CN107667167B - 人造皮肤培养容器及使用其制造人造皮肤的方法 - Google Patents
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Abstract
本发明的人造皮肤培养容器,通过采用琼脂以及将一部分琼脂进行疏水改性,可以解决在制造人造皮肤过程中,由人造皮肤真皮层中存在的胶原和成纤维细胞之间的相互作用导致的人造皮肤真皮层的收缩以及真皮层与培养容器分离的问题。因此,使用该培养容器能够稳定地培养人造皮肤并制造出与人体皮肤相似的人造皮肤。此外,本发明的人造皮肤培养容器包含琼脂,因此可以通过培养容器的侧部和下部供给培养液,从而可以有效地培养人造皮肤。
Description
技术领域
本发明涉及一种培养人造皮肤的培养容器及使用其制造人造皮肤的方法。
背景技术
皮肤是覆盖身体外部的器官,从外到里由表皮、真皮和皮下层三层组成。表皮主要由复层扁平上皮的角化细胞组成。真皮由基质蛋白如胶原纤维和弹性纤维组成,其位于表皮之下。真皮包含血管、神经、汗腺等。皮下层由脂肪细胞组成。这些不同的细胞和组成物质相互作用以保持皮肤形状,使皮肤能够执行各种各样的功能,如温度调节、对外部环境的屏障等。
利用构成皮肤的皮肤细胞、胶原和弹性蛋白等,通过三维构建皮肤替代物来制造人造皮肤。它由活的成纤维细胞和角化细胞组成,具有与真实皮肤相似的结构和功能特性。因此,它也被称为皮肤替代物或重建的皮肤。人造皮肤是一种高分子复合物,其主要在弹性、强度和材料渗透性等方面具有与皮肤相似的特性。它与皮肤的不同之处在于它不涉及皮肤的生命现象。人造皮肤不仅用于替代(永久性植入型)或再生(临时涂层型)由于烧伤或伤口等而受损的皮肤,还应用于各种领域,例如皮肤生理学研究、皮肤刺激评估和对皮肤的影响评估。
然而,现有的人造皮肤的制造过程是,将胶原和真皮成纤维细胞混合之后使其固化以获得真皮层,然后进行培养。在组织培养过程中,胶原和成纤维细胞相互作用,这不可避免地会导致真皮逐渐收缩。结果,在人造皮肤的制造过程中人造皮肤的真皮层与培养容器分离。此外,当真皮收缩严重时,人造皮肤组织的结构完全畸形,组其织在生理上也变得异常,因此该人造皮肤不得不被丢弃。
[引用列表]
[专利文献]
日本专利号1996-243156
发明内容
技术问题
为解决上述问题,本发明提供了一种新颖的人造皮肤培养容器,其可以防止在人造皮肤制造过程中发生真皮收缩。本发明还提供一种使用该培养容器制造的具有与真皮相似的结构和功能的人造皮肤。
技术方案
本发明的实施方式提供一种人造皮肤培养容器,其包含凝胶状的琼脂,其中一部分琼脂被疏水改性。
本发明的实施方式还提供一种在人造皮肤培养容器中制造人造皮肤的方法,其包括以下步骤:将脱细胞胶原溶液导入人造皮肤培养容器中;培养脱细胞胶原溶液以使脱细胞胶原溶液侵入人造皮肤培养容器的琼脂中;将人真皮成纤维细胞溶液引入人造皮肤培养容器中并使胶原溶液凝胶化;并在从人造皮肤培养容器外部供给培养液的同时,培养人真皮成纤维细胞溶液。
本发明的有益效果
本发明的人造皮肤培养容器通过琼脂,琼脂的一部分被疏水改性并且包括其中形成的三维微孔,从而可以解决在制造人造皮肤过程中,由人造皮肤真皮层中存在的胶原和成纤维细胞之间的相互作用导致的人造皮肤真皮层的收缩及其与培养容器分离的问题。因此,使用该培养容器而不是现有的细胞培养容器,能够稳定地培养人造皮肤并制造出与人体皮肤相似的人造皮肤。
此外,本发明的人造皮肤培养容器包含琼脂,因此可以通过培养容器的侧部和下部供给培养液。这使得能够三维地供应培养液,从而有效地培养人造皮肤。
附图说明
图1示出了琼脂糖的凝胶结构。
图2示出了人造皮肤培养容器的模拟图和表示在结构中形成的三维微孔形状的SEM(扫描电子显微镜)照片。
