[박상규]BAE cells isolation , storage and Maintenance

1. BAE cells isolation , storage and Maintenance

 

1. MATERIALS REQUIRED

 

DMEM-20, sterile D-PBS with antibiotics, sterile razor blade, X1 trypsin-EDTA solution, 100Æ tissue culture, scissor, ice, 5L beaker, DMSO, 10ml disposable pipettes, and pipette aid.

a)      Bovine aorta: Freshly isolated from cow.

b)      Serum: heat inactivated Fetal bovine serum (Invitrogen Life Technologies catalog #16000-044).*

 

* Serum must be thawed and heat inactivated, unless this has already been done by the

manufacturer. Immerse the bottle of thawed serum in a 56°C water bath for 30 minutes,

making sure that the level of the water is the same as the level of the serum in the bottle.

Allow the serum to cool. Small aliquots can then be frozen for future use. Serum should be

filtered before use. The easiest way to do this is to add the appropriate amount of DMEM (with other components, if needed), and filter the medium through a 0.22 mM filter.

 

c)      Culture medium: DMEM-20: DMEM (Invitrogen Life Technologies catalog #12100-038) with L-glutamine and high glucose. DMEM with 3.7g/L sodium bicarbonate 80%; fetal bovine serum 20%.

d)     fungizone^TM :Invitrogen Life Technologies catalog # 15290-018.

e)      Penicillin-Streptomycin: Invitrogen Life Technologies catalog #15140-122.

f)       Dulbecco’s Phosphate-Buffered Saline: D-PBS; Invitrogen Life Technologies catalog #14190-144.

g)      1X Trypsin-EDTA: Invitrogen Life Technologies catalog # 25300-062.

h)      Freeze Medium: culture medium, 95%, DMSO, 5%

 

 

2. INSTRUMENTS

a)  Laminar flow hood for animal cell culture.

b)  Centrifuge capable of spinning at 200 x g for washing cells.

b)  Microscope.

c)  37°C, CO2 incubator with greater than 95% humidity.

d)  Liquid N₂ tank for cell stock storage.

 

3. DETAILED PROTOCOL

 

1.      Cut out 2 or 3 fresh aorta from cows (about 30cm each) and immerse immediately in ice- cold PBS containing 1% penicillin-stretomycin and 0.25ug/ml fungizone (sterile 3.5L buffer in 5L beaker). It is important to keeping 4 °C during transportation from slaughterhouse to laboratory.

 

NOTE: SUBSEQUENT PROCEDURES SHOULD BE PERFORMED IN A LAMINAR FLOW HOOD USING STERILE TECHNIQUE.

2.      Cut aorta lengthways with sterile scissor from the 5L beaker and wash inside of aorta 3 times with ice- cold PBS containing 1% penicillin-stretomycin and 0.25ug/ml fungizone.

3.      You can isolate endothelial cells by clearing from descending thoracic aortic surface using razor blade.

4.      Detach cells on the surface of razor blade by tapping on empty animal cell culture dish (100Æ) and add DMEM-20. 

5.      Repeat step 1-4 for every aorta prepared and pool isolated cells in a sterile 50ml tubes.

6.       Centrifuging at 200 x g for 5 minutes at room temperature and remove supernatant.  After washing, isolated cells are resuspended in DMEM-20% FBS, 1% antibiotics and divided into 100Æ culture dishes in ratio of 2 dishes per 1 aorta. Incubate cell culture dishes at 37°C in a CO2 incubator

7.      After overnight incubation remove media containing unattached cells and replenish with new media.

8.      When endothelial cells are grown about 80% of confluency after 5~6 days incubation (change with new medium every 2 days), remove medium, add fresh trypsin-EDTA solution, rinse, remove trypsin and let the culture sit at room temperature for 2 to 4 minutes**. Add fresh medium (These are passage 1 cells).

9.      Aspirate and dispense into 10 culture dishes.

10.  Repeat step 8 ~ 9 once or twice.  Sufficiently amplified endothelial cells are stored as frozen stock (We usually make stock with passage 3 cells) 

11.  For long term storage, after detachment of cells with trypsin-EDTA treatment and addition of medium, centrifuging at 200 X g for 5 minutes at room temperature and remove supernatant. Resuspend cell pallet in freeze medium and aliquot into cryogenic storage vials. (3 frozen stocks with nearly confluent cells from 100Æ dish)

12.  Cells are frozen slowly at 1°C/min.  This can be done by programmable coolers or by placing vials in an insulated box in a –70°C to –90°C freezer, then transferring to liquid nitrogen storage.

13.  Remove cells from storage and thaw quickly in a 37°C water bath.

14.  Place 1ml of frozen cells in 10ml of growth medium.  Mix very gently.

15.  Centrifuge the cells at 100 X g for 3min and discard supernatant.

16.  Resuspend and plate cells.

17.  Subcultivation ratio: A subcultivation ratio of 1:3 to 1:5 is recommended.( Medium renewal: Every 2 days)

18.  For bioactivity assay cultured cells between passage 5 and 10 are used.

 

 

** Minimize exposure to trypsin.