CN109385396A - Clinical application grade umbilical cord mesenchymal stem cells and its method for separating and preparing - Google Patents
Clinical application grade umbilical cord mesenchymal stem cells and its method for separating and preparing Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Abstract
The invention belongs to technical field of bioengineering, and in particular to the method for obtaining high-purity, high vigor mescenchymal stem cell is separated from umbilical cord China Tong Shi glue.This method obtains umbilical cord tissue by screening, sterilized cleaning, the separation of China Tong Shi glue, tissue block preparation, tissue block inoculation, cell primary culture and secondary culture, the umbilical cord mesenchymal stem cells of acquisition high-purity, high vigor.The present invention makes umbilical cord mesenchymal stem cells production technology more standards and specifications, especially suitable for clinic.
Description
Technical field
The invention belongs to technical field of bioengineering, it is related specifically between separation clinical application grade in umbilical cord China Tong Shi glue
The method of mesenchymal stem cells.
Background technique
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is present in a variety of groups of marrow, fat, umbilical cord etc.
It knits in organ, because it is with height self-renewing and multi-lineage potential, in clinical hematopoiesis support, promotes stem cell implantation and exempt from
The application of epidemic disease regulation etc. is increasingly subject to the concern of people.In burn, fracture, defect Soft-tissue operation, End-stage liver disease, the heart
The treatment of the wounds such as flesh infarct, diabetes or disease confirms its safety and validity.
Umbilical cord mesenchymal stem cells (umbilical cord mesenchymal stem cells, UC-MSCs) refer to and deposit
It is one of neonatal umbilical cord tissue versatile stem cell, derives from the mesoderm and ectoderm of mesoderm growing early stage.Umbilical cord
Abandon after being taken from perinatal period health puerpera production, materials are convenient, do not influence on puerpera and infantile health, ethics is striven
It discusses small.Umbilical cord is better than marrow in terms of mescenchymal stem cell content and proliferative capacity, and the cellular immunity in umbilical cord source is former
Property is lower than marrow.Umbilical cord mesenchymal stem cells have the possibility of infinite multiplication, keep caryogram normal within external 12 generation and telomere
Enzymatic activity illustrates umbilical cord mesenchymal stem cells without oncogenicity.Its surface antigen is unobvious, does not express MHC- class Ⅱ antigens, low table
Up to MHC- class Ⅰ antigens, allograft rejection is lighter, and clinical use is not necessarily to distribution type.Umbilical cord mesenchymal stem cells can inhibit T, B, NK,
The immunoregulation effect of DC cell, alloimmune originality and immunological rejection are very low.Umbilical cord mesenchymal stem cells can be divided into
Various Tissues cell can be used as seed cell and be stored, for repairing for injuries of tissues and organs caused by aging and lesion etc.
It is multiple.Can secretion of VEGF, the cell factors such as bFGF, HGF, promote revascularization, the neurotrophies such as NGF secretion, BDGF, G-CSF because
Son promotes the Regeneration and Repair of nerve cell.
Currently, having multiple unit applications umbilical cord mesenchymal stem cells separates relevant patent, wherein having using collagen
Enzyme or compound enzymic digestion carry out cell separation (ZL201611173913.9, ZL201611174018.9,
ZL201611037484.2, ZL201610910565.2, ZL201510395470.7 etc.), have and is cut after umbilical cord is carried out blood vessel
It is broken or carry out after directly shredding the adherent acquisition cell of tissue (ZL201610910134.6, ZL201410751410.X,
ZL201410515345.0, ZL201010605542.3, ZL201410515345.0, ZL201510191059.8 etc.), also have logical
Cross separation China's Tong Shi glue shred rear adherent acquisition cell (ZL201710067279.9, ZL201610615998.5,
ZL201610311244.0,ZL201510945799.6,ZL201510423138.7).The time of cell is obtained by enzymic digestion
It is shorter, but enzyme can have a certain impact to the vigor of cell.Directly shred adherent easy to operate, but the purity of cell is low, unfavorable
In the cell for obtaining purity is high.Separation China's Tong Shi glue shreds adherent method, obtains cell purity height, but the time that cell climbs out of
It is longer.Therefore, it is necessary to improve to the method that umbilical cord is handled, it can just get that vigor is stronger, the higher cell of purity.
