CN106957815B - 一种用于人类多潜能干细胞的无血清培养基的配方 - Google Patents
一种用于人类多潜能干细胞的无血清培养基的配方 Download PDFInfo
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Abstract
本发明公开了一种用于人类多潜能干细胞的无血清培养基的配方，包括以下原料：无机盐成分、有机物成分、氨基酸及氨基酸盐、能量物质及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽、微量元素和显色物质；其培养流程分为：选定基础配方、进行组合筛选、结果鉴定评估、新配方试培养；并按以下方法配比：将上述原料加注射用水至950毫升，并轻微搅拌至溶解，最后加入2.438克碳酸氢钠，并轻微搅拌溶解，再加注射用水至1升，再用1mol/L氢氧化钠溶液或1mol/L盐酸溶液调pH至所需值，最后用0.1μm直径滤膜正压过滤除菌，并将培养基溶液放置2℃‑8℃下避光存储，解决了国内进口无血清配方成本高的问题。
Description
技术领域
本发明涉及生物制药技术领域，具体为一种用于人类多潜能干细胞的无血清培养基的配方。
背景技术
生物制药是国家发展的重点项目之一。其独特之处在于生物药物只能用活性载体如细胞来表达。传统的化学合成方式不能用于生物药及疫苗的生产。作为生物制药所必需的支柱性原材料，细胞培养基的等级和相应生产工艺流程的放大直接反映了国家生物制药工业的发展水平及药物成本的高低。目前高等级无血清培养基的开发与生产技术及全球细胞培养基市场基本由Sigma、Life、HYCLONE 等美国公司所垄断，因此国内生物制药企业在供货时间及成本上受制于人，这也是阻碍国内企业大规模产业化和以低成本与国际生物制药企业竞争的主要因素之一。中国2010版药典及GMP法规对国内生物制药企业所用细胞培养基提出了更高的要求，尽管已批准的一些老产品还准许使用含血清培养基，无血清培养基已是当前新药物报批的必要条件。但由于国内尚无高质量的、产业化的无血清细胞培养基产品，国内企业所使用的高等级无血清细胞培养基还基本全部依赖进口。本发明开发的无血清细胞培养基产品居于国际先进水平，将填补国内无血清细胞培养基高端产品的空白。
由于研发技术人员和工业化生产技术的匮乏，目前国内的无血清细胞培养基研发和生产技术还处在较低水平。根据被默克（Merck）公司收购的国内培养基公司，北京清大天一生物技术有限公司，2008年4 月公布的数据显示，清大天一的无血清培养基可以支持大约300 mg/L 的抗体产量。与目前国外培养基支持的平均2-3g/L 的产量相比还有10 倍的差距。这个差距意味着使用目前国内最大的培养基公司的产品所生产的生物药，要比使用国外培养基公司所生产的生物药高10 倍的价钱才能达到同样的盈利。市场上另外一家国内企业仅能生产配方公开并需配合血清一起使用的低端商业化产品，年产量不到30 吨，供已批准的老的疫苗产品使用。其余生物仿制药及高端疫苗所使用的高附加值无血清细胞培养基全部依赖进口。因此有必要自主研发属于我国自己的无血清细胞大规模工业化培养技术。
发明内容
本发明的目的在于提供一种用于人类多潜能干细胞的无血清培养基的配方，其无血清细胞培养基产品居于国际先进水平，将填补国内高等级细胞培养基高端产品的空白，以解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：一种用于人类多潜能干细胞的无血清培养基的配方，包括以下原料：无机盐成分、有机物成分、氨基酸及氨基酸盐、能量物质及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽、微量元素和显色物质；其培养流程分为：选定基础配方、进行组合筛选、结果鉴定评估、新配方试培养；并按以下方法配比：
