CN103045537A - Method for obtaining mesenchymal cell of umbilical cord - Google Patents
Method for obtaining mesenchymal cell of umbilical cord Download PDFInfo
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- CN103045537A CN103045537A CN2012105311741A CN201210531174A CN103045537A CN 103045537 A CN103045537 A CN 103045537A CN 2012105311741 A CN2012105311741 A CN 2012105311741A CN 201210531174 A CN201210531174 A CN 201210531174A CN 103045537 A CN103045537 A CN 103045537A
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Abstract
The invention relates to the technical field of biological medicine, and in particular relates to a method for obtaining mesenchymal cell of umnilical cord, and the method comprises the following steps: step1, washing the umbilical cord living tissue under an aseptic condition, and cutting into tissue fragments; and step 2, putting the tissue fragments into a culture dish, adding mixed culture solution with low sugar, and then putting the culture dish into a CO2 incubator for cultivation, wherein the mixed culture solution with low sugar in the culture dish is changed every 3-4 days. According to the method, the cells in the umnilical cord can be removed repeatedly through repeated transmission, and the cells are cultivated repeatedly, so that the purpose of obtaining a large amount of cells is achieved, on the basis of production effect above 100%, the yield of the cells is improved, 4.04*10<10> cells are obtained by cultivating cells of the third generation, and 1.56*10<12> cells are obtained by cultivating cells of the sixth generation, so that sufficient cell resources are provided for clinical tissue engineering and gene therapy.
Description
Technical field
The present invention relates to the biological medicine technology field, is a kind of preparation method of human umbilical mesenchymal cell.
Background technology
Mescenchymal stem cell (MSC) is to be derived from a mesoblastic class stem cell, and recent researches finds that MSC has good plasticity-and amplification ability, can provide progenitor cell for the reparative regeneration of disease damage tissue.Arnold I. caplan thinks: " a large amount of reconstructions or the reparation that utilize the mescenchymal stem cell of culture method amplification that particular organization is carried out, head and shoulders above their normal utilization and usage range." therefore, the mescenchymal stem cell of different tissue sources becomes the study hotspot of each study group.The MSC in umbilical cord source is sufficient and non-invasive because originating, and amplification technique is ripe, becomes the focus point that many research groups design a large amount of amplification schemes.The 3rd generation acquisition＞7 * 10 is being cultivated by C é cile DB study group
9Individual cell; The production effect of the culture scheme umbilical cord MSC of Fong CY study group is 100%, and the cell count of acquisition is about 4 * 10
6To 5 * 10
6Individual cell/cm
2Umbilical cord; Some investigative technique makes the production effect of umbilical cord can reach 100% but the cell count of a results enough or inadequate patient's therapeutic dose only; The increase cell quantity in the 3rd generation of some investigative technique umbilical cord is the most nearly 7 * 10
9, the cell harvesting amount of these amplification schemes does not all break through 10
9Individual cell.
Summary of the invention
The invention provides a kind of preparation method of human umbilical mesenchymal cell, overcome the deficiency of above-mentioned prior art, it is very few and cause the problem that can't satisfy therapeutic dose that it can effectively solve the amount of the method results human umbilical mesenchymal cell that obtains at present human umbilical mesenchymal cell.
Technical scheme of the present invention realizes by following measures: a kind of preparation method of human umbilical mesenchymal cell, carry out in the steps below: the first step, people's umbilical cord living tissue under aseptic condition is cleaned, and is chopped into and organizes fragment; Second step will be organized fragment to put into culture dish, and to wherein adding the low sugar mixed-culture medium, then culture dish be put into CO
2Cultivate in the incubator, culture dish changed the low sugar mixed-culture medium one time in per 3 days to 4 days; The 3rd step, in culture dish organize the fragment periphery to have spindle cell to climb out of the time after, will organize in fragment moves in the new culture dish, and in new culture dish, add the low sugar mixed-culture medium, put into CO
2Proceed in the incubator to cultivate; The 4th step repeated for the 3rd step repeatedly, until organize the fragment periphery to no longer include spindle cell to grow in the last culture dish that shifts.
