CN112342192A - Continuous tangential flow stem cell separation and purification method - Google Patents
Continuous tangential flow stem cell separation and purification method Download PDFInfo
- Publication number
- CN112342192A CN112342192A CN202011165699.9A CN202011165699A CN112342192A CN 112342192 A CN112342192 A CN 112342192A CN 202011165699 A CN202011165699 A CN 202011165699A CN 112342192 A CN112342192 A CN 112342192A
- Authority
- CN
- China
- Prior art keywords
- serum
- liquid
- tangential flow
- concentration
- free complete
- 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.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims abstract description 34
- 210000000130 stem cell Anatomy 0.000 title claims abstract description 26
- 238000000746 purification Methods 0.000 title claims abstract description 19
- 238000000926 separation method Methods 0.000 title claims abstract description 16
- 239000007788 liquid Substances 0.000 claims abstract description 90
- 239000001963 growth medium Substances 0.000 claims abstract description 45
- 238000005406 washing Methods 0.000 claims abstract description 43
- 210000004027 cell Anatomy 0.000 claims abstract description 35
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 24
- 230000029087 digestion Effects 0.000 claims abstract description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 15
- 239000002699 waste material Substances 0.000 claims abstract description 15
- 108010019160 Pancreatin Proteins 0.000 claims abstract description 10
- 229940055695 pancreatin Drugs 0.000 claims abstract description 10
- 238000004140 cleaning Methods 0.000 claims abstract description 6
- 238000010992 reflux Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 51
- 239000000463 material Substances 0.000 claims description 23
- 239000002609 medium Substances 0.000 claims description 15
- 239000000654 additive Substances 0.000 claims description 10
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 8
- 230000000996 additive effect Effects 0.000 claims description 7
- 230000004907 flux Effects 0.000 claims description 7
- 238000003860 storage Methods 0.000 claims description 7
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 claims description 5
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 5
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 5
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims description 5
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 claims description 5
- 102000008100 Human Serum Albumin Human genes 0.000 claims description 5
- 108091006905 Human Serum Albumin Proteins 0.000 claims description 5
- 229940116978 human epidermal growth factor Drugs 0.000 claims description 5
- 229960000890 hydrocortisone Drugs 0.000 claims description 5
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 claims description 5
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 claims description 5
- 230000001079 digestive effect Effects 0.000 claims description 4
- 238000002955 isolation Methods 0.000 claims description 4
- 238000002347 injection Methods 0.000 claims description 2
- 239000007924 injection Substances 0.000 claims description 2
- 230000005540 biological transmission Effects 0.000 claims 3
- 239000007640 basal medium Substances 0.000 claims 2
- 239000012679 serum free medium Substances 0.000 claims 1
- 239000012466 permeate Substances 0.000 abstract description 17
- 238000012258 culturing Methods 0.000 abstract description 6
- 238000011084 recovery Methods 0.000 abstract description 6
- 230000004083 survival effect Effects 0.000 abstract description 3
- 238000003756 stirring Methods 0.000 description 22
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 22
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 16
- 239000011324 bead Substances 0.000 description 16
- 235000010410 calcium alginate Nutrition 0.000 description 16
- 239000000648 calcium alginate Substances 0.000 description 16
- 229960002681 calcium alginate Drugs 0.000 description 16
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=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](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 16
- 239000000499 gel Substances 0.000 description 15
- 239000008213 purified water Substances 0.000 description 12
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 8
- 239000011259 mixed solution Substances 0.000 description 8
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 6
- 230000000052 comparative effect Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 235000010413 sodium alginate Nutrition 0.000 description 6
- 239000000661 sodium alginate Substances 0.000 description 6
- 229940005550 sodium alginate Drugs 0.000 description 6
- 230000012010 growth Effects 0.000 description 5
- 239000002504 physiological saline solution Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 108010010803 Gelatin Proteins 0.000 description 4
- 239000004471 Glycine Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000011248 coating agent Substances 0.000 description 4
- 238000000576 coating method Methods 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 239000008273 gelatin Substances 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 239000012510 hollow fiber Substances 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- 230000005855 radiation Effects 0.000 description 4
- 230000003833 cell viability Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000002791 soaking Methods 0.000 description 3
- 230000001502 supplementing effect Effects 0.000 description 3
- GUTLYIVDDKVIGB-OUBTZVSYSA-N Cobalt-60 Chemical compound [60Co] GUTLYIVDDKVIGB-OUBTZVSYSA-N 0.000 description 2
