CN103114073B - Method for removing cells from human amnion - Google Patents

Method for removing cells from human amnion Download PDF

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CN103114073B
CN103114073B CN 201310023898 CN201310023898A CN103114073B CN 103114073 B CN103114073 B CN 103114073B CN 201310023898 CN201310023898 CN 201310023898 CN 201310023898 A CN201310023898 A CN 201310023898A CN 103114073 B CN103114073 B CN 103114073B
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pbs
translucent
amnion
amniotic
solution
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CN103114073A (en
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竺亚斌
史佩娜
沈秋霞
高梦娜
卢珍珍
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宁波大学
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Abstract

本发明公开了一种人羊膜的脱细胞方法,特点是先得到干净的半透明羊膜,然后用PBS漂洗半透明羊膜,再将半透明羊膜置于综合PBS溶液中浸泡震荡,取出羊膜后用PBS漂洗,再将羊膜先后浸泡在胰脂酶/PBS溶液和DNA酶/PBS溶液中处理,最后用双抗/PBS溶液漂洗羊膜,最后将脱细胞后的羊膜取出放到PBS中保存待用;优点在于该方法采用表面活性剂结合胰脂酶和DNA酶共同来脱除羊膜中的细胞,将二者结合起来使用可以促使羊膜中的细胞被高效彻底地脱去,同时最大程度地保留细胞外基质,使其不受损伤。 The present invention discloses a method acellular human amnion, characterized translucent to get clean amnion, amniotic translucent then rinsed with PBS and then placed in a translucent amnion was immersed in PBS integrated shock, taken with PBS amnion rinsed, and then immersed in amniotic membrane has pancreatic lipase / PBS solution and the enzyme DNA / PBS solution, and finally with a double antibody / PBS solution was rinsed amnion, amniotic finally taken into acellular stored in PBS stand; advantages in that the method uses a surfactant and DNA binding pancreatic lipase enzymes work together to remove amnion cells to use the two together may cause amnion cells are efficiently removed completely, while maximizing the retention of extracellular matrix make it from damage.

Description

一种人羊膜的脱细胞方法 One human amnion acellular method

技术领域 FIELD

[0001] 本发明涉及组织工程技术领域,尤其涉及一种人羊膜的脱细胞方法。 [0001] The present invention relates to the field of tissue engineering, particularly to a method of acellular human amnion.

背景技术 Background technique

[0002] 脱细胞技术是指通过一系列方法处理人和动物的组织和/或器官,使该组织和/或器官的细胞完全去除,而保留相应的细胞外基质的方法。 [0002] technology is the acellular tissue of humans and animals through a series of processing methods and / or organs, the tissue and / or organs, cells completely removed, while retaining the corresponding method of extracellular matrix. 它在组织工程支架的构建中起着重要的作用。 It plays an important role in the construction of tissue engineering scaffolds. 羊膜,即人胎盘粘膜,是一光滑的、无血管、神经及淋巴的、具有弹性的半透明膜,厚约0.02、.5mm,由单层上皮细胞层、致密的基底膜以及无血管的基质等成份组成。 Translucent amniotic membrane, i.e. the mucosal human placenta, is a smooth, non-vascular, lymphatic and nerve, having elasticity, thickness 0.02, .5mm, by a single epithelial cell layer, and a dense basement membrane matrix avascular and other ingredients. 羊膜基质中含有大量的胶原、纤粘连蛋白和层粘连蛋白等成份,是一种廉价易得、性能优良的组织工程支架材料。 Amniotic membrane matrix contains large amounts of collagen, fibronectin and laminin and other ingredients, it is an inexpensive, readily available, high performance tissue engineering scaffold. 但将其作为一种切实可用的支架材料应用于组织构建的主要问题在于要克服其内在细胞所带来的免疫原性。 But the main problem as a practical scaffold material available to build organizations that applied to overcome the inherent cells brought immunogenicity.

