CN114958734B - 一种人间充质干细胞培养基 - Google Patents

一种人间充质干细胞培养基 Download PDF

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CN114958734B
CN114958734B CN202111233082.0A CN202111233082A CN114958734B CN 114958734 B CN114958734 B CN 114958734B CN 202111233082 A CN202111233082 A CN 202111233082A CN 114958734 B CN114958734 B CN 114958734B
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黄鹏羽
何润邦
刘立会
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Institute of Biomedical Engineering of CAMS and PUMC
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Abstract

本发明公开了一种人间充质干细胞培养基，包括胎牛血清、胰岛素、转铁蛋白、成纤维生长因子‑2、胰岛素样生长因子2、牛磺酸、盐酸腐胺、甲基b‑D‑木吡喃糖苷、α‑酮戊二酸、牛血清白蛋白、亚油酸、胆固醇、α‑生育酚、N‑乙酰半胱氨酸、抗坏血酸、亚硒酸钠、甘氨酸、L‑丙氨酸、L‑天门冬酰胺、L‑天门冬氨酸、L‑谷氨酸、L‑丝氨酸、L‑脯氨酸、丙酮酸钠和DMEM/F12，本发明细胞培养基培养人间充质干细胞，可以传代培养30代以上，到第30代所述干细胞仍然能保持比较好的形态，具有增殖能力，并能继续维持人间充质干细胞的分化属性，即具备向脂肪、软骨和骨的分化潜能。

