原代培养中成纤维细胞的抑制

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发布日期：2012-02-16 15:35 文章来源：丁香园
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关键词： 丁香园 生物专题 义翘神州 细胞培养 成纤维细胞 　 点击次数：

材料与方法：

材料 :

1. 培养皿、培养瓶、烧瓶、试管，玻璃针等。

2. 眼科剪、眼科镊、手术刀片。

3. 5%CO₂孵箱、PH计。

4. 恒温水浴箱。

5. RPMi1640培养基，D-Hank’s缓冲液 、胎牛血清，内皮生长因子（ECGF），青霉素，链霉素,戊巴比妥钠等。

方法：

1.取材：4～6w Wistar大鼠，大剂量2%戊巴比妥钠麻醉处死，将处死后的大鼠浸入75%酒精中，放入超净台。在无菌状态下剪开胸腹腔，分离胸主动脉，用2ml注射器从主动脉弓处向胸主动脉注入D-Hank’s液,驱除残血,取主动脉弓至肾动脉一段，两端结扎剪断，放入60℃预热无菌水中2秒。然后置于准备好的RPMi1640培养基（含双抗）中。

2.剪切：在超净台上，将血管置于培养皿中,用眼科镊小心剥离外膜面的结缔组织,并从根部剪去所有肋间动脉。无血清培养基冲洗数遍，去除残血后，将动脉转移至另一含少量培基的无菌培养皿中，用刀片将主动脉两末端切去,剩下的主动脉切成宽1～1.5mm的环。

3.接种：将动脉环竖直放入35mm培养皿(1%明胶4℃预置过夜，用前2h移入CO₂ 培养箱,用前培养液冲洗)中。置CO₂ 培养箱2h后，加入1.5ml培养基。放入37℃，5%CO₂ 培养箱中培养。72h后细胞从环内外生长迁出,环内为内皮细胞，环外为成纤维细胞。将动脉环除去后，可看到内膜面细胞集落与外膜面之间有明显的无细胞区界限，用玻璃针剔除成纤维细胞，得到纯的内皮细胞生长克隆。继续培养10～15d，此时FBS浓度为20％，形成细胞单层。

4.置于5%CO₂孵箱中培养，每隔3天换液，培养时添加15% FBS，5～10μg/ml的内皮细胞生长因子（ECGF）以及100μg/ml的肝素。待细胞90%~95%融合时，0.25胰蛋白酶消化传代。

5.内皮细胞鉴定：相差显微镜下,细胞呈单层铺路石状；免疫组化证明有vWF相关抗原存在

6.注意事项：将分离出来的主动脉热处理时温度以60℃ 2秒为宜，太低则成纤维细胞多，太高或时间久则内皮细胞迁出减少。

【tomandmikes】如果原代培养后，脐静脉内皮细胞和成纤维细胞并存，如何消除后者？尽管通过消化时间的调整可以避免成纤维细胞的存在，但是消化时间过短，收集的细胞数量较少，过长又会增加成纤维细胞污染的机会。

然而一旦污染之后，如何分离纯化内皮细胞呢？有文献报道，可以用消化法，根据消化时间的不同，来筛选内皮细胞，我一知半解，还请高手进来指教。

【Goodluck_XD】Simple, fast and efficient mouse endothelial cell isolation with Dynabeads. Isolated cells are pure and free of contaminating fibroblasts

Introduction

Endothelial cells line the entire vascular system, from the heart to the smallest capillary and control the passage of materials and the transit of leucocytes into and out of the bloodstream. Leucocytes interact with endothelial cells during trafficking, immune responses, inflammatory reactions and haemostasis. A pure population of cells, free from contaminating fibroblasts is required to study endothelial cells. Murine tissue is digested to obtain a single cell suspension. Dynabeads coated with an anti-murine endothelial cell antibody are added directly to the cell suspension and the bead-captured cells are separated with a magnet, Dynal MPC.

1.Endothelial cell isolation from murine tissues

A rapid, reproducible method for the isolation of murine tissue endothelial cells (EC) has been developed by Dong et al (1) using Dynabeads M-450 Sheep anti-Rat IgG coupled with a monoclonal rat anti-mouse CD31 antibody. After releasing the beads with trypsin/EDTA, the released cells were centrifuged and resuspended in growth medium for cell culture and further cloning.

Of 300 cells plated, 29 clones were obtained after two weeks and all clones were positive for CD31. To evaluate the specificity and recovery efficiency of this method, the CD31+ murine endothelial cell line H5V was mixed with a melanoma cell line and/or mixed with L929 fibroblasts. In both cases the percentage of isolated cells was > 98% and the recovery was 65% of the original CD31+ murine endothelial cells.

2.Primary mouse lung endothelial cell isolation from cultures

Hartwell et al (2) used lung tissue as plated cells. Dynabeads M-450 Sheep anti-Rat IgG were coated with rat anti-mouse intercellular adhesion molecule-2 IgG and added to the plated cells. After incubation, the cells were trypsinized to release all cells before isolation with the Dynabeads. Endothelial cells were stained for P-selectin, an adhesion receptor for leucocytes.