[0003]目前对羊膜组织的脱细胞处理的方法,主要在于采用表面活性剂或表面活性剂结合机械力刮除等,这些方法无法完整地保留羊膜细胞外基质,对羊膜基质的力学和化学等性能均有不同程度的损伤,而且脱细胞效率比较低,适用范围比较窄。 [0003] The method currently decellularized amniotic membrane tissue, primarily using a surfactant or surfactant combination of mechanical scraping force, etc., these methods are not intact amniotic cells outside the matrix, the mechanical and chemical amniotic mesenchymal like properties in varying degrees of damage, and the efficiency is relatively low acellular, relatively narrow range.

发明内容 SUMMARY

[0004] 本发明所要解决的技术问题是提供一种高效脱细胞的同时可完整地保留细胞外基质的人羊膜的脱细胞方法。 [0004] The present invention solves the technical problem while providing acellular an efficient method can be decellularized extracellular matrix intact human amniotic membrane.

[0005] 本发明解决上述技术问题所采用的技术方案为:一种人羊膜的脱细胞方法,包括以下具体过程: [0005] aspect of the present invention to solve the above technical problem is: one human decellularized amniotic method comprises the following procedure:

[0006] (I)、将新鲜的人胎盘羊膜除去血块和绒毛膜,留下包含上皮细胞层、基底膜和无血管基质的完整羊膜组织,并以PH值为7.4的磷酸缓冲液(简称PBS)清洗,得到一干净半透明的羊膜; [0006] (I), fresh human placental amniotic and chorionic clot removed, leaving the tissue which contains a complete amniotic epithelium layer, basement membrane and avascular matrix, and phosphate buffer PH value to 7.4 (referred to as PBS ) washing to obtain a clean, translucent amniotic membrane;

[0007] (2)、取体积浓度为75%的酒精将上述得到的半透明羊膜漂洗I~2次,再用PBS漂洗2~4次,然后将青霉素和链霉素混合到PBS中形成双抗/PBS溶液,所述的青霉素和链霉素又叫双抗,其中青霉素与链霉素在PBS中的含量均为100~300U/L,再将半透明羊膜按体积比为1:1~3的比例置于双抗/PBS溶液中浸泡震荡,每12小时换液一次,共两次; [0007] (2), take the concentration of 75% by volume of alcohol obtained above was rinsed translucent amniotic I ~ 2 times, rinsed with PBS to 4 times, then mixed into PBS penicillin and streptomycin forming a double anti / PBS solution, the double antibody called penicillin and streptomycin, penicillin and streptomycin, wherein the content in PBS were 100 ~ 300U / L, then the translucent amniotic volume ratio of 1: 1 Comparative Example 3 was placed in double antibody / PBS solution soak shock, medium was changed every 12 hours, a total of two;

[0008] (3)、将双抗、表面活性剂混合到PBS中形成综合PBS溶液,其中:青霉素与链霉素在PBS中的含量均为100~300U/L,表面活性剂占PBS的重量比为1%,并将半透明羊膜按体积比为1:1~3的比例置于综合PBS溶液中浸泡震荡,每6~8小时换液一次,共两次,然后将半透明羊膜取出,用PBS漂洗I~2次; [0008] (3), a double-antibody, surfactant mixed to the integrated PBS PBS solution is formed, wherein: the content of penicillin and streptomycin in PBS were 100 ~ 300U / L, the surfactant comprises by weight of the PBS ratio was 1% and the volume ratio of the translucent amnion 1: 1 to 3 was placed in PBS solution soak integrated shock, every 6 to 8 hours the medium was changed once, a total of two, then the translucent amniotic removed I ~ 2 were rinsed with PBS once;

[0009] (4)、将半透明羊膜浸泡在胰脂酶含量为500~5000U/L的胰脂酶/PBS溶液中,半透明羊膜与胰脂酶/PBS溶液的体积比为1:1~3,并在溶液温度为30~40°C下处理10~20小时,然后将半透明羊膜取出用PBS漂洗I~2次,再将半透明羊膜浸泡在DNA酶(又叫脱氧核糖核酸酶)含量为1000~3000U/L的DNA酶/PBS溶液中,半透明羊膜与DNA酶/PBS溶液的体积比为1:1~3,在溶液温度为30~40°C下处理3~5小时; [0009] (4), the amnion was immersed in a translucent pancreatic lipase content of 500 ~ 5000U / L of pancreatic lipase / PBS solution in a volume ratio of translucent amnion pancreatic lipase / PBS solution of 1: 1 3, and for 10 to 20 hours at 30 ~ 40 ° C, and then rinsed with PBS and taken translucent amnion I ~ 2 times, and then soaked in translucent amnion DNA enzyme (also known as DNase) solution at a temperature of content of 1000 ~ 3000U / L of the enzyme DNA / PBS solution, the translucent volume of amniotic membrane and the enzyme DNA / PBS solution ratio of 1: 1 to 3, the solution treatment temperature at 30 ~ 40 ° C for 3-5 hours;

