Eccles:CB Cell Culture

Culturing CB Cells (Mouse Embryonic Kidney Cells)

Background

These cell lines were made from e18.5 mouse embryonic kidneys of various pkd1, pax2 and immorto genotypes.
Refer to (Making Primary Cells from Mouse Tissue protocol) for details on how this was done.
We were aiming for epithelial cells (particularly of the collecting duct) positive for the SV40 large T "immorto" gene that were either wt, pkd1-/-, pkd1-/- pax2+/- or pax2+/- in genotype. Some epithelial-like cells from mouse embryos that were immorto positive were cloned using cloning rings and bulked up at the permissive temperature of 33 degrees C in the presence of interferon-gamma. All cells stemming from cloned cells are designated with "ex cloned". During the first passage of cells not cloned we found that the fibroblast-like cells were more sensitive to trypsinisation than the epithelial-like cells meaning they would lift off after 2-3mins whereas the epithelial-like cells required 5mins to lift. To enrich for epithelial-like cell populations we performed differential trypsinisation. The lifted fibroblast-like cells were frozen down and are designated as "fib lift or lifted cells". The epithelial-like cells enriched by differential trypsinisation are designated with "differential lift eps"
All cell lines designated with "ex 33'C" have been grown at 33 degrees C with interferon-gamma (and are immorto positive). Those without this designation have been established at 37 degrees C only.
The cell lines that have been cloned are CB163E4 (IM+ pkd1-/- pax2+/+), CB167E3 (IM+ pkd1-/- pax2+/+) and CB167E2 (IM+ pkd1-/- pax2+/-). Note that the early passages are pre-cloning and contain a mixed population of cells. Remember all cells stemming from cloned cells are designated with "ex cloned". Future cloning can be performed on any of the passage 0 cells frozen down.
Some immorto positive cells have been grown at 33'C for various analyses that are not cloned such as the wt lines.
All cells were grown on collagen coated plates unless otherwise indicated eg: by "plastic". Some cells are designated with "2nd plating". This means that 24h-48h post initial plating of the cells the non-adhered cells were replated on a new cell culture vessel surface. The cells that successfully established were plated onto plastic (no collagen coating). These cells should now be grown on collagen coated cell culture vessels.

As of October 2007 - Lana Ellis.

Grow Cells on Collagen Coated Surfaces

Refer to (Collagen Coating of Cell Culture Vessels) for protocol.

Cell Culture Media used for mouse embryonic kidney cells (with additives)

Reagent	50mL	500mL	Final Concentration
DMEM/F-12 (1:1)	46.85mL
insulin-transferrin-selenium (100x)	375uL	3.75mL	0.75x
dexamethasone (20ug/mL)	250uL	2.5mL	100ng/mL
triiodotyronine (20ug/mL)	15uL	150uL	6ng/uL
murine EGF (0.1mg/mL)	5uL	50uL	10ng/mL
pen/strep	0.5mL	5mL
FCS	2.5mL	25mL	5%

Interferon-gamma

• When culturing cells in SV40 large T antigen up-regulation conditions (33 degrees C) add interferon-gamma to a final concentration of 10units/mL in cell culture media.
• Cells are grown at 33 degrees C with interferon-gamma at 5% CO₂. If you wish to down-regulate SV40 large T antigen expression, replace media with that not containing interferon-gamma and grow cells at 37 degrees C, 5% CO₂.

