原代内皮细胞的分离与培养

原代内皮细胞的分离与培养
材料
1. PBS
3. DMEM/F12.
5. 青霉素G (5000 U/mL) +链霉素(5000 μg/mL).
6. 内皮细胞生长添加剂（ECGS）.
7. 肝素.
8. 热处理FBS。

56°C，30 min (see Note 1).
9. HUVEC培养基(Table 1).使用前加温到室温或37°C 。

建议分装到50-mL 管，4°C储存，直至使用。

避免重复加温和冷却。

10. 胶原酶A(Sigma). 13mg/100mL 溶解到无血清的DMEM/F-12，含青霉素和链霉素分别50 U/mL 和50 μg/mL。

0.2-μm filter过滤。

应该现用现配。

11. 胰蛋白酶EDTA.A5X储备液（Table2）.0.2-μm filter过滤消毒。

以10-mL 分装入50-mL 管，-20°C储存。

使用前加40 mL无菌去离子水稀释到1X。

4°C保存可达到4 周。

12. 纱布(高压灭菌).
13. 套管。

14. 双向开关活塞。

15. 止血钳。

16. 尼龙绳。

17. 手术刀片。

18. 烧杯。

19. 酒精。

20. O.2-μm 滤膜。

21. 漂白粉。

22. 30-mL 注射器。

23. 0.2%明胶。

，2% 明胶储备液1 mL 稀释到9 mL PBS制成0.2%明胶工作液10 mL。

24. 50-mL管。

25.100-mm 组织培养板(Falcon).
26. Ficoll-Paque (Amersham).
29. HUVEC冷培养基。

需要容量的45% FBS、45% HUVEC培养基和10%二甲基亚砜(DMSO)混合，需要时现配。

使用前必须是冰冷的。

30. 抗体：兔抗人vWF) (Dako), 羊抗人VE-cadherin (Santa Cruz).
31. 4%多聚甲醛(Fisher)。

在通风橱内，加热到65°e，每100mL PBS溶解4g多聚甲醛。

逐滴加入1 M NaOH 直至溶液变清。

用0.5 M HCI调整pH 7.0 ，S 4°C储存。

32. Dil-conjugated 乙酰化的LDL(AcLDL)(Molecular Probes). (光敏感)。

用无血清的MCDB稀释成10μg/mL。

33. DAPI(4',6-diamidino-2-phenyindole)(Sigma) (光敏感). 水溶解成2 mg/mL 的储备液。

储备液分装，-20°e储存。

每10mL PBS溶解5μL储备液制成l μg/mL 的工作液。

工作液避光储存于4°C，最长2周。

Table 1 Preparation of HUVEC Medium
DMEM/F-12 To final volume of 500ml
Table 2 Preparation of Trypsin EDTA (5X)
Methods
HUVEC的分离。