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 210000004504 adult stem cell Anatomy 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 210000003995 blood forming stem cell Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000013043 cell viability test Methods 0.000 description 1
- 210000003701 histiocyte Anatomy 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000003094 microcapsule Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 210000003954 umbilical cord Anatomy 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J3/00—Processes of treating or compounding macromolecular substances
- C08J3/12—Powdering or granulating
- C08J3/126—Polymer particles coated by polymer, e.g. core shell structures
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2305/00—Characterised by the use of polysaccharides or of their derivatives not provided for in groups C08J2301/00 or C08J2303/00
- C08J2305/04—Alginic acid; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2489/00—Characterised by the use of proteins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/44—Thiols, e.g. mercaptoethanol
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/38—Hormones with nuclear receptors
- C12N2501/39—Steroid hormones
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N2531/00—Microcarriers
Abstract
The invention provides a continuous tangential flow stem cell separation and purification method, which mainly comprises the following steps: s1: placing mesenchymal stem cells into a reactor, adding a microcarrier and a serum-free complete culture medium for culturing for 7 days, cleaning with normal saline, adding pancreatin for digestion for 30min, backfilling the serum-free complete culture medium to stop digestion, and preparing a pretreatment solution; s2: placing the pretreatment liquid in a liquid feeding bottle, connecting the liquid feeding bottle to a TFF system, connecting a permeate collecting bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the liquid feeding bottle, adding an equal amount of washing liquid into the liquid feeding bottle when the permeate collecting bottle collects a certain volume of waste liquid, emptying the permeate collecting bottle after the washing liquid is added, and stopping the system to obtain mesenchymal stem cells when the feed liquid in the liquid feeding bottle is left in a predicted volume; the cell recovery rate obtained by the continuous tangential flow stem cell separation and purification method provided by the invention is more than 80%, and the cell survival rate can reach more than 85%.
Description
Technical Field
The invention belongs to the technical field of stem cells, and particularly relates to a continuous tangential flow stem cell separation and purification method.
Background
Mesenchymal Stem cells (Mesenchymal Stem cells) are adult Stem cells with self-replicating renewal capacity and multidirectional differentiation potential, and can be differentiated into various histiocytes such as fat, cartilage, bone and the like under in vitro specific induction conditions. Its functions include immunosuppression, promoting the repair of body tissues and organs, promoting angiogenesis/regeneration, and supporting the proliferation and differentiation of hemopoietic stem cells. The umbilical cord derived mesenchymal stem cells are convenient to obtain materials, have no moral and ethical disputes, have a large number of cells and are fast in proliferation, and easy to passage, amplification and culture. In order to meet clinical requirements, mesenchymal stem cells need to be cultured in vitro in large scale. The collection of cells after culture is also an important part of obtaining stem cells for therapeutic use. The traditional centrifugation method needs manual operation and large-scale centrifugation equipment for collecting cells, and has the disadvantages of complicated process, long time consumption and pollution risk.
Disclosure of Invention
In order to solve the technical problems, the invention provides a continuous tangential flow stem cell separation and purification method.
The specific technical scheme of the invention is as follows:
the invention provides a continuous tangential flow stem cell separation and purification method, which mainly comprises the following steps:
s1: placing the mesenchymal stem cells into a reactor, adding a microcarrier and a serum-free complete culture medium for culture, cleaning with normal saline, adding pancreatin for digestion, backfilling the serum-free complete culture medium to stop digestion, and preparing a pretreatment solution;
s2: placing the pretreatment liquid in a liquid feeding bottle, connecting the liquid feeding bottle to a TFF system, connecting a permeate collecting bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the liquid feeding bottle, adding an equal amount of washing liquid into the liquid feeding bottle when the permeate collecting bottle collects a certain volume of waste liquid, emptying the permeate collecting bottle after the washing liquid is added, and stopping the system to obtain mesenchymal stem cells when the feed liquid in the liquid feeding bottle is left in a predicted volume;
wherein the incubation time with the addition of microcarriers and serum-free complete medium is preferably 7 days and the time for pancreatin digestion is preferably 30min, but other incubation times or digestion times are within the scope of the invention, and the same amount of wash solution can be added when a volume of 1/4-1/2 pretreatment solution waste is collected in the permeate collection bottle, the expected volume being 1/5-1/15 of the volume of waste emptied from the permeate collection bottle.
