人脑微血管内皮细胞查看源代码讨论查看历史
| 人脑微血管内皮细胞 |
人脑微血管内皮细胞是血脑屏障的主要组成成分，能够限制可溶性物质和细胞等从血液进入大脑。大脑微血管内皮细胞与外周内皮细胞相比具有一些相同特性。
目录
概括
细胞培养说明
概括
英文名：HBMEC (Human Brain Microvascular Endothelial Cells 如：
1）脑微血管内皮细胞存在许多细胞间紧密连接，产生很高的跨内皮阻抗，延迟细胞旁的通量；
2）脑微血管内皮细胞缺乏内皮细胞的窗孔结构，其液相物质胞饮水平较低；
3）脑微血管内皮细胞具有不对称定位酶和载体介导转运系统，从而产生 “两极分化”的表现型。 与外周内皮细胞相同，大脑微血管内皮细胞表面表达细胞粘附分子，调控白细胞进入大脑。由于微血管内皮细胞的器官特异性，内皮细胞通常取源于疾病研究的相关组织。
推荐培养基
The brain microvascular endothelial cells are the major element of the blood-brain barrier and comprises the primary limitation to passage of substances, both soluble and cellular, from the blood into the brain. Brain microvascular endothelial cells utilize unique features that distinguish themselves from those of peripheral endothelial cells such as 1) numerous intercellular'tight junctions' that display high transendothelial electrical resistance and retard paracellular flux ; 2) absence of fenestrae and a reduced level of fluid-phase endocytosis and 3) asymmetrically-localized enzymes and carrier-mediated transport systems that engender a truly 'polarized' phenotype . Like peripheral endothelial cells, however, brain microvacular endothelial cells express cell adhesion molecules on their surface that regulate the extravasation of leukocytes into the brain. In view of the organ specificity of microvascular endothelial cells, endothelial cells should be derived from the tissue involved in the diseases one wishes to study.
细胞培养说明
注意：冷冻保存的细胞非常脆弱。将小瓶置于37°C水浴融化细胞，然后尽快移入培养物(液)中，尽量减少操作。
准备
1.准备纤维连接蛋白包被的培养瓶(2 μg /cm2，推荐用T-75的培养瓶)。向培养瓶中加入10 ml无菌Dulbecco's磷酸盐缓冲液（DPBS），然后加入150 μl 纤维连接蛋白原液(1 mg/ml, Sigma cat. no. F1141)。将培养瓶放入培养箱中孵育过夜。
2.准备完全培养基：用70%的酒精消毒培养基和添加物的外表面，然后放到无菌的地方。在无菌环境下打开每一个添加物管并用吸管将其加入到基本培养基中。以保证添加物全部加入基本培养基中。
3.吸出纤维连接蛋白溶液并向瓶内加入20 ml完全培养基。将培养瓶放入超净台中，然后准备融化细胞。纤维连接蛋白溶液可以使用两次。
4.将小瓶放入37°C水浴中，握住并轻轻旋转小瓶直到其内细胞完全融化。将小瓶立刻移出水浴，擦干，用70%的酒精冲洗小瓶，然后放到无菌环境中。小心地打开盖子，注意手指不要碰到里面的螺纹。eppendorf吸管轻轻重悬管内容物。
5.将管内容物均匀的放入纤维连接蛋白包被的培养瓶中。推荐的接种密度为7,500 cells/cm2。
注意：不推荐在细胞融化后进行稀释和离心，因为这些操作比培养基中的DMSO残留物对细胞的伤害更大。需要注意的是，内皮细胞接种在纤维连接蛋白包被的培养瓶中能够促进细胞的帖壁。
6.盖好培养瓶的盖子，轻轻地摇动培养瓶以使细胞分布均匀，如需气体交换则适当放松瓶盖。
7.将培养瓶放入培养箱中。
8.为了得到良好的结果，在培养开始后至少16个小时内不要动培养物。第二天更换培养基以去除残留的DMSO和未贴壁的细胞，然后每隔一天进行如上操作。培养良好的细胞将呈现鹅卵石形或纺锤体形，细胞质无颗粒且细胞数量在培养2-3天后会倍增。
培养的维持
1.应用冰冻的细胞构建培养物后，可于次日早晨更换新鲜的含有添加物的培养基。随后的传代培养中，每隔48小时更换一次培养基。 2.此后每隔一天更换一次培养基，直到细胞约达到50%融合。
3.一旦细胞达到50%融合，则要每天更换培养基直到细胞大约达到90%融合。
传代培养：[1]
1.细胞达到90%融合时进行传代培养。
2.传代培养的前一天准备纤维连接蛋白包被的培养瓶(2 μg/cm2)。
3.将培养基，胰酶/EDTA消化液，胰酶中和液和DPBS(磷酸盐缓冲液) 加热至室温。我们不推荐在使用前用37°C水浴加热试剂和培养基。 4.用DPBS冲洗细胞。
5.用10 ml胰酶/EDTA消化液消化细胞(以T-75培养瓶为例)，直到80%细胞呈现圆形(在显微镜下观察)。立刻加入10 ml 胰酶中和液并轻轻摇动培养瓶。 注意：应用ScienCell研究室的胰酶/EDTA消化液能使由于过度消化导致的细胞死亡降到最小程度。
6.收取细胞并将其移入50ml离心管中。另外用10ml生长培养基冲洗培养瓶以收集残留的细胞。在显微镜下观察剩余细胞数量以确定是否收集成功。剩余细胞数应小于5%。
7.以1000转/分（1000rpm）离心收取的细胞悬液5分钟，然后在生长培养基中重悬细胞。
8. 计数细胞，然后将它们以推荐的细胞密度接种在新的纤维连接蛋白包被过的培养瓶中。
注意：
处理人类来源的产品存在潜在的生物风险。尽管每一株细胞经检测为艾滋病病毒，乙肝病毒，丙肝病毒阴性，但检测并不能达到100%准确，因此，必须采取适当的保护措施以避免无意中的暴露。操作这些产品时请带手套和安全镜。不要用嘴吸。我们推荐采用通用的程序来处理人源性产品，从而达到以最小的预防来对抗污染。
相关资料
Instruction for culturing cells Caution: Cryopreserved cells are very delicate. Thaw the vial in a 37oC waterbath and return them to culture as quickly as possible with minimal handling! Set up culture after receiving the order: 1. Prepare a fibronectin coated flask (2 μg/cm2, T-75 flask is recommended). Add 10 ml of sterile Dulbecco’s phosphate buffered saline (DPBS) to a T-75 flask and then add 150 μl of fibronectin stock solution (1 mg/ml, Sigma cat. no. F1141). Leave the flask in incubator overnight. 2. Prepare complete medium: decontaminate the external surfaces of medium and medium supplements with 70% ethanol and transfer them to sterile field. Aseptically open each supplement tube and add them to the basal medium with a pipette. Rinse each tube with medium to recover the entire volume. 3. Aspirate fibronectin solution and add 20 ml of complete medium to the flask. Leave the flask in the hood and go to thaw the cells. The fibronectin solution can be used twice. 4. Place the vial in a 37oC waterbath, hold and rotate the vial gently until the contents are completely thawed. Remove the vial from the waterbath immediately, wipe it dry, rinse the vial with 70% ethanol and transfer it to a sterile field. Remove the cap, being careful not to touch the interior threads with fingers. Using a 1 ml eppendorf pipette gently resuspend the contents of the vial. 5. Dispense the contents of the vial into the equilibrated, fibronectin-coated flask. A seeding density of 7,500 cells/cm2 is recommended. Note: Dilution and centrifugation of cells after thawing are not recommended since these actions are more harmful to the cells than the effect of DMSO residue in the culture. It is also important that endothelial cells are plated in fibronectin coated flask that promotes cell attachment. 6. Replace the cap or cover of flask, and gently rock the flask to distribute the cells evenly. Loosen cap if necessary to permit gas exchange. 7. Return the culture vessels to the incubator. 8. For best results, do not disturb the culture for at least 16 hours after the culture has been initiated. Change the growth medium the next day to remove the residual DMSO and unattached cells, then every other day thereafter. A healthy culture will display cobblestone or spindle shaped morphology, nongranular cytoplasm and the cell number will be double after two to three days in culture. Maintenance of Culture: 1. Change the medium to fresh supplemented medium the next morning after establishing a culture from cryopreserved cells. For subsequent subcultures, change medium 48 hours after establishing the subculture. 2. Change the medium every other day thereafter, until the culture is approximately 50% confluent. 3. Once the culture reaches 50% confluence, change medium every day until the culture is approximately 90% confluent. Subculture: 1. Subculture the cells when they are over 90% confluent. 2. Prepare fibronectin coated flasks (2 μg/cm2) one day before subculture. 3. Warm medium, trypsin/EDTA solution, trypsin neutralization solution, and DPBS to room temperature. We do not recommend warming the reagents and medium at 37oC waterbath prior to use. 4. Rinse the cells with DPBS. 5. Incubate cells with 10 ml of trypsin/EDTA solution (in the case of T-75 flask) until 80% of cells are rounded up (monitored with microscope). Add 10 ml of trypsin neutralization solution to the digestion immediately and gently rock the culture vessel. Note: Use ScienCell Research Laboratories’ trypsin/EDTA solution that is optimized to minimize the killing of the cells by over trypsinization. 6. Harvest and transfer released cells into a 50 ml centrifuge tube. Rinse the flask with another 10 ml of growth medium to collect the residue cells. Examine the flask under microscope to make sure the harvesting is successful by looking at the number of cells left behind. There should be less than 5%. 7. Centrifuge the harvested cell suspension at 1000 rpm for 5 min and resuspend cells in growth medium. 8. Count cells and plate them in a new, fibronectin-coated flask with cell density as recommended. Caution: Handling human derived products is potentially bioharzadous. Although each cell strain testes negative for HIV, HBV and HCV DNA, diagnostic tests are not necessarily 100% accurate, therefore, proper precautions mush be taken to avoid inadvertent exposure. Always wear gloves and safety glasses when working these materials. Never mouth pipette. We recommend following the universal procedures for handling products of human origin as the minimum precaution against contamination [1]. [1]. Grizzle, W. E., and Polt, S. S. (1988) Guidelines to avoid personal contamination by infective agents in research laboratories that use human tissues. J Tissue Culture Methods. 11