The below is the further optimization and/or improvements to the foregoing invention technical scheme:
Above-mentioned CO
2The culture condition of incubator can be 37 ℃, 3.6% CO
2, saturated humidity.
The living tissue cleaning and chopping can be become 2 mm in the above-mentioned the first step
3To 3mm
3Fritter.
Above-mentioned after the 4th step, can cultivate with the low sugar mixed-culture medium migrate out the spindle cell that adheres at the bottom of the culture dish of organizing behind the fragment at every turn, when converging, Growth of Cells obtains cell with tryptic digestion after being 80% to 95%, collect the cell after obtaining, centrifugal, abandon supernatant liquor, counting cells, regulate cell concn on request the density renewed vaccination enter in the new culture dish and to wherein adding the low sugar mixed-culture medium, the Growth of Cells in new culture dish converges and reaches 80% to 95% rear continuation and digest by this step.
Above-mentioned tryptic concentration can be 0.5g/L to 2.5g/L, wherein also is added with the EDTA-Na of 0.53mmol/L
2
Above-mentioned centrifugal condition is that to can be 1000r/min to 1500r/min, time be 5min to 10min to rotating speed.
The above-mentioned density that requires when carrying out renewed vaccination can be 5 * 10
3Individual cell/cm
2Culture dish floorage to 10 * 10
3Individual cell/cm
2The culture dish floorage.
Above-mentioned low sugar mixed-culture medium can be and adds respectively foetal calf serum, L-glutaminate, sodium bicarbonate, L-alpha-phosphate xitix and green grass or young crops-Streptomycin sulphate solution in the low sugar DMEM nutrient solution and form mixed-culture medium, and the concentration that the volumetric concentration of foetal calf serum is 15% in the mixed-culture medium, the concentration of L-glutaminate is 2mM, sodium bicarbonate is that the concentration of 10.2mM, L-alpha-phosphate xitix is that the concentration of 50 μ g/mL, penicillin is that the concentration of 100u/ml, Streptomycin sulphate is 100ug/ml.
Utilization of the present invention is repeatedly shifted the cell that makes in the umbilical cord tissue and is repeatedly shifted out, and this cell is cultivated repeatedly again, thereby is reached the purpose of a large amount of acquisition cells, and the production effect improves the harvest yield of cell on 100% basis, cultivates for the 3rd generation to obtain 4.04 * 10
10Individual cell is cultivated the 6th generation acquisition 1.56 * 10
12Individual cell is for clinical tissue engineering and gene therapy provide sufficient cell source.
Embodiment
The present invention is not subjected to the restriction of following embodiment, can determine concrete embodiment according to technical scheme of the present invention and practical situation.
The invention will be further described below in conjunction with embodiment:
Embodiment 1, and the preparation method of this human umbilical mesenchymal cell carries out in the steps below: the first step, and people's umbilical cord living tissue under aseptic condition is cleaned, and is chopped into and organizes fragment; Second step will be organized fragment to put into culture dish, and to wherein adding the low sugar mixed-culture medium, then culture dish be put into CO
2Cultivate in the incubator, culture dish changed the low sugar mixed-culture medium one time in per 3 days to 4 days; The 3rd step, in culture dish organize the fragment periphery to have spindle cell to climb out of the time after, will organize in fragment moves in the new culture dish, and in new culture dish, add the low sugar mixed-culture medium, put into CO
2Proceed in the incubator to cultivate; The 4th step repeated for the 3rd step repeatedly, until organize the fragment periphery to no longer include spindle cell to grow in the last culture dish that shifts.
Can be according to actual needs, the preparation method of above-mentioned human umbilical mesenchymal cell is made further optimization and/or improvements:
Embodiment 2, preferred as above-described embodiment, CO
2The culture condition of incubator is 37 ℃, 3.6% CO
2, saturated humidity.3.6% CO
2In percentage ratio be percent by volume.
Embodiment 3, and preferred as above-described embodiment becomes 2 mm with the living tissue cleaning and chopping in the first step
3To 3mm
3Fritter.
