CN109468274A - A kind of technique using cell factory preparation clinical grade umbilical cord mesenchymal stem cells - Google Patents
A kind of technique using cell factory preparation clinical grade umbilical cord mesenchymal stem cells Download PDFInfo
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- CN109468274A CN109468274A CN201811638660.7A CN201811638660A CN109468274A CN 109468274 A CN109468274 A CN 109468274A CN 201811638660 A CN201811638660 A CN 201811638660A CN 109468274 A CN109468274 A CN 109468274A
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- 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
- C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
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- 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
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- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
Abstract
The invention discloses a kind of techniques using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, described method includes following steps: step (1), cell inoculation: taking 1 dress stem cell in stem cell seed bank, progress expansion of stem cells culture in 12 layer cell factory is inoculated into after being recovered；Step (2), cell factory prepare stem cell: when cell fusion degree is up to 90~95%, the stem cell of 2 layer cell factories digest, is inoculated into 1 10 layer cell factory and carries out expansion of stem cells culture；Step (3), stem cell products preparation: when cell fusion degree reaches 90~95% in 10 layer cell factories, stem cell is digested, obtains a large amount of stem cell.The present invention can be with pilot scale culture stem cell, and it is the direction of Mirae Corp. that not only cell yield is big for the stem cell of production, process stabilizing, product quality are easy to control, and can save personnel's spending, workshop building.
Description
Technical field
The invention belongs to biological study technical fields, are related to a kind of preparation process of umbilical cord mesenchymal stem cells, especially relate to
And a kind of technique using cell factory preparation clinical grade umbilical cord mesenchymal stem cells.
Background technique
Cell factory (Cell Factory) is a kind of cell culture apparatus of deft design, and material is to meet " the U.S.
Pharmacopeia " as defined in polystyrene material, the hydrophilic treated adsorptivity for improving cell of culture surface, it is ensured that cell it is adherent more
One, more stable, it is easier to operate in large-scale culture, more meets the requirement of GMP, and due to the simplification of its technological operation,
The pollution risk for greatly reducing incubation cell enhances the controllability of pollution.In process of production, cell factory can be with
Culture surface is utilized in a limited space to greatest extent, saves (the culture of 10 layer cell factories of incubator usable floor area
The culture area of area=36.2 T175 culture bottle, and an incubator can place 4 10 layer cell factories or 50
T175 culture bottle, i.e., using cell factory can reduce incubator usable floor area more than half), without carrying out any old factory rehabilitation
The purpose enhanced production capacities can be realized.If be used in combination with cell factory automatic operation equipment, it can realize that cell is trained comprehensively
Automation is supported, to substantially reduce labor intensity and closeness, quickly substitutes traditional cell bottle culture technique, is realized extensive
Cell culture enhances the quality controllability to production environment, while can reduce differences between batches.
Human umbilical cord mesenchymal stem cells (Human Umbilical cord-mesenchymal stem cells, hUC-
MSCs) refer to and be present in one of neonatal umbilical cord tissue versatile stem cell.HUC-MSCs form, immunophenotype and more
It is very much like to the various biologicals such as differentiation potential characteristic and bone marrow MSCs, and hUC-MSCs expression SSEA4, Oct-4,
The embryonic stem cell markers such as Nanog and Sox-2, it may be possible to the more original stem cell of a group.In addition, hUC-MSCs also takes
Material convenience, safe virus-free infection risk；Content is high in umbilical cord, and ability of cell proliferation is stablized, can massive amplification in a short time,
It is easy to preparation of industrialization；Do not express/low expression HLA-I class molecule, HLA-II class molecule and costimulatory molecules are not expressed, therefore exempt from
Epidemic focus is extremely low, is not easy to cause immunological rejection；It can be directed differentiation in tissue local microenvironment after feeding back in vivo a variety of
Histocyte is low with Tumor formation with high security, not by advantages such as ethics and legal restrictions.Exogenous hUC-MSCs can be used as
The Regeneration and Repair of seed cell participation damaged tissues；HUC-MSCs can secrete large amount of cell factor, be adjusted by paracrine action
The reparation of damaged tissues cell, or even promote the proliferation and activation of the stem cell of damaged tissues itself；And also there is immune adjust
Section effect, is adjustable a variety of congenital or acquired immunity dysfunction；There is chemotaxis simultaneously, inflammatory mediator can be prevented
Release, mitigate inflammatory reaction, mitigate tissue damage, be conducive to prevent diseases associated with inflammation progression of the disease.HUC-MSCs these
Characteristic gathers around it in the treatment of regenerative medicine industry and immunity disease to hold out broad prospects.
