CN108865986A - For repairing articular cartilage damage/defect mescenchymal stem cell preparation and its preparation method and application - Google Patents
For repairing articular cartilage damage/defect mescenchymal stem cell preparation and its preparation method and application Download PDFInfo
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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
- C12N5/0668—Mesenchymal stem cells from other natural sources
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2502/00—Coculture with; Conditioned medium produced by
- C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
Abstract
The invention discloses/mescenchymal stem cell the preparation and its preparation method and application of defect is damaged for repairing articular cartilage.Mescenchymal stem cell and synovial cell are carried out Indirect co-culture by the present invention, the mesenchymal stem cell cryopreserving after harvest co-cultivation；Synovial cell and umbilical cord mesenchymal stem cells are according to 4:Umbilical cord mesenchymal stem cells obtained are easier to break up to cartilage direction under identical inductive condition after 1 density content inoculation is co-cultured.Animal knee articular cartilage defect/defect model that the present invention passes through building, it was demonstrated that the umbilical cord mesenchymal stem cells after co-culturing with synovial cell promote articular cartilage damage/defect repair effect obvious.Invention further provides a kind of preparations using the mescenchymal stem cell preparation after the co-cultivation, it can be applied to clinical repair articular cartilage damage/defect, the reparation of articular cartilage damage/defect is effectively facilitated by joint cavity injection said preparation, therapeutic effect is substantially better than simple mescenchymal stem cell preparation.
Description
Technical field
The present invention relates to repairing articular cartilage damage/defect preparation, more particularly, to repairing articular cartilage is damaged/is lacked
Mescenchymal stem cell preparation of damage and preparation method thereof, the invention further relates to the medicinal usages of the mescenchymal stem cell preparation
That is repairing articular cartilage damage/defect, belongs to mescenchymal stem cell preparation and its preparation and application field.
Background technique
Articular cartilage is one layer of hyaline cartilage for being covered in articular surface, is made of cartilage cell and matrix.Articular cartilage
Surface is smooth, can reduce friction, and Saving cortilage is not easy to wear while bearing sport dynamics load, rises in joint motion
Important function.The reason of causing articular cartilage damage/defect mainly includes that long-term a large amount of, high load capacity movement and person in middle and old age's bone close
Save inflammation etc..Since articular cartilage lacks blood vessel, nerve and lymphoid tissue, so self-repairing capability is limited, once impaired remove shadow
It rings outside normal joint motion, other progressive lesions also easy to happen.
Currently, common articular cartilage damage/defect repair method mainly includes：Operative treatment, as debridement arthroscopy,
Microfrature etc.；Skin grafing and mending, such as bone cartilage transplantation；Organization engineered cartilage, such as chondrocyte cell transplantation, mescenchymal stem cell
Transplanting, MACI, the transplanting of gel-like cartilage repair material for having loaded seed cell etc..
It has a good application prospect with cellular transplantation therapy cartilage damage/defect.Intra-articular primary structure includes closing
Save cartilage, meniscus, synovial membrane and subpatellar fat pad.Directly carrying out injury repair using cartilage cell can no doubt obtain preferably
Effect, but cartilage cell must be taken from donor health cartilaginous tissue, and wound is larger for patients, and cartilage cell is in vitro
Proliferative capacity is limited and is easy to dedifferente, therefore is difficult the biggish cartilage damage/defect of repaired area.Meniscus is mainly by fiber
Cartilage composition.Synovial cell is broadly divided into A type and two kinds of Type B, and wherein type B cell is the source of Synovial Mesenchymal Stem Cells, grinds
Study carefully and show that Synovial Mesenchymal Stem Cells and cartilage cell have similar express spectra, derives from same precursor, filled between synovial membrane
Matter stem cell is in cartilage damage/defect synovial membrane microenvironment with the potential of differentiating cartilage-forming cell.In addition, synovial cell produces
Raw synovia is the important component of environment in articular cavity, and Synovial Mesenchymal Stem Cells can secrete many kinds of substance to synovia
In.Existing research confirms that Synovial Mesenchymal Stem Cells are capable of forming the cartilage rich in II collagen type and sulfated glycosaminoglycan
Matrix.Therefore, synovial cell plays a significant role the formation of environment in articular cavity.
