CN111821317A - 一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法及应用 - Google Patents
一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法及应用 Download PDFInfo
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Abstract
本发明公开了一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法及应用，包括胎盘蜕膜干细胞的分离培养，胎盘蜕膜间充质干细胞上清液收集、间充质干细胞因子的纯化、间充质干细胞因子冻干粉制备。本发明采用无血清培养体系培养胎盘蜕膜间充质干细胞。避免了FBS中的成分对皮肤的刺激。此外，在低氧的条件下培养间充质干细胞，刺激细胞分泌大量的细胞因子。通过低温冻干技术，使产品可在常温下保存，方便运输，且有延长分泌因子活性保存期限。得到的胎盘蜕膜间充质干细胞分泌因子冻干粉，能通过焕活衰老细胞来达到肌肤抗皱、美白、抗氧化、淡化痘印等多重功效。
Description
技术领域
本发明涉及生物技术领域，具体为一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法及应用。
背景技术
间充质干细胞(MSC,mesenchymal stem cells)是干细胞家族的重要成员，来源于发育早期的中胚层，属于多能干细胞，MSC因其具有多向分化潜能、促进造血干细胞归巢、修复损伤或病变的组织器官、免疫调控和自我复制等功能。MSC最初发现在骨髓中，胎盘蜕膜间充质干细胞与骨髓间充质干细胞相比，具有来源充足、免疫原性低、病毒污染率低，且无社会伦理争议等方面的有点，使其具有更好的应用前景。
随着对间充质干细胞研究的不断深入，其作用机理越来越清晰明确，主要有二方面：一、间充质干细胞分泌细胞因子。二、间充质干细胞替代或修复死亡或受损的组织细胞。越来越多的研究表明，间充质干细胞在培养过程中分泌上百种细胞活性因子，如表皮生长因子(EGF)、血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、角质细胞生长因子(KGF)、胰岛素样生长因子(IGF)、肝细胞生长因子(HGF)、转化生长因子β1(TGF-β1)、血小板衍生因子(PDGF-BB)白介素6(IL-6)等。这些细胞因子通过直接或间接地参与细胞信号转导、免疫调节过程，能够促进血管形成、促进细胞新陈代谢、加速伤口愈合，修复细胞损伤、促进胶原蛋白合成、缓解细胞衰老等，因此市场上急需一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法及应用来解决这些问题。
发明内容
本发明的目的在于提供一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法及应用，以解决上述背景技术中提出的目前市面上的油茶籽油营养价值不够高的问题。
为实现上述目的，本发明提供如下技术方案：一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法，包括以下步骤：
步骤1：取足月健康的新生儿胎盘，置于含无菌保存液的储运盒中，待分离制备；
步骤2：将羊膜去除，将胎膜全部剪下，放置培养皿中，用0.9％氯化钠注射液清洗2-3次，直至无明显血液，从胎膜上分离蜕膜组织，分离下来的蜕膜组织收集于离心管中，加入适量0.9％氯化钠注射液，离心洗涤1-2遍。弃上清，剪碎成1-4mm3大小的组织块；
步骤3：加入胶原酶消化1.5h；
步骤4：消化使用输血器滤网将消化后的混合液马上过滤，组织块需要洗涤1-2次；
步骤5：细胞滤液离心后，用移液管轻轻吸取去掉上清；
步骤6：加入适量完全培养基吹打后转入培养瓶，培养瓶水平放入37℃，5％CO2培养箱中培养；