The method disclosed by the invention is used for separating and purifying the mesenchymal stem cells, the recovery rate of the cells can reach 80%, and the cell survival rate can reach 85%. And collecting the concentrated mesenchymal stem cells, adding a fresh culture medium for continuous culture, and observing the growth state of the cells on the 2 nd day. The cells adhere well and are in a slender fiber shape. The concentration, washing and filtering process has no influence on the activity and growth and proliferation capacity of cells, the microcarrier is honeycomb-shaped, the diameter is 0.5-10mm, the pore diameter of the microcapsule is 10-10000 nm, and the preparation method of the microcarrier comprises the following steps:
the preparation method of the microcarrier comprises the following steps:
(1) preparing balls: dripping 5L of 1% nanocrystallized sodium alginate solution into 0.5% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 3;
(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises adding purified water, stirring at 50rpm for 5min, discarding washing solution to remove redundant liquid, and the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(3) gelatin coating: uniformly mixing 10L of 0.05% gelatin solution and 10mL of 0.3% glutaraldehyde solution to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 30min at the temperature of 20 ℃ and the stirring speed of 50rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1: 1;
(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture at the rotating speed of 50rpm for 5min, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(5) neutralizing: adding 0.5% glycine solution, neutralizing for 2h at 40 deg.C and stirring speed of 50rpm, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);
(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture at a rotating speed of 50rpm for 5min, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(7) coupling DEAE-HCl: adding 1mol/L NaOH solution into the washed calcium alginate micro-gel beads, stirring, and adding 0.5mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:1: 1; the specific conditions of the two times of stirring are as follows: the stirring speed is 50rpm, the stirring temperature is 60 ℃, and the stirring time is 0.5 h;
(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free2+And Mg2+Washing with PBS buffer solution (5 min) at 50rpm, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free solution2+、Mg2+The volume ratio of the PBS buffer solution to the calcium alginate micro-gel beads is 1:1, and a wet microcarrier is prepared;
(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8), and performing radiation sterilization by using cobalt 60 gamma rays, wherein the radiation dose is 20KGy, so as to obtain the microcarrier.
Further, serum-free complete culture medium is added into the bioreactor, then the mesenchymal stem cells and the microcarriers are inoculated into the bioreactor together,the density of the mesenchymal stem cells in a serum-free complete culture medium is 0.5-1x1010The density of the microcarrier in a serum-free complete culture medium is 4-6g/L, the microcarrier is cultured for 7 days, the temperature is controlled to be 37 ℃, the concentration of CO2 is 5%, the rotating speed of a bioreactor is 4-6rpm, the serum-free complete culture medium is respectively supplemented in days 2, 4 and 6, the volume ratio of the supplemented volume to the first serum-free complete culture medium is 1-3:3, 1-3:3 and 2-4:3, the culture solution is extracted into a liquid storage bottle in day 8, the microcarrier is cleaned by physiological saline for 5-20 minutes, the volume of the physiological saline is larger than the volume of the serum-free culture medium, pancreatin digestion cells are added after the physiological saline is removed, the temperature is set to be 37 ℃ during digestion, the rotating speed of the bioreactor is 4-6rpm, the digestion time is 20-40 minutes, the serum-free complete culture medium is refilled to terminate digestion, the ratio of the volume of the backfilled serum-free complete culture medium to the volume of the serum-free complete culture medium added for the first time is 1-3:2-4, and then the pretreatment solution is obtained.
Wherein, the step of washing the microcarrier with normal saline refers to the step of immersing the microcarrier with normal saline for 5-20 minutes, and pancreatin needs to be ensured to submerge all carriers during digestion.
Further, the washing solution is serum-free complete medium or normal saline.
Further, the washing solution in step S2 is serum-free complete medium or normal saline, and the washing time is 10-20 minutes.
Further, the serum-free complete medium comprises a basic medium and additives, wherein the basic medium is DMEM/F12 medium; the additive is human fibronectin with the concentration of 20-30 mug/ml, basic fibroblast growth factor with the concentration of 5-15ng/ml, human epidermal growth factor with the concentration of 5-15ng/ml, ITS with the concentration of 0.5-1.5% and human serum albumin with the concentration of 4-6%; NEAA with the concentration of 0.5 to 1.5 percent, hydrocortisone with the concentration of 0.05 to 0.15 mu mol/L and beta-mercaptoethanol with the concentration of 0.05 to 0.15 percent.
