CN108635372A - A kind of preparation method of the biological agent of human mesenchymal stem cell source excretion body - Google Patents
A kind of preparation method of the biological agent of human mesenchymal stem cell source excretion body Download PDFInfo
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- 230000029142 excretion Effects 0.000 title claims abstract description 87
- 210000002901 mesenchymal stem cell Anatomy 0.000 title claims abstract description 41
- 239000003124 biologic agent Substances 0.000 title claims abstract description 30
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 239000006228 supernatant Substances 0.000 claims abstract description 43
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 39
- 239000000725 suspension Substances 0.000 claims abstract description 15
- CYDQOEWLBCCFJZ-UHFFFAOYSA-N 4-(4-fluorophenyl)oxane-4-carboxylic acid Chemical compound C=1C=C(F)C=CC=1C1(C(=O)O)CCOCC1 CYDQOEWLBCCFJZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 150000001413 amino acids Chemical class 0.000 claims abstract description 14
- 239000001540 sodium lactate Substances 0.000 claims abstract description 14
- 229940005581 sodium lactate Drugs 0.000 claims abstract description 14
- 235000011088 sodium lactate Nutrition 0.000 claims abstract description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 12
- 210000002966 serum Anatomy 0.000 claims abstract description 11
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims abstract description 10
- 102000009027 Albumins Human genes 0.000 claims abstract description 9
- 108010088751 Albumins Proteins 0.000 claims abstract description 9
- 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 9
- 239000008156 Ringer's lactate solution Substances 0.000 claims abstract description 9
- BMLSTPRTEKLIPM-UHFFFAOYSA-I calcium;potassium;disodium;hydrogen carbonate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].OC([O-])=O BMLSTPRTEKLIPM-UHFFFAOYSA-I 0.000 claims abstract description 9
- 239000008103 glucose Substances 0.000 claims abstract description 9
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 6
- 239000001110 calcium chloride Substances 0.000 claims abstract description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims abstract description 6
- 239000011780 sodium chloride Substances 0.000 claims abstract description 6
- 239000001103 potassium chloride Substances 0.000 claims abstract description 5
- 235000011164 potassium chloride Nutrition 0.000 claims abstract description 5
- 238000012545 processing Methods 0.000 claims abstract description 4
- 239000012679 serum free medium Substances 0.000 claims abstract description 4
- 210000004027 cell Anatomy 0.000 claims description 82
- 239000007788 liquid Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 21
- 108090000623 proteins and genes Proteins 0.000 claims description 15
- 238000001514 detection method Methods 0.000 claims description 12
- 239000012530 fluid Substances 0.000 claims description 12
- 235000015097 nutrients Nutrition 0.000 claims description 12
- 239000012528 membrane Substances 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 238000003760 magnetic stirring Methods 0.000 claims description 10
- 238000005406 washing Methods 0.000 claims description 9
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 claims description 8
- 239000012634 fragment Substances 0.000 claims description 8
- 238000011002 quantification Methods 0.000 claims description 8
- 238000001556 precipitation Methods 0.000 claims description 7
- 238000004321 preservation Methods 0.000 claims description 6
- 239000012267 brine Substances 0.000 claims description 5
- 239000006285 cell suspension Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 229910021529 ammonia Inorganic materials 0.000 claims description 4
- 230000035755 proliferation Effects 0.000 claims description 4
- 230000001640 apoptogenic effect Effects 0.000 claims description 3
- 230000007910 cell fusion Effects 0.000 claims description 3
- 238000004140 cleaning Methods 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 235000002639 sodium chloride Nutrition 0.000 abstract description 5
- 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 abstract 1
- 235000011148 calcium chloride Nutrition 0.000 abstract 1
- 235000001727 glucose Nutrition 0.000 abstract 1
- 230000006698 induction Effects 0.000 description 16
- 238000010586 diagram Methods 0.000 description 10
- 230000004069 differentiation Effects 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 8
- 210000002950 fibroblast Anatomy 0.000 description 7
- 230000001737 promoting effect Effects 0.000 description 7
- 210000001612 chondrocyte Anatomy 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 6
- 238000011081 inoculation Methods 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 238000000034 method Methods 0.000 description 5
- 210000000845 cartilage Anatomy 0.000 description 4
- 230000004663 cell proliferation Effects 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 230000002293 adipogenic effect Effects 0.000 description 3
- 230000012292 cell migration Effects 0.000 description 3