Description

一种人间充质干细胞培养基
技术领域
本发明涉及一种人间充质干细胞培养基，属于生物材料领域。
背景技术
间充质干细胞(MSC,mesenchymal stem cells)是一种多能干细胞，具有一定的分化潜能，可以分化为骨骼肌、心肌、脂肪、软骨、骨骼等多种细胞类型。应用于临床，可用于目标细胞群的替换和损伤病变组织器官的修复。具有免疫调节功能，可以发挥免疫重建的功能。异体移植排异反应较轻，对配型的要求不严格，因而应用广泛。
间充质干细胞主要存在于骨髓中，也能够从脂肪、滑膜、骨骼、肌肉、肺、肝、胰腺等组织以及羊水、脐带血中分离和制备间充质干细胞。
分离人间充质干细胞后，需要培养人间充质干细胞到足够量，用于分化为目标组织。也需要在培养过程中保持分化潜能。我们发现用传统细胞培养基，以(DMEM+10％FBS)为例，培养人间充质干细胞，细胞到第三代就会出现明显的衰老特征，因此，亟需一种能较多代数培养人间充质干细胞，并且保持其分化潜能的培养基。
发明内容
本发明的目的是克服现有技术的不足，提供一种人间充质干细胞培养基。
本发明的技术方案概述如下：
一种人间充质干细胞培养基，包括胎牛血清：25ml、胰岛素：5mg、转铁蛋白：2.75mg、成纤维生长因子-2：5ug、胰岛素样生长因子2：5ug、牛磺酸：0.625mg、盐酸腐胺：0.5mg、甲基b-D-木吡喃糖苷：50mg、α-酮戊二酸：7.3mg、牛血清白蛋白：0.625g、亚油酸：2.675mg、胆固醇：2.5mg、α-生育酚：终浓度为9×10-7M、N-乙酰半胱氨酸：5mg、抗坏血酸：25mg、亚硒酸钠：3.35ug、甘氨酸：3.75mg、L-丙氨酸：4.45mg、L-天门冬酰胺：6.6mg、L-天门冬氨酸：6.65mg、L-谷氨酸：7.35mg、L-丝氨酸：5.25mg、L-脯氨酸：5.75mg、丙酮酸钠：27.5mg，用DMEM/F12补齐到500ml。
本发明的优点：
本发明细胞培养基培养人间充质干细胞，可以传代培养30代以上，到第30代所述干细胞仍然能保持比较好的形态，具有增殖能力，并能继续维持人间充质干细胞的分化属性，即具备向脂肪、软骨和骨的分化潜能。
附图说明
图1为培养的人间充质干细胞形态；
图2为人间充质干细胞增殖曲线；
图3为人间充质干细胞分化能力。
具体实施方式
DMEM/F12：杜尔贝科改良Eagle培养基/营养混合物F-12(生产厂家：赛默飞)；其它原料均为市售。
下面通过具体实施例对本发明作进一步的说明。
实施例1、一种人间充质干细胞培养基，包括胎牛血清：25ml、胰岛素：5mg、转铁蛋白：2.75mg、成纤维生长因子-2：5ug、胰岛素样生长因子2：5ug、牛磺酸：0.625mg、盐酸腐胺：0.5mg、甲基b-D-木吡喃糖苷：50mg、α-酮戊二酸：7.3mg、牛血清白蛋白：0.625g、亚油酸：2.675mg、胆固醇：2.5mg、α-生育酚：终浓度为9×10-7M、N-乙酰半胱氨酸：5mg、抗坏血酸：25mg、亚硒酸钠：3.35ug、甘氨酸：3.75mg、L-丙氨酸：4.45mg、L-天门冬酰胺：6.6mg、L-天门冬氨酸：6.65mg、L-谷氨酸：7.35mg、L-丝氨酸：5.25mg、L-脯氨酸：5.75mg、丙酮酸钠：27.5mg，用DMEM/F12补齐到500ml。
实施例2
人间充质干细胞的培养：
(1)P0代细胞的获得：
人脐带组织贴壁法分离人间充质干细胞。用生理盐水充分洗涤脐带，并剪成小段。将脐带纵向剪开,用镊子剥去脐带中的两根脐动脉和一根脐静脉。将脐带剪碎，均匀接种于装有15mL实施例1一种人间充质干细胞培养基的T75培养瓶中，37℃，5％二氧化碳培养，得到的细胞即为P0代细胞。
(2)换液
根据步骤(1)的最终的培养基颜色(由原来的红色变成淡黄色)决定更换新的一种人间充质干细胞培养基，更换方法是用巴氏吸管吸掉旧培养基，用移液管，缓慢加入37℃预热的等量新的一种人间充质干细胞培养基。
(3)传代
待细胞密度在85％以上时，分瓶培养即为传代。传代1次即为P1代细胞，以此类推。具体操作方法为：
a吸掉旧培养基，用37℃预热的pH＝7.4的PBS洗2遍细胞；
b每瓶加TrypLETM Express胰酶(生产厂家：赛默飞)3ml，室温消化细胞1分钟左右，待细胞变圆后，每瓶加实施例1制备的人间充质干细胞培养基10ml；
c吸出细胞悬液至50ml离心管中，1000rpm，离心3min，弃上清；
d用10ml一种人间充质干细胞培养基重悬沉淀，经细胞筛过滤，5ml人间充质干细胞培养基冲洗筛网，计数；
e调整细胞浓度，根据实际需要铺板或瓶，置于37℃、5％二氧化碳培养箱中培养。
见图1、2和3。
图1为培养的人间充质干细胞形态，其中：
A.DMEM+10％FBS培养人间充质干细胞第3代细胞形态，第3代开始，可以观察到细胞体积增大，出现明显的细胞衰老特征；
B.人间充质干细胞培养基培养人间充质干细胞第1代细胞形态；
C.人间充质干细胞培养基培养人间充质干细胞第3代细胞形态；
D.人间充质干细胞培养基培养人间充质干细胞第30代细胞形态；细胞在第30代依然维持良好的梭形形态，没有明显的细胞衰老特征。
图2为人间充质干细胞增殖曲线。
其中，菱形点表示人间充质干细胞培养基(系列1)培养细胞的增殖曲线，圆形点表示DMEM+10％FBS(系列2)。如图2中所示，人间充质干细胞培养基培养的人间充质干细胞到第30代仍然增殖很好，DMEM+10％FBS培养的人间充质干细胞到第7代已经丢失增殖力。
图3为人间充质干细胞分化能力。
图中所示为人间充质干细胞培养基培养的人间充质干细胞到第30代以后的细胞诱导分化的图片。
A未进行成脂肪诱导，进行油红O染色(脂肪会被染成红色)；
B成脂肪诱导，进行油红O染色(脂肪会被染成红色)；
C未进行成骨诱导，进行茜素红染色(骨会被染成红色)；
D成骨诱导，进行茜素红染色(骨会被染成红色)；
E未进行成软骨诱导，进行阿利新蓝染色(软骨会被染成蓝色)；
F成软骨诱导，进行阿利新蓝染色(软骨会被染成蓝色)。

Claims (1)

1.一种人间充质干细胞培养基，其特征是包括胎牛血清：25ml、胰岛素：5mg、转铁蛋白：2.75mg、成纤维生长因子-2：5ug、胰岛素样生长因子2：5ug、牛磺酸：0.625mg、盐酸腐胺：0.5mg、甲基b-D-木吡喃糖苷：50mg、α-酮戊二酸：7.3mg、牛血清白蛋白：0.625g、亚油酸：2.675mg、胆固醇：2.5mg、α-生育酚：终浓度为9×10-7M、N-乙酰半胱氨酸：5mg、抗坏血酸：25mg、亚硒酸钠：3.35ug、甘氨酸：3.75mg、L-丙氨酸：4.45mg、L-天门冬酰胺：6.6mg、L-天门冬氨酸：6.65mg、L-谷氨酸：7.35mg、L-丝氨酸：5.25mg、L-脯氨酸：5.75mg、丙酮酸钠：27.5mg，用DMEM/F12补齐到500ml。
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