CN109207420B - 人造皮肤的制造方法及人造皮肤 - Google Patents

人造皮肤的制造方法及人造皮肤 Download PDF

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CN109207420B
CN109207420B CN201810713481.9A CN201810713481A CN109207420B CN 109207420 B CN109207420 B CN 109207420B CN 201810713481 A CN201810713481 A CN 201810713481A CN 109207420 B CN109207420 B CN 109207420B
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medium
artificial skin
culturing
skin
simulated
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CN109207420A (zh
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李成训
裵日弘
闵大珍
吴秀晶
李泰龙
曹恩敬
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Amorepacific Corp
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Abstract

一实现例提供一种人造皮肤的制造方法，该方法包括以下步骤：通过对成纤维细胞及胶原蛋白进行混合而形成仿真皮层；在所述仿真皮层上应用角质形成细胞后进行培养；在第一培养基中培养所述仿真皮层，所述第一培养基为角质形成细胞培养基；及将在所述第一培养基中培养的仿真皮层在混合DMEM培养基及F12培养基而成的第二培养基中进行培养。

Description

人造皮肤的制造方法及人造皮肤
技术领域
本记载涉及一种制造人造皮肤的方法。本记载具体提供一种产生色素沉着的人造皮肤的制造方法。此外，本记载提供一种人造皮肤，所述人造皮肤包括角质形成细胞及黑色素形成细胞。
背景技术
皮肤为覆盖身体外部的器官，从外面由表皮、真皮和皮下脂肪层这三层构成。在表皮中，中层鳞状上皮的角质形成细胞占大部分。由胶原纤维和弹性纤维等基质蛋白质来构成的真皮位于表皮之下，真皮具有血管、神经和汗腺等。皮下脂肪层由脂肪细胞来构成。皮肤通过如上所述各种细胞和构成物质的相互作用来保持其形状，并且起到调节体温的功能及作为对外部环境的屏障的功能等各种功能。
人造皮肤(artificial skin)为利用皮肤细胞及皮肤构成物质即胶原蛋白及弹性蛋白等来三维重构如上所述皮肤的的物质，其由存活的成纤维细胞和角质形成细胞来构成并且表现出与实际皮肤相似的结构及功能特征，因此还称为皮肤仿形体(skin equivalent或reconstructed skin)。人造皮肤为一种主要在弹力、强度及物质渗透等方面上与皮肤表现出相似的物理性质的高分子复合物，区别在于人造皮肤不会表现出与皮肤同样的生命现象。人造皮肤不仅用于取代烧伤或外伤等受损的皮肤(永久性植入型)或这种皮肤的再生(暂时包覆型)，而且在皮肤生理研究、皮肤刺激评价及皮肤功效评价等多种领域中得到应用。
诱导了色素沉着的人造皮肤模型例如可用于评价化妆品的美白效果。皮肤的色素沉着主要是表皮内黑色素的存在引起的，黑色素由位于表皮基底层的树枝状细胞—黑色素形成细胞来合成。先前已知的色素沉着人造皮肤采用以下方法来获得：将角质形成细胞和黑色素形成细胞分别分开培养后，对其进行共播种(co-seeding)并培养，从而诱导表皮层的发达。
发明内容
要解决的技术问题
在形成表皮层时，将角质形成细胞和黑色素形成细胞分别分开培养后对其进行共播种(co-seeding)的方法可能会导致人造皮肤制造过程的复杂化。此外，由于实质上难以设定能够一起培养角质形成细胞和黑色素形成细胞的培养条件，因此黑色素细胞不能完整地存在于最终的人造皮肤模型中，具有难以起到作为正常的色素沉着人造皮肤模型的功能的问题。