- 239000002537 cosmetic Substances 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 230000035479 physiological effects, processes and functions Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 108010019160 Pancreatin Proteins 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 230000002016 colloidosmotic effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 210000001808 exosome Anatomy 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 239000006193 liquid solution Substances 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 230000003534 oscillatory effect Effects 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000004072 osteoblast differentiation Effects 0.000 description 2
- 230000002188 osteogenic effect Effects 0.000 description 2
- 229940055695 pancreatin Drugs 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 230000018199 S phase Effects 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000002659 cell therapy Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000003328 fibroblastic effect Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000011187 glycerol Nutrition 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 230000035992 intercellular communication Effects 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000036560 skin regeneration Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
- Cell Biology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Dermatology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Hematology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Virology (AREA)
- Birds (AREA)
- Urology & Nephrology (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of preparation methods of the biological agent of human mesenchymal stem cell source excretion body, include the following steps：（1）The culture that human mesenchymal stem cell is carried out using serum free medium, takes supernatant；（2）To step（1）After obtained supernatant carries out Treatment with Ultrafiltration, required excretion body is obtained；（3）Using sodium lactate ringer's injection to step（2）Obtained excretion body carries out resuspension processing, obtains excretion body suspension；（4）In step（3）Sodium chloride, potassium chloride, sodium lactate, calcium chloride, glucose, necessary amino acid, nonessential amino acid, human serum albumins is added in example in mass ratio in obtained excretion body suspension, as prepared excretion body biological agent, the excretion body biological agent prepared by the invention have preferable stability, biocompatibility, biological stability.
Description
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of biological agent of human mesenchymal stem cell source excretion body
Preparation method.
Background technology
Excretion body（Exosome it) is most found earlier than nineteen eighty-three, Johnstone is named as " exosome " within 1987.
It is as a kind of important vesica in human body, by a kind of vesicles of double-deck membrane structure of living cells secretion, diameter about 40-
150nm, carries the important signaling molecule of mother cell, such as protein, mRNA, miRNA and lipid etc., by with target cell
In conjunction with and regulating cell function, participate in intercellular communication.
At present have multiple studies have shown that, human mesenchymal stem cell source excretion body can participate in the signal during skin regeneration
It transmits, there is very strong antioxidation, and the signaling molecule of the regenerative process under the physiology of multiple organs and pathological state
Cooperation in play an important role.
The patent application of 201710262634 .8 of Patent No. is about human mesenchymal stem cell source property bioactivity system
Agent obtains the patent application of preparation method and application, it discloses excretion body biologically active agents are made freeze-dried powder form, and it is molten
Solution in glycerine etc. form in solution, finally obtain the suspension of excretion body；It is dry that suspension is subjected to centrifugal treating collection precipitation
Cosmetic formulation is obtained after dry.
But there are clearly disadvantageous for this method：1. excretion body can damage excretion body in freezing dry process repeatedly
Imitated vesicle structure reduces the action activity of excretion body, to influence the therapeutic effect in later stage；2. being added in this cosmetic formulation too many
Other compositions, and these ingredients itself do not have therapeutic effect, can influence or weaken the therapeutic effect of excretion body.
Invention content
The skill that the purpose of the invention is to overcome the preservation effect in prior art problem about biological agent undesirable
Art defect.
For this purpose, the present invention provides a kind of preparation methods of the biological agent of human mesenchymal stem cell source excretion body, including
Following steps：
（1）The culture that human mesenchymal stem cell is carried out using serum free medium, takes supernatant；
（2）To step（1）After obtained supernatant carries out Treatment with Ultrafiltration, required excretion body is obtained；
（3）Using sodium lactate ringer's injection to step（2）Obtained excretion body carries out resuspension processing, obtains excretion body suspension；
（4）In step（3）Sodium chloride is added in example in mass ratio in obtained excretion body suspension（0.1-2%）, potassium chloride（0.1-
1%）, sodium lactate（0.1-1%）, calcium chloride（0.1-1%）, glucose（0.05-5%）, must amino acid（0.01-5%）, it is nonessential
Amino acid（0.01-5%）, human serum albumins（0.1-10%）, as prepared excretion body biological agent.