Summary of the invention
It is described it is a primary object of the present invention to establish the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells
Magnificent Tong Shi glue of the umbilical cord mesenchymal stem cells from separation.
Technical scheme is as follows:
The present invention provides the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells, and the method includes following steps
It is rapid:
(1) obtaining source of people specificity virus blood examination report result is negative umbilical cord；
(2) after taking tissue preserration liquid to carry out detection of mycoplasma, umbilical cord is handled, separates China's Tong Shi glue, and sufficiently washed with physiological saline
Wash magnificent Tong Shi glue；
(3) magnificent Tong Shi glue is shredded with sterile scissors, it is dry thin using tissue adherent method culture umbilical cord mesenchyma after washing centrifugation
Born of the same parents after digestive ferment progress digestion process is added, is collected by filtration single cell suspension, obtains when cell fusion degree reaches 80% or more
Obtain the primary cell of umbilical cord mesenchymal stem cells.
Above-mentioned preparation method is further comprising the steps of:
(4) primary cell for the umbilical cord mesenchymal stem cells that step (3) obtain is frozen.
Above-mentioned blood examination report project includes AIDS virus (HIV), hepatitis B (HBV), hepatitis C virus (HCV), treponemal
Body (TP), nerpes vinrus hominis (EB) and cytomegalovirus (CMV) source of people specificity virus.
Above-mentioned blood examination is reported as the blood examination report of puerpera's peripheral blood and Cord blood.
Detection of mycoplasma result is feminine gender in above-mentioned steps (2), washs umbilical cord with phosphate buffer, the phosphate is slow
Fliud flushing contains 100,000 units/L Aminopenicillin and the streptomycin sulphate of 100mg/L；Step (3) described digestive ferment is
0.125% trypsase.
The magnificent Tong Shi glue that removing obtains first is transferred to centrifuge tube in above-mentioned steps (3) and carries out centrifugal treating, takes upper layer
Cleaning solution carries out Sterility testing；Magnificent Tong Shi glue is cut into 1-3mm with sterile scissors again3Multiple tissue blocks of size, the China are logical
Before the inoculation of family name's glue tissue block, the special culture media washing of culture umbilical cord mesenchymal stem cells is added, after centrifugation, takes upper layer culture medium
Sterility testing is carried out, lower-hierarchy block is for being inoculated with.
Above-mentioned steps (3) are added dedicated with the culture umbilical cord mesenchymal stem cells of tissue block volume equivalent into tissue block
Culture medium, and sufficiently piping and druming is uniformly mixed；By tissue suspension according to the every 75cm of 4ml tissue suspension2The ratio of culture bottle connects respectively
Kind into several culture bottles, wait organize it is adherent after, then add isometric special culture media.
Above-mentioned steps (3) cell culture condition is 37 DEG C, 5%CO2；When culture medium colour changed into yellow in the culture bottle,
Replace full dose culture medium.
Above-mentioned culture medium is serum free medium.
Above-mentioned virus and detection of mycoplasma result are feminine gender.
The clinical application grade umbilical cord that the method for separating and preparing of above-mentioned clinical application grade umbilical cord mesenchymal stem cells is prepared
Mescenchymal stem cell.
Above-mentioned stem cell is the uniform spindle shape cell of form, and the positive ratio of CD73, CD90, CD105 are more than or equal to
95%, the positive ratio that CD34, CD45, HLA-DR are is less than or equal to 2%.
The above method is further comprising the steps of (5): the primary cell for the umbilical cord mesenchymal stem cells that step (3) are obtained is again
It after secondary resuspension, is inoculated in culture bottle, carries out had digestive transfer culture culture, density is 6000-8000/cm when cell secondary culture2。
In above-mentioned steps (5) when secondary culture, when cell fusion degree reaches 90%, the interior addition digestive ferment into culture bottle
Processing, then terminated with special culture media；Piping and druming bottom of bottle is until cell and culture bottle disengaging, are trained with brine repeatedly
Bottle is supported, cleaning solution is collected and is mixed with cell suspension, sample and count after constant volume；After cell suspension centrifugation, supernatant is abandoned, with dedicated culture
Base weight hangs cell, and according to certain cell density, cell suspension is assigned in several culture bottles, and mixing is placed on training in incubator
It supports, through had digestive transfer culture culture repeatedly, culture is to P2-P4 for umbilical cord mesenchymal stem cells.
