Baltimore PKD Center - Core D
Cell Culture Protocols and Handling
Instructions For Pkd1V/V and Pkd1WT Clonal Cell Lines
Drs. Erik Schwiebert and Eric Seales
Revised January, 2013
Background on the Development of Pkd1V/V and Pkd1WT Clonal Cell Lines: We have
developed a panel of clonal cell lines in collaboration with Feng Qian (Core B). We performed
clonal selection on immortalized and lectin- immunoselected cell cultures from a mouse model
of PKD bearing a mutation at the GPS cleavage site of polycystin 1 (PKD1, PCL1) and from wildtype (WT) littermate controls ( Pkd1V/V and Pkd1WT, respectively).
Specific information about these panels of clonal mouse renal epithelial cell lines is provided
below:
There are 8-24 clonal cell lines for each series below and they are currently being provided to the
Research Base for further testing, while Dr. Feng Qian’s lab and Core D also engage in
characterization. The clones are numbered – it is imperative that you keep track of clone number
and culture each clone in parallel.
DBA-WT clonal cell lines are distal convoluted tubule/collecting duct cell lines from wild-type
(WT) mouse littermates.
DBA-WT ‘Mono’ clonal cell lines are distal convoluted tubule/collecting duct cell lines from wildtype (WT) mouse littermates that were derived/cloned on permeable filter supports to maintain
the ability to form well polarized epithelial cell monolayers on filter supports. Only this culture
had a phenotype when received from the investigator’s lab that suggested that such cell lines
could be derived.
DBA-v/v-2 clonal cell lines are distal convoluted tubule/collecting duct cell lines that bear the
PCL-1 GPS cleavage mutant (Pkd1V/V).
LTL-WT clonal cell lines are proximal tubule cell lines from WT mouse littermates.
LTL-v/v-2 clonal cell lines are proximal tubule cell lines that bear the GPS cleavage mutant
(Pkd1V/V).
Protocols for Pkd1V/V and Pkd1 WT Cell lines
These immortalized cell cultures and clonal cell lines were derived from the kidneys of Pkd1V/V
and Pkd1WT mice that were bred to a mouse line bearing the temperature sensitive SV40 large
T antigen (immortomouse ® bred to homozygosity). immortalized clonal cell lines that also
require interferon-gamma selection or not.
When expanding the cell cultures for experimentation and/or cryopreservation, these clonal cell
lines need to be grown at 32-33 oC with interferon gamma to maintain immortalization. Please
see the following paper for more details regarding this method: Jat PS, Noble MD, Ataliotis P, et
al. Direct derivation of conditionally immortal cells lines from H-2kb-tsA58 transgenic mouse.
PNAS 88: 5096-5100, 1991.
Cell Culture Medium for 33 oC Growth (when received from Core D or revived from
cryopreservation)
For a 500 ml bottle of DMEM/F12 medium, add…
- FBS, 50 mls
- Pen/Strep, 5-10 mls (note we do 2X Pen/Strep when shipping or after revival for a time)
-L-Glutamine, 5 mls
-Amphotericin B, 0.5-1.0 ml (note we do 2X Fungizone when shipping or after revival for a time)
-Gentamicin Solution, 0.125-0.25 mls (note we do 2X Gent when shipping or after revival for a
time)
Note: For the antibiotics and antifungals and for L-glutamine, the amounts are added from
standard cell culture stock solutions
-Insulin-Transferrin-Selenium solution, 2 mls (from 20 ml standard cell culture stock vial)
-Dexamethasone, 5 nM
-T3, 1 nM
-EGF, 10 ng/ml
-IFNgamma, 1 ng/ml
Filter sterilize with a 0.2 micron filter bottle system – This medium is provided in the package
with a filter bottle system – The living T25 flasks (if provided) are also filled to the neck with this
medium (it can be pipetted off and filtered for use as well).
37-39 oC Switch for Experimentation When one ‘passages’ and sets up these clonal cell
lines for an experiment, one needs to transfer the cell lines to 37-39 oC and omit the
IFNgamma from the medium above.
Note: At the outset of using these cells, one may wish to study your biological or physiological
endpoint(s) of interest at both 32-33 oC and at 37-39 oC and with or without IFNgamma or other
supplements for an initial comprehensive assessment.
Passaging of Cell Cultures
(1) Aspirate the serum-containing cell culture medium from the flask, well or plate.
(2) Wash 1X with excess CaMg-free PBS or HBSS. Aspirate.
(3) Incubate in an appropriate volume of CaMg-free PBS or HBSS in the cell culture
incubator at 33 oC for 15-30 minutes. (Note: The ‘Mono’ clones require at least 30
minutes and then addition of trypsin-EDTA solution or the equivalent to promote full
detachment – the other clones do not in our hands and detach in CaMg-free saline)
(4) Pellet the cells using a centrifuge set at 3,000 RPM for 5 minutes. Aspirate supernatant
while leaving a bit of volume to break up the pellet by finger flicking/vortexing or by
gentle vortexing with a vortexer.
(5) Resuspend in cell culture medium desired and plate as desired for your experiment.
Cryopreservation of Cell Cultures
(1) Aspirate the serum-containing cell culture medium from the flask, well or plate.
(2) Wash 1X with excess CaMg-free PBS or HBSS. Aspirate.
(3) Incubate in an appropriate volume of CaMg-free PBS or HBSS in the cell culture
incubator at 33 oC for 15-30 minutes. (Note: The ‘Mono’ clones require at least 30
minutes and then addition of trypsin-EDTA solution or the equivalent to promote full
detachment – the other clones do not in our hands and detach in CaMg-free saline)
(4) Pellet the cells using a centrifuge set at 3,000 RPM for 5 minutes. Aspirate supernatant
while leaving a bit of volume to break up the pellet by finger flicking/vortexing or by
gentle vortexing with a vortexer.
(5) Resuspend in Crypreservation Medium (see recipe below). Note: DBM routinely freezes
6 vials from a confluent T75 flask of cells or a vial per 24 mm filter support (the large
filter supports held in wells of a 6-well plate or 6 vials for 6 such filter supports).
Cryopreservation Medium (-80 oC freezer or liquid nitrogen dewar)
Core D does recommend that you ‘cryopreserve’ multiple vials of each clone for safekeeping
at your facility – The Cores have only finite resources for sending packages to investigators in
the Research Base. This also mitigates risk of loss of the clonal cell lines due to unforeseen
events.
For a 500 ml bottle of Advanced MEM medium, add 20% DMSO cryoprotectant by volume and
add:
- FBS, 100 mls
- Pen/Strep, 10 mls
-L-Glutamine, 5 mls
-Amphotericin B, 1.0 ml
-Gentamicin Solution, 0.25 mls
Feel free to e-mail Dr. Erik Schwiebert, Core D Director, at erik@discoverybiomed.com or Dr.
Eric Seales, Core D co-Director, at eric.seales@discoverybiomed.com or call (205) 918-8138 with
questions and/or feedback.