80 ℃, thawing after 24 hours, and staining trypan blue to check the survival rate (see table 1).
TABLE 1 human umbilical cord mesenchymal stem cell cryopreservation survival rate
| Numbering | Cryopreservation liquid | Survival rate |
| Application example 1 | A | 76.5％ |
| Application example 2 | B | 78.0％ |
| Comparative example 1 | Freezing fluid 1# | 76.4％ |
As can be seen from the data in Table 1, the cryopreservation liquid disclosed by the invention does not need to add a stem cell culture medium, so that the introduction of a culture medium with complex components is avoided, and only a bionic ice control material and a small amount of DMSO are added, so that the survival rate of stem cells can reach about 80% when the human umbilical cord mesenchymal stem cells are subjected to cryopreservation, which shows that the cryopreservation liquid has the effectiveness equivalent to that of the existing cryopreservation liquid with the culture medium added, and the components are simpler and more controllable, so that the cryopreservation liquid has a wide application prospect.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. 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 (10)
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