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85%\uff0c\u7b26\u5408\u6b63\u5e38\u7ec6\u80de\u590d\u82cf\u7279\u6027\uff09\uff0c\u4e14\u6b63\u5e38\u4e0a\u76ae\u5f62\u6001\u3001\u65e0\u81f4\u7624\u6027\u7279\u6027\u65e0\u663e\u8457\u6539\u53d8\uff1bT25 \u74f6\u53d1\u8d27\u65f6\u7ec6\u80de\u5bc6\u5ea6\u8fbe 70%-80%\uff0c\u786e\u4fdd\u5230\u8fbe\u540e\u53ef\u76f4\u63a5\u7528\u4e8e\u5b9e\u9a8c\u6216\u6309\u6b63\u5e38\u7ec6\u80de\u8282\u594f\u4f20\u4ee3\uff09<\/li>\n<\/ul>\n<ul>\n<li class=\"temp-li bullet1\" data-line=\"true\" data-list=\"bullet1\">\u652f\u539f\u4f53\u68c0\u6d4b\uff1a\u65e0\uff08\u6bcf\u6279\u6b21\u7ec6\u80de\u901a\u8fc7 PCR \u6cd5\u68c0\u6d4b\u652f\u539f\u4f53\uff08\u8986\u76d6\u80ba\u708e\u652f\u539f\u4f53\u3001\u4eba\u578b\u652f\u539f\u4f53\u3001\u89e3\u8132\u652f\u539f\u4f53\uff09\uff0c\u7ed3\u679c\u4e3a\u9634\u6027\uff0c\u907f\u514d\u652f\u539f\u4f53\u6c61\u67d3\u5f71\u54cd\u7ec6\u80de\u6b63\u5e38\u751f\u957f\u53ca\u751f\u7406\u529f\u80fd\uff0c\u4fdd\u969c\u4e0e AGS \u201c\u6b63\u5e38 &#8211; \u80bf\u7624\u201d \u5bf9\u6bd4\u5b9e\u9a8c\u7684\u53ef\u9760\u6027\uff09<\/li>\n<\/ul>\n<ul>\n<li class=\"temp-li bullet1\" data-line=\"true\" data-list=\"bullet1\">\u57f9\u517b\u57fa\uff1a90% RPMI-1640+10% FBS+PS\uff08\u9752\u9709\u7d20 &#8211; \u94fe\u9709\u7d20\uff09\uff08\u6838\u5fc3\u7279\u8272\uff1a\u2460\u91c7\u7528 RPMI-1640 \u57f9\u517b\u57fa\uff0c\u533a\u522b\u4e8e AGS \u7684 F12K\u3001WPMY-1 \u7684 DMEM\uff1bRPMI-1640 \u542b\u6709\u7684\u6c28\u57fa\u9178\u53ca\u7ef4\u751f\u7d20\u7ec4\u5408\uff0c\u9002\u914d\u80ce\u513f\u80c3\u9ecf\u819c\u4e0a\u76ae\u7ec6\u80de\u7684\u4ee3\u8c22\u9700\u6c42\uff0c\u7ef4\u6301\u5176\u6b63\u5e38\u4e0a\u76ae\u529f\u80fd\uff1b\u246110% FBS \u9700\u9009\u62e9\u4f4e\u5185\u6bd2\u7d20\u3001\u6279\u6b21\u95f4\u5dee\u5f02\u5c0f\u7684\u4f18\u8d28\u80ce\u725b\u8840\u6e05\uff0c\u907f\u514d\u8840\u6e05\u4e2d\u80bf\u7624\u76f8\u5173\u56e0\u5b50\u8bf1\u5bfc\u7ec6\u80de\u5f02\u5e38\u589e\u6b96\uff1b\u2462PS \u7ec8\u6d53\u5ea6\u4e3a 100U\/mL \u9752\u9709\u7d20\u3001100\u03bcg\/mL \u94fe\u9709\u7d20\uff0c\u6709\u6548\u9884\u9632\u7ec6\u83cc\u6c61\u67d3\uff0c\u4e0d\u5f71\u54cd\u7ec6\u80de\u6c38\u751f\u5316\u7279\u6027\u53ca\u6b63\u5e38\u751f\u7406\u529f\u80fd\uff09<\/li>\n<\/ul>\n<ul>\n<li class=\"temp-li bullet1\" data-line=\"true\" data-list=\"bullet1\">\u57f9\u517b\u6761\u4ef6\uff1a\u6c14\u76f8\uff1a95% \u7a7a\u6c14 + 5% \u4e8c\u6c27\u5316\u78b3\uff1b\u6e29\u5ea6\uff1a37\u2103\uff08\u57f9\u517b\u7bb1\u6e7f\u5ea6\u7ef4\u6301\u5728 70%-80%\uff0c\u5b9a\u671f\u66f4\u6362\u6c34\u76d8\uff0c\u9632\u6b62\u57f9\u517b\u57fa\u84b8\u53d1\u5bfc\u81f4\u6e17\u900f\u538b\u5347\u9ad8\uff1b\u57f9\u517b\u7bb1\u5185\u6258\u76d8\u9700\u6c34\u5e73\u653e\u7f6e\uff0c\u907f\u514d\u57f9\u517b\u57fa\u503e\u659c\u5bfc\u81f4\u7ec6\u80de\u5206\u5e03\u4e0d\u5747\uff1b\u4e0e AGS\u3001TE-1\u3001WPMY-1 \u7b49\u6240\u6709\u5df2\u68b3\u7406\u7ec6\u80de\u7684\u57f9\u517b\u6761\u4ef6\u5b8c\u5168\u4e00\u81f4\uff0c\u4ec5\u9700\u9002\u914d\u6b63\u5e38\u7ec6\u80de\u589e\u6b96\u8282\u594f\u8c03\u6574\u4f20\u4ee3\u95f4\u9694\uff0c\u4fbf\u4e8e\u591a\u7ec6\u80de\u682a\u5e76\u884c\u5b9e\u9a8c\uff09<\/li>\n<\/ul>\n<ul>\n<li class=\"temp-li bullet1\" data-line=\"true\" data-list=\"bullet1\">\u51bb\u5b58\u6761\u4ef6\uff1a\u65e0\u8840\u6e05\u51bb\u5b58\u6db2\uff0c\u6db2\u6c2e\u50a8\u5b58\uff08\u51bb\u5b58\u64cd\u4f5c\u9002\u914d\u6b63\u5e38\u6c38\u751f\u5316\u7ec6\u80de\u7279\u6027\uff1a\u2460\u9009\u62e9\u5bf9\u6570\u751f\u957f\u671f\u7ec6\u80de\uff08\u63a5\u79cd\u540e 3-4 \u5929\uff09\uff0c\u7528 0.25% \u80f0\u9176 &#8211; EDTA \u5b75\u80b2 2-3 \u5206\u949f\uff08\u957f\u4e8e AGS \u7684 1-2 \u5206\u949f\uff09\uff0c\u955c\u4e0b\u89c1\u7ec6\u80de\u95f4\u9699\u589e\u5927\u3001\u8fb9\u7f18\u6536\u7f29\u5373\u53ef\u7ec8\u6b62\uff0c\u8f7b\u67d4\u5439\u6253\u907f\u514d\u635f\u4f24\u4e0a\u76ae\u7ec6\u80de\u95f4\u8fde\u63a5\u7ed3\u6784\uff1b\u24611000rpm \u79bb\u5fc3 5 \u5206\u949f\u6536\u96c6\u7ec6\u80de\uff0c\u7528\u9884\u51b7\u7684\u65e0\u8840\u6e05\u51bb\u5b58\u6db2\u91cd\u60ac\uff0c\u8c03\u6574\u6d53\u5ea6\u81f3 1\u00d710\u2076-2\u00d710\u2076cells\/mL\uff1b\u2462\u51bb\u5b58\u6d41\u7a0b\uff1a4\u2103\u5e73\u8861 