Above-mentioned method for separating and preparing is further comprising the steps of:
(6) the primary freeze-stored cell for the umbilical cord mesenchymal stem cells for obtaining step (4), secondary culture after recovery, inoculum density
For in 6000-8000/cm2。
In above-mentioned steps (6) when freeze-stored cell recovery culture, primary cell is taken out from cell bank, is transferred to 37 DEG C of water rapidly
Recovery cell in bath；After cell is completely dissolved, it is transferred to Biohazard Safety Equipment, with brine cell, is used after centrifugation special
It is resuspended with culture medium, according to certain cell density, cell suspension is assigned in several culture bottles, mixing is placed in incubator
Culture, cell can be cultivated to P2-P4 through had digestive transfer culture culture repeatedly for umbilical cord mesenchymal stem cells.
The clinical application grade umbilical cord that the method for separating and preparing of above-mentioned clinical application grade umbilical cord mesenchymal stem cells is prepared
Mescenchymal stem cell.
Above-mentioned cell is the uniform spindle shape cell of form, and the positive ratio of CD73, CD90, CD105 are more than or equal to
95%, the positive ratio that CD34, CD45, HLA-DR are is less than or equal to 2%.
The screening of umbilical cord tissue: the umbilical cord of screening young healthy puerpera comes as the main material of separating mesenchymal stem cell
Source, and the blood examination of puerpera's peripheral blood and Cord blood is reported and carries out screening, it is ensured that every source of people specificity virus is feminine gender.
The separation of umbilical cord mesenchymal stem cells: the umbilical cord tissue of both ends ligation is taken out from storage and transportation bottle, uses phosphate-buffered
Liquid cleans umbilical cord tissue, and from ligation place inside, 1-2 centimeters cut umbilical cord, discard the umbilical cord tissue of ligation；Remaining umbilical cord group
Several sections that truncation is about 5 centimetres of sizes are knitted, is placed in phosphate buffer and cleans, until blood stains are cleaned substantially；With sterile medical
Scissors is cut off in tissue ends, then is gently torn apart umbilical cord with tweezers, and is walked formula by the spiral of blood vessel and rejected 2 arteries and 1
Vein, tries not to pull apart blood vessel；Magnificent Tong Shi glue is torn with buckle tweezers with teeth, is put into sterilized petri dishes, uses physiology salt
Water washing China Tong Shi glue, until colloid becomes pure white；Magnificent Tong Shi glue is transferred in plate or centrifuge tube again, is taken after centrifugation
It is clear to carry out Sterility testing, then magnificent Tong Shi glue is cut into 1-3mm with sterile scissors3Tissue block, with culture medium wash resuspension after, connect
Kind is into culture bottle, according to the method culture of tissue block adherent culture.
The culture of umbilical cord mesenchymal stem cells: tissue adherent method culture umbilical cord mesenchymal stem cells are reached to cell fusion degree
To after 80-90%, had digestive transfer culture culture is carried out, passage density is 6000-8000/cm2；Each stage of cell culture needs
Sterile, mycoplasma is carried out to cell and endotoxin detects, continue to cultivate after qualified；When cell reach P2 for when, take appropriate thin
Born of the same parents carry out Phenotypic examination can be using cell as seed cell after phenotypic results meet the minimum standard of international cell therapy association
It freezes in liquid nitrogen and saves for a long time.
Advantages of the present invention:
That the present invention provides a kind of pair of cellular damages is small, with high purity, entirely from magnificent Tong Shi glue umbilical cord mesenchyma it is dry thin
The preparation method of born of the same parents, at least has the advantage that compared with prior art
Umbilical cord screening process of the invention, can be effectively reduced the pollution of humanized's virus, be acquisition high quality, available
It is effectively ensured in clinical umbilical cord mesenchymal stem cells offer.
The present invention does not use any digestive ferment during separating funicle mesenchyme stem cell primary cell, obtained thin
Born of the same parents are free of any heterologous protein, safer, can satisfy clinical demand, also provide technology guarantor to establish umbilical cord stem cells library
Barrier.
The present invention uses tissue adherent method, and mescenchymal stem cell is actively climbed out of from tissue block, and cell viability is strong, and is reduced
Isolated cost.