With the rapid development of the cell therapy research based on stem cell, in treatment degenerative disease, ischemic cardiac
Multinomial clinical experimental study has been carried out in the fields such as cranial vascular disease, cirrhosis and diabetes, autoimmune disease, domestic
The industrialization base Duo Jia is established, breakthrough achievement is achieved.Foreign countries have multinomial stem cell injection and obtain drug batch number,
Domestic Ye Youduojia biotech firm submits the new drug of stem cell injection to declare, and the industrialization pattern of stem cell new drug is just in shape
At.In the process of stem cell industrialization, dry cell mass stability is the key link of quality control, to obtain stabilised quality
Stem cell, yield, the stable processing technique of the large-scale serial production of stem cell are preconditions.Use traditional T175
Culture bottle produces hUC-MSCs, and that there is production capacities is low, high, easy to pollute, each intercellular difference of bottle of personnel's energy consumption greatly, stability not
The problems such as easy to control, workshop is greatly, Quality Control point is more.The cell factory that especially 10 layers/40 layers of cell factory, primary culture can
A large amount of stem cells are produced, production site is not only reduced, while also greatly reducing the operation link of technical staff, that reduces pollution can
Energy property, and the cell of cell factory production can be used as the uniform product of quality, greatly reduce the work of Quality Control personnel
Work amount and job costs, meanwhile, cell factory will realize full-automatic production in conjunction with automation equipment, be following industrialization
Direction.
Summary of the invention
Traditional stem cell culture method cell quantity when turning to clinical application is insufficient, product quality in order to solve by the present invention
The inhomogenous and inadequate problem of stability provides a kind of work using cell factory preparation clinical grade umbilical cord mesenchymal stem cells
Skill.The present invention can be with pilot scale culture stem cell, and not only cell yield is big for the stem cell of production, process stabilizing, product quality
It is easy to control, and can save personnel spending, workshop building, if the equipment such as combination cell factory automation shaking table, it can be achieved that
Feed liquor, cell mix, digest the automatic operations such as liquid out, can automatically, efficiently carry out extensive stem cell culture, reduce heavy
Manual operation and human factor bring error, be the direction of Mirae Corp..
The purpose of the present invention is what is be achieved through the following technical solutions:
A kind of technique using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, includes the following steps:
Step (1), cell inoculation:
1 is taken to fill 5~10 × 10 in stem cell seed bank6The P2 of cells/ml/ pipe, will for umbilical cord mesenchymal stem cells
It presses 5~10 × 10 after recovering3cells/cm2Density be inoculated into 12 layer cell factory (NEST, Cat No.771101)
Carry out expansion of stem cells culture, in which: the source of human stem cell of each 2 layer cell factory inoculation is in same branch cell cryopreservation tube
Cell, each 2 layer cell factory inoculating cell sum are 6.395 × 106It is a, every layer addition cell suspension volume be 150~
200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity；
Step (2), cell factory prepare stem cell:
When cell fusion degree is up to 90~95%, the stem cell of 2 layer cell factories is digested, by 5~10 ×
103cells/cm2Density be inoculated into 1 10 layer cell factory progress expansion of stem cells culture, in which: each 10 confluent monolayer cells
The source of human stem cell of factory's inoculation is in the stem cell of same 2 layer cell factory culture, the cell of each 10 layer cell factory inoculation
Sum is 3.17 × 107A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2
With adhere-wall culture 3~5 days in the incubator of saturated humidity；
Step (3), stem cell products preparation:
When cell fusion degree reaches 90~95% in 10 layer cell factories, stem cell is digested, is obtained a large amount of
Stem cell.
In the present invention, the stem cell of the step (2) and step (3) is in 2 layer cell factories and 10 layer cell factory cultures
When, inoculum density is controlled 30%, makes to reach within stem cell 3~5 days 90~95% degrees of fusion, centre is without changing liquid.
In the present invention, the method also includes step (4): by the dry thin of 10 layer cell factory each in step (3) production
Born of the same parents sampling, send qualified third party to do Quality Control detection after, using serum-free cell frozen stock solution (ZENOAQ, Cat No:
Cellbanker2 it) is resuspended, then presses 5~10 × 106The effective cell cryopreservation tube of cells/ml/ is dispensed, and is cooled down using program
Box is placed at least 24 hours at -80 DEG C and is stored in postposition liquid nitrogen container, and stem cell work library is established.
