CN112604031A - 一种眼部干细胞膜片及其制备方法 - Google Patents

一种眼部干细胞膜片及其制备方法 Download PDF

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CN112604031A
CN112604031A CN202011504302.4A CN202011504302A CN112604031A CN 112604031 A CN112604031 A CN 112604031A CN 202011504302 A CN202011504302 A CN 202011504302A CN 112604031 A CN112604031 A CN 112604031A
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徐哲
曾皓宇
刘园月
刘素娜
刘又瑜
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Guangdong Prokairong Biomedical Technology Co ltd
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Abstract

本发明提供一种眼部干细胞膜片及其制备方法，包括以下步骤：1)用完全培养基培养和扩增脐带干细胞；2)在培养皿中加入明胶溶液，将呈碗状的硅水凝胶载体扣在培养皿中孵育；3)另取新的培养皿，加入完全培养基，将孵育后的硅水凝胶载体翻面取出，开口朝上垂直放入完全培养基中；4)将培养的脐带干细胞消化，重悬均匀，吸取细胞悬液滴入呈碗状的硅水凝胶载体的凹面处，放入培养箱培养；5)定期清洗和换液，待脐带干细胞铺满后，清洗，将硅水凝胶载体转移到加有完全培养基的膜片取用盒，封上封口膜。本发明的眼部干细胞膜片可以与人眼生理相容，可用于抑制眼部炎症反应，减轻眼部纤维化修复。