Further, the flow rate of the sample injection of the TFF system is 90-160 ml/min.
Further, the inlet end pressure of the TFF system is below 8 psi.
Further, the shear force of the TFF system is 1100-1800s-1。
Further, the flux of the TFF system is 15-22L/h/m2。
Further, the pump speed of the TFF system was 110-.
Further, the filtration time of the TFF system is 15-20 min.
The cell recovery rate of the cells obtained by the continuous tangential flow stem cell separation and purification method provided by the invention is more than 80%, the cell survival rate can reach more than 85%, the concentrated mesenchymal stem cells are collected, a fresh culture medium is added for continuous culture, and the cell growth state is observed on day 2. The cells adhere well and are in a slender fiber shape. The concentration and washing process has no influence on the activity and growth and proliferation capacity of the cells.
Drawings
FIG. 1 is a microscopic image of cells purified by the method of example 1 after culturing in a medium.
Detailed Description
Example 1
Example 1 provides a continuous tangential flow stem cell isolation and purification method, which comprises the following steps:
s1: 2x1010Inoculating the mesenchymal stem cells and 15g of microcarrier into a bioreactor, adding a serum-free complete culture medium to 3L, culturing for 7 days, controlling the temperature to be 37 ℃, the concentration of CO2 to be 5 percent and the rotating speed of the bioreactor to be 5rpm, respectively supplementing 2L, 2L and 3L of serum-free complete culture medium on the days 2, 4 and 6, extracting the culture solution into a liquid storage bottle on the day 8, soaking and cleaning the microcarrier for 10 minutes by using physiological saline, removing the physiological saline, adding pancreatin digestive cells, setting the temperature to be 37 ℃ during digestion, setting the rotating speed of the bioreactor to be 5rpm, digesting for 30 minutes, backfilling the culture solution in the 2L liquid storage bottle, and stopping digestion to obtain a pretreatment solution;
s2: placing the pretreatment liquid in a material liquid bottle, connecting the material liquid bottle to a TFF system, connecting a permeate collection bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the material liquid bottle, adding 5L of serum-free complete culture medium into the material liquid bottle when the permeate collection bottle collects 5L of waste liquid, emptying the permeate collection bottle (namely emptying 5L of waste liquid) after the serum-free complete culture medium is completely added, and stopping the system when 500ml of feed liquid in the material liquid bottle is left, so as to obtain mesenchymal stem cells;
the preparation method of the microcarrier comprises the following steps:
(1) preparing balls: dripping 5L of 1% nanocrystallized sodium alginate solution into 0.5% calcium chloride solution to react to generate calcium alginate gel beads, wherein the volume ratio of the nanocrystallized sodium alginate solution to the calcium chloride solution is 1: 3;
(2) washing: removing redundant calcium chloride solution, washing with sterile purified water once, wherein the washing method comprises adding purified water, stirring at 50rpm for 5min, discarding washing solution to remove redundant liquid, and the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(3) gelatin coating: uniformly mixing 10L of 0.05% gelatin solution and 10mL of 0.3% glutaraldehyde solution to obtain a mixed solution, soaking calcium alginate micro-gel beads in the mixed solution, and reacting for 30min at the temperature of 20 ℃ and the stirring speed of 50rpm, wherein the volume ratio of the mixed solution to the nano sodium alginate solution is 1: 1;
(4) washing: after the coating reaction is finished, removing redundant mixed liquid, washing the mixture for three times by using sterile purified water, adding purified water, stirring the mixture at the rotating speed of 50rpm for 5min, and removing the redundant liquid by using the washing liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(5) neutralizing: adding 0.5% glycine solution, neutralizing for 2h at 40 deg.C and stirring speed of 50rpm, wherein the volume of glycine solution is the same as that of the mixed solution in step (3);
(6) washing: discarding the waste liquid after the reaction, washing the waste liquid for three times by using sterile purified water, adding the purified water, stirring the mixture at a rotating speed of 50rpm for 5min, discarding the washing liquid to remove redundant liquid, wherein the volume ratio of the purified water to the calcium alginate micro-gel beads is 1: 1;
