人腦微血管內皮細胞檢視原始碼討論檢視歷史
| 人腦微血管內皮細胞 |
人腦微血管內皮細胞是血腦屏障的主要組成成分，能夠限制可溶性物質和細胞等從血液進入大腦。大腦微血管內皮細胞與外周內皮細胞相比具有一些相同特性。
目錄
概括
細胞培養說明
概括
英文名：HBMEC (Human Brain Microvascular Endothelial Cells 如：
1）腦微血管內皮細胞存在許多細胞間緊密連接，產生很高的跨內皮阻抗，延遲細胞旁的通量；
2）腦微血管內皮細胞缺乏內皮細胞的窗孔結構，其液相物質胞飲水平較低；
3）腦微血管內皮細胞具有不對稱定位酶和載體介導轉運系統，從而產生 「兩極分化」的表現型。 與外周內皮細胞相同，大腦微血管內皮細胞表面表達細胞粘附分子，調控白細胞進入大腦。由於微血管內皮細胞的器官特異性，內皮細胞通常取源於疾病研究的相關組織。
推薦培養基
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.
細胞培養說明
注意：冷凍保存的細胞非常脆弱。將小瓶置於37°C水浴融化細胞，然後儘快移入培養物(液)中，儘量減少操作。
準備
1.準備纖維連接蛋白包被的培養瓶(2 μg /cm2，推薦用T-75的培養瓶)。向培養瓶中加入10 ml無菌Dulbecco's磷酸鹽緩衝液（DPBS），然後加入150 μl 纖維連接蛋白原液(1 mg/ml, Sigma cat. no. F1141)。將培養瓶放入培養箱中孵育過夜。
2.準備完全培養基：用70%的酒精消毒培養基和添加物的外表面，然後放到無菌的地方。在無菌環境下打開每一個添加物管並用吸管將其加入到基本培養基中。以保證添加物全部加入基本培養基中。
3.吸出纖維連接蛋白溶液並向瓶內加入20 ml完全培養基。將培養瓶放入超淨台中，然後準備融化細胞。纖維連接蛋白溶液可以使用兩次。
4.將小瓶放入37°C水浴中，握住並輕輕旋轉小瓶直到其內細胞完全融化。將小瓶立刻移出水浴，擦乾，用70%的酒精沖洗小瓶，然後放到無菌環境中。小心地打開蓋子，注意手指不要碰到裡面的螺紋。eppendorf吸管輕輕重懸管內容物。
5.將管內容物均勻的放入纖維連接蛋白包被的培養瓶中。推薦的接種密度為7,500 cells/cm2。
注意：不推薦在細胞融化後進行稀釋和離心，因為這些操作比培養基中的DMSO殘留物對細胞的傷害更大。需要注意的是，內皮細胞接種在纖維連接蛋白包被的培養瓶中能夠促進細胞的帖壁。
6.蓋好培養瓶的蓋子，輕輕地搖動培養瓶以使細胞分布均勻，如需氣體交換則適當放鬆瓶蓋。
7.將培養瓶放入培養箱中。
8.為了得到良好的結果，在培養開始後至少16個小時內不要動培養物。第二天更換培養基以去除殘留的DMSO和未貼壁的細胞，然後每隔一天進行如上操作。培養良好的細胞將呈現鵝卵石形或紡錘體形，細胞質無顆粒且細胞數量在培養2-3天後會倍增。
培養的維持
1.應用冰凍的細胞構建培養物後，可於次日早晨更換新鮮的含有添加物的培養基。隨後的傳代培養中，每隔48小時更換一次培養基。 2.此後每隔一天更換一次培養基，直到細胞約達到50%融合。
3.一旦細胞達到50%融合，則要每天更換培養基直到細胞大約達到90%融合。
傳代培養：[1]
1.細胞達到90%融合時進行傳代培養。
2.傳代培養的前一天準備纖維連接蛋白包被的培養瓶(2 μg/cm2)。
3.將培養基，胰酶/EDTA消化液，胰酶中和液和DPBS(磷酸鹽緩衝液) 加熱至室溫。我們不推薦在使用前用37°C水浴加熱試劑和培養基。 4.用DPBS沖洗細胞。
5.用10 ml胰酶/EDTA消化液消化細胞(以T-75培養瓶為例)，直到80%細胞呈現圓形(在顯微鏡下觀察)。立刻加入10 ml 胰酶中和液並輕輕搖動培養瓶。 注意：應用ScienCell研究室的胰酶/EDTA消化液能使由於過度消化導致的細胞死亡降到最小程度。
6.收取細胞並將其移入50ml離心管中。另外用10ml生長培養基沖洗培養瓶以收集殘留的細胞。在顯微鏡下觀察剩餘細胞數量以確定是否收集成功。剩餘細胞數應小於5%。
7.以1000轉/分（1000rpm）離心收取的細胞懸液5分鐘，然後在生長培養基中重懸細胞。
8. 計數細胞，然後將它們以推薦的細胞密度接種在新的纖維連接蛋白包被過的培養瓶中。
注意：
處理人類來源的產品存在潛在的生物風險。儘管每一株細胞經檢測為艾滋病病毒，乙肝病毒，丙肝病毒陰性，但檢測並不能達到100%準確，因此，必須採取適當的保護措施以避免無意中的暴露。操作這些產品時請帶手套和安全鏡。不要用嘴吸。我們推薦採用通用的程序來處理人源性產品，從而達到以最小的預防來對抗污染。
相關資料
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