In the present invention, the method for building up of the stem cell seed bank is as follows:
(1) it is obtained under aseptic condition through the qualified umbilical cord tissue of detection (puerpera's inspection the third plum of second Chinese mugwort, TORCH, HTLV disease
Poison, Epstein-Barr virus, poor Genotyping, hepatic and renal function etc. it is without exception), while take the inspection of 10ml Cord blood (the third plum of second Chinese mugwort, TORCH,
HTLV virus, Epstein-Barr virus, aseptic detection), umbilical cord tissue is saved into 4 DEG C of sterile salines, is sealed, cold chain transportation in 4h
To the workshop GMP；
(2) in Biohazard Safety Equipment, umbilical cord is gone in 10cm culture dish, is dipped in 2~5min of disinfection in 75~80% ethyl alcohol,
Sodium chloride injection cleans 3~5 times, removes bloodstain；Sterile scissors cut off the surgeon's knot binding of umbilical cord both ends, then by preparation navel
Band is cut to 2~3cm number section, moves towards to reject 2 arteria umbilicalis and 1 umbilical vein by blood vessel spiral；With pincers will be located at amnion with
Intervascular white tissues Wharton colloid strips down, and sodium chloride injection washing colloids 3~5 times；
(3) colloid of acquisition is transferred to 50ml centrifuge tube, is added 20~30ml sodium chloride injection, centrifugation 5~
10min, abandons supernatant, and weighing record is added 1~2ml culture medium, is cut with sterile tissue and cut colloid to 1~4mm3It organizes even
Lumps；Culture medium, which is added, makes tissue homogenate block concentration be set to 0.5~1.0g/ml, after electric pipettor piping and druming uniformly, each T175
It is inoculated with 2.33ml tissue homogenate, i.e. 1.17g Wharton colloid in culture bottle, adds 25ml culture medium；
(4) culture medium full dose being carried out after originally culture 5~6 days and changing liquid, half amount culture medium is sopped up after 9~10 days, is supplied
Amount fresh culture carries out half amount and changes liquid；According to cell growth state, culture carried out primary half amount again to 12~13 days and changes liquid, trained
It supports to the 14th~17 day, when cell clone rolls into a ball area percentage up to 80~85%, harvests cell (P0 generation) after being digested with pancreatin；
(5) cell is pressed 5~10 × 103/cm2Density be seeded in 2 layer cell factories (NEST, Cat No.771101)
Carry out expansion of stem cells culture, in which: the source of human stem cell of each 2 layer cell factory inoculation is in thin in same T175 culture bottle
Born of the same parents, each 2 layer cell factory inoculating cell sum are 6.395 × 106It is a, every layer addition cell suspension volume be 150~
200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity；After culture 3~5 days
When cell fusion to 90~95%, stem cell (P1 generation) is digested, is continued by 5~10 × 103/cm2Density be seeded to it is 10 layers thin
Expansion of stem cells culture is carried out in born of the same parents factory (NEST, Cat No.771302), in which: each 10 layer cell factory inoculation is done
Cell origin in the stem cell of same 2 layer cell factory culture, the total number of cells of each 10 layer cell factory inoculation are 3.17 ×
107A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With saturated humidity
Adhere-wall culture 3~5 days in incubator；When cell fusion to 90~95% after culture 3~5 days, stem cell (P2 generation) is digested, often
Quality Control detection is done in the stem cell sampling of a 10 layer cell factory production, then uses serum-free cell frozen stock solution (ZENOAQ, Cat
No:Cellbanker2 it) is resuspended, presses 5~10 × 106The density of cells/ml/ pipe is sub-packed in cell cryopreservation tube, is put at -80 DEG C
It sets at least 24 hours and is placed on deep-bed drying in liquid nitrogen container, establish stem cell seed bank (P2 generation), and press Chinese Pharmacopoeia (2015
Version) carry out seed bank calibrating；
(6) recovery stem cell, after centrifugation removes frozen stock solution, after sodium chloride injection washs cell precipitation 1 time, by 5~10
×103/cm2Density stem cell is inoculated in 2 layer cell factories progress expansion of stem cells culture, in which: each 2 confluent monolayer cells
In the cell in same cell cryopreservation tube, each 2 layer cell factory inoculating cell sum is the source of human stem cell of factory's inoculation
6.395×106A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2And saturation
Adhere-wall culture 3~5 days in the incubator of humidity；After culture 3~5 days when cell fusion is to 90~95%, stem cell is digested
After (P3 generation), piping and druming mix, single cell suspension is pressed 5~10 × 103/cm2Density be inoculated in 10 layer cell factories and done
Cell amplification cultivation, in which: the source of human stem cell of each 10 layer cell factory inoculation is in the dry thin of same 2 layer cell factory culture
The total number of cells of born of the same parents, each 10 layer cell factory inoculation are 3.17 × 107It is a, every layer addition cell suspension volume be 150~
200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity；To cell fusion degree
Reach 90~95%, stem cell is digested, recycles stem cell (P4 generation), the stem cell sampling of each 10 layer cell factory production,
Quality Control detection is done, be then resuspended with serum-free cell frozen stock solution (ZENOAQ, Cat No:Cellbanker2), by 5~10 ×
106The packing of cells/ml/ pipe, freezes, and establishes stem cell work library (P4 generation), and press (2015 editions) progress stem cells of Chinese Pharmacopoeia
The calibrating in work library.
The present invention, the stem cell are umbilical cord mesenchymal stem cells.
The present invention, the cell cryopreservation tube are to print to be pasted on cryopreservation tube after software editing, generation two dimensional code, often
Solencyte has a unique ID, and when recovery provides after barcode scanning is checked.
