CN101508971B - 一种组织工程人角膜内皮的重建方法 - Google Patents
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Abstract
本发明涉及一种组织工程人角膜内皮的重建方法，是利用20％小牛血清-DMEM/F12培养液将角膜内皮细胞体外培养至对数生长期，采用胰蛋白酶消化获得完全脱上皮层的羊膜载体支架，平铺在培养板孔中牢固干贴后，将悬浮于含有IV型胶原蛋白和20％小牛血清的DMEM/F12培养液的对数生长期人角膜内皮细胞，接种至在平铺有羊膜载体支架的培养板孔中启动体外培养，进行组织工程人角膜内皮的体外重建。本发明工艺科学合理，所重建的组织工程人角膜内皮可用于批量生产，满足角膜内皮盲临床角膜移植治疗对组织工程人角膜内皮的大量需求，且组织工程人角膜内皮体外重建和临床治疗的成本低。
Description
技术领域
本发明涉及一种利用人角膜内皮细胞和脱上皮层羊膜重建组织工程人角膜内皮的方法。
背景技术
高等动物的角膜内皮在维持角膜透明、角膜厚度以及为角膜提供营养等方面具有不可替代的作用。角膜受到感染或手术等创伤后，将引起角膜内皮细胞发生不同程度的减少，一旦角膜内皮细胞密度低于维持角膜内皮生理功能的临界密度，角膜内皮将出现不可逆之病变，形成角膜内皮盲。目前，我国约有角膜内皮盲患者80万人，尽管他们可以通过角膜移植来治愈，但由于捐献角膜的高度匮乏致使绝大多数患者因得不到移植角膜而无法重见光明。近年来，角膜组织工程的兴起为组织工程角膜内皮的体外重建及其应用于角膜内皮盲的临床治疗带来了新的希望，但如何利用角膜内皮细胞和适宜的载体支架在体外重建出组织工程人角膜内皮已经成为研究热点。
利用对人角膜内皮的体外重建研究开始于1992年。此后，May Griffith等(1999)利用癌基因转染的永生化人角膜上皮细胞、基质细胞、内皮细胞与醛交联胶原蛋白在体外成功重建出形态、透明度和组织结构与正常角膜大体相近的功能性人角膜组织类似物，为人角膜组织的体外重建开辟了道路，但由于所用角膜细胞均为转染了癌基因、具有潜在致瘤性的永生化细胞，从而限制了其在人角膜内皮重建和临床中的应用。2004年，日本学者Yutaka Ishino等和Tatsuya Mimura等利用体外培养至第4-5代的人角膜内皮细胞分别与去上皮层羊膜和胶原膜片分别重建出与正常功能接近的人角膜内皮细胞薄片。2007年，Jui-Yang Lai等将取自眼库成人角膜的人角膜内皮细胞原代培养在修饰后的N-(1-甲基乙基)-2-丙烯酰胺均聚物载体支架上获得了近似于正常功能的人角膜内皮细胞薄片。2008年，日本东京大学医学院Kouichiro Hitani等又利用薄膜培养法获得了近似于正常功能的人角膜内皮细胞薄片。这些研究结果为利用非转染人角膜内皮细胞重建人组织工程角膜内皮开辟了道路，但由于所用种子细胞均为来源于眼库角膜或在24孔培养板中原代培养或仅传4-5代的人角膜内皮细胞，故细胞数量非常有限，所能重建出的组织工程人角膜内皮的数量极少，即使成功后能用于临床角膜移植的数量也极其有限，故仍只限于实验研究，更无法满足我国约80万、全世界1000余万角膜内皮盲患者临床治疗的大量需要。
2005年，樊廷俊等在国际上首次成功建立了没有任何致瘤性的非转染人角膜内皮细胞系，成功解决了人组织工程角膜内皮规模化重建所需大量种子细胞的来源问题。因此，尽早建立理想载体支架的制备技术，进而建立一种利用非转染人角膜内皮细胞和理想载体支架重建组织工程角膜内皮的方法，已成为全世界眼科专家们的主攻目标，也是组织工程角膜内皮临床应用以及造福全世界角膜盲患者的关键之所在。
发明内容
