CN1539963A - Method for cltivating cartilage cells in knee joint of BALB/c chmice - Google Patents
Method for cltivating cartilage cells in knee joint of BALB/c chmice Download PDFInfo
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- CN1539963A CN1539963A CNA2003101107737A CN200310110773A CN1539963A CN 1539963 A CN1539963 A CN 1539963A CN A2003101107737 A CNA2003101107737 A CN A2003101107737A CN 200310110773 A CN200310110773 A CN 200310110773A CN 1539963 A CN1539963 A CN 1539963A
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Abstract
A method for culturing the cartilage cells of BALB/c mouse knee joint is disclosed. Its advantage is high surviral rate (80-90%).
Description
Technical field
The invention provides a kind of cultural method of BALB/c mouse knee cartilage cell, specifically, is to be the cell culture processes that the chondrocyte originates with the BALB/c mouse knee joint, belongs to biological technical field.
Background technology
The damage of repairing articular cartilage is clinical one big, the bioengineered tissue technology of utilization biomaterial and cellular engineering provides a kind of selection for the biological reparation of cartilage at present, prior art only has the making and the cultural method of knee cartilage cell of rabbit a kind of, the material that this method is selected for use is: the DMEM substratum, II Collagen Type VI enzyme, newborn calf serum.The separating step of concrete rabbit knee cartilage cell is: knee cartilage is taken from 4 newborn Japan large ear rabbits, cuts the bilateral knee joint under the aseptic condition, and (PBS) washes and peel off cartilage with phosphate buffered saline buffer, is trimmed to 1mm repeatedly
3Size is at first carried out 2.5g/L tryptic digestion 30min, and the centrifugal Digestive system of abandoning adds 2g/LII Collagen Type VI enzyme, 37 ℃ of concussion digestion 1～2h.Microscopically control digestion time, in time collecting cell is inoculated in the DMEM substratum, includes 10% calf serum.The report that does not have other animal knee cartilage cell culture processes so far.
Summary of the invention
In order to address the above problem, technical scheme of the present invention is: the cultural method that a kind of BALB/c mouse knee cartilage cell is provided.
The invention provides a kind of cultural method of BALB/c mouse knee cartilage cell, it is made up of following steps:
A, get the cartilaginous tissue sheet from BALB/c mouse knee joint surface;
B, with the tissue block of a step gained and Digestive system mixture slaking vibration, liquid must vibrate;
C, with b step vibration liquid filter, centrifugal, isolate chondrocyte group and place nutrient solution, make cell suspension, standby;
After d, the cell suspension filtration, regulate cell density to 1 * 10 with c step gained
5Individual/ml, with this cell suspension inoculation in 36.7～36.9 ℃, 4.9～5.1%v/v CO
2, the CO of saturated humidity
2Cultivate in the incubator, get culturing cell;
E, will make inoculating cell after the culturing cell deproteinated of d step gained, the digestion, divide bottle to be inoculated in the nutrient solution and cultivates, promptly get BALB/c mouse knee cartilage cell.
Wherein step b, c, the described digestion Digestive system of e are: 0.15～0.18%w/w (weight percent) II Collagen Type VI enzyme solution; The described nutrient solution of step c, e is: contain 12～18%w/w newborn calf serum DMEM nutrient solution (Dulbecco ' the s Modified EagleMedium of tissue culture medium).
Described tissue block of b step and Digestive system blended volume ratio are: 1: 20～30, described digestion vibration is digestion stage by stage, the i.e. every digestion of all tissue block 1h just filters, centrifugal 1 time, the condition of vibration digestion is: slightly vibration in the 36.7-36.9 ℃ of water-bath, digested 2.5～4 hours, oscillation frequency is 20-30 time/min.
Centrifugal 13～the 17min of the described centrifugal employing high speed freezing centrifuge 800～1200r/min of step c.The described deproteinated of step e is 0.15～0.2%w/w trypsin solution, and Digestive system is 0.15～0.18%w/wII Collagen Type VI enzyme solution, 36-37 ℃ of digestion down.
