CN115181719B - 一种用于培养组织工程表皮的无血清培养基 - Google Patents
一种用于培养组织工程表皮的无血清培养基 Download PDFInfo
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Abstract
本发明公开了一种用于培养组织工程表皮的无血清培养基。所述无血清培养基由DMEM培养基、F12培养基、人血白蛋白、人胰岛素、人转铁蛋白、亚硒酸钠、氨基乙醇、谷氨酰胺、环磷酸腺苷、人骨形态发生蛋白4、α‑黑色素细胞刺激素、肝细胞生长因子、粒细胞巨噬细胞集落刺激因子、碱性成纤维细胞生长因子、干细胞生长因子、三碘甲状腺原氨酸、表皮生长因子、异丙肾上腺素、内皮素‑1、内皮素‑3、氢化可的松和ROCK抑制剂组成。所述无血清培养基,避免了使用异种或异体血清可能引起的传染病风险以及免疫反应的风险,还避免了使用tpa、ct、bpe等有害物质,且培养得到的组织工程表皮质量稳定、黑色素含量极高,临床应用前景良好。
Description
技术领域
本发明属于细胞与组织体外培养领域,尤其涉及一种用于培养组织工程表皮的无血清培养基。
背景技术
白癜风是一种发病机制不明的皮肤病,全球每200人中就有1人受到白癜风的影响。目前已经提出了几种潜在的白癜风发病机制,包括自身免疫、神经体液因素和自身细胞毒性等,不过白癜风可能是由多种因素引起的,发病部位覆盖全身,常见于面部、躯干、四肢。白癜风是局部黑色素细胞破坏引起的色素脱失的皮肤病,发病区域为边界清晰的皮肤白斑,临床上较常见的以皮肤颜色减退、变白,境界鲜明,无自觉症状为特征。目前白癜风治疗方案主要为药物、紫外照射及表皮移植等。传统的表皮移植方案是从健康皮肤上取下一部分正常肤色的皮肤,然后移植到患病的白斑区,移植比例一般都小于1:1。大面积的白斑患者需要多次重复进行移植手术,过程比较痛苦。
组织工程表皮的培养可实现皮肤面积由小变大,很好地解决了移植时需大面积取皮的问题。组织工程表皮主要由角质细胞和黑色素细胞组成。现阶段,将组织工程表皮应用于临床主要面临两个问题:1.使用无血清培养基让角质细胞和黑色素细胞共同有效扩增,2.黑色素细胞在普通培养基中很难生长,而为了在细胞扩增过程中保持黑色素细胞的功能,目前商业化的黑色素培养基中会添加十四烷酞佛波醇乙酯(TPA)、霍乱毒素 (cT)、牛垂体提取物(BPE)、胎牛血清(FBS)等其中一种或多种物质,但是TPA是公认的致癌物,CT对人体是有毒性的,BPE和FBS来源于异种,使用含有这些物质培养出来的组织工程表皮应用于临床是有极大潜在风险的。因此,现在亟需开发一种不含这些物质的无血清培养基。
发明内容
本发明的目的是提供一种无血清、化学成分清晰的培养基,其适用于体外扩增培养组织工程表皮。
为实现上述目的,本发明采用如下技术方案:
本发明首先提供了一种用于培养组织工程表皮的无血清培养基,所述无血清培养基包括基础培养基、血清替代物和细胞因子;所述基础培养基包括DMEM培养基和F12培养基;所述血清替代物包括人血白蛋白、人胰岛素、人转铁蛋白、亚硒酸钠、氨基乙醇和谷氨酰胺;所述细胞因子包括环磷酸腺苷、人骨形态发生蛋白4、α-黑色素细胞刺激素、肝细胞生长因子、粒细胞巨噬细胞集落刺激因子、碱性成纤维细胞生长因子、干细胞生长因子、三碘甲状腺原氨酸、表皮生长因子、异丙肾上腺素、内皮素-1、内皮素-3、氢化可的松和ROCK抑制剂;所述ROCK抑制剂为Y-27632。