S1：将上述原料加水温为20℃-30℃ 的注射用水至950毫升，并轻微搅拌至溶解；
S2：加入2.438克碳酸氢钠，并轻微搅拌溶解，再加注射用水至1升；
S3：选择用1mol/ L氢氧化钠溶液或1mol/L盐酸溶液调pH至所需值；
S4：用0.1μm直径滤膜正压过滤除菌，并将培养基溶液放置2℃-8℃下避光存储。
优选的，无机盐成分具体的重量配比包括有：无水氯化钙116.6mg、氯化钾311.8mg、氯化镁28.64mg、无水硫酸镁48.84mg、氯化钠6999.5mg、无水磷酸二氢钠54.35mg和磷酸氢二钠71.02mg。
优选的，有机物成分具体的重量配比包括有：4-丁二胺二盐酸盐0.081mg、 丙酮酸钠55mg、盐酸吡哆醛2mg、盐酸吡哆醇0.031mg。
优选的，氨基酸及氨基酸盐具体的重量配比包括有：L-亮氨酸59.05mg、 L-赖氨酸盐酸盐91.25mg、L-蛋氨酸17.24mg、L-苯丙氨酸35.48mg、L-丝氨酸26.25mg、L-苏氨酸53.45mg、L-丙氨酸4.45mg、L-天门冬酰胺7.5mg、L-天门冬氨酸6.65mg、L-半胱氨酸盐酸盐26.34mg、L-谷氨酸11.03mg、L-精氨酸盐酸盐 147.5mg、L-脯氨酸17.25mg、L-胱氨酸盐酸盐31.29mg、L-色氨酸9.02mg、L-谷氨酰胺365mg、L-酪氨酸38.4mg、甘氨酸28.13mg、L-缬氨酸52.85mg、L-组氨酸盐酸盐31.48mg、L-异亮氨酸54.47mg、L-羟脯氨酸6.82mg。
优选的，能量物质及代谢中间产物具体添加的重量配比包括有：亚油酸 0.042mg、肌醇12.6mg、氯化胆碱8.98mg、烟酰胺2.02mg、D-葡萄糖3151mg、胸苷0.365mg、次黄嘌呤2mg。
优选的，维生素及抗氧化剂具体的重量配比包括有：硫辛酸0.105mg、维生素H0.0035mg、D-泛酸钙2.24mg、叶酸2.65mg、核黄素0.219mg、盐酸硫胺2.17mg、 维生素B20.4mg、维生素B120.68mg、维生素C-2-磷酸盐87.54mg。
优选的，蛋白质及多肽具体的重量配比包括有：转铁蛋白4000mg、胰岛素 1200mg、碱性成纤维细胞生长因子2.0mg、表皮生长因子1.65mg、人血清白蛋白856.36mg、孕酮150mg。
优选的，微量元素具体的重量配比包括有：五水硫酸铜0.0013mg、九水硝酸铁0.05mg、七水硫酸亚铁0.417mg、七水硫酸锌0.432mg、二水氟化银0.0085mg、 十八水硫酸铝0.049mg、二水氯化钡0.0092mg、八水硫酸镉0.0008mg、七水硫酸钴0.0057mg、九水硝酸铬0.0038mg、水合偏锗酸钠0.0009mg、五水亚硒酸钠0.013mg、溴化钾0.0071mg、二水碘化钠0.0007mg、七水硫酸锰0.0025mg、九水硅酸钠0.034mg、二水偏钒酸钠0.06mg、二水钼酸钠0.074mg、六水二氯化镍 0.0086mg、氯化铷0.0017mg、三水锡酸钾0.0054mg、二水硝酸氧锆0.004mg。
优选的，显色物质为8.1mg的酚红溶液。
优选的，针对S4中培养基溶液的性状为：溶解性、完全溶解、溶液澄明。
优选的，针对S3中PH值调配：加NaHCO3为6.60-7.20，不加NaHCO3为5.50-6.10；渗透压为：加NaHCO3为277-312，不加NaHCO3为234-258；其细菌内毒素≤10EU/ml；微生物检查≤1000CFU/g。
与现有技术相比，本发明的有益效果是：
本用于人类多潜能干细胞的无血清培养基的配方，其无血清细胞培养基产品居于国际先进水平，将填补国内高等级细胞培养基高端产品的空白，其具备市场大、用途广等优点，所有以细胞为平台生产的疫苗、大分子生物药物（包括：单克隆抗体、重组蛋白药）都需要使用无血清细胞培养基，解决了国内进口配方成本高的问题。