(7) coupling DEAE-HCl: adding 1mol/L NaOH solution into the washed calcium alginate micro-gel beads, stirring, and adding 0.5mol/L DEAE-HCl solution, and stirring; the ratio of the volume of the calcium alginate micro-gel beads to the volume of the NaOH solution and the volume of the DEAE-HCl solution is 1:1: 1; the specific conditions of the two times of stirring are as follows: the stirring speed is 50rpm, the stirring temperature is 60 ℃, and the stirring time is 0.5 h;
(8) washing: removing excess NaOH solution and DEAE-HCl solution, sequentially using sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free2+And Mg2+Washing with PBS buffer solution (5 min) at 50rpm, discharging the washing solution, and washing with sterile purified water, 0.1M hydrochloric acid, 0.1mM hydrochloric acid, and Ca-free solution2+、Mg2+The volume ratio of the PBS buffer solution to the calcium alginate bead is 1:1, preparing a wet microcarrier;
(9) freeze-drying: freeze-drying the microcarrier obtained in the step (8), and performing radiation sterilization by using cobalt 60 gamma rays, wherein the radiation dose is 20KGy, so as to prepare the microcarrier;
the serum-free complete culture medium comprises a basic culture medium and additives, wherein the basic culture medium is DMEM/F12 culture medium; the additive is human fibronectin with the concentration of 25 mug/ml, basic fibroblast growth factor with the concentration of 10ng/ml, human epidermal growth factor with the concentration of 10ng/ml, ITS with the concentration of 1 percent and human serum albumin with the concentration of 5 percent; NEAA with the concentration of 0.1 percent, hydrocortisone with the concentration of 0.1 mu mol/L and beta-mercaptoethanol with the concentration of 0.1 percent;
the parameters of the hollow fiber column of the TFF system are shown in Table 1, and the operating parameters of the TFF system are shown in Table 2.
Table 1 parameters of hollow fiber column.
TABLE 2 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 110ml/min | 132rpm | 18L/h/m2 | 0.3psi | 1300s-1 | 18min |
Example 2
Example 2 provides a continuous tangential flow stem cell separation and purification method, which comprises the following steps:
s1: 2x1010Inoculating the mesenchymal stem cells and 12g of microcarriers into a bioreactor, adding a serum-free complete culture medium to 3L, culturing for 7 days, controlling the temperature to be 37 ℃, the concentration of CO2 to be 5 percent and the rotating speed of the bioreactor to be 5rpm, respectively supplementing 2L, 2L and 3L of serum-free complete culture medium on the days 2, 4 and 6, extracting the culture solution into a liquid storage bottle on the day 8, cleaning the microcarriers for 10 minutes by using normal saline, removing the normal saline, adding pancreatin digestive cells, setting the temperature to be 37 ℃ during digestion, setting the rotating speed of the bioreactor to be 4rpm, digesting for 20 minutes, backfilling 2L of serum-free complete culture to terminate digestion, and obtaining a pretreatment solution;
s2: placing the pretreatment liquid in a material liquid bottle, connecting the material liquid bottle to a TFF system, connecting a permeate collection bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the material liquid bottle, adding 5L of serum-free complete culture medium into the material liquid bottle when the permeate collection bottle collects 5L of waste liquid, emptying the permeate collection bottle (namely emptying 5L of waste liquid) after the serum-free complete culture medium is completely added, and stopping the system when 1000ml of feed liquid in the material liquid bottle is left, so as to obtain mesenchymal stem cells;
the preparation method of the microcarrier is the same as that of the microcarrier in the embodiment 1, and the serum-free complete culture medium comprises a basal culture medium and an additive, wherein the basal culture medium is DMEM/F12 culture medium; the additive is human fibronectin with the concentration of 20 mu g/ml, basic fibroblast growth factor with the concentration of 5ng/ml, human epidermal growth factor with the concentration of 5ng/ml, ITS with the concentration of 0.5 percent and human serum albumin with the concentration of 4 percent; NEAA with the concentration of 0.5 percent, hydrocortisone with the concentration of 0.05 mu mol/L and beta-mercaptoethanol with the concentration of 0.05 percent;
the parameters of the hollow fiber column of the TFF system are shown in Table 1, and the operating parameters of the TFF system are shown in Table 3.