步骤7：原代培养24-48h后，进行第一次全量换液。此后每隔3天全量换液，放置培养箱进行培养。
步骤8：将收集的浓缩原液用0.22um滤器过滤除菌，加入终浓度5％甘露醇，2％海藻糖，2％右旋糖苷，调节蛋白溶度至1mg/ml，混匀；
步骤9：按照2.5ml/支分装到无菌、无热源7ml西林瓶中，在洁净环境下，冻干30小时，冻干结束后，真空压盖，去除西林瓶，制备得到胎盘蜕膜间充质干细胞分泌因子冻干粉。
优选的，所述储运液为150ml的DMEM；II型胶原酶为2mg/ml；无血清培养液为5％血清替代物和无酚红无血清培养基。
优选的，所述胎盘蜕膜间充质干细胞选用P4、P5代的细胞，将检测合格的细胞按6-7*104/ml(以活细胞计)的浓度转至细胞工厂，做好标记(日期、代次)，置37℃，5％CO2，5％O2陪养箱中进行低氧培养。经过72h培养，细胞融合度达到85％-95％时，收集细胞上清液。
优选的，所述胎盘蜕膜间充质干细胞分泌因子的分离纯化；将收集的细胞上清液放入离心管中，4℃离心，3000g离心10分钟，取上清液，出去沉淀物，将去沉淀物后的上清液转移至5KD超滤浓缩离心管中，将上清液体积浓缩至一半体积。
一种人胎盘蜕膜间干细胞分泌因子冻干粉的应用，所述该冻干粉能够促进血管形成、促进细胞新陈代谢、加速伤口愈合，修复细胞损伤、促进胶原蛋白合成、缓解细胞衰老。
与现有技术相比，本发明的有益效果是：本发明本发明采用无血清培养体系培养胎盘蜕膜间充质干细胞。避免了FBS中的成分对皮肤的刺激。此外，在低氧的条件下培养间充质干细胞，刺激细胞分泌大量的细胞因子。通过低温冻干技术，使产品可在常温下保存，方便运输，且有延长分泌因子活性保存期限。得到的胎盘蜕膜间充质干细胞分泌因子冻干粉，能通过焕活衰老细胞来达到肌肤抗皱、美白、抗氧化、淡化痘印等多重功效。
具体实施方式
下面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。
实施例1：
一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法，包括以下步骤：
步骤1：取足月健康的新生儿胎盘，置于含无菌保存液的储运盒中，待分离制备；
步骤2：将羊膜去除，将胎膜全部剪下，放置培养皿中，用0.9％氯化钠注射液清洗2次，直至无明显血液，从胎膜上分离蜕膜组织，分离下来的蜕膜组织收集于离心管中，加入适量0.9％氯化钠注射液，离心洗涤1-2遍。弃上清，剪碎成1mm3大小的组织块；
步骤3：加入胶原酶消化1.5h；
步骤4：消化使用输血器滤网将消化后的混合液马上过滤，组织块需要洗涤1次；
步骤5：细胞滤液离心后，用移液管轻轻吸取去掉上清；
步骤6：加入适量完全培养基吹打后转入培养瓶，培养瓶水平放入37℃，5％CO2培养箱中培养；
步骤7：原代培养24h后，进行第一次全量换液。此后每隔3天全量换液，放置培养箱进行培养。
步骤8：将收集的浓缩原液用0.22um滤器过滤除菌，加入终浓度5％甘露醇，2％海藻糖，2％右旋糖苷，调节蛋白溶度至1mg/ml，混匀；
步骤9：按照2.5ml/支分装到无菌、无热源7ml西林瓶中，在洁净环境下，冻干30小时，冻干结束后，真空压盖，去除西林瓶，制备得到胎盘蜕膜间充质干细胞分泌因子冻干粉。
进一步，储运液为150ml的DMEM；II型胶原酶为2mg/ml；无血清培养液为5％血清替代物和无酚红无血清培养基。
进一步，胎盘蜕膜间充质干细胞选用P4、P5代的细胞，将检测合格的细胞按6-7*104/ml(以活细胞计)的浓度转至细胞工厂，做好标记(日期、代次)，置37℃，5％CO2，5％O2陪养箱中进行低氧培养。经过72h培养，细胞融合度达到85％-95％时，收集细胞上清液。
进一步，胎盘蜕膜间充质干细胞分泌因子的分离纯化；将收集的细胞上清液放入离心管中，4℃离心，3000g离心10分钟，取上清液，出去沉淀物，将去沉淀物后的上清液转移至5KD超滤浓缩离心管中，将上清液体积浓缩至一半体积。
一种人胎盘蜕膜间干细胞分泌因子冻干粉的应用，该冻干粉能够促进血管形成、促进细胞新陈代谢、加速伤口愈合，修复细胞损伤、促进胶原蛋白合成、缓解细胞衰老。
实施例2：
一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法，包括以下步骤：
步骤1：取足月健康的新生儿胎盘，置于含无菌保存液的储运盒中，待分离制备；
步骤2：将羊膜去除，将胎膜全部剪下，放置培养皿中，用0.9％氯化钠注射液清洗2-3次，直至无明显血液，从胎膜上分离蜕膜组织，分离下来的蜕膜组织收集于离心管中，加入适量0.9％氯化钠注射液，离心洗涤2遍。弃上清，剪碎成4mm3大小的组织块；
步骤3：加入胶原酶消化1.5h；
步骤4：消化使用输血器滤网将消化后的混合液马上过滤，组织块需要洗涤2次；
步骤5：细胞滤液离心后，用移液管轻轻吸取去掉上清；
步骤6：加入适量完全培养基吹打后转入培养瓶，培养瓶水平放入37℃，5％CO2培养箱中培养；
步骤7：原代培养48h后，进行第一次全量换液。此后每隔3天全量换液，放置培养箱进行培养。
步骤8：将收集的浓缩原液用0.22um滤器过滤除菌，加入终浓度5％甘露醇，2％海藻糖，2％右旋糖苷，调节蛋白溶度至1mg/ml，混匀；
步骤9：按照2.5ml/支分装到无菌、无热源7ml西林瓶中，在洁净环境下，冻干30小时，冻干结束后，真空压盖，去除西林瓶，制备得到胎盘蜕膜间充质干细胞分泌因子冻干粉。
进一步，储运液为150ml的DMEM；II型胶原酶为2mg/ml；无血清培养液为5％血清替代物和无酚红无血清培养基。
进一步，胎盘蜕膜间充质干细胞选用P4、P5代的细胞，将检测合格的细胞按6-7*104/ml(以活细胞计)的浓度转至细胞工厂，做好标记(日期、代次)，置37℃，5％CO2，5％O2陪养箱中进行低氧培养。经过72h培养，细胞融合度达到85％-95％时，收集细胞上清液。
进一步，胎盘蜕膜间充质干细胞分泌因子的分离纯化；将收集的细胞上清液放入离心管中，4℃离心，3000g离心10分钟，取上清液，出去沉淀物，将去沉淀物后的上清液转移至5KD超滤浓缩离心管中，将上清液体积浓缩至一半体积。
一种人胎盘蜕膜间干细胞分泌因子冻干粉的应用，该冻干粉能够促进血管形成、促进细胞新陈代谢、加速伤口愈合，修复细胞损伤、促进胶原蛋白合成、缓解细胞衰老。
对于本领域技术人员而言，显然本发明不限于上述示范性实施例的细节，而且在不背离本发明的精神或基本特征的情况下，能够以其他的具体形式实现本发明。因此，无论从哪一点来看，均应将实施例看作是示范性的，而且是非限制性的，本发明的范围由所附权利要求而不是上述说明限定，因此旨在将落在权利要求的等同要件的含义和范围内的所有变化囊括在本发明内。
Claims (5)
1.一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法，其特征在于，包括以下步骤：
步骤1：取足月健康的新生儿胎盘，置于含无菌保存液的储运盒中，待分离制备；
步骤2：将羊膜去除，将胎膜全部剪下，放置培养皿中，用0.9％氯化钠注射液清洗2-3次，直至无明显血液，从胎膜上分离蜕膜组织，分离下来的蜕膜组织收集于离心管中，加入适量0.9％氯化钠注射液，离心洗涤1-2遍。弃上清，剪碎成1-4mm3大小的组织块；
步骤3：加入胶原酶消化1.5h；
步骤4：消化使用输血器滤网将消化后的混合液马上过滤，组织块需要洗涤1-2次；
步骤5：细胞滤液离心后，用移液管轻轻吸取去掉上清；
步骤6：加入适量完全培养基吹打后转入培养瓶，培养瓶水平放入37℃，5％CO2培养箱中培养；
步骤7：原代培养24-48h后，进行第一次全量换液。此后每隔3天全量换液，放置培养箱进行培养。
步骤8：将收集的浓缩原液用0.22um滤器过滤除菌，加入终浓度5％甘露醇，2％海藻糖，2％右旋糖苷，调节蛋白溶度至1mg/ml，混匀；
