CN114891736A - 一种三维培养的间充质干细胞外泌体增强软骨细胞生长与特性维持的方法及其应用 - Google Patents

一种三维培养的间充质干细胞外泌体增强软骨细胞生长与特性维持的方法及其应用 Download PDF

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CN114891736A
CN114891736A CN202210764877.2A CN202210764877A CN114891736A CN 114891736 A CN114891736 A CN 114891736A CN 202210764877 A CN202210764877 A CN 202210764877A CN 114891736 A CN114891736 A CN 114891736A
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chondrocytes
mesenchymal stem
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刘广
吴琼
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China Origin Biotechnology Co ltd
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Abstract

本发明公开了一种三维培养的间充质干细胞外泌体增强软骨细胞生长与特性维持的方法及其应用，属于软骨损伤修复的组织工程领域。本发明利用三维培养的不同组织来源的间充质干细胞，分离纯化其培养后的外泌体，加入组织工程用软骨细胞的培养体系，模拟体内三维环境干细胞促进软骨细胞生长微环境，其软骨细胞生长速度快，且能够有效分泌软骨细胞相关因子，维持软骨细胞的特性，减少软骨细胞培养、传代造成的分化，可高速、稳定的获得更多数量的软骨细胞，该细胞具有强劲的透明软骨组织形成能力。所述制备方法可促进软骨细胞的高效、快速、稳定的增殖，不同组织来源的间充质干细胞经过三维培养后，分离纯化其外泌体，外泌体鉴定后按照一定浓度加入软骨细胞的原代培养过程中，模拟体内三维软骨细胞所处环境。软骨组织来源的软骨细胞在原代培养10‑21天后，进行鉴定，其能够稳定表达、分泌二型胶原蛋白。该方法在临床病人自体软骨细胞供应数量有限的情况下具有潜在的应用价值。

Description

一种三维培养的间充质干细胞外泌体增强软骨细胞生长与特 性维持的方法及其应用
技术领域
本发明属于软骨损伤修复的组织工程用软骨细胞快速高效制备领域，涉及到三维培养间充质干细胞的外泌体制备技术，特别涉及到模拟体内三维环境的软骨细胞培养技术领域。
背景技术
软骨组织没有血管、神经组织、淋巴管，自我修复能力很差。关节软骨一旦损伤不能自愈，成为医学界公认的难题，关节软骨损伤就如“破了洞的衣服，不及时修补，破损就会越磨越大”，而关节软骨损伤若不及时得到早期、有效的救治，最终结局将发展为骨关节炎不得不接受人工关节置换，专家普遍认为早期修复关节软骨损伤非常必要。关节软骨损伤是骨性关节炎最早的病理表现,骨关节炎发病率高，60岁以上达60%,70岁以上达70%,是严重影响人类生活质量的重大慢性疾病之一。
在软骨组织工程修复中，一般包括三大要素：种子细胞，生物材料及细胞因子，其中种子细胞则是其中最为核心的要素。理想的组织工程种子细胞须符合以下条件：1、含量丰富；2、易于获取；3、自体或异体移植具有高效性和安全性；4、操作符合现有医疗实践指南及伦理要求。目前软骨组织工程常用的种子细胞包括：软骨细胞和间充质干细胞，其中软骨细胞体外培养增殖效率不高，且易出现分化等问题，在临床应用特别是自体组织工程软骨细胞移植过程中受到一定的限制。间充质干细胞属于中胚层细胞，可在体外诱导分化成软骨细胞，但其诱导效率不确定，诱导时间长，诱导效果不稳定及干细胞本身的应用均极大的限制了其在临床软骨损伤治疗中的应用。
事实上，在软骨细胞修复中间充质干细胞特别是关节软骨周围的干细胞（如滑膜间充质干细胞、膑下脂肪垫来源的间充质干细胞）在维持软骨微环境中起重要作用。间充质干细胞通常通过调节免疫，旁分泌作用等促进软骨细胞的增殖和软骨细胞外基质的分泌。
在干细胞分泌的众多因子中，外泌体已被确定为在几种疾病适应症中介导内源性或移植细胞的治疗功效的因子。人们发现外泌体含有各种类型的生物活性如 microRNA、核酸、蛋白质和独特的基因产物。据报道外泌体可以转移、营养邻近细胞的因子并充当细胞间通讯。与传统的干细胞相比，外泌体的使用可以提供现货型和无细胞型的再生医学方法。因此，一种使用三维培养间充质干细胞外泌体促进软骨细胞高效、快速、稳定增殖的方法成为临床的迫切需要。
发明内容
发明人经过研究分析发现:在哺乳动物胚胎发育过程中，间充质干细胞，特别是处在人体三维环境中的间充质干细胞对软骨细胞的稳定、增殖、分化及其分泌细胞外基质具有重要意义。要实现体外软骨细胞的高效、快速、稳定的增殖而不分化，就既需要合理的软骨细胞的微环境，又不干扰其氧气、营养成分等物质交换，该种采用添加10-60μg/ml外泌体的培养体系能够在短时间内形成直径大约100-500μm的软骨样细胞团，该细胞团不仅形成了类似于体内的软骨细胞三维空间结构，而且能够分泌软骨相关的细胞外基质，并在短时期内高效、快速、稳定的获得大量软骨细胞。
针对现有技术存在的问题，本发明的目的是提供一种三维培养的间充质干细胞外泌体增强软骨细胞生长与特性维持的方法，该方法具有速度快——从软骨组织取出计算能够在14-20天之内形成2-5*107的软骨细胞，高效——均有形成透明软骨能力，可分泌软骨外细胞基质，稳定——未出现分化。
本发明所述的透明软骨样细胞团，其包含大量增殖且处于三维环境的软骨细胞，能够分泌天然软骨成分二型胶原蛋白和粘多糖，可用于进一步形成天然软骨相接近成分、结构及性质的组织工程软骨。
作为可选方式，在上述透明软骨样细胞团中，所述软骨细胞呈现出球状或者椭圆状形态，与体内三维环境中的软骨细胞接近。
作为可选方式，在上述透明软骨样细胞团中，所述透明软骨样细胞团直径大小为100-500μm。
作为可选方式，在上述透明软骨样细胞团中，在干细胞外泌体的作用下，所述软骨细胞通过聚集及相互作用形成类似于体内软骨细胞所处的三维环境。
本发明所提出的透明软骨样细胞团软骨细胞，可形成和天然软骨相接近的成分、结构以及性质，透明软骨样细胞团软骨细胞分泌大量的二型胶原和粘多糖，细胞团内的软骨细胞呈现出与天然软骨细胞相接近的球状或椭圆状形态，大体观察可以发现组织呈现半透明样，本发明透明软骨样细胞团能够分泌大量的二型胶原（ColⅡ）和粘多糖（GAG）。软骨样细胞团呈现三维结构排列。
本发明所述透明软骨样细胞团的制备方法为：三维培养第3-5代的不同组织来源的包括但不限于滑膜间充质干细胞、膑下脂肪垫来源的间充质干细胞、骨髓间充质干细胞、牙髓来源的间充质干细胞、皮下脂肪来源的间充质干细胞等，经过无血清培养48-72小时后分离纯化其外泌体，置于-80℃保存以备后续使用；选择性获得直径为20- 150微米的外泌体按照10-60μg/ml的浓度，模拟体内微环境制成无血清软骨细胞培养体系。原代获得的二维贴壁软骨细胞消化后重悬于加入外泌体的无血清软骨细胞培养体系中，并置于低粘附培养板中培养，软骨细胞能够稳定表达分泌二型胶原蛋白和粘多糖，具有透明软骨形成能力的软骨细胞。反转录酶-聚合酶链锁反应（RT-PCR）显示软骨细胞中的 ColⅡ,Sox9等透明软骨相关基因呈现高表达。
作为可选方式，在上述制备方法中，所述用于透明软骨样细胞团的细胞通过软骨组织原代分离、二维培养获得。
作为可选方式，在上述制备方法中，将所得透明软骨样细胞团可马上储存于液氮中以待后期的检测和应用。
作为可选方式，所述制备方法包括以下步骤：
1）将哺乳动物来源的关节透明软骨经混合胶原酶消化10-20小时后分离出的软骨细胞在体外二维环境使用含自体血清的培养体系培养至原代达到80-90%融合，用不含EDTA的胰酶消化；
2）使用400g离心10分钟，取上清，留软骨细胞沉淀，使用含10-60μg/ml的三维培养间充质干细胞外泌体浓度的无血清软骨细胞培养基重悬细胞，调整软骨细胞浓度为3-8×104cells/ml，然后将此细胞悬浮液接种于低粘附六孔板内；
3）每隔2-3天换一次液，直到培养细胞团直径约100-500μm，收集生长出来的透明软骨样细胞团。
作为可选方式，在上述制备方法中，整个制备过程所用的培养基配方为：DF12、DMEM基础培养基、50μg/ml抗坏血酸、50μg/ml脯氨酸、1%ITS、1%的青霉素和链霉素混合液和10%的自体血清。
本发明还提供了一种所述透明软骨样细胞团的应用，其特征在于，将其用于多种原因造成的软骨损伤进行组织工程软骨移植。
本说明书中公开的所有特征，或公开的所有方法或过程中的步骤，除了互相排斥的特征和/或步骤以外，均可以以任何方式组合。
本发明的有益效果：
本发明所制备的透明软骨样细胞团其包含大量增殖且处于三维环境的软骨细胞，能够分泌天然软骨成分二型胶原蛋白和粘多糖，可用于进一步形成天然软骨相接近成分、结构及性质的组织工程软骨。本发明结合了三维细胞培养、有效获得三维培养外泌体模拟体内软骨细胞环境，且不额外使用生物材料，所需要的初始软骨组织少，软骨细胞增殖高效、快速、稳定，在临床病人自体软骨细胞供应数量有限的情况下具有潜在的临床应用价值。

