CN115418344A - 一种简单并快速制备图案化细胞薄膜的方法 - Google Patents

一种简单并快速制备图案化细胞薄膜的方法 Download PDF

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CN115418344A
CN115418344A CN202210975083.0A CN202210975083A CN115418344A CN 115418344 A CN115418344 A CN 115418344A CN 202210975083 A CN202210975083 A CN 202210975083A CN 115418344 A CN115418344 A CN 115418344A
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关淑文
高博韬
肖将尉
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Institute of Biological and Medical Engineering of Guangdong Academy of Sciences
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Abstract

本发明属于细胞再生医学技术领域，公开了一种简单并快速制备图案化细胞薄膜的方法，具体公开了一种图案化细胞薄膜的制备方法，包括以下步骤：将不同图案性状的封口膜紧贴容器底部，接种细胞，培养，去除封口膜，得到图案化细胞薄膜。本发明通过控制封口膜的形状和大小从而实现细胞薄膜的图案化处理，与传统方法相比，无需特殊加工定制培养皿或者细胞培养辅助模具，所用封口膜为常见实验用品且价格低廉，可根据实际需要设置图案。

Description

一种简单并快速制备图案化细胞薄膜的方法
技术领域
本发明属于细胞再生医学技术领域，具体涉及一种简单并快速制备图案化细胞薄膜的方法。
背景技术
细胞薄膜技术是一种无支架的组织工程技术，在组织工程和再生医学领域应用前景广泛。细胞薄膜由细胞和细胞外基质组成，细胞密度高，可完整地保留细胞与细胞之间的结合蛋白以及细胞与基侧间的粘合蛋白，有效地维持细胞间相互作用。此外，少量的细胞外基质可以起到类似于胶水的作用，有利于粘接和叠层，方便后续的组织修复和构建三维立体模型。
目前，制备细胞薄膜普遍采用温度敏感型培养皿。温度敏感型培养皿表面涂有聚(N-异丙基丙烯酰胺)[poly(N-isopropylacrylamide)，PIPAAm]，当温度低于32℃时，PIPAAm由疏水性变为亲水性，导致培养皿上的细胞自行脱落，形成完整的细胞薄膜。从温度敏感型培养皿剥离得到的细胞薄膜形状与培养皿外形一致，通常为圆形，但在实际的应用中针对不同场景会有不同的形状需求。传统的方法是定制特定形状的温度敏感型培养皿或定制特定形状的模具，将模具固定在培养皿底部，再向模具中接种细胞。也有文献报道在温度敏感型培养皿上利用光栅，通过光引发化学接枝聚丙烯酰胺，无接枝聚丙烯酰胺的部分可以贴附细胞，从而实现细胞薄膜的图案化调控。由此发现，现有制作图案化细胞薄膜的方法需要特殊定制模具或需要较为复杂的化学方法，费时费力，操作复杂且成本高。
发明内容
本发明第一方面的目的，在于提供一种图案化细胞薄膜的制备方法。
本发明第二方面的目的，在于提供本发明第一方面的制备方法或本发明第一方面的图案化细胞薄膜的制备方法制备得到的图案化细胞膜在伤口修复中的应用。
本发明第三方面的目的，在于提供本发明第一方面的图案化细胞薄膜的制备方法或本发明第一方面的图案化细胞薄膜的制备方法备得到的图案化细胞膜在构建仿生组织中的应用。
本发明第四方面的目的，在于提供本发明第一方面的图案化细胞薄膜的制备方法或本发明第一方面的图案化细胞薄膜的制备方法备得到的图案化细胞膜在生物材料或药物的安全性评价以及毒性检测中的应用。
为了实现上述目的，本发明所采取的技术方案是：
本发明的第一个方面，提供一种图案化细胞薄膜的制备方法，包括以下步骤：将不同图案性状的封口膜紧贴容器底部，接种细胞，培养，去除封口膜，得到图案化细胞薄膜。
优选地，所述封口膜紧贴容器前进行灭菌处理。
优选地，所述灭菌处理包括酒精溶液浸泡和紫外照射。
