1、原代內(nèi)皮細(xì)胞的分離與培養(yǎng)材料PBSDMEM/F12.青霉素 G (5000 U/mL) + 鏈霉素(5000 卩g/mL).內(nèi)皮細(xì)胞生長添加劑(ECGS).肝素.熱處理 FBS。56C, 30 min (seeNote 1).HUVEC培養(yǎng)基(Table 1)使用前加溫到室溫或37C。建議分裝到50-mL管，4C儲(chǔ)存， 直至使用。避免重復(fù)加溫和冷卻。膠原酶A (Sigma). 13mg/100mL溶解到無血清的DMEM/F-12,含青霉素和鏈霉素分別 50 U/mL 和 50 yg/mL。0.2-gm filter 過濾。應(yīng)該現(xiàn)用現(xiàn)配。胰蛋白酶EDTA.A5X儲(chǔ)備液(Table2) .0.2屮

2、m filter過濾消毒。以10-mL分裝入50-mL 管, -20C儲(chǔ)存。使用前加40 mL無菌去離子水稀釋到IX。4C保存可達(dá)到4周。紗布 (高壓滅菌).套管。雙向開關(guān)活塞。止血鉗。尼龍繩。手術(shù)刀片。燒杯。酒精。O.2-ym 濾膜。漂白粉。30-mL 注射器。0.2%明膠。，2%明膠儲(chǔ)備液1 mL稀釋到9 mL PBS制成0.2%明膠工作液10 mL。50-mL 管。25.100-mm 組織培養(yǎng)板 (Falcon).26. Ficoll-Paque (Amersham).HUVEC冷培養(yǎng)基。需要容量的45% FBS、5% HUVEC培養(yǎng)基和10%二甲基亞砜(DMSO) 混合，需要時(shí)現(xiàn)配。使

3、用前必須是冰冷的。抗體： 兔抗人vWF) (Dako),羊抗人VE-cadherin (Santa Cruz).31.4%多聚甲醛(Fisher)。在通風(fēng)櫥內(nèi)，加熱至l65e，每100mL PBS溶解4g多聚甲醛。逐滴加 入1 MNaOH直至溶液變清。用0.5 MHCI調(diào)整pH 7.0，S 4C儲(chǔ)存。Dil-conjugated 乙酰化的LDL(AcLDL)(Molecular Probes).(光敏感)。用無血清的MCDB稀 釋成 10yg/mL。DAPI(4,6-diamidino-2-phenyindole)(Sigma) (光敏感). 水溶解成2 mg/mL 的儲(chǔ)備液。儲(chǔ)備 液分裝，-

4、20e儲(chǔ)存。每10mL PBS溶解5yL儲(chǔ)備液制成l yg/mL的工作液。工作液避光儲(chǔ)存于 4C,最長2周。Table 1 Preparation of HUVEC MediumCcmpcncnLVoiunie added for 5(X) inLFinial concentrationFeta bovine semm100 mL20%Penicillin (5000 1 J/mL)-5 mL50 U/mL penicillinstrep itniycin (予000 pg/mL)50streptomycinEndothelial cell growth1 ml.20 |Jtg/rnLsupp

5、kmcnL (10 ing/mLlHeparin (8000 U/mL)1 mL.16 U/mLDMEM/F-12To final volume of 500mlTable 2 Preparation of Trypsin EDTA (5X)ComponentQuantity added for 200 ml.Final concentration (IX)Irypsin25 g0.25%EDTA (250 mA/)4 nil.1 m/WPBS (i OX)100 mLIXEk ionized waterTo final volume of 200 mLMethodsHUVEC的分離。下面的方

6、法7-10 d在100mm的培養(yǎng)皿融合成單細(xì)胞層。(seeNote 2).新鮮的臍靜脈(less than 24 h old)置于無菌容器，儲(chǔ)存于4C直至使用。(see Note 3).開始之前打開生物安全罩至少15 min,噴灑70%酒精風(fēng)干。準(zhǔn)備膠原酶溶液，預(yù)溫至37C。長13-20CM的臍帶需要約25 mL膠原酶溶液。在生物安全罩內(nèi)從容器取出臍帶。70%酒精輕微浸泡紗布，包裹臍帶的一端。輕輕捏住 紗布擦拭臍帶到另一端，清除表面血液和清除腔內(nèi)的凝血塊。(seeNote4).將導(dǎo)管插入臍靜脈的一端；用尼龍線在適當(dāng)位置固定導(dǎo)管。連接雙向開關(guān)到導(dǎo)管。關(guān)閉雙向閥。拔出注射器的活塞并把注射器連接到雙

