CN117210398A - Stem cell washing liquid and use method thereof - Google Patents
Stem cell washing liquid and use method thereof Download PDFInfo
- Publication number
- CN117210398A CN117210398A CN202311176481.7A CN202311176481A CN117210398A CN 117210398 A CN117210398 A CN 117210398A CN 202311176481 A CN202311176481 A CN 202311176481A CN 117210398 A CN117210398 A CN 117210398A
- Authority
- CN
- China
- Prior art keywords
- stem cells
- stem cell
- washing liquid
- umbilical cord
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000005406 washing Methods 0.000 title claims abstract description 85
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 83
- 239000007788 liquid Substances 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 21
- 229920001184 polypeptide Polymers 0.000 claims abstract description 24
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 24
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 24
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims abstract description 22
- 210000003056 antler Anatomy 0.000 claims abstract description 22
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 22
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 22
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 18
- 239000008103 glucose Substances 0.000 claims abstract description 18
- 238000004113 cell culture Methods 0.000 claims abstract description 11
- 230000012010 growth Effects 0.000 claims abstract description 9
- 108091005804 Peptidases Proteins 0.000 claims abstract description 8
- 239000004365 Protease Substances 0.000 claims abstract description 5
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims abstract description 5
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 58
- 210000003954 umbilical cord Anatomy 0.000 claims description 55
- 239000000243 solution Substances 0.000 claims description 28
- 239000006285 cell suspension Substances 0.000 claims description 17
- 239000000644 isotonic solution Substances 0.000 claims description 12
- 239000006143 cell culture medium Substances 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 7
- 238000004115 adherent culture Methods 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 6
- 102000004142 Trypsin Human genes 0.000 claims description 5
- 108090000631 Trypsin Proteins 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000009210 therapy by ultrasound Methods 0.000 claims description 5
- 239000012588 trypsin Substances 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000000872 buffer Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 102000035195 Peptidases Human genes 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 claims description 3
- 239000008280 blood Substances 0.000 claims description 3
- 239000008213 purified water Substances 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 2
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 50
- 230000006907 apoptotic process Effects 0.000 abstract description 14
- 230000000694 effects Effects 0.000 abstract description 14
- 238000000338 in vitro Methods 0.000 abstract description 12
- 239000003102 growth factor Substances 0.000 abstract description 9
- 230000008439 repair process Effects 0.000 abstract description 9
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- ZIIUUSVHCHPIQD-UHFFFAOYSA-N 2,4,6-trimethyl-N-[3-(trifluoromethyl)phenyl]benzenesulfonamide Chemical compound CC1=CC(C)=CC(C)=C1S(=O)(=O)NC1=CC=CC(C(F)(F)F)=C1 ZIIUUSVHCHPIQD-UHFFFAOYSA-N 0.000 abstract description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 3
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 abstract description 3
- 102000015439 Phospholipases Human genes 0.000 abstract description 3
- 108010064785 Phospholipases Proteins 0.000 abstract description 3
- 229910052799 carbon Inorganic materials 0.000 abstract description 3
- 230000035699 permeability Effects 0.000 abstract description 3
- 230000026731 phosphorylation Effects 0.000 abstract description 3
- 238000006366 phosphorylation reaction Methods 0.000 abstract description 3
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 abstract description 3
- 102000001253 Protein Kinase Human genes 0.000 abstract description 2
- 239000003226 mitogen Substances 0.000 abstract description 2
- 108060006633 protein kinase Proteins 0.000 abstract description 2
- 230000019491 signal transduction Effects 0.000 abstract 2
- 239000002953 phosphate buffered saline Substances 0.000 description 15
- 239000007853 buffer solution Substances 0.000 description 10
- 230000000052 comparative effect Effects 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 7
- 230000003833 cell viability Effects 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 4
- 230000008054 signal transmission Effects 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 230000035899 viability Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 230000002900 effect on cell Effects 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- XJRBAMWJDBPFIM-UHFFFAOYSA-N methyl vinyl ether Chemical compound COC=C XJRBAMWJDBPFIM-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 210000002894 multi-fate stem cell Anatomy 0.000 description 2
- 239000008188 pellet Substances 0.000 description 2
- 210000005059 placental tissue Anatomy 0.000 description 2
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 206010009900 Colitis ulcerative Diseases 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000009329 Graft vs Host Disease Diseases 0.000 description 1
- 102000043136 MAP kinase family Human genes 0.000 description 1
- 108091054455 MAP kinase family Proteins 0.000 description 1