In the present invention, the culture medium is the serum-free mescenchymal stem cell culture medium for being added to 2% serum substitute,
I.e.: UltraCULTURE SF Medium (Lonza, Cat No:12-725F)+2%Ultroser G serum substitute (Pall,
Cat No:15950-017)。
Compared with the prior art, the present invention has the advantage that
1, the present invention replaces traditional T bottle training method, is having using cell factory come large scale preparation stem cell products
It maximum culture area is utilized in the space of limit, saves a large amount of hall space, be realized with a low cost and enhance production capacities
Purpose, and many experiments room technical staff can be saved, while avoiding operating a large amount of T175 trainings in narrow Biohazard Safety Equipment
The risk of bring cell contamination when supporting bottle, and the mescenchymal stem cell quantity produced is trained with the culture bottle of identical culture surface product
For feeding stem cell sum compared to there was no significant difference, Linear Amplifer will not change the dynamic conditions of cell growth, same cell
The stem cell produced in factory is able to maintain higher identity, avoids the difference of the stem cell between each bottle in a large amount of different culture bottles
The opposite sex, and can avoid the unstability of cell quality between each bottle.
2, the present invention has preferable stability, and the stem cell produced has still maintained good stem cell properties, will
It is applied to have good therapeutic effect in the treatment of some chronic diseases, while cell factory is set as 10 layers, can reach people
The maximum production (40 layer cell factories are not easy manual operation) of work operation, meets the production principle of three lot sample of biological products, is
The exploitation for the production technology that stem cell is declared as drug and commercial applications are laid a good foundation.
3, whole process using serum-free mescenchymal stem cell culture medium and adds when the present invention cultivates stem cell in cell factory
Increase serum substitute avoids ingredient in animal derived serum from causing the possibility of Human immune responses and animal derived pathogen contamination
Property, the safety of product is improved, and meet pharmacopoeial requirements.
Detailed description of the invention
Fig. 1 is that the UC-MSCs form under T175 culture bottle and cell factory culture compares, Figure 1A: T175 culture bottle culture
Stem cell；The stem cell of Figure 1B: 10 layer cell factory cultures；
Fig. 2 is the flow cytometer detection of the stem cell of 10 layer cell factory cultures as a result, Fig. 2 a:UC-MSCs CD19/CD34/
CD14 blank control；Fig. 2 b:UC-MSCs CD19/CD34/CD14 sample detection；Fig. 2 c:UC-MSCs CD45/HLA-DR blank
Control；D:UC-MSCs CD45/HLA-DR sample detection；
Fig. 3 is that the P4 of 10 layer cell factory cultures for stem cell three is Analytical Chemical Experiment as a result, Fig. 3 a:MSC differentiation front and back shape
State comparison, A: undifferentiated hUC-MSC (Day 0), Osteoblast Differentiation 6 days (Day6), 9 days (Day9) bright field forms
Figure, B: undifferentiated hUC-MSC (Day 0) breaks up 6 days (Day6), 9 days (Day9) bright field aspect graphs at rouge, C:
Undifferentiated hUC-MSC (Day 0), at cartilage differentiation 6 days (Day6), 9 days (Day9) bright field aspect graphs；Fig. 3 b:
HUC-MSC osteoblast differentiation and identification, A: undifferentiated hUC-MSC bright field aspect graph, B:hUC-MSC do not divide
When change, alizarin red S dyeing, the red S dyeing of C:hUC-MSC induction differentiation hystazarin；The induction point of Fig. 3 c:hUC-MSC chondroblast
Change identification, A: undifferentiated hUC-MSC bright field aspect graph, B: undifferentiated hUC-MSC alcian blue (Alcian blue)
Negative staining control, C: induction 14 days alcian blue (Alcian blue) coloration results (400 times) of differentiation, D: induction differentiation 12 days
Alcian blue (Alcian blue) coloration result (200 times)；Fig. 3 d:hUC-MSC lipoblast differentiation and identification, A-G:
After hUC-MSC breaks up 14 days at rouge, oil red O stain (100 times, 400 times), after H:hUC-MSC breaks up 14 days at rouge, it is seen that obvious
Fat drips form (100 times, 400 times)；
Fig. 4 such as is at the cell harvest yield comparison of the T175 culture bottle and 1 10 layer cell factory under culture areas；
Fig. 5 is Nanog (N), OCT-4 of the P4 for umbilical cord mesenchymal stem cells that RT-PCR detects 10 layer cell factory cultures
(O), SOX-2 (S) gene expression.
Specific embodiment
Further description of the technical solution of the present invention with reference to the accompanying drawing, and however, it is not limited to this, all to this
Inventive technique scheme is modified or replaced equivalently, and without departing from the spirit and scope of the technical solution of the present invention, should all be covered
Within the protection scope of the present invention.