The present invention using the method for removing China's Tong Shi glue, effectively removes blood vessel and umbilical cord amniotic membrane tissue during the separation process,
The heteroproteose cells such as vascular endothelial cell, amniotic epithelial cells are reduced, the purity of umbilical cord mesenchymal stem cells is effectively improved.
Detailed description of the invention
The human umbilical cord mesenchymal stem cells aspect graph that Fig. 1 is cultivated by the specific embodiment of the invention；
The human umbilical cord mesenchymal stem cells growth activity period map that Fig. 2 is cultivated by the specific embodiment of the invention；
The human umbilical cord mesenchymal stem cells Phenotypic examination streaming figure that Fig. 3 is cultivated by the specific embodiment of the invention；
The human umbilical cord mesenchymal stem cells that Fig. 4 is cultivated by the specific embodiment of the invention at rouge, skeletonization, at chondrocyte induction
Differentiation figure.
Specific embodiment
With reference to the accompanying drawing and by specific embodiment to further illustrate the technical scheme of the present invention.Of the invention
Technical solution will all describe clearer.But as examples are merely exemplary, the scope of the present invention is not constituted
Any restrictions.Relevant technical staff in the field should be understood that without departing from the spirit and scope of the invention, all similar
Replacement and change be intended to be included in the scope of the present invention.Biochemical reagents used in embodiment are commercially available.
Embodiment: human umbilical cord mesenchymal stem cells are separately cultured
The separation of magnificent Tong Shi glue: opening constant temperature storage box, observes Hygrothermograph in case, it is ensured that the entire transportational process of umbilical cord
In safe slave mode.Umbilical cord storage and transportation bottle is taken out, after the situations such as observation well-tended appearance, no liquid leakage, uses medical wine
Essence is placed into Biohazard Safety Equipment the processing that carries out disinfection outside storage and transportation bottle.Two are taken out from storage and transportation bottle using aseptic nipper
The umbilical cord tissue of head ligation, is placed in the sterile petri dish of 15mm, and from ligation place inside, 1-2 centimeters cut umbilical cord, discard
The umbilical cord tissue of ligation.Remaining umbilical cord tissue is cut to several sections of about 5 centimetres of sizes, is placed in phosphate buffer clear
It washes, removal blood clot etc., repeated washing is until blood stains are cleaned substantially.It is cut off with sterile medical scissors in tissue ends, then uses tweezer
Son gently tears apart umbilical cord, and walks formula by the spiral of blood vessel and reject 2 arteries and 1 vein, tries not to pull apart blood vessel.This
When, magnificent Tong Shi glue is torn using long handle sawtooth buckle tweezers, is put into sterilized petri dishes by the exposure safely of magnificent Tong Shi glue.
The adhere-wall culture of tissue block: the magnificent Tong Shi glue in sterilized petri dishes is washed using physiological saline, until colloid
Become pure white.It shifts in China's Tong Shi glue to 50ml centrifuge tube, adding physiological saline to total volume is 40ml.It turns upside down centrifugation
Pipe, after be centrifuged with 600g, 5min.It draws supernatant liquid and carries out Sterility testing, then be cut into magnificent Tong Shi glue with sterile scissors
1-3mm3The tissue block of size, after being resuspended with the isometric serum free medium of magnificent Tong Shi glue, every 4ml tissue fluid is seeded to one
Culture bottle is placed into 37 DEG C, 5%CO by T75 culture bottle2The adhere-wall culture of tissue block is carried out in incubator.
The originally culture of umbilical cord mesenchymal stem cells: liquid is changed in the 3-7 days progress full doses of tissue block adherent culture, is most
It is likely to reduced tissue block to slide because of frequent operation, handle with care when changing liquid, culture bottle slightly lifts, and culture medium is allowed to flow into bottom
Abandoning is inhaled, then new culture medium is added by 4mL/ bottles in portion.The 10-14 days or so under the microscope, 50% or more tissue block week
It encloses and climbs out of cell and meromixis degree after 80%-90%, carry out cell dissociation passage.