In recent years, umbilical cord mesenchymal stem cells (Umbilical Cord Mesenchymal Stem Cells, UC-
MSCs) since tissue obtains simply, cell is easily isolated the ability and tissue cultivated and had to Various Tissues cell differentiation
Repair function has become the seed cell of a variety of disease model applications.Studies have shown that application MSCs carries out articular cartilage damage
Wound/defect repair has certain curative effect.It wherein, by joint cavity injection is directly the simple and easy operating method of one kind, but
The MSCs of direct injection may cause transplanted cells quantity constantly to reduce, cell due to being not suitable with articular cavity microenvironment etc.
Differentiation function state weakens, and cell concentration and quality directly affect repairing effect, and a large amount of injections is thus needed repeatedly to move
Cell is planted to improve curative effect.
Therefore, it urgently needs to reduce when application MSCs carries out articular cartilage damage/defect repair in clinical practice
The loss late of MSCs cell and the function of effectively activating its injury repair.
Summary of the invention
An object of the present invention is to provide the mesenchyma that a kind of pair of articular cartilage damage/defect has significant repair
Stem cell medicine；
The second object of the present invention is to provide a kind of preparation method applied to clinical mescenchymal stem cell preparation；
The third object of the present invention is to repair the mescenchymal stem cell formulation application in articular cartilage damage/defect
It is multiple.
Above-mentioned purpose of the invention is achieved through the following technical solutions：
A kind of preparation method for repairing articular cartilage damage/defect mescenchymal stem cell, by mescenchymal stem cell
Through with synovial cell's Indirect co-culture, harvest co-culture after mescenchymal stem cell, freeze.
Wherein, it is preferred that the Indirect co-culture is the upper chamber that synovial cell is inoculated into Transwell culture plate,
Umbilical cord mesenchymal stem cells are inoculated into the lower room of Transwell culture plate, Transwell culture plate is placed in CO2Incubator
In co-cultured；Wherein the conditions for the training include：Cultivation temperature is 37 DEG C, CO2Concentration be 5%.
The present invention passes through largely test discovery, when co-culturing, the inoculative proportion of synovial cell and mescenchymal stem cell
Influence for mesenchymal stem cells into chondrocytes differentiation is very big；The present invention by synovial cell and mescenchymal stem cell according to
(10-1)：The inoculative proportion of (1-10) is inoculated into the upper chamber of Transwell culture plate respectively or lower room is co-cultured, in vitro
Each group co-cultured cell is detected at progress aminoglycan content after chondrocyte induction and II collagen mRNA expression quantity, amino is poly-
Sugared content testing result discovery, when synovial cell and umbilical cord mesenchymal stem cells inoculum density ratio are 4:When 1, co-cultured cell
At aminoglycan content highest after chondrocyte induction, and there is extremely significant sex differernce compared with other inoculum density ratios；II type glue
Original mRNA expression quantity testing result discovery, when synovial cell and umbilical cord mesenchymal stem cells inoculum density ratio are 4:When 1, train altogether
Feeding cell has extremely significant property at II collagen mRNA expression quantity highest after chondrocyte induction and compared with other inoculum density ratios
Difference.
Preferably, heretofore described synovial cell or mescenchymal stem cell are P2 for cell；Wherein, the synovial membrane is thin
Born of the same parents are preferably derived from people's health synovial tissue, and the mescenchymal stem cell is preferably umbilical cord mesenchymal stem cells.
Wherein, the preparation method of the synovial cell of the co-cultivation includes：It obtains people's health synovial tissue and uses enzyme
Digestion method separation obtains primary synovial cell (P0), and culture withholds collection synovial cell to P2 after passage inoculation, is co-cultured
Use synovial cell.
The preparation method of the umbilical cord mesenchymal stem cells of the co-cultivation includes：It is obtained by tissue block adherent cultivation
Primary human umbilical cord mesenchymal stem cells (P0) are obtained, collection cell is withheld in culture to P2, obtains co-cultivation umbilical cord mesenchymal stem cells.
The present invention is further provided for repairing articular cartilage damage/defect co-cultivation mescenchymal stem cell preparation
Preparation method, including：
(1) mescenchymal stem cell after the co-cultivation frozen is recovered through water-bath, after being washed with physiology salt, is centrifuged, in abandoning
Clearly；(2) cell is resuspended by adjuvant of physiological saline, it is stand-by in inhalation syringe after adjustment cell concentration.
Wherein, step (1) recovers the mescenchymal stem cell after the co-cultivation frozen through 37 DEG C of water-baths；Institute in step (1)
The centrifugation stated is that 2000rpm is centrifuged 5min；Step (2) adjusts cell concentration to (1~10) × 106A/mL.