Embodiment 4, be with the difference of above-described embodiment, after the 4th step, cultivate with the low sugar mixed-culture medium migrate out the spindle cell that adheres at the bottom of the culture dish of organizing behind the fragment at every turn, when converging, Growth of Cells obtains cell with tryptic digestion after being 80% to 95%, collect the cell after obtaining, centrifugal, abandon supernatant liquor, counting cells, regulate cell concn on request the density renewed vaccination enter in the new culture dish and to wherein adding the low sugar mixed-culture medium, the Growth of Cells in new culture dish converges and reaches 80% to 95% rear continuation and digest by this step.80% and 95% is respectively the percentage ratio that the cell total area behind the confluent growth accounts for the culture dish floorage.
Embodiment 5, preferred as above-described embodiment, and tryptic concentration is 0.5g/L to 2.5g/L, wherein also is added with the EDTA-Na of 0.53mmol/L
2
Embodiment 6, preferred as above-described embodiment, and centrifugal condition is that rotating speed is that 1000r/min to 1500r/min, time are 5min to 10min.
Embodiment 7, preferred as above-described embodiment, and the density that requires when carrying out renewed vaccination is 5 * 10
3Individual cell/cm
2Culture dish floorage to 10 * 10
3Individual cell/cm
2The culture dish floorage.
Embodiment 8, preferred as above-described embodiment, the low sugar mixed-culture medium forms mixed-culture medium for adding respectively foetal calf serum, L-glutaminate, sodium bicarbonate, L-alpha-phosphate xitix and green grass or young crops-Streptomycin sulphate solution in the low sugar DMEM nutrient solution, and the concentration that the volumetric concentration of foetal calf serum is 15% in the mixed-culture medium, the concentration of L-glutaminate is 2mM, sodium bicarbonate is that the concentration of 10.2mM, L-alpha-phosphate xitix is that the concentration of 50 μ g/mL, penicillin is that the concentration of 100u/ml, Streptomycin sulphate is 100ug/ml.
Embodiment 9, and a kind of preparation method of human umbilical mesenchymal cell carries out in the steps below: the first step is chopped into 2 mm with people's umbilical cord living tissue under the aseptic condition
3To 3mm
3Organize fragment; Second step will organize fragment to add in the low sugar mixed-culture medium, insert 37 ℃, 3.6%CO
2Cultivate in the incubator, culture dish changed the low sugar mixed-culture medium one time in per 3 days to 4 days; The 3rd step, cultivate and see after 15 days to 20 days and organize the fragment periphery to have cell to shift out, and will organize fragment to move in the new culture dish when being grown to spindle cell, add in the new culture dish and partly measure new low sugar mixed-culture medium to organizing fragment to continue to cultivate, organize the fragment periphery to have again emigrated cells to grow up to fusiformis after 3 days to 5 days; In the 4th step, will organize fragment again to move in the new culture dish this moment, and tissue block repeatedly shifts at least and can reach more than 20 times, grow up to until the tissue block periphery of cultivating no longer includes spindle cell; The 5th step migrated out the culture dish of organizing fragment and adds and partly to measure new low sugar mixed-culture medium cell is continued to cultivate, when cell cultures to 80% to 95% and when becoming the whirlpool shape, use trypsin digestion and cell, will press 8 * 10 behind the harvested cell counting
3/ cm
2Dividing the ware inoculation, is the first-generation cell of cultivating, and repeatedly digests, divides ware inoculation and cultivation by this, and every wheel cells was cultured to for the 3rd generation in generation to 6.3.6% CO
2In percentage ratio be percent by volume, 80% and 95% is respectively the percentage ratio that the cell total area behind the confluent growth accounts for the culture dish floorage.
Claims (8)
1. the preparation method of a human umbilical mesenchymal cell is characterized in that carrying out in the steps below:
The first step, people's umbilical cord living tissue under aseptic condition is cleaned, and is chopped into and organizes fragment;
Second step will be organized fragment to put into culture dish, and to wherein adding the low sugar mixed-culture medium, then culture dish be put into CO
2Cultivate in the incubator, culture dish changed the low sugar mixed-culture medium one time in per 3 days to 4 days;
The 3rd step, in culture dish organize the fragment periphery to have spindle cell to climb out of the time after, will organize in fragment moves in the new culture dish, and in new culture dish, add the low sugar mixed-culture medium, put into CO
2Proceed in the incubator to cultivate;
The 4th step repeated for the 3rd step repeatedly, until organize the fragment periphery to no longer include spindle cell to grow in the last culture dish that shifts.