图3为利用本发明的一个实施方式的人造皮肤培养容器制造人造皮肤的方法的顺序图。
图4为在一般人造皮肤培养容器(比较例2)、纯琼脂培养容器(比较例1)或本发明的疏水性琼脂培养容器(实施例1)中培养7天的人造皮肤细胞的图片。
图5示出了在比较例1的一般琼脂培养容器中培养的人造皮肤(左侧两个图),其与容器分离,和在实施例1的疏水改性琼脂培养容器中培养的人造皮肤(右侧两个图),其紧密地附着在容器上而不脱离。
图6为在实施例1的疏水改性琼脂培养容器中培养之后冷冻干燥的人造皮肤的SEM(扫描电子显微镜)图片。这表明人造皮肤真皮层的胶原纤维朝向培养容器的内部。
优选实施方式
在本文中,术语“人造皮肤”是指利用构成皮肤的皮肤细胞、胶原等,通过三维构建皮肤替代物而制造的一种皮肤替代物。这意味着包括具有与真实皮肤相似的结构和功能特性的所有高分子复合物。
下面将具体描述本发明。
本发明的实施例提供一种人造皮肤培养容器,其包含凝胶状的琼脂,其中一部分琼脂被疏水改性。
琼脂是一种细胞间粘多糖,从红藻如石花菜中获得。它包括硫酸,主要由半乳糖组成。琼脂来源于属于蜈蚣藻属、江蓠属、沙菜属和杉藻属以及其他属的红藻类,还有石花菜属。琼脂主要由琼脂糖(约70%)和琼脂胶(约30%)组成。其呈白色,透明,有光泽。其不溶于冷水,但溶于热水形成溶胶。具体地,当将琼脂粉末在水溶液中混合,在约100℃-140℃的恒温下加压,然后在室温下冷却或熟化时,琼脂粉末中分子的物理结合使其凝胶化。该凝胶在约85℃以下不溶解。其仅与特定细菌相互作用,且不溶于盐。其耐酸性弱,但耐碱性强。以下式1表示琼脂中琼脂糖的结构,图1显示了琼脂糖的凝胶结构。图1显示了1975年发表在LaasT.,Acta University上的琼脂糖的凝胶结构,这里引用了其全部内容。
【式1】
本发明的实施例可以包括疏水改性的琼脂,其通过用疏水性分子取代构成人造皮肤培养容器的琼脂中的一部分琼脂糖的一个或多个氢原子而获得。例如,疏水性分子可以选自C12-C18烷基、烯基和酰基链中的一个或多个;聚丙二醇(PPG);和聚己内酯(PCL)的组合。然而,疏水性分子不限于此,只要其可以疏水改性琼脂即可。
在一个实施例中,当将琼脂凝胶化前的100个琼脂粉末定义为一个单位时,将一个单位琼脂粉末中取代的疏水性分子的数量定义为取代度(DS)(%),进入构成人造皮肤培养容器的琼脂中的疏水性分子的取代度可以为1.0%-40%,更具体为5.0%-20%。如果疏水性分子的取代度小于5.0%时,则疏水性低,导致与胶原凝胶的结合力降低。如果疏水性分子的取代度大于20%,则琼脂和胶原凝胶之间的亲和力低。
在另一个实施例中,疏水改性琼脂占人造皮肤培养容器的琼脂总重量的比例可以为5.0wt%-50wt%,更具体地5.0wt%-20wt%。人造皮肤培养容器的疏水性由上述重量比确定。疏水改性琼脂的比例也可以根据所需的疏水性程度而调整,而不限于上述重量比例。疏水改性琼脂的比例可以根据待培养的人造皮肤的成纤维细胞的数量来选择。例如,如果使用具有高比例的疏水改性琼脂的培养容器,则可以通过培养大量成纤维细胞来制造人造皮肤而不发生收缩,因为该容器与胶原具有高的结合力。
在一个实施例中,例如,人造皮肤培养容器的侧部和下部的琼脂凝胶的厚度为0.5-50mm,培养人造皮肤的内底部的直径为0.3-20.0mm,但不限于此(参见图2中的培养容器的模拟图)。
根据本发明的实施例,琼脂可以包含微孔,并且微孔的平均直径可以为0.1-10μm。人造皮肤培养容器的孔隙率可以为人造皮肤培养容器的总琼脂体积的5~50%。然而,微孔的孔隙率和平均直径不限于此,并且可以根据本发明中包含的琼脂的浓度来调整。
