癌相关成纤维细胞提取

外科手术切除恶性肿瘤组织块及离恶性肿瘤原发处两厘米左右的组织块，取下后放置于干净的封口膜袋内（或者无菌培养皿），然后放在含有冰袋的密闭盒内，为保证细胞提取成功率，一定要在术后尽快提取。

The cancer-associated regions were selected to be minimally necrotic regions of the tumor mass. Non-cancer associated stroma, which was isolated from tissue at least 2 cm. distal to the outer margin of the cancer mass, exhibited normal epithelial and stromal breast histology. Normal fibroblasts were extracted from the breast stroma of a sample obtained from a reduction mammoplasty. Tissues were digested with collagenase type I (1 mg/ml; Boehringer Mannheim) and hyaluronidase (125 units/ml; Sigma) at 37°C with agitation for 12-18 hrs in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal calf serum (FCS). The dissociated tissues were incubated without shaking for 5 min at room temperature, followed by the separation of stromal cell-enriched supernatant to a new tube. The stromal fraction was centrifuged at 250 X g for 5 min and the pellet was resuspended in DMEM with 10% FCS and the cells were cultured on tissue culture plates. These primary cultured fibroblasts were isolated in the same way, by tissue dissociation followed by differential sedimentation, plating, and growth in high serum media conditions which select for fibroblast growth. Each fibroblast was then expanded into two 15 cm petri dishes and stored as cells passaged for 2-3 population doublings (PDs) within total 8-10 days after tissue dissociation. We used fibroblasts passaged for up to 5 PDs for subsequent experiments, in order to minimize clonal selection and culture stress which could occur during extended tissue culture.

Collagenase Solution: 0.2% trypsin

0.2 % Collagenase A

5% FCS (fetal calf serum)

5ug/ml Gentamycin

47.5 mls DMEM/F12 media

细胞提取液配制：按照上述配方，47.5mlMEM培养液中加入0.1g 胰酶，0.1g胶原酶，无菌滤膜过滤后加入1ml双抗，提取细胞前再加入2.5ml胎牛血清。

拿到的组织块首先用酒精擦拭干净，用无菌PBS冲洗几遍，先在台子外操作，然后移入超净台内，将组织块置于PBS中（大号培养皿），使用高压灭菌过的镊子和剪刀将组织块剪成很小块，然后将小组织块收集至15ml离心管（提前加入了细胞提取液），置于培养箱中开始消化，中间要经常摇晃离心管，以利于快速消化。

1．消化4-5小时后，待组织块基本消化完全后，静止5min后，取上清液进行离心1300rpm 4min，然后将上清丢掉，细胞重悬于MEM+10%FBS中进行培养，两小时后换液去除血细胞等。同时对底部细胞进行提取

2．第二天待细胞贴壁长满后进行消化，重新置于新的培养皿中，通过差速贴壁的方法进行细胞的纯化和分离。