The supernatant carries out Treatment with Ultrafiltration, specifically comprises the following steps:
A, the supernatant being collected into is sub-packed in the centrifuge tube of 50mL, at 4 DEG C, 300g centrifuges 10min, removes residual cells；
2000g centrifuges 20min, removes cell fragment；It collects supernatant and is filtered with the filter in 0.22 μm of aperture；
B, by step（1）The supernatant that is collected into is added in the rotation ultrafiltration instrument equipped with ultrafiltration membrane, connect nitrogen and by maximum into
Atmospheric pressure control is in 517.125kPa hereinafter, opening magnetic stirring apparatus；After waiting for supernatant ultrafiltration, 200-500mL lifes are added
Brine and ultrafiltration again are managed, is repeated 3-5 times.
The step（3）Obtained excretion body suspension, after the detection that need to carry out albumen concentration, then adds step（4）It is described
Substance.
The specific preparation method of the biological agent is:
1) human mesenchymal stem cell is utilized and contains 10%FBS（GIBCO）Alpha-MEM fluid nutrient mediums carry out suspend be cell
Suspension, and it is 1 × 106/ml-2 × 106/ml to adjust cell concentration；
2) be inoculated with according to 3E7/ cell factories, culture to cell fusion degree to 70%-80%, cell be in proliferation it is best when
Machine, incubation time are 2-3 days；
3) by liquid pouring in cell factory, alpha-MEM fluid nutrient mediums is used in combination to wash cell factory, in this mistake
Washing is carried out in journey using alpha-MEM fluid nutrient mediums on the one hand to remove FBS remaining in cell factory；
4) the alpha-MEM fluid nutrient mediums of 1250ml are added in cell factory；
5) it is cultivated 96 hours under the conditions of 37 DEG C, 5% CO2, collection obtains MSC culture solutions；
6) 5 are taken）The supernatant of middle collection carries out centrifugal treating（300-1000g/min, 5min), cell fragment is removed, in collection
Clear liquid discards precipitation；
7）By 6）The supernatant of middle collection carries out secondary centrifuging（1000g-2000g/min,15min)；Apoptotic body is removed, is collected
Supernatant discards precipitation；
8）By 7）The supernatant of middle collection carries out hyperfiltration treatment using rotation ultrafiltration instrument, to collect the excretion secreted in culture solution
Body；
9）Using the excretion body on sodium lactate ringer's injection washing ultrafiltration membrane, washing three times, utilizes after cleaning solution is mixed
BCA kits carry out protein quantification；
10）According to 9）After the result of middle protein quantification calculates concentration, example adds glucose, human serum albumins, ammonia in mass ratio
Base acid, nonessential amino acid is placed on magnetic stirring apparatus and is stirred 20min, to make excretion body be dispersed in preservation liquid
In after, be placed in -20 degree preserve.
Beneficial effects of the present invention：The biology system of this novel human mesenchymal stem cell source excretion body provided by the invention
The preparation method of agent, has the following advantages compared with prior art：（1）Excretion body biological agent prepared by the present invention has preferable
Stability, the excretion somatocyst bubble after -20 degree preserve 1 year 80% or more still keeps complete structure, after being preserved 1 month at 4 degree
70% or more excretion somatocyst bubble still keeps complete structure；（2）Excretion body biological agent prepared by the present invention has preferable biology
Compatibility, because, comprising the various ions and albumen for keeping crystal osmotic pressure and colloid osmotic pressure, having in excretion body biological agent
Preferable biocompatibility, can be added to cosmetic formulation, tissue engineering product, in pharmaceutical preparation；（3）It is outer prepared by the present invention
Secreting body biological agent has preferable bioactivity, because the separation of excretion body is carried out using ultrafiltration, to vesica various composition
Damage is smaller, and therefore, the activity of this factor, albumen, nucleic acid is barely affected.