本发明的目的就是利用人角膜内皮细胞和脱上皮层羊膜载体支架，提供一种组织工程人角膜内皮的重建方法。
本发明的方法；取人角膜内皮细胞，用含20％小牛血清的DMEM/F12培养液在底面积25平方厘米培养瓶中置37℃扩增培养至对数生长期，用0.25％胰蛋白酶溶液消化和滴管吹打法悬浮细胞，离心收集细胞沉淀，用5毫升组织工程人角膜内皮重建专用培养液悬浮所得细胞沉淀制成人角膜内皮细胞悬液，经血球计数板计数后，用上述专用培养液调整细胞浓度至4.2×106～4.2×107个细胞/毫升。
然后，利用0.25％胰蛋白酶溶液对冻干羊膜的上皮面进行倒置消化，以得到完全脱去上皮层的羊膜，用D-hanks溶液漂洗2次后，用打孔器将脱上皮层羊膜打成与培养板孔直径相同的羊膜圆片，使其上皮面朝上平铺于48孔培养板孔的底部，放置于5％CO2培养箱中在37℃条件下牢固干贴后制成羊膜载体支架备用。
最后，在贴有脱上皮层羊膜载体支架的培养板孔中，轻轻加入上述准备好的人角膜内皮细胞悬液0.1～0.2毫升，使细胞悬液均匀分散于培养孔中，置5％CO2培养箱中37℃培养20～24小时待细胞完全贴附到羊膜载体支架上后，补加上述专用培养液使其液面达到培养孔高度的3/4，待人角膜内皮细胞在羊膜载体支架上长成单层后即为重建的组织工程人角膜内皮。
本发明所用组织工程人角膜内皮重建专用培养液的配方为：DMEM/F12培养液，20％小牛血清，0.05％～0.1％IV型胶原蛋白；
为了提高人角膜内皮细胞在脱上皮层羊膜载体支架上的贴附效果，本发明又添加了0.05％～0.1％纤连蛋白。
本发明的方法所重建出的组织工程人角膜内皮在形态结构和透明度上与正常角膜内皮相似并具有正常角膜内皮的功能，本发明所重建的组织工程人角膜内皮移植到撕除角膜内皮和后弹力层的新西兰兔眼中可使兔眼角膜透明长达39天以上。本发明的主要特点是：利用无致瘤性、非转染的人角膜内皮细胞，能通过连续传代为组织工程人角膜内皮的批量重建提供出大量的种子细胞；利用商品化冻干羊膜所制备的脱上皮层羊膜，从而满足组织工程人角膜内皮批量重建所需要的大量载体支架材料；而且可直接应用于批量生产和临床角膜移植；且其生产和临床治疗的成本低。
具体实施方式
1、组织工程人角膜内皮重建专用培养液的配制：取常规配制的DMEM/F12培养液70毫升，加入IV型胶原蛋白40～80毫克，完全溶解后用0.22微米的微孔滤膜过滤除菌，加入20毫升小牛血清，补加常规配制的DMEM/F12培养液至100毫升，即为本发明的组织工程人角膜内皮重建专用培养液。
为了提高人角膜内皮细胞在脱上皮层羊膜载体支架上的贴附效果，本发明又添加了0.05％～0.1％纤连蛋白。
2、人角膜内皮细胞系种子细胞的制备：取人角膜内皮细胞，用含20％小牛血清的DMEM/F12培养液在底面积25平方厘米培养瓶中置37℃进行扩增培养，细胞繁殖60～84小时至对数生长期后，用玻璃滴管吸出培养液，加入0.25％胰蛋白酶溶液消化1～2分钟，加入此前吸出的培养液终止消化，1000～1500转/分离心10～15分钟，细胞沉淀用5毫升上述专用培养液悬浮均匀制成人角膜内皮细胞悬液；利用血球计数板进行细胞计数，用上述专用培养液调整细胞浓度至4.2×106～4.2×107个细胞/毫升。
3、脱上皮层羊膜载体支架制备的倒置消化：利用0.25％胰蛋白酶溶液对冻干羊膜的上皮面进行倒置消化，以得到完全脱去上皮层的羊膜，用D-hanks溶液漂洗2次后，用打孔器将脱上皮层羊膜打成与培养板孔直径相同的羊膜圆片。
4、组织工程人角膜内皮的体外重建：脱上皮层羊膜圆片按照上皮面朝上的方向平铺于48孔培养板孔的底部，放置于37℃培养箱中牢固干贴后制成羊膜载体支架。在贴有脱上皮层羊膜载体支架的培养板孔中，轻轻加入上述准备好的细胞悬液，注意接种时使滴管口尽量接近但不要碰到膜，从膜的中央开始缓慢滴加细胞悬液，接种量为0.1～0.2毫升，用滴管轻轻吹打使细胞悬液均匀分散于培养孔中，置5％CO2细胞培养箱中在37℃条件下培养20～24小时待细胞完全贴附到羊膜载体支架上后，补加上述专用培养液使其液面达到培养孔高度的3/4，待人角膜内皮细胞在羊膜载体支架上长成单层后即为重建的组织工程人角膜内皮。
实施例1
取人角膜内皮细胞，利用含20％小牛血清的DMEM/F12培养液在底面积25平方厘米培养瓶中置37℃进行扩增培养84小时。取常规配制的DMEM/F12培养液70毫升，加入IV型胶原蛋白40～80毫克，完全溶解后用0.22微米的微孔滤膜过滤除菌，加入20毫升小牛血清，补加常规配制的DMEM/F12培养液至100毫升，即为本发明的组织工程人角膜内皮重建专用培养液。对扩增培养后的培养瓶，用玻璃滴管吸出培养液，加入0.25％胰蛋白酶溶液消化1.5分钟，加入此前吸出的培养液终止消化，1500转/分离心10分钟，获得细胞沉淀。细胞沉淀用5毫升上述专用培养液悬浮均匀制成人角膜内皮细胞悬液；利用血球计数板进行细胞计数，用上述专用培养液调整细胞浓度至4.7×106个细胞/毫升。
利用0.25％胰蛋白酶溶液对冻干羊膜的上皮面进行倒置消化，以得到完全脱去上皮层的羊膜，用D-hanks溶液漂洗2次后，用打孔器将脱上皮层羊膜打成与培养板孔直径相同的羊膜圆片，将该羊膜圆片按照上皮面朝上的方向平铺于48孔培养板孔的底部，放置于37℃培养箱中牢固干贴后制成羊膜载体支架。在贴有脱上皮层羊膜载体支架的培养板孔中，轻轻加入上述准备好的人角膜内皮细胞悬液，注意接种时使滴管口尽量接近但不要碰到膜，从膜的中央开始缓慢滴加细胞悬液，接种量为0.2毫升，用滴管轻轻吹打使细胞悬液均匀分散于培养孔中，置5％CO2细胞培养箱中在37℃条件下培养24小时待细胞完全贴附到羊膜载体支架上后，补加上述专用培养液使其液面达到培养孔高度的3/4，待人角膜内皮细胞在羊膜载体支架上长成单层后即为重建的组织工程人角膜内皮。
实施例2
取人角膜内皮细胞，利用含20％小牛血清的DMEM/F12培养液在底面积25平方厘米培养瓶中置37℃进行扩增培养72小时。取常规配制的DMEM/F12培养液70毫升，加入IV型胶原蛋白40～80毫克，完全溶解后用0.22微米的微孔滤膜过滤除菌，加入20毫升小牛血清，补加常规配制的DMEM/F12培养液至100毫升，即为本发明的组织工程人角膜内皮重建专用培养液。对扩增培养后的培养瓶，用玻璃滴管吸出培养液，加入0.25％胰蛋白酶溶液消化1分钟，加入此前吸出的培养液终止消化，1000转/分离心15分钟，细胞沉淀用5毫升上述专用培养液悬浮均匀制成人角膜内皮细胞悬液；利用血球计数板进行细胞计数，用上述专用培养液调整细胞浓度至4.2×107个细胞/毫升。
利用0.25％胰蛋白酶溶液对冻干羊膜的上皮面进行倒置消化，以得到完全脱去上皮层的羊膜，用D-hanks溶液漂洗2次后，用打孔器将脱上皮层羊膜打成与培养板孔直径相同的羊膜圆片，将该羊膜圆片按照上皮面朝上的方向平铺于48孔培养板孔的底部，放置于37℃培养箱中牢固干贴后制成羊膜载体支架。在贴有脱上皮层羊膜载体支架的培养板孔中，轻轻加入上述准备好的人角膜内皮细胞悬液，注意接种时使滴管口尽量接近但不要碰到膜，从膜的中央开始缓慢滴加细胞悬液，接种量为0.1毫升，用滴管轻轻吹打使细胞悬液均匀分散于培养孔中，置5％CO2细胞培养箱中在37℃条件下培养22小时待细胞完全贴附到羊膜载体支架上后，补加上述专用培养液使其液面达到培养孔高度的3/4，待人角膜内皮细胞在羊膜载体支架上长成单层后即为重建的组织工程人角膜内皮。
实施例3
取人角膜内皮细胞，利用含20％小牛血清的DMEM/F12培养液在底面积25平方厘米培养瓶中置37℃进行扩增培养60小时。取常规配制的DMEM/F12培养液70毫升，加入IV型胶原蛋白40～80毫克，完全溶解后用0.22微米的微孔滤膜过滤除菌，加入20毫升小牛血清，补加常规配制的DMEM/F12培养液至100毫升，即为本发明的组织工程人角膜内皮重建专用培养液。对扩增培养后的培养瓶，用玻璃滴管吸出培养液，加入0.25％胰蛋白酶溶液消化2分钟，加入此前吸出的培养液终止消化，1500转/分离心12分钟，细胞沉淀用5毫升上述专用培养液悬浮均匀制成人角膜内皮细胞悬液；利用血球计数板进行细胞计数，用上述专用培养液调整细胞浓度至4.4×106个细胞/毫升。
利用0.25％胰蛋白酶溶液对冻干羊膜的上皮面进行倒置消化，以得到完全脱去上皮层的羊膜，用D-hanks溶液漂洗2次后，用打孔器将脱上皮层羊膜打成与培养板孔直径相同的羊膜圆片，将该羊膜圆片按照上皮面朝上的方向平铺于48孔培养板孔的底部，放置于37℃培养箱中牢固干贴后制成羊膜载体支架。在贴有脱上皮层羊膜载体支架的培养板孔中，轻轻加入上述准备好的人角膜内皮细胞悬液，注意接种时使滴管口尽量接近但不要碰到膜，从膜的中央开始缓慢滴加细胞悬液，接种量为0.2毫升，用滴管轻轻吹打使细胞悬液均匀分散于培养孔中，置5％CO2细胞培养箱中在37℃条件下培养20小时待细胞完全贴附到羊膜载体支架上后，补加上述专用培养液使其液面达到培养孔高度的3/4，待人角膜内皮细胞在羊膜载体支架上长成单层后即为重建的组织工程人角膜内皮。
Claims (4)
1.一种组织工程人角膜内皮的重建方法，首先利用含20％小牛血清的DMEM/F12培养液对人角膜内皮细胞进行扩增培养，经0.25％胰蛋白酶溶液消化和1000～1500转/分离心10～15分钟获得人角膜内皮细胞沉淀，用DMEM/F12培养液、20％小牛血清和0.05％～0.1％IV型胶原蛋白组成的组织工程人角膜内皮重建专用培养液将该细胞沉淀悬浮成人角膜内皮细胞悬液；然后，利用0.25％胰蛋白酶对冻干羊膜的上皮面进行倒置消化以制备完全脱去上皮层的羊膜，并用打孔器将该脱上皮层羊膜打成与培养板孔直径相同的羊膜圆片；最后，按上皮面朝上将脱上皮层羊膜圆片平铺于48孔培养板孔的底部，放在37℃培养箱中干贴牢固后制成羊膜载体支架，将上述人角膜内皮细胞悬液0.1～0.2毫升接种在平铺有上述羊膜载体支架的培养板中，待人角膜内皮细胞全部贴附在上述羊膜载体支架上后，再补加上述专用培养液并使液面达到培养孔高度的3/4，待人角膜内皮细胞在羊膜载体支架上长成单层即为重建的组织工程人角膜内皮。
2.如权利要求1所述的组织工程人角膜内皮的重建方法，其特征是所述组织工程人角膜内皮重建专用培养液中又添加了0.05％～0.1％纤连蛋白。
3.如权利要求1所述的组织工程人角膜内皮的重建方法，其特征在于上述扩增培养方法是将人角膜内皮细胞扩增培养至对数生长期。
4.如权利要求3所述的组织工程人角膜内皮的重建方法，其特征是上述已达到对数生长期的人角膜内皮细胞的浓度为4.2×106～4.2×107个细胞/毫升。
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| CN101843923B (zh) * | 2010-01-22 | 2013-05-08 | 中国海洋大学 | 利用新鲜羊膜制备组织工程人工角膜内皮载体支架的方法 |
| CN102166375B (zh) * | 2011-02-16 | 2013-04-10 | 中国海洋大学 | 一种组织工程人角膜上皮的重建方法 |
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