人主动脉内皮细胞(HAOEC)培养方案

I. 储存

A. 冻存管 (304-05aS304-05a)

收到后立即将冻存管保存在液氮储存罐中。

*从液氮储存罐中取出冻存管时,务必佩戴防护面罩和手套。从液氮罐到房间的剧烈温度变化可能会导致冻存管中滞留的液氮爆炸并造成伤害。

304-05a-cells

人主动脉内皮细胞(HAOEC)

 

II. 细胞培养准备工作

1. 确保具有HEPA过滤层流的II级生物安全柜处于良好的工作状态。

2. 使用70%酒精对生物安全柜进行消毒。

3.  开始细胞培养工作之前,提前10分钟打开生物安全柜吹风机。

4.  确保所有血清学移液器、移液管和试剂溶液都是无菌的。

5.  遵守标准无菌技术和安全规则:

a. 不要用嘴吸移液管。
b.在处理人类细胞时始终佩戴手套和护目镜,即使所有菌株均已经过测试为HIV、乙肝和丙肝病毒阴性。
c.在无菌超净台内处理所有细胞培养工作。

 

III.  HAOEC培养

A. 为HAOEC培养准备细胞培养瓶

1.   从冰箱中取出 内皮细胞生长培养基(211-500)。在无菌超净台内使用70%酒精对培养基瓶身消毒。

1.   吸取15 ml内皮细胞生长培养基(211-500)。*置于T-75培养瓶 (SIAL0641)中。
*保持培养基与表面积的比例为1ml:5 cm2
例如:
- 一个 T-25培养瓶 (SIAL0639)或一个60 mm组织培养皿 (SIAL0166)使用5ml培养基。
- 一个 T-75培养瓶 (SIAL0641)或一个  100 mm组织培养皿 (SIAL0167)使用15ml培养基。

 

B. 解冻和接种HCF

1. 佩戴合适的护目镜和手套,从液氮罐中取出HCF冻存管。

2. 将管盖旋开四分之一周,将可能滞留在管盖螺纹处的液氮释放出来,然后再次拧紧管盖。

3.  迅速将冻存管下半部分放在37°C水浴中解冻细胞,并在解冻过程中密切观察冻存管。

4.  当冻存管内仅剩下少量冰时,从水浴中取出冻存管。不要让细胞完全解冻

5.  在无菌生物安全柜中,用70%酒精对管身消毒。

6.  小心取下管盖。不要触碰管盖或冻存管的边缘,以避免污染。

7.  使用2 ml移液管轻轻吹打管内细胞5次,使细胞重悬。小心不要吹打过猛,以免产生泡沫。

8.  用移液管从冻存管中取出细胞悬液(1ml)并轻轻转移至装有15 ml 内皮细胞生长培养基(211-500)的 T-75培养瓶 (SIAL0641)中中。

9.  盖上培养瓶盖并轻轻摇动,使细胞均匀分布。

10. 将 T-75培养瓶 (SIAL0641)中放在37oC、5% CO2恒湿培养箱中。松开瓶盖,以便气体交换。为获得最佳结果,在接种后24小时内不要挪动培养物。

11. 接种24小时后或过夜后,更换新鲜的内皮细胞生长培养基(211-500),以去除所有残留的DMSO。

12. 每隔一天更换一次内皮细胞生长培养基(211-500),直至细胞达到60%融合。

13. 当培养细胞融合率达到60%以上或周末无换液培养时,将内皮细胞生长培养基(211-500)用量加倍。

14. 当HCAEC达到85融合时,进行细胞传代。

IV. Subculturing HAOEC

A. Preparing Subculture Reagents

  1. Remove the Trypsin-EDTA solution (T3924) and Trypsin Inhibitor (T6414) from the -20°C freezer and thaw overnight in a refrigerator.
  2. Make sure all the subculture reagents are thawed. Swirl each bottle gently several times to form homogeneous solutions.
  3. Store all the subculture reagents at 4°C for future use.
  4. Aliquot Trypsin/EDTA solution (T3924) and store the unused portion at -20°C if only a portion of the Trypsin/EDTA (T3924) is needed.

B. Preparing Culture Flask

  1. Take the Endothelial Cell Growth Medium (211-500) from the refrigerator. Decontaminate the bottle with 70% alcohol in a sterile hood.
  2. Pipette 30 ml of Endothelial Cell Growth Medium (211-500) to a T-175 flask (SIAL1080) (to be used in Section IV C Step 15.)

C. Subculturing HAOEC

Trypsinize Cells at Room Temperature. Do Not Warm Any Reagents to 37°C.

  1. Remove the medium from culture flasks by aspiration.
  2. Wash the monolayer of cells with HBSS (H6648) and remove the solution by aspiration.
  3. Pipette 5 ml of Trypsin/EDTA Solution (T3924) into the T-75 flask (SIAL0641). Rock the flask gently to ensure the solution covers all the cells.
  4. Remove 4.5 ml of the solution immediately.
  5. Re-cap the flask tightly and monitor the trypsinization progress at room temperature under an inverted microscope. It usually takes about 1 minute for the cells to become rounded.
  6. Release the rounded cells from the culture surface by hitting the side of the flask against your palm until most of the cells are detached.
  7. Pipette 5 ml of Trypsin Inhibitor Solution (T6414) to the flask to inhibit further tryptic activity.
  8. Transfer the cell suspension from the flask to a 50 ml sterile conical tube.
  9. Rinse the flask with an additional 5 ml of Trypsin Inhibitor Solution (T6414) and transfer the solution into the same conical tube.
  10. Examine the T-75 flask (SIAL0641) under a microscope. If there are >20% cells left in the flask, repeat Steps 2-9.
  11. Centrifuge the conical tube at 220 x g for 5 minutes to pellet the cells.
  12. Aspirate the supernatant from the tube without disturbing the cell pellet.
  13. Flick the tip of the conical tube with your finger to loosen the cell pellet.
  14. Resuspend the cells in 2 ml of Endothelial Cell Growth Medium (211-500) by gently pipetting the cells to break up the clumps.
  15. Count the cells with a hemocytometer or cell counter. Inoculate at 10,000 cells per cm2 for rapid growth, or at 5,000 cells per cm2 for regular subculturing.

Materials