Cell Culture Vessel	Volume of stock IFN-gamma to add (1x104U/mL)	Final [IFN-gamma]	Media volume
24 well plate	5ul of 1/10 dilution of stock IFN-gamma	10U/mL	0.5mL
6 well plate	2uL	10U/mL	2mL
T25 flask	5uL	10U/mL	5mL
T75 flask	25uL	10U/mL	25mL
T175 flask	50uL	10U/mL	50mL

Trypsinisation

• Some of the cell lines are quite difficult to trypsinise.
• Aspirate media from cells in dish.
• Add warmed trypsin to cells and incubate at 33 degrees C for 5min.
• Pipette trypsin up and down in dish to remove cells from the dish surface and add to media (5% FCS, no additives), (3-4x volume of trypsin used) in 15ml tube. Can rinse dish with media and add to tube to maximise cell recovery.
• DO NOT trypsinise for longer. This destroys cell integrity. I have tried 7.5mins and 10mins and both led to the cells becoming sticky and sparse.
• Pellet cells at 250g, 5mins.
• Aspirate off media.
• Resuspend cells in suitable volume of warmed media + additives.
• Split cells 1:6 maximum.

Getting Cells up from Freeze Down

• Defrost cells by placing vial at 37 degrees C (water bath) until there is a small lump of ice left.
• Spray vial with ethanol, wipe and place vial in cell culture hood. Pipette contents into 5mls warmed media (5% FCS, no additives) in 15ml tube (by this time cells will be completely defrosted).
• Pellet cells at 250g, 5mins.
• Aspirate off freeze media/media mix.
• Resuspend cells in suitable volume of warmed media + additives and seed onto collagen coated dish.
• This method means you don't need to change media next day.

Handy Hint

• When inactivating trypsin or getting cells up from freeze down use DMEM/F-12 1:1 containing 5% FCS and pen strep so that you don't waste media with additives.

Media Additive Preparation

Insulin-Transferrin-Selenium (100x)

Insulin-transferrin-selenium, 10mL, Invitrogen #41400-045
Store at 4 degrees C
Ready to use

Dexamethasone (20ug/mL)

Dexamethasone, 1mg, Sigma #D8893
Lyophilised reagent stored at 4 degrees C

• Dissolve 1mg dexamethasone in 1mL of absolute ethanol to give a 1mg/mL solution
• Dilute 1:50 by adding 49mL media (DMEM/F-12 1:1 without additives) to give a final stock concentration of 20ug/mL
• Aliquot and store at -20
• Use between 20-200ng/mL final (50-500uL in 50mL media)

Triiodotyronine (20ug/mL)

Triiodotyronine, 1mg, Sigma #T5516

• Dilute 1mg in 1mL sterile 1M NaOH.
• Dilute 1:50 by adding 49mL media (DMEM/F-12 1:1 without additives) to give a final stock concentration of 20ug/mL
• Aliquot and store at -20
• Use between 0.6-6ng/mL final (1.5-15ul in 50mL media

Murine Epidermal Growth Factor

mEGF, 0.1mg, Sigma #E4127

• Add 1mL media containing 10% FCS (0.9mL DMEM/F-12 1:1 without additives + 0.1ml FCS)
• Aliquot into 6uL lots to avoid freeze/thawing
• Store at -20
• 0.1mg/mL stock solution is stable for up to 2 weeks at 4 degrees C once made up
• Use between 2-20ng/mL final (1-10ul in 50mL media)

Interferon-gamma, mouse recombinant

0.1mg, Sigma #I4777

After contacting Sigma technical help I identified that this batch of interferon-gamma contained not less than 1x10⁶ units
It seems that with any new batch of IFN-gamma ordered in one would need to check this (details didn't come with IFN-gamma)

• Make 1mg/mL solution in 1x PBS (0.1mg in 0.1mL of 1x PBS). This gives a 1x10⁷U/mL solution.
• Further dilute in DMEM/F-12 (1:1) no additives containing 10% FCS to 1x10⁵U/mL.
• Aliquoted into 100uL lots and stored at -20.
• Each time a 1x10⁵U/mL. aliquot is thawed it will need to be further diluted 1/10 to 1x10⁴U/mL in DMEM/F-12 (1:1) no additives containing 10% FCS.
• Aliquot 1x10⁴U/mL interferon-gamma and store at -20 to prevent repeated freeze/thawing.
• Once thawed the interferon-gamma solution is stable for 1 week at 4 degrees C