TABLE 3 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 90ml/min | 110rpm | 15L/h/m2 | 0.2psi | 1100s-1 | 20min |
Example 3
Example 3 provides a continuous tangential flow stem cell isolation and purification method, which comprises the following steps:
s1: 2x1010Inoculating the mesenchymal stem cells and 18g of microcarriers into a bioreactor, adding a serum-free complete culture medium to 3L, culturing for 7 days, controlling the temperature to be 37 ℃, the concentration of CO2 to be 5 percent and the rotating speed of the bioreactor to be 5rpm, respectively supplementing 2L, 2L and 3L of serum-free complete culture medium on the days 2, 4 and 6, extracting the culture solution into a liquid storage bottle on the day 8, cleaning the microcarriers for 10 minutes by using normal saline, removing the normal saline, adding pancreatin digestive cells, setting the temperature to be 37 ℃ during digestion, setting the rotating speed of the bioreactor to be 6rpm, digesting for 40 minutes, backfilling the culture solution in the 2L liquid storage bottle, and stopping digestion to obtain a pretreatment solution;
s2: placing the pretreatment liquid in a material liquid bottle, connecting the material liquid bottle to a TFF system, connecting a permeate collection bottle to the tail end of a liquid outlet pipeline of the TFF system, connecting a reflux pipeline to the material liquid bottle, adding 5L of serum-free complete culture medium into the material liquid bottle when the permeate collection bottle collects 5L of waste liquid, emptying the permeate collection bottle (namely emptying the 5L of waste liquid) after the serum-free complete culture medium is completely added, and stopping the system when the material liquid in the material liquid bottle is 250ml, thus obtaining mesenchymal stem cells;
the preparation method of the microcarrier is the same as that of the microcarrier in the embodiment 1, and the serum-free complete culture medium comprises a basal culture medium and an additive, wherein the basal culture medium is DMEM/F12 culture medium; the additive is human fibronectin with the concentration of 30 mu g/ml, basic fibroblast growth factor with the concentration of 15ng/ml, human epidermal growth factor with the concentration of 15ng/ml, ITS with the concentration of 1.5 percent and human serum albumin with the concentration of 6 percent; 1.5% NEAA, 0.15% micromole/L hydrocortisone, 0.15% beta-mercaptoethanol;
the parameters of the hollow fiber column of the TFF system are shown in Table 1, and the operating parameters of the TFF system are shown in Table 4.
TABLE 4 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 160ml/min | 173rpm | 22L/h/m2 | 0.5psi | 1800s-1 | 15min |
Comparative example 1
The present comparative example provides a continuous tangential flow stem cell separation and purification method, and the difference from example 1 is that the working parameters of the TFF system are shown in Table 5.
TABLE 5 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 60ml/min | 70rpm | 10L/h/m2 | 0psi | 650s-1 | 40min |
Comparative example 2
The present comparative example provides a continuous tangential flow stem cell separation and purification method, and the difference from example 1 is that the working parameters of the TFF system are shown in Table 6.
TABLE 6 operating parameters of TFF System
| Flow rate of sample introduction | Speed of pump | Flux (W) | Transmembrane pressure | Shear force | Time of filtration |
| 200ml/min | 220rpm | 31L/h/m2 | 1.1psi | 2300s-1 | 10min |
Test example 1
Mesenchymal stem cells were isolated and purified by the methods provided in examples 1 to 3 and comparative examples 1 to 2 of the present invention, the number of cells harvested by each set of methods, the cell viability rate, and the cell recovery rate were calculated, and the number of cells before isolation after culturing in examples 1 to 3 and comparative examples 1 to 2 was 1.78 x1011The calculation formula of the cell recovery rate is as follows: cell count/1.78 × 1011The cell number is measured by counting under a microscope; the cell viability rate, which is (total number of cells-number of dead cells)/total number of cells, was calculated according to the following formula, and the test results are shown in table 7; the cells obtained in example 1 after 24 hours of culture were observed under a microscope, and the results are shown in FIG. 1 at a magnification of 100.
TABLE 7 results of cell viability test by each set of methods.
As can be seen from Table 1, the continuous tangential flow stem cell separation and purification method provided by the invention can be used for recovering cells, the cell recovery rate is more than 80%, the cell viability can reach more than 85%, the concentrated mesenchymal stem cells are collected, fresh culture medium is added for continuous culture, and the growth state of the cells is observed on day 2. The cells adhere well, the shape is in a slender fiber shape, and the activity and the growth and proliferation capacity of the cells are not influenced in the process of concentration and washing.
Therefore, the invention is not limited to the specific embodiments and examples, but rather, all equivalent variations and modifications are within the scope of the invention as defined in the claims and the specification.