优选地，所述容器包括细胞培养皿。
优选地，所述细胞培养皿包括温度敏感型培养皿和细胞培养皿。
优选地，所述细胞的接种数为8～15万/cm2；优选为10～15万/cm2；更优选为15万/cm2。
优选地，所述细胞通过细胞复苏或常规培养传代得到。
优选地，所述细胞的来源优选为人。
优选地，所述细胞包括成纤维细胞和骨髓间充质干细胞中至少一种。
优选地，所述培养基包括血清、双抗和基础培养基。
优选地，所述基础培养基包括DMEM、alpha-MEM中的至少一种。
优选地，所述培养的条件为30～37℃、3％～7％CO2浓度培养3～10天。
进一步优选，所述培养的条件为35～37℃、3％～5％CO2浓度培养5～8天。
更进一步优选地，所述培养的条件为35～37℃、3％～5％CO2浓度培养5～8天。
优选地，在所述培养过程中，每2～3天换液一次。
优选地，所述制备方法还包括剥离。
优选地，当采用温度敏感型培养皿时，所述剥离包括以下步骤：将去除封口膜的容器置于10～32℃下30～45min；当采用普通细胞培养皿时，用移液枪轻轻吹打容器边缘。
进一步优选地，当采用温度敏感型培养皿时，所述剥离包括以下步骤：将去除封口膜的容器置于15～25℃下30～45min。
本发明的第二个方面，提供本发明第一方面的制备方法或本发明第一方面的制备方法制备得到的图案化细胞膜在制备伤口修复的产品中的应用。
本发明的第三个方面，提供本发明第一方面的图案化细胞薄膜的制备方法或本发明第一方面的制备方法制备得到的图案化细胞膜在构建仿生组织中的应用。
优选地，所述仿生组织包括仿生血管组织、仿生骨组织。
本发明的第四个方面，提供本发明第一方面的图案化细胞薄膜的制备方法或本发明第一方面的制备方法制备得到的图案化细胞膜在生物材料或药物的安全性评价以及毒性检测中的应用。
本发明的有益效果是：
本发明通过控制封口膜的形状和大小从而实现细胞薄膜的图案化处理，与传统方法相比，无需特殊加工定制培养皿或者细胞培养辅助模具，所用封口膜为常见实验用品且价格低廉，可根据实际需要设置图案。
本发明提供的制备方法具有普遍通用性，适用于任何细胞培养皿，也适用于任何贴壁细胞。所得到的图案化细胞薄膜具有广泛的运用场景，有望用于构建小口径人工血管、体外仿生组织或器官、用于伤口修复的个性化“创可贴”等，使细胞薄膜技术更好地运用到组织工程和再生医学领域。
将通过本发明提供的制备方法得到的细胞薄膜直接贴敷于伤口部位，使大部分细胞保留在病灶处，通过细胞旁分泌的作用，用于伤口修复，促进伤口的愈合。
附图说明
图1为图案化细胞薄膜；其中，A为长条状细胞薄膜，左：剥离前，右：剥离后；B为三角形细胞薄膜，左：剥离前，右：剥离后；C为正方形细胞薄膜，左：剥离前，右：剥离后；D为四叶草形细胞薄膜，左：剥离前，右：剥离后。
图2为实施例1的长条状细胞薄膜卷成管状组织。
图3为实施例2的“铜钱状”细胞薄膜叠层。
图4为实施例3的正方形细胞薄膜和三角形细胞薄膜用于大鼠伤口修复的结果图。
具体实施方式
以下通过具体的实施例对本发明的内容作进一步详细的说明。
应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。
本实施例中所使用的材料、试剂等，如无特别说明，为从商业途径得到的材料和试剂。
实施例1长条状细胞薄膜/NHDF/温度敏感型培养皿
一种长条状NHDF细胞薄膜的制备方法，包括以下步骤：
(1)根据35mm温度敏感型培养皿(购自CellSeed)底部内径大小，在封口膜(购自SEP)上画出直径约34mm的圆，用剪刀将圆剪下，然后在圆形封口膜中画出对角线长30mm的长方形。将圆中间的长方形剪去，形成一张中间镂空的圆形封口膜。将镂空的圆形封口膜浸泡于75％酒精中，紫外照射过夜。次日，从酒精中取出封口膜，紫外照射直至风干。将镂空的圆形封口膜贴于温度敏感型培养皿底部，用镊子头部轻轻按压，使其与培养皿底部完全贴合，不留缝隙。
(2)正常人皮肤成纤维细胞(NHDF)进行复苏、传代，将第7代NHDF接种到底部贴有封口膜的温度敏感型培养皿上，接种密度为100万/皿，加入2mL培养基(含有10v/v％胎牛血清、1v/v％双抗(青链霉素)的低糖DMEM(购自康宁))，置于37℃、5％CO2培养箱内培养6天，每2天用上述培养基换液一次。
