CN103114073A - Method for removing cells from human amnion - Google Patents
Method for removing cells from human amnion Download PDFInfo
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- CN103114073A CN103114073A CN201310023898XA CN201310023898A CN103114073A CN 103114073 A CN103114073 A CN 103114073A CN 201310023898X A CN201310023898X A CN 201310023898XA CN 201310023898 A CN201310023898 A CN 201310023898A CN 103114073 A CN103114073 A CN 103114073A
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Abstract
The invention discloses a method for removing cells from a human amnion. The method is characterized by comprising the following steps of: obtaining a clean semitransparent amnion, rinsing the semitransparent amnion by using a PBS (Phosphate Buffer Solution), soaking the semitransparent amnion in a comprehensive PBS liquid, shocking, rinsing the amnion by using the PBS after the amnion is taken out, successively soaking the amnion in steapsin/PBS liquid and deoxyribonuclease/PBS liquid for treatment, rinsing the amnion by using double-antibody/PBS liquid, taking out the decellularized amnion and putting the decellularized amnion in the PBS for preservation and standby. The method has the advantages that the cells are removed from the amnion in a manner that a surfactant is combined with steapsin and deoxyribonuclease, the cells can be prompted to be completely and efficiently removed from the amnion due to the combined use of the steapsin and the deoxyribonuclease, and meanwhile, the extracellular matrix is reserved to the maximum and is enabled to be free from damage.
Description
Technical field
The present invention relates to the tissue engineering technique field, relate in particular to a kind of method for removing cells of people's amnion.
Background technology
Take off tissue and/or organ that cell technology refers to process by serial of methods humans and animals, the cell of this tissue and/or organ is removed fully, and keep the method for corresponding extracellular matrix.It plays an important role in the structure of tissue engineering bracket.Amnion, i.e. people's placenta mucous membrane, be one smooth, without blood vessel, nerve and lymph, have elastic semi-transparent film, thick approximately 0.02 ~ 0.5mm is comprised of compositions such as adhesion layer, fine and close basilar membrane and avascular matrix.Containing the compositions such as a large amount of collagen, fibronectin and ln in amnion stroma, is a kind of tissue engineering bracket material of cheap and easy to get, excellent property.But it is to overcome the immunogenicity that its inherent cell brings as the subject matter that a kind of available timbering material conscientiously is applied to tissue construction.
The present method that cell is processed of taking off to amnion tissue, mainly be to adopt that tensio-active agent or tensio-active agent strike off in conjunction with mechanical force etc., these methods can't intactly keep characteristics of amniotic extracellular matrix, the performances such as the mechanics of amnion stroma and chemistry all there is in various degree damage, and it is lower to take off the cell efficiency ratio, and the scope of application is narrow.
Summary of the invention
Technical problem to be solved by this invention is to provide the method for removing cells that a kind of efficient cell free while can intactly keep people's amnion of extracellular matrix.
The present invention solves the problems of the technologies described above the technical scheme that adopts: a kind of method for removing cells of people's amnion comprises following detailed process:
(1), fresh people's placenta amnion is removed clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and the phosphoric acid buffer take the pH value as 7.4 (being called for short PBS) cleans, and obtains a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is with translucent amnion rinsing obtained above 1～2 time, use again the PBS rinsing 2～4 times, then penicillin and Streptomycin sulphate are mixed into formation pair anti-/PBS solution in PBS, described penicillin and Streptomycin sulphate are named again two anti-, wherein penicillin and the Streptomycin sulphate content in PBS is 100～300U/L, again translucent amnion is soaked concussion, changed liquid once, twice totally in every 12 hours for the ratio of 1:1～3 is placed in two resisting/PBS solution by volume;
(3), will be two anti-, tensio-active agent is mixed into and forms comprehensive PBS solution in PBS, wherein: penicillin and the Streptomycin sulphate content in PBS is 100～300U/L, the weight ratio that tensio-active agent accounts for PBS is 1%, and translucent amnion for being placed in comprehensive PBS solution, the ratio of 1:1～3 is soaked concussion by volume, changed liquid once in every 6～8 hours, totally twice, then translucent amnion is taken out, with PBS rinsing 1～2 time;
(4), translucent amnion is immersed in the steapsin that steapsin content is 500～5000U/L/PBS solution, the volume ratio of translucent amnion and steapsin/PBS solution is 1:1～3, and be to process under 30～40 ℃ 10～20 hours at solution temperature, then translucent amnion is taken out with PBS rinsing 1～2 time, again translucent amnion is immersed in the DNA enzyme that DNA enzyme (being again deoxyribonuclease) content is 1000～3000U/L/PBS solution, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:1～3, be to process under 30～40 ℃ 3～5 hours at solution temperature,
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and with translucent amnion be placed in penicillin and content of streptomycin be 100～300U/L two anti-/PBS solution shakes rinsing, changed liquid once in every 12 hours, twice totally, to take off at last translucent amnion after cell and take out and be put in PBS, preserve stand-by at the temperature of 4 ℃.