30 \u5206\u949f\u2192-20\u2103\u653e\u7f6e 2 \u5c0f\u65f6\uff08\u7a0b\u5e8f\u964d\u6e29\u76d2\uff0c\u51cf\u5c11\u4f4e\u6e29\u5bf9\u7ec6\u80de\u7684\u635f\u4f24\uff09\u2192-80\u2103\u8fc7\u591c\u2192\u6db2\u6c2e\u4fdd\u5b58\uff1b\u590d\u82cf\u65f6 37\u2103\u6c34\u6d74 1 \u5206\u949f\u878d\u5316\uff0c\u79bb\u5fc3\u540e\u7528\u9884\u70ed\u7684 RPMI-1640 \u57f9\u517b\u57fa\u91cd\u60ac\uff0c\u76f4\u63a5\u63a5\u79cd\uff0c\u907f\u514d DMSO \u5bf9\u6b63\u5e38\u7ec6\u80de\u7684\u6bd2\u6027\u5f71\u54cd\uff09<\/li>\n<\/ul>\n<ul>\n<li class=\"temp-li bullet1\" data-line=\"true\" data-list=\"bullet1\">\u7ec6\u80de\u8d27\u671f\uff1a\u73b0\u8d27\uff0c1 \u5468\u5de6\u53f3\uff08\u5e93\u5b58\u7ec6\u80de\u5747\u7ecf\u8fc7 RPMI-1640 \u57f9\u517b\u57fa\u9002\u5e94\u6027\u57f9\u517b\u3001\u6b63\u5e38\u4e0a\u76ae\u5f62\u6001\u9a8c\u8bc1\u53ca\u65e0\u81f4\u7624\u6027\u9884\u5b9e\u9a8c\uff08\u5982\u8f6f\u743c\u8102\u514b\u9686\u5f62\u6210\u5b9e\u9a8c\u9634\u6027\uff09\uff0c\u786e\u4fdd\u6838\u5fc3\u7279\u6027\u7a33\u5b9a\uff1b\u4e0b\u5355\u540e 48 \u5c0f\u65f6\u5185\u53ef\u5b89\u6392\u53d1\u8d27\uff0c\u53ef\u4e0e AGS \u540c\u6b65\u4e0b\u5355\uff0c\u4fdd\u969c \u201c\u6b63\u5e38 &#8211; \u80bf\u7624\u201d \u5e73\u884c\u5b9e\u9a8c\u7684\u65f6\u95f4\u4e00\u81f4\u6027\uff09<\/li>\n<\/ul>\n<ul>\n<li class=\"temp-li bullet1\" data-line=\"true\" data-list=\"bullet1\">\u8fd0\u8f93\u65b9\u5f0f\uff1a\u590d\u82cf\u53d1\u8d27\uff08T25 \u74f6\u514d\u8fd0\u8f93\u8d39\u7528\uff0c\u91c7\u7528\u9632\u9707\u6052\u6e29\u6ce1\u6cab\u7bb1\uff0c\u5185\u7f6e 2-8\u2103\u51b0\u888b\u53ca\u6e29\u5ea6\u76d1\u6d4b\u5361\uff0c\u8fd0\u8f93\u8fc7\u7a0b\u4e2d\u907f\u514d\u5267\u70c8\u98a0\u7c38\uff0c\u51cf\u5c11\u8d34\u58c1\u7ec6\u80de\u8131\u843d\uff1b\u5230\u8fbe\u540e\u7acb\u5373\u653e\u5165 37\u2103\u57f9\u517b\u7bb1\uff0c24 \u5c0f\u65f6\u540e\u89c2\u5bdf\u7ec6\u80de\u8d34\u58c1\u60c5\u51b5\uff0c\u65e0\u9700\u7279\u6b8a\u9759\u7f6e\u5904\u7406\uff0c3 \u5929\u5185\u9996\u6b21\u6362\u6db2\uff09\/ \u51bb\u5b58\u53d1\u8d27\uff08\u9700\u52a0\u5e72\u51b0\u8fd0\u8f93\u8d39\u7528\uff0c\u5e72\u51b0\u7528\u91cf\u6309\u8fd0\u8f93\u65f6\u957f\u8ba1\u7b97\uff0c\u56fd\u5185\u8fd0\u8f93\u4e00\u822c\u914d\u5907 500g-1kg \u5e72\u51b0\uff0c\u786e\u4fdd\u5230\u8fbe\u65f6\u5e72\u51b0\u5269\u4f59\u91cf\u226550%\uff0c\u7ef4\u6301\u7ec6\u80de\u51bb\u5b58\u72b6\u6001\uff09 \u987a\u4e30\u5feb\u9012\uff08\u4f18\u5148\u9009\u62e9\u6b21\u65e5\u8fbe\u670d\u52a1\uff0c\u7f29\u77ed\u8fd0\u8f93\u65f6\u95f4\uff0c\u964d\u4f4e\u7ec6\u80de\u6d3b\u6027\u635f\u5931\u53ca\u6c38\u751f\u5316\u7279\u6027\u6539\u53d8\u98ce\u9669\uff09<\/li>\n<\/ul>\n<ul>\n<li class=\"temp-li bullet1\" data-line=\"true\" data-list=\"bullet1\">\u4f9b\u5e94\u9650\u5236\uff1a\u4ec5\u9650\u4e8e\u79d1\u5b66\u7814\u7a76\uff0c\u7edd\u4e0d\u53ef\u4f5c\u4e3a\u52a8\u7269\u6216\u4eba\u7c7b\u75be\u75c5\u7684\u6cbb\u7597\u4ea7\u54c1\u4f7f\u7528\uff08\u4e0e AGS\u3001WPMY-1 \u7b49\u7ec6\u80de\u9650\u5236\u6761\u6b3e\u4e00\u81f4\uff0c\u4e25\u7981\u7528\u4e8e\u4e34\u5e8a\u6cbb\u7597\u3001\u7ec6\u80de\u6cbb\u7597\u3001\u7f8e\u5bb9\u53ca\u5176\u4ed6\u975e\u79d1\u7814\u7528\u9014\uff1b\u56e0\u542b SV40 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\u9700\u9009\u62e9\u80ce\u513f\u6765\u6e90\u6216\u4f4e\u4ee3\u6570\u725b\u8840\u6e05\uff0c\u907f\u514d\u6210\u4eba\u8840\u6e05\u4e2d\u590d\u6742\u56e0\u5b50\u5f71\u54cd\u80ce\u513f\u7ec6\u80de\u7279\u6027\uff1b\u57f9\u517b\u57fa\u5f00\u5c01\u540e 4\u2103\u4fdd\u5b58\uff0c1 \u4e2a\u6708\u5185\u4f7f\u7528\u5b8c\u6bd5\uff0c\u9632\u6b62\u8425\u517b\u6210\u5206\u964d\u89e3\uff1b\u2461\u4f20\u4ee3\u4e0e\u6362\u6db2\uff1a\u56e0\u589e\u6b96\u8282\u594f\u5e73\u7f13\uff0c\u4f20\u4ee3\u95f4\u9694\u5efa\u8bae 3-4 \u5929\uff08\u957f\u4e8e AGS \u7684 1 \u5929\uff09\uff0c\u4f20\u4ee3\u6bd4\u4f8b 1:2-1:3\uff08\u4f4e\u4e8e AGS \u7684 1:4-1:5\uff09\uff0c\u63a5\u79cd\u5bc6\u5ea6\u63a7\u5236\u5728 2\u00d710\u2075cells\/T25\uff0c\u786e\u4fdd\u7ec6\u80de\u8d34\u58c1\u540e\u7f13\u6162\u8fdb\u5165\u5bf9\u6570\u751f\u957f\u671f\uff0c\u7ef4\u6301\u6b63\u5e38\u4e0a\u76ae\u5f62\u6001\uff1b\u6362\u6db2\u9891\u7387\u5efa\u8bae 2-3 \u5929 1 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