因此，一实现例的人造皮肤的制造方法旨在提供一种人造皮肤，该方法通过仅使用角质形成细胞来制造表现出色素沉着的人造皮肤，从而提供进一步接近色素沉着的实际皮肤而仿形的人造皮肤。
技术方案
一实现例提供一种人造皮肤的制造方法，该方法包括以下步骤：通过对成纤维细胞及胶原蛋白进行混合而形成仿真皮层；在所述仿真皮层上应用角质形成细胞后进行培养；在第一培养基中培养所述仿真皮层，所述第一培养基为角质形成细胞培养基；及将在所述第一培养基中培养的仿真皮层在混合DMEM培养基及F12培养基而成的第二培养基中进行培养。
所述角质形成细胞可包括黑色素形成细胞的干细胞。
所述第二培养基可包括用于诱导所述黑色素形成细胞的干细胞分化的成分。
将所述第一培养基可设置为与所述仿真皮层的底面相接，并且使与设置有所述第一培养基的表面相反的表面暴露在空气中。
可在结束所述第一培养基中的培养步骤后直接进行所述第二培养基中的培养步骤。
在所述第一培养基中的培养期和在所述第二培养基中的培养期合计可为20天内。
在所述第一培养基中的培养期可以等于或长于在所述第二培养基中的培养期。
所述第二培养基可通过以1:1至4:1的比例混合所述DMEM培养基和所述F12培养基而成。
所述第二培养基可进一步包括选自氢化可的松(hydrocortisone)、三碘甲状腺原氨酸(Triiodothyronine)、霍乱毒素(Cholera toxin)、抗坏血酸(ascorbic acid)及氯化钙(CaCl2)中的一种或两种以上。
所述第二培养基中的培养步骤可在暴露在空气中的状态下进行。
另一实现例可提供一种人造皮肤，所述人造皮肤包括仿真皮层及位于所述仿真皮层上的仿表皮层，所述仿表皮层包括与所述仿真皮层相邻的表皮基底层部分及暴露在空气中的角质层部分，并且存在于所述角质层中的角质形成细胞和存在于所述表皮基底层中的黑色素形成细胞来自同一个人。
另一实现例可提供一种色素化调节剂的功效评价方法，该方法包括以下步骤：在根据上述制造方法制造的人造皮肤或在上述结构的人造皮肤中应用所述色素化调节剂；及对应用所述色素化调节剂的人造皮肤的色素沉着程度和未应用所述色素化调节剂的人造皮肤的色素沉着程度进行比较。
所述色素化调节剂可通过与所述人造皮肤直接接触来应用。
发明效果
一实现例的制造方法能够诱导存在于角质形成细胞之间的黑色素形成细胞的干细胞分化，从而在不使用黑色素形成细胞的情况下也能够制造表现出色素沉着的人造皮肤。根据所述方法制造的人造皮肤更加接近实际皮肤，因此当用作皮肤美白评价模型时，能够提供可靠性得到提高的评价数据。
附图说明
图1为用于说明根据一实现例的人造皮肤的制造方法的顺序图；
图2表示从实施例1中获得的人造皮肤的剖面，即为经H&E组织染色后的显微镜照片；
图3表示从比较例1中获得的人造皮肤的剖面，即为经H&E组织染色后的显微镜照片。
具体实施方式
下面，对本发明的实现例进行详细说明，以使本发明所属技术领域的技术人员能够容易实施。但本发明可用多种方式来实现，并不局限于在此说明的实现例。
在本申请文件中所称的“人造皮肤(artificial skin)”是指利用皮肤细胞和皮肤构成物质即胶原蛋白等来三维重构皮肤的物质，其为最广义的含义，只要是表现出与实际皮肤相似的结构及功能特性的高分子复合物均包含在所述人造皮肤。
在本申请文件中，当表述为层、膜、区域、板等部分在另一部分的“上面”时，不仅包括所述层、膜、区域、板等部分“直接”位于该另一部分的“上方”的情况，还包括中间还有其它部分的情况。与此相反，当表述为某部分“直接”设置在另一部分的“上方”时，表示两者之间不存在其它部分。
下面，参照图1对根据一实现例的人造皮肤的制造方法进行说明。
图1为用于说明根据一实现例的人造皮肤的制造方法的顺序图。
参照图1，一实现例的人造皮肤的制造方法包括：形成仿真皮层的步骤(S1)；在所述仿真皮层上应用角质形成细胞后进行培养的步骤(S2)；在第一培养基中培养所述仿真皮层的步骤(S3)；及将在所述第一培养基中培养的仿真皮层在第二培养基进行培养的步骤(S4)。
首先，对形成仿真皮层的步骤(S1)进行说明。
真皮(dermis)为由连接组织来构成的表皮下面的皮肤层，起到缓冲作用，从而在压力和张力(stress and strain)中保护身体。真皮通过基底膜(basement membrane)与表皮牢固连接。真皮供给用于支撑其结构中内置的血管及神经等各种附属物的基质。