(3)培养6天后将温度敏感型培养皿从培养箱中取出，用镊子移除培养皿底部封口膜。将温度敏感型培养皿置于20℃低温培养箱中孵育45min，使NHDF完全脱离培养皿底部，即得到长条状NHDF细胞薄膜。
用塑料片将长条状NHDF细胞薄膜转移到圆形塑料管上，使其包裹在塑料管外围，如图2所示，形成一个管状组织。该结构有望未来用于构建人工血管等管状仿生组织，用于模拟体外人工血管。
实施例2“铜钱状”细胞薄膜/hBMSC/温度敏感型培养皿
一种铜钱状hBMSC细胞薄膜的制备方法，包括以下步骤：
(1)根据35mm温度敏感型培养皿(购自CellSeed)底部内径大小，在封口膜(购自SEP)上画出直径约12mm的圆，用剪刀将圆剪下，形成一张圆形封口膜。将圆形封口膜浸泡于75％酒精中，紫外照射过夜。次日，从酒精中取出封口膜，紫外照射直至风干。将圆形封口膜贴于温度敏感型培养皿底部中央处，用镊子头部轻轻按压，使其与养皿底部完全贴合，不留缝隙。
(2)对hBMSC细胞进行复苏、传代，将第6代细胞接种到底部贴有封口膜的温度敏感型培养皿上，接种密度为100万/皿，加入2mL培养基(含有10v/v％胎牛血清、1v/v％双抗(青链霉素)的alpha-MEM(购自康宁))，置于37℃、5％CO2培养箱内培养6天，每2天用上述培养基换液一次。
(3)培养5天后从培养箱中取出，用镊子移除培养皿底部封口膜。将温度敏感型培养皿置于20℃低温培养箱中孵育30min，使细胞完全脱离培养皿底部，即得到中间具有缺损的“铜钱状”细胞薄膜。
将铜钱状hBMSC细胞薄膜叠层，共叠3层，如图3所示，可形成一个中间有缺损的三维立体组织。该组织有望用于构建骨缺损模型，体外模拟有缺损的人骨膜组织，进而对骨修复材料或药物进行体外评价。
实施例3任意形状的细胞薄膜/NHDF/普通细胞培养皿
一种正方形NHDF细胞薄膜的制备方法，包括以下步骤：
(1)根据35mm普通细胞培养皿(购自康宁)底部内径大小，在封口膜(购自SEP)上画出直径约34mm的圆，用剪刀将圆剪下，然后在圆形封口膜中画出对角线长30～32mm的正方形(还可以是边长为29mm的等边三角形或长对称轴为32mm的四叶草形)。将圆中间的图形剪去，形成中间镂空的圆形封口膜。将镂空的圆形封口膜浸泡于75％酒精中，紫外照射过夜。次日，从酒精中取出封口膜，紫外照射直至风干。将镂空的圆形封口膜贴于普通细胞培养皿底部，用镊子头部轻轻按压，使其与培养皿底部完全贴合，不留缝隙。
(2)NHDF进行复苏、传代，将第7代细胞接种到底部贴有封口膜的普通细胞培养皿上，接种密度为100万/皿，加入2mL培养基(NHDF细胞薄膜专用培养基：包含10v/v％胎牛血清、1v/v％青链霉素的DMEM高糖培养基，1v/v％的ITS细胞培养添加物，0.04μg/mL地塞米松，50μg/mL维生素C，25mg/mL Ficoll 400)，置于37℃、5％CO2培养箱内培养6天，每2天用上述培养基换液一次。
(3)NHDF细胞培养6天后从培养箱中取出，用镊子移除培养皿底部封口膜。用移液枪吸取约1mL培养基，轻轻吹打培养皿底部，使细胞与培养皿底部完全脱离，即得到正方形(三角形或四叶草形)NHDF细胞薄膜。
效果实施例
根据实例3所得到的正方形和三角形NHDF细胞薄膜大小和形状，在大鼠背部创建形状与之对应的皮肤缺损伤口，正方形伤口边长1.2cm，三角形伤口边长1.2cm。
具体操作如下：将大鼠进行麻醉处理，用手术钳在大鼠背部剪出边长1.2cm的正方形伤口和边长为1.2cm的三角形伤口。进一步将大鼠分为两组，处理组和对照组，处理组：在大鼠伤口处贴上实例3得到的正方形NHDF细胞薄膜和三角形NHDF细胞薄膜；对照组不做任何处理。将处理后的大鼠常规养殖，于第1天、第5天、第8天和第13天观察大鼠背部创口的修复情况，并拍照记录。
如图4所示，在修复的第5天，处理组与对照组差异并不明显；但在第8天时，可以看出处理组伤口大小明显小于对照组；在第13天，处理组伤口基本愈合，而对照组还可以看到明显的结痂。因此，在实际应用中，可根据伤口大小和形状调整细胞薄膜的形态，从而实现个性化伤口修复。
上面对本发明实施例作了详细说明，但是本发明不限于上述实施例，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下作出各种变化。此外，在不冲突的情况下，本发明的实施例及实施例中的特征可以相互组合。