微孔包括通过疏水改性一部分琼脂形成的微孔,但其直径不受琼脂疏水性的影响。当在培养容器中培养人造皮肤时,人造皮肤的胶原纤维通过微孔侵入培养容器,并且通过与疏水性分子的三维结合将胶原纤维固定在培养容器中,从而使结合力增强。因此,可以防止在人造皮肤的制造过程中由于存在于真皮层中的胶原纤维和成纤维细胞之间的相互作用而导致的真皮层收缩和真皮层从培养容器脱离的问题。此外,使用培养容器进行人造皮肤制造时可以通过微孔自由提供培养液,从而能够有效地培养具有正常组织和生理功能的人造皮肤(参见图2的右图)。图2示出了本发明的实施例的人造皮肤培养容器的模拟图及其包括三维微孔的内部结构的扫描电子显微镜(SEM)照片。本发明的人造皮肤培养容器的制造方法可以采用本领域通常使用的琼脂凝胶的制造方法,但不限于此。
在其他实施例中,本发明还提供一种在人造皮肤培养容器中制造人造皮肤的方法,其包括以下步骤:
将脱细胞胶原溶液导入人造皮肤培养容器中;
培养脱细胞胶原溶液以使脱细胞胶原溶液侵入人造皮肤培养容器的琼脂中;
将人真皮成纤维细胞溶液引入人造皮肤培养容器中,并使胶原溶液凝胶化;和
在从人造皮肤培养容器外部供给培养液的同时,培养人真皮成纤维细胞溶液。
图3阐明了利用根据本发明的实施例的人造皮肤培养容器制造人造皮肤的方法的一个例子。参见图3,导入的胶原的浓度例如为3mg/ml-6mg/ml。然而,浓度不限于此,可以进行调整。此外,培养引入的胶原溶液的步骤可以包含在1-10℃或3-5℃下过夜培养。在一个实施例中,使引入的脱细胞胶原溶液侵入人造皮肤培养容器的琼脂中的步骤,可以包括使胶原溶液的胶原纤维侵入人造皮肤培养容器中的微孔,并通过与人造皮肤培养容器的疏水改性琼脂结合,从而固定的步骤。在图3第三步的方框下方的照片显示了胶原溶液的胶原纤维侵入由琼脂制成的容器,同时胶原纤维朝向容器。
此外,从人造皮肤培养容器外部供给培养液的步骤,可以包括将人造皮肤培养容器放置在一个腔室内,并将培养液供给到该腔室中,通过人造皮肤培养容器的侧部和下部供给培养液,从而实现三维供应,这与仅允许通过底部供给的现有容器不同。在上述步骤之后,还可以包括在培养容器中培养人造皮肤约7天的步骤。培养的人造皮肤的成纤维细胞的数量可以为1.0x 103-1.0x 105(细胞/培养插入物),但不限于此。
具体实施方式
下面,将结合以下实施例描述本发明。以下实施例仅用于解释本发明,但不用于限制本发明的范围。此外,以下比较例仅用于与实施例进行比较。其不视为现有技术。在不脱离本发明构思的情况下,可以对本发明进行各种修改和变化,这对于本领域技术人员来说是显而易见的。
实施例1
在本发明的一个实施例中,制备由琼脂凝胶组成的人造皮肤培养容器,其包括未被疏水改性的琼脂(超纯试剂,日本Yakuri纯化学品有限公司)和用疏水性十八烷基分子(Sigma Aldrich)改性的琼脂,其重量比为10:2,其中琼脂基于人造皮肤培养容器的总含量为3%(w/v)。该琼脂培养容器的厚度为2mm,内底直径为12mm。容器内的微孔尺寸约为0.5μm。
比较例1
在本发明的一个比较例中,制备由纯琼脂组成的人造皮肤培养容器,其中,琼脂(超纯试剂,日本Yakuri纯化学品有限公司)基于人造皮肤培养容器的含量为3%(w/v)。该琼脂培养容器的厚度为2mm,内底直径为12mm。容器内的微孔尺寸约为0.5μm。比较例2
在本发明的另一个比较例中,制备直径为12mm,底部孔径为0.4μm的Transwell(Corning,NY,14831,USA),它是一种常规使用的人造皮肤培养容器。
[试验例1]人造皮肤的培养1
根据以下方法分别使用实施例1和比较例1和2的人造皮肤培养容器培养人造皮肤。
所制造的人造皮肤模型是AmoReSkinTM(AMOREPACIFIC Group,Inc.),它是由真皮层和表皮层组成的重建皮肤。在人造皮肤模型中,胶原和人类真皮成纤维细胞用于形成真皮层,角化细胞在其上增殖和分化,从而获得具有与真实皮肤相似的结构/功能特性的人造皮肤。