在与实际皮肤的人体相关性的角度上，使用将成纤维细胞及胶原蛋白混合而成的材料来形成所述仿真皮层。所述仿真皮层可构成为两层以上，例如可包括，可分成含有胶原蛋白的第一真皮层及含有成纤维细胞的第二真皮层来构成。此时，能够进一步缓和人造皮肤的真皮收缩现象。
所述第一真皮层可含有胶原蛋白，并且可进一步含有构成真皮的细胞外基质(extracellular matrix)，如弹性蛋白(Elastin)、壳聚糖(Chitosan)、糖胺聚糖(GAGs；glycosaminoglycans)和透明质酸(HA；hyaluronic acid)。
所述第二真皮层可位于所述第一真皮层的上部，所述第二真皮层含有成纤维细胞，并且可进一步含有构成真皮的细胞外基质(extracellular matrix)，如弹性蛋白(Elastin)、壳聚糖(Chitosan)、糖胺聚糖(GAGs；glycosaminoglycans)和透明质酸(HA；hyaluronic acid)。
所使用的胶原蛋白可为来自牛的胶原蛋白、来自大鼠尾巴或鱼类的胶原蛋白、天然胶原蛋白、或通过基因工程生成的胶原蛋白中的任何不同来源的胶原蛋白，所述胶原蛋白可在成纤维细胞的存在下进行收缩。
仿真皮层的厚度一般为0.05cm以上，尤其约为0.05至2cm，但只要不损害本发明的人造皮肤的有利特性，就可增加或减小。
如上所述，使用将成纤维细胞及胶原蛋白混合而成的材料来制作仿真皮层后，例如可培养约5天至9天、约6天至约8天或约7天。
在形成仿真皮层后，经过在其上应用角质形成细胞后进行培养的步骤(S2)。
角质形成细胞(keratinocyte)构成表皮细胞的约80％至90％，所述角质形成细胞通过在表皮层的最深层即在基底层中的有丝分裂而形成后朝向皮肤表面上升至上层。
在应用角质形成细胞后进行培养的步骤(S2)为用于形成人造皮肤的仿表皮层的第一步骤，在此使用的角质形成细胞例如可为来自人类的角质形成细胞。所述角质形成细胞可使用常用的任何角质形成细胞，不仅可以使用从人体直接分离的角质形成细胞或者从人体分离后进行培养的角质形成细胞，还可以使用从其它细胞中诱导的角质形成细胞。作为能够在商业上获取的人类角质形成细胞可以列举：龙沙(Lonza)提供的NHEK-Neo，Pooled(新生儿正常人表皮角质形成细胞，混合(Neonatal Normal Human EpidermalKeratinocytes，Pooled)：产品编号00192906，组织获得编号P867，白人们)、NHEK-Neo(新生儿正常人表皮角质形成细胞，产品编号00192907，组织获得编号：20647，白人)、NHEK-Adult(成人正常人表皮角质形成细胞，产品编号00192627,组织获得编号：21155，白人)及NHEK-Neo(新生儿正常人表皮角质形成细胞，产品编号00192907，组织获得编号：18080，黑人)。此外可以列举：赛默飞世尔(Thermo Fisher)提供的HEKn(新生儿人表皮角质形成细胞(HumanEpidermal keratinocytes,neonatal)：产品编号C0015C，组织获得编号1781129，白人)及HEKn(产品编号C0015C，组织获得编号1803827，黑人)。为了保持角质形成细胞附着于基底膜(base membrane)增殖的性质，培养皿(dish)优选由0.1～0.2％明胶或0.1～0.2mg/ml的IV型胶原蛋白涂覆后使用。所述细胞可在35～37℃及5～10％CO2孵化器中培养，当达到约70～80％的密度时，可继代培养。
所述角质形成细胞例如可通过播种(seeding)来在所述仿真皮层上应用，培养期例如可为1天至4天、1天至3天或1天至2天，但并不局限于此。
在所述角质形成细胞之间可包括少量的黑色素形成细胞(melanocyte)的干细胞。黑色素形成细胞的干细胞经过将在后面描述的第一培养基及第二培养基的培养步骤，分化为黑色素形成细胞。
经过角质形成细胞的应用及培养步骤(S2)后，经过在角质形成细胞培养基即第一培养基中进行培养的步骤(S3)。