附图说明
图1为本发明的整体流程图；
图2为本发明实施例二示意图；
图3为本发明实施例二示意图；
图4为本发明实施例三示意图；
图5为本发明实施例三示意图。
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，本发明提供一种技术方案：一种用于人类多潜能干细胞的无血清培养基的配方，包括以下原料：无机盐成分、有机物成分、氨基酸及氨基酸盐、能量物质及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽、微量元素和显色物质；其中，无机盐成分具体的重量配比包括有：无水氯化钙116.6mg、氯化钾311.8mg、氯化镁28.64mg、无水硫酸镁48.84mg、氯化钠6999.5mg、无水磷酸二氢钠54.35mg和磷酸氢二钠71.02mg；有机物成分具体的重量配比包括有：4-丁二胺二盐酸盐0.081mg、丙酮酸钠55mg、盐酸吡哆醛2mg、盐酸吡哆醇0.031mg；氨基酸及氨基酸盐具体的重量配比包括有：L-亮氨酸59.05mg、 L-赖氨酸盐酸盐91.25mg、L-蛋氨酸17.24mg、L-苯丙氨酸35.48mg、L-丝氨酸26.25mg、L-苏氨酸53.45mg、L-丙氨酸4.45mg、L-天门冬酰胺7.5mg、L-天门冬氨酸6.65mg、L-半胱氨酸盐酸盐26.34mg、L-谷氨酸11.03mg、L-精氨酸盐酸盐 147.5mg、L-脯氨酸17.25mg、L-胱氨酸盐酸盐31.29mg、L-色氨酸9.02mg、L-谷氨酰胺365mg、L-酪氨酸38.4mg、甘氨酸28.13mg、L-缬氨酸52.85mg、L-组氨酸盐酸盐31.48mg、L-异亮氨酸54.47mg、L-羟脯氨酸6.82mg；能量物质及代谢中间产物具体添加的重量配比包括有：亚油酸 0.042mg、肌醇12.6mg、氯化胆碱8.98mg、烟酰胺2.02mg、D-葡萄糖3151mg、胸苷0.365mg、次黄嘌呤2mg；维生素及抗氧化剂具体的重量配比包括有：硫辛酸0.105mg、维生素H0.0035mg、D-泛酸钙2.24mg、叶酸2.65mg、核黄素0.219mg、盐酸硫胺2.17mg、维生素B20.4mg、维生素B120.68mg、维生素C-2-磷酸盐87.54mg；蛋白质及多肽具体的重量配比包括有：转铁蛋白4000mg、胰岛素 1200mg、碱性成纤维细胞生长因子2.0mg、表皮生长因子1.65mg、人血清白蛋白856.36mg、孕酮150mg；微量元素具体的重量配比包括有：五水硫酸铜0.0013mg、九水硝酸铁0.05mg、七水硫酸亚铁0.417mg、七水硫酸锌0.432mg、二水氟化银0.0085mg、十八水硫酸铝0.049mg、二水氯化钡0.0092mg、八水硫酸镉0.0008mg、七水硫酸钴0.0057mg、九水硝酸铬0.0038mg、水合偏锗酸钠0.0009mg、五水亚硒酸钠0.013mg、溴化钾0.0071mg、二水碘化钠0.0007mg、七水硫酸锰0.0025mg、九水硅酸钠0.034mg、二水偏钒酸钠0.06mg、二水钼酸钠0.074mg、六水二氯化镍 0.0086mg、氯化铷0.0017mg、三水锡酸钾0.0054mg、二水硝酸氧锆0.004mg；显色物质为8.1mg的酚红溶液。
其培养流程分为：在配方库中选定基础配方、在培养箱中进行组合筛选、用软件进行结果鉴定评估、新配方试培养；并按以下方法配比：
第一步：将上述原料加水温为20℃-30℃ 的注射用水至950毫升，并轻微搅拌至溶解；
第二步：加入2.438克碳酸氢钠，并轻微搅拌溶解，再加注射用水至1升；