Embodiment 1
Present embodiments provide it is a kind of establish safely and effectively, the methods of the enough cell banks of quantity, the cell in cell bank
Third party by profession is qualified, and presses Chinese Pharmacopoeia (2015 editions) progress stem cell seed bank/work library calibratings, and
And cell concentration can satisfy being continuing to supply for clinical research.
Umbilical cord tissue is obtained after newborn's birth, wherein donor has to pass through stringent physical examination, including every viral diagnosis,
Infectious disease detection, signs informed consent form and agreement of donation.In aseptic operating room, turn rapidly from the umbilical cord after human body fresh separated
It moves in 4 DEG C of sterile NaCl injections, seal, center is prepared by the cell that cold chain is transported to profession in 4h.
In the present embodiment, the specific establishment step of cell bank is as follows:
(1) umbilical cord tissue is transferred to the workshop drug rank GMP, is strictly handled, 75% ethanol disinfection, is transferred in advance
According in 30 minutes Biohazard Safety Equipments of ultraviolet light.
(2) in Biohazard Safety Equipment, umbilical cord is gone in 10cm culture dish, is dipped in 75% ethyl alcohol and sterilizes 2min, sodium chloride
Injection cleans 3 times, removes bloodstain.Sterile scissors cut off the surgeon's knot binding of umbilical cord both ends, then by preparation umbilical cord scissors to 2~
3cm number section walks formula by blood vessel spiral and rejects 2 arteria umbilicalis and 1 umbilical vein.With pincers will be located at amnion with it is intervascular white
Colour cell is knitted Wharton colloid and is stripped down, and sodium chloride injection washing colloids 3 times.
(3) colloid obtained is transferred to 50ml centrifuge tube, and 20~30ml sodium chloride injection is added, and 840g is centrifuged 5min,
Supernatant is abandoned, weighing record is added 1~2ml culture medium, is cut with sterile tissue and cut colloid to 1~4mm3Tissue homogenate block.Add
Enter appropriate culture medium and tissue homogenate block concentration is set to about 0.5g/ml, after electric pipettor piping and druming uniformly, each T175 culture bottle
Middle inoculation 2.33ml tissue homogenate, i.e. 1.17g Wharton colloid, add 25ml culture medium.
(4) culture medium full dose being carried out after originally culture 5~6 days and changing liquid, half amount culture medium is sopped up after 9~10 days, is supplied
Amount fresh culture carries out half amount and changes liquid.According to cell growth state, culture carried out primary half amount again to 12~13 days and changes liquid, trained
It supports to the 14th~17 day, when cell clone rolls into a ball area percentage up to 80% or so, with harvesting cell after the digestion of 0.125% pancreatin
(P0 generation).
(5) cell is pressed 5~10 × 103/cm2Density be seeded in 2 layer cell factories (NEST, Cat No.771101)
Carry out expansion of stem cells culture, in which: the source of human stem cell of each 2 layer cell factory inoculation is in thin in same T175 culture bottle
Born of the same parents, each 2 layer cell factory inoculating cell sum are 6.395 × 106It is a, every layer addition cell suspension volume be 150~
200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity；After culture 3~5 days
When cell fusion is to 90% or so, stem cell (P1 generation) is digested, is continued by 5~10 × 103/cm2Density be seeded to 10 layers
Expansion of stem cells culture is carried out in cell factory (NEST, Cat No.771302), in which: each 10 layer cell factory inoculation
For source of human stem cell in the stem cell of same 2 layer cell factory culture, the total number of cells of each 10 layer cell factory inoculation are 3.17
×107A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2And saturated humidity
Incubator in adhere-wall culture 3~5 days；After culture 3~5 days when cell fusion is to 90% or so, stem cell (P2 generation) is disappeared
Change, Quality Control detection is done in the stem cell sampling of each 10 layer cell factory production, then presses 5~10 × 106Cells/ml/ pipe
Density is resuspended with serum-free cell frozen stock solution (ZENOAQ, Cat No:Cellbanker2), is sub-packed in cell cryopreservation tube, is placed in
It is frozen at least 24 hours in program temperature reduction box at -80 DEG C and is placed on deep-bed drying in liquid nitrogen container, establish stem cell seed bank (P2
Generation), and by the calibrating of Chinese Pharmacopoeia (2015 editions) progress seed banks.
