CN102628028A - Preparation method for carrier-free mesenchymal stem cell patch - Google Patents
Preparation method for carrier-free mesenchymal stem cell patch Download PDFInfo
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- CN102628028A CN102628028A CN2012100907950A CN201210090795A CN102628028A CN 102628028 A CN102628028 A CN 102628028A CN 2012100907950 A CN2012100907950 A CN 2012100907950A CN 201210090795 A CN201210090795 A CN 201210090795A CN 102628028 A CN102628028 A CN 102628028A
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Abstract
The invention discloses a preparation method for a carrier-free mesenchymal stem cell patch. The preparation method comprises the following steps: firstly, culturing a mesenchymal stem cell in a culture dish by using an attachment method; after the cell is fused, continuously culturing for over 7 days; flushing the cell with phosphate buffer solution (PBS) or D-Hank's solution; adding 100mu L of 0.01-0.004 percent ethylene diamine tetraacetic acid (EDTA) into the dish, wherein the cell patch is separated from the culture dish from the surrounding to the center; after the cell path is completely separated, absorbing the EDTA solution; and slightly flushing the cell patch for two times by using a culture medium and then adding the flushed cell path in the culture medium for later use. Compared with a traditional carrier cell path, the patch prepared by the preparation method disclosed by the invention has the advantages that the patch is large in cell density; an operation method is simple and economic; the cell on the patch can be easily observed by microscope; the cell patch is transplanted in the body to avoid the adverse action caused by carrier metabolism; and the separated cell is digested and cultured by pancreatin and the multiplication capacity, the morphology and the activity of the cell have no remarkable change.
Description
Affiliated technical field
Patent of the present invention belongs to mesenchymal stem cells MSCs and cultivates the field, is specifically related to a kind of carrier free mesenchymal stem cells MSCs diaphragm-operated preparation method.
Background technology
Mesenchymal stem cells MSCs is arranged in the stromal cell lines of marrow; Belong to the multipotent adult stem cell; Can not only be divided into mesoderm tissues such as scleroblast, chondrocyte, sarcoplast, adipocyte, and can neuralward ectoderm and entoderm tissue differentiation such as cell, liver cell, islet cells.Mesenchymal stem cells MSCs owing to have is prone to separatedly to obtain, high division proliferative, many tissue differentiation potential, inhibitive ability of immunity, be convenient to plurality of advantages such as legalizing of autotransplantation, ethics, is one of the focus in current stem-cell research field.
Disease such as eye alkali burn, skin heat burn etc. that stem cell lacks are quite thorny in clinical treatment.The discovery of stem cell and the application of stem cell transplantation bring a gleam of hope for these patients.And mesenchymal stem cells MSCs becomes one of essential species daughter cell of this type of disease of treatment because of the characteristics of its multipotency.How it is transplanted the focus that becomes research in body surface.At present, more sophisticated method is it to be inoculated in form the compound stem cell diaphragm of amnion on the cell free amnion.But amnion is not complete transparent tissue, therefore sees through difficulty of amnion observation of cell state and cell density, and amnion has the defective work that degradation speed is fast, degraded product is piled up in addition.
At present, field of tissue engineering technology is being sought complete the coming off of a kind of stem cell that can make cultivation always and is being formed the material that diaphragm is used for clinical treatment.Studying more is temperature sensing material, hope to make cell detachment through the change of temperature, though obtained certain effect, but still immature.
YD 30 (EDTA) is a kind of divalent cation chelators, and cell surface much all has calcium ions and magnesium ions with petridish or culturing bottle bonded albumen, and therefore, adding behind the EDTA can these divalent ion of chelating, makes the disengaging of cell and petridish.
Summary of the invention
The purpose of this invention is to provide a kind of carrier free mesenchymal stem cells MSCs diaphragm-operated preparation method, be the clinical simple cell that supply the to transplant diaphragm that provides.
Preparing method of the present invention is following: at first; With adherent method mesenchymal stem cells MSCs is incubated in the petridish; Full dose is changed stem cell media once every other day, treat that mesenchymal stem cells MSCs merges fully after, cultivate again more than 7 days as mesenchymal stem cells MSCs for use;
Then; Get mesenchymal stem cells MSCs for use; More than 2 times, in petridish, add the divalent cation chelators that configures with the damping fluid flushing that does not contain divalent cation, treat that the mesenchymal stem cells MSCs diaphragm breaks away from petridish; Remove the sequestrant in the petridish, promptly obtain carrier free mesenchymal stem cells MSCs diaphragm;
At last, carrier free mesenchymal stem cells MSCs diaphragm, is added stem cell training base again and preserves more than 2 times with the flushing of stem cell training base.