Claims (10)

1.一种图案化细胞薄膜的制备方法，包括以下步骤：将不同图案性状的封口膜紧贴容器底部，接种细胞，培养，去除封口膜，得到图案化细胞薄膜。
2.根据权利要求1所述的制备方法，其特征在于，所述细胞的接种数为8～15万/cm2。
3.根据权利要求2所述的制备方法，其特征在于，所述容器包括细胞培养皿。
4.据权利要求1～3中任一项所述的制备方法，其特征在于，所述培养皿包括温度敏感型培养皿和普通细胞培养皿。
5.据权利要求1～3中任一项所述的制备方法，其特征在于，所述培养的条件为30～37℃、3％～7％CO2浓度培养3～10天。
6.据权利要求1～3中任一项所述的制备方法，其特征在于，所述封口膜紧贴容器前进行灭菌处理。
7.据权利要求1～3中任一项所述的制备方法，其特征在于，所述制备方法还包括剥离；优选地，当采用温度敏感型培养皿时，所述剥离包括以下步骤：将去除封口膜的容器置于10～32℃下30～45min；当采用普通细胞培养皿时，所述剥离包括以下步骤：用移液枪轻轻吹打容器边缘。
8.权利要求1～7中任一项所述的制备方法或权利要求1～7中任一项所述的制备方法制备得到的图案化细胞膜在制备伤口修复的产品中的应用。
9.权利要求1～7中任一项所述的制备方法或权利要求1～7中任一项所述的制备方法制备得到的案化细胞薄膜在构建仿生组织中的应用。
10.权利要求1～7中任一项所述的制备方法或权利要求1～7中任一项所述的制备方法制备得到的图案化细胞薄膜在生物材料或药物的安全性评价以及毒性检测中的应用。
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