In order to examine mescenchymal stem cell preparation provided by the invention for articular cartilage damage/defect repairing effect,
The present invention constructs animal knee articular cartilage defect/defect model and carries out injection with mescenchymal stem cell preparation of the invention and controls
It treats, by finding that high dose co-culture experiments group cartilage defect repair effect is substantially better than blank control to sample gross examination of skeletal muscle
Group, mescenchymal stem cell control group and low dosage co-culture experiments group, repair place cartilage color and hardness and surrounding cartilage are basic
Identical, surface is smooth；The experimental results showed that the umbilical cord mesenchymal stem cells repairing articular cartilage after co-culturing with synovial cell damages
Wound/defect effect is obvious.
It is soft that mescenchymal stem cell preparation after co-cultivation provided by the invention by joint cavity injection can effectively facilitate joint
The reparation of bone injury/defect, therapeutic effect are substantially better than simple mescenchymal stem cell preparation.Provided by the present invention is filled
Matter stem cell medicine can be applied to allogeneic or heterogenous animal cell transplantation repairing articular cartilage damage/defect.
Detailed description of the invention
Aminoglycan content detection result after Fig. 1 co-cultured cell breaks up at chondrocyte induction in vitro；
II collagen mRNA expression quantity testing result after Fig. 2 co-cultured cell breaks up at chondrocyte induction in vitro；
Fig. 3 blank control group experiment front and back gross examination of skeletal muscle comparison diagram；
Fig. 4 mescenchymal stem cell control group experiment front and back gross examination of skeletal muscle comparison diagram；
Fig. 5 low dosage co-culture experiments group experiment front and back gross examination of skeletal muscle comparison diagram；
Fig. 6 high dose co-culture experiments group experiment front and back gross examination of skeletal muscle comparison diagram；
Fig. 7 cartilage surface injury repair Wakitani appraisal result.
Specific embodiment
Further describe the present invention below in conjunction with specific embodiment, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
Member it should be understood that can modify without departing from the spirit and scope of the invention to details and form of the invention or
Replacement, but these modifications and replacement are fallen within the protection scope of the present invention.
Separation and culture of the Preparative Example 1 for the synovial cell of co-cultivation
1) patient without immunity diseases such as rheumatism, rheumatoid and osteoarthritis is screened, by arthrocsopic surgery, collects half
Health synovial tissue at the autologous patient or synovial membrane contributor's knee joint of month plate or cruciate ligament, and by tissue preserration in containing
Have in 1% dual anti-PBS buffer solution.
2) synovial tissue is placed in culture dish and is shredded under aseptic condition, with PBS buffer solution rinse 3 times, collect to from
In heart pipe.
3) 4% Type I collagen enzyme of 5 times of tissue volumes is added, is placed in constant-temperature table, 37 DEG C of 150rpm digest 2~3h.
4) after tissue block digests completely, cell suspension is filtered through 70 μm of cell strainers, collects filtrate 1500rpm centrifugation
5min。
5) supernatant is abandoned, centrifugation bottom of the tube cell DMEM/F12 culture medium is resuspended, 1500rpm is centrifuged 5min, washing two
It is secondary.
6) it has washed for the last time, cell is resuspended with complete medium, obtains primary synovial cell (P0 generation).
7) cell density is adjusted to 1.5~2.5 × 105A/mL, is inoculated in 25cm2In culture bottle, every bottle of inoculation 5mL is set
In 37 DEG C, 5%CO2Progress P1 is commissioned to train feeding in incubator.
8) the non-attached cell of liquid reject is about changed after 48h, is changed the liquid once within hereafter every 2-3 days, until cell confluency reaches
80% or more is passed on.
9) secondary culture：Former culture medium is discarded, every bottle of 0.25% trypsase 2mL of addition, digest 3min, become to cell
Circle is added isometric complete medium and terminates digestion after floating, by cell piping and druming at being transferred in centrifuge tube after individual cells,
1500rpm is centrifuged 5min.Supernatant is abandoned, cell precipitation is collected, washs cell 1 time with DMEM/F12 culture medium and be centrifuged.It will receive afterwards
The cell of collection is resuspended in complete medium, by 5000 cell/cm2It is seeded to 75cm2Culture bottle progress P2 is commissioned to train feeding.
10) P2 is collected for synovial cell, is frozen spare.
Separation and culture of the Preparative Example 2 for the umbilical cord mesenchymal stem cells of co-cultivation
1) 1, aseptic collection term fetus umbilical cord, and by tissue preserration in containing 1% dual anti-PBS buffer solution.
2) umbilical cord tissue is taken out into culture dish, each 0.5cm in both ends is cut off under aseptic condition and is discarded, remaining tissue cut to
1.5cm-2cm length squeezes umbilical cord section and clears up vasculature residence blood.
3) umbilical cord arteriovenous is rejected in sterile dissecting pan, removes jelly of Wharton, the jelly of Wharton of collection is transferred to centrifuge tube
In, it shreds to 2-5mm3Size.
4) tissue block is collected, washed once with DMEM/F12 culture medium, then by tissue block with 3-5 block/cm2It is uniformly distributed
In culture bottle, complete medium is added to be advisable with covering whole tissue blocks, is placed in 37 DEG C, 5%CO2It is carried out in incubator primary
It cultivates (P0 generation).
5) cultivate 12~15 days, when observed climb out of tissue block compared with many cells when, tissue block is washed from, is added new complete
Full culture medium continues to cultivate, and passes on when cell confluency is up to 80% or more.
6) secondary culture：It operates identical as synovial cell's secondary culture.
7) cell reaches P2 generation, collects umbilical cord mesenchymal stem cells, freezes spare.
Since umbilical cord tissue obtains simply, cell is separately cultured technology maturation, and primary culture can get a large amount of cells, because
This is with the obvious advantage as seed cell.
The co-cultivation of embodiment 1 synovial cell and umbilical cord mesenchymal stem cells
1) P2 frozen is recovered in 37 DEG C of water-baths for synovial cell and P2 for umbilical cord mesenchymal stem cells respectively, it is each to use
Culture medium is washed 1 time, and 2000rpm is centrifuged 5min, and cell then is resuspended with complete medium respectively.
2) Transwell culture plate (aperture 0.4um) is taken, if 5000 cells/cm2 is basic inoculum density, synovial cell
It is inoculated into upper chamber (area about 4.5cm2), umbilical cord mesenchymal stem cells are inoculated into lower room (area about 9.5cm2), not according to table 1
It is divided into 11 test groups with inoculum density ratio, is respectively placed in 37 DEG C, 5%CO2It is co-cultured in incubator.
The different vaccination density content of 1 synovial cell of table and umbilical cord mesenchymal stem cells
| Group | Synovial cell:The inoculum density ratio of umbilical cord mesenchymal stem cells |
| 1 | 10:1 |
| 2 | 8:1 |
| 3 | 6:1 |
| 4 | 4:1 |
| 5 | 2:1 |
| 6 | 1:1 |
| 7 | 1:2 |
| 8 | 1:4 |
| 9 | 1:6 |
| 10 | 1:8 |
| 11 | 1:10 |
3) it co-cultures 5~7 days, to lower room umbilical cord mesenchymal stem cells fusion rate up to 80% or more, harvests lower room navel respectively
Band mescenchymal stem cell.
4) umbilical cord mesenchymal stem cells of the harvest after 11 groups of co-cultivations are frozen spare.
The preparation of 2 preparation of embodiment
11 groups of co-cultivation umbilical cord mesenchymal stem cells that embodiment 1 is frozen are handled as follows respectively：Through 37 DEG C of water-baths
Recovery is washed 1 time with physiology salt, and 2000rpm is centrifuged 5min, abandons supernatant.
1) cell, adjustment cell concentration to 1 × 10 is resuspended by adjuvant of physiological saline6/ mL, every 1mL, inhalation syringe
In it is stand-by.
2) cell, adjustment cell concentration to 10 × 10 is resuspended by adjuvant of physiological saline6/ mL, every 1mL, sucking injection
It is stand-by in device.
1 co-cultured cell of test example breaks up at chondrocyte induction in vitro to be tested
11 groups of mescenchymal stem cells after embodiment 1 is co-cultured are inoculated in six orifice plates, adjustment cell density to 2.5 respectively
×104A/mL, every hole are inoculated with 2mL.It is adherent when cell and up to 60% or more merge when, discard former culture medium, be changed into cartilage
Induced medium (contains TGF-β 1, dexamethasone, ascorbic acid, Sodium Pyruvate, insulin, transferrins, selenous acid, cow's serum
Albumen and linoleic DMEM/F12 culture medium), it was changed every 2~3 days liquid 1 time, Fiber differentiation 21 days.
Aminoglycan (GAG) content detection
DMMB (1,9 dimethylated methylene is blue) method：Group of cells is collected, cell precipitation is placed in papain digestion liquid
(pH6.8) in, 60 DEG C of digestion 16h.Supernatant is taken after centrifugation, DMMB dye liquor is added, is mixed, the colorimetric at 525nm wavelength, and measurement is inhaled
Shading value.Make standard curve (0~100ug/mL) with chondroitin sulfate, GAG content in group of cells is calculated according to standard curve.
The detection of II collagen mRNA expression quantity
Real-time quantitative PCR method：Illustrate according to related kit, Total RNAs extraction is carried out to group of cells respectively, reverse transcription obtains
CDNA is obtained, qRT-PCR reaction is carried out, measures II collagen mRNA expression quantity in group of cells.
To each group co-cultured cell at chondrocyte induction after, aminoglycan content detection result is analyzed：Work as synovial cell
It is 4 with umbilical cord mesenchymal stem cells inoculum density ratio:When 1, co-cultured cell at aminoglycan content highest after chondrocyte induction,
And there is extremely significant sex differernce compared with other inoculum density ratios.
To each group co-cultured cell at chondrocyte induction after, II collagen mRNA expression quantity testing result is analyzed：Work as cunning
Theca cell and umbilical cord mesenchymal stem cells inoculum density ratio are 4:When 1, co-cultured cell is at II Collagen Type VI after chondrocyte induction
Mrna expression amount highest, and there is extremely significant sex differernce compared with other inoculum density ratios.
The above results show that when synovial cell and umbilical cord mesenchymal stem cells inoculum density ratio be 4:When 1, after co-cultivation
Umbilical cord mesenchymal stem cells be easier under identical inductive condition to cartilage differentiation.
2 animal knee articular cartilage defect of test example/defect model and reparation application test
Animal Model：16 adult White Rabbits are chosen, knee articular cartilage defect/defect model is manufactured.It is quiet through ear edge
Arteries and veins injecting medicinal chloraldurate (2mL/kg weight) anesthetized animal chooses both legs knee joint inner incision, exposure knee joint, in stock
The abrasive drilling of condyle middle and lower part makes 1 diameter about 3mm in bone, and the defect of deep about 1-1.5mm is sewed up a wound.
16 modeling White Rabbits are randomly divided into 4 groups：
1) blank control group, injecting normal saline.
2) mescenchymal stem cell control group, injection 10 × 106Common umbilical cord mesenchymal stem cells.
3) low dosage co-culture experiments group, the co-cultivation umbilical cord mesenchymal stem cells of injection 2 step of embodiment (1) preparation
(concentration is 1 × 106/mL)。
4) high dose co-culture experiments group, the co-cultivation umbilical cord mesenchymal stem cells of injection 2 step of embodiment (2) preparation
(concentration is 10 × 106/mL)。
After animal model 1 week, injection treatment is carried out.Blank control group is in the intracavitary injecting normal saline respectively of double knee joint
1mL；Mescenchymal stem cell control group is in the common umbilical cord mesenchymal stem cells 1mL of double knee joint intracavitary administration；Experimental group is in double knees
Each dosage is injected in articular cavity respectively and co-cultures umbilical cord mesenchymal stem cells 1mL；Injection treatment every two weeks 1 time, continuous injection 5 times
Complete treatment.Experimental animal is put to death in last 1 injection treatment after two weeks, carries out gross examination of skeletal muscle and tissue disease to articular cartilage surface
Neo-Confucianism observation scores to articular cartilage surface injury repair degree according to Wakitani standards of grading.It the results are shown in Table 2.
2 articular cartilage surface injury repair degree appraisal result of table
After carrying out cell transplantation, by finding high dose co-culture experiments group cartilage defect repair effect to sample gross examination of skeletal muscle
Fruit is substantially better than blank control group, mescenchymal stem cell control group and low dosage co-culture experiments group, repair place cartilage color and
Hardness and surrounding cartilage are essentially identical, and surface is smooth.Wakitani scoring display, each cell therapy group repair of cartilage degree are bright
It is aobvious to be better than blank control group, experimental group and mescenchymal stem cell control group compared to blank control group Wakitani integral with significant
Sex differernce；In addition, high dose co-culture experiments group repair of cartilage degree is substantially better than mescenchymal stem cell in the case of same dose
Control group is also better than low dosage co-culture experiments group, the experimental results showed that the umbilical cord mesenchyma after co-culturing with synovial cell is dry
Cell promotes articular cartilage damage/defect repair effect obvious (Fig. 3-Fig. 7).
Claims (10)
1. a kind of preparation method for repairing articular cartilage damage/defect mescenchymal stem cell, it is characterised in that：It is filled by between
Matter stem cell and synovial cell carry out Indirect co-culture, harvest co-culture after mescenchymal stem cell, freeze to get.
2. preparation method described in accordance with the claim 1, it is characterised in that：The Indirect co-culture is using Transwell
The mode of culture plate carries out Indirect co-culture；Preferably, the Indirect co-culture is that synovial cell is inoculated into Transwell
Umbilical cord mesenchymal stem cells are inoculated into the lower room of Transwell culture plate, after inoculating cell by the upper chamber of culture plate
Transwell culture plate is placed in CO2It is co-cultured in incubator.
3. preparation method according to claim 2, it is characterised in that：By synovial cell and mescenchymal stem cell according to (10-
1)：The inoculative proportion of (1-10) is inoculated into the upper chamber of Transwell culture plate respectively or lower room is co-cultured；Preferably, will
Synovial cell and umbilical cord mesenchymal stem cells are according to 6:1 or 4:1 inoculative proportion is inoculated into the upper of Transwell culture plate respectively
Room or lower room are co-cultured.
4. preparation method described in accordance with the claim 1, it is characterised in that：The synovial cell is P2 for synovial cell；Institute
The mescenchymal stem cell stated is P2 for umbilical cord mesenchymal stem cells.
5. preparation method according to claim 4, it is characterised in that：The P2 includes for the preparation method of synovial cell：
Obtaining people's health synovial tissue uses enzyme digestion separation to obtain P0 for synovial cell, cultivates after passage inoculation to P2 and withholds collection
Cell obtains to co-culture and uses synovial cell；
The P2 of the co-cultivation includes for the preparation method of umbilical cord mesenchymal stem cells：It is obtained by tissue block adherent cultivation
P0 is obtained for human umbilical cord mesenchymal stem cells, collection cell is withheld in culture to P2, obtains co-cultivation umbilical cord mesenchymal stem cells.
6. the mescenchymal stem cell after the co-cultivation be prepared by any one of claim 1-5 method.
7. a kind of preparation method for repairing articular cartilage damage/defect mescenchymal stem cell preparation, including：
(1) mescenchymal stem cell after the co-cultivation as claimed in claim 6 frozen is recovered through water-bath, is washed with physiology salt
Afterwards, it is centrifuged, abandons supernatant；(2) cell is resuspended by adjuvant of physiological saline, it is stand-by in inhalation syringe after adjustment cell concentration.
8. preparation method according to claim 7, it is characterised in that：Step (1) is by the mesenchyma after the co-cultivation frozen
Stem cell recovers through 37 DEG C of water-baths；Centrifugation described in step (1) is that 2000rpm is centrifuged 5min；Step (2) adjusts cell concentration
To (1~10) × 106A/mL.
9. the mescenchymal stem cell preparation obtained by the preparation method of claim 7 or 8.
10. mescenchymal stem cell or mescenchymal stem cell preparation as claimed in claim 9 after co-cultivation as claimed in claim 6
Preparing the purposes in repairing articular cartilage damage/defect drug.
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| CN111826343A (en) * | 2020-07-23 | 2020-10-27 | 北京中卫医正科技有限公司 | Cell culture solution for enhancing induced cartilage differentiation, method and application |
| CN112870229A (en) * | 2021-03-01 | 2021-06-01 | 河南省银丰生物工程技术有限公司 | Mesenchymal stem cell preparation for treating knee osteoarthritis and research method thereof |
| CN113699100A (en) * | 2021-08-27 | 2021-11-26 | 王伟 | Construction method of stem cell and articular chondrocyte co-culture system for simulating in-vivo microenvironment |
| CN114149959A (en) * | 2021-11-15 | 2022-03-08 | 山西中医药大学 | Method for establishing cell model for researching autoimmune hepatitis |
| CN114191451A (en) * | 2021-12-16 | 2022-03-18 | 上海华颜医药科技有限公司 | Method for delaying chronic bone injury by adopting umbilical cord mesenchymal stem cells |
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| CN114149959A (en) * | 2021-11-15 | 2022-03-08 | 山西中医药大学 | Method for establishing cell model for researching autoimmune hepatitis |
| CN114191451A (en) * | 2021-12-16 | 2022-03-18 | 上海华颜医药科技有限公司 | Method for delaying chronic bone injury by adopting umbilical cord mesenchymal stem cells |
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