在此，“角质形成细胞培养基”意味着用于培养人类的角质形成细胞的培养基，该培养基可使用在本领域中众所周知的任何培养基。例如，所述角质形成细胞培养基可为包括牛脑垂体提取物(Bovine Pitutary Extract，BPE)、人上皮生长因子(human epidermalgrowth factor，hEGF)、牛胰岛素(bovine Insulin)、氢化可的松(Hydrocortisone)及庆大霉素及两性霉素-B(GA-1000(Gentamicin，Amphotericin-B))的培养基。此外，也可为在上述成分的基础上进一步包括肾上腺素(Epinephrine)及转铁蛋白(Transferrin)的培养基。作为所述培养基的能够在商业上获取的例子，可以列举CnT-3D-PR(CellnTEC公司)。
可将所述第一培养基设置为与所述仿真皮层的底面相接，并且使与设置有所述第一培养基的表面相反的表面暴露在空气中。在这种第一培养基中的培养例如可进行例如3天至10天、4天至10天、5天至9天或4至8天，但并不局限于此。
接下来，经过将在所述第一培养基中培养的仿真皮层在混合DMEM培养基及F12培养基而成的第二培养基中进行培养的步骤(S4)。
所述混合培养基为将常用的DMEM培养基及F12培养基混合而成的培养基，这些培养基例如可以1:1至4:1的比例混合，更具体地可以2:1至3:1的比例混合，但并不局限于此。DMEM(杜尔伯科改良伊格尔培养基，Dubelco's modified Eagle medium)培养基为通常使用的动物细胞培养用培养基，可包括氨基酸、维生素、无机盐、葡萄糖、脂质、指示剂和血清等。
所述第二培养基以总培养液为基准包括100％的DMEM培养基及F12培养基，并且可进一步包括选自氢化可的松(hydrocortisone)、三碘甲状腺原氨酸(Triiodothyronine)、霍乱毒素(Cholera toxin)、抗坏血酸(ascorbic acid)及氯化钙(CaCl2)中的一种或两种以上。
在所述第二培养基中的培养步骤可以与在第一培养基中的培养步骤同样地在暴露在空气中的状态下进行。
可在结束第一培养基中的培养步骤后直接进行所述第二培养基中的培养步骤，所述第二培养基中的培养步骤的培养期例如可为4天至15天、5天至13天、6天至10天或7天至9天，但并不局限于此。不过，所述第一培养基中的培养期及所述第二培养基中的培养期的总和例如可在20天内调节。例如，所述第一培养基中的培养期可设定为等于或长于所述第二培养基中的培养期。
经过所述步骤S2至S4，获得在仿真皮层上形成有仿表皮层的人造皮肤结构。
一实现例的人造皮肤的制造方法依次经过第一培养基中的培养步骤(S3)和第二培养基中的培养步骤(S4)，从而能够将存在于在此之前播种在仿真皮层上的角质形成细胞之间的黑色素形成细胞的干细胞分化为黑色素形成细胞。
因此，在根据上述方法制造人造皮肤时，能够获得如下的人造皮肤模型：在该人造皮肤模型的表皮基底层中均匀地分布有黑色素形成细胞，这些黑色素形成细胞的分布密度在相对于真皮取代物表面的平行面上实质上恒定，并且与在人类实际皮肤中发现的情况实质上相似。
即，可在不另行经过对黑色素形成细胞进行培养的过程的情况下，诱导角质形成细胞中存在的黑色素形成细胞的干细胞分化，从而能够实现存在有效的角质形成细胞和黑色素形成细胞的人造皮肤模型。
本发明的另一实现例提供一种人造皮肤，该人造皮肤包括仿真皮层及位于所述仿真皮层上的仿表皮层，所述仿表皮层包括与所述仿真皮层相邻的表皮基底层部分及暴露在空气中的角质层部分，此外，存在于所述角质层中的角质形成细胞和存在于所述表皮基底层中的黑色素形成细胞来自同一个人。
所述人造皮肤的角质形成细胞和黑色素形成细胞来自同一个人意味着在所述人造皮肤的制造过程中不是分别培养角质形成细胞及黑色素形成细胞，而是通过存在于角质形成细胞内的黑色素形成细胞的干细胞的分化来获得黑色素形成细胞。
本发明的又一实现例提供一种色素化调节剂的功效评价方法，该方法包括以下步骤：在上述人造皮肤中或在通过上述方法获得的人造皮肤中应用所述色素化调节剂；及对应用所述色素化调节剂的人造皮肤的色素沉着程度和未应用所述色素化调节剂的人造皮肤的色素沉着程度进行比较。
术语“色素化调节”意味着通过直接或间接的方式增加、减少或抑制来改变皮肤的结构性或诱导性色素化程度。所述色素化调节剂可为色素化增加剂，也可为色素化减少剂。