下面的方法7-10 d 在100mm的培养皿融合成单细胞层。

(see Note 2).
1新鲜的脐静脉(less than 24 h old) 置于无菌容器，储存于4°C 直至使用。

(see Note 3). 2开始之前打开生物安全罩至少15 min，喷洒70%酒精风干。

3准备胶原酶溶液，预温至37°C。

长13-20CM的脐带需要约25 mL胶原酶溶液。

4在生物安全罩内从容器取出脐带。

70%酒精轻微浸泡纱布，包裹脐带的一端。

轻轻捏住纱布擦拭脐带到另一端，清除表面血液和清除腔内的凝血块。

(see Note4).
5将导管插入脐静脉的一端；
6用尼龙线在适当位置固定导管。

7连接双向开关到导管。

8关闭双向阀。

拔出注射器的活塞并把注射器连接到双向开关。

25 mL PBS充满注射器。

插入活塞，打开双向阀并放脐带的开口端在大废物烧杯上。

慢慢用PBS冲洗脐带。

冲掉红细胞和小血凝块，直至洗出的液体变得清亮。

(see Note 5).
9关闭双向阀，分离注射器，拔出活塞，重新连接注射器到双向开关。

不要在连接到注射器时拔出活塞，这样产生的真空就会把组织和血液吸入导管。

用预温的胶原酶溶液充满注射器。

(see Note 6)在插入活塞，打开双向阀。

用胶原酶溶液冲洗掉脐带内的PBS。

用止血钳夹紧脐带末端。

慢慢用胶原酶充盈脐静脉。

一旦脐静脉膨胀，关闭双向阀，轻轻搓揉脐带，使胶原酶分布均匀。

室温放置30min。

10. 放明胶溶液进入培养瓶以完全覆盖底部。

建议：60-mm 的培养皿3 mL，100 mm 的7 mL 。

在生物安全罩内，室温下静置20 min。

吸出明胶溶液并让参与溶液蒸发直至无液体残留。

一旦表面干燥盖回盖子。

(see Note 7).
11.轻轻搓揉脐带挤出内皮细胞。

(see Note 8).
12. 把钳夹的一端放在50-mL管上。

打开阀门，轻轻用剩余的胶原溶液冲出内皮细胞。

13. 用3倍于脐带内的胶原溶液的PBS灌注脐带，收集溶液在同一管内。

14. 在一个吊桶式转头中，以4°C离心细胞悬液，300g，5min。

15. 在生物安全厨内，用无菌巴斯德吸管吸出上清，把小细胞粒留在管中。

16. 15mL PBS重悬浮细胞粒并离心5min。

17. 轻轻的重新悬浮细胞粒在HUVEC培养基。

悬浮容量依赖于细胞粒的大小和选择的培养皿。

例如，用20cm长的脐带，以5-mL容量平铺细胞在60-mm 培养皿。

两个20cm长的脐带，以10-mL容量平铺细胞在100-mm 培养皿。

18. 平铺细胞在之前用0.2%明胶包被的培养皿上。

孵育培养皿在37°C in 5% CO2+95% air.
19. 过夜孵育后，其培养基，用PBS洗，移除漂浮的红细胞。

加入新鲜的HUVEC培养基。

应可见粘附细胞的集落。

20. 每3天更换新鲜培养基，直至融合的单细胞层形成。

原代内皮细胞的传代培养
Passaging Primary Endothelial Cells
HUVECs一般最多能传8-10 代。

使用前，PBS、胰蛋白酶-EDTA和HUVEC 培养基应该加温到室温或37°C。

1. 在溶合或接近溶合时，从原代培养的HUVECs培养皿内吸出培养基。

2. 用5mLPBS洗100-mm培养皿内的单层细胞。

(2mL，60-mm) (see Note 13).
3. 吸出。

4. 加2mL溶解于PBS的胰蛋白酶-EDTA，覆盖单层细胞表面。

5. 37°C孵育细胞1-3 min。

6. 显微镜检查细胞是否分离，如果细胞呈圆形但没有分离，可轻轻敲打培养皿使细胞分离。

7. 直接加5mL全成分HUVEC培养基，其的血清将消除胰蛋白酶的活性。

8. 把稀释的细胞悬液移入明胶预包被的培养皿。

9. 用全成分HUVEC培养基把每个培养皿最终容量加到8-10mL (see Note 14).
10. 孵育培养皿37°C-5%CO2 +95% air。

每2-3 d更换培养基。

原代内皮细胞的低温储藏（Cryopreservation）
推荐：尽可能传几代就要冷冻HUVECs ，以确保储备足够量的低代细胞以备后续试验。

1. 用适当数字标记好冷冻管，包括冷冻日期和代数，并冰上预冷。

2. 选择未完全融合的培养皿(最好是第二代HUVEC)，向前面描述的用胰蛋白酶消化。

悬浮细胞在10 mL新鲜的HUVEC培养基，转移到15-mL管。

3. 300g 离心5min，重悬浮细胞在1mL HUVEC培养基。

4. 准备细胞计数：转移50μL细胞悬液在微离心管，加50μL 0.4% 台盼蓝溶液。

5. 混合并用移液管加到细胞计数器的两个小室，计数并计算细胞浓度。

6. 用5mL HUVEC培养基加入试管，并300g离心5min。

7. 吸出大多数培养基并在冰上冷却(1-2min)。

8.重悬浮细胞在冰冷的培养基，理想浓度：5x 105~1x 106/mL.
9.按l-mL分装至遇冷的标记好的冷冻管。

10. 把冷冻管放在聚苯乙烯泡沫架上，在-80°C冷冻24 h (see Note 15).
11. 转移冷冻管至液氮nitrogen长期保存。

解冻
HUVEC培养基加温至室温或37°C。

1从液氮中取出冷冻管，立即放在搁架上，37°C水浴。

2把细胞转至15-mL 离心管，加10mL HUVEC培养基（逐滴）(see Note 15)。

3300g离心5 min ，弃上清。

4加8mL HUVEC培养基，轻轻吹打成单细胞悬液并铺到100-mm培养皿。

537°C孵育，更换培养基。

Arteries
Fig. 1.Cross-sectionofanumbilicalcorddisplayingthetwoarteries(left)andvein (right), which has a largerlumen. Note that the lowerartery is sectioned tangentially.
Characterization of Primary Endothelial Cells
特异性的内皮细胞标志包括:vWF, CD31, CD34, VE-cadherin, VEGF R1-2, AcLDL强摄取, staining with 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.
3.4.1. HUVEC形态的评估
单层EC具有"鹅卵石"特征。

(see Fig. 3 and Note 16).
3.4.2. EC的免疫荧光染色
vonWillebrandfactor,CD31 (PECAM-I),CD34, VE-cadherin, and the VEGF receptors I and 2 (fit-I, KDR). 这些标志物并不是内皮细胞特异性的.
1. 胰蛋白酶消化的细胞(as described in Subheading 3.1.1.) ，铺到无菌的多孔玻璃板(usually 75,000 cells on a 4-well glass slide 1 d prior to staining).
2. 过夜孵育37°C in 5%CO2+95% air. 染色前细胞达到80% 融合。

3. 用PBS洗一次。

(每个腔加1mL，所有随后洗的步骤都如此)。

4. PBS配制4% 多聚甲醛室温固定细胞5 min。

5. PBS洗1次。

6. 室温下，甲醇固定(预冷至-20°C) 1 min 帮助透化。

7. PBS洗两次。

8. 含4%血清的PBS阻断透化(see Note 17)，0.1%Triton X-100, 10min,室温。

9. 准备一抗溶液，用含4%血清的PBS和0.1%Triton X-100稀释。

(浓度根据制造商的详细说明)。

同种型的免疫球蛋白用作阴性对照。

10. 室温孵育细胞和一抗1h (see Note 18).
11.含4%血清的PBS和0.1%TritonX-100洗2次。

.
12. 准备二抗溶液(连接荧光染料)在含4%血清的PBS和0.1%Triton X-100(通常1:64到1:100).
13. 避光孵育二抗30 min (see Note 19).
14. 含4%血清的PBS和0.1%TritonX-100洗1次。

15. PBS洗2次。

16.用1μg/mL DAPI-PBS孵育1min衬染细胞核。

17. PBS洗2次。

18. 加抗荧光衰减封片剂在盖玻片上用指甲油封闭盖玻片的边缘。

19. Visualization of stained cells is performed using fluorescein (for fluorescein isothiocyanate, Alexa 488) or rodhamine (for phycoerythrin [PE], Alexa 594, Texas red) standard
excitation/emission filters (see Notes 20 and 21).
3.4.3. AcLDL摄取
用荧光染料1,l'-dioctadecyl-3,3,3',3'-tetramethyl indocarbocyanine (Dil)标记的AcLDL的摄取是快速、方便的识别培养内皮细胞的方法。

然而，巨噬细胞也用这种方法检测。

1. 胰蛋白酶消化的细胞(as described in Subheading 3.1.1.) ，铺到无菌的多孔玻璃板(usually 75,000 cells on a 4-well glass slide 1 d prior to staining).
2. 过夜孵育37°C in 5%CO2+95% air.
3.在无血清的MCDB中，用10μg/mL 的有DiI连接的AcLDL（来自人类的血浆）孵育细胞4h。

4. 标记后，用无探针的MCDB 1mL 洗2次。

然后用PBS 1mL洗2次。

5. 福尔马林固定5 min。

6. PBS1mL洗两次。

7. 用1μg/mL DAPI-PBS孵育1min衬染细胞核。

8. PBS 洗2次
9. 加抗荧光衰减封片剂在盖玻片上用指甲油封闭盖玻片的边缘。

10. Visualization of cells that incorporate AcLDL can be performed using a rodhamine standard excitation/emission filter (see Note 20).
4. Notes
1. 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 culture 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, whereas 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 incubated 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% water). 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.
2. Two common problems exist in the establishment ofHUVEC 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.
3. Itisimportanttonotethat,aswithisolation ofanycellsfromhumantissue,there is a potential risk ofinfection. Precautions for working with human tissues, such as wearing gloves, alaboratory coat, and safetygoggles, mustbe usedatall times.
4. Discardcord iflengthislessthan 12cmorifthecordhascrushedareasorpierced segments, as it might result in a low HUVEC 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.
5. Ifthe cord is clogged and the blood clot is not dislodged by flushing, discard the cord. Do not force PBS through a clotted umbilical cord.
6. Use only freshly prepared collagenase solution, as it tends to precipitate, and precipitates ofcollagenase can be cytotoxic.
7. If gelatin-coated dishes are not be ingused immediately, they can be stored at 4°C for up to 2 wk, provided that they remain sterile by keeping them in a sealed bag.
8. Do not overmassage, as it can increase contamination with fibroblasts. Massaging the whole length ofthe cord twice is usually sufficient.
9. Umbilical cord blood is to be harvested in 200-mL plastic bottles containing 40 mL of IMDM medium containing 800 U/mL heparin. 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.
10. It is important to turn off the brake of the centrifuge at this point, as breaking might disrupt the Ficoll interface.
11. When harvesting the buffy coat interface 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.
12. ItisimportantthatHUVECpassageisdoneregularlybecauseovergrowth ofthe cultures can induce cell 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 replace the original number. Usually, a typical HUVEC preparation can be splitfor up to 3-4 wk in this fashion before becoming senescent.
13. When passaging the cells, it is important to remove the serum from the medium, as itinhibits trypsin activity. During trypsinization, itis important to monitorthe cellscarefullyto avoidexcessiveexposure to trypsin, as this mightresult in lower cell viability.
14. 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 105cellsper 100-mmdish is a good starting point. Watch the cultures and adjust cell numbers accordingly.
15. 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 celldeath. Similarly, when thawing the cells, it is important to add the medium slowly, as sudden dilution of DMSO can cause severe osmotic damage.
16. Morphological identification (Subheading 3.4.1.) is not sufficient for the determination ofthe endothelial phenotype, as endothelialcells can change their 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, andgrowth requirements in culture. Itis therefore necessary to use acombination ofmarkers, visual identification, and/or functional assays to confirm the endothelial phenotype.
17. 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 substituted.
18. Both the incubation time and the required dilution of the primary antibody can vary withdifferentantibodies. Whenevera new primary antibody is used, adilution series should be performed to assay the optimal concentration for that antibody.Agoodstartingpointistouse 1:10, I:100,and 1:1000dilutions.Another seriesofdilutions(e.g., 1:50, I:100,and
I:200)shouldbeusedoncetheappropriate range is found.
19. Itisimportanttocovertheslideswithfoiltopreventexposuretolight,asfluorescent dyes are light sensitive.
20. Thechoiceoffiltertovisualizethefluorescenceisdependentonthefluorochrome conjugated with the secondary antibody. Some ofthe mostcommonly used fluorochromes are fluorescein isothiocyanate (FITC), tetramethyl rodhamine isothiocyanate (TRITC), Texas red, and DiI. The ideal filter to visualize FITC is excitation450-490 nm and barrier520-560nm. ForTRITC, Texas red, and DiI, the ideal filters combination are excitation 510-560 nm and barrier 590 nm.
21. Phenotypical characterization of endothelial cells can also be assessed by standard immunohistochemistry rather than by immunofluorescence if access to a fluorescent microscope is limited. In this case, the secondary antibody will be conjugatedwith horseradish peroxidase, and the antibody will be visualized with DAB staining.。