Claims (10)
1. A continuous tangential flow stem cell separation and purification method is characterized by mainly comprising the following steps:
s1: placing the mesenchymal stem cells into a reactor, adding a microcarrier and a serum-free complete culture medium for culture, cleaning with normal saline, adding pancreatin for digestion, backfilling the serum-free complete culture medium to stop digestion, and preparing a pretreatment solution;
s2: placing the pretreatment liquid in a material liquid bottle, connecting the material liquid bottle to a TFF system, connecting the tail end of a liquid outlet pipeline of the TFF system with a transmission liquid collecting bottle, connecting a reflux liquid pipeline to the material liquid bottle, adding an equivalent amount of washing liquid into the material liquid bottle when the transmission liquid collecting bottle collects waste liquid with a certain volume, emptying the transmission liquid collecting bottle after the washing liquid is added, and stopping the system operation when the material liquid in the material liquid bottle has a residual preset volume to obtain the mesenchymal stem cells.
2. The continuous tangential flow stem cell separation and purification method of claim 1, wherein the pretreatment solution of step S1 is prepared by the following steps: adding a serum-free complete culture medium into a bioreactor, and then inoculating the MSC cells and the microcarriers into the bioreactor together, wherein the density of the mesenchymal stem cells in the serum-free complete culture medium is 0.5-1x1010The density of the microcarrier in a serum-free complete culture medium is 4-6g/L, the microcarrier is cultured for 7 days, the temperature is controlled to be 37 ℃, the concentration of CO2 is 5%, the rotating speed of a bioreactor is 4-6rpm, and the microcarrier is respectively supplemented with serum-free complete culture on days 2, 4 and 6And (3) a medium, wherein the volume ratio of the supplemented volume to the volume of the serum-free complete medium added for the first time is 1-3:3, 1-3:3 and 2-4:3, on the 8 th day, the culture solution is extracted into a liquid storage bottle, the microcarrier is washed by using normal saline for 5-20 minutes, the volume of the normal saline is larger than the volume of the serum-free medium, pancreatin digestive cells are added after the normal saline is removed, the temperature is set at 37 ℃ during digestion, the rotating speed of the bioreactor is set at 4-6rpm, the digestion time is 20-40 minutes, the serum-free complete medium is refilled to terminate the digestion, and the volume ratio of the refilled serum-free complete medium to the volume of the serum-free complete medium added for the first time is 1-3:2-4, so that the pretreatment.
3. The method for separating and purifying continuous tangential flow stem cells of claim 1, wherein the washing solution in step S2 is serum-free complete culture medium or normal saline, and the washing time is 10-20 minutes.
4. The continuous tangential flow stem cell separation and purification method of claims 1-2, wherein the serum-free complete medium comprises a basal medium and additives, and the basal medium is DMEM/F12 medium; the additive is human fibronectin with the concentration of 20-30 mug/ml, basic fibroblast growth factor with the concentration of 5-15ng/ml, human epidermal growth factor with the concentration of 5-15ng/ml, ITS with the concentration of 0.5-1.5% and human serum albumin with the concentration of 4-6%; NEAA with the concentration of 0.5 to 1.5 percent, hydrocortisone with the concentration of 0.05 to 0.15 mu mol/L and beta-mercaptoethanol with the concentration of 0.05 to 0.15 percent.
5. The continuous tangential flow stem cell separation and purification method of claim 1, wherein the sample injection flow rate of the TFF system is 90-160 ml/min.
6. The method for separating and purifying continuous tangential flow stem cell as claimed in claim 1, wherein the shear force of the TFF system is 1100-1800s-1。
7. The method for the isolation and purification of continuous tangential flow stem cells according to claim 1, wherein the pressure at the inlet end of the TFF system is lower than 8 psi.
8. The method for separating and purifying the continuous tangential flow stem cells of claim 1, wherein the TFF system has a flux of 15-22L/h/m2。
9. The method for separating and purifying continuous tangential flow stem cells of claim 1, wherein the pump speed of the TFF system is 110-.
