CN103114073B - Method for removing cells from human amnion - Google Patents
Method for removing cells from human amnion Download PDFInfo
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- CN103114073B CN103114073B CN201310023898.XA CN201310023898A CN103114073B CN 103114073 B CN103114073 B CN 103114073B CN 201310023898 A CN201310023898 A CN 201310023898A CN 103114073 B CN103114073 B CN 103114073B
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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 tissue engineering technique field, relate in particular to a kind of method for removing cells of people's amnion.
Background technology
De-cell technology refers to tissue and/or the organ of processing humans and animals by serial of methods, and the cell of this tissue and/or organ is removed completely, and retains 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, there is 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.In amnion stroma, containing the compositions such as a large amount of collagen, fibronectin and ln, is a kind of tissue engineering bracket material of cheap and easy to get, excellent property.But the subject matter that it is applied to tissue construction as a kind of available timbering material is conscientiously to overcome the immunogenicity that its inherent cell brings.
The method of at present the de-cell of amnion tissue being processed, mainly be to adopt tensio-active agent or tensio-active agent in conjunction with mechanical force, to strike off etc., these methods cannot intactly retain characteristics of amniotic extracellular matrix, the performances such as the mechanics of amnion stroma and chemistry are all had to damage in various degree, and de-cell efficiency ratio is lower, 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 retain people's amnion of extracellular matrix.
The present invention solves the problems of the technologies described above adopted technical scheme: a kind of method for removing cells of people's amnion, comprises following detailed process:
(1), fresh people's placenta amnion is removed to clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and take the phosphoric acid buffer that pH value is 7.4 (being called for short PBS) and clean, obtain a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is by translucent amnion rinsing obtained above 1～2 time, use again PBS rinsing 2～4 times, then penicillin and Streptomycin sulphate are mixed into and in PBS, form dual anti-/PBS solution, described penicillin and Streptomycin sulphate are named again dual anti-, wherein penicillin and the Streptomycin sulphate content in PBS is 100～300U/L, again translucent amnion is soaked to concussion for the ratio of 1:1～3 is placed in dual anti-/PBS solution by volume, within every 12 hours, change liquid once, twice totally;
(3), dual anti-, tensio-active agent are mixed into and in PBS, form comprehensive PBS solution, 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 to concussion by volume, within every 6～8 hours, change liquid once, totally twice, then translucent amnion is taken out, with PBS rinsing 1～2 time;
(4), translucent amnion is immersed in steapsin/PBS solution that steapsin content is 500～5000U/L, the volume ratio of translucent amnion and steapsin/PBS solution is 1:1～3, and be at 30～40 ℃, to process 10～20 hours at solution temperature, then translucent amnion is taken out with PBS rinsing 1～2 time, again translucent amnion is immersed in DNA enzyme/PBS solution that DNA enzyme (being again deoxyribonuclease) content is 1000～3000U/L, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:1～3, at solution temperature, be at 30～40 ℃, to process 3～5 hours,
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and by translucent amnion be placed in penicillin and content of streptomycin be 100～300U/L dual anti-/PBS solution shakes rinsing, within every 12 hours, change liquid once, twice totally, finally the translucent amnion after de-cell is taken out and is put in PBS, at the temperature of 4 ℃, preserve stand-by.