优选的,上述一种用于培养组织工程表皮的无血清培养基中,所述基础培养基为DMEM培养基和F12培养基按照3:1体积比例的混合液;所述人血白蛋白浓度为1-20 g/L,所述人胰岛素浓度为1-20 mg/L,所述人转铁蛋白浓度为0.5-15 mg/L,所述亚硒酸钠浓度为1-15 μg/L,所述氨基乙醇浓度为0.1-15 mg/L,所述谷氨酰胺浓度为0.5-10 mmol/L;所述环磷酸腺苷浓度为0.05-2 mg/mL,所述人骨形态发生蛋白4浓度为1-60 ng/mL,所述α-黑色素细胞刺激素浓度为0.1-5 nmol/L,所述肝细胞生长因子浓度为1-20 ng/mL,所述粒细胞巨噬细胞集落刺激因子浓度为0.1-10 μg/mL,所述碱性成纤维细胞生长因子浓度为0.1-1.5 μg/mL,所述干细胞生长因子浓度为1-100 μmol/L,所述三碘甲状腺原氨酸浓度为0.1-8 mg/L,所述表皮生长因子浓度为1-20 ng/mL,所述异丙肾上腺素浓度为0.1-1 μg/mL,所述内皮素-1浓度为1-20 nmol/L,所述内皮素-3浓度为1-20 nmol/L,所述氢化可的松浓度为0.05-1 μg/mL,所述ROCK抑制剂浓度为1-20 μmol/L。
优选的,上述一种用于培养组织工程表皮的无血清培养基中,所述人血白蛋白浓度为5-15 g/L,所述人胰岛素浓度为5-15 mg/L,所述人转铁蛋白浓度为3-10 mg/L,所述亚硒酸钠浓度为5-10 μg/L,所述氨基乙醇浓度为1-5 mg/L,所述谷氨酰胺浓度为1-7 mmol/L;所述环磷酸腺苷浓度为0.1-1 mg/mL,所述人骨形态发生蛋白4浓度为15-45 ng/mL,所述α-黑色素细胞刺激素浓度为0.5-3 nmol/L,所述肝细胞生长因子浓度为5-15 ng/mL,所述粒细胞巨噬细胞集落刺激因子浓度为0.5-5 μg/mL,所述碱性成纤维细胞生长因子浓度为0.3-1 μg/mL,所述干细胞生长因子浓度为30-70 μmol/L,所述三碘甲状腺原氨酸浓度为0.5-5 mg/L,所述表皮生长因子浓度为5-15 ng/mL,所述异丙肾上腺素浓度为0.3-0.8 μg/mL,所述内皮素-1浓度为5-15 nmol/L,所述内皮素-3浓度为5-15 nmol/L,所述氢化可的松浓度为0.1-0.7 μg/mL,所述ROCK抑制剂浓度为5-15 μmol/L。
优选的,上述一种用于培养组织工程表皮的无血清培养基中,所述人血白蛋白浓度为10 g/L,所述人胰岛素浓度为10 mg/L,所述人转铁蛋白浓度为6.5 mg/L,所述亚硒酸钠浓度为7.5 μg/L,所述氨基乙醇浓度为3 mg/L,所述谷氨酰胺浓度为4 mmol/L;所述环磷酸腺苷浓度为0.5 mg/mL,所述人骨形态发生蛋白4浓度为30 ng/mL,所述α-黑色素细胞刺激素浓度为1 nmol/L,所述肝细胞生长因子浓度为10 ng/mL,所述粒细胞巨噬细胞集落刺激因子浓度为1 μg/mL,所述碱性成纤维细胞生长因子浓度为0.6 μg/mL,所述干细胞生长因子浓度为50 μmol/L,所述三碘甲状腺原氨酸浓度为2 mg/L,所述表皮生长因子浓度为10ng/mL,所述异丙肾上腺素浓度为0.5 μg/mL,所述内皮素-1浓度为10 nmol/L,所述内皮素-3浓度为10 nmol/L,所述氢化可的松浓度为0.4 μg/mL,所述ROCK抑制剂浓度为10 μmol/L。
本发明还提供了一种培养组织工程表皮的方法,所述方法为使用上述的无血清培养基进行组织工程表皮培养。
本发明的优点在于:
(1)本发明提供的是一种无血清培养基,使用该培养基培养细胞,避免了使用异种或异体血清可能引起的传染病风险以及免疫反应的风险;
(2)血清或血浆批次之间差异明显,而本发明所提供的培养基化学成分明确,因而所培养得到的组织工程表皮质量更稳定;
(3)本发明使用其他因子替代TPA、CT等物质,不仅去除了这些物质所带来的安全隐患,而且所培养的组织工程表皮黑色素含量极高,非常适合于白癜风的临床应用。
附图说明
图1为三种培养基的组织工程表皮细胞传代前的细胞形态示意图。
图2为三种培养基的组织工程表皮细胞融合过程的示意图。
图3为三种培养基的组织工程表皮细胞完全融合的示意图。
图4为组织工程表皮剥离的示意图。
图5为三种培养基的组织工程表皮DOPA染色的示意图。
图6为三种培养基的组织工程表皮HE染色的示意图。
具体实施方式
下面通过具体实施例对本发明的技术方案进行详细的介绍和说明,但是应当理解的是,下述实施例并不限制本发明范围。