The present invention is described in further details below with reference to attached drawing.
Description of the drawings
Fig. 1 a are human mesenchymal stem cell morphology schematic diagrames.
Fig. 1 b are the streaming identification schematic diagrames of human mesenchymal stem cell.
Fig. 2 a are the schematic diagrames that human mesenchymal stem cell is broken up under inducing culture induction.
Fig. 2 b be human mesenchymal stem cell osteogenic induction liquid induction under, at cartilage differentiation gene expression count
Figure.
Fig. 2 c be human mesenchymal stem cell adipogenic induction liquid induction under, at fat differentiation associated gene expression count
Figure.
Fig. 2 d be human mesenchymal stem cell at chondrocyte induction liquid induce under, at the expression of cartilage differentiation related gene
Statistical chart.
Fig. 3 a are that excretion body is promoting cell migration Ergonomy detects schematic diagram.
Fig. 3 b are that excretion body is promoting cell migration Ergonomy detection quantitative statistics schematic diagram.
Fig. 4 a are in the culture medium for being added to excretion body for control group in Period of Fibroblast testing result schematic diagram.
Fig. 4 b are for UV irradiation groups in the culture medium for being added to excretion body in Period of Fibroblast testing result schematic diagram.
Fig. 4 c are in the culture medium for being added to excretion body for WMT groups in Period of Fibroblast testing result schematic diagram.
Fig. 4 d are the results of statistical analysis schematic diagram of cycle detection result.
Fig. 5 a are under different storage temperatures, and excretion body puts the testing result in terms of promoting cell Proliferation in different times
Schematic diagram.
Fig. 5 b are lower under different storage temperatures to be preserved 1 month, testing result of the excretion body in terms of promoting cell Proliferation.
Specific implementation mode
Reach the technological means and effect that predetermined purpose is taken for the present invention is further explained, below in conjunction with attached drawing and reality
Example is applied to specific implementation mode, structure feature and its effect of the present invention, detailed description are as follows.
Embodiment 1
For the technological deficiency for overcoming the preservation effect in prior art problem about biological agent undesirable；The present invention provides one
The preparation method of the biological agent of kind human mesenchymal stem cell source excretion body, includes the following steps：
（1）The culture that human mesenchymal stem cell is carried out using serum free medium, takes supernatant；
（2）To step（1）After obtained supernatant carries out Treatment with Ultrafiltration, required excretion body is obtained；
（3）Using sodium lactate ringer's injection to step（2）Obtained excretion body carries out resuspension processing, obtains excretion body suspension；
（4）In step（3）Sodium chloride is added in example in mass ratio in obtained excretion body suspension（0.1-2%）, potassium chloride（0.1-
1%）, sodium lactate（0.1-1%）, calcium chloride（0.1-1%）, glucose（0.05-5%）, must amino acid（0.01-5%）, it is nonessential
Amino acid（0.01-5%）, human serum albumins（0.1-10%）, as prepared excretion body biological agent.
The supernatant carries out Treatment with Ultrafiltration, specifically comprises the following steps:
A, the supernatant being collected into is sub-packed in the centrifuge tube of 50mL, at 4 DEG C, 300g centrifuges 10min, removes residual cells；
2000g centrifuges 20min, removes cell fragment；It collects supernatant and is filtered with the filter in 0.22 μm of aperture；
B, by step（1）The supernatant that is collected into is added in the rotation ultrafiltration instrument equipped with ultrafiltration membrane, connect nitrogen and by maximum into
Atmospheric pressure control is in 517.125kPa hereinafter, opening magnetic stirring apparatus；After waiting for supernatant ultrafiltration, 200-500mL lifes are added
Brine and ultrafiltration again are managed, is repeated 3-5 times.
The step（3）Obtained excretion body suspension, after the detection that need to carry out albumen concentration, then adds step（4）It is described
Substance.