7、向開關(guān)。25 mL PBS充滿注射器。 插入活塞，打開雙向閥并放臍帶的開口端在大廢物燒杯上。慢慢用PBS沖洗臍帶。沖掉紅 細(xì)胞和小血凝塊，直至洗出的液體變得清亮。(seeNote 5).關(guān)閉雙向閥，分離注射器，拔出活塞，重新連接注射器到雙向開關(guān)。不要在連接到注射 器時(shí)拔出活塞，這樣產(chǎn)生的真空就會(huì)把組織和血液吸入導(dǎo)管。用預(yù)溫的膠原酶溶液充滿注射 器。(seeNote 6)在插入活塞，打開雙向閥。用膠原酶溶液沖洗掉臍帶內(nèi)的PBS。用止血鉗夾 緊臍帶末端。慢慢用膠原酶充盈臍靜脈。一旦臍靜脈膨脹，關(guān)閉雙向閥，輕輕搓揉臍帶，使 膠原酶分布均勻。室溫放置30min。放明膠溶液進(jìn)入培養(yǎng)瓶以完全覆蓋底部。建議

8、：60-mm的培養(yǎng)皿3 mL，100 mm的7mL。在生物安全罩內(nèi)，室溫下靜置20 min。吸出明膠溶液并讓參與溶液蒸發(fā)直至無液體殘 留。一旦表面干燥蓋回蓋子。(see Note 7).輕輕搓揉臍帶擠出內(nèi)皮細(xì)胞。 (see Note 8).把鉗夾的一端放在50-mL管上。打開閥門，輕輕用剩余的膠原溶液沖出內(nèi)皮細(xì)胞。用3倍于臍帶內(nèi)的膠原溶液的PBS灌注臍帶，收集溶液在同一管內(nèi)。在一個(gè)吊桶式轉(zhuǎn)頭中，以4C離心細(xì)胞懸液，300g, 5min。在生物安全廚內(nèi)，用無菌巴斯德吸管吸出上清，把小細(xì)胞粒留在管中。15mL PBS重懸浮細(xì)胞粒并離心5min。輕輕的重新懸浮細(xì)胞粒在HUVEC培養(yǎng)基。懸浮容量依賴于

9、細(xì)胞粒的大小和選擇的培養(yǎng) 皿。例如，用20cm長的臍帶，以5-mL容量平鋪細(xì)胞在60-mm培養(yǎng)皿。兩個(gè)20cm長的臍帶， 以10-mL容量平鋪細(xì)胞在100-mm培養(yǎng)皿。平鋪細(xì)胞在之前用0.2%明膠包被的培養(yǎng)皿上。孵育培養(yǎng)皿在37C in 5% CO2+95% air.過夜孵育后，其培養(yǎng)基，用PBS洗，移除漂浮的紅細(xì)胞。加入新鮮的HUVEC培養(yǎng)基。應(yīng) 可見粘附細(xì)胞的集落。每3天更換新鮮培養(yǎng)基，直至融合的單細(xì)胞層形成。原代內(nèi)皮細(xì)胞的傳代培養(yǎng)Passaging Primary Endothelial CellsHUVECs一般最多能傳8-10代。使用前，PBS、胰蛋白酶-EDTA和HUVEC培養(yǎng)基應(yīng)

10、該加溫到室溫或37C。在溶合或接近溶合時(shí)，從原代培養(yǎng)的HUVECs培養(yǎng)皿內(nèi)吸出培養(yǎng)基。用5mLPBS洗 100-mm培養(yǎng)皿內(nèi)的單層細(xì)胞。(2mL, 60-mm) (see Note 13).吸出。力口2mL溶解于PBS的胰蛋白酶-EDTA，覆蓋單層細(xì)胞表面。37C 孵育細(xì)胞 1-3 min。顯微鏡檢查細(xì)胞是否分離，如果細(xì)胞呈圓形但沒有分離，可輕輕敲打培養(yǎng)皿使細(xì)胞分離。直接加5mL全成分HUVEC培養(yǎng)基，其的血清將消除胰蛋白酶的活性。把稀釋的細(xì)胞懸液移入明膠預(yù)包被的培養(yǎng)皿。用全成分HUVEC培養(yǎng)基把每個(gè)培養(yǎng)皿最終容量加至O8-10mL (see Note 14).孵育培養(yǎng)皿37C-5%CO2+9

11、5% air。每2-3 d更換培養(yǎng)基。原代內(nèi)皮細(xì)胞的低溫儲(chǔ)藏(Cryopreservation)推薦：盡可能傳幾代就要冷凍HUVECs，以確保儲(chǔ)備足夠量的低代細(xì)胞以備后續(xù)試驗(yàn)。用適當(dāng)數(shù)字標(biāo)記好冷凍管，包括冷凍日期和代數(shù)，并冰上預(yù)冷。選擇未完全融合的培養(yǎng)皿(最好是第二代HUVEC),向前面描述的用胰蛋白酶消化。懸浮 細(xì)胞在10 mL新鮮的H UVEC培養(yǎng)基，轉(zhuǎn)移到15-mL管。300g離心5min，重懸浮細(xì)胞在1mL HUVEC培養(yǎng)基。準(zhǔn)備細(xì)胞計(jì)數(shù)：轉(zhuǎn)移50yL細(xì)胞懸液在微離心管，加50yL 0.4%臺(tái)盼藍(lán)溶液。混合并用移液管加到細(xì)胞計(jì)數(shù)器的兩個(gè)小室，計(jì)數(shù)并計(jì)算細(xì)胞濃度。用5mL HUVEC培養(yǎng)

12、基加入試管，并300g離心5min。吸出大多數(shù)培養(yǎng)基并在冰上冷卻 (1-2min)。重懸浮細(xì)胞在冰冷的培養(yǎng)基，理想濃度： 5x 1051x 106/mL.按1-mL分裝至遇冷的標(biāo)記好的冷凍管。把冷凍管放在聚苯乙烯泡沫架上，在-80C冷凍24 h (see Note 15).轉(zhuǎn)移冷凍管至液氮nitrogen長期保存。解凍HUVEC培養(yǎng)基加溫至室溫或37C。從液氮中取出冷凍管，立即放在擱架上，37C水浴。把細(xì)胞轉(zhuǎn)至15-mL離心管，加10mL HUVEC培養(yǎng)基(逐滴)(see Note 15)。300g離心5 min，棄上清。4力口 8mL HUVEC培養(yǎng)基，輕輕吹打成單細(xì)胞懸液并鋪到100-m

13、m培養(yǎng)皿。537C孵育，更換培養(yǎng)基。Fig. 1.Cross-sectionofanumbilicalcorddisplayingthetwoarteries(left)andvein (right), which has a largerlumen. Note that the lowerartery is sectioned tangentially.Characterization of Primary Endothelial Cells特異性的內(nèi)皮細(xì)胞標(biāo)志包括:vWF, CD31, CD34, VE-cadherin, VEGF R1-2, AcLDL強(qiáng)攝 取, staining wi

14、th Ulex europaeus lectin type I, and morphology. The steps described in this subsection outline the procedure used to characterize the endothelial phenotype of primary endothelial cells, such as HUVECs.HUVEC形態(tài)的評(píng)估單層EC具有鵝卵石特征。(see Fig. 3 and Note 16).EC的免疫熒光染色vonWillebrandfactor,CD31 (PECAM-I),CD34, V

15、E-cadherin, and the VEGF receptors I and 2 (fit-I, KDR). 這些標(biāo)志物并不是內(nèi)皮細(xì)胞特異性的.胰蛋白酶消化的細(xì)胞(as described in Subheading 3.1.1.)，鋪至U無菌的多孑L玻璃板(usually 75,000 cells on a 4-well glass slide 1 d prior to staining).過夜孵育37C in 5%CO2+95% air.染色前細(xì)胞達(dá)到80%融合。用PBS洗一次。(每個(gè)腔加1mL,所有隨后洗的步驟都如此)。PBS配制4%多聚甲醛室溫固定細(xì)胞5 min。PBS 洗 1 次