本发明中所用到的试剂和原料均可由市场购得。如无特别说明,本发明中的制备方法均为常规制备方法,不再详述。
实施例1
实验一 人组织工程表皮的培养及形态学观察
本实施例之中,采用的组织工程表皮培养基为:在DMEM/F12培养基中加入10g/L人血白蛋白,10mg/L人胰岛素,6.5mg/L人转铁蛋白,7.5μg/L亚硒酸钠,3mg/L氨基乙醇,4mM谷氨酰胺,0.5mg/mL环磷酸腺苷(cAMP),30ng/mL人骨形态发生蛋白4(BMP-4),1nM α-黑色素细胞刺激素(α-MSH),10ng/mL肝细胞生长因子(HGF),1μg/mL粒细胞巨噬细胞集落刺激因子(GM-CSF),0.6μg/mL碱性成纤维细胞生长因子(bFGF),50μM干细胞生长因子(SCF),2mg/L三碘甲状腺原氨酸(T3),10ng/mL表皮生长因子(EGF),0.5μg/mL异丙肾上腺素,10nM内皮素-1(ET-1),10nM内皮素-3(ET-3),0.4μg/mL氢化可的松和10μM ROCK抑制剂Y-27632;其中,所述DMEM/F12培养基为DMEM和F12按照3:1体积比例的混合液。组织工程表皮培养基具体按照表1中A组用量进行配置。
另参照表1中B组用量和C组用量配制两种培养基作为对比。
表1 组织工程表皮培养基及其配方
1 准备滋养层细胞
在组织工程表皮细胞原代培养及传代培养前,要进行滋养层细胞(人成纤维细胞)复苏操作及MMC处理。具体操作步骤如下:
复苏冻存的人成纤维细胞,制成细胞悬液,接种到成纤维细胞培养基(DMEM+5%血小板裂解液)中,调整密度为5×104cells/mL,接种至六孔板或60mm培养皿里,六孔板每孔接种2mL,60mm培养皿每皿接种4mL。置37℃条件下培养。三天后,镜下观察细胞融合度达到90%时,向培养基中加入1/24倍体积的MMC(MMC的初始浓度为0.1mg/mL),置于37℃孵育90min。孵育完成后,吸走上清,用α-MEM培养基(Gibco)清洗细胞两遍,清洗完成后加入相应体积的成纤维细胞培养基。
2 人组织工程表皮细胞的分离
取人大腿内侧正常皮肤3cm*3cm,用75%酒精消毒1min,然后用剪刀和镊子将结缔组织和脂肪组织剔除。接着用生理盐水清洗7遍,用剪刀将皮肤剪碎,加入8mL 0.1wt% I型胶原酶(215U/mg)酶液,37℃摇床消化过夜。第二天加入终浓度为2mL 0.05wt%的胰蛋白酶(Gibco),37℃摇床消化2h。随后加入等体积的胰酶抑制剂(Gibco,R002100)终止消化,吹打,将细胞悬液过70μm细胞筛,500g离心5min,弃上清,加入生理盐水重悬细胞,得到人组织工程表皮细胞悬液,取样计数。
3 组织工程表皮细胞的培养及观察
将细胞悬液分成三组A、B和C,500g离心5min,弃上清。A组加入表1中的培养基A,B组加入表1中的培养基B,C组加入表1中的培养基C。按照5×105 cells/孔的量接种到含有MMC处理过的滋养层细胞的六孔板里,置于37℃条件下培养,每隔两天镜下观察一次,培养四天后首次换液,随后每两天换液一次。当镜下观察到角质细胞克隆约每个克隆含有100-150个细胞时即可执行传代步骤。此时组织工程表皮细胞的形态如图1所示。
4 组织工程表皮细胞的传代
去除培养基,加入0.02wt%的EDTA清洗细胞两次,然后再加入0.02wt%的EDTA于37℃孵育20min。接着加入0.05wt%的胰蛋白酶消化1-3min,再加入等体积的胰酶抑制剂终止消化。500g离心5min,弃上清,向A、B、C三组细胞沉淀中分别加入各自与表1中相对应的培养基,接种到含有MMC处理过的滋养层细胞的60mm培养皿中。按照1个孔传1个60mm培养皿的比例传代。
5 组织工程表皮细胞的融合
传代后每两天换一次液,直至表皮细胞完全融合。接着改为每天换液,表皮细胞开始重叠生长,并分化增厚成多层的层状表皮,如图2、图3所示。
6 组织工程表皮的剥离