- FZWLAAWBMGSTSO-UHFFFAOYSA-N Thiazole Chemical compound C1=CSC=N1 FZWLAAWBMGSTSO-UHFFFAOYSA-N 0.000 description 1
- 201000006704 Ulcerative Colitis Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 210000004413 cardiac myocyte Anatomy 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003412 degenerative effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 210000001723 extracellular space Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000004700 fetal blood Anatomy 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 208000024908 graft versus host disease Diseases 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000027866 inflammatory disease Diseases 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 210000005229 liver cell Anatomy 0.000 description 1
- 230000001050 lubricating effect Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000010899 nucleation Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000010298 pulverizing process Methods 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000010008 shearing Methods 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000012192 staining solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000002137 ultrasound extraction Methods 0.000 description 1
Abstract
The application provides a stem cell washing liquid and a use method thereof. Hyaluronic acid has high permeability, and can promote tyrosine phosphorylation, activate phospholipase PLGamma 1, activate protease signal transduction pathway and mitogen to activate protein kinase signal transduction system, repair damaged cells, and enhance cell activity. Glucose can provide energy, carbon source, growth factors and the like for the culture of stem cells and promote the in-vitro growth of the stem cells. The pilose antler polypeptide contains various growth factors, can promote human body to secrete various growth factors, repair damaged cells, strengthen cell activity, reduce apoptosis, play a better role in the subsequent cell culture process, and promote in-vitro culture effect. The stem cell washing liquid provided by the application can repair damaged cells in the cell washing process, enhance the cell activity, reduce the apoptosis and is beneficial to the expansion culture of stem cells in vitro.
Description
Technical Field
The application belongs to the technical field of stem cells, and relates to a stem cell washing liquid and a use method thereof.
Background
Mesenchymal stem cells are multipotent stem cells having self-renewal and multipotent differentiation capabilities, and are mainly present in various tissues such as bone marrow, umbilical cord blood, umbilical cord tissue, placenta tissue, skeletal muscle, etc. In recent years, mesenchymal stem cells have attracted public and academic attention due to their unique biological properties and potential therapeutic value. More and more data prove that the mesenchymal stem cells can play an immunoregulatory function under the regulation of inflammatory environment, so that the mesenchymal stem cells can be used for treating a wide range of degenerative and inflammatory diseases, such as ulcerative colitis, arthritis, systemic lupus erythematosus, graft-versus-host disease and other diseases.
The umbilical cord mesenchymal stem cells (Human umbilical cord mesenchymal stem cell, abbreviated as hUC-MSC) are multipotent stem cells which can induce differentiation into various tissue cells such as liver cells, cardiac muscle cells, nerve cells and the like under specific in-vivo and in-vitro conditions, and are mainly derived from umbilical cord and placenta tissues of newborns. Umbilical cord mesenchymal stem cells are subjected to separation subculture, and are frozen at a deep low temperature in a gas-phase liquid nitrogen tank at-196 ℃ and MVE (English full name: methyl Vinyl Ether; chinese name: vinyl methyl ether). Compared with adult stem cells, umbilical mesenchymal stem cells are more original, have stronger proliferation capacity and differentiation capacity, are purer due to protection of a barrier of a fetal disc, have low immunogenicity, do not need to be matched, are not limited by race, and are used for chemotactically repairing body injury through a homing mechanism, so that the safety is high, and the disputes in the aspects of society, ethics and law are not related. The umbilical cord mesenchymal stem cells have the name of universal cells, have unique immunoregulation function, have huge medical clinical application value, and are the optimal seed cells for cell therapy.
The number of stem cells collected in an organism that can be used for transplantation therapy is very small, so that the collected umbilical cord mesenchymal stem cells are generally subjected to expansion culture in vitro. In the process of subculture, cells are mostly washed by using normal saline or PBS (English name: phosphate buffered saline; chinese name: phosphate buffer solution) buffer solution, but the two solutions only have buffer effect and have no repair effect on cells. In the multiple washing process, the umbilical cord mesenchymal stem cells are influenced by suspension and centrifugal shearing force, and the cell death occurs in a certain proportion, so that the number of the umbilical cord mesenchymal stem cells is greatly reduced, and the activity of the cells in the subsequent culture process is also greatly reduced. In order to perform high quality regenerative medicine, it is important to provide a sufficient number of stem cells while providing high quality stem cells, i.e., stem cells/stem cell populations with high viability and high proportion of viable cells, and therefore it is urgent to develop a stem cell washing solution.