[0010] (5)、将半透明羊膜从DNA酶/PBS溶液中取出,并将半透明羊膜置于青霉素与链霉素含量均为100~300U/L的双抗/PBS溶液中震荡漂洗,每12小时换液一次,共两次,最后将脱细胞后的半透明羊膜取出放到PBS中,在4°C的温度下保存待用。 [0010] (5), the amniotic membrane was removed from the translucent enzyme DNA / PBS solution, and the amniotic membrane is placed penicillin and streptomycin translucent contents were 100 ~ 300U / L of double-antibody / PBS solution was rinsed shock, medium was changed every 12 hours, a total of two, and finally a translucent acellular amniotic taken into PBS and stored under the temperature at 4 ° C.

[0011] 所述的表面活性剂采用Triton X-100 (曲拉通100)。 [0011] The use of the surfactant Triton X-100 (Triton 100).

[0012] 与现有技术相比,本发明的优点在于该方法采用表面活性剂结合胰脂酶和DNA酶共同来脱除羊膜中的细胞,脂类是细胞膜的主要成份,DNA存在于细胞核中,是遗传信息的携带者,胰脂酶和DNA酶能针对性地分别降解细胞膜和细胞核中的DNA,将二者结合起来使用可以促使羊膜中的细胞被高效彻底地脱去,同时最大程度地保留细胞外基质,使其不受损伤。 [0012] Compared with the prior art, advantages of the present invention is that the method using a surfactant and a DNA binding pancreatic lipase enzymes work together to remove amnion cells, are major components of membrane lipids, DNA present in the nucleus , a carrier, and pancreatic lipase enzyme capable of DNA degradation were targeted to the cell membrane, and DNA in the nucleus, to use the two together may cause amnion cells are efficiently removed completely, while maximizing the genetic information reserved extracellular matrix, it is not damaged. 另一方面,本发明针对真核细胞共同具有的结构成份进行处理,因此本发明适用于任何真核细胞的脱除,其使用具有普适性。 Another aspect, the present invention for structural components in common eukaryotic cell treatment, thus the present invention is applicable to any removal of eukaryotic cells, having universality.

具体实施方式 Detailed ways

[0013] 以下对本发明作进一步详细描述。 [0013] The following further detailed description of the invention.

[0014] 实施例一:一种人羊膜的脱细胞方法,包括以下具体过程: [0014] Example a: one human acellular amniotic membrane method, comprises the following procedure:

[0015] (I)、将新鲜的人胎盘羊膜除去血块和绒毛膜,留下包含上皮细胞层、基底膜和无血管基质的完整羊膜组织,并以PH值为7.4的磷酸缓冲液(简称PBS)清洗,得到一干净半透明的羊膜; [0015] (I), fresh human placental amniotic and chorionic clot removed, leaving the tissue which contains a complete amniotic epithelium layer, basement membrane and avascular matrix, and phosphate buffer PH value to 7.4 (referred to as PBS ) washing to obtain a clean, translucent amniotic membrane;

[0016] (2)、取体积浓度为75%的酒精将上述得到的半透明羊膜漂洗2次,再用PBS漂洗2次,然后将青霉素和链霉素混合到PBS中形成双抗/PBS溶液,其中青霉素与链霉素在PBS中的含量均为100U/L,再将半透明羊膜按体积比为1:1的比例置于双抗/PBS溶液中浸泡震荡,每12小时换液一次,共两次; [0016] (2), take the concentration of 75% by volume of alcohol obtained above was rinsed twice translucent amnion, rinsed twice with PBS, penicillin and streptomycin and then mixing the formed double-antibody in PBS / PBS solution wherein the content of penicillin and streptomycin in PBS were 100U / L, then the translucent amnion by volume ratio of 1: 1 was placed in double antibody / PBS solution soak shock, medium was changed every 12 hours, a total of two;

[0017] (3)、将双抗、表面活性剂混合到PBS中形成综合PBS溶液,其中:青霉素与链霉素在PBS中的含量均为100U/L,表面活性剂占PBS的重量比为1%,并将半透明羊膜按体积比为1:1的比例置于综合PBS溶液中浸泡震荡,每6小时换液一次,共两次,然后将半透明羊膜取出,用PBS漂洗2次; [0017] (3), a double-antibody, surfactant mixture is formed integrated into PBS PBS solution, wherein: the content of penicillin and streptomycin in PBS were 100U / L, the surfactant comprises a weight ratio of PBS 1%, and the translucent amnion by volume ratio of 1: 1 was placed in PBS soaked integrated shock, medium was changed every 6 hours, a total of two, then the translucent amnion removed, rinsed twice with PBS;

[0018] (4)、将半透明羊膜浸泡在胰脂酶含量为1000U/L的胰脂酶/PBS溶液中,半透明羊膜与胰脂酶/PBS溶液的体积比为1:2,并在溶液温度为30°C下处理20小时,然后将半透明羊膜取出用PBS漂洗I次,再将半透明羊膜浸泡在DNA酶含量为1000U/L的DNA酶/PBS溶液中,半透明羊膜与DNA酶/PBS溶液的体积比为1:2,在溶液温度为40°C下处理3小时; [0018] (4), the amnion was immersed in a translucent pancreatic lipase content of 1000U / L of pancreatic lipase / PBS solution, the translucent amnion pancreatic lipase / PBS solution in a volume ratio of 1: 2, and the solution temperature was 20 hours at 30 ° C, and then rinsed with PBS and taken translucent amnion I times, and then soaked in translucent amnion DNA enzyme content of 1000U / L of the enzyme DNA / PBS solution, the translucent amnion DNA volume of enzyme / PBS solution ratio of 1: 2, treatment at 40 ° C 3 hours at a temperature of the solution;

[0019] (5)、将半透明羊膜从DNA酶/PBS溶液中取出,并将半透明羊膜置于青霉素与链霉素含量均为100U/L的双抗/PBS溶液中震荡漂洗,每12小时换液一次,共两次,最后将脱细胞后的半透明羊膜取出放到PBS中,在4°C的温度下保存待用。 [0019] (5), the amniotic membrane was removed from the translucent enzyme DNA / PBS solution, and the amniotic membrane is placed penicillin and streptomycin translucent contents of both 100U / L of double-antibody / PBS solution was rinsed shock every 12 h medium was changed, a total of two, and finally a translucent acellular amniotic taken into PBS, at a preservation temperature at 4 ° C until use.

[0020] 实施例二:一种人羊膜的脱细胞方法,包括以下具体过程: [0020] Second Embodiment: one human decellularized amniotic method comprises the following procedure:

[0021] (I)、将新鲜的人胎盘羊膜除去血块和绒毛膜,留下包含上皮细胞层、基底膜和无血管基质的完整羊膜组织,并以PH值为7.4的磷酸缓冲液(简称PBS)清洗,得到一干净半透明的羊膜; [0021] (I), fresh human placental amniotic and chorionic clot removed, leaving the tissue which contains a complete amniotic epithelium layer, basement membrane and avascular matrix, and phosphate buffer PH value to 7.4 (referred to as PBS ) washing to obtain a clean, translucent amniotic membrane;

[0022] (2)、取体积浓度为75%的酒精将上述得到的半透明羊膜漂洗I次,再用PBS漂洗4次,然后将青霉素和链霉素混合到PBS中形成双抗/PBS溶液,其中青霉素与链霉素在PBS中的含量均为200U/L,再将半透明羊膜按体积比为1:3的比例置于双抗/PBS溶液中浸泡震荡,每12小时换液一次,共两次; [0022] (2), take the concentration of 75% by volume of alcohol obtained above was translucent amniotic I rinsed twice, rinsed four times with PBS, penicillin and streptomycin and then mixing the formed double-antibody in PBS / PBS solution wherein the content of penicillin and streptomycin in PBS were 200U / L, then the translucent amnion by volume ratio of 1: 3 ratio in double anti / PBS solution soak shock, medium was changed every 12 hours, a total of two;

[0023] (3)、将双抗、表面活性剂混合到PBS中形成综合PBS溶液,其中:青霉素与链霉素在PBS中的含量均为300U/L,表面活性剂占PBS的重量比为1%,并将半透明羊膜按体积比为1:2的比例置于综合PBS溶液中浸泡震荡,每7小时换液一次,共两次,然后将半透明羊膜取出,用PBS漂洗I次; [0023] (3), a double-antibody, surfactant mixture is formed integrated into PBS PBS solution, wherein: the content of penicillin and streptomycin in PBS were 300U / L, the surfactant comprises a weight ratio of PBS 1%, and the translucent amnion by volume ratio of 1: 2 ratio of the PBS solution was placed in soaking integrated shock, medium was changed every seven hours, a total of two, then the translucent amnion removed, rinsed with PBS I times;

[0024] (4)、将半透明羊膜浸泡在胰脂酶含量为3000U/L的胰脂酶/PBS溶液中,半透明羊膜与胰脂酶/PBS溶液的体积比为1: 1,并在溶液温度为35°C下处理15小时,然后将半透明羊膜取出用PBS漂洗2次,再将半透明羊膜浸泡在DNA酶含量为2000U/L的DNA酶/PBS溶液中,半透明羊膜与DNA酶/PBS溶液的体积比为1:3,在溶液温度为30°C下处理5小时; [0024] (4), the amnion was immersed in a translucent pancreatic lipase content of 3000U / L of pancreatic lipase / PBS solution, the translucent amnion pancreatic lipase / PBS solution in a volume ratio of 1: 1, and solution temperature was 15 hours at 35 ° C, then removed with PBS translucent amnion rinsed twice, then soaked in translucent amnion DNA enzyme content of 2000U / L of the enzyme DNA / PBS solution, the translucent amnion DNA volume of enzyme / PBS solution ratio of 1: 3, treatment at 30 ° C 5 hours at a temperature of the solution;

[0025] (5)、将半透明羊膜从DNA酶/PBS溶液中取出,并将半透明羊膜置于青霉素与链霉素含量均为200U/L的双抗/PBS溶液中震荡漂洗,每12小时换液一次,共两次,最后将脱细胞后的半透明羊膜取出放到PBS中,在4°C的温度下保存待用。 [0025] (5), the amniotic membrane was removed from the translucent enzyme DNA / PBS solution, and the amniotic membrane is placed penicillin and streptomycin translucent contents of both 200U / L of double-antibody / PBS solution was rinsed shock every 12 h medium was changed, a total of two, and finally a translucent acellular amniotic taken into PBS, at a preservation temperature at 4 ° C until use.

[0026] 实施例三:一种人羊膜的脱细胞方法,包括以下具体过程: [0026] Example Three: one human decellularized amniotic method comprises the following procedure:

[0027] (I)、将新鲜的人胎盘羊膜除去血块和绒毛膜,留下包含上皮细胞层、基底膜和无血管基质的完整羊膜组织,并以PH值为7.4的磷酸缓冲液(简称PBS)清洗,得到一干净半透明的羊膜; [0027] (I), fresh human placental amniotic and chorionic clot removed, leaving the tissue which contains a complete amniotic epithelium layer, basement membrane and avascular matrix, and phosphate buffer PH value to 7.4 (referred to as PBS ) washing to obtain a clean, translucent amniotic membrane;

[0028] (2)、取体积浓度为75%的酒精将上述得到的半透明羊膜漂洗2次,再用PBS漂洗3次,然后将青霉素和链霉素混合到PBS中形成双抗/PBS溶液,其中青霉素与链霉素在PBS中的含量均为300U/L,再将半透明羊膜按体积比为1:2的比例置于双抗/PBS溶液中浸泡震荡,每12小时换液一次,共两次; [0028] (2), take the concentration of 75% by volume of alcohol obtained above was rinsed twice translucent amnion, rinsed three times with PBS, penicillin and streptomycin and then mixing the formed double-antibody in PBS / PBS solution wherein the content of penicillin and streptomycin in PBS were 300U / L, then the translucent amnion by volume ratio of 1: 2 ratio in double anti / PBS solution soak shock, medium was changed every 12 hours, a total of two;

[0029] (3)、将双抗、表面活性剂混合到PBS中形成综合PBS溶液,其中:青霉素与链霉素在PBS中的含量均为200U/L,表面活性剂占PBS的重量比为1%,并将半透明羊膜按体积比为1:3的比例置于综合PBS溶液中浸泡震荡,每8小时换液一次,共两次,然后将半透明羊膜取出,用PBS漂洗2次; [0029] (3), a double-antibody, surfactant mixture is formed integrated into PBS PBS solution, wherein: the content of penicillin and streptomycin in PBS were 200U / L, the surfactant comprises a weight ratio of PBS 1%, and the translucent amnion by volume ratio of 1: 3 ratio of the PBS solution was placed in soaking integrated shock, medium was changed every 8 hours, a total of two, then the translucent amnion removed, rinsed twice with PBS;

[0030] (4)、将半透明羊膜浸泡在胰脂酶含量为5000U/L的胰脂酶/PBS溶液中,半透明羊膜与胰脂酶/PBS溶液的体积比为1:3,并在溶液温度为40°C下处理10小时,然后将半透明羊膜取出用PBS漂洗2次,再将半透明羊膜浸泡在DNA酶含量为3000U/L的DNA酶/PBS溶液中,半透明羊膜与DNA酶/PBS溶液的体积比为1:1,在溶液温度为35°C下处理4小时; [0030] (4), the amnion was immersed in a translucent pancreatic lipase content of 5000U / L of pancreatic lipase / PBS solution, the translucent amnion pancreatic lipase / PBS solution in a volume ratio of 1: 3, and the solution temperature was 10 hours at 40 ° C, then removed with PBS translucent amnion rinsed twice, then soaked in translucent amnion DNA enzyme content of 3000U / L of the enzyme DNA / PBS solution, the translucent amnion DNA enzyme / PBS solution in a volume ratio of 1: 1, for 4 hours at 35 ° C in the temperature of the solution;

[0031] (5)、将半透明羊膜从DNA酶/PBS溶液中取出,并将半透明羊膜置于青霉素与链霉素含量均为300U/L的双抗/PBS溶液中震荡漂洗,每12小时换液一次,共两次,最后将脱细胞后的半透明羊膜取出放到PBS中,在4°C的温度下保存待用。 [0031] (5), the amniotic membrane was removed from the translucent enzyme DNA / PBS solution, and the amniotic membrane is placed penicillin and streptomycin translucent contents of both 300U / L of double-antibody / PBS solution was rinsed shock every 12 h medium was changed, a total of two, and finally a translucent acellular amniotic taken into PBS, at a preservation temperature at 4 ° C until use.

[0032] 上述实施例中,所用到的PBS (又叫磷酸缓冲液)的pH值均为7.4,表面活性剂采用北京中杉金桥生物技术有限公司提供的Triton X-100。 [0032] The above-described embodiments, pH values ​​used in PBS (phosphate buffer called) are 7.4, using a surfactant Triton X-100 Beijing Zhongshan Golden Bridge Biotechnology Co. provided.

Claims (2)

1.一种人羊膜的脱细胞方法,其特征在于包括以下具体过程: (1)、将新鲜的人胎盘羊膜除去血块和绒毛膜,留下包含上皮细胞层、基底膜和无血管基质的完整羊膜组织,并以PH值为7.4的PBS清洗,得到一干净半透明的羊膜; (2)、取体积浓度为75%的酒精将上述得到的半透明羊膜漂洗I~2次,再用PBS漂洗2~4次,然后将青霉素和链霉素混合到PBS中形成双抗/PBS溶液,所述的青霉素和链霉素又叫双抗,其中青霉素与链霉素在PBS中的含量均为100~300U/L,再将半透明羊膜按体积比为1:1~1:3的比例置于双抗/PBS溶液中浸泡震荡,每12小时换液一次,共两次; (3)、将双抗、表面活性剂混合到PBS中形成综合PBS溶液,其中:青霉素与链霉素在PBS中的含量均为100~300U/L,表面活性剂占PBS的重量比为1%,并将半透明羊膜按体积比为1:1~1:3的比例置于综合PBS溶液中浸泡震荡,每6~8小时换液一次 An acellular human amnion, characterized in that the process comprises the following: (1), fresh human placental amniotic and chorionic clot removed, leaving a layer comprising epithelial cells, basement membrane and matrix complete avascular amnion, and washed with PBS PH value to 7.4, to obtain a clean, translucent amnion; (2), take the concentration of 75% by volume of alcohol obtained above was rinsed translucent amniotic I ~ 2 times, rinsed with PBS 2 to 4 times, penicillin and streptomycin and then mixing the formed double-antibody in PBS / PBS solution, the double antibody called penicillin and streptomycin, penicillin and streptomycin in which the content of the PBS 100 are ~ 300U / L, then the translucent amniotic volume ratio of 1: 1 to 1: 3 was placed in double antibody / PBS solution soak shock, once, twice every 12 hours for a total solution; (3), the double-antibody, surfactant mixture is formed integrated into PBS PBS solution, wherein: the content of penicillin and streptomycin in PBS were 100 ~ 300U / L, the surfactant comprises a weight ratio of 1% PBS, and half transparent amniotic volume ratio of 1: 1 to 1: 3 PBS solution is placed in an integrated shock soaking, every 6 to 8 hours medium was changed 共两次,然后将半透明羊膜取出,用PBS漂洗I~2次; (4)、将半透明羊膜浸泡在胰脂酶含量为500~5000U/L的胰脂酶/PBS溶液中,半透明羊膜与胰脂酶/PBS溶液的体积比为1:1~1: 3,并在溶液温度为30~40°C下处理10~20小时,然后将半透明羊膜取出用PBS漂洗I~2次,再将半透明羊膜浸泡在DNA酶含量为1000~3000U/L的DNA酶/PBS溶液中,半透明羊膜与DNA酶/PBS溶液的体积比为1:1~1:3,在溶液温度为30~40°C下处理3~5小时; (5)、将半透明羊膜从DNA酶/PBS溶液中取出,并将半透明羊膜置于青霉素与链霉素含量均为100~300U/L的双抗/PBS溶液中震荡漂洗,每12小时换液一次,共两次,最后将脱细胞后的半透明羊膜取出放到PBS中,在4°C的温度下保存待用。 A total of two, then the translucent amnion removed, rinsed with PBS I ~ 2 times; (4), the amnion was immersed in a translucent pancreatic lipase content of 500 ~ 5000U / L of pancreatic lipase / PBS solution, the translucent amniotic pancreatic lipase / PBS solution in a volume ratio of 1: 1 to 1: 3, and for 10 to 20 hours at 30 ~ 40 ° C solution temperature, and then rinsed with PBS and taken translucent amnion I ~ 2 times and then soaked in translucent amnion DNA enzyme content of 1000 ~ 3000U / L of the enzyme DNA / PBS solution, the ratio of the volume of amniotic membrane translucent with DNA enzyme / PBS solution is 1: 1 to 1: 3, the temperature of the solution 30 ~ 40 ° C for 3 to 5 hours treatment; (5), the amniotic membrane was removed from the translucent enzyme DNA / PBS solution, and the amniotic membrane is placed penicillin and streptomycin translucent contents were 100 ~ 300U / L of double antibody / PBS solution was rinsed shock, medium was changed every 12 hours, a total of two, and finally a translucent acellular amniotic taken into PBS and stored under the temperature at 4 ° C.
2.如权利要求1所述的一种人羊膜的脱细胞方法,其特征在于所述的表面活性剂采用Triton X-1OO0 2 is a person according to claim 1 decellularized amniotic method, characterized in that the employed surfactant Triton X-1OO0
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