CN110551677A - 一种黑色素体外细胞模型及其制备方法 - Google Patents

一种黑色素体外细胞模型及其制备方法 Download PDF

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CN110551677A
CN110551677A CN201810549315.XA CN201810549315A CN110551677A CN 110551677 A CN110551677 A CN 110551677A CN 201810549315 A CN201810549315 A CN 201810549315A CN 110551677 A CN110551677 A CN 110551677A
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Abstract

一种黑色素体外模型的制备方法。所述方法包括：包被多孔琼脂糖微球作为细胞基质，并在其上构建含黑色素的真皮层和表皮层。所构建的模型与人体皮肤结构相似，且包含部分炎症应激因子，更有利于黑色素代谢疾病相关产品的筛选及黑色素形成或抑制机理的研究。

Description

一种黑色素体外细胞模型及其制备方法
技术领域
本发明涉及生物材料领域，尤其涉及一种黑色素体外细胞模型及其制备方法。
背景技术
黑色素是决定人类皮肤和毛发颜色的主要色素，由黑素细胞合成并储存，黑素细胞在皮肤中终生保持活性。黑色素代谢障碍会引起皮肤色素障碍性疾病，包括色素增多如色斑、色痣和色素减退如白发、白化病、白癜风。色素代谢性疾病发病较为普遍，且随环境的恶化有有升趋势，至今仍是无法解决的疾病。因此，进行色素代谢性疾病的发病机理研究、治疗药物、治疗化妆品的研究成为人们关注的课题。
色素代谢性疾病治疗产品在上市前通常都要进行大量的测试以保证产品的有效性。传统的检测一般通过动物试验进行，但是黑色素调节研究中可供选择的动物种类少、动物试验的偏差性以及3R理论的提出，促使研究者寻找更合适的检测方法。例如，在祛斑美白化妆品开发中，功能验证多采用豚鼠进行验证，但试验动物与人体结果会出现一定的偏差。而且欧盟对化妆品的管理条例中，将逐渐取消动物试验。目前已基于组织工程原理构建出多种皮肤模型用于替代动物模型来研究皮肤的不同生物学功能。
专利200910138703.X公开了一种采用胶原、层粘连蛋白、成纤维细胞制备的支架结构即真皮层，然后接种角质形成细胞、黑色素细胞，并通过体外培养制备出功能性色素等同物。这种方法制备的皮肤模型可包含真皮层和表皮层。但是这种皮肤模型含有的真皮层中的细胞外基质主要来源于动物，后其培养中这些胶原会有较快的降解，易发生结构收缩，从而导致整体模型损坏。
专利201019018002.2中采用生物可降解材料支撑膜模拟真皮层，表皮层得用角质形成细胞、黑色素细胞共同接种，构建的含黑色素细胞的皮肤模型。此种含黑色素的细胞模型只含有表皮层。
虽然黑色素体外模型已经被开发，但是这些模型基底层主要来源于外源细胞外基质或惰性载体，这样的基底层不能很好地模拟天然皮肤的基底层结构，因此存在一定的局限性，导致黑色素模型后期培养的稳定性较差。
发明内容
本发明的目的在于提供一种黑色素体外模型的制备方法，所制备的黑色素模型及其应用，以解决现有技术中与天然皮肤结构差异或稳定性差而带来的不易保持的缺陷。
为实现上述目的，本发明提出一种黑色素体外模型的制备方法，包括：
步骤1：
将交联琼脂糖微球放入0.01-2％的透明质酸钠中，浸泡10-60min后取出，室温晾干。将含有透明质酸钠的琼脂糖微球放入成纤维细胞生长因子中，成纤维细胞生长因子与透明质酸钠通过电荷作用结合。
步骤2：
将载有成纤维生长因子的琼脂糖微球放入0.001-0.05mg/ml胶原溶液中，浸泡30min后，加入成纤维细胞，细胞接种密度为1*105/cm2,用加入10％的10*DMEM培和胎牛血清培养，于37℃浸没培养12小时；重复接种2-4次成纤维细胞，并培养。在其表面接种1*107/cm2的肥大细胞并培养24小时。重复接种2-4次成纤维细胞，并培养至形成厚度为0.2cm的成纤维细胞层。
步骤3：
将角质形成细胞与黑色素细胞混合成混合细胞悬液，色素细胞和角质形成细胞的接种比例在1:5-1:30之间，接种的细胞密度为2.5×106/cm2，进行浸没式培养，培养基为10％的10*DMEM培和胎牛血清培养，含胰岛素0.5-5ng/ml，表皮细胞生长因子0.1-1ng/ml，浸没式培养2天，每天换液。在其表面以1*107/cm2密度接种角质形成细胞，继续培养3天。
步骤4：
将步骤3中液下培养后的体外测试皮肤模型置于气液面培养支架上，加入10％的10*DMEM培和胎牛血清培养，含胰岛素0.5-5ng/ml，成纤维细胞生长因子0.1-1ng/ml培养液进行气液面培养，使液面与皮肤模型表面平齐，每天更换新鲜的培养液，培养2-3天。之后更换10％的10*DMEM培和胎牛血清培养，含胰岛素0.5-5ng/ml，表皮细胞生长因子0.1-1ng/ml培养液进行气液面培养3-6天。即获得黑色素组织工程皮肤。
本发明所制备的黑色素体外模型与天然皮肤结构类似，包含有真皮层、表皮层，真皮层在培养过程中添加了肥大细胞，使真皮层的结构与皮肤结构更为接近，并且可产生一定炎症应激反应。多孔琼脂糖基质中包埋的透明质酸钠，透明质酸钠能过电荷作用与成纤维生长因子结合，可以促进真皮区和表皮区连接处的成熟，为黑色素细胞活性及功能发挥提供良好的环境。多孔琼脂糖基质的微孔结构有利于透明质酸钠和成纤维细胞生长因子的释放，可以持续提供稳定的环境。并且琼脂糖微球具有很好的机械性能，后期培养稳定性好。
技术效果：
本发明所制备的黑色素体外模型与天然皮肤结构类似。多孔琼脂糖基质中包埋的透明质酸钠可以促进真皮区和表皮区连接处的成熟，为黑色素细胞活性及功能发挥提供良好的环境。并且琼脂糖微球具有很好的机械性能，后期培养稳定性好。本发明所制备的黑色素体外模型可用于药品、化妆品中黑色素相关疾病的筛选和验证。
实施例
实施例1
将交联琼脂糖微球放入0.01％的透明质酸钠中，浸泡60min后取出，室温晾干。将含有透明质酸钠的琼脂糖微球放入0.05ng/ml的成纤维细胞生长因子中。将载有成纤维生长因子的琼脂糖微球放入0.01mg/ml胶原溶液中，浸泡30min后，加入成纤维细胞，细胞接种密度为1*105/cm2,用加入10％的10*DMEM培和胎牛血清培养，于37℃浸没培养12小时；重复接种4次成纤维细胞，并培养。在其表面接种1*107/cm2的肥大细胞并培养24小时。重复接种3次成纤维细胞。
将角质形成细胞与黑色素细胞按比例在1:5混合，接种的细胞密度为2.5×106/cm2，进行浸没式培养，培养基为10％的10*DMEM培和胎牛血清培养，含胰岛素0.5ng/ml，表皮细胞生长因子0.1ng/ml，浸没式培养2天，每天换液。在其表面以1*107/cm2密度接种角质形成细胞，继续培养3天。
将皮肤模型置于气液面培养支架上，加入10％的10*DMEM培和胎牛血清培养，含胰岛素0.5ng/ml，成纤维细胞生长因子0.1ng/ml培养液进行气液面培养，使液面与皮肤模型表面平齐，每天更换新鲜的培养液，培养3天。之后更换10％的10*DMEM培和胎牛血清培养，含胰岛素0.5ng/ml，表皮细胞生长因子0.1ng/ml培养液进行气液面培养6天。即获得黑色素组织工程皮肤。