Claims (8)

1.一种三维培养的间充质干细胞外泌体增强软骨细胞生长与特性维持的方法及其应用，其特征在于，所述方法是利用三维培养间充质干细胞外泌体，可高效、快速、稳定的获得可形成透明软骨的软骨细胞体外制备方法，具体为：
三维培养第3-5代的不同组织来源的包括但不限于滑膜间充质干细胞、膑下脂肪垫来源的间充质干细胞、骨髓间充质干细胞、牙髓来源的间充质干细胞、皮下脂肪来源的间充质干细胞等，经过无血清培养48-72小时后分离纯化其外泌体，置于-80℃保存以备后续使用；
选择性获得直径为20- 150微米的外泌体按照10-60μg/ml的浓度，模拟体内微环境制成无血清软骨细胞培养体系。
2.原代获得的二维贴壁软骨细胞消化后重悬于加入外泌体的无血清软骨细胞培养体系中，并置于低粘附培养板中培养，软骨细胞将形成能够稳定表达分泌二型胶原蛋白和粘多糖，具有透明软骨形成能力的软骨细胞。
3.根据权利要求1所述制备方法，其特征在于，具体步骤包括：1）将哺乳动物来源的关节透明软骨经混合胶原酶消化10-20小时后分离出的软骨细胞在体外二维环境使用含自体血清的培养体系培养至原代达到80-90%融合，用不含EDTA的胰酶消化；2）使用400g离心10分钟，取上清，留软骨细胞沉淀，使用含10-60μg/ml的三维培养间充质干细胞外泌体浓度的无血清软骨细胞培养基重悬细胞，调整软骨细胞浓度为3-8×104cells/ml，然后将此细胞悬浮液接种于低粘附六孔板内； 3）每隔2-3天换一次液，直到培养细胞团直径约100-500μm，收集生长出来的透明软骨样细胞团。
4.一种采用权利要求1所述方法获得的透明软骨样细胞团，其特征在于，包括大量增殖处于三维环境的软骨细胞，能够分泌丰富的天然软骨成分二型胶原蛋白和粘多糖。
5.根据权利要求3所述的透明软骨样细胞团，其特征在于，所述软骨细胞呈现出球状或者椭圆状形态。
6.根据权利要求3所述的透明软骨样细胞团，其特征在于，所述透明软骨样细胞球直径约为100-500μm。
7.根据权利要求 3 所述的透明软骨样细胞团，其特征在于，在干细胞外泌体的作用下，所述软骨细胞通过聚集及相互作用形成类似于体内软骨细胞所处的三维环境。
8.一种根据权利要求 3 所述的透明软骨细胞团的应用，其特征在于，将其用于多种原因造成的软骨损伤进行组织工程软骨移植。
CN202210764877.2A 2022-07-01 2022-07-01 一种三维培养的间充质干细胞外泌体增强软骨细胞生长与特性维持的方法及其应用 Pending CN114891736A (zh)

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