Disclosure of Invention
The application aims to provide a stem cell washing liquid and a using method thereof, so as to solve the problem that the existing washing liquid has no repairing effect on cells.
In order to achieve the above purpose, the present application adopts the following technical scheme:
the application provides a stem cell washing liquid, which comprises an isotonic solution containing hyaluronic acid, glucose and pilose antler polypeptide. Wherein the isotonic solution is PBS buffer solution or physiological saline. Preferably, the isotonic solution is PBS buffer. The stem cell washing liquid is mainly used for a cell washing process.
Hyaluronic acid is also called hyaluronic acid, and plays roles of preserving water, maintaining extracellular space, regulating osmotic pressure, lubricating and promoting cell repair in vivo. In the application, the molecular weight of the hyaluronic acid is 40 ten thousand, and the hyaluronic acid of the molecular section has high permeability, can promote tyrosine phosphorylation, activate phospholipase PLC gamma 1, thereby activating a protease signal transmission path and a mitogen-activated protein kinase signal transmission system, repairing damaged cells and enhancing cell viability.
Glucose is a nutrient source and can provide energy, carbon source, growth factors and the like for the culture of stem cells so as to promote the in-vitro growth of the stem cells.
The pilose antler polypeptide contains various growth factors, can promote human body to secrete various growth factors, repair damaged cells, strengthen cell activity, reduce apoptosis, play a better role in the subsequent cell culture process, and promote in-vitro culture effect.
In the application, the pilose antler polypeptide is extracted by adopting ultrasonic extraction biochemical technology, and the specific extraction method is as follows:
s01: peeling fresh cornu Cervi Pantotrichum, slicing, and washing with purified water at-4deg.C until no blood color is present.
Peeling fresh cornu Cervi Pantotrichum, and slicing into slices with thickness of about 1 cm. The mixture was rinsed several times with purified water at-4℃until there was no blood color.
S02: adding 50% ethanol solution into cornu Cervi Pantotrichum until the cornu Cervi Pantotrichum is 5-10cm, and performing ultrasonic treatment for 24 hr.
Adding 50% ethanol solution into sliced cornu Cervi Pantotrichum until the cornu Cervi Pantotrichum is 5-10cm. Placing cornu Cervi Pantotrichum into which alcohol solution is added into an ultrasonic instrument, and performing ultrasonic treatment at 20deg.C for 24 hr.
S03: after the ultrasonic treatment is finished, filtering, concentrating at the temperature of below 80 ℃, drying and crushing to obtain the pilose antler polypeptide.
After the sonication was completed, the filtrate was filtered and concentrated in a rotary evaporator. In order to prevent the denaturation of the proteins, the rotary evaporation temperature was chosen below 80 ℃. Concentrating the filtrate, and drying in a drying oven at 80deg.C until the water is completely evaporated. Pulverizing the dried material with pulverizer, and sieving with 80 mesh sieve to obtain cornu Cervi Pantotrichum polypeptide. The cornu Cervi Pantotrichum polypeptide is preserved at-20deg.C.
In the application, the addition amount of hyaluronic acid, glucose and pilose antler polypeptide is 0.1-3wt% of hyaluronic acid, 0.1-2wt% of glucose and 0.5-50 mug/mL of pilose antler polypeptide respectively, and the balance is isotonic solution. More preferably, the addition amounts of hyaluronic acid, glucose and pilose antler polypeptide are 0.1wt% of hyaluronic acid, 0.5wt% of glucose and 30 mug/mL of pilose antler polypeptide respectively, and the balance is isotonic solution. When the stem cell washing liquid provided by the application is prepared, hyaluronic acid, glucose, pilose antler polypeptide and isotonic solution are weighed according to the proportion and then uniformly mixed, so that the stem cell washing liquid is obtained.
In addition, the application also provides a using method of the stem cell washing liquid, which comprises the following steps:
s01: and taking out the umbilical cord mesenchymal stem cells subjected to the adherent culture, removing the cell culture medium, and washing by using a stem cell washing liquid.
And taking out the umbilical cord mesenchymal stem cells subjected to the adherent culture, and removing a cell culture medium, wherein the cell culture medium is a special culture medium for stem cells commonly used in the field. After removal of the medium, the stem cell washing liquid provided by the application is used for washing to remove the residual cell culture medium. The umbilical cord mesenchymal stem cells in the application are umbilical cord mesenchymal stem cells in the logarithmic phase of P3-P5 generation growth.