10. The method for separating and purifying the continuous tangential flow stem cells of claim 1, wherein the filtration time of the TFF system is 15-20 min.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011165699.9A CN112342192B (en) | 2020-10-27 | 2020-10-27 | Continuous tangential flow stem cell separation and purification method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011165699.9A CN112342192B (en) | 2020-10-27 | 2020-10-27 | Continuous tangential flow stem cell separation and purification method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112342192A true CN112342192A (en) | 2021-02-09 |
| CN112342192B CN112342192B (en) | 2022-08-05 |
Family
ID=74359141
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011165699.9A Active CN112342192B (en) | 2020-10-27 | 2020-10-27 | Continuous tangential flow stem cell separation and purification method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112342192B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002218747A1 (en) * | 2000-07-12 | 2002-01-21 | Argaet, Victor Peter | Use of dammarane-type tritepenoid saporins |
| US20050189297A1 (en) * | 2003-11-24 | 2005-09-01 | Northwest Biotherapeutics, Inc. | Tangential flow filtration devices and methods for stem cell enrichment |
| US20120294836A1 (en) * | 2010-01-22 | 2012-11-22 | Lonza Walkersville, Inc. | High yield method and apparatus for volume reduction and washing of therapeutic cells using tangential flow filtration |
| CN109234231A (en) * | 2018-11-29 | 2019-01-18 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of scale amplification of mesenchymal stem cells |
-
2020
- 2020-10-27 CN CN202011165699.9A patent/CN112342192B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002218747A1 (en) * | 2000-07-12 | 2002-01-21 | Argaet, Victor Peter | Use of dammarane-type tritepenoid saporins |
| US20050189297A1 (en) * | 2003-11-24 | 2005-09-01 | Northwest Biotherapeutics, Inc. | Tangential flow filtration devices and methods for stem cell enrichment |
| US20120294836A1 (en) * | 2010-01-22 | 2012-11-22 | Lonza Walkersville, Inc. | High yield method and apparatus for volume reduction and washing of therapeutic cells using tangential flow filtration |
| CN109234231A (en) * | 2018-11-29 | 2019-01-18 | 广州赛莱拉干细胞科技股份有限公司 | A kind of method of scale amplification of mesenchymal stem cells |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112342192B (en) | 2022-08-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN112522191B (en) | Culture method of mesenchymal stem cells | |
| CN107354129B (en) | A kind of Fibroblast cell-culture method in autologous skin source | |
| CN111925982A (en) | Human bone marrow mesenchymal stem cell culture process applying three-dimensional cells | |
| CN114540298A (en) | Stem cell serum-free medium and preparation method thereof | |
| CN108179137B (en) | Preparation method of recombinant adeno-associated virus of serum 8 type | |
| CN113930393A (en) | Method for extracting exosome from umbilical cord stem cells and preparing hydrogel | |
| CN112342192B (en) | Continuous tangential flow stem cell separation and purification method | |
| CN110628706B (en) | Method for extracting and culturing embryonic neural stem cells in vitro and preparation of culture medium | |
| EP2456855B1 (en) | Drain down and re-feed of microcarrier bioreactor | |
| CN115975918A (en) | Method for extracting skin tissue source exosomes | |
| CN116970553A (en) | Preparation method of yak precursor fat cells | |
| CN108034634B (en) | Method for separating endometrial mesenchymal stem cells from menstrual blood | |
| CN115820540A (en) | Endothelial differentiation inducer for mesenchymal stem cells | |
| CN101451122B (en) | Construction method of Epinephelus fuscoguttatus swim bladder cell line | |
| CN115531297A (en) | Injectable hydrogel system loaded with platelet-rich plasma and umbilical cord mesenchymal stem cell spheres, and preparation method and application thereof | |
| CN114948998A (en) | Polysaccharide biomedical colloidal fluid rich in human adipose-derived mesenchymal stem cell factor compound and preparation method and application thereof | |
| CN101451121A (en) | Construction method of Epinephelus fuscoguttatus heart cell line | |
| CN110106142B (en) | Production process for preparing billion-grade fat source regenerative cells | |
| CN111733161B (en) | Application of circ6148 and recombinant vector thereof in promoting angiogenesis | |
| CN114164164A (en) | In-vitro culture kit for epidermal stem cells and application thereof | |
| CN111394303A (en) | Culture medium containing stem cell activator and culture method of mesenchymal stem cells | |
| CN112708594B (en) | Umbilical cord mesenchymal stem cell recovery culture medium and preparation method and application thereof | |
| CN112587718B (en) | Method for preparing scar gel by using active factors secreted by mesenchymal stem cells | |
| CN104371969A (en) | Improved stem/progenitor cell and regenerative porcine islet cell co-culturing method | |
| CN113462636B (en) | Improved method for differentiating epidermal stem cells into liver cells |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |