CN113862214A - 用于构建人阴道粘膜模型培养基的配制方法 - Google Patents
用于构建人阴道粘膜模型培养基的配制方法 Download PDFInfo
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- CN113862214A CN113862214A CN202110575428.9A CN202110575428A CN113862214A CN 113862214 A CN113862214 A CN 113862214A CN 202110575428 A CN202110575428 A CN 202110575428A CN 113862214 A CN113862214 A CN 113862214A
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Abstract
本发明提供了一种用于构建人阴道粘膜模型培养基的配制方法,以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,0.5~2.0ng/mL表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸和5~10nmol/mL异丙肾上腺素。
Description
本分案申请是基于申请号为202110356820.4,申请日为2021年4月1日,发明名称为“一种体外重建人阴道粘膜模型构建方法”的中国专利申请的分案申请。
技术领域
本发明属于生物医学领域,尤其涉及一种用于构建人阴道粘膜模型培养基的配制方法。
背景技术
人的阴道粘膜由厚的,非角化的,分层的鳞状上皮组成,富含糖原,以及少量其他类型的细胞,例如巨噬细胞和朗格汉斯细胞。上皮细胞下面为固有层,包含许多弹性纤维和密集的血管网络,因此阴道粘膜是为局部和全身治疗输送药物的绝佳途径。但是由于长期暴露于生殖道管腔内的各种病原体中,阴道上皮也是许多病原体进入体内的潜在部位,例如在使用女性护理和美容产品,避孕药具或杀微生物剂之后,可能会造成轻度伤害,这可能会引起组织刺激,并使阴道上皮特别容易受到各种类型的感染。药品,化妆品和个人护理产品等专门配制用于人阴道粘膜的配方有时也会引起不良的局部或全身性副作用。因此,在产品投放市场之前评估新开发的化妆品、个人护理产品或局部应用药物与人阴道粘膜表面的相容性是制造商需要解决的关键问题。
分层分化的人类阴道上皮模型具有克服细胞单层膜的某些缺点的潜力,因为前者包含屏障层并允许局部施用活性成分和最终制剂,包括那些非水溶性的成分。除了可以避免动物福利风险和物种差异问题之外,体外组织模型通常还可以区分动物模型对其不敏感的非常温和的产品。
法国SkinEthic公司的RHVE模型采用外阴表皮样癌细胞系A431为种子细胞,细胞系稳定可靠,可以解决种子细胞来源困难问题,但只包含由A431细胞组成的上皮层,缺少基质层细胞和基质,因此在组织结构上还是与天然阴道组织存在较大差异。美国MatTek公司的EpiVaginaL阴道模型和中国专利CN201710048227.7中公开的人阴道上皮3D模型组织结构与天然阴道粘膜高度相似,然而由于可用的阴道组织来源有限,而且阴道黏膜上皮为复层鳞状上皮,大部分细胞属于分化成熟细胞,增殖能力较低,仅少部分基底层细胞具有较强的增殖能力,因此体外大量扩增培养条件要求较高,无法实现体外大量生产,满足市场需求。中国专利CN201710048227.7中公开的人阴道上皮3D模型还存在一些缺陷,如培养体系单一不能适应细胞不同阶段的营养需求,导致模型复层化较差,屏障功能较弱,而且该模型构建时间长,体外构建一批模型需要长达一个月时间,过长的生产周期会增加过程中的不可控因素,影响模型的稳定性,目前该模型仅限于实验室培养,未进行产业化生产,所以国内目前尚未有商业化的人阴道粘膜模型。
发明内容
针对现有技术存在的问题,本发明提供了一种人阴道粘膜模型的体外构建方法。
为实现上述发明目的,本发明采用下述技术方案予以实现:
一种人阴道粘膜模型的体外构建方法,包括如下步骤:
A、含成纤维细胞的基质层构建:
(1)取第5~10代阴道成纤维细胞,用含20%胎牛血清的DMEM配成细胞悬液;
(2)将细胞悬液和中性胶原支架按照1:9的体积比例混匀,接种到细胞培养小室中首次培养,然后在小室内外分别添加HVE-1培养液,二次培养后形成阴道粘膜模型中的基质层;
所述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加10%胎牛血清,添加2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL 氢化可的松,25~100μg/mL维生素C,1~10ng/mL成纤维细胞生长因子和5~20ng/mL转铁蛋白;
B、细胞的接种:
吸去基质层表面的HVE-1培养液,将阴道上皮细胞或外阴表皮样癌细胞系A431细胞用HVE-2培养液配制成细胞悬液,接种到细胞培养小室中的基质层上,将培养基更换成HVE-2培养液进行浸没式培养;
所述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL 氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,0.5~2.0ng/mL 表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸和5~10nmol/mL异丙肾上腺素;
C、阴道粘膜模型的培养:
吸去培养物表面的HVE-2培养液,将细胞培养小室提升至空气-液体界面进行气液面培养;将培养基更换成HVE-3培养液,培养后得到双层阴道粘膜模型。所述双层阴道粘膜模型为基质层中含有成纤维细胞,上皮层中含有阴道上皮细胞的双层阴道粘膜模型。
所述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,2.0~5.0ng/mL 表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸,5~10nmol/mL异丙肾上腺素,25~100μg/mL维生素C和1.0~1.5μmol/mL CaCl2。
优选地,步骤A中阴道成纤维细胞的分离和培养步骤如下:
(1)将分离的阴道组织固有层放入胶原酶消化液中消化,然后使用含10%胎牛血清的DMEM培养液终止消化,过滤后离心去上清,收集阴道成纤维细胞;
(2)PBS清洗阴道成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,传代培养至第五代后用于进行阴道粘膜模型中基质层的构建。
优选地,步骤B中阴道上皮细胞的分离和培养步骤如下:
(1)将人体阴道组织置于培养皿中, PBS溶液清洗6次,除去粘膜下组织,将组织块切块加入分散酶中消化;
(2)眼科镊分离出阴道粘膜上皮层和固有层,将阴道粘膜上皮层放入胰酶-EDTA中消化;
(3)使用含10%胎牛血清的DMEM培养液终止消化,过滤后离心去上清,收集阴道粘膜上皮细胞;
(4)PBS清洗阴道粘膜上皮细胞,重悬细胞沉淀于无血清上皮细胞培养液中,获得分离成单个细胞的阴道上皮细胞悬液,传代培养至第二代得到纯度达95%以上的阴道上皮细胞。
优选地,所述无血清上皮细胞培养液为以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加2.0~6.0μmol/mL的谷氨酰胺,2.0~5.0ng/mL 表皮生长因子,15~30μg/mL 牛垂体提取物,10~15ng/mL胰岛素,0.5~2.0μg/mL 氢化可的松和0.1~0.4μmol/mL CaCl2。
本发明中的无血清上皮细胞培养液以DMEM/F12为基础液,包含谷氨酰胺、牛垂体提取物(BPE)、表皮生长因子(EGF)、胰岛素、氢化可的松、氯化钙等适宜浓度的因子和蛋白,适合用于培养阴道上皮细胞,包括从正常组织中提取的原代细胞以及各种永生化和转化的阴道上皮细胞系。本发明中的无血清上皮细胞培养液由于不含血清成份,可有效减少阴道上皮细胞提取过程中成纤维细胞的污染,传代培养至第二代阴道上皮细胞纯度达95%以上。此外,还可以适用于多种永生化和转化的阴道上皮细胞系的培养,有利于进行大规模生产,可以很好的节约人工和材料成本。
优选地,步骤B中外阴表皮样癌细胞系A431细胞的培养过程为:重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、2.0~6.0μmol/mL谷氨酰胺、0.1~1μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,传代培养至第七代后用于进行上皮层的构建。
通过外阴表皮样癌细胞系A431细胞培养构建的双层阴道粘膜模型为基质层中含有成纤维细胞,上皮层中含有外阴表皮样癌细胞系A431的双层阴道粘膜模型。
优选地,步骤A中中性胶原支架的制备方法为:称量胶原置于0.1%醋酸溶液中配制成4~10mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.2~7.4。
优选地,步骤A中首次培养温度为37℃,首次培养时间为2~3小时,二次培养温度为37℃,二次培养时间为2~4天。
优选地,步骤B中培养温度为37℃,培养时间为4天。
优选地,步骤C中培养温度为37℃,培养时间为8~12天。
优选地,所述PBS溶液中含有100U/mL青霉素和100U/mL链霉素。
优选地,所述EDTA浓度为0.2mg/mL。
优选地,步骤C中采用去离子水将冰醋酸稀释为0.1%醋酸溶液,过滤除菌后使用。
优选地,本发明中用于阴道粘膜上皮层构建的种子细胞为正常原代阴道上皮细胞、永生化阴道上皮细胞系Ect1、E6E7、End1 、E6E7、Vk2 、E6E7或外阴表皮样癌细胞系A431中的一种或多种。种子细胞选择多样,体外构建重复性佳,解决种子细胞来源困难、细胞稳定性差等问题,可实现产业化制备。
相对于现有技术,本发明的有益效果在于:
本发明公开了一种人阴道粘膜模型的体外构建方法,采用阴道成纤维细胞和胶原构建基质层,在此基础上接种阴道上皮细胞,形成具有双层结构的体外阴道粘膜模型,在结构上与天然组织高度相似,在该模型上进行产品的阴道刺激性实验更加接近于体内真实情况,实验结果更准确可靠。本发明中阴道上皮细胞和基质层共培养时采用液下培养和气液面培养两个阶段,并且在不同的培养阶段针对细胞增殖和分化的不同需求对培养液的营养成分进行了精细化的调节,这种分段式培养法,既能保证细胞在不同发育阶段的营养需求,有利于复层化阴道粘膜上皮结构的形成,又能缩短构建时间、降低生产成本。
附图说明
图1A为本发明中使用正常人阴道上皮细胞培养构建的双层阴道粘膜上皮模型表观图片;
图1B为本发明中使用正常人阴道上皮细胞培养构建的双层阴道粘膜上皮模型组织学H&E染色图片;
图2A为本发明中使用外阴表皮样癌细胞系A431细胞培养构建的双层阴道粘膜上皮模型表观图片;
图2B为本发明中使用外阴表皮样癌细胞系A431细胞培养构建的双层阴道粘膜上皮模型组织学H&E染色图片。
具体实施方式
下面通过具体实施方式来进一步说明本发明的技术方案。本领域技术人员应该明了,所述实施例仅仅是帮助理解本发明,不应视为对本发明的具体限制。
实施例1
一种人阴道粘膜模型的体外构建方法,包括如下步骤:
A、阴道上皮细胞的分离和培养:
(1)将人体阴道组织置于培养皿中,使用4℃预冷的PBS溶液清洗6次,除去粘膜下组织,将组织块切成0.2cm×0.2cm的大小,加入1.5U/mL的分散酶中,4℃消化过夜;
(2)眼科镊分离出阴道粘膜上皮层和固有层,将阴道粘膜上皮层放入0.25%胰酶-EDTA中,37℃消化30min,所述EDTA的浓度为0.2mg/mL;
(3)使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集阴道粘膜上皮细胞;
(4)PBS清洗阴道粘膜上皮细胞,重悬细胞沉淀于无血清上皮细胞培养液中,获得分离成单个细胞的阴道上皮细胞悬液,接种于T75培养瓶培养,37℃,5%CO2培养箱培养,传代培养至第二代得到纯度达95%以上的阴道上皮细胞;
上述无血清上皮细胞培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加2.0μmol/mL的谷氨酰胺,2.0ng/mL EGF,15μg/mL BPE,10ng/mL胰岛素,0.5μg/mL 氢化可的松,0.1μmol/mL CaCl2。
B、阴道成纤维细胞的分离和培养:
(1)将分离的阴道组织固有层放入200U/mL胶原酶消化液中,37℃消化3h,使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集阴道成纤维细胞;
(2)PBS清洗阴道成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,接种于T75培养瓶培养,37℃,5%CO2条件下传代培养至第五代后用于进行阴道粘膜模型中基质层的构建;
C、中性胶原支架的制备:
称量胶原置于0.1%醋酸溶液中配制成4mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.2;
D、含成纤维细胞的基质层构建:
(1)取第5代阴道成纤维细胞,用含20%胎牛血清的DMEM配成密度为2×106个/mL细胞悬液;
(2)将细胞悬液和中性胶原支架按照1:9的体积比例混匀,接种到Transwell小室中,置于37℃条件下培养2小时,然后在Transwell小室内外分别添加HVE-1培养液,37℃,5%CO2培养箱培养2天,形成阴道粘膜模型中的基质层;
上述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加10%胎牛血清,2.0μmol/mL的谷氨酰胺,15μg/mL腺嘌呤,5μg/mL胰岛素,0.5μg/mL 氢化可的松,加入25μg/mL维生素C,1ng/mL成纤维细胞生长因子,5ng/mL转铁蛋白。
E、阴道上皮细胞的接种:
(1)吸去基质层表面的培养基,将上述得到的阴道上皮细胞或A431细胞系用HVE-2培养液配制成2.0×106个/mL细胞悬液,接种到Transwell小室中的基质层上;
(2)将培养基更换成HVE-2培养液进行浸没式培养,37℃、5%CO2培养箱中培养4天;
上述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0μmol/mL的谷氨酰胺,15μg/mL腺嘌呤,5μg/mL胰岛素,0.5μg/mL 氢化可的松,1ng/mL成纤维细胞生长因子,5ng/mL转铁蛋白,0.5ng/mLEGF,0.1nmol/mL三碘甲状腺原氨酸,5nmol/mL异丙肾上腺素。
F、阴道粘膜模型的培养:
(1)吸去Transwell小室中培养物表面的HVE-2培养液,将Transwell小室提升至空气-液体界面进行气液面培养;
(2)将HVE-2培养液更换成HVE-3培养液, 37℃、5%CO2培养箱中培养8天,得到基质层中含有成纤维细胞、上皮层中含有阴道上皮细胞的双层阴道粘膜模型。
上述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比1:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0μmol/mL的谷氨酰胺,15μg/mL腺嘌呤,5μg/mL胰岛素,0.5μg/mL 氢化可的松,1ng/mL成纤维细胞生长因子,5ng/mL转铁蛋白,2.0ng/mLEGF,0.1nmol/mL三碘甲状腺原氨酸,5nmol/mL异丙肾上腺素,25μg/mL维生素C,1.0μmol/mLCaCl2。
实施例2
一种人阴道粘膜模型的体外构建方法,包括如下步骤:
A、阴道上皮细胞的分离和培养:
(1)将人体阴道组织置于培养皿中,使用4℃预冷的PBS溶液清洗6次,除去粘膜下组织,将组织块切成0.2cm×0.2cm的大小,加入1.5U/mL的分散酶中,4℃消化过夜;
(2)眼科镊分离出阴道粘膜上皮层和固有层,将阴道粘膜上皮层放入0.25%胰酶-EDTA中,37℃消化45min,所述EDTA的浓度为0.2mg/mL;
(3)使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集阴道粘膜上皮细胞;
(4)PBS清洗阴道粘膜上皮细胞,重悬细胞沉淀于无血清上皮细胞培养液中,获得分离成单个细胞的阴道上皮细胞悬液,接种于T75培养瓶培养,37℃,5%CO2培养箱培养,传代培养至第二代得到纯度达95%以上的阴道上皮细胞;
上述无血清上皮细胞培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加4.0μmol/mL的谷氨酰胺,3.0ng/mL EGF,20μg/mL BPE,13ng/mL胰岛素,1.5μg/mL 氢化可的松,0.2μmol/mL CaCl2;
B、阴道成纤维细胞的分离和培养:
(1)将分离的阴道组织固有层放入200U/mL胶原酶消化液中,37℃消化3h,使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集阴道成纤维细胞;
(2)PBS清洗阴道成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,接种于T75培养瓶培养,37℃,5%CO2条件下传代培养至第五代后用于进行阴道粘膜模型中基质层的构建;
C、中性胶原支架的制备:
称量胶原置于0.1%醋酸溶液中配制成6mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.3;
D、含成纤维细胞的基质层构建:
(1)取第8代阴道成纤维细胞,用含20%胎牛血清的DMEM配成密度为5×106个/mL细胞悬液;
(2)将细胞悬液和中性胶原支架中性胶原支架按照1:9的体积比例混匀,接种到Transwell小室中,置于37℃条件下培养2.5小时,然后在Transwell小室内外分别添加HVE-1培养液,37℃,5%CO2培养箱培养3天,形成阴道粘膜模型中的基质层;
上述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加10%胎牛血清,4.0μmol/mL的谷氨酰胺,20μg/mL腺嘌呤,10μg/mL胰岛素,1.2μg/mL 氢化可的松,加入75μg/mL维生素C,5ng/mL成纤维细胞生长因子,15ng/mL转铁蛋白;
E、阴道上皮细胞的接种:
(1)吸去基质层表面的培养基,将上述得到的阴道上皮细胞或A431细胞系用HVE-2培养液配制成2.0×106个/mL细胞悬液,接种到Transwell小室中的基质层上;
(2)将培养基更换成HVE-2培养液进行浸没式培养,37℃、5%CO2培养箱中培养4天;
上述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加5%胎牛血清,4.5μmol/mL的谷氨酰胺,22μg/mL腺嘌呤,10μg/mL胰岛素,1.5μg/mL 氢化可的松,6ng/mL成纤维细胞生长因子,12ng/mL转铁蛋白,1.5ng/mLEGF,0.2nmol/mL三碘甲状腺原氨酸,8nmol/mL异丙肾上腺素;
F、阴道粘膜模型的培养:
(1)吸去Transwell小室中培养物表面的HVE-2培养液,将Transwell小室提升至空气-液体界面进行气液面培养;
(2)将HVE-2培养液更换成HVE-3培养液, 37℃、5%CO2培养箱中培养10天,得到基质层中含有成纤维细胞、上皮层中含有阴道上皮细胞的双层阴道粘膜模型;
上述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比2:1混合的培养基为基础液,在其中添加5%胎牛血清,3.0μmol/mL的谷氨酰胺,18μg/mL腺嘌呤,12μg/mL胰岛素,1.5μg/mL 氢化可的松,5.0ng/mL成纤维细胞生长因子,15ng/mL转铁蛋白,3.5ng/mLEGF,0.25nmol/mL三碘甲状腺原氨酸,8nmol/mL异丙肾上腺素,60μg/mL维生素C,1.2μmol/mL CaCl2。
实施例3
本实施例与实施例1的区别之处在于:
步骤A中所述无血清上皮细胞培养液以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加6.0μmol/mL的谷氨酰胺,5.0ng/mL EGF,30μg/mL BPE,15ng/mL胰岛素,2.0μg/mL 氢化可的松,0.4μmol/mL CaCl2。
实施例4
一种人阴道粘膜模型的体外构建方法,包括如下步骤:
A、外阴表皮样癌细胞系A431细胞培养:
重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、2.0μmol/mL谷氨酰胺、0.1μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,37℃,5%CO2培养箱传代培养至第七代后用于进行上皮层的构建;
B、阴道成纤维细胞的分离和培养:
(1)将分离的阴道组织固有层放入200U/mL胶原酶消化液中,37℃消化3h,使用含10%胎牛血清的DMEM培养液终止消化,200目筛网过滤,离心去上清,收集阴道成纤维细胞;
(2)PBS清洗阴道成纤维细胞,重悬细胞沉淀于含10%新生牛血清的DMEM培养液中,接种于T75培养瓶培养,37℃,5%CO2条件下传代培养至第五代后用于进行阴道粘膜模型中基质层的构建;
C、中性胶原支架的制备:
称量胶原置于0.1%醋酸溶液中配制成10mg/mL的胶原溶液,待完全溶解后置于冰上,将胶原溶液和含10%胎牛血清DEME培养液按体积比1:8混匀,加入0.1M的NaOH溶液调节PH至7.4;
D、含成纤维细胞的基质层构建:
(1)取第10代阴道成纤维细胞,用含20%胎牛血清的DMEM配成密度为8×106个/mL细胞悬液;
(2)将细胞悬液和中性胶原支架按照1:9的体积比例混匀,接种到Transwell小室中,置于37℃条件下培养3小时,然后在Transwell小室内外分别添加HVE-1培养液,37℃,5%CO2培养箱培养4天,形成阴道粘膜模型中的基质层;
上述HVE-1培养液按照以下方法配制而成:以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加10%胎牛血清,在其中添加6.0μmol/mL的谷氨酰胺,30μg/mL腺嘌呤,15μg/mL胰岛素,2.0μg/mL 氢化可的松,加入100μg/mL维生素C,10ng/mL成纤维细胞生长因子,20ng/mL转铁蛋白;
E、外阴表皮样癌细胞系A431细胞的接种:
(1)吸去基质层表面的培养基,将上述得到的A431细胞系用HVE-2培养液配制成5.0×106个/mL细胞悬液,接种到Transwell小室中的基质层上;
(2)将培养基更换成HVE-2培养液进行浸没式培养,37℃、5%CO2培养箱中培养4天;
上述HVE-2培养液按照以下方法配制而成:以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加5%胎牛血清,6.0μmol/mL的谷氨酰胺,30μg/mL腺嘌呤,15μg/mL胰岛素,2.0μg/mL 氢化可的松,2.0ng/mL成纤维细胞生长因子,20ng/mL转铁蛋白,2.0ng/mLEGF,0.4nmol/mL三碘甲状腺原氨酸,10nmol/mL异丙肾上腺素;
F、阴道粘膜模型的培养:
(1)吸去Transwell小室中培养物表面的HVE-2培养液,将Transwell小室提升至空气-液体界面进行气液面培养;
(2)将HVE-2培养液更换成HVE-3培养液, 37℃、5%CO2培养箱中培养8~12天,得到基质层中含有成纤维细胞、上皮层中含有外阴表皮样癌细胞系A431的双层阴道粘膜模型;
上述HVE-3培养液按照以下方法配制而成:以DMEM和F12按体积比3:1混合的培养基为基础液,在其中添加5%胎牛血清,6.0μmol/mL的谷氨酰胺,30μg/mL腺嘌呤,15μg/mL胰岛素,2.0μg/mL 氢化可的松,10ng/mL成纤维细胞生长因子,20ng/mL转铁蛋白,5.0ng/mLEGF,0.4nmol/mL三碘甲状腺原氨酸,10nmol/mL异丙肾上腺素,100μg/mL维生素C,1.5μmol/mL CaCl2。
实施例5
本实施例与实施例4的区别在于:
步骤A:外阴表皮样癌细胞系A431细胞培养:
重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、4.5μmol/mL谷氨酰胺、0.5μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,37℃,5%CO2培养箱传代培养至第七代后用于进行上皮层的构建。
实施例6
本实施例与实施例4的区别在于:
步骤A:外阴表皮样癌细胞系A431细胞培养:
重悬外阴表皮样癌细胞系A431细胞沉淀于含10%胎牛血清、6.0μmol/mL谷氨酰胺、1μmol/mL丙酮酸钠和4.5g/L高糖的DMEM培养液,37℃,5%CO2培养箱传代培养至第七代后用于进行上皮层的构建。
由图1A和图1B可知,使用正常人阴道上皮细胞培养构建的阴道粘膜模型具有与正常阴道组织相似的结构特征,其上皮层主要分为三层,最上层为分化成熟的角质层,中间为移行层,最下层为基底层,基质层为包含有成纤维细胞的胶原层,表明本发明所建立的3D阴道粘膜模型可以在体外正常分化,适用于人正常生殖道上皮的生理学研究、药物毒性安全性评价和体外阴道刺激性实验。
由图2A和图2B可知,使用外阴表皮样癌细胞系A431所构建的阴道粘膜模型其上皮层不能分化出成熟的角质层,但是该模型具有一定的屏障功能,且构建周期短,种子细胞来源广泛,性能比较稳定,能够满足体外阴道刺激性实验的基本要求。
以上所述,仅为本申请的具体实施方式,但本申请的保护范围并不局限于此,任何在本申请揭露的技术范围内的变化或替换,都应涵盖在本申请的保护范围之内。因此,本申请的保护范围应以所述权利要求的保护范围为准。
Claims (1)
1.用于构建人阴道粘膜模型培养基的配制方法,其特征在于,包括如下步骤:
以DMEM和F12按体积比1~3:1混合的培养基为基础液,在其中添加5%胎牛血清,2.0~6.0μmol/mL的谷氨酰胺,15~30μg/mL腺嘌呤,5~15μg/mL胰岛素,0.5~2.0μg/mL 氢化可的松,1~10ng/mL成纤维细胞生长因子,5~20ng/mL转铁蛋白,0.5~2.0ng/mL 表皮生长因子,0.1~0.4nmol/mL三碘甲状腺原氨酸和5~10nmol/mL异丙肾上腺素。
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