Clamping the mesenchymal stem cells MSCs diaphragm for preparing with tweezers during use perhaps sucks the whole sheet of planting in the rifle head for one jiao; Place the position that needs transplanting; Add a small amount of saline water or balanced salt solution, with its flattening, sew up then/be adhesively fixed; Cover with contact lense, semi-permeable membranes or synthetic material etc., routine observation is by the transplantation site state.
The principle that the present invention utilizes the calcium ions and magnesium ions on divalent cation chelators chelating cell surface and petridish or the culturing bottle bonded albumen that cell and petridish are broken away from realizes; Compare cell patch in the past, the undesirable action that the present invention has avoided the carrier metabolism to be produced; This working method is simple, economy, pair cell are harmless, obtains pure cell patch, and cell density is big; Cell on the diaphragm can be observed through microscope easily.
Embodiment
Patent of the present invention is that material is processed DNAcarrier free mesenchymal stem cells MSCs diaphragm with the mesenchymal stem cells MSCs of BN rat, has the characteristic of mesenchymal stem cells MSCs, is used to treat the transplantation treatment of diseases such as stem cell shortage.
Preparing method of the present invention:
One, the cultivation of mesenchymal stem cells MSCs
Getting BN rat bilateral femur, the shin bone of 100g～150g under the aseptic condition goes in the aseptic ware to bring in the aseptic technique platform.0.9% sodium chloride injection flushing Lower limb bone, the sterile tissue scissors is removed femur, shin bone surface soft tissue clean, 0.9% sodium chloride injection flushing femur, shin bone.The 20ml asepsis injector is drawn the medullary space that 10ml mesenchymal stem cells MSCs training base (containing 10% foetal calf serum FBS+90%DMED liquid) washes femur, shin bone repeatedly, and washing fluid is gone into 37 ℃, 5%CO in the petridish
2Cultivate under the saturated humidity condition, cultivate and changed liquid in 48 hours first, changed liquid 1 time in later per 2 days, remove not adherent cell, treat cytogamy after, use tryptic digestion, by the cultivation of going down to posterity in 1: 3, changed liquid 1 time in per 2 days, after the fusion, continue to go down to posterity.This invention is taken is to be incubated at P2 in the 35mm plastic culture dish for mesenchymal stem cells MSCs, and change liquid continue to cultivate subsequent use more than seven days every day after the cytogamy.
Two, the preparation of YD 30 (EDTA)
20mgEDTA is dissolved in 100ml, and not contain divalent cation, pH value be to make 0.02%EDTA in 7.4 phosphate buffered saline buffer (PBS) or D-Hank ' the s liquid; The filter membrane aperture is that the sterile filters of 0.22 μ m places sterile chamber with above-mentioned EDTA liquid filtering; Time spent, concentration range all could between 0.01%～0.004% with aseptic PBS or the dilution of D-Hank ' s liquid.
Three, carrier free mesenchymal stem cells MSCs diaphragm-operated preparation
The petridish of getting ready that has mesenchymal stem cells MSCs is taken out from incubator; Place the aseptic technique platform; Stem cell training base in the exhaustion petridish; Aseptic PBS or D-Hank ' s liquid 2ml are added in the petridish with the divalent cation in the remaining training base in the flush away petridish, exhaustion, repeat once more than.Add good YD 30 (EDTA) the liquid 100 μ L of dilution; The mesenchymal stem cells MSCs of petridish periphery at first breaks away from petridish, and the mesenchymal stem cells MSCs of disengaging links to each other with the adjacent bone bone marrow-drived mesenchymal stem and forms sheet, and mesenchymal stem cells MSCs breaks away from scope to be developed to central authorities from periphery gradually; Finally break away from fully; Form carrier free mesenchymal stem cells MSCs diaphragm, with the disengaging of mesenchymal stem cells MSCs, carrier free mesenchymal stem cells MSCs diaphragm dwindles thickening gradually.Fully the separation time is 40 seconds～1 minute, breaks away from back carrier free mesenchymal stem cells MSCs diaphragm area size and be 1/3 at the bottom of the ware, and the contained cell quantity of carrier free mesenchymal stem cells MSCs diaphragm is 10
6The order of magnitude.
Claims (7)
1. carrier free mesenchymal stem cells MSCs diaphragm-operated preparation method is characterized in that:
At first, with adherent method mesenchymal stem cells MSCs is incubated in the petridish, full dose is changed stem cell media once every other day, treat that mesenchymal stem cells MSCs merges fully after, cultivate again more than 7 days as mesenchymal stem cells MSCs for use;
Then; Get mesenchymal stem cells MSCs for use; More than 2 times, in petridish, add the divalent cation chelators that configures with the damping fluid flushing that does not contain divalent cation, treat that the mesenchymal stem cells MSCs diaphragm breaks away from petridish; Remove the sequestrant in the petridish, promptly obtain carrier free mesenchymal stem cells MSCs diaphragm;
At last, carrier free mesenchymal stem cells MSCs diaphragm, is added stem cell training base again and preserves more than 2 times with the flushing of stem cell training base.