The specific preparation method of the biological agent is:
1) human mesenchymal stem cell is utilized and contains 10%FBS（GIBCO）Alpha-MEM fluid nutrient mediums carry out suspend be cell
Suspension, and it is 1 × 106/ml-2 × 106/ml to adjust cell concentration；
2) be inoculated with according to 3E7/ cell factories, culture to cell fusion degree to 70%-80%, cell be in proliferation it is best when
Machine, incubation time are 2-3 days；
3) by liquid pouring in cell factory, alpha-MEM fluid nutrient mediums is used in combination to wash cell factory, in this mistake
Washing is carried out in journey using alpha-MEM fluid nutrient mediums on the one hand to remove FBS remaining in cell factory；
4) the alpha-MEM fluid nutrient mediums of 1250ml are added in cell factory；
5) it is cultivated 96 hours under the conditions of 37 DEG C, 5% CO2, collection obtains MSC culture solutions；
6) 5 are taken）The supernatant of middle collection carries out centrifugal treating（300-1000g/min, 5min), cell fragment is removed, in collection
Clear liquid discards precipitation；
7）By 6）The supernatant of middle collection carries out secondary centrifuging（1000g-2000g/min,15min)；Apoptotic body is removed, is collected
Supernatant discards precipitation；
8）By 7）The supernatant of middle collection carries out hyperfiltration treatment using rotation ultrafiltration instrument, to collect the excretion secreted in culture solution
Body；
9）Using the excretion body on sodium lactate ringer's injection washing ultrafiltration membrane, washing three times, utilizes after cleaning solution is mixed
BCA kits carry out protein quantification；
10）According to 9）After the result of middle protein quantification calculates concentration, example adds glucose, human serum albumins, ammonia in mass ratio
Base acid, nonessential amino acid is placed on magnetic stirring apparatus and is stirred 20min, to make excretion body be dispersed in preservation liquid
In after, be placed in -20 degree preserve.
In conclusion the preparation method of the biological agent of the human mesenchymal stem cell source excretion body, has compared with prior art
It has the advantage that：（1）Excretion body biological agent prepared by the present invention has preferable stability, after -20 degree preserve 1 year
80% or more excretion somatocyst bubble still keeps complete structure, and 70% or more excretion somatocyst bubble after being preserved 1 month at 4 degree still keeps complete
Structure；（2）Excretion body biological agent prepared by the present invention has preferable biocompatibility, because being wrapped in excretion body biological agent
Containing the various ions and albumen for keeping crystal osmotic pressure and colloid osmotic pressure, there is preferable biocompatibility, U.S. can be added to
Hold preparation, tissue engineering product, in pharmaceutical preparation；（3）Excretion body biological agent prepared by the present invention has preferable biology
Activity, it is smaller to the damage of vesica various composition because the separation of excretion body is carried out using ultrafiltration, therefore, and this factor, egg
In vain, the activity of nucleic acid is barely affected.
Embodiment 2
1. cell prepares
（1）Recovery：The cell recovered needed for choosing, quickly dissolves in 37 DEG C of preheated in advance thermostat water baths, time control
System is put into 1000r/min in ready culture solution in advance at 1 minute or so, by the suction of dissolved cell suspension and centrifuges 5 points
Clock,
（2）Motility rate calculates and cell count, and carries out cell inoculation.It slightly should back and forth shake and cultivate by mark signature method after inoculation
Bottle/ware makes cell be uniformly distributed, and has marked cell batch, algebraically, after the passage date, has been placed in cell incubator and continues to cultivate,
Cell confluency is waited for 85% or so passage, next day changes liquid.
2. cell passes on
（1）After recovery cell three days, morphological observation is taken pictures.Culture supernatant is discarded, using pipette, extract physiological saline
It to Tissue Culture Flask, keeps flat, slight oscillatory, washes away culture medium remaining in culture bottle, discard.
（2）Digestion：Suitable 0.125% pancreatin is added, is positioned over incubator after shaken well, 37 DEG C of vitellophags 1~
3min.The termination digestion of equivalent serum-containing media is added after being all condensed to circle in microscopic observation cell.
（3）It collects：Culture bottle attached cell one side is uniformly blown and beaten with suction pipe, is moved into disposable 50ml centrifuge tubes, 1000-
1500r/min is centrifuged 5 minutes.