Description

一种眼部干细胞膜片及其制备方法
技术领域
本发明属于生物技术领域，特别地，涉及一种眼部干细胞膜片及其制备方法。
背景技术
眼睛是心灵的一扇窗户，这扇窗户如今正在被一系列问题困扰。过度用眼、年龄老化和先天性基因突变导致的青光眼、白内障、红眼病、屈光不正和视网膜退行性疾病等眼科疾病正在不断增加。
根据世界卫生组织发布的首份《世界视力报告》，目前全球有超过22亿人视力受损或失明，其中有超过10亿人是因近视、远视、青光眼和白内障等问题未得到必要的治疗所导致。目前针对眼科疾病的治疗方法主要有药物治疗、基因治疗、手术治疗和细胞治疗。其中药物治疗一般只能缓解病程，需要持续用药。基因治疗有一定的局限性，对单基因控制的眼科疾病是比较有效的，但是大多数眼科疾病都是由于先天和后天多种因素造成的。手术治疗可以根除或减轻疾病，但同时有一定的风险性和局限性，很多眼科疾病无法进行手术。细胞治疗是通过干细胞移植或定向分化的感光细胞替代损伤的方式治疗眼科疾病。
在治疗眼科疾病中，干细胞是目前最具有应用前景和临床转化潜能的策略之一。干细胞不仅有很强的自我更新能力也具有很强的调控作用，主要表现为通过影响免疫细胞的增殖、分化和免疫因子的分泌而抑制免疫反应，因此，干细胞会显示出较强的抗炎能力。
发明内容
本发明的目的在于提供一种眼部干细胞膜片，该膜片负载有脐带干细胞，可以与人眼生理相容，具有良好的透光性和透氧率，脐带干细胞可通过分泌生长因子来抑制眼部疾病的炎症反应，减轻眼部纤维化。
本发明的技术方案如下：
一种眼部干细胞膜片的制备方法，其特征在于,包括以下步骤：
1)用完全培养基培养和扩增脐带干细胞；
2)在培养皿中加入明胶溶液，将呈碗状的硅水凝胶载体扣在培养皿中，放入30～40℃培养箱中孵育0.5～2h；
3)另取新的培养皿，加入带有抗生素的完全培养基，将步骤2)孵育后的硅水凝胶载体翻面取出，开口朝上垂直放入完全培养基中；
4)将步骤1)培养的脐带干细胞消化，重悬均匀，吸取细胞悬液滴入呈碗状的硅水凝胶载体的凹面处，待细胞沉底后放入培养箱培养；
5)定期清洗和换液，待脐带干细胞铺满所述硅水凝胶载体后，清洗，将硅水凝胶载体转移到加有完全培养基的膜片取用盒，封上封口膜。
脐带干细胞(MSC)是多能干细胞，具有高度自我更新能力和多向分化潜能的特征。MSC具有免疫调节特性和低免疫源性，以及较少的副作用。脐带干细胞具有平衡免疫促进和免疫抑制的特性。脐带干细胞可以通过旁分泌机质以及脐带干细胞与免疫细胞的相互作用，介导调节免疫反应平衡，最终促进局部炎症反应下调，减轻眼部各组织炎症损伤，脐带干细胞对炎症免疫反应的调节主要包括对先天免疫和获得性免疫反应的调节；免疫炎症对于组织再生是不利的因素，尽管炎症可能加速修复反应，带来的负面效应却是纤维化形成，阻碍再生。因此，减轻损伤眼部的炎症反应程度，可以减轻纤维化修复，有利于组织再生修复发展。
硅水凝胶是一种具有较大体积的硅氧基团的亲水性有机高分子材料。有机硅是一种具有较好透氧能力的物质，因此，硅水凝胶既有有机硅材料的高透氧性，又有水凝胶材料的柔软和亲水特性，其可通过有机硅相和水凝胶相两种通道输送氧气，呈“蜂窝状”结构。另外，有机硅的加入，也同时起到了增强材料机械性能的作用。
本发明通过特定的制备方法，将脐带干细胞负载到了硅水凝胶上制备成眼部膜片，将膜片放入眼睛后，其可以与人眼生理性相容，脐带干细胞可通过分泌生长因子来抑制眼部疾病的炎症反应，减轻眼部纤维化。
作为上述技术方案的进一步改进，所述呈碗状的硅水凝胶载体是由硅氧烷单体、亲水性单体、交联剂、亲水性添加剂混合聚合成。硅氧烷单体可以是三羟甲基氨基甲烷，亲水性可以是甲基丙烯酸酯或二甲基乙酰胺，交联剂可以是乙二醇二甲基丙烯酸酯,亲水性添加剂可以是乙烯基吡咯烷酮。
作为上述技术方案的进一步改进，所述呈碗状的硅水凝胶载体的直径为12～15mm，曲率半径为8.3～8.6mm，中心厚度为0.07～0.09mm，透氧率为135DK/t以上，以便于与人眼更好的相容。
作为上述技术方案的进一步改进，重悬均匀后的细胞悬液的浓度为5～10×105个/mL。
本发明还提供了一种眼部干细胞膜片，其特征在于,包括呈碗状的硅水凝胶载体和贴附生长在所述硅水凝胶载体的脐带干细胞。
作为上述技术方案的进一步改进，所述呈碗状的硅水凝胶载体中间设置了一个孔洞，孔洞的直径为2～6mm。
本发明通过特定的制备方法将脐带干细胞负载在硅水凝胶载体上制备成膜片，其可以与人眼生理相容，具有良好的透光性和透氧率，脐带干细胞可通过分泌生长因子来抑制眼部疾病的炎症反应，减轻眼部纤维化修复。
附图说明
图1为本发明的眼部干细胞膜片在不同生长阶段的状态图；
图2为本发明一个实施例的眼部干细胞膜片。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1
1.MSC(脐带干细胞)的培养和扩增
提前在T75培养瓶中铺上6mL猪皮明胶，放入37℃培养箱孵育1h。从液氮罐中取出脐带干细胞，在37℃水浴快速溶解，转移至装有10mL DF(10)(DMEM/F12：9mL，FBS：1mL,青霉素/链霉素/新霉素混合物100μL)的15mL离心管，1000rpm离心5min，离心结束后用15mL带有三抗(青霉素-链霉素-两性霉素B)的完全培养基(可商业化购买)重悬细胞，转移到铺好胶的T75培养瓶中，隔天换液。
MSC长到80％左右传代冻存，提前在T75培养瓶中铺好胶，弃去旧培养基，用DPBS(杜氏磷酸缓冲盐溶液)清洗两遍，加入0.25％胰蛋白酶消化液消化2.5min，用6mL DF(10)终止消化，并将壁上细胞吹下来，用6mL DF(-)(不含有FBS的DF(10))清洗培养瓶，转移到50mL离心管，计数。取100万细胞到15mL离心管，1000rpm离心5min，离心结束后用15mL带有三抗的完全培养基重悬，转移到铺好胶的T75培养瓶中，隔天换液。50mL离心管中的细胞用2mL冻存液重悬为单细胞悬液，按200万/管冻存。
2.取单细胞悬液
显微镜下观察细胞生长情况并拍照记录：细胞状态正常，贴壁80％左右。
细胞传代过程中，将消化后的细胞悬液计数，离心。离心后去上清，用一定量完全培养基重悬，使细胞密度为500万/mL，待用。
3.MSC种入膜片
将完全培养基放至室温。
在35mm培养皿中加入1mL猪皮明胶溶液，用硅水凝胶材料专用镊子和托棒将呈碗状的硅水凝胶材料倒扣放入培养皿中，将膜片与眼睛接触的一侧包被明胶，放入37℃培养箱中孵育1h。
另取新的35mm培养皿，加入2mL完全培养基。将呈碗状的硅水凝胶材料从培养箱中小心拿出，将碗口朝上，垂直放入培养基中。
将所培养增值的脐带干细胞细胞悬液重悬均匀，吸取100μL(约50万个细胞)细胞悬液滴入硅水凝胶材料的凹面处，待细胞沉底后，在显微镜下拍照记录，小心放入培养箱培养。1～2h后，在培养皿中加入500μL完全培养基，使呈碗状的硅水凝胶材料的外培养基液面与边缘持平。次日换液。
4.换液
将完全培养基放至室温。
显微镜下观察细胞生长情况并拍照记录：细胞已贴壁，形成多层贴壁细胞如图1所示。
将培养皿中旧培养基吸弃掉，加入2mL DPBS清洗，重复1次。在12孔板中加入3mL完全培养基，将清洗后眼部干细胞膜片完全浸入培养基中，继续培养。隔天换液。
5.眼部干细胞膜片取用
将完全培养基放至室温。
显微镜下观察材料中细胞生长情况并拍照记录：细胞状态好，整体密度大，无空隙，无细胞团。
吸弃旧培养基，加入2mL DPBS，轻轻摇晃清洗，弃掉。重复1次。在膜片取用盒中加入2.5mL完全培养基，将清洗后的膜片完全浸入培养基中，封上封口膜。
实施例2
实施例2的操作方式与实施例1相同，只是在呈碗状的硅水凝胶载体中间设置了一个孔洞，孔洞的直径为4mm，具体如图2所示。
眼部干细胞膜片放入人眼后，由于细胞会在膜片上形成一层薄薄的膜，可能会导致人眼视野有些模糊。本实施例在硅水凝胶载体中间设置了一个孔洞，使瞳孔可以露出，视野不受膜片的影响。人眼可以一只眼睛放入不带有孔洞的干细胞膜片，一只眼睛放入带有孔洞的干细胞膜片，或者两只眼睛均放入带有孔洞的干细胞膜片，这样既能保证干细胞膜片对眼睛的治疗或缓解效果，也能保证人眼视野清晰，不会影响到正常生活。
以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。