2. carrier free mesenchymal stem cells MSCs diaphragm-operated preparation method according to claim 1 is characterized in that: the said damping fluid that does not contain divalent cation is that pH value is 7.4 phosphate buffered saline buffer (PBS) or D-Hank ' s liquid.
3. carrier free mesenchymal stem cells MSCs diaphragm-operated preparation method according to claim 1 is characterized in that: the said divalent cation chelators that configures is YD 30 (EDTA) solution of concentration between 0.01%～0.004%.
4. method according to claim 3; It is characterized in that: said YD 30 (EDTA) collocation method is that to be configured to concentration in 7.4 phosphate buffered saline buffer (PBS) or D-Hank ' the s liquid be 0.02% YD 30 (EDTA) solution for 20mg YD 30 (EDTA) being dissolved in the 100mlPH value; With 0.02% YD 30 (EDTA) solution dilution, obtain YD 30 (EDTA) solution of concentration range between 0.01%～0.004% again.
5. carrier free mesenchymal stem cells MSCs diaphragm-operated preparation method according to claim 1; It is characterized in that: after adding YD 30 (EDTA) solution; The mesenchymal stem cells MSCs of petridish periphery at first breaks away from petridish, and the mesenchymal stem cells MSCs of disengaging links to each other with the adjacent bone bone marrow-drived mesenchymal stem and forms sheet, and mesenchymal stem cells MSCs breaks away from scope to be developed to central authorities from periphery gradually; Finally break away from fully; Form carrier free mesenchymal stem cells MSCs diaphragm, with the disengaging of mesenchymal stem cells MSCs, carrier free mesenchymal stem cells MSCs diaphragm dwindles thickening gradually.
6. method according to claim 5; It is characterized in that: the complete separation time of petridish of 35mm size is 40 seconds～1 minute; Break away from back carrier free mesenchymal stem cells MSCs diaphragm area size and be 1/3 at the bottom of the ware, the contained cell quantity of carrier free mesenchymal stem cells MSCs diaphragm is 10
6The order of magnitude.
7. carrier free mesenchymal stem cells MSCs diaphragm that makes like any one method in the claim 1～6, it is characterized in that: carrier free mesenchymal stem cells MSCs diaphragm-operated cell density is big; Can effectively avoid the undesirable action that produced because of the carrier metabolism; Carrier free mesenchymal stem cells MSCs diaphragm is after trysinization, cultivating, and its multiplication capacity, form and active nothing obviously change.
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| CN2012100907950A CN102628028A (en) | 2012-03-31 | 2012-03-31 | Preparation method for carrier-free mesenchymal stem cell patch |
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| CN2012100907950A CN102628028A (en) | 2012-03-31 | 2012-03-31 | Preparation method for carrier-free mesenchymal stem cell patch |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106924817A (en) * | 2017-03-02 | 2017-07-07 | 浙江大学 | A kind of ultra-thin carrier cell piece and preparation method thereof |
| CN112292447A (en) * | 2019-02-28 | 2021-01-29 | 京东方科技集团股份有限公司 | Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof |
| WO2021146994A1 (en) * | 2020-01-22 | 2021-07-29 | 京东方科技集团股份有限公司 | Mesenchymal stem cell sheet and use thereof |
Citations (1)
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| US20050221476A1 (en) * | 2004-03-18 | 2005-10-06 | Arindom Sen | Chemical dissociation of cell aggregates |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20050221476A1 (en) * | 2004-03-18 | 2005-10-06 | Arindom Sen | Chemical dissociation of cell aggregates |
Non-Patent Citations (4)
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| NORIAKI MATSUDA等: "Tissue Engineering Based on Cell Sheet Technology", 《ADV. MATER.》 * |
| 盛伟华等编: "《医用细胞工程实验指导》", 31 March 2008, 化学出版社 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106924817A (en) * | 2017-03-02 | 2017-07-07 | 浙江大学 | A kind of ultra-thin carrier cell piece and preparation method thereof |
| CN106924817B (en) * | 2017-03-02 | 2019-12-17 | 浙江大学 | Ultrathin carrier cell sheet and preparation method thereof |
| CN112292447A (en) * | 2019-02-28 | 2021-01-29 | 京东方科技集团股份有限公司 | Umbilical cord mesenchymal stem cell and preparation method of cell membrane thereof |
| WO2021146994A1 (en) * | 2020-01-22 | 2021-07-29 | 京东方科技集团股份有限公司 | Mesenchymal stem cell sheet and use thereof |
| CN113891933A (en) * | 2020-01-22 | 2022-01-04 | 京东方科技集团股份有限公司 | Mesenchymal stem cell membrane and application thereof |
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Application publication date: 20120808 |