Described tensio-active agent adopts Triton X-100(Triton X-100).
Compared with prior art, the invention has the advantages that the method adopts tensio-active agent jointly to remove cell in amnion in conjunction with steapsin and DNA enzyme, lipid is the major ingredient of cytolemma, DNA is present in nucleus, the carrier of genetic information, steapsin and DNA enzyme can be distinguished the DNA in degradation of cell film and nucleus pointedly, can impel the cell in amnion efficiently to be sloughed up hill and dale with both combining use, simultaneously farthest keep extracellular matrix, make it injury-free.On the other hand, the present invention is directed to the structure component that eukaryotic cell has jointly and process, so the present invention is applicable to any eukaryotic removing, its use has universality.
Embodiment
Below the present invention is described in further detail.
Embodiment one: a kind of method for removing cells of people's amnion comprises following detailed process:
(1), fresh people's placenta amnion is removed clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and the phosphoric acid buffer take the pH value as 7.4 (being called for short PBS) cleans, and obtains a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is with translucent amnion rinsing obtained above 2 times, use again the PBS rinsing 2 times, then penicillin and Streptomycin sulphate are mixed into formation pair anti-/PBS solution in PBS, wherein penicillin and the Streptomycin sulphate content in PBS is 100U/L, again translucent amnion for being placed in two resisting/PBS solution, the ratio of 1:1 is soaked concussion by volume, changed liquid once, twice totally in every 12 hours;
(3), will be two anti-, tensio-active agent is mixed into and forms comprehensive PBS solution in PBS, wherein: penicillin and the Streptomycin sulphate content in PBS is 100U/L, the weight ratio that tensio-active agent accounts for PBS is 1%, and translucent amnion for being placed in comprehensive PBS solution, the ratio of 1:1 is soaked concussion by volume, changed liquid once in every 6 hours, totally twice, then translucent amnion is taken out, with PBS rinsing 2 times;
(4), translucent amnion is immersed in the steapsin that steapsin content is 1000U/L/PBS solution, the volume ratio of translucent amnion and steapsin/PBS solution is 1:2, and be to process 20 hours under 30 ℃ at solution temperature, then translucent amnion is taken out with PBS rinsing 1 time, again translucent amnion is immersed in the DNA enzyme that DNA enzyme content is 1000U/L/PBS solution, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:2, is to process 3 hours under 40 ℃ at solution temperature;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and with translucent amnion be placed in penicillin and content of streptomycin be 100U/L two anti-/PBS solution shakes rinsing, changed liquid once in every 12 hours, twice totally, to take off at last translucent amnion after cell and take out and be put in PBS, preserve stand-by at the temperature of 4 ℃.
Embodiment two: a kind of method for removing cells of people's amnion comprises following detailed process:
(1), fresh people's placenta amnion is removed clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and the phosphoric acid buffer take the pH value as 7.4 (being called for short PBS) cleans, and obtains a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is with translucent amnion rinsing obtained above 1 time, use again the PBS rinsing 4 times, then penicillin and Streptomycin sulphate are mixed into formation pair anti-/PBS solution in PBS, wherein penicillin and the Streptomycin sulphate content in PBS is 200U/L, again translucent amnion for being placed in two resisting/PBS solution, the ratio of 1:3 is soaked concussion by volume, changed liquid once, twice totally in every 12 hours;
(3), will be two anti-, tensio-active agent is mixed into and forms comprehensive PBS solution in PBS, wherein: penicillin and the Streptomycin sulphate content in PBS is 300U/L, the weight ratio that tensio-active agent accounts for PBS is 1%, and translucent amnion for being placed in comprehensive PBS solution, the ratio of 1:2 is soaked concussion by volume, changed liquid once in every 7 hours, totally twice, then translucent amnion is taken out, with PBS rinsing 1 time;
(4), translucent amnion is immersed in the steapsin that steapsin content is 3000U/L/PBS solution, the volume ratio of translucent amnion and steapsin/PBS solution is 1:1, and be to process 15 hours under 35 ℃ at solution temperature, then translucent amnion is taken out with PBS rinsing 2 times, again translucent amnion is immersed in the DNA enzyme that DNA enzyme content is 2000U/L/PBS solution, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:3, is to process 5 hours under 30 ℃ at solution temperature;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and with translucent amnion be placed in penicillin and content of streptomycin be 200U/L two anti-/PBS solution shakes rinsing, changed liquid once in every 12 hours, twice totally, to take off at last translucent amnion after cell and take out and be put in PBS, preserve stand-by at the temperature of 4 ℃.