所述色素化调节剂可为通过与人造皮肤直接接触来应用的色素化调节剂。所述色素化调节剂例如可为美白化妆品，但并不局限于此。
下面，通过实施例对上述本发明的实现例进行更加详细的说明。不过，下面的实施例仅出于说明目的而提供的，并不用于限制本发明的范围。
人造皮肤的制作
实施例1
(1)步骤1：仿真皮层的制造
通过混合成纤维细胞和胶原蛋白来制作仿真皮层后培养一周。
(2)步骤2：角质形成细胞的应用及培养步骤
在所述步骤1后，在所述仿真皮层上放置角质形成细胞(HEKn，Thermo Fisher)并培养两天。
(3)步骤3：第一培养基中的培养步骤
在所述步骤2后，将第一培养基(CnT-3D-PR，CellnTEC)设置为与所述真皮仿形体的底部相接，并将表皮层部分暴露于空气中，从而在空气中暴露的状态下培养八天。
(4)步骤4：第二培养基中的培养步骤
在所述步骤3后，在空气中暴露的状态下进一步在第二培养基中培养六天而获得人造皮肤模型。第二培养基的组成如下：将DMED培养基(Welgene，LM001-05)和F12培养基(Welgene，LM010-01)以2.5:1的比例混合，并且在其中包括氢化可的松(hydrocortisone)、三碘甲状腺原氨酸(Triiodothyronine)、霍乱毒素(Cholera toxin)、抗坏血酸(ascorbicacid)及氯化钙(CaCl2)。
在图2中示出所获得的人造皮肤模型的显微镜剖面照片。
比较例1
除了不经过第二培养基中的培养步骤(步骤4)之外，与实施例1相同的方法制作人造皮肤。
在图3中示出所获得的人造皮肤模型的显微镜剖面照片。
评价：观察人造皮肤模型的剖面
通过显微镜观察在实施例1及比较例1中获得的人造皮肤模型的剖面。
图2及图3表示从实施例1及比较例1中获得的人造皮肤的剖面，即为H&E组织染色后的显微照片。
参照图2及图3可知，可通过调节用于培养人造皮肤的培养基并且仅使用角质形成细胞来制造表现出色素沉着的人造皮肤。更加详细说明如下，在本发明的实施例1中即使使用了相同的角质形成细胞，也能够在表皮层基底部中观察到从黑色素形成细胞的干细胞分化的黑色素形成细胞(图2)，相反在常规方法下的比较例1中，在表皮层基底部中未发现黑色素形成细胞(图3)。
虽然在上面对本发明的优选实施例进行了详细说明，但本发明的保护范围并不局限于此，本领域技术人员在所附的权利要求书中定义的本发明基本概念的基础上所进行的各种变形及改良形式也属于本发明的保护范围。

Claims (7)

1.一种人造皮肤的制造方法，包括以下步骤：
通过对成纤维细胞及胶原蛋白进行混合而形成仿真皮层；
在所述仿真皮层上应用包括黑色素形成细胞的干细胞的角质形成细胞后进行培养；
在第一培养基中培养所述仿真皮层，所述第一培养基为角质形成细胞培养基；及
将在所述第一培养基中培养的仿真皮层在包括DMEM培养基及F12培养基的第二培养基中进行培养，
其中，所述第二培养基通过以1:1至4:1的比例混合所述DMEM培养基和所述F12培养基而成，并且包括氢化可的松、三碘甲状腺原氨酸、霍乱毒素、抗坏血酸及氯化钙；
其中，所述角质形成细胞使用Thermo Fisher的HEKn；其中所述角质形成细胞培养基包括牛脑垂体提取物(BPE)、人上皮生长因子(hEGF)、牛胰岛素、氢化可的松、庆大霉素和两性霉素-B(GA-1000)。
2.根据权利要求1所述的人造皮肤的制造方法，其中，
所述第二培养基包括用于诱导所述黑色素形成细胞的干细胞分化的成分。
3.根据权利要求1所述的人造皮肤的制造方法，其中，
将所述第一培养基设置为与所述仿真皮层的底面相接，并且使与设置有所述第一培养基的表面相反的表面暴露在空气中。
4.根据权利要求1所述的人造皮肤的制造方法，其中，
在结束所述第一培养基中的培养步骤后直接进行所述第二培养基中的培养步骤。
5.根据权利要求1所述的人造皮肤的制造方法，其中，
在所述第一培养基中的培养期和在所述第二培养基中的培养期合计为20天内。
6.根据权利要求5所述的人造皮肤的制造方法，其中，
在所述第一培养基中的培养期等于或长于在所述第二培养基中的培养期。
7.根据权利要求1所述的人造皮肤的制造方法，其中，
所述第二培养基中的培养步骤在暴露在空气中的状态下进行。
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