(6) recovery stem cell, after centrifugation removes frozen stock solution, after sodium chloride injection washs cell precipitation 1 time, by 5~10
×103/cm2Density stem cell is inoculated in 2 layer cell factories progress expansion of stem cells culture, in which: each 2 confluent monolayer cells
In the cell in same cell cryopreservation tube, each 2 layer cell factory inoculating cell sum is the source of human stem cell of factory's inoculation
6.395×106A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2And saturation
Adhere-wall culture 3~5 days in the incubator of humidity；After culture 3~5 days when cell fusion is to 90% or so, stem cell is digested
After (P3 generation), piping and druming mix, single cell suspension is pressed 5~10 × 103/cm2Density be inoculated in 10 layer cell factories and done
Cell amplification cultivation, in which: the source of human stem cell of each 10 layer cell factory inoculation is in the dry thin of same 2 layer cell factory culture
The total number of cells of born of the same parents, each 10 layer cell factory inoculation are 3.17 × 107It is a, every layer addition cell suspension volume be 150~
200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity；To cell fusion degree
When reaching 90% or so, pancreatin digestion attracts recycling stem cell (P4 generation) with negative pressure of vacuum pipeline, and each 10 layer cell factory is raw
Quality Control detection is done in the stem cell sampling of production, and then with serum-free cell frozen stock solution, (ZENOAQ, Cat No:Cellbanker2 press 5
~10 × 106Cells/ml/ pipe is resuspended, dispenses, and freezes, and establishes stem cell work library (P4 generation), and press Chinese Pharmacopoeia (2015
Version) carry out stem cell work library calibrating.
In the present embodiment, the stem cell is umbilical cord mesenchymal stem cells；The culture medium used is UltraCULTURE SF
Medium+2%Ultroser G serum substitute.Stem cell special culture media+serum substitute can utmostly keep dry thin
The undifferentiated state of born of the same parents avoids animal derived pathogen contamination and human body caused by fetal calf serum is added for animal-based protein
Immune response, prepare for clinical application.
In the present embodiment, the cell cryopreservation tube used is to print to be pasted on after software editing, generation two dimensional code to freeze
Guan Shang, every solencyte have a unique ID, and when recovery provides after barcode scanning is checked.
In the present embodiment, the inoculum density of each stem cell, which is controlled, can reach 90~95% degrees of fusion at 30%, 3~5 day,
Midway is without changing liquid.
Embodiment 2
Another important technology difficult point of the invention is a large amount of preparations and the stem cell of clinical grade umbilical cord mesenchymal stem cells
Quality Identification, after the main large scale preparation for carrying out cell products using cell factory, each 10 layer cell factory production is done
Cell sampling send qualified third party to do Quality Control detection, and applies Flow cytometry stem cell surface marker, simultaneously
Embryonic stem cell molecular marker is identified using RT-PCR method, and detecting stem cell three is differentiation capability, it is international by 2006
The mesenchyma that cell therapy association (International Society for Cellular Therapy, ISCT) formulates is dry thin
After the minimum standard of perfection of born of the same parents carries out dry cell mass identification, clinical grade stem cell products are finally prepared.
In the present embodiment, using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, specific step is as follows:
(1) 1 is taken to fill 5~10 × 10 in stem cell seed bank6The P2 of cells/ml/ pipe is dry thin for umbilical cord mesenchyma
Born of the same parents, by 5~10 × 10 after being recovered3/cm2Density be inoculated into 12 layer cell factory (NEST, Cat No.771101)
Expansion of stem cells culture is carried out, stem cell is made to be expanded to certain amount (P3 generation), in which: each 2 layer cell factory inoculation
For source of human stem cell in the cell in same branch cell cryopreservation tube, each 2 layer cell factory inoculating cell sum is 6.395 × 106
A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With the training of saturated humidity
It supports in case adhere-wall culture 3~5 days；
(2) when cell fusion degree is up to 90% or so, the stem cell of 2 layer cell factories is digested, by 5~10 ×
103/cm2Density be inoculated into 1 10 layer cell factory progress expansion of stem cells culture, in which: each 10 layer cell factory connects
Kind source of human stem cell in the stem cell of same 2 layer cell factory culture, the total number of cells of each 10 layer cell factory inoculation are
3.17×107A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2And saturation
Adhere-wall culture 3~5 days in the incubator of humidity；
(3) when cell fusion degree reaches 90% or so in 10 layer cell factories, pancreatin digestion is inhaled with negative pressure of vacuum pipeline
It leads back and receives stem cell (P4 generation), obtain a large amount of stem cell.
In the present embodiment, culture medium used in incubation is UltraCULTURE SF Medium+2%Ultroser
G serum substitute.It is whole in incubation to use mescenchymal stem cell culture medium and add serum substitute, avoid clinical application
In in animal derived serum ingredient potential insecurity (such as virus, fungal contamination, animal-based protein).
2006, international cell therapy association (International Society for Cellular Therapy,
ISCT the standard of perfection of mescenchymal stem cell) has been determined are as follows:
(1) adherent growth in plastic culture dish, in the culture medium inner height proliferation containing serum；
(2) expression cell surface marker CD105, CD73, CD90 do not express CD45, CD34, CD14 or CD11b, CD79a
Or CD19；
(3) it can be divided into osteoblast, fat cell, cartilage cell in vitro.
The present embodiment carries out the phenotypic evaluation of stem cell according to this minimum standard.
After stem cells hyperplasia is fused to 90% in 10 layer cell factories, cell is observed using multi-layer cellular incubator observation platform
Form, as shown in Figure 1B, Figure 1A are the stem cell morphology of T175 culture bottle culture, be can be seen that both from Figure 1A and Figure 1B
In adherent growth, shuttle shape is normal stem cell morphology, and the stem cell of indifference, cell factory production meets mescenchymal stem cell
Minimum standard of perfection morphological criteria.