S02: umbilical cord mesenchymal stem cells are peeled from the cell culture flask using a proteolytic enzyme and suspended in a stem cell washing solution to obtain a stem cell suspension.
Umbilical cord mesenchymal stem cells are treated with a proteolytic enzyme to detach the umbilical cord mesenchymal stem cells from the cell culture flask. The protease of the present application is trypsin. Suspending the stripped umbilical cord mesenchymal stem cells in the stem cell washing liquid provided by the application to obtain a stem cell suspension.
S03: and after the stem cell suspension is centrifuged, the stem cell is resuspended by using a special culture medium for stem cells, so as to obtain a resuspension.
The stem cell suspension was centrifuged at 1000r/min for 5min to remove the cell suspension solution. After removing the stem cell suspension and centrifuging, the cells are resuspended in a medium specific for stem cells to obtain a resuspension.
S04: the resuspension was incubated at 37℃with 5% CO 2 Culturing under culture conditions to obtain umbilical cord mesenchymal stem cells.
Inoculating the resuspension in cell culture flask at 37deg.C with 5% CO 2 Culturing under culture conditions to obtain umbilical cord mesenchymal stem cells.
The application has the following beneficial effects:
the application provides a stem cell washing liquid and a use method thereof. Wherein, the hyaluronic acid has high permeability, can promote tyrosine phosphorylation, activate phospholipase PLCgamma 1, thereby activating protease signal transmission path and mitogen to activate protein kinase signal transmission system, repairing damaged cells, and enhancing cell activity. Glucose can provide the stem cells with the energy, carbon source, growth factors, etc. required to promote stem cell growth in vitro. The pilose antler polypeptide contains various growth factors, can promote human body to secrete various growth factors, repair damaged cells, strengthen cell activity, reduce apoptosis, play a better role in the subsequent cell culture process, and promote in-vitro culture effect. Therefore, the stem cell washing liquid provided by the application can repair damaged cells in the cell washing process, enhance the cell activity, reduce the apoptosis and is beneficial to the expansion culture of stem cells in vitro.
Drawings
FIG. 1 is a graph showing the comparison result of apoptosis rate of umbilical cord mesenchymal stem cells treated with different washing solutions in the embodiment of the application;
FIG. 2 is a graph showing the comparison of the viability of umbilical cord mesenchymal stem cells after treatment with different washing solutions according to the present application;
FIG. 3 is a graph showing the results of comparing the viability of umbilical cord mesenchymal stem cells after treatment with different washing solutions according to the embodiment of the present application.
Detailed Description
The technical scheme of the application is further explained and illustrated by specific examples.
Example 1
The embodiment of the application provides a stem cell washing liquid, which comprises 0.1wt% of hyaluronic acid, 0.5wt% of glucose, 30 mug/mL of pilose antler polypeptide and PBS buffer solution, wherein the rest of the washing liquid is complemented by the PBS buffer solution. The stem cell wash is labeled wash a.
The using method of the stem cell washing liquid provided by the embodiment of the application comprises the following steps:
s01: and taking out the umbilical cord mesenchymal stem cells in the logarithmic phase of P3-P5 generation growth of the adherent culture, and removing the cell culture medium. Washing was performed using washing liquid a to remove residual cell culture medium.
S02: umbilical cord mesenchymal stem cells are treated with trypsin to detach umbilical cord mesenchymal stem cells from a cell culture flask. Suspending the stripped umbilical cord mesenchymal stem cells in the washing liquid A to obtain a stem cell suspension.
S03: the stem cell suspension was centrifuged at 1000r/min for 5min to remove the cell suspension solution. After removing the stem cell suspension and centrifuging, the cells are resuspended in a medium specific for stem cells to obtain a resuspension.
S04: inoculating the resuspension in cell culture flask at 37deg.C with 5% CO 2 Culturing under culture conditions to obtain umbilical cord mesenchymal stem cells.
Example 2
The embodiment of the application provides a stem cell washing liquid, which comprises 3wt% of hyaluronic acid, 0.1wt% of glucose, 0.5 mug/mL of pilose antler polypeptide and PBS buffer solution, wherein the rest of the washing liquid is complemented by the PBS buffer solution. The stem cell wash is labeled wash a.