16、。室溫下，甲醇固定(預(yù)冷至-20C) 1 min幫助透化。PBS洗兩次。含4%血清的PBS阻斷透化(see Note 17)，0.1%Triton X-100, 10min,室溫。準(zhǔn)備一抗溶液，用含4%血清的PBS和0.1%Triton X-100稀釋。(濃度根據(jù)制造商的詳細(xì)說 明)。同種型的免疫球蛋白用作陰性對(duì)照。室溫孵育細(xì)胞和一抗lh (see Note 18).含 4% 血清的 PBS 和 0.1%TritonX-100 洗2次。.準(zhǔn)備二抗溶液(連接熒光染料)在含4%血清的PBS和0.1%Triton X-100(通常1:64到1:100).避光孵育二抗30 min (see Note

17、19).含4%血清的PBS和0.1%TritonX-100洗 1 次。PBS 洗 2 次。用1卩g/mL DAPI-PBS孵育1min襯染細(xì)胞核。PBS 洗 2 次。加抗熒光衰減封片劑在蓋玻片上用指甲油封閉蓋玻片的邊緣。Visualization of stained cells is performed using fluorescein (for fluorescein isothiocyanate, Alexa 488) or rodhamine (for phycoerythrin PE, Alexa 594, Texas red) standard excitation/emissi

18、on filters (see Notes 20 and 21).3.4.3. AcLDL 攝 取用熒光染料 1,l-dioctadecyl-3,3,3,3-tetramethyl indocarbocyanine (Dil)標(biāo)記的 AcLDL 的攝取是 快速、方便的識(shí)別培養(yǎng)內(nèi)皮細(xì)胞的方法。然而，巨噬細(xì)胞也用這種方法檢測(cè)。胰蛋白酶消化的細(xì)胞(as described in Subheading 3.1.1.)，鋪至U無菌的多孑L玻璃板(usually 75,000 cells on a 4-well glass slide 1 d prior to staining).過夜孵育37C in 5

19、%CO2+95% air.在無血清的MCDB中，用10卩g/mL的有DiI連接的AcLDL (來自人類的血漿)孵育細(xì)胞4h。標(biāo)記后，用無探針的MCDB 1mL洗2次。然后用PBS 1mL洗2次。福爾馬林固定5 min。PBS1mL洗 兩次。用lyg/mL DAPI-PBS孵育1min襯染細(xì)胞核。PBS 洗2次加抗熒光衰減封片劑在蓋玻片上用指甲油封閉蓋玻片的邊緣。Visualization of cells that incorporate AcLDL can be performed using a rodhamine standard excitation/emission filter (

20、see Note 20).Notes1. It is very important that all material that comes in contact with cells be sterile and endotoxin-free because endotoxin interferes with normal cell proliferation and growth. The effects of endotoxin are not completely predictable and can actually artificially enhance cell cultur

21、e growth and activate endothelial cells. Because there can be variability between batches of FBS, it is advisable to test batches of FBS for the ability to support the viability and proliferation of HUVECs. The most sensitive indicators of FBS quality are provided by plating efficiency assays, where

22、as multiple passage tests are more reliable and correlate with long-term culture performance. The plating efficiency assay consists of inoculating cells at a density that will yield 100-300 discrete colonies per 100-mm tissue culture dish in HUVEC medium (made with test FBS). The dishes are incubate

23、d for 10-14 d until colonies of cells are visible. To count the colonies, the dishes are fixed (2% formaldehyde, 5 min), rinsed with water, stained with Coomassie brilliantbluedye(0.1%Coomassiein 10% aceticacid:50%methanol:40%water) and rinsed with wash solution (10% acetic acid:50% methanol:40% wat

24、er). The total number ofcolonies is counted and the relative plating efficiency ofthe test FBS lot is compared against reference FBS. The FBS lot qualification testing should be quantifiable and statistically relevant toensureinterpretativeobjectivity.Two common problems exist in the establishment o

25、fHUVEC primary cell cultures. First, bacterial and/or fungal contamination is a major cause of failure. The second problem relates to the freshness of the umbilical cord at the commencement ofprocessing. Cords should be handled within 24 h after delivery.Itisimportanttonotethat,aswithisolation ofany

26、cellsfromhumantissue,there is a potential risk ofinfection. Precautions for working with human tissues, such as wearing gloves, alaboratory coat, and safetygoggles, mustbe usedatall times.Discardcord iflengthislessthan 12cmorifthecordhascrushedareasorpierced segments, as it might result in a low HUV

27、EC yield. Occasionally, a cord may be severely clogged by clotted blood. Ifpossible, cut off the clotted section. Trim the ends with a scalpel blade to get even edges and proceed following the outlined protocol.Ifthe cord is clogged and the blood clot is not dislodged by flushing, discard the cord.

28、Do not force PBS through a clotted umbilical cord.Use only freshly prepared collagenase solution, as it tends to precipitate, and precipitates ofcollagenase can be cytotoxic.If gelatin-coated dishes are not be ingused immediately, they can be stored at 4C for up to 2 wk, provided that they remain st

29、erile by keeping them in a sealed bag.Do not overmassage, as it can increase contamination with fibroblasts. Massaging the whole length ofthe cord twice is usually sufficient.Umbilical cord blood is to be harvested in 200-mL plastic bottles containing 40 mL of IMDM medium containing 800 U/mL heparin

30、. The volume of cord blood collected per bottle should not exceed 120 mL, as the final concentration ofheparin should not decrease below 200 U/mL.It is important to turn off the brake of the centrifuge at this point, as breaking might disrupt the Ficoll interface.When harvesting the buffy coat inter

31、face band, avoid disturbing the red blood cell pellet to reduce red blood cell contamination. Moreover, do not collect more than 5 mLof Ficoll, as the Ficoll might make itdifficult to pelletthecells afterward.ItisimportantthatHUVECpassageisdoneregularlybecauseovergrowth ofthe cultures can induce cel

32、l cycle arrest ofthe cells and will have deleterious effects on the subsequentability ofthe cells to proliferate. We usually passage HUVECs in 100-mm tissue culture dishes. Working with three dishes allows two plates to be used for experimental studies and the remaining dish to be split 1:3 to repla

33、ce the original number. Usually, a typical HUVEC preparation can be splitfor up to 3-4 wk in this fashion before becoming senescent.When passaging the cells, it is important to remove the serum from the medium, as itinhibits trypsin activity. During trypsinization, itis important to monitorthe cells

34、carefullyto avoidexcessiveexposure to trypsin, as this mightresult in lower cell viability.HUVECsdonotgrowwell iftheyaresetupattoo Iowadensity.It isbesttoaim for a situation in which cells cover approx 40 to 50% of the surface area of the culture vessel at 24hafterplating. Usually, plating5x 105cell

35、sper 100-mmdish is a good starting point. Watch the cultures and adjust cell numbers accordingly.Cell viability can be severely compromised if the procedures for freezing and thawing are not carried out carefully. Placing the cryovials in a styrofoam rack ensuresthatfreezing occurs slowly andreduces

36、 celldeath. Similarly, when thawing the cells, it is important to add the medium slowly, as sudden dilution of DMSO can cause severe osmotic damage.Morphological identification (Subheading 3.4.1.) is not sufficient for the determination ofthe endothelial phenotype, as endothelialcells can change the

37、ir morphology depending on the growth supplements in the medium orthe matrix onto which the cells are seeded. Indeed, studies have shown that endothelial cells isolated from different organs or different-sized vessels can differ in their antigens, expression ofcellularadhesion molecules, metabolism,

38、 andgrowth requirements in culture. Itis therefore necessary to use acombination ofmarkers, visual identification, and/or functional assays to confirm the endothelial phenotype.Blocking serum from another species, which is not recognized by the secondary antibodies, should be chosen. Alternatively, another blocking agent such as immunohistochemical-grade bovine serum albumin (BSA) can be substi