往培养液里加入终浓度为0.1mg/mL的分散酶,37℃孵育15-20min,用镊子从边缘开始往中间方向将组织工程表皮从培养皿上剥离下来,用1× PBS缓冲液(pH 7.2)清洗两遍后,将表皮转移到新的60mm培养皿中,4℃冷藏,待质检合格后即可用于临床移植。
7 结果及分析
A、B、C三个处理组人组织工程表皮细胞生长状态均为良好。如图2、3所示,角质细胞形成大面积的克隆过程中,滋养层细胞逐步凋亡脱落,等到角质细胞完全融合后,滋养层细胞就完全脱落消失不见了。角质细胞融合成片后开始分化增厚成多层的层状表皮。如图4所示,皮片具有一定的厚度,易于剥离,且剥离后皮片完整无破损。
实验二 DOPA染色
切取部分组织工程表皮,用1× PBS缓冲液(pH 7.2)清洗2遍,加入4%多聚甲醛固定10min,然后再用1× PBS缓冲液(pH 7.2)液清洗2次,自然干燥2h,加入1mg/mL的左旋多巴,37℃孵育4-5h。镜下观察计数,结果如图5所示:A组黑色素细胞分布均匀,细胞数大于120个/mm2,黑色素细胞得到有效扩增;B组黑色素细胞分布不均一,细胞数大于40个/mm2;C组黑色素细胞分布不均一,细胞数大于60个/mm2。由上述结果可见,黑色素细胞扩增效力以A组最强,C组次之,B组最差。
实验三 HE染色
切取部分组织工程表皮,经过脱蜡至水、组织包埋、组织切片、组织切片脱蜡至水、HE染色及脱蜡至二甲苯等步骤,最终镜下观察并拍照,结果如图6所示:A、B、C三个处理组组织工程表皮都具有人正常表皮的分层结构,A组分层最厚,C组次之,B组最差。
实验四 内毒素检测
取组织工程表皮成品的上清液,按照药典2020版规定的内毒素凝胶限度试验进行检测,结果如表2所示:组织工程表皮内毒素检测结果为阴性。
表2 内毒素检测结果
注:“+”表示“阳性”,“-”表示“阴性”。
实验五 无菌性检测
取组织工程表皮成品一周前的上清液,按照药典2020版规定的无菌检查法进行检测,结果如表3所示:组织工程表皮无菌性检测结果为阴性。
表3 无菌性检测结果
注:“+”表示“阳性”,“-”表示“阴性”。
实验六 支原体检测
取组织工程表皮成品的上清液,按照一步法支原体检测试剂盒(购自上海易色医疗科技有限公司 ,货号:MD001)的说明书进行操作,检测结果如表4所示:组织工程表皮支原体检测结果为阴性。
表4 支原体检测结果
注:“+”表示“阳性”,“-”表示“阴性”。
结论:采用本发明表1中的培养基A和C培养的人组织工程表皮具有人正常表皮完整的分层结构,且含有大量形态完整的黑色素细胞,与含10% FBS的DMEM/F12培养基相比,无血清培养基A和C所培养的组织工程表皮分层层数更多,表皮更厚,更有利于移植操作;黑色素细胞含量也显著增加,分布更均匀,更有利于白癜风白斑均匀复色,更美观,并且表皮通过内毒素检测、无菌性检测、支原体检测等安全性检测。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (2)
1.一种用于培养组织工程表皮的无血清培养基,其特征在于:所述无血清培养基为在基础培养基中添加10g/L人血白蛋白、10mg/L人胰岛素、6.5mg/L人转铁蛋白、7.5μg/L亚硒酸钠、3mg/L氨基乙醇、4mmol/L谷氨酰胺、0.5mg/mL环磷酸腺苷、30ng/mL人骨形态发生蛋白4、1nmol/L α-黑色素细胞刺激素、10ng/mL肝细胞生长因子、1μg/mL粒细胞巨噬细胞集落刺激因子、0.6μg/mL碱性成纤维细胞生长因子、50μmol/L干细胞生长因子、2mg/L三碘甲状腺原氨酸、10ng/mL表皮生长因子、0.5μg/mL异丙肾上腺素、10nmol/L内皮素-1、10nmol/L内皮素-3、0.4μg/mL氢化可的松、10μmol/L ROCK抑制剂Y-27632;所述基础培养基为DMEM培养基和F12培养基按照3:1体积比例的混合液。
2.一种培养组织工程表皮的方法,其特征在于:使用权利要求1所述的无血清培养基进行组织工程表皮培养。
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