The method for using the stem cell washing liquid provided by the embodiment of the application is the same as that of embodiment 1, and will not be described here again.
Example 3
The embodiment of the application provides a stem cell washing liquid, which comprises 1wt% of hyaluronic acid, 2wt% of glucose, 50 mug/mL of pilose antler polypeptide and PBS buffer solution, wherein the rest of the mass is complemented by the PBS buffer solution. The stem cell wash is labeled wash a.
The method for using the stem cell washing liquid provided by the embodiment of the application is the same as that of embodiment 1, and will not be described here again.
Example 4
The embodiment of the application provides a stem cell washing liquid, which comprises 2wt% of hyaluronic acid, 1wt% of glucose, 10 mug/mL of pilose antler polypeptide and PBS buffer solution, wherein the rest of the mass is complemented by the PBS buffer solution. The stem cell wash is labeled wash a.
The method for using the stem cell washing liquid provided by the embodiment of the application is the same as that of embodiment 1, and will not be described here again.
The stem cell washing liquid (washing liquid a) prepared in example 1, the stem cell washing liquid provided in comparative example 1, and the stem cell washing liquid provided in comparative example 2 according to the present application were used to culture umbilical cord mesenchymal stem cells, and the apoptosis rate, the cell survival rate, and the cell viability were measured, respectively, and are described in detail below. Wherein, comparative example 1 is sodium chloride solution with concentration of 0.9%, and is marked as washing liquid B; comparative example 2 is PBS buffer, labeled wash C.
1. Apoptosis rate and cell survival rate detection of umbilical cord mesenchymal stem cells
The umbilical cord mesenchymal stem cells in the logarithmic phase of P3-P5 generation growth are cultured by adopting different detergents, and the culture method comprises the following steps:
s01: and taking out the umbilical cord mesenchymal stem cells in the logarithmic phase of P3-P5 generation growth of the adherent culture, and removing the cell culture medium. Washing was performed with washing liquid a, washing liquid B, and washing liquid C, respectively, to remove the residual cell culture medium.
S02: umbilical cord mesenchymal stem cells are treated with trypsin to detach umbilical cord mesenchymal stem cells from a cell culture flask. And suspending the stripped umbilical cord mesenchymal stem cells in a washing liquid A, a washing liquid B and a washing liquid C respectively to obtain different stem cell suspensions.
S03: the stem cell suspension was centrifuged at 1000r/min for 5min and the supernatant was removed. Then 1mL of 1 Xbuffer was added to resuspend the cells, and the cells were centrifuged at 1000r/min for 5min to remove the supernatant. Cell pellet was obtained after repeated cell resuspension once. To the cell pellet, 100. Mu.L of 1 Xbuffer was added to suspend the cells, and 5. Mu.L of Annexin V and PI staining solution were added to each sample, and after mixing, the mixture was incubated at room temperature for 20 minutes in the dark.
S04: after the incubation, 100. Mu.L of 1 Xbuffer was added to suspend the cells, and the cell suspension was passed through a 100 mesh screen and then added to a flow tube, followed immediately by detection by a flow cytometer, and the experiment was repeated 3 times to obtain the results shown in FIGS. 1 and 2.
As can be seen from fig. 1 and 2, in the process of cell washing, the apoptosis rate of umbilical cord mesenchymal stem cells washed by the washing solution a provided in example 1 is about 6%, and the cell survival rate is 94%; the apoptosis rate of umbilical cord mesenchymal stem cells washed by the washing liquid B provided in the comparative example 1 is about 14%, and the cell survival rate is 86%; the apoptosis rate of umbilical cord mesenchymal stem cells washed by the washing solution C provided in comparative example 2 was about 12%, and the cell viability was 88%.
It can be seen that the umbilical cord mesenchymal stem cells washed by the washing solution a provided in example 1 have a lower apoptosis rate and a higher cell survival rate compared with the washing solutions B and C of comparative examples 1 and 2. This shows that, under the condition of using PBS isotonic solution as base, the damaged cells can be repaired after hyaluronic acid, glucose and pilose antler polypeptide are added, so that apoptosis is obviously reduced, and cell survival rate is improved.