Claims (11)

1.一种黑色素体外模型的制备方法，其特征在于：由多孔琼脂糖构建皮肤模型的基质，在其上包被透明质酸钠和成纤维生长因子，作为成纤维细胞生长的支撑，并在其上构建含黑色素的真皮层和表皮层。
其制备过程包括以下步骤：
步骤1：将交联琼脂糖微球放入透明质酸钠中浸泡后取出，室温晾干。将含有透明质酸钠的琼脂糖微球放入成纤维细胞生长因子中，成纤维细胞生长因子与透明质酸钠通过电荷作用结合。
步骤2：将载有成纤维生长因子的琼脂糖微球放入胶原溶液中，浸泡30min后，加入成纤维细胞，于37℃浸没培养12小时；重复接种成纤维细胞，并培养。在其表面接种肥大细胞并培养24小时。重复接种纤维细胞，并培养至形成厚度为0.2cm的成纤维细胞层。
步骤3：将角质形成细胞与黑色素细胞混合成混合细胞悬液，接种后进行浸没式培养，培养2天，每天换液。在其表面接种角质形成细胞，继续培养3天。
步骤4：将步骤3中液下培养后的体外测试皮肤模型置于气液面培养支架上，加入培养液进行气液面培养，使液面与皮肤模型表面平齐，每天更换新鲜的培养液，培养2-3天。之后更换培养液进行气液面培养3-6天。即获得黑色素组织工程皮肤。
2.根据权利要求1所述的黑色素体外模型制备方法，其特征在于透明质酸钠的浓度为0.01-2％。
3.根据权利要求1所述的黑色素体外模型制备方法，其特征在于透明质酸钠浸泡时间为10-60min。
4.根据权利要求1所述的黑色素体外模型制备方法，其特征在于构建真皮层所用胶原溶液浓度为0.001-0.05mg/ml。
5.根据权利要求1所述的黑色素体外模型制备方法，其特征在于成纤维细胞接种密度为1*105/cm2.
6.根据权利要求1所述的黑色素体外细胞模型制行方法，其特征在于培养液为加入10％的10*DMEM培和胎牛血清。
7.根据权利要求1所述的黑色素体外细胞模型，其特征在于在成纤维细胞层表面接种肥大细胞。
8.根据权利要求1所述的黑色素体外细胞模型，其特征在于肥大细胞的接种密度为1*107/cm2。
9.根据权利要求1所述的黑色素体外细胞模型，其特征在于角质形成细胞和色素细胞的接种密度为2.5×106/cm2。
10.根据权利要求1所述的黑色素体外细胞模型，其特征在于角质细胞和色素细胞的培养液为10％的10*DMEM培和胎牛血清培养，含胰岛素0.5-5ng/ml，表皮细胞生长因子0.1-1ng/ml。
11.根据权利要求1所述的黑色素体外细胞模型，其特征在于色素细胞层表面接种角质形成细胞，接种密度1*107/cm2。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111117945A (zh) * 2019-12-31 2020-05-08 广东博溪生物科技有限公司 含黑色素的表皮皮肤模型及其构建方法和应用

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111117945A (zh) * 2019-12-31 2020-05-08 广东博溪生物科技有限公司 含黑色素的表皮皮肤模型及其构建方法和应用
CN111117945B (zh) * 2019-12-31 2023-11-07 广东博溪生物科技有限公司 含黑色素的表皮皮肤模型及其构建方法和应用

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