Protocols of cell culture and seeding cells on CytoVu

I got some experiences in dealing with cells. This is for your reference.

 Aseptic Technique and Good Cell Culture Practice 

1. Sanitize the cabinet using 70% ethanol before commencing work.
2.  Sanitize gloves by washing them in 70% ethanol
3.  Put all materials and equipment into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol.
4.  While working, do not contaminate hands or gloves by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol.
5. Equipment in the cabinet or that which will be taken into the cabinet during cell culture procedures (media bottles, pipette tip boxes, pipette aids) should be wiped with tissue soaked with 70% ethanol prior to use.
6. Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly.
7. After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue.
8. Sanitize the cabinet with UV light.

Make the media (10% FBS media) 

1. Autoclave a 250ml flask
2. The complete media consists of:

•                  250ml 1X DMEM (Cellgro: 10-013-CV)
•                  25ml FBS (Gibco: 26140-087)
•                  1ml L-Glutamine ( Gibco: 25030-081)
•                  1ml penicillin streptomycin (Gibco: 15140-122)

Use a Nalgene rapid-flow sterile disposable filter to put those contents in the flask. 

Thawing Cells

1. Pipette 9 mL of warmed complete media into the T75 flask.
2. Take the cryogenic vial from the liquid nitrogen and place in the 37℃ water bath to thaw.  Keep the cap above the water level, and move the vial through the water to aid in the thawing process.  Thawing should take less than a minute.
3. Pipette contents of the vial into the flask.
4. Rock the flask back and forth to distribute the cells.
5. Place the flask in the incubator.
6. Label T75 flask with cell type, passage number (this could be #0), date, and group member’s initials.

Changing Media 

1. Put the warmed complete media in the hood.
2. Aspirate the media in the flask using the glass Pasteur pipette.  Place the pipette a little away from the growth surface, to reduce the chance of sucking up cells.
3. Pipette 10 mL warmed complete media into the flask.
4. Close the lid and put the flask back in the incubator.

Passaging Cells (1:4 split)

Cell Culture Splits – a 1:4 indicates that one fourth of the cells are transferred into a new flask. There is no magic number or formula for how to do this.

1. Pipette 7.5 mL of warmed complete media into the T75 flask. Incubate the flask in the incubator for a while to achieve good CO2 content and pH.
2. View cultures using an inverted microscope to assess the degree of confluency and confirm the absence of bacterial and fungal contaminants.
3. Aspirate the media in the flask using the glass Pasteur pipette.
4. (Omitted) Wash the cell monolayer with 1-2 ml of PBS without Ca2+/Mg2+ (CMF-PBS).
5. Pipette trypsin onto the cells (eg. 1 ml for T25 flask, 2 ml for T75 flask). Rotate flask to cover the monolayer with trypsin and incubate for 3-4 minutes.  Check your flask and if the cells are not detached, incubate for another 2 minutes.
   • 0.25% Trypsin (Gibco: 15050-065)
   • Detached cells will have a rounded morphology and will float in the trypsin.
   • You can gently tap the flask to encourage the detachment process.
   • Cells should only be exposed to trypsin long enough to detach cells. Prolonged exposure could damage surface receptors
   • You may examine the cells using a microscope to ensure that many (~40%) the cells are detached and floating.
6. Pipette 8ml fresh medium to inactivate the trypsin. Pipette the solution up and down several times. (CAREFUL not to aspirate your media into the pipet aid)
7. Check the flask to ensure very few cells are left behind.  If there are remaining cells, wash the surface again with media.
8. Transfer the required number of cells (2.5ml for 1:4 split) to a new flask containing pre-warmed medium.
9. Pipette enough media to have a total of 10 mL of volume in your flask.
10. Rock the flask back and forth to distribute the cells in the flask, and then place in the incubator.
11. Label the flask with the cell and media type, passage number, cell split rate, date, and your initials.

Seeding cells on CytoVu 

1. Pre-condition the membrane by applying media (10% FBS), 25 μl to the basal wells and 10 μl to apical wells.
2. Replace lid and incubate at least 2 hours.
3. The same procedure with cell passaging (step 1-7). Suspended cells could be pipette to 15 mL conical vial.
4. Aspirate media using hand-held pipettes.
5. Add 10 μl of suspended cells to apical well.
6. Add 25 μl of media in basal well.
7. Replace lid and allow cells to attach to membrane.
8. Incubate to let cells grow to reach confluence. Change media every day. 

Results:

These are the pictures of cells on cytovu. Cells in 3 wells were alive. At first, the cells seemed to be very unhappy with the membrane. But things were better after day2 (I changed media every day).