首先,在室温下干燥实施例1和比较例1的培养容器,将水分含量降低至80%或以下。将0.5ml的6mg/ml胶原溶液引入干燥的琼脂培养容器中,并在4℃下培养过夜。通过培养,将胶原溶液侵入琼脂培养容器后,将1.0×104个人皮肤成纤维细胞与胶原溶液混合,然后加入60μl胶原溶液(重建溶液,0.22g NaHCO3,0.477g HEPES,0.4mL 1N的NaOH在10mL H2O中)以使胶原凝胶化。将人造皮肤培养容器放入一腔室中。然后,在向腔室内供给培养液(DMEM)的同时,三维供给培养液,培养7天,获得人造皮肤的真皮层。
在比较例2中,引入0.5ml的6mg/ml胶原溶液,并将胶原溶液(重建溶液,0.22gNaHCO3,0.477g HEPES,0.4mL 1N的NaOH在10mL H2O中)与1.0×104个人真皮成纤维细胞混合,以使胶原凝胶化。将Transwell放入腔室中,然后在向腔室内供给培养液(DMEM)的同时,通过Transwell的下部供应培养液,培养7天,获得人造皮肤的真皮层。
将1.5×105个角化细胞(人类新生儿角化细胞,HEKn,Cascade Biologics)种植在获得的真皮层上。然后,在培养插入物内部和外部加入一个角化细胞培养基(EpiLife,Cascade Biologics)。过夜培养后,检查细胞的粘附性。然后,每隔一天更换培养基,培养约1周,以增殖表皮细胞。当表皮细胞充分增殖到覆盖真皮层的整个上部时,向培养基中加入1.2mM Ca2+,使角化细胞分化2天。然后除去所有培养基,再将培养基只添加到培养插入物外部,使角化细胞暴露于空气中。每天更换培养基,在空气中培养一周,形成表皮层,从而获得最终的人造皮肤。
在人造皮肤的真皮层的7天培养过程中观察到了真皮层的收缩程度。图4显示了最终获得的人造皮肤的照片。
如图4所示,在涉及普通人造皮肤培养容器的比较例2中,由于真皮层的严重收缩,不能维持人造皮肤的组织形状。在比较例1的纯琼脂制的培养容器中,由于胶原和成纤维细胞之间的相互作用,人造皮肤从培养容器中脱离。相反,由于胶原纤维和培养容器之间的结合力强,本发明的实施例1的疏水性琼脂培养容器防止了真皮层的收缩和真皮层与培养容器的分离。
[试验例2]人造皮肤的培养2
根据以下方法分别使用实施例1和比较例1的人造皮肤培养容器培养人造皮肤。
所制造的人造皮肤模型是AmoReSkinTM(AMOREPACIFIC Group,Inc.),它是由真皮层和表皮层组成的重建皮肤。在人造皮肤模型中,胶原和人真皮成纤维细胞用于形成真皮层,角化细胞在其上增殖和分化,从而获得具有与真实皮肤相似的结构/功能特性的人造皮肤。
首先,在室温下干燥实施例1和比较例1的培养容器,将水分含量降低至80%或以下。将0.5ml的6mg/ml胶原溶液引入干燥的琼脂培养容器中,并在4℃下过夜培养。通过培养,将胶原溶液侵入琼脂培养容器后,将1.0×104个人真皮成纤维细胞与胶原溶液混合,然后加入60μl胶原溶液(重建溶液,0.22g NaHCO3,0.477g HEPES,0.4mL 1N的NaOH在10mL H2O中)以使胶原凝胶化。将人造皮肤培养容器放入腔室中。然后,在向腔室内供给培养液(DMEM)的同时,三维供给培养液,培养7天,获得人造皮肤的真皮层。
将1.5×105个角化细胞(人类新生儿角化细胞,HEKn,Cascade Biologics)种植在获得的真皮层上。然后,在培养插入物内部和外部加入角化细胞培养基(EpiLife,CascadeBiologics)。过夜培养后,检查细胞的粘附性。然后,每隔一天更换培养基,培养约1周,以增殖表皮细胞。当表皮细胞充分增殖到覆盖真皮层的整个上部时,向培养基中加入1.2mM Ca2 +,使角化细胞分化2天。然后除去所有培养基,再将培养基只添加到培养插入物外部,使角化细胞暴露于空气中。每天更换培养基,在空气中培养一周,形成表皮层,从而获得最终的人造皮肤。