（4）Cell inoculation：Cell density is adjusted to by 2-5E4/ml using culture solution, is inoculated in 10 layer cell factories,
It is slightly back and forth shaken by mark signature method, so that cell is uniformly distributed and marked cell batch, algebraically, after the passage date, be placed in cell
In incubator, air pump is accessed, continues to cultivate.
3, culture solution is collected
（1）Cell in cell factory is carried out morphological observation and taken pictures by third day after passage.
（2）Liquid in cell factory is collected as in liquid collecting container.
4, culture solution hyperfiltration treatment
（1）The cell supernatant being collected into is sub-packed in 50mL centrifuge tubes by the culture solution that will be collected in 3,4 DEG C of 300g centrifugations
10min removes residual cells, and 2000g centrifuges 20min and removes cell fragment, careful collection supernatant and with 0.22 μm of bore filter
Device is filtered.
（2）The supernatant being collected into is added in the rotation ultrafiltration instrument equipped with ultrafiltration membrane, connects nitrogen and by full admission
Pressure control is in 517.125kPa hereinafter, opening magnetic stirring apparatus.After waiting for supernatant ultrafiltration, 200-500mL physiology is added
Brine and again ultrafiltration repeat 3-5 times.After ultrafiltration, with the excretion on 50ml sodium lactate ringer's injection suspension ultrafiltration membranes
Body.
5. determination of protein concentration
It utilizes BCA detection kits to carry out with reference to specification the detection of albumen concentration excretion liquid solution obtained in 4, measures
The sample obtained is marked afterwards.
6. according to 5）After the result calculating concentration of middle protein quantification sodium chloride is added in mass concentration ratio（0.1%）, chlorine
Change potassium（0.1%）, sodium lactate（0.1%）, calcium chloride（0.1%）, glucose（0.05%）, must amino acid（0.01%）, nonessential ammonia
Base acid（0.01%）, human serum albumins（0.1%）It is placed on after magnetic stirring apparatus is stirred 20min, is placed in -20 degree and preserves.
Embodiment 3
1. cell prepares
（1）Recovery：The cell recovered needed for choosing, quickly dissolves in 37 DEG C of preheated in advance thermostat water baths, time control
System is put into 1000r/min in ready culture solution in advance at 1 minute or so, by the suction of dissolved cell suspension and centrifuges 5 points
Clock,
（2）Motility rate calculates and cell count, and carries out cell inoculation.It slightly should back and forth shake and cultivate by mark signature method after inoculation
Bottle/ware makes cell be uniformly distributed, and has marked cell batch, algebraically, after the passage date, has been placed in cell incubator and continues to cultivate,
Cell confluency is waited for 85% or so passage, next day changes liquid.
2. cell passes on
（1）After recovery cell three days, morphological observation is taken pictures.Culture supernatant is discarded, using pipette, extract physiological saline
It to Tissue Culture Flask, keeps flat, slight oscillatory, washes away culture medium remaining in culture bottle, discard.
（2）Digestion：Suitable 0.125% pancreatin is added, is positioned over incubator after shaken well, 37 DEG C of vitellophags 1~
3min.The termination digestion of equivalent serum-containing media is added after being all condensed to circle in microscopic observation cell.
（3）It collects：Culture bottle attached cell one side is uniformly blown and beaten with suction pipe, is moved into disposable 50ml centrifuge tubes, 1000-
1500r/min is centrifuged 5 minutes.
（4）Cell inoculation：Cell density is adjusted to by 2-5E4/ml using culture solution, is inoculated in 10 layer cell factories,
It is slightly back and forth shaken by mark signature method, so that cell is uniformly distributed and marked cell batch, algebraically, after the passage date, be placed in cell
In incubator, air pump is accessed, continues to cultivate.
3, culture solution is collected
（1）Cell in cell factory is carried out morphological observation and taken pictures by third day after passage.
（2）Liquid in cell factory is collected as in liquid collecting container.
4, culture solution hyperfiltration treatment
（1）The cell supernatant being collected into about is sub-packed in 50mL centrifuge tubes by the culture solution that will be collected in 3,4 DEG C of 300g from
Heart 10min removes residual cells, and 2000g centrifuges 20min and removes cell fragment, careful collection supernatant and with 0.22 μm of aperture mistake
Filter is filtered.