Claims (8)

1.一种眼部干细胞膜片的制备方法，其特征在于,包括以下步骤：
1)用完全培养基培养和扩增脐带干细胞；
2)在培养皿中加入明胶溶液，将呈碗状的硅水凝胶载体扣在培养皿中，放入30～40℃培养箱中孵育0.5～2h；
3)另取新的培养皿，加入完全培养基，将步骤2)孵育后的硅水凝胶载体取出，碗口朝上垂直放入完全培养基中；
4)将步骤1)培养的脐带干细胞消化，重悬均匀，吸取细胞悬液滴入呈碗状的硅水凝胶载体的凹面处，待细胞沉底后放入培养箱培养；
5)定期清洗和换液，待脐带干细胞铺满所述硅水凝胶载体后，清洗，将硅水凝胶载体转移到加有完全培养基的膜片取用盒，封上封口膜。
2.如权利要求1所述的眼部干细胞膜片的制备方法，其特征在于，所述呈碗状的硅水凝胶载体是由硅氧烷单体、亲水性单体、交联剂、亲水性添加剂混合聚合成。
3.如权利要求2所述的眼部干细胞膜片的制备方法，其特征在于，所述呈碗状的硅水凝胶载体的直径为12～15mm，曲率半径为8.3～8.6mm，中心厚度为0.07～0.09mm，透氧率为135DK/t以上。
4.如权利要求1所述的眼部干细胞膜片的制备方法，其特征在于，重悬均匀后的细胞悬液的浓度为5～10×105个/mL。
5.一种眼部干细胞膜片，其特征在于,包括呈碗状的硅水凝胶载体和贴附生长在所述硅水凝胶载体的脐带干细胞。
6.如权利要求5所述的眼部干细胞膜片，其特征在于，所述呈碗状的硅水凝胶载体是由硅氧烷单体、亲水性单体、交联剂、亲水性添加剂混合聚合成。
7.如权利要求6所述的眼部干细胞膜片，其特征在于，所述呈碗状的硅水凝胶载体的直径为12～15mm，曲率半径为8.3～8.6mm，中心厚度为0.07～0.09mm，透氧率为135DK/t以上。
8.如权利要求5所述的眼部干细胞膜片，其特征在于，所述呈碗状的硅水凝胶载体中间设置了一个孔洞，孔洞的直径为2～6mm。
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