图5和图6为最终获得的人造皮肤的照片,其显示了人造皮肤的真皮层与不同琼脂培养容器的脱离和附着。
图5的左侧两张照片显示了在比较例1的普通琼脂培养容器中培养的人造皮肤与容器脱离。图5的右侧两张照片显示了在实施例1的疏水改性琼脂容器中培养的人造皮肤紧密地附着在容器上而不脱离。图6为在实施例1的疏水改性琼脂培养容器中培养的人造皮肤的照片,其中真皮层的胶原纤维朝向培养容器的内部。
结果表明,本发明的人造皮肤培养容器可以防止由于人造皮肤在培养过程中的收缩而导致人造皮肤从容器中脱离,由于容器在人造皮肤培养过程中与人造皮肤紧密粘连,从而能够有效地培养人造皮肤。
Claims (9)
1.一种人造皮肤培养容器,其包含凝胶状的琼脂,其中,一部分所述琼脂经过疏水改性,当将琼脂凝胶化前的100个琼脂粉末定义为一个单位时,将被取代到一个单位所述琼脂粉末中的疏水性分子的数量定义为取代度(%),进入构成所述人造皮肤培养容器的所述琼脂中的疏水性分子的所述取代度为5.0%-20%,以及
其中,所述琼脂包含微孔。
2.根据权利要求1所述的人造皮肤培养容器,其特征在于,疏水改性琼脂通过用疏水性分子取代琼脂中琼脂糖的一个或多个氢原子来制备。
3.根据权利要求2所述的人造皮肤培养容器,其特征在于,所述疏水性分子选自:
C12-C18烷基、烯基和酰基链中的一个或多个;
聚丙二醇(PPG);和
聚己内酯(PCL)。
4.根据权利要求1所述的人造皮肤培养容器,其特征在于,疏水改性琼脂占人造皮肤培养容器的所述琼脂总重量的比例为5.0%-50%。
5.根据权利要求1所述的人造皮肤培养容器,其特征在于,所述微孔的平均直径为0.1-10μm。
6.根据权利要求1所述的人造皮肤培养容器,其特征在于,所述人造皮肤培养容器的孔隙率为所述人造皮肤培养容器的总琼脂体积的5%-50%。
7.一种在权利要求1-6任一项所述的人造皮肤培养容器中制造人造皮肤的方法,其特征在于,包括以下步骤:
将脱细胞胶原溶液导入所述人造皮肤培养容器中;
培养所述脱细胞胶原溶液以使脱细胞胶原溶液侵入所述人造皮肤培养容器的琼脂中;
将人真皮成纤维细胞溶液引入所述人造皮肤培养容器中,并使所述胶原溶液凝胶化;和
在从人造皮肤培养容器外部供给培养液的同时,培养所述人真皮成纤维细胞溶液。
8.根据权利要求7所述的方法,其特征在于,所述使脱细胞胶原溶液侵入所述人造皮肤培养容器的琼脂中的步骤,包括以下步骤:使胶原溶液的胶原纤维侵入所述人造皮肤培养容器中的所述微孔中,并通过与所述人造皮肤培养容器的疏水改性琼脂结合而被固定。
9.根据权利要求7所述的方法,其特征在于,所述从人造皮肤培养容器外部供给培养液的步骤,包括将所述人造皮肤培养容器放置在一腔室内,并将培养液供给到该腔室中,以通过所述人造皮肤培养容器的侧部和下部供给所述培养液。
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CN113101009B (zh) * | 2021-04-19 | 2023-05-30 | 河南亚都实业有限公司 | 一种医疗用人工皮肤涂敷器装置 |
KR102622424B1 (ko) * | 2021-07-19 | 2024-01-09 | 한스바이오메드 주식회사 | 무세포 진피 제조장치 및 그의 운전방법 |
KR20230077302A (ko) | 2021-11-25 | 2023-06-01 | 쓰리이솔루션 주식회사 | 기능성 혈관이 도입된 인공피부 및 그 제조방법 |