Fig. 2 the results show that stem cell CD73+, CD90+, CD105+ >=95.0%, CD14- of cell factory culture,
CD19-, CD34-, CD45-, HLA-DR-≤2.0% meet the identification of the cell surface marker in the minimum standard of stem cell
Standard.
Fig. 3 the results show that stem cell under Incubation Condition can using directed differentiation as osteoblast, chondroblast,
Lipoblast meets the three systems differentiation standard in the minimum standard of perfection of stem cell.
Stem cell collection in 10 layer cell factories is counted, while the cell harvest yield in order to be cultivated with conventional T175 bottles
It compares, equally stem cell (1 NEST10 confluent monolayer cells in the T175 bottle of collection same culture conditions, identical culture surface product
Factory=36.2 T175 culture bottle), two groups of parallel testings, counting compares.As shown in figure 4, under identical culture surface product, 10
The cell harvest yield of layer cell factory is close with T bottles, and there was no significant difference between two groups (P > 0.05), while having saved mass propgation
Space and human cost.And Quality Control detection, phase are done since the stem cell of each 10 layer cell factory production need to only sample portion
The cell of than 36.2 T175 culture bottles production needs every bottle of sampling to do Quality Control detection, greatly reduce the workload of Quality Control personnel with
The use of reagent consumptive material.
Fig. 5 is the results show that the P4 of cell factory production is dry for embryos such as stem cell strongly expressed Oct-4, Nanog and Sox-2
The culture of cell factory is passed through in cell sign object, display, and stem cell still maintains its good undifferentiated state, answers for its clinic
With providing reliable basis.
Claims (10)
1. a kind of technique using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, it is characterised in that the technique includes
Following steps:
Step (1), cell inoculation:
1 is taken to fill 5~10 × 10 in stem cell seed bank6The stem cell of cells/ml/ pipe, after being recovered by 5~10 ×
103cells/cm2Density be inoculated into 12 layer cell factory progress expansion of stem cells culture；
Step (2), cell factory prepare stem cell:
When cell fusion degree is up to 90~95%, the stem cell of 2 layer cell factories is digested, by 5~10 × 103cells/
cm2Density be inoculated into 1 10 layer cell factory progress expansion of stem cells culture；
Step (3), stem cell products preparation:
When cell fusion degree reaches 90~95% in 10 layer cell factories, stem cell is digested, is obtained largely dry thin
Born of the same parents.
2. the technique according to claim 1 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, feature
It is in the step (1), stem cell is P2 for umbilical cord mesenchymal stem cells.
3. the technique according to claim 1 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, feature
Be in the step (2), the source of human stem cell of each 2 layer cell factory inoculation in the cell in same branch cell cryopreservation tube,
Each 2 layer cell factory inoculating cell sum is 6.395 × 106A, the cell suspension volume of every layer of addition is 150~200ml,
Condition of culture are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity；In the step (3), each
In the stem cell of same 2 layer cell factory culture, each 10 layer cell factory connects the source of human stem cell of 10 layer cell factories inoculation
The total number of cells of kind are 3.17 × 107A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: 37
DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity.
4. the technique according to claim 1 or 3 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, special
Sign is in the step (2) and step (3) that stem cell is in 2 layer cell factories and 10 layer cell factory culture, inoculum density
Control makes to reach within stem cell 3~5 days 90~95% degrees of fusion, centre is without changing liquid 30%.
5. the technique according to claim 1 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, feature
It is that the technique further includes step (4): by the stem cell sampling of 10 layer cell factory each in step (3) production, does Quality Control
After detection, it is resuspended using with serum-free cell frozen stock solution, then presses 5~10 × 106The effective cell cryopreservation tube of cells/ml/ carries out
Packing is placed at least 24 hours at -80 DEG C using program temperature reduction box and is stored in postposition liquid nitrogen container, and stem cell work library is established.
6. the technique according to claim 1 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, feature
It is that culture medium used in the incubation is the serum-free mescenchymal stem cell culture for being added to 2% serum substitute
Base.