步骤9：按照2.5ml/支分装到无菌、无热源7ml西林瓶中，在洁净环境下，冻干30小时，冻干结束后，真空压盖，去除西林瓶，制备得到胎盘蜕膜间充质干细胞分泌因子冻干粉。
2.根据权利要求1所述的一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法，其特征在于，所述储运液为150ml的DMEM；II型胶原酶为2mg/ml；无血清培养液为5％血清替代物和无酚红无血清培养基。
3.根据权利要求1所述的一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法，其特征在于，所述胎盘蜕膜间充质干细胞选用P4、P5代的细胞，将检测合格的细胞按6-7*104/ml(以活细胞计)的浓度转至细胞工厂，做好标记(日期、代次)，置37℃，5％CO2，5％O2陪养箱中进行低氧培养。经过72h培养，细胞融合度达到85％-95％时，收集细胞上清液。
4.根据权利要求1所述的一种人胎盘蜕膜间干细胞分泌因子冻干粉制备方法，其特征在于，所述胎盘蜕膜间充质干细胞分泌因子的分离纯化；将收集的细胞上清液放入离心管中，4℃离心，3000g离心10分钟，取上清液，出去沉淀物，将去沉淀物后的上清液转移至5KD超滤浓缩离心管中，将上清液体积浓缩至一半体积。
5.根据权利要求1所述的一种人胎盘蜕膜间干细胞分泌因子冻干粉的应用，其特征在于，所述该冻干粉能够促进血管形成、促进细胞新陈代谢、加速伤口愈合，修复细胞损伤、促进胶原蛋白合成、缓解细胞衰老。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112442480A (zh) * | 2020-11-17 | 2021-03-05 | 焕生汇生物基因技术(北京)有限公司 | 一种来源于人底蜕膜间充质干细胞外泌体的制备方法及应用 |
| CN114470002A (zh) * | 2022-03-18 | 2022-05-13 | 北京岳淘生物科技有限公司 | 胎盘干细胞冻干粉在制备药物及化妆品中的应用 |
| WO2023053065A1 (en) * | 2021-09-29 | 2023-04-06 | Osiris Therapeutics, Inc. | Compositions of decidua tissue and uses thereof |
| WO2023092479A1 (zh) * | 2021-11-26 | 2023-06-01 | 金湧长生医学生物科技股份有限公司 | 人类蜕膜间充质干细胞的细胞培养上清液的用途 |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112442480A (zh) * | 2020-11-17 | 2021-03-05 | 焕生汇生物基因技术(北京)有限公司 | 一种来源于人底蜕膜间充质干细胞外泌体的制备方法及应用 |
| WO2023053065A1 (en) * | 2021-09-29 | 2023-04-06 | Osiris Therapeutics, Inc. | Compositions of decidua tissue and uses thereof |
| WO2023092479A1 (zh) * | 2021-11-26 | 2023-06-01 | 金湧长生医学生物科技股份有限公司 | 人类蜕膜间充质干细胞的细胞培养上清液的用途 |
| CN114470002A (zh) * | 2022-03-18 | 2022-05-13 | 北京岳淘生物科技有限公司 | 胎盘干细胞冻干粉在制备药物及化妆品中的应用 |
| CN114470002B (zh) * | 2022-03-18 | 2023-10-03 | 河北意和医学检验实验室有限公司 | 胎盘间充质干细胞冻干粉在制备药物中的应用 |
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