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CA2083494A1 (en) | 1990-06-13 | 1991-12-14 | Maxine M. Dumais | Derivatized agarose products and processes therefor |
JP2858066B2 (ja) * | 1993-04-06 | 1999-02-17 | グンゼ株式会社 | 組織培養法並びにこれに用いる培養基材 |
JP3543869B2 (ja) | 1995-03-07 | 2004-07-21 | 株式会社メニコン | 培養皮膚およびその製造法 |
US6905694B1 (en) * | 1997-05-12 | 2005-06-14 | Hercules Incorporated | Hydrophobically modified polysaccharide in personal care products |
JP4664646B2 (ja) | 2004-10-20 | 2011-04-06 | 一般社団法人オンチップ・セロミクス・コンソーシアム | 細胞培養用マイクロチャンバーおよび細胞構造構築法 |
CA2637663C (en) | 2006-01-24 | 2015-06-02 | Brown University | Cell aggregation and encapsulation device and method |
US8097274B2 (en) * | 2006-10-27 | 2012-01-17 | National Defense Medical Center | Skin substitutes, preparation methods and uses thereof |
US20100190255A1 (en) * | 2009-01-28 | 2010-07-29 | Theresa Chang | Cross-linked gums for hepatocyte culture |
JP5458259B2 (ja) * | 2009-06-25 | 2014-04-02 | 富士フイルム株式会社 | 細胞積層体 |
JP2012080874A (ja) | 2010-09-15 | 2012-04-26 | National Institute Of Advanced Industrial Science & Technology | 擬微小重力環境下での三次元組織構築方法 |
KR101739330B1 (ko) * | 2010-10-29 | 2017-05-24 | (주)아모레퍼시픽 | 진피 모사체를 이용한 피부 탄력 증진력 평가법 |
JP2012235921A (ja) * | 2011-05-12 | 2012-12-06 | Shiseido Co Ltd | 三次元皮膚モデルの製造方法 |
CN108478866B (zh) * | 2013-06-28 | 2021-08-03 | 广州迈普再生医学科技股份有限公司 | 组织修复支架、其制备方法和用途 |
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US10669527B2 (en) | 2020-06-02 |
JP6813498B2 (ja) | 2021-01-13 |
KR20160116982A (ko) | 2016-10-10 |
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US20180087030A1 (en) | 2018-03-29 |
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