3.Pure fibroblast and endothelial cell colony cultures from murine bone marrow

The adherent stromal cells of bone marrow consist of endothelial cells, fibroblasts and macrophages. Endothelial cells and macrophages are phagocytic but fibroblasts are not. These differences can be used to isolate the different cell populations. Fei et al (3) used magnetic separation to obtain cultures of EC and fibroblasts (3). Bone marrow cells were cultured and non-adherent cells poured off. The adherent cells were subsequently used in two ways:

i) To obtain endothelial cells, adherent cells were released from the culture plates with trypsin and Dynabeads M-450 Epoxy were added at the ratio 3 beads to 1 cell and incubated for 2 hours at 37°C. At this temperature, phagocytic cells in culture engulf the beads. The bead-captured cells were collected with the magnet and non-phagocytic fibroblasts discarded. The bead/cell complexes were then cultured in low concentration for 10 days to reduce macrophage growth. Endothelial cell clones (> 20 cells) were then picked and cultured again to confluent growth.

ii) To achieve pure fibroblasts more magnetic beads were used. A 40 bead to 1 cell ratio was added to trypsinized adherent cells to ensure all phagocytic cells (endothelial cells and macrophages) engulf beads. The bead-captured cells were collected and the non-phagocytic fibroblasts were collected and cultured. The fibroblasts were re-passaged to ensure there were no contaminating cells and then grown to confluence.

1.Q. G. Dong, et al., A general strategy for isolation of endothelial cells from murine tissues -Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. Arterioscler.Thromb.Vasc.Biol. 1997; 17:1599-1604.

2.D. W. Hartwell, et al., Role of P-selectin cytoplasmic domain in granular targeting in vivo and in early inflammatory responses. Journal of Cell Biology 1998; 143 4:1129-1141.

3.R-G. Fei, et al., A method to establish pure fibroblast and endothelial cell colony cultures from murine bone marrow. Exp.Hematol. 1990, 18:953-957.

【zhaoqingshan】Goodluck_XD 斑竹的解决方案可能比较费银子。

我也曾遇到过类似的问题，一般来说，原代的时候一定要尽可能的纯，宁可错杀一千，不可放过一个成纤维细胞，否则传代后细胞就会比较杂。所以原代不够纯就放弃。另外，给足生长因子让内皮充分的生长起来。

以前一个老师提供过采用胶原酶的方法，差速消化，不知道是否有效，楼主可以试试，有结果告诉我。另外，有些差速贴壁的方法，好像并不合适，因为内皮贴壁也很快的。

我也是听说的，并不可靠啊，您最好试验一下，就是用0.1%I型或II型胶原酶消化，试试，据说成纤维细胞会先掉下来。要是有结果，一定要告诉我啊！呵呵！

说实话，我养过好几种内皮，就是没有正儿八经的养过HUVEC，帮别人养过几次，纯度还可以(其实消化得好的话，不存在杂细胞的问题的，传2-3代进行实验没有问题的)，就是传不了几代。

【tomandmikes】是啊，纯度的问题好说，可以摸索控制时间，但是要不加生长因子等刺激物，仅用培养基和血清，很难多次传代。其实消化后的成纤维细胞不是很多，但见了很烦，我且试试你说的方法。不知道用胰酶可以不可以？

【zhaoqingshan】应该是不可以的，我试过，换低浓度的胰酶也不可以的。内皮和成纤维被消化下来的时间差不多。用ECGF 20ug/ml也不是很贵的，我倒觉得胎牛太贵了。因为我以前养脑微血管内皮用ECGF 150ug/ml。

混合星形细胞

【midas】混合星形细胞的培养

1、取材：取新生4天SD大鼠，颈动脉放血，无菌操作取出 脑组织，切除脑干和海马，解剖显微镜下仔细剔除脑膜和血管等结 缔组织。

2、机械分散：取大脑皮质转移至试管中，并将其快速剪成 1mm³ 左右小块，加适量培养液，用吸管轻轻吹打，使细胞分散，制成细 胞悬液，再经74 um网筛过滤，取滤液，离心，1500r/min, Smin，重悬细胞，进行细胞计数。

3、细胞计数：台盼蓝:细胞悬液=1: 9染色，在显微镜下计数。

4、接种与培养：调整细胞密度，以1 X 10⁵/cm²的密度接种于 预先涂有多聚赖氨酸(poly-L-lysine, PLL)的培养瓶中，于培养 箱中孵育30min,翻转瓶并吸出细胞悬液再接种于培养瓶中，以除去成纤维细胞。370C，5% CO₂ 培养，每3天换液一次，培养10天。倒置相差显微镜下观察细胞形态细胞，见细胞分为两层：层是 TIA，上层是0-2A祖细胞。

人胚嗅鞘细胞

【lvranbo1010】 人胚嗅鞘细胞的原代培养

将胚胎头部剪下，浸泡在75％的酒精内3分钟，大量D-Hanks平衡盐溶液冲洗。手术显微镜下，超净台内，迅速完整取出两侧嗅球，置于4℃的D-Hanks平衡盐溶液中，小心除去软脑膜，血管及与其相连的组织。然后用显微器械剪取嗅球最外两层(嗅神经层和小球层，ONGL)，再用D-Hanks平衡盐溶液洗3遍。置于含3－4ml DMEM/F12培养基的35mm²培养皿内，将剪取的ONGL组织块用显微器械小心撕开，再用火焰抛光后的移液管小心反复吹打至尽可能小（勿使培养基产生过多的气泡），放入CO₂培养箱(5%CO₂,37℃)中培养。

编辑: cq

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