第三步：选择用1mol/ L氢氧化钠溶液或1mol/L盐酸溶液调pH至所需值；其PH值调配：加NaHCO3为6.60-7.20，不加NaHCO3为5.50-6.10；渗透压为：加NaHCO3为277-312，不加NaHCO3为234-258；其细菌内毒素≤10EU/ml；微生物检查≤1000CFU/g；
第四步：用0.1μm直径滤膜正压过滤除菌，并将培养基溶液放置2℃-8℃下避光存储；其培养基溶液的性状为：溶解性、完全溶解、溶液澄明。
实施例一：
一种用于人类多潜能干细胞的无血清培养基的配方，包括以下原料：无机盐成分、有机物成分、氨基酸及氨基酸盐、能量物质及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽、微量元素和显色物质；按上述方法进行人多能干细胞的扩增培养。
人多能干细胞的扩增培养：在配制好的培养基溶液中加入新鲜配制的bFGF(即：碱性成纤维细胞生长因子，每升培养基加入bFGF 2mg)、β-巯基乙醇(每升培养液加入β-巯基乙醇7μl)；维持人多能干细胞的未分化状态，通过扩增培养，使多能干细胞自我更新、增殖，从而达到增加细胞数量、扩大储备、可进行后续分装、保种的目的；与当前市场同类产品(DMEM/F12+Knockout SR(Invitrogen公司)、StemPro hESC SFM培养基、KnockOutTM CTSTMZenoFree培养基) 相比，在细胞集落形态方面并无差异；人多能干细胞在未分化状态下表达一种特异性蛋白，即SSEA-4 (stage specific embryonic antigen-4，阶段特异性胚胎抗原-4)，本配方培养基与市场同类产品之间就SSEA-4+阳性细胞占比而言，并无显著性差异。
实施例二：
请参阅图2-3：一种用于人类多潜能干细胞的无血清培养基的配方，包括以下原料：无机盐成分、有机物成分、氨基酸及氨基酸盐、能量物质及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽、微量元素和显色物质；按上述方法进行心肌诱导培养，其培养流程分为：悬滴培养3天+悬浮培养2天+贴壁培养5天，同时加入维生素C (每升培养液加入ascorbic acid 128.05 mg)。
心肌诱导培养：利用人类多能干细胞系IMR90-1 (Wicell Research Institute,Madison, WI, USA)，诱导培养之前先通过电转染的手段将报告基因MHC-eGFP (肌球蛋白重链启动子驱动的绿色荧光蛋白表达，在诱导培养开始之后，一旦干细胞向心肌分化，开始表达心肌特异性的MHC之时，就会有绿色荧光出现)转染至IMR90-1，随后用不同的诱导培养系统(分别为IMEM+15%SR(Invitrogen公司)、IMEM+15%胎牛血清、本配方培养基（无血清）、DMEM/F12+15%胎牛血清、DMEM(高糖)+15% 胎牛血清) 对其进行诱导培养，按照图2标示的日程，在不同时间点取样，以资对比不同培养基的诱导效能；从对比结果显示，以第3、5、7天细胞的存活率及第7天生成的心肌细胞占比为评价指标 (图3)，可知本培养基优于当前市场上的同类产品 (* p<0.05 差异具有显著性，与其他任一相比)。
实施例三：
请参阅图4-5：一种用于人类多潜能干细胞的无血清培养基的配方，包括以下原料：无机盐成分、有机物成分、氨基酸及氨基酸盐、能量物质及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽、微量元素和显色物质；按上述方法进行神经诱导：在配制好的培养基溶液以HS-5细胞系作为饲养层，同时加入NT-3(每升培养基加入NT-3 1342mg)。
神经诱导培养：利用人类多能干细胞系iPS-1 (Wicell Research Institute,Madison, WI, USA)，通过将其与人骨髓基质细胞系HS-5共培养 (图4)，来达到将多能干细胞向神经细胞方向诱导培养的目的；拟对比的当前市场同类产品有DMEM/F12+10% 胎牛血清、Knockout DMEM+10% SR(Invitrogen 公司)、Neurobasal培养基；按照图4的培养法诱导培养2周后，以细胞存活率、Nestin+细胞 (Nestin是由神经前体细胞特异性表达出的一种细胞表面标志物) 的占比作为评价指标，来对比不同培养基对多能干细胞神经诱导培养的效能；对比结果显示，尽管在细胞存活率方面，多种培养基并无显著性差异，但就Nestin+细胞占比而言，本配方培养基优于当前市场同类产品(图5；* p<0.05 差异具有显著性，与其他任一相比)。
综上所述：本用于人类多潜能干细胞的无血清培养基的配方，其无血清细胞培养基产品居于国际先进水平，将填补国内高等级细胞培养基高端产品的空白，其具备市场大、用途广等优点，所有以细胞为平台生产的疫苗、大分子生物药物（包括：单克隆抗体、重组蛋白药）都需要使用无血清细胞培养基，解决了国内进口配方成本高的问题。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
Claims (4)
1.一种用于人类多潜能干细胞的无血清培养基，其特征在于，包括以下原料：无机盐成分、有机物成分、氨基酸及氨基酸盐、能量物质及代谢中间产物、维生素及抗氧化剂、蛋白质及多肽、微量元素和显色物质；
其中，所述蛋白质及多肽具体的重量配比包括有：转铁蛋白4000mg、胰岛素1200mg、碱性成纤维细胞生长因子2.0mg、表皮生长因子1.65mg、人血清白蛋白856.36mg、孕酮150mg；
所述微量元素具体的重量配比包括有：五水硫酸铜0.0013mg、九水硝酸铁0.05mg、七水硫酸亚铁0.417mg、七水硫酸锌0.432mg、二水氟化银0.0085mg、十八水硫酸铝0.049mg、二水氯化钡0.0092mg、八水硫酸镉0.0008mg、七水硫酸钴0.0057mg、九水硝酸铬0.0038mg、水合偏锗酸钠0.0009mg、五水亚硒酸钠0.013mg、溴化钾0.0071mg、二水碘化钠0.0007mg、七水硫酸锰0.0025mg、九水硅酸钠0.034mg、二水偏钒酸钠0.06mg、二水钼酸钠0.074mg、六水二氯化镍0.0086mg、氯化铷0.0017mg、三水锡酸钾0.0054mg、二水硝酸氧锆0.004mg；
所述无机盐成分具体的重量配比包括有：无水氯化钙116.6mg、氯化钾311.8mg、氯化镁28.64mg、无水硫酸镁48.84mg、氯化钠6999.5mg、无水磷酸二氢钠54.35mg和磷酸氢二钠71.02mg；
所述有机物成分具体的重量配比包括有：4-丁二胺二盐酸盐0.081mg、丙酮酸钠55mg、盐酸吡哆醛2mg、盐酸吡哆醇0.031mg；
所述氨基酸及氨基酸盐具体的重量配比包括有：L-亮氨酸59.05mg、L-赖氨酸盐酸盐91.25mg、L-蛋氨酸17.24mg、L-苯丙氨酸35.48mg、L-丝氨酸26.25mg、L-苏氨酸53.45mg、L-丙氨酸4.45mg、L-天门冬酰胺7.5mg、L-天门冬氨酸6.65mg、L-半胱氨酸盐酸盐26.34mg、L-谷氨酸11.03mg、L-精氨酸盐酸盐147.5mg、L-脯氨酸17.25mg、L-胱氨酸盐酸盐31.29mg、L-色氨酸9.02mg、L-谷氨酰胺365mg、L-酪氨酸38.4mg、甘氨酸28.13mg、L-缬氨酸52.85mg、L-组氨酸盐酸盐31.48mg、L-异亮氨酸54.47mg、L-羟脯氨酸6.82mg；
所述能量物质及代谢中间产物具体添加的重量配比包括有：亚油酸0.042mg、肌醇12.6mg、氯化胆碱8.98mg、烟酰胺2.02mg、D-葡萄糖3151mg、胸苷0.365mg、次黄嘌呤2mg；
所述维生素及抗氧化剂具体的重量配比包括有：硫辛酸0.105mg、维生素H 0.0035mg、D-泛酸钙2.24mg、叶酸2.65mg、核黄素0.219mg、盐酸硫胺2.17mg、维生素B2 0.4mg、维生素B12 0.68mg、维生素C-2-磷酸盐87.54mg；
所述的无血清培养基的培养流程分为：选定基础配方、进行组合筛选、结果鉴定评估、新配方试培养；并按以下方法配比：
S1：将上述原料加水温为20℃-30℃的注射用水至950毫升，并轻微搅拌至溶解；
S2：加入2.438克碳酸氢钠，并轻微搅拌溶解，再加注射用水至1升；
S3：选择用1mol/L氢氧化钠溶液或1mol/L盐酸溶液调pH至所需值；
S4：用0.1μm直径滤膜正压过滤除菌，并将培养基溶液放置2℃-8℃下避光存储。
2.根据权利要求1所述的无血清培养基，其特征在于，所述显色物质为8.1mg的酚红溶液。
3.根据权利要求1所述的无血清培养基，其特征在于，针对S4中培养基溶液的性状为：溶解性、完全溶解、溶液澄明。
4.一种如权利要求1～3中任一项所述的无血清培养基在人类多潜能干细胞培养、心肌诱导培养和神经细胞诱导培养中的用途。
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| BR102017018191A2 (pt) * | 2017-08-24 | 2019-03-26 | Can Technologies, Inc. | Método para reduzir a incidência de miopatias peitorais em aves de corte, ração para reduzir a incidência de miopatias peitorais em aves de corte, ração para aves de corte e uso de um aditivo |
| CN109749997B (zh) * | 2018-05-11 | 2020-03-17 | 中山大学中山眼科中心 | 一种角膜缘干细胞无血清培养基及其培养方法 |
| CN109112099A (zh) * | 2018-08-30 | 2019-01-01 | 丰泽康生物医药（深圳）有限公司 | 一种提高单核细胞向多潜能细胞转化的无血清培养基 |
| EP3858980A4 (en) * | 2018-09-28 | 2021-12-08 | Ajinomoto Co., Inc. | CULTURE ADDITIVE, CULTURE MEDIA AND CULTURE PROCESS FOR ANIMAL CELLS |
| CN109370988A (zh) * | 2018-11-03 | 2019-02-22 | 章毅 | 干细胞体外扩增培养体系及其方法 |
| CN112795532A (zh) * | 2019-11-13 | 2021-05-14 | 苏州易迈吉生物医药科技有限公司 | 一种人牙周膜干细胞无血清培养基 |
| CN111467372B (zh) * | 2020-03-03 | 2022-09-30 | 中山大学附属第一医院 | 间充质干细胞外泌体在制备延缓脊髓小脑性共济失调3型病程进展的药物中的应用 |
| CN113512521B (zh) * | 2021-05-26 | 2023-06-23 | 江苏普瑞康生物医药科技有限公司 | 无血清培养基的添加剂，无血清培养基及其应用 |
| CN115616097B (zh) * | 2021-07-16 | 2024-10-25 | 深圳奥萨制药有限公司 | 叶酸类物质的新用途 |
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