The secondary culture of umbilical cord mesenchymal stem cells: when cell needs to pass in culture bottle, preparation disappears to cell
Change processing.0.125% trypsase is added into culture bottle, room temperature should be restored before digestive ferment use.Every bottle of addition digestive ferment
2-5ml, digestion time 1.5-3min.After digestion, the culture medium that 5ml is added into culture bottle terminates digestion.Use liquid relief
Pipe collects cell suspension into 50ml centrifuge tube, primary using brine.After cell washs, 10-40ml is used
Physiological saline is resuspended, and sampling counts cell.With 6000-8000/cm2Density passed on, cell culture
Each stage require to carry out cell sterile, mycoplasma and endotoxin detection, continue to cultivate after qualified.P2 is reached to cell
Dai Shi, taking appropriate cell to carry out Phenotypic examination can be by cell after phenotypic results meet the minimum standard of international cell therapy association
It freezes in liquid nitrogen as seed cell and saves for a long time.
As a result: by handling umbilical cord, we have obtained the uniform spindle shape cell (Fig. 1) of form, to cell into
Row streaming phenotypic analysis, wherein the positive rate of CD73, CD90 and CD105 are far longer than 95%, the sun of CD34, CD45 and HLA-DR
Property rate be far smaller than 2% (Fig. 3), meet the minimum standard about mescenchymal stem cell of international cell therapy association setting.
The P2 of culture is collected for cell, the detection of cell cycle is carried out using flow cytometer, testing result is as schemed, cell
Period profile be the G0/G1 phase be 89.9%, the S phase be 8.1% 1.4%, the G2/M phase.It is not less than 80% in G0/G1 phase cell,
Cell in the phase has differentiation effect, can be used for realizing therapeutic effect.
Induction differentiation potential experiment is carried out to the obtained umbilical cord mesenchymal stem cells of culture, mainly investigate to fat cell,
The potential of osteoblast, Chondrocyte Differentiation, experimental result is respectively as Fig. 4 is from left to right shown, it was demonstrated that acquired in this explanation
The potential that umbilical cord mesenchymal stem cells have stem cell properties and break up to adult cell.
Claims (20)
1. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells, which comprises the following steps:
(1) obtaining source of people specificity virus blood examination report result is negative umbilical cord；
(2) after taking tissue preserration liquid to carry out detection of mycoplasma, umbilical cord is handled, separates China's Tong Shi glue, and sufficiently washed with physiological saline
Wash magnificent Tong Shi glue；
(3) magnificent Tong Shi glue is shredded with sterile scissors, it is dry thin using tissue adherent method culture umbilical cord mesenchyma after washing centrifugation
Born of the same parents after digestive ferment progress digestion process is added, is collected by filtration single cell suspension, obtains when cell fusion degree reaches 80% or more
Obtain the primary cell of umbilical cord mesenchymal stem cells.
2. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as described in claim 1, which is characterized in that also wrap
Include following steps:
(4) primary cell for the umbilical cord mesenchymal stem cells that step (3) obtain is frozen.
3. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 1 or 2, which is characterized in that
The blood examination report project includes HIV, HBV, HCV, TP, EB and CMV source of people specificity virus.
4. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 3, which is characterized in that described
Blood examination is reported as the blood examination report of puerpera's peripheral blood and Cord blood.
5. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 1 or 2, which is characterized in that
Detection of mycoplasma result is feminine gender in step (2), washs umbilical cord with phosphate buffer, the phosphate buffer contains 100,000
The streptomycin sulphate of unit/L Aminopenicillin and 100mg/L；The trypsase that step (3) digestive ferment is 0.125%.
6. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as described in right wants 5, which is characterized in that
First magnificent Tong Shi glue is transferred in centrifuge tube in step (3) and is centrifuged, the cleaning solution on upper layer is taken to carry out Sterility testing；It again will be magnificent
Tong Shi glue is cut into 1-3mm with sterile scissors3Culture umbilical cord is added before China's Tong Shi glue tissue block inoculation in the tissue block of size
The special culture media of mescenchymal stem cell washs, and after centrifugation, takes upper layer culture medium to carry out Sterility testing, lower-hierarchy block is for connecing
Kind.
7. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 6, which is characterized in that
The special culture media with the culture umbilical cord mesenchymal stem cells of tissue block volume equivalent is added into tissue block for step (3),
And sufficiently piping and druming is uniformly mixed；By tissue suspension according to the every 75cm of 4ml tissue suspension2The ratio of culture bottle is seeded to several respectively
In a culture bottle, wait organize it is adherent after, then add isometric special culture media.
8. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claims 6 or 7, which is characterized in that
Step (3) cell culture condition is 37 DEG C, 5%CO2；When culture medium colour changed into yellow in the culture bottle, replacement full dose training
Support base.
9. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells, feature exist as described in claim 1,2,6 or 7
In the culture medium is serum free medium.
10. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells, feature as described in claim 1,4,6 or 7
It is, the virus, mycoplasma or Sterility testing result are feminine gender.
11. the clinic that the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 9 is prepared
Application layer umbilical cord mesenchymal stem cells.
12. the clinic that the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 10 is prepared
Application layer umbilical cord mesenchymal stem cells.
13. the clinical application grade umbilical cord mesenchymal stem cells as described in claim 11 or 12, which is characterized in that the cell is shape
The uniform spindle shape cell of state, and the positive ratio of CD73, CD90, CD105 are more than or equal to 95%, CD34, CD45, HLA-DR are
Positive ratio be less than or equal to 2%.
14. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells, feature exist as described in claim 1,4,6,7
In the method also includes following steps:
After the primary cell for the umbilical cord mesenchymal stem cells that step (3) obtain is resuspended again, it is inoculated in culture bottle, is passed
It is commissioned to train feeding, it is 6000-8000/cm that cell, which passes on density,2。
15. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 14, which is characterized in that institute
When stating secondary culture in step (5), when cell fusion degree reaches 90%, into culture bottle, interior addition digests enzymatic treatment, then uses
Special culture media is terminated；Piping and druming bottom of bottle is until cell and culture bottle are detached from, with brine culture bottle, collection repeatedly
Cleaning solution is mixed with cell suspension, is sampled and is counted after constant volume；After cell suspension centrifugation, supernatant is abandoned, is resuspended with special culture media thin
Born of the same parents assign to cell suspension in several culture bottles, and mixing is placed on culture in incubator, through had digestive transfer culture culture repeatedly, culture
To P2-P4 for umbilical cord mesenchymal stem cells.
16. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 2, which is characterized in that institute
It is further comprising the steps of to state method:
(6) the primary freeze-stored cell for the umbilical cord mesenchymal stem cells for obtaining step (4), secondary culture after recovery, inoculum density
For in 6000-8000/cm2。
17. the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 16, which is characterized in that institute
When stating freeze-stored cell recovery culture in step (6), primary cell is taken out from cell bank, is transferred to rapidly in 37 DEG C of water-baths and recovers
Cell；After cell is completely dissolved, it is transferred to Biohazard Safety Equipment, with brine cell, with special culture media weight after centrifugation
It is outstanding, cell suspension is assigned in several culture bottles, mixing is placed on culture, cell in incubator and can train through had digestive transfer culture repeatedly
It supports, culture is to P2-P4 for umbilical cord mesenchymal stem cells.
18. the clinic that the method for separating and preparing of clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 14 is prepared
Application layer umbilical cord mesenchymal stem cells.
19. the separation preparation side of clinical application grade umbilical cord mesenchymal stem cells as described in claim 15-17 any claim
The clinical application grade umbilical cord mesenchymal stem cells that method is prepared.
20. clinical application grade umbilical cord mesenchymal stem cells as claimed in claim 19, which is characterized in that the cell is that form is equal
One spindle shape cell, and the positive ratio of CD73, CD90, CD105 are more than or equal to 95%, the sun that CD34, CD45, HLA-DR are
Sex ratio is less than or equal to 2%.
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| CN111346051A (en) * | 2020-03-19 | 2020-06-30 | 瑞太干细胞中心(沈阳)有限公司 | Preparation method of umbilical cord mesenchymal stem cell injection for treating cerebral infarction |
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| CN115323498A (en) * | 2022-08-18 | 2022-11-11 | 宁波希诺赛生物科技有限公司 | Construction method and application of ready-to-use clinical-grade umbilical cord mesenchymal stem cell working library |
| CN116270405A (en) * | 2023-02-28 | 2023-06-23 | 深圳市领呈健康生物科技有限公司 | A preparation method of cell active ingredient raw material with the function of repairing blemishes |
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