Embodiment three: a kind of method for removing cells of people's amnion comprises following detailed process:
(1), fresh people's placenta amnion is removed clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and the phosphoric acid buffer take the pH value as 7.4 (being called for short PBS) cleans, and obtains a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is with translucent amnion rinsing obtained above 2 times, use again the PBS rinsing 3 times, then penicillin and Streptomycin sulphate are mixed into formation pair anti-/PBS solution in PBS, wherein penicillin and the Streptomycin sulphate content in PBS is 300U/L, again translucent amnion for being placed in two resisting/PBS solution, the ratio of 1:2 is soaked concussion by volume, changed liquid once, twice totally in every 12 hours;
(3), will be two anti-, tensio-active agent is mixed into and forms comprehensive PBS solution in PBS, wherein: penicillin and the Streptomycin sulphate content in PBS is 200U/L, the weight ratio that tensio-active agent accounts for PBS is 1%, and translucent amnion for being placed in comprehensive PBS solution, the ratio of 1:3 is soaked concussion by volume, changed liquid once in every 8 hours, totally twice, then translucent amnion is taken out, with PBS rinsing 2 times;
(4), translucent amnion is immersed in the steapsin that steapsin content is 5000U/L/PBS solution, the volume ratio of translucent amnion and steapsin/PBS solution is 1:3, and be to process 10 hours under 40 ℃ at solution temperature, then translucent amnion is taken out with PBS rinsing 2 times, again translucent amnion is immersed in the DNA enzyme that DNA enzyme content is 3000U/L/PBS solution, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:1, is to process 4 hours under 35 ℃ at solution temperature;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and with translucent amnion be placed in penicillin and content of streptomycin be 300U/L two anti-/PBS solution shakes rinsing, changed liquid once in every 12 hours, twice totally, to take off at last translucent amnion after cell and take out and be put in PBS, preserve stand-by at the temperature of 4 ℃.
In above-described embodiment, PBS(used is again phosphoric acid buffer) the pH value be 7.4, the Triton X-100 that tensio-active agent adopts Bioisystech Co., Ltd of China fir in Beijing Golden Bridge to provide.
Claims (2)
1. the method for removing cells of people's amnion is characterized in that comprising following detailed process:
(1), fresh people's placenta amnion is removed clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and the phosphoric acid buffer take the pH value as 7.4 (being called for short PBS) cleans, and obtains a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is with translucent amnion rinsing obtained above 1～2 time, use again the PBS rinsing 2～4 times, then penicillin and Streptomycin sulphate are mixed into formation pair anti-/PBS solution in PBS, described penicillin and Streptomycin sulphate are named again two anti-, wherein penicillin and the Streptomycin sulphate content in PBS is 100～300U/L, again translucent amnion is soaked concussion, changed liquid once, twice totally in every 12 hours for the ratio of 1:1～3 is placed in two resisting/PBS solution by volume;
(3), will be two anti-, tensio-active agent is mixed into and forms comprehensive PBS solution in PBS, wherein: penicillin and the Streptomycin sulphate content in PBS is 100～300U/L, the weight ratio that tensio-active agent accounts for PBS is 1%, and translucent amnion for being placed in comprehensive PBS solution, the ratio of 1:1～3 is soaked concussion by volume, changed liquid once in every 6～8 hours, totally twice, then translucent amnion is taken out, with PBS rinsing 1～2 time;
(4), translucent amnion is immersed in the steapsin that steapsin content is 500～5000U/L/PBS solution, the volume ratio of translucent amnion and steapsin/PBS solution is 1:1～3, and be to process under 30～40 ℃ 10～20 hours at solution temperature, then translucent amnion is taken out with PBS rinsing 1～2 time, again translucent amnion is immersed in the DNA enzyme that DNA enzyme content is 1000～3000U/L/PBS solution, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:1～3, is to process under 30～40 ℃ 3～5 hours at solution temperature;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and with translucent amnion be placed in penicillin and content of streptomycin be 100～300U/L two anti-/PBS solution shakes rinsing, changed liquid once in every 12 hours, twice totally, to take off at last translucent amnion after cell and take out and be put in PBS, preserve stand-by at the temperature of 4 ℃.
2. the method for removing cells of a kind of people's amnion as claimed in claim 1, is characterized in that described tensio-active agent adopts Triton X-100.
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104013998A (en) * | 2014-05-26 | 2014-09-03 | 宁波大学 | Preparation method of artificial esophagus with histological structure |
| CN105797212A (en) * | 2016-04-22 | 2016-07-27 | 黑龙江天晴干细胞股份有限公司 | Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal |
| CN106540323A (en) * | 2016-10-27 | 2017-03-29 | 江西瑞诺健医学科技有限公司 | A kind of preparation method of bioamnion |
| CN107583110A (en) * | 2017-10-24 | 2018-01-16 | 杭州恩格生物医疗科技有限公司 | A kind of preparation method of the artificial esophagus with biological structure and function |
| CN108048391A (en) * | 2017-12-20 | 2018-05-18 | 上海睿泰生物科技股份有限公司 | One-way fashion amnion method for removing cells |
| CN109069867A (en) * | 2016-03-14 | 2018-12-21 | 无痛疗法股份有限公司 | Cell-free placental preparations |
| CN109771697A (en) * | 2018-12-29 | 2019-05-21 | 江苏艾尔康生物医药科技有限公司 | A kind of dermal fibroblast skin graft and its construction method and application |
| CN111053949A (en) * | 2019-12-16 | 2020-04-24 | 杭州恩格生物医疗科技有限公司 | Preparation method and application of endometrial stem cell composite acellular amniotic membrane |
| CN111380750A (en) * | 2020-04-13 | 2020-07-07 | 北京科技大学 | Amnion tissue non-contact full-field deformation measurement method using methylene blue to make spots |
| US11806370B2 (en) | 2017-02-01 | 2023-11-07 | Plakous Therapeutics, Inc. | Methods of preparing and using placental tissue compositions |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104013998A (en) * | 2014-05-26 | 2014-09-03 | 宁波大学 | Preparation method of artificial esophagus with histological structure |
| CN109069867A (en) * | 2016-03-14 | 2018-12-21 | 无痛疗法股份有限公司 | Cell-free placental preparations |
| CN105797212B (en) * | 2016-04-22 | 2019-05-10 | 天晴干细胞股份有限公司 | A kind of de- cell amnion preparation method and application for the refractory conjunction wound repair of skin |
| CN105797212A (en) * | 2016-04-22 | 2016-07-27 | 黑龙江天晴干细胞股份有限公司 | Preparing method and application of acellular amniotic membrane used for repairing skin wound difficult to heal |
| CN106540323A (en) * | 2016-10-27 | 2017-03-29 | 江西瑞诺健医学科技有限公司 | A kind of preparation method of bioamnion |
| US11806370B2 (en) | 2017-02-01 | 2023-11-07 | Plakous Therapeutics, Inc. | Methods of preparing and using placental tissue compositions |
| CN107583110A (en) * | 2017-10-24 | 2018-01-16 | 杭州恩格生物医疗科技有限公司 | A kind of preparation method of the artificial esophagus with biological structure and function |
| CN108048391B (en) * | 2017-12-20 | 2021-02-19 | 上海睿泰生物科技股份有限公司 | One-way amnion decellularization method |
| CN108048391A (en) * | 2017-12-20 | 2018-05-18 | 上海睿泰生物科技股份有限公司 | One-way fashion amnion method for removing cells |
| CN109771697A (en) * | 2018-12-29 | 2019-05-21 | 江苏艾尔康生物医药科技有限公司 | A kind of dermal fibroblast skin graft and its construction method and application |
| CN109771697B (en) * | 2018-12-29 | 2021-09-07 | 江苏艾尔康生物医药科技有限公司 | Dermal fibroblast skin sheet and construction method and application thereof |
| CN111053949A (en) * | 2019-12-16 | 2020-04-24 | 杭州恩格生物医疗科技有限公司 | Preparation method and application of endometrial stem cell composite acellular amniotic membrane |
| CN111380750A (en) * | 2020-04-13 | 2020-07-07 | 北京科技大学 | Amnion tissue non-contact full-field deformation measurement method using methylene blue to make spots |
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