2. Cell viability detection of umbilical cord mesenchymal stem cells
Culturing umbilical cord mesenchymal stem cells in the logarithmic phase of P3-P5 generation growth by adopting different detergents, and detecting the cell viability of the umbilical cord mesenchymal stem cells by adopting an MTT method. The specific operation is as follows:
s01: and taking out the umbilical cord mesenchymal stem cells in the logarithmic phase of P3-P5 generation growth of the adherent culture, and removing the cell culture medium. Washing was performed with washing liquid a, washing liquid B, and washing liquid C, respectively, to remove the residual cell culture medium.
S02: umbilical cord mesenchymal stem cells are treated with trypsin to detach umbilical cord mesenchymal stem cells from a cell culture flask. And suspending the stripped umbilical cord mesenchymal stem cells in a washing liquid A, a washing liquid B and a washing liquid C respectively to obtain different stem cell suspensions.
S03: the stem cell suspension was centrifuged at 1000r/min for 5min and the supernatant was removed. Adding culture medium to blow the cells, and dispersing the stem cells uniformly.
S04: according to 10 3 -10 4 Seeding ratio of individual cells/well stem cells were seeded into 96-well plates at 37℃with 5% CO 2 Culturing under culture conditions for 3 days.
S05: after the completion of the culture, 20. Mu.L of MTT (English name: 3- (4, 5-Dimethyl-2-Thiazolyl) -2,5-Diphenyl Tetrazolium Bromide; chinese name: thiazole blue) solution was added to each well, and incubation was continued for 4 hours.
S06: after the incubation was completed, 150. Mu.L of DMSO (English name: dimethyl sulfoxide; chinese name: dimethyl sulfoxide) solution was added to each well after the culture solution in the wells was aspirated, and the 96-well plate was shaken on a microplate shaker at room temperature for 10min to dissolve the crystals.
S07: under the condition of 570nm wavelength, putting the 96-well plate on an enzyme-labeled instrument to detect the OD (optical density) value of each well, wherein only the OD value of the culture medium well is added as a control group, recording the detection result, and repeating each group of experiments for 3 times to obtain the result shown in the figure 3.
As can be seen from fig. 3, the OD value of umbilical cord mesenchymal stem cells washed by the washing solution a provided in example 1 was about 1.2 during the cell washing process; the OD value of the umbilical cord mesenchymal stem cells washed by the washing liquid B provided in the comparative example 1 is about 0.9; the OD value of the umbilical cord mesenchymal stem cells washed with the washing solution C provided in comparative example 2 was about 0.8.
It can be seen that the washing solution A of example 1 provided the washing solution A had the highest OD value and the corresponding umbilical cord mesenchymal stem cells had the highest cell viability, as compared with the washing solutions B and C of comparative examples 1 and 2. The method shows that under the condition of taking PBS isotonic solution as a base, damaged cells can be repaired after hyaluronic acid, glucose and pilose antler polypeptide are added, the cell activity is obviously enhanced, the stem cells are maintained to have better biological activity, and the method is more beneficial to the expansion culture of the stem cells in vitro.
In conclusion, the stem cell washing liquid provided by the embodiment of the application can repair damaged cells in the cell washing process, enhance the cell activity, reduce the apoptosis and is beneficial to the expansion culture of stem cells in vitro.
The above description is only of the preferred embodiments of the present application and is not intended to limit the present application, but various modifications and variations can be made to the present application by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the protection scope of the present application.
Claims (9)
1. A stem cell washing solution, which is characterized by comprising an isotonic solution containing hyaluronic acid, glucose and pilose antler polypeptide.
2. The stem cell washing liquid according to claim 1, comprising an isotonic solution containing 0.1-3wt% of hyaluronic acid, 0.1-2wt% of glucose and 0.5-50 μg/mL of hairy antler polypeptide.
3. The stem cell washing solution of claim 1, comprising an isotonic solution containing 0.1 wt.% hyaluronic acid, 0.5 wt.% glucose and 30 μg/mL of pilose antler polypeptide.
4. The stem cell washing liquid of claim 1, wherein the pilose antler polypeptide is prepared by the following method:
peeling and slicing fresh cornu Cervi Pantotrichum, and washing with purified water at-4deg.C until no blood color exists;
adding 50% ethanol solution into cornu Cervi Pantotrichum until the cornu Cervi Pantotrichum is 5-10cm, and performing ultrasonic treatment for 24 hr;
after the ultrasonic treatment is finished, filtering, concentrating at the temperature of below 80 ℃, drying and crushing to obtain the pilose antler polypeptide.