7. the technique according to claim 1 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, feature
It is that the method for building up of the stem cell seed bank is as follows:
(1) obtain the umbilical cord tissue qualified through detection under aseptic condition, while taking 10ml Cord blood inspection, umbilical cord tissue save to
In 4 DEG C of sterile salines, sealing, cold chain transportation is to the workshop GMP in 4h；
(2) in Biohazard Safety Equipment, umbilical cord is gone in 10cm culture dish, is dipped in 2~5min of disinfection, chlorination in 75~80% ethyl alcohol
Sodium injection cleans 3~5 times, removes bloodstain；Sterile scissors cut off the surgeon's knot binding of umbilical cord both ends, then by preparation umbilical cord scissors
To 2~3cm number section, move towards to reject 2 arteria umbilicalis and 1 umbilical vein by blood vessel spiral；Amnion and blood vessel will be located at pincers
Between white tissues Wharton colloid strip down, sodium chloride injection washing colloids 3~5 times；
(3) colloid of acquisition is transferred to 50ml centrifuge tube, 20~30ml sodium chloride injection is added, be centrifuged 5~10min, abandoned
Supernatant, weighing record, is added 1~2ml culture medium, is cut with sterile tissue and cut colloid to 1~4mm3Tissue homogenate block；It is added
Culture medium makes that homogenate block concentration will be organized to be set to 0.5~1.0g/ml, after electric pipettor piping and druming uniformly, each T175 culture bottle
Middle inoculation 2.33ml tissue homogenate, adds 25ml culture medium；
(4) culture medium full dose being carried out after originally culture 5~6 days and changing liquid, half amount culture medium is sopped up after 9~10 days, it is new to supply equivalent
Fresh culture medium carries out half amount and changes liquid；According to cell growth state, culture carried out primary half amount again to 12~13 days and changes liquid, and culture is extremely
14th~17 day, when cell clone rolls into a ball area percentage up to 80~85%, cell was harvested after being digested with pancreatin；
(5) cell is pressed 5~10 × 103/cm2Density be seeded in 2 layer cell factories progress expansion of stem cells culture；Culture 3
When cell fusion to 90~95% after~5 days, stem cell is digested, is continued by 5~10 × 103/cm2Density be seeded to 10 layers
Expansion of stem cells culture is carried out in cell factory；When cell fusion to 90~95% after culture 3~5 days, stem cell is digested, often
Quality Control detection is done in the stem cell sampling of a 10 layer cell factory production, is then resuspended with serum-free cell frozen stock solution, is pressed 5~10
×106The density of cells/ml/ pipe is sub-packed in cell cryopreservation tube, is placed at least 24 hours and is placed in liquid nitrogen container at -80 DEG C
Deep-bed drying establishes stem cell seed bank, and carries out the calibrating of seed bank；
(6) recovery stem cell, after centrifugation removes frozen stock solution, after sodium chloride injection washs cell precipitation 1 time, by 5~10 × 103/
cm2Density stem cell is inoculated in 2 layer cell factories progress expansion of stem cells culture；Culture worked as cell fusion after 3~5 days
When to 90~95%, after stem cell digestion, piping and druming are mixed, single cell suspension is pressed 5~10 × 103/cm2Density be inoculated in
Expansion of stem cells culture is carried out in 10 layer cell factories；Reach 90~95% to cell fusion degree, stem cell is digested, recycling is dry
Quality Control detection is done in cell, the stem cell sampling of each 10 layer cell factory production, be then resuspended with serum-free cell frozen stock solution,
By 5~10 × 106The packing of cells/ml/ pipe, freezes, and establishes stem cell work library, and carry out the calibrating in stem cell work library.
8. the technique according to claim 7 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, feature
It is in the step (5), the source of human stem cell of each 2 layer cell factory inoculation is in the cell in same T175 culture bottle, often
A 2 layer cell factory inoculating cell sum is 6.395 × 106A, the cell suspension volume of every layer of addition is 150~200ml, training
The condition of supporting are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity；Each 10 layer cell factory inoculation
Source of human stem cell in the stem cell of same 2 layer cell factory culture, the total number of cells of each 10 layer cell factory inoculation are
3.17×107A, the cell suspension volume of every layer of addition is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2And saturation
Adhere-wall culture 3~5 days in the incubator of humidity；In the step (6), the source of human stem cell of each 2 layer cell factory inoculation in
Cell in same cell cryopreservation tube, each 2 layer cell factory inoculating cell sum are 6.395 × 106It is a, every layer addition it is thin
Born of the same parents' suspension volume is 150~200ml, condition of culture are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3 in the incubator of saturated humidity
~5 days；The source of human stem cell of each 10 layer cell factory inoculation is in the stem cell of same 2 layer cell factory culture, and each 10 layers
The total number of cells of cell factory inoculation are 3.17 × 107A, the cell suspension volume of every layer of addition is 150~200ml, cultivates item
Part are as follows: in 37 DEG C, 5%CO2With adhere-wall culture 3~5 days in the incubator of saturated humidity.
9. the technique of clinical grade umbilical cord mesenchymal stem cells is prepared using cell factory according to claim 5,7 or 8,
It is characterized in that the cell cryopreservation tube is to print to be pasted on cryopreservation tube after software editing, generation two dimensional code, every solencyte
There is a unique ID, when recovery provides after barcode scanning is checked.
10. the technique according to claim 7 using cell factory preparation clinical grade umbilical cord mesenchymal stem cells, feature
It is that the culture medium is the serum-free mescenchymal stem cell culture medium for being added to 2% serum substitute.
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