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 the 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, the two is combined to use can impel the cell in amnion efficiently to be sloughed up hill and dale, farthest retain extracellular matrix, make it injury-free simultaneously.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 mono-: a kind of method for removing cells of people's amnion, comprises following detailed process:
(1), fresh people's placenta amnion is removed to clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and take the phosphoric acid buffer that pH value is 7.4 (being called for short PBS) and clean, obtain a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is by translucent amnion rinsing obtained above 2 times, use again PBS rinsing 2 times, then penicillin and Streptomycin sulphate are mixed into and in PBS, form dual anti-/PBS solution, wherein penicillin and the Streptomycin sulphate content in PBS is 100U/L, again translucent amnion for being placed in dual anti-/PBS solution, the ratio of 1:1 is soaked to concussion by volume, within every 12 hours, change liquid once, twice totally;
(3), dual anti-, tensio-active agent are mixed into and in PBS, form comprehensive PBS solution, 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 to concussion by volume, within every 6 hours, change liquid once, totally twice, then translucent amnion is taken out, with PBS rinsing 2 times;
(4), translucent amnion is immersed in steapsin/PBS solution that steapsin content is 1000U/L, the volume ratio of translucent amnion and steapsin/PBS solution is 1:2, and be at 30 ℃, to process 20 hours at solution temperature, then translucent amnion is taken out with PBS rinsing 1 time, again translucent amnion is immersed in DNA enzyme/PBS solution that DNA enzyme content is 1000U/L, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:2, at solution temperature, is at 40 ℃, to process 3 hours;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and by translucent amnion be placed in penicillin and content of streptomycin be 100U/L dual anti-/PBS solution shakes rinsing, within every 12 hours, change liquid once, twice totally, finally the translucent amnion after de-cell is taken out and is put in PBS, at the temperature of 4 ℃, preserve stand-by.
Embodiment bis-: a kind of method for removing cells of people's amnion, comprises following detailed process:
(1), fresh people's placenta amnion is removed to clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and take the phosphoric acid buffer that pH value is 7.4 (being called for short PBS) and clean, obtain a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is by translucent amnion rinsing obtained above 1 time, use again PBS rinsing 4 times, then penicillin and Streptomycin sulphate are mixed into and in PBS, form dual anti-/PBS solution, wherein penicillin and the Streptomycin sulphate content in PBS is 200U/L, again translucent amnion for being placed in dual anti-/PBS solution, the ratio of 1:3 is soaked to concussion by volume, within every 12 hours, change liquid once, twice totally;
(3), dual anti-, tensio-active agent are mixed into and in PBS, form comprehensive PBS solution, 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 to concussion by volume, within every 7 hours, change liquid once, totally twice, then translucent amnion is taken out, with PBS rinsing 1 time;
(4), translucent amnion is immersed in steapsin/PBS solution that steapsin content is 3000U/L, the volume ratio of translucent amnion and steapsin/PBS solution is 1:1, and be at 35 ℃, to process 15 hours at solution temperature, then translucent amnion is taken out with PBS rinsing 2 times, again translucent amnion is immersed in DNA enzyme/PBS solution that DNA enzyme content is 2000U/L, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:3, at solution temperature, is at 30 ℃, to process 5 hours;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and by translucent amnion be placed in penicillin and content of streptomycin be 200U/L dual anti-/PBS solution shakes rinsing, within every 12 hours, change liquid once, twice totally, finally the translucent amnion after de-cell is taken out and is put in PBS, at the temperature of 4 ℃, preserve stand-by.
Embodiment tri-: a kind of method for removing cells of people's amnion, comprises following detailed process:
(1), fresh people's placenta amnion is removed to clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and take the phosphoric acid buffer that pH value is 7.4 (being called for short PBS) and clean, obtain a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is by translucent amnion rinsing obtained above 2 times, use again PBS rinsing 3 times, then penicillin and Streptomycin sulphate are mixed into and in PBS, form dual anti-/PBS solution, wherein penicillin and the Streptomycin sulphate content in PBS is 300U/L, again translucent amnion for being placed in dual anti-/PBS solution, the ratio of 1:2 is soaked to concussion by volume, within every 12 hours, change liquid once, twice totally;
(3), dual anti-, tensio-active agent are mixed into and in PBS, form comprehensive PBS solution, 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 to concussion by volume, within every 8 hours, change liquid once, totally twice, then translucent amnion is taken out, with PBS rinsing 2 times;
(4), translucent amnion is immersed in steapsin/PBS solution that steapsin content is 5000U/L, the volume ratio of translucent amnion and steapsin/PBS solution is 1:3, and be at 40 ℃, to process 10 hours at solution temperature, then translucent amnion is taken out with PBS rinsing 2 times, again translucent amnion is immersed in DNA enzyme/PBS solution that DNA enzyme content is 3000U/L, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:1, at solution temperature, is at 35 ℃, to process 4 hours;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and by translucent amnion be placed in penicillin and content of streptomycin be 300U/L dual anti-/PBS solution shakes rinsing, within every 12 hours, change liquid once, twice totally, finally the translucent amnion after de-cell is taken out and is put in PBS, at the temperature of 4 ℃, preserve stand-by.
In above-described embodiment, PBS(used is again phosphoric acid buffer) pH value be 7.4, the Triton X-100 that tensio-active agent adopts Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge to provide.
Claims (2)
1. a method for removing cells for people's amnion, is characterized in that comprising following detailed process:
(1), fresh people's placenta amnion is removed to clot and chorion, stay and comprise epithelium layer, basilar membrane and without the complete amnion tissue of vascular stroma, and take the PBS that pH value is 7.4 and clean, obtain a clean translucent amnion;
(2), getting volumetric concentration is that 75% alcohol is by translucent amnion rinsing obtained above 1～2 time, use again PBS rinsing 2～4 times, then penicillin and Streptomycin sulphate are mixed into and in PBS, form dual anti-/PBS solution, described penicillin and Streptomycin sulphate are named again dual anti-, wherein penicillin and the Streptomycin sulphate content in PBS is 100～300U/L, again translucent amnion is soaked to concussion for the ratio of 1:1～1:3 is placed in dual anti-/PBS solution by volume, within every 12 hours, change liquid once, twice totally;
(3), dual anti-, tensio-active agent are mixed into and in PBS, form comprehensive PBS solution, 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 1:1～1:3 is soaked to concussion by volume, within every 6～8 hours, change liquid once, totally twice, then translucent amnion is taken out, with PBS rinsing 1～2 time;
(4), translucent amnion is immersed in steapsin/PBS solution that steapsin content is 500～5000U/L, the volume ratio of translucent amnion and steapsin/PBS solution is 1:1～1:3, and be at 30～40 ℃, to process 10～20 hours at solution temperature, then translucent amnion is taken out with PBS rinsing 1～2 time, again translucent amnion is immersed in DNA enzyme/PBS solution that DNA enzyme content is 1000～3000U/L, the volume ratio of translucent amnion and DNA enzyme/PBS solution is 1:1～1:3, at solution temperature, is at 30～40 ℃, to process 3～5 hours;
(5), translucent amnion is taken out from DNA enzyme/PBS solution, and by translucent amnion be placed in penicillin and content of streptomycin be 100～300U/L dual anti-/PBS solution shakes rinsing, within every 12 hours, change liquid once, twice totally, finally the translucent amnion after de-cell is taken out and is put in PBS, at the temperature of 4 ℃, preserve stand-by.
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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| CN104013998B (en) * | 2014-05-26 | 2015-06-24 | 宁波大学 | Preparation method of artificial esophagus with histological structure |
| CN109069867B (en) * | 2016-03-14 | 2022-02-01 | 无痛疗法股份有限公司 | Cell-free placenta preparation |
| 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 |
| 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 |
| US10960028B2 (en) | 2017-02-01 | 2021-03-30 | Plakous Therapeutics, Inc. | 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 |
| 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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Effective date of registration: 20170928 Address after: Hangzhou City, Zhejiang province 310018 poplar economic and Technological Development Zone Street No. 6 Street No. 452 Building 2 room C2001-2005/C2017 Patentee after: Hangzhou Engel biological medical technology Co Ltd Address before: 315211 Zhejiang Province, Ningbo Jiangbei District Fenghua Road No. 818 Patentee before: Ningbo University |
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