5. The stem cell washing liquid of claim 1, wherein the hyaluronic acid has a molecular weight of 40 ten thousand.
6. The stem cell wash solution of any one of claims 1-5, wherein the isotonic solution is PBS buffer or physiological saline.
7. A method of using a stem cell wash comprising:
taking out the umbilical cord mesenchymal stem cells subjected to the adherent culture, and washing the umbilical cord mesenchymal stem cells with the stem cell washing liquid according to any one of claims 1 to 6 after removing the cell culture medium;
stripping umbilical cord mesenchymal stem cells from a cell culture bottle by using protease, and suspending in a stem cell washing liquid to obtain a stem cell suspension;
after the stem cell suspension is centrifuged, the stem cell is resuspended by a special culture medium for stem cells to obtain a resuspension;
the resuspension was incubated at 37℃with 5% CO 2 Culturing under culture conditions to obtain umbilical cord mesenchymal stem cells.
8. The method of claim 7, wherein the umbilical cord mesenchymal stem cells are P3-P5 generation growth log phase umbilical cord mesenchymal stem cells.
9. The method of claim 7, wherein the proteolytic enzyme is trypsin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311176481.7A CN117210398A (en) | 2023-09-13 | 2023-09-13 | Stem cell washing liquid and use method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311176481.7A CN117210398A (en) | 2023-09-13 | 2023-09-13 | Stem cell washing liquid and use method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117210398A true CN117210398A (en) | 2023-12-12 |
Family
ID=89038285
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311176481.7A Pending CN117210398A (en) | 2023-09-13 | 2023-09-13 | Stem cell washing liquid and use method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117210398A (en) |
-
2023
- 2023-09-13 CN CN202311176481.7A patent/CN117210398A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105765060B (en) | Method for producing amniotic mesenchymal cell composition, cryopreservation method, and therapeutic agent | |
| US7122371B1 (en) | Modular cell culture bioreactor | |
| CN102660497B (en) | Method for freezing and reviving umbilical cord tissues and for separating and increasing stem cells | |
| CN106754674B (en) | The method and its application of amnion mesenchymal stem cell are prepared from Human plactnta amnion | |
| CN109234229B (en) | Method for separating mesenchymal stem cells from placental blood vessels and digestive enzyme composition used in same | |
| WO2007024441A2 (en) | Compositions of cells enriched for combinations of various stem and progenitor cell populations, methods of use thereof and methods of private banking thereof | |
| CN108456657B (en) | Canine umbilical cord mesenchymal stem cells and preparation method and cryopreservation method thereof | |
| WO2022217878A1 (en) | Preparation method and recovery method for pariduval mesenchymal stem cells | |
| US20150231244A1 (en) | Regenerative cell and adipose-derived stem cell processing system and method | |
| TW201510223A (en) | Method for separating living cell and constructing cell bank by means of tissue homogenate method | |
| Bianco et al. | Characterization of a novel decellularized bone marrow scaffold as an inductive environment for hematopoietic stem cells | |
| Romanov et al. | Optimized protocol for isolation of multipotent mesenchymal stromal cells from human umbilical cord | |
| CN109628388B (en) | Isolation of mesenchymal stem cells from placental blood vessels with digestive enzyme composition | |
| CN108728408B (en) | Canine fetal membrane mesenchymal stem cell, preparation method and culture medium used by same | |
| CN110964693A (en) | Separation method of umbilical cord mesenchymal stem cells | |
| CN104630135B (en) | The extensive method and purposes for preparing liver stem cells | |
| CN113322231A (en) | Method for separating and culturing mesenchymal stem cells and preparation | |
| CN114703120B (en) | Separation method of animal nervous system vascular smooth muscle cell single cells | |
| CN117210398A (en) | Stem cell washing liquid and use method thereof | |
| CN111961640A (en) | Construction method and culture system of three-dimensional differentiation model of urinary renal stem cells | |
| JP6721504B2 (en) | Process for producing pluripotent stem and progenitor cells | |
| RU2645255C1 (en) | Method for obtaining of biosafe culture of mesenchimal stem cells from human chorionic villae | |
| CN108795853B (en) | Method for preparing canine fetal membrane mesenchymal stem cells and canine fetal membrane mesenchymal stem cells | |
| CN112852727A (en) | Culture method of human umbilical cord-derived Muse cells | |
| TWI742399B (en) | A method for the isolation and the expansion of adipose tissue-derived stromal cells using non-enzymatic pre-treatment procedures |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |