CN114525246A - 一种用于化妆品脐带间充质干细胞提取物的制备方法 - Google Patents
一种用于化妆品脐带间充质干细胞提取物的制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于化妆品脐带间充质干细胞提取物的制备方法，包括脐带间充质干细胞分离制备和鉴定、脐带间充质干细胞培养上清液获取、脐带间充质干细胞提取物的分离纯化、脐带间充质干细胞蛋白浓度检测这些步骤。该用于化妆品脐带间充质干细胞提取物的制备方法，采用ELISA法测定蛋白浓度准确，保证批次产品均一性，同时使用先进的细胞工厂培养容器作为载体，模拟体内环境，刺激细胞大量分泌细胞因子，提高脐带间充质干细胞提取物的质量，并且所得细胞提取物采用了无血清、无酚红培养体系培养人脐带间充质干细胞，避免了酚红对皮肤的刺激，特别是过敏皮肤，安全性更有保证，适合各种人群使用。
Description
技术领域
本发明涉及生物化妆品技术领域，具体为一种用于化妆品脐带间充质干细胞提取物的制备方法。
背景技术
间充质干细胞是来源于发育期早期中胚层和外胚成的一类多能干细胞。具有免疫调控、能够修复损伤或病变的组织器官、具备多向分化潜能等功能，因此具有广泛的应用前景。
干细胞抗衰老基于其两种基本功能，一是干细胞的增殖分化能力，可以分化成不同的体细胞以代替衰老死亡的机体细胞，二是干细胞的旁分泌功能，可以产生许多生物活性分子，间充质干细胞一个强大的功能就是分泌众多的营养性细胞因子。越来越多的研究表明，间充质干细胞旁分泌作用在其发挥治疗效果中起主要作用。脐带间充质干细胞在培养过程中可分泌多种细胞因子，如白介素(IL-6)：免疫调节；肿瘤坏死因子-α(TNF-α)：减退黑色素；血管内皮生长因子(VEGF):诱导血管生成；转化生长因子-β(TGF-β)：促进细胞增殖，分化，修复；血小板源性生长因子(PDGF):诱导胶原蛋白生成；肝细胞生长因子(THGF)：修复疤痕；碱性成纤维细胞生长因子(b-FGF)：促进细胞增殖与血管生成；角质细胞生长因子(KGF)：刺激毛囊再生；胰岛素生长因子(IGF)：促进细胞分化与增殖；纤连蛋白：激活细胞；I型胶原蛋白：保湿、美白、防皱、祛斑等。间充质干细胞所分泌的因子具有改善细胞生长微环境、调节机体免疫、促进血管再生、促进细胞增殖、抑制细胞凋亡等功能，融抗皱、美白、抗氧化、淡化痘印、促进毛发再生等多重功效于一体，通过焕活衰老的细胞来达到肌肤、毛发再生的功效。
目前市面上添加到化妆品中的成分多为干细胞培养上清液，该上清液分泌因子含量低、均一性差、稳定性差，美容效果一般。本申请提供了一种新的间充质干细胞提取物的制备方法，具体表现为一种用于化妆品脐带间充质干细胞提取物的制备方法。
发明内容
(一)解决的技术问题
针对现有技术的不足，本发明提供了一种用于化妆品脐带间充质干细胞提取物的制备方法，采用ELISA法测定蛋白浓度准确，保证批次产品均一性。使用先进的细胞工厂培养容器作为载体，模拟体内环境，刺激细胞大量分泌细胞因子，提高脐带间充质干细胞提取物的质量。
(二)技术方案
为实现以上目的，本发明通过以下技术方案予以实现：一种用于化妆品脐带间充质干细胞提取物的制备方法，包括脐带间充质干细胞分离制备和鉴定、脐带间充质干细胞培养上清液获取、脐带间充质干细胞提取物的分离纯化、脐带间充质干细胞蛋白浓度检测这些步骤。
优选的，所述的脐带间充质干细胞分离制备和鉴定方法为：
(1)取健康足月的新生脐带15-30cm，置于含保存液的无菌储存容器中，待分离制备；
(2)将脐带置于培养皿中，用生理盐水充分洗涤脐带，将脐带两端结扎部分去除；再反复洗涤脐带组织，直至血液全部洗净；将脐带剪成3-5cm长度小段；将脐带剖开，去除血管，撕取华通氏胶；用生理盐水充分洗涤华通氏胶，后将其剪碎成约1mm3大小组织块；将组织块接种于T75的培养瓶中，每T75培养瓶中添加10ml无血清培养基，放置于37℃，5％的CO2培养箱中培养，培养第5d后，去除旧培养基，添加新的无血清培养基；8d在显微镜下观察，可见组织块周围有成纤维状细胞爬出；培养第10d后，去除旧培养基，添加新的无血清培养基，第13天细胞数量较多，去除组织块，
用胰蛋白酶消化将贴壁的细胞消化转移至新的T175培养瓶中传代培养。
优选的，所述保存液为100ml的DMEM，所述无血清培养基为10％的血清替代物，其余为无酚红干细胞培养基。
优选的，所述脐带间充质干细胞培养上清液获取的方法为：
(1)将检测合格的细胞按5×104/ml的溶度转至T175培养瓶，做好标记，置37℃，5％的CO2培养箱中进行扩增培养；
(2)当扩增培养的细胞融合度达到60％时，调整培养箱参数37℃，5％的CO2，5％的O2继续培养，24小时后以上收集细胞上清液；
(3)将上述细胞收集，用每10ml盐水重悬5000万细胞数，将细胞悬液放置零下80℃的超低温冰箱，反复冻融2-3次，离心去掉死细胞，将1：49添加到细胞上清液中。
优选的，所述间充质干细胞因子的分离纯化的方法为：
(1)收集的间充质干细胞培养上清液放入离心管中，4℃，2000g离心10分钟，取上清，去除细胞沉渣；
(2)将去除沉渣的上清液转至100KD超滤浓缩离心管中，4℃，4000g离心10min，去除大于100KD分子量的物质，调节总蛋白浓度至1mg/ml。
优选的，所述脐带间充质干细胞蛋白浓度检测的方法为用ELISA法测定代表因子KGF、VEGF、EGF、bFGF、TGF-β、PDGF以及总蛋白浓度和毒理学实检测。
(三)有益效果
本发明提供了一种用于化妆品脐带间充质干细胞提取物的制备方法，具备以下有益效果：
(1)、该用于化妆品脐带间充质干细胞提取物的制备方法，采用了无血清、无酚红培养体系培养人脐带间充质干细胞，避免了酚红对皮肤的刺激，特别是过敏皮肤，安全性更有保证，适合各种人群使用。
(2)、该用于化妆品脐带间充质干细胞提取物的制备方法，采用贴壁细胞专业细胞培养瓶，能让细胞增殖速度稳定，提高收集上清液的效率。
(3)、该用于化妆品脐带间充质干细胞提取物的制备方法，通过低氧环境中培养，模拟干细胞体内环境，刺激大量的细胞因子分泌。
(4)、该用于化妆品脐带间充质干细胞提取物的制备方法，通过细胞的反复冻融，使得细胞破碎，同样可以分泌出大量的细胞因子。
(5)、该用于化妆品脐带间充质干细胞提取物的制备方法，采用ELISA方法测定蛋白浓度，保证批次产品的均一性，有利于产品后续的应用。
附图说明
图1为本发明中ELISA检测细胞因子浓度示意图；
图2为本发明中人脐带间充质干细胞第3代次细胞的示意图；
图3为本发明中人脐带间充质干细胞表明标志物CD14检测结果图；
图4为本发明中人脐带间充质干细胞表明标志物CD34检测结果图；
图5为本发明中人脐带间充质干细胞表明标志物CD45检测结果图；
图6为本发明中人脐带间充质干细胞表明标志物CD79a检测结果图；
图7为本发明中人脐带间充质干细胞表明标志物HLA-DR检测结果图；
图8为本发明中人脐带间充质干细胞表明标志物CD73检测结果图；
图9为本发明中人脐带间充质干细胞表明标志物CD90检测结果图；
图10为本发明中人脐带间充质干细胞表明标志物CD105检测结果图；
图11为本发明中提取物的生产流程图；
图12为本发明中提取物皮肤抗皱实验示意图；
图13为本发明中提取物皮肤美白实验示意图；
图14为本发明中提取物抗氧化实验中超氧化物歧化酶活性实验示意图；
图15为本发明中提取物抗氧化实验中谷胱甘肽过氧化酶活性实验示意图；
图16为本发明中提取物皮肤保湿实验示意图。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。请参阅图1-16，本发明提供一种技术方案：一种用于化妆品脐带间充质干细胞提取物的制备方法，包括脐带间充质干细胞分离制备和鉴定、脐带间充质干细胞培养上清液获取、脐带间充质干细胞提取物的分离纯化、脐带间充质干细胞蛋白浓度检测这些步骤。
1.脐带间充质干细胞分离制备和鉴定：
(1)取健康足月的新生人脐带15-30cm，置于含保存液的无菌储存容器中，待分离制备；
(2)将脐带置于培养皿中，用生理盐水充分洗涤脐带，将脐带两端结扎部分去除；再反复洗涤脐带组织，直至血液全部洗净；将脐带剪成3-5cm长度小段；将脐带剖开，去除血管，撕取华通氏胶；用生理盐水充分洗涤华通氏胶，后将其剪碎成约1mm3大小组织块；将组织块接种于T75的培养瓶中，每T75培养瓶中添加10ml无血清培养基，放置于37℃，5％的CO2培养箱中培养，培养第5d后，去除旧培养基，添加新的无血清培养基；8d在显微镜下观察，可见组织块周围有成纤维状细胞爬出；培养第10d后，去除旧培养基，添加新的无血清培养基。第13天细胞数量较多，去除组织块，用胰蛋白酶消化将贴壁的细胞消化转移至新的T175培养瓶中传代培养。其中保存液为100ml的DMEM；无血清培养基为10％的血清替代物，其余为无酚红干细胞培养基。
2.脐带间充质干细胞上清液获取：
将检测合格的细胞(一般采用P3-P4代)按5×104/ml的溶度转至T175培养瓶，做好标记，置37℃，5％的CO2培养箱中进行扩增培养；当扩增培养的细胞融合度达到60％时，调整培养箱参数37℃，5％的CO2，5％的O2继续培养，24小时后以上收集细胞上清液。并将细胞收集，用每10ml盐水重悬5000万细胞数。将细胞悬液放置零下80℃的超低温冰箱，反复冻融2-3次，离心去掉死细胞，将得到的液体1：49添加到细胞上清液中。
3.间充质干细胞提取物的分离纯化：
将收集的间充质干细胞培养上清液放入离心管中，4℃，2000g离心10分钟，取上清，去除细胞沉渣；将去除沉渣的上清液转至100KD超滤浓缩离心管中，4℃，4000g离心10min，去除大于100KD分子量的物质，调节总蛋白浓度至1mg/ml。
4.混匀取样：
用ELISA法测定代表因子KGF、VEGF、EGF、bFGF、TGF-β、PDGF以及总蛋白浓度，并将收集好的提取物放入4℃冰箱储存。同时取样进行毒理实验。毒理实验：
皮肤刺激：无皮肤刺激性。
兔：原液试验，单次体内皮肤刺激试验(4小时)(OECD 404)无皮肤刺激性。
人：原液试验，单次刺激贴斑试验(24小时)极轻微的刺激。
兔：原液试验，多次体内皮肤刺激试验(14天)极轻微的刺激。
眼睛刺激：无眼睛刺激。
兔：原液试验，(OECD 405)。
皮肤致敏性：无皮肤致敏性。(OECD 406)。
急毒性：LD50>2000mg/Kg(小鼠，经口，UDP法限量试验)
致突变性：无致突变性。(CHO细胞，染色体畸变试验)
重金属：铅、砷、汞、镉检测合格。
激素：性激素-雌三醇、雌酮、乙烯雌酚、雌二醇、睾丸酮、甲基睾丸酮、黄体酮检测合格。
糖皮质：糖皮质技术41项检测合格。
5.化妆品中添加：
5.1适用剂型：
洁面、爽肤水、修复原液、精华液、喷雾、乳液、面霜、眼霜、面膜、眼膜、冻干粉等
5.2配方指导：
1.本液为水溶性液体。若与水溶性基质配伍，则直接加入后，混匀即可。若与脂溶性基质配伍，则需加入相应的表面活性剂进行配伍优化。
2.本液中所含活性物有不耐受高温特性。作为配料使用过程中，避免其使用温度超过40℃。若配料过程中有步骤的温度超过该温度时，请先进行高温度反应步骤，待反应完成后，温度降至40℃以下后，加入本品进行混匀。
3.本液为无菌液。保存时需要无菌保存。若打开后应用，不能一次性用完的，请在无菌条件下使用。
需要说明的是，在本文中，诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来，而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者设备所固有的要素。在没有更多限制的情况下。由语句“包括一个......限定的要素，并不排除在包括所述要素的过程、方法、物品或者设备中还存在另外的相同要素”。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。
Claims (6)
1.一种用于化妆品脐带间充质干细胞提取物的制备方法，其特征在于：包括脐带间充质干细胞分离制备和鉴定、脐带间充质干细胞培养上清液获取、脐带间充质干细胞提取物的分离纯化、脐带间充质干细胞蛋白浓度检测这些步骤。
2.根据权利要求1所述的一种用于化妆品脐带间充质干细胞提取物的制备方法，其特征在于：所述的脐带间充质干细胞分离制备和鉴定方法为：
(1)取健康足月的新生脐带15-30cm，置于含保存液的无菌储存容器中，待分离制备；
(2)将脐带置于培养皿中，用生理盐水充分洗涤脐带，将脐带两端结扎部分去除；再反复洗涤脐带组织，直至血液全部洗净；将脐带剪成3-5cm长度小段；将脐带剖开，去除血管，撕取华通氏胶；用生理盐水充分洗涤华通氏胶，后将其剪碎成约1mm3大小组织块；将组织块接种于T75的培养瓶中，每T75培养瓶中添加10ml无血清培养基，放置于37℃，5％的CO2培养箱中培养，培养第5d后，去除旧培养基，添加新的无血清培养基；8d在显微镜下观察，可见组织块周围有成纤维状细胞爬出；培养第10d后，去除旧培养基，添加新的无血清培养基，第13天细胞数量较多，去除组织块，用胰蛋白酶消化将贴壁的细胞消化转移至新的T175培养瓶中传代培养。
3.根据权利要求2所述的一种用于化妆品脐带间充质干细胞提取物的制备方法，其特征在于：所述保存液为100ml的DMEM，所述无血清培养基为10％的血清替代物，其余为无酚红干细胞培养基。
4.根据权利要求2所述的一种用于化妆品脐带间充质干细胞提取物的制备方法，其特征在于：所述脐带间充质干细胞培养上清液获取的方法为：
(1)将检测合格的细胞按5×104/ml的溶度转至T175培养瓶，做好标记，置37℃，5％的CO2培养箱中进行扩增培养；
(2)当扩增培养的细胞融合度达到60％时，调整培养箱参数37℃，5％的CO2，5％的O2继续培养，24小时后以上收集细胞上清液；
(3)将上述细胞收集，用每10ml盐水重悬5000万细胞数，将细胞悬液放置零下80℃的超低温冰箱，反复冻融2-3次，离心去掉死细胞，将1：49添加到细胞上清液中。
5.根据权利要求2所述的一种用于化妆品脐带间充质干细胞提取物的制备方法，其特征在于：所述间充质干细胞因子的分离纯化的方法为：
(1)收集的间充质干细胞培养上清液放入离心管中，4℃，2000g离心10分钟，取上清，去除细胞沉渣；
(2)将去除沉渣的上清液转至100KD超滤浓缩离心管中，4℃，4000g离心10min，去除大于100KD分子量的物质，调节总蛋白浓度至1mg/ml。
6.根据权利要求5所述的一种用于化妆品脐带间充质干细胞提取物的制备方法，其特征在于：所述脐带间充质干细胞蛋白浓度检测的方法为用ELISA法测定代表因子KGF、VEGF、EGF、bFGF、TGF-β、PDGF以及总蛋白浓度和毒理学实检测。
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