急需人软骨原代培养方法

骨道边

今天从手术室拿来那了一个人的股骨头要提取软骨细胞，不知道如何个取法？前几次都是手术刀切下来剪碎然后胰蛋白酶消化后再用二型胶原酶消化之后得到细胞用F12培养一直出不来，请高手们指点一下。谢谢了！

ytumuyangren

一、将软骨组织用手术刀切成小块，尽可能的小，加入0.15%的II 型胶原酶消化10-12h。) @+ I# i5 Z, u! f
二、 1000rpm/min离心5min。. R4 Z* r3 U( d  Z' B4 y; E
三、生理盐水冲洗，重复步骤二至少3次。" V5 I- L" G( \8 W  ~+ D  h, T; J
四、将细胞种植到培养瓶中，用含有10%胎牛血清的高糖DMEM培养。
/ o1 Y! z' M5 u不过用DF12的效果会更好一些，用DF12培养，细胞生长增殖的能力会快一些。祝你好运。呵呵！

valentiboa

回复 骨道边 的帖子1 h8 l' f5 w) X! q5 s' X
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这是我用来消化培养软骨细胞的方法，屡试不爽/ [+ T3 P8 u1 X+ J9 P
1.        The tissue was minced with scalpels, being taken care to exclude subchondral bone or connective tissue. This tissue was finely minced into 1 mm3 pieces. The pieces were then washed three times in sterile phosphate buffered saline.
" ^3 x# H  B/ k3 U2.        The tissues were transferred into a centrifuge tube and pre-digested in 10mL(per gram) alpha MEM(100U/mL penicillin, 100mg/mL streptomycin, 10% FBS) containing 25,000 IU/ml trypsin(1%), , for 15min in room temperature.
9 R( [4 R$ U; \  a8 J3.        After pre-digestion, the cartilage pieces were allowed to settle and the supernatant was discarded. The partially digested cartilage pieces were washed twice in MEM with 10%FBS to neutralize any remaining proteolytic enzymes and then subjected to overnight digestion with collagenase I and II, both at a concentration of 800IU/ml of MEM at 27℃ gives the optiamal combination of matrix digestion and cellular preservation.
4 I; z3 R( Q: e: M4.        After overnight digestion, the solution was filtered through a sterile nylon mesh to remove undigested debris. The filtrate was centrifuged at 400xg for 6min to pellet the cells. The supernatant was discarded and the pelleted cells were washed in MEM, and centrifuged at 400xg for 6min. The cells were then resuspended in 5 ml of the same media.
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jlzheng1224

valentiboa 发表于 2012-11-13 01:45
6 d: |) |% X' h% P+ i/ ^& A回复 骨道边 的帖子
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- {" l6 ?1 \2 m" i# n这是我用来消化培养软骨细胞的方法，屡试不爽

7 X$ M' F! n5 H2 x* Z' K8 e9 EThe tissues were transferred into a centrifuge tube and pre-digested in 10mL(per gram) alpha MEM(100U/mL penicillin, 100mg/mL streptomycin, 10% FBS) containing 25,000 IU/ml trypsin(1%), , for 15min in room temperature.5 p* F% d, t& `. a% X& r

& M) d4 O1 S: @5 |$ N2 v4 C您好，这一步同时加入胰酶和FBS，FBS不会抑制胰酶活性吗？？