（2）The supernatant being collected into is added in the rotation ultrafiltration instrument equipped with ultrafiltration membrane, connects nitrogen and by full admission
Pressure control is in 517.125kPa hereinafter, opening magnetic stirring apparatus.After waiting for supernatant ultrafiltration, 200-500mL physiology is added
Brine and again ultrafiltration repeat 3-5 times.After ultrafiltration, outside on 100ml sodium lactate ringer's injection suspension ultrafiltration membranes
Secrete body.
5. determination of protein concentration
It utilizes BCA detection kits to carry out with reference to specification the detection of albumen concentration excretion liquid solution obtained in 4, measures
The sample obtained is marked afterwards.
According to 5）The result of middle protein quantification adds sodium chloride in proportion after calculating concentration（1%）, potassium chloride（1%）, lactic acid
Sodium（5%）, calcium chloride（1%）, glucose（5%）, must amino acid（10.01%）, nonessential amino acid（1%）, human serum albumins
（10%）It is placed on after magnetic stirring apparatus is stirred 20min, sets -20 degree and preserve.
Embodiment 4
Above-described embodiment 1, embodiment 2, embodiment are further detailed in conjunction with attached drawing.Fig. 1 is human mesenchymal stem cell
Identify picture.Wherein Fig. 1 a are human mesenchymal stem cell morphology photo, it can be seen from the figure that human mesenchymal stem cell is length
Fusiformis attached cell, Fig. 1 b are the streaming qualification figure of human mesenchymal stem cell, and streaming qualification result meets international cell therapy association
Requirement of the meeting to mescenchymal stem cell.
Fig. 2 a are the lower differentiation of human mesenchymal stem cell inducing culture induction as a result, aobvious to the result of Adipocyte Differentiation
Show more fat drop after dyeing, obtained human mesenchymal stem cell can be to Adipocyte Differentiation；To the result of osteoblast differentiation
There are more calcium tubercle, obtained human mesenchymal stem cell can be to osteoblast differentiation after display dyeing；To chondroblast point
The result of change shows that obtained human mesenchymal stem cell can be to Chondrocyte Differentiation.Fig. 2 b are that human mesenchymal stem cell is lured in skeletonization
Under drain induction, at the expression of cartilage differentiation gene, show that human mesenchymal stem cell can be under osteogenic induction liquid to skeletonization
Cell differentiation.Fig. 2 c are human mesenchymal stem cell under the induction of adipogenic induction liquid, at the expression of fat differentiation associated gene, knot
Fruit shows that human mesenchymal stem cell can break up under the induction of adipogenic induction liquid to lipoblast.Fig. 2 d are that human mesenchymal stem cell exists
At chondrocyte induction liquid induce under, at the expression of cartilage differentiation related gene, the results showed that human mesenchymal stem cell can at
Break up to chondroblast under the induction of chondrocyte induction liquid.
Fig. 3 a figures are that excretion body is promoting cell migration Ergonomy detection picture.It can relative to control group excretion body from figure
Promote fibroblastic migration.Fig. 3 b figures are the quantitative statistics of mobility in Fig. 3 a figures as a result, its result is shown for control
The mobility of group, excretion body group are prompted by higher mobility, prompt excretion body that may be risen during the injury repair of skin
To important role.
Fig. 4 a figures are control group in the culture medium for being added to excretion body in Period of Fibroblast testing result, Fig. 4 b figures
For UV irradiation groups in the culture medium for being added to excretion body in Period of Fibroblast testing result, Fig. 4 c figures are that WMT groups are being added
For the culture medium of excretion body in Period of Fibroblast testing result, Fig. 4 d figures are the statistical analysis knot of cycle detection result
Fruit, result show that excretion body biological agent can promote the raising of fibroblast S phases, can promote the proliferation of cell.
Fig. 5 a are under different storage temperatures, and excretion body puts the detection knot in terms of promoting cell Proliferation in different times
Fruit, Fig. 5 b are to be preserved 1 month under different preservations, and testing result of the excretion body in terms of promoting cell Proliferation, as a result display should
The activity of excretion body can be kept under different temperatures storage by preserving liquid.