The mouse chondrocyte that the inventive method obtains is used for the repairing articular cartilage damage, increases chondrocyte's new source.The inventive method BALB/c mouse knee cartilage cell cultures, can obtain competent, WD mouse chondrocyte, mouse chondrocyte surviving rate one Zhou Houke that cultivates reaches 80%～90%, its high-survival rate helps cell cultures, and cost is lower, because mouse chondrocyte's antigenicity is low, for further medical research and clinical demand have been established condition.
Obviously, according to foregoing of the present invention,,, can also make modification, replacement or the change of other various ways not breaking away under the above-mentioned basic fundamental thought of the present invention prerequisite according to the ordinary skill knowledge and the customary means of this area.
The embodiment of form is described in further detail foregoing of the present invention again by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following example.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
The concrete steps of embodiment 1 the inventive method:
1, main experiment material
(1) chondrocyte source: animal material can be with the only female BALB/c of 8-16 (h-2d) mouse knee cartilage sample in 6～8 age in week.
(2) Digestive system: 0.15-0.2%w/wII Collagen Type VI enzyme solution, (Sigma, U.S.A), filtration sterilization.
(3) nutrient solution: can select DMEM substratum (Gibco, U.S.A.) substratum.Generally in nutrient solution, add the 12-18%w/w newborn calf serum.
(4) screen cloth: 100-200 order copper mesh.
2, operation steps
(1) draws materials and isolated cell
1) cut down the cartilaginous tissue sheet with knife blade from the knee joint surface of BALB/c mouse, be collected among the phosphate buffered saline buffer PBS, clean repeatedly the blood remove cartilage sheet surface and may be by mistake with synovial tissue.Fresh cartilage be with in the milky white light blue, translucent, tool elasticity slightly.
2) the cartilage sheet fully is cut into 1～2mm
3Following tissue block, clean 3～5 times with PBS, by the volume ratio of tissue block and Digestive system is that 1: 20～30 amount adds Digestive system, be placed at 20～30 times/min of slight vibration in 36.7～36.9 ℃ of water-baths, digestion method is stage by stage adopted in vibration digestion, digestion time 2.5-4h, be that every digestion 1h just filters, centrifugal 1 time, adopt the centrifugal 13-17min of high speed freezing centrifuge (Heraeus) 800～1200r/min, collecting cell, chondrocyte after separating put into immediately contain 12～18%w/w calf serum DMEM nutrient solution and preserve and get up to make cell suspension, filter with 100～200 order copper mesh, collection contains chondrocyte's filtrate.
(2) culturing process:
1) counting cells, increase contains 12-18%w/w calf serum DMEM nutrient solution adjusting cell density to 1 * 10 as required
5The density inoculation of individual/ml places 36.7～36.9 ℃, 4.9～5.1%v/vCO
2, the CO of saturated humidity
2Cultivate in the incubator, every 3d changes nutrient solution 1 time.
2) after cell covers with bottle wall, can go down to posterity.When the time comes, clean culture, add 0.15～0.2%w/w trypsin solution, 36～37 ℃ of digestion down with PBS.Mirror is observed visible most of intercellular matrix dissolving down and is disappeared.When cell rounding, add and contain 12～18%w/w calf serum DMEM nutrient solution, the piping and druming cell dispersion.
3) dividing bottle to be inoculated in contains in 12～18%w/w calf serum DMEM nutrient solution and cultivation.
(3) cultivation results:
Observe under inverted phase contrast microscope, it is spherical rigidly connecting the chondrocyte who plants, and begins adherent growth about 6h.After former generation, cultured cells was converged in flakes, rounded or ellipse can be secreted the small amounts of cells epimatrix.Can obtain competent, WD BALB/c mouse knee cartilage cell.
The cultural method that the cultural method of embodiment 2 usefulness rabbit knee cartilage cells is cultivated BALB/c mouse knee cartilage cell and BALB/c mouse knee cartilage cell of the present invention compares
With the cultural method of the knee cartilage cell of rabbit, cultivate the knee cartilage cell of mouse, the surviving rate after the week is 0～20%, surviving rate is lower;
Chondrocyte with the preparation of the cultural method of BALB/c mouse knee cartilage cell of the present invention can obtain competent, WD Balb/c mouse knee cartilage cell, and one week of surviving rate can reach 80%～90%, the surviving rate height.