2. the preparation method of human umbilical mesenchymal cell according to claim 1 is characterized in that CO
2The culture condition of incubator is 37 ℃, 3.6% CO
2, saturated humidity.
3. the preparation method of human umbilical mesenchymal cell according to claim 1 and 2 is characterized in that in the first step living tissue cleaning and chopping being become 2 mm
3To 3mm
3Fritter.
4. according to claim 1 and 2 or the preparation method of 3 described human umbilical mesenchymal cells, it is characterized in that after the 4th step, cultivate with the low sugar mixed-culture medium migrate out the spindle cell that adheres at the bottom of the culture dish of organizing behind the fragment at every turn, when converging, Growth of Cells obtains cell with tryptic digestion after being 80% to 95%, collect the cell after obtaining, centrifugal, abandon supernatant liquor, counting cells, regulate cell concn on request the density renewed vaccination enter in the new culture dish and to wherein adding the low sugar mixed-culture medium, the Growth of Cells in new culture dish converges and reaches 80% to 95% rear continuation and digest by this step.
5. the preparation method of human umbilical mesenchymal cell according to claim 4 is characterized in that tryptic concentration is 0.5g/L to 2.5g/L, wherein also is added with the EDTA-Na of 0.53mmol/L
2
6. according to claim 4 or the preparation method of 5 described human umbilical mesenchymal cells, it is characterized in that centrifugal condition is that rotating speed is that 1000r/min to 1500r/min, time are 5min to 10min.
7. according to claim 4 or the preparation method of 5 or 6 described human umbilical mesenchymal cells, the density that requires when it is characterized in that carrying out renewed vaccination is 5 * 10
3Individual cell/cm
2Culture dish floorage to 10 * 10
3Individual cell/cm
2The culture dish floorage.
8. according to claim 1 and 2 or the preparation method of 3 or 4 or 5 or 6 or 7 described human umbilical mesenchymal cells, it is characterized in that the low sugar mixed-culture medium is for to add respectively foetal calf serum in low sugar DMEM nutrient solution, L-glutaminate, sodium bicarbonate, L-alpha-phosphate xitix and green grass or young crops-Streptomycin sulphate solution forms mixed-culture medium, and the volumetric concentration of foetal calf serum is 15% in the mixed-culture medium, the concentration of L-glutaminate is 2mM, the concentration of sodium bicarbonate is 10.2mM, the concentration of L-alpha-phosphate xitix is 50 μ g/mL, the concentration of penicillin is 100u/ml, the concentration of Streptomycin sulphate is 100ug/ml.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592008A (en) * | 2019-09-26 | 2019-12-20 | 新疆医科大学第一附属医院 | Method for culturing bone marrow mesenchymal stem cells of canine animals |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321575A (en) * | 2011-10-08 | 2012-01-18 | 南方医科大学珠江医院 | Separation and culture method of human umbilical mesenchymal cell |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321575A (en) * | 2011-10-08 | 2012-01-18 | 南方医科大学珠江医院 | Separation and culture method of human umbilical mesenchymal cell |
Non-Patent Citations (3)
| Title |
|---|
| 宋立新: "不同月龄胎儿脐带和不同培养方法对分离人脐带间充质干细胞的影响", 《中国优秀硕士学位论文全文数据库》 * |
| 李铎: "人脐带间充质干细胞分离培养方法的研究", 《中国优秀硕士学位论文全文数据库》 * |
| 鄂玲玲等: "贴壁组织块反复消化法培养新生大鼠下颌骨成骨细胞及鉴定", 《华西口腔医学杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110592008A (en) * | 2019-09-26 | 2019-12-20 | 新疆医科大学第一附属医院 | Method for culturing bone marrow mesenchymal stem cells of canine animals |
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Application publication date: 20130417 |