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
The specific implementation of the present invention is confined to these explanations.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to the present invention's
Protection domain.
Claims (4)
1. a kind of preparation method of the biological agent of human mesenchymal stem cell source excretion body, which is characterized in that include the following steps：
（1）The culture that human mesenchymal stem cell is carried out using serum free medium, takes supernatant；
（2）To step（1）After obtained supernatant carries out Treatment with Ultrafiltration, required excretion body is obtained；
（3）Using sodium lactate ringer's injection to step（2）Obtained excretion body carries out resuspension processing, obtains excretion body suspension；
（4）In step（3）Sodium chloride is added in example in mass ratio in obtained excretion body suspension（0.1-2%）, potassium chloride（0.1-
1%）, sodium lactate（0.1-1%）, calcium chloride（0.1-1%）, glucose（0.05-5%）, must amino acid（0.01-5%）, it is nonessential
Amino acid（0.01-5%）, human serum albumins（0.1-10%）, as prepared excretion body biological agent.
2. a kind of preparation method of the biological agent of human mesenchymal stem cell source excretion body as described in claim 1, feature
It is, the supernatant carries out Treatment with Ultrafiltration, specifically comprises the following steps:
A, the supernatant being collected into is sub-packed in the centrifuge tube of 50mL, at 4 DEG C, 300g centrifuges 10min, removes residual cells；
2000g centrifuges 20min, removes cell fragment；It collects supernatant and is filtered with the filter in 0.22 μm of aperture；
B, by step（1）The supernatant that is collected into is added in the rotation ultrafiltration instrument equipped with ultrafiltration membrane, connect nitrogen and by maximum into
Atmospheric pressure control is in 517.125kPa hereinafter, opening magnetic stirring apparatus；After waiting for supernatant ultrafiltration, 200-500mL lifes are added
Brine and ultrafiltration again are managed, is repeated 3-5 times.
3. a kind of preparation method of the biological agent of human mesenchymal stem cell source excretion body as described in claim 1, feature
It is, the step（3）Obtained excretion body suspension, after the detection that need to carry out albumen concentration, then adds step（4）The object
Matter.
4. a kind of preparation method of the biological agent of human mesenchymal stem cell source excretion body as described in claim 1, feature
It is, the specific preparation method of the biological agent is:
1) human mesenchymal stem cell is utilized and contains 10%FBS（GIBCO）Alpha-MEM fluid nutrient mediums carry out suspend be cell
Suspension, and it is 1 × 106/ml-2 × 106/ml to adjust cell concentration；
2) be inoculated with according to 3E7/ cell factories, culture to cell fusion degree to 70%-80%, cell be in proliferation it is best when
Machine, incubation time are 2-3 days；
3) by liquid pouring in cell factory, alpha-MEM fluid nutrient mediums is used in combination to wash cell factory, in this mistake
Washing is carried out in journey using alpha-MEM fluid nutrient mediums on the one hand to remove FBS remaining in cell factory；
4) the alpha-MEM fluid nutrient mediums of 1250ml are added in cell factory；
5) it is cultivated 96 hours under the conditions of 37 DEG C, 5% CO2, collection obtains MSC culture solutions；
6) 5 are taken）The supernatant of middle collection carries out centrifugal treating（300-1000g/min, 5min), cell fragment is removed, in collection
Clear liquid discards precipitation；
7）By 6）The supernatant of middle collection carries out secondary centrifuging（1000g-2000g/min,15min)；Apoptotic body is removed, is collected
Supernatant discards precipitation；
8）By 7）The supernatant of middle collection carries out hyperfiltration treatment using rotation ultrafiltration instrument, to collect the excretion secreted in culture solution
Body；
9）Using the excretion body on sodium lactate ringer's injection washing ultrafiltration membrane, washing three times, utilizes after cleaning solution is mixed
BCA kits carry out protein quantification；
10）According to 9）After the result of middle protein quantification calculates concentration, example adds glucose, human serum albumins, ammonia in mass ratio
Base acid, nonessential amino acid is placed on magnetic stirring apparatus and is stirred 20min, to make excretion body be dispersed in preservation liquid
In after, be placed in -20 degree preserve.
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