By above-mentioned embodiment as can be known, the inventive method obtains competent, WD mouse chondrocyte, mouse chondrocyte surviving rate one Zhou Houke that cultivates reaches 80%～90%, its high-survival rate helps cell cultures, and cost is lower, because this bright method gained mouse chondrocyte antigenicity is low, for further medical research and clinical application have been established condition.
Claims (7)
1, a kind of cultural method of BALB/c mouse knee cartilage cell is characterized in that it is made up of following steps:
A, get the cartilaginous tissue sheet from BALB/c mouse knee joint surface;
B, with the tissue block of a step gained and Digestive system mixture slaking vibration, liquid must vibrate;
C, with b step vibration liquid filter, centrifugal, isolate chondrocyte group and place nutrient solution, make cell suspension, standby;
After d, the cell suspension filtration, regulate cell density to 1 * 10 with c step gained
5Individual/ml, with this cell suspension inoculation in 36.7～36.9 ℃, 4.9～5.1%v/v CO
2, the CO of saturated humidity
2Cultivate in the incubator, get culturing cell;
E, will make inoculating cell after the culturing cell deproteinated of d step gained, the digestion, divide bottle to be inoculated in the nutrient solution and cultivates, promptly get BALB/c mouse knee cartilage cell.
2, the cultural method of BALB/c mouse knee cartilage cell according to claim 1 is characterized in that: step b, c, the described digestion Digestive system of e are: 0.15～0.18%w/wII Collagen Type VI enzyme solution.
3, the cultural method of BALB/c mouse knee cartilage cell according to claim 1 is characterized in that: the described nutrient solution of step c, e is: the DMEM nutrient solution that contains 12～18%w/w newborn calf serum.
4, the cultural method of BALB/c mouse knee cartilage cell according to claim 1 is characterized in that: described tissue block of b step and Digestive system blended volume ratio are: 1: 20～30.
5, the cultural method of BALB/c mouse knee cartilage cell according to claim 1, it is characterized in that: the described digestion vibration of b step is digestion stage by stage, the i.e. every digestion of all tissue block 1h just filters, centrifugal 1 time, the condition of vibration digestion is: slightly vibration in the 36.7-36.9 ℃ of water-bath, digested 2.5～4 hours, oscillation frequency is 20-30 time/min.
6, the cultural method of BALB/c mouse knee cartilage cell according to claim 1 is characterized in that: the centrifugal 13～17min of the described centrifugal employing high speed freezing centrifuge 800～1200r/min of c step.
7, the cultural method of BALB/c mouse knee cartilage cell according to claim 1, it is characterized in that: the described deproteinated of step e is 0.15～0.2%w/w trypsin solution, Digestive system is 0.15～0.18%w/wII Collagen Type VI enzyme solution, 36～37 ℃ of digestion down.
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| Application Number | Priority Date | Filing Date | Title |
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| CNA2003101107737A CN1539963A (en) | 2003-10-27 | 2003-10-27 | Method for cltivating cartilage cells in knee joint of BALB/c chmice |
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| CNA2003101107737A CN1539963A (en) | 2003-10-27 | 2003-10-27 | Method for cltivating cartilage cells in knee joint of BALB/c chmice |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN108699521A (en) * | 2016-11-30 | 2018-10-23 | 再生生物科学私人有限公司 | Prepare the method and application thereof of cartilage cell's suspension |
| CN113528454A (en) * | 2021-07-12 | 2021-10-22 | 福州载基生物科技有限公司 | Construction method of rat cartilage immortalized cell line |
-
2003
- 2003-10-27 CN CNA2003101107737A patent/CN1539963A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN104911145B (en) * | 2015-05-25 | 2018-11-09 | 上海中医药大学附属曙光医院 | A kind of preparation method of high vigor Primary chondrocyte |
| 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 |
| CN108699521A (en) * | 2016-11-30 | 2018-10-23 | 再生生物科学私人有限公司 | Prepare the method and application thereof of cartilage cell's suspension |
| CN113528454A (en) * | 2021-07-12 | 2021-10-22 | 福州载基生物科技有限公司 | Construction method of rat cartilage immortalized cell line |
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