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.

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.

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