CN105907707A - Isolation and culture method for primary mice or rat cartilage cells - Google Patents
Isolation and culture method for primary mice or rat cartilage cells Download PDFInfo
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- CN105907707A CN105907707A CN201610218208.XA CN201610218208A CN105907707A CN 105907707 A CN105907707 A CN 105907707A CN 201610218208 A CN201610218208 A CN 201610218208A CN 105907707 A CN105907707 A CN 105907707A
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- cartilage cell
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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
- C12N5/0655—Chondrocytes; Cartilage
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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
- C12N2500/05—Inorganic components
- C12N2500/10—Metals; Metal chelators
- C12N2500/20—Transition metals
- C12N2500/24—Iron; Fe chelators; Transferrin
- C12N2500/25—Insulin-transferrin; Insulin-transferrin-selenium
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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
- C12N2500/70—Undefined extracts
- C12N2500/80—Undefined extracts from animals
- C12N2500/84—Undefined extracts from animals from mammals
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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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N2509/10—Mechanical dissociation
Abstract
The invention discloses an isolation and culture method for primary mice or rat cartilage cells. According to the method, a step of polishing the surface of cartilage is carried out in acquisition of the cartilage cells, so contamination by subsequent synovial cells is maximally eliminated; the method innovatively employs a biopsy needle to cut interior cartilage tissue, so sampling is directed at target cells, the method is direct and effective, and potential bacterial and fungal contamination is ingeniously prevented; meanwhile, the isolation and culture method employs a specially-prepared compound enzyme digestive juice, so compared with traditional single collagenase preheating and digestion, the method provided by the invention overcomes the problems of low cell viability and poor yield caused by excess digestion and excess blowing and beating of tissue, and enables digestion to be more uniform and thorough.
Description
Technical field
The present invention relates to technical field of cell culture, particularly relate to a kind of Primary mouse or rat cartilage cell point
From cultural method.
Background technology
Chondrocyte is positioned in cartilage, and Main Function is that the high score of some collagen protein of synthesis and non-collagen is careful
Extracellular matrix, including: II collagen type, proteoglycan, link albumen, IX collagen type and XI type
Collagen protein.The propagation of regulation and control chondrocyte and differentiation, most important for coordinating vertebrate skeleton development.
Chondrocyte can produce substantial amounts of peptide growth factor and cytokine, and reacts therewith, such as: islets of langerhans
Element like growth factor-1 and il-1.The regeneration of Investigating Cartilage and reparation, cytokine and growth because of
The sub effect to cartilage, specific gene are in arthritic regulating and controlling effect and arthritic pathophysiological process, soft
The cultivation of osteocyte is very important external model.
At present, substantial amounts of researcher has been had to carry out cultivation and the pharmacology of mice/rat cartilage cell
Learning the research in terms of characteristic, research previously uses the 37 degree of water-baths of 0.25% pancreatin to digest 1 hour more, then
Digest about 5 hours with II Collagenase Type 37 degree.It is disadvantageously, most of enzyme is at 37 degree
Under the conditions of violent Digestion all can occur, therefore, on the one hand can accelerate the speed of digestion, but another
Aspect, can cause again injury greatly to cell, and especially pancreatin is all the more so.Thus the result caused is,
The bad grasp of digestion time, often digestion time is long, and cell debris is in the majority, and heteroproteose cell is relatively more and cartilage is thin
Born of the same parents are fewer, and the cell viability obtained is poor, along with the prolongation of incubation time and pass on, great-hearted cartilage
Cell is fewer and feweri, and cell is the most dead.Meanwhile, in the gatherer process of chondrocyte, other cells such as synovial membrane
The pollution of cell is the most common, the most above-mentioned enzymic digestion process, it is easy to cause chondrocyte quantity not to be dominant
Gesture, and the higher heteroproteose cell isolated on the contrary of other tolerations is more.
Summary of the invention
The present invention solves a kind of high-purity that the problems referred to above of the prior art propose, high motility rate, high activity with
Time oligosaprobic Primary mouse or the isolated culture method of rat cartilage cell.
In order to realize above-mentioned technical purpose, the technical measures that the present invention is taked are:
A kind of Primary mouse or the isolated culture method of rat cartilage cell, it is characterised in that comprise the following steps:
Step 1: the pretreatment of cartilaginous tissue
Clear up in vitro meniscus, and utilize the D-hanks solution flushing containing dual anti-and amphotericin B, then
Polishing meniscal surface, reuses and rinses containing dual anti-and amphotericin B D-hanks solution；
Step 2: take chondrocyte
Use small-sized biopsy needle to thrust the meniscus after step 1 processes, cut meniscus internal cartilage cell；
Step 3: enzymic digestion
Chondrocyte step 2 obtained is placed in compound enzyme Digestive system digestion, wherein wraps in compound enzyme Digestive system
Include type i collagen enzyme, II Collagenase Type, IV Collagenase Type and Dispase enzyme that mass ratio is 1:1:1:1；
Step 4: separation and Culture chondrocyte
Step 3 is digested complete chondrocyte and crosses 70um screen cloth, be centrifuged washing, then carry out resuspended
And recentrifuge, finally the most resuspended with serum-free medium, isolated chondrocyte, use containing 10%-20%
Hyclone, 2% dual anti-culture medium are at 37 DEG C of 5%CO2Incubator in cultivate.
In order to optimize technique scheme further, the technical measures that the present invention is taked also include:
Preferably, the small-sized biopsy needle used in above-mentioned steps 2 is small semiautomatic or full-automatic biopsy needle.
Preferably, in above-mentioned steps 3, in compound enzyme Digestive system, the initial concentration of each enzyme is 1mg/ml.
Preferably, in above-mentioned steps 3, enzymic digestion process is 4 DEG C of compound enzyme Digestive systems digestion 4-20h of use；Or
Person uses 37 DEG C of compound enzyme Digestive system digestion 10min-2h preheated.
Preferably, the culture medium of above-mentioned steps 4 also includes the transferrins of 5ug/ml and the islets of langerhans of 10ug/ml
Element.
Preferably, the culture medium of above-mentioned steps 4 is cultivated selected from DMEM culture medium, Williams'medium E
Base or DMEM or F12 culture medium；More preferably DMEM culture medium.
Preferably, above-mentioned dual anti-for mycillin.
On the other hand, the present invention also provides for a kind of separation training according to above-mentioned Primary mouse or rat cartilage cell
Mice that breeding method is cultivated or rat cartilage cell.
The present invention uses technique scheme, compared with prior art, has the following technical effect that
First, the present invention, during gathering chondrocyte, has the step of polishing cartilage surface, maximum limit
Eliminate the pollution of follow-up synovial cell degree；Secondly, the biopsy needle that have employed taking process initiative cuts interior
Portion's cartilaginous tissue so that sampling is directed to purpose cell, not only the most effectively, avoids latent dexterously simultaneously
Antibacterial and fungal contamination；Meanwhile, the isolated culture method of the Primary mouse/rat cartilage cell of the present invention,
The compound enzyme Digestive system of the special preparation used, compound enzyme Digestive system is by type i collagen enzyme, II Collagenase Type, IV
Collagenase Type and Dispase enzyme composition, and use 4 DEG C of condition digested overnight (4-20h), compare tradition single
Collagenase preheating digestion (in particular cases can be with 37 DEG C of digestion), not only avoid and excessive tissue digested
The cytoactive that caused of degree piping and druming is low, the problem of rate variance, and digest the most thorough.In sum,
The present invention the isolated culture method of Primary mouse/rat cartilage cell, it is possible to obtain high-purity, high motility rate,
High activity Primary mouse/rat cartilage cell that pollution risk is extremely low simultaneously, the cell of cultivation breaks up, cell
Vigor is high, has bigger dissemination.
Accompanying drawing explanation
Fig. 1 is Mouse cartilage cell microphotograph (10X) that the isolated culture method of the present invention obtains；
Fig. 2 is Mouse cartilage cell microphotograph (20X) that the isolated culture method of the present invention obtains.
Detailed description of the invention
The invention provides the isolated culture method of a kind of Primary mouse or rat cartilage cell, including following step
Rapid:
A kind of Primary mouse or the isolated culture method of rat cartilage cell, it is characterised in that comprise the following steps:
Step 1: the pretreatment of cartilaginous tissue
Clear up in vitro meniscus, and utilize the D-hanks solution flushing containing dual anti-and amphotericin B, then
Polishing meniscal surface, reuses and rinses containing dual anti-and amphotericin B D-hanks solution；
Step 2: take chondrocyte
Use small-sized biopsy needle to thrust the meniscus after step 1 processes, cut meniscus internal cartilage cell；
Step 3: enzymic digestion
Chondrocyte step 2 obtained is placed in compound enzyme Digestive system digestion, wherein wraps in compound enzyme Digestive system
Include type i collagen enzyme, II Collagenase Type, IV Collagenase Type and Dispase enzyme that mass ratio is 1:1:1:1；
Step 4: separation and Culture chondrocyte
Step 3 is digested complete chondrocyte and crosses 70um screen cloth, be centrifuged washing, then carry out resuspended
And recentrifuge, finally the most resuspended with serum-free medium, isolated chondrocyte, use containing 10%-20%
Hyclone, 2% dual anti-culture medium are at 37 DEG C of 5%CO2Incubator in cultivate.
Below by specific embodiment, the present invention is carried out detailed and concrete introduction, so that being better understood from this
Bright, but following embodiment is not limiting as the scope of the invention.
Embodiment 1
This enforcement uses SD rat, and the method collection of the application present invention and cultured cartilage cell, concrete steps are such as
Under:
1. the extraction of cartilage of rats tissue
First newborn SD rat (freezing 10 minutes or cervical dislocation) is put to death, alcohol-pickled 5 minutes.Cut
Take extremity, the extremity taken off are immersed in the PBS containing 2% mycillin, put and be kept on ice.
The most meniscal stripping
Under anatomic microscope, carefully divest skin and the muscle of extremity with eye scissors, remove completely as far as possible,
Avoid to limits being mixed into of heteroproteose cell.By arthrotomy, expose articular surface, it can be seen that half in articular cavity
Month plate, is drawn off collecting.
3. the pretreatment of cartilaginous tissue
Clear up in vitro meniscus, and utilize the D-hanks solution flushing containing dual anti-and amphotericin B, then
Polishing meniscal surface, reuses and rinses containing dual anti-and amphotericin B D-hanks solution；Fully clean
Remove the synovial tissue that the cartilage surface after polishing may carry by mistake.
4. enzymic digestion
The cartilaginous tissue bleached after processing shreds, and adds compound enzyme Digestive system (type i collagen enzyme+II Collagen Type VI
Enzyme+IV Collagenase Type+Dispase enzyme=1:1:1:1), the initial concentration of 4 kinds of enzymes is 1mg/ml.4
Degree refrigerator overnight, it is simple to enzyme fully combines with tissue, makes tissue loose, and cell is easily peeled off.So can keep away
Exempt from digestion process, excessively digestion and the excessively piping and druming to cell, causes injury greatly to cell.2nd day,
Being taken out from refrigerator by tissue, somewhat blow and beat several times, put into 37 degree of water-baths 30 minutes, slight piping and druming is not again
More than 10 times.According to circumstances, can be placed again into 37 degree of water-baths 30 minutes, finally, slight piping and druming is less than
10 times, cross 70um screen cloth.
5. centrifugal purification
Take the cell filtrate after sieving, carry out resuspended and recentrifuge, finally the most resuspended with serum-free medium,
Isolated chondrocyte, then expects blue living cell counting number with 0.2%.
6. culture medium preparation
Due to the particularity of chondrocyte, when preparing complete medium, need to add cytokine, i.e. basis
Culture medium is containing 10% hyclone, the DMEM culture medium of 1% mycillin, adds turning of 5ug/ml before using
Ferritin and the insulin of 10ug/ml.It addition, in order to promote the adherent of cell, primary, pass on and multiple
The 1st day of Soviet Union, brings up to 20% by the serum-concentration in culture medium, within the 2nd day, is replaced by 10%.
7. two dimension is cultivated
The rat cartilage cell of purification is resuspended by above-mentioned culture medium, prepare chondrocyte re-suspension liquid, join T
Cultivating in 25 culture bottles, inoculum density is 0.5*106/ bottle.It is placed in 37 degree, in the incubator of 5%CO2
Cultivate.Every culture medium more renewed for 3 days, every day in basis of microscopic observation cell growth status, to cell
Degrees of fusion reaches 70%～80%, carries out Secondary Culture.
8. Secondary Culture
When cell degrees of fusion reaches 70%～80%, every bottle of cell adds 0.25% pancreas enzyme-EDTA 1ml, 37 degree of trainings
Support and case is hatched digestion 2 minutes.Basis of microscopic observation, when intercellular substance increases, by the culture medium containing serum
Terminate digestion, and cell is blown and beaten.400g is centrifuged 5 minutes, it is thus achieved that cell add containing cell because of
Son and serum-concentration are the complete medium of 20%, are inoculated in T25 culture bottle, and inoculum density is 0.5*106/
Bottle.It is placed in 37 degree, the incubator of 5%CO2 continues amplification cultivation.Within 2nd day, replace medium to containing cell
The factor and serum-concentration are the complete medium of 10%.Every culture medium more renewed for 3 days, every day is under microscope
Observation of cell growing state.
9. cellular morphology is observed
The chondrocyte of isolated through cultivation adherent after, use microscope observation of cell form, such as Fig. 1 and
Shown in Fig. 2, it is seen that full full, the form health vigor height of cell.
10. other indexs of correlation measure
Measure antibacterial and fungal contamination situation, cell contamination (synovial cell etc.) situation during cultivation, add up thin
Born of the same parents' motility rate etc..
Comparative example
Comparative example uses traditional piece of tissue digestion method, separates after obtaining meniscus, peels off and removes other even
Band tissue, uses and rinses containing dual anti-PBS, shred, and use 0.25% pancreatin 37 DEG C under sterile working
Digestion 1h, the most again with II Collagenase Type 37 DEG C digestion 5h, use buffer piping and druming tissue, cell dispersion,
Cross screen cloth, be centrifuged washing, then carry out resuspended and recentrifuge, finally again weigh with serum-free medium
Outstanding, isolated Mouse cartilage cell, after cell counting, join in T25 culture bottle and cultivate, connect
Planting density is 0.5*106/ bottle, is placed in 37 degree, 5%CO2Incubator in carry out cultivating and observing and measure phase
Close index.
The above embodiments and comparative example experiment, applicant carried out many experiments, and carried out index of correlation
Comparison and data statistics.As shown in table 1:
Table 1
Applicant uses mice to carry out testing several times simultaneously, and experimental result is identical with mice, it was demonstrated that the present invention
Method good result that the chondrocyte isolation of rat and mice is cultivated authentic and valid.
In sum, the Primary mouse of the present invention or the isolated culture method of rat cartilage cell, use special
Compound enzyme carries out long-term ingestion so that obtain cell total amount big, and separating degree is high, and motility rate is high；Use owing to gathering
The method of polishing also make use of biopsy needle, chondrocyte antibacterial that the method for the present invention obtains, fungus and other
The contamination probability of cell is extremely low, can also carry out Secondary Culture, for the phase based on cultured chondrocytes simultaneously
Close research and provide good cell cultivation solution.
Being described in detail the specific embodiment of the present invention above, but it is intended only as example, the present invention is also
It is not restricted to particular embodiments described above.To those skilled in the art, any the present invention is carried out
Equivalent modifications and substitute the most all among scope of the invention.Therefore, without departing from the spirit of the present invention and model
Enclose lower made impartial conversion and amendment, all should contain within the scope of the invention.
Claims (10)
1. a Primary mouse or the isolated culture method of rat cartilage cell, it is characterised in that comprise the following steps:
Step 1: the pretreatment of cartilaginous tissue
Clear up in vitro meniscus, and utilize the D-hanks solution flushing containing dual anti-and amphotericin B, then polish
Meniscal surface, reuses and rinses containing dual anti-and amphotericin B D-hanks solution；
Step 2: take chondrocyte
Use small-sized biopsy needle to thrust the meniscus after step 1 processes, cut meniscus internal cartilage cell；
Step 3: enzymic digestion
Chondrocyte step 2 obtained is placed in compound enzyme Digestive system digestion, and wherein compound enzyme Digestive system includes matter
Amount is than type i collagen enzyme, II Collagenase Type, IV Collagenase Type and the Dispase enzyme for 1:1:1:1；
Step 4: separation and Culture chondrocyte
Step 3 is digested complete chondrocyte and crosses 70um screen cloth, be centrifuged washing, then carry out resuspended and again
Secondary centrifugal, finally the most resuspended with serum-free medium, isolated chondrocyte, use containing 10%-20%
Hyclone, 2% dual anti-culture medium are at 37 DEG C of 5%CO2Incubator in cultivate.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat cartilage cell, it is special
Levying and be, the small-sized biopsy needle used in described step 2 is small semiautomatic or full-automatic biopsy needle.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat cartilage cell, it is special
Levying and be, in described step 3, in compound enzyme Digestive system, the initial concentration of each enzyme is 1mg/ml.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat cartilage cell, it is special
Levying and be, in described step 3, enzymic digestion process is for using 4 DEG C of compound enzyme Digestive system digestion 4-20h.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat cartilage cell, it is special
Levying and be, in described step 3, enzymic digestion process is to use the compound enzyme Digestive system digestion of 37 DEG C of preheatings
10min-2h。
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat cartilage cell, it is special
Levying and be, the culture medium of described step 4 also includes the transferrins of 5ug/ml and the insulin of 10ug/ml.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat cartilage cell, it is special
Levy and be, the culture medium of described step 4 selected from DMEM culture medium, Williams'medium E culture medium or
DMEM/F12 culture medium.
A kind of Primary mouse the most according to claim 7 or the isolated culture method of rat cartilage cell, it is special
Levying and be, the culture medium of described step 4 is DMEM culture medium.
A kind of Primary mouse the most according to claim 1 or the isolated culture method of rat cartilage cell, it is special
Levy and be, described dual anti-for mycillin.
10. one kind according to the Primary mouse described in claim 1-9 any one or the separation and Culture of rat cartilage cell
Mice that method is cultivated or rat cartilage cell.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011055A (en) * | 2016-06-29 | 2016-10-12 | 广东省第二中医院 | Preparation method of human primary cartilage cells with high yield rate |
| CN112342188A (en) * | 2019-08-06 | 2021-02-09 | 中晶生物技术股份有限公司 | Human primary chondrocyte separation kit |
| CN113403266A (en) * | 2021-06-03 | 2021-09-17 | 上海派森诺生物科技有限公司 | Preparation method of single cell suspension of skeletal tissue |
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| CN101829362A (en) * | 2010-04-12 | 2010-09-15 | 山西医科大学第二医院 | Experimental method for establishing tissue engineering cartilage in vitro by taking knee-joint cartilage unit of rabbit as seed cell |
| CN104480066A (en) * | 2014-12-30 | 2015-04-01 | 陕西瑞盛生物科技有限公司 | Chondrocyte culture medium and chondrocyte culture method |
| CN104911145A (en) * | 2015-05-25 | 2015-09-16 | 上海中医药大学附属曙光医院 | High-activity primary cartilage cell preparing method |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101829362A (en) * | 2010-04-12 | 2010-09-15 | 山西医科大学第二医院 | Experimental method for establishing tissue engineering cartilage in vitro by taking knee-joint cartilage unit of rabbit as seed cell |
| CN104480066A (en) * | 2014-12-30 | 2015-04-01 | 陕西瑞盛生物科技有限公司 | Chondrocyte culture medium and chondrocyte culture method |
| CN104911145A (en) * | 2015-05-25 | 2015-09-16 | 上海中医药大学附属曙光医院 | High-activity primary cartilage cell preparing method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106011055A (en) * | 2016-06-29 | 2016-10-12 | 广东省第二中医院 | Preparation method of human primary cartilage cells with high yield rate |
| CN112342188A (en) * | 2019-08-06 | 2021-02-09 | 中晶生物技术股份有限公司 | Human primary chondrocyte separation kit |
| CN113403266A (en) * | 2021-06-03 | 2021-09-17 | 上海派森诺生物科技有限公司 | Preparation method of single cell suspension of skeletal tissue |
| WO2022252753A1 (en) * | 2021-06-03 | 2022-12-08 | 上海派森诺生物科技有限公司 | Method for preparing single cell suspension of skeletal tissue |
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Application publication date: 20160831 |