Preparation and Immortalization of
Primary Murine Cells
The generation of immortal cell lines from genetically
defined mice has proven useful in the understanding
of molecular pathways in mammalian
biology. This article describes the preparation and
immortalization of murine embryo fibroblasts (MEFs),
the workhorse cell type of genetically defined mice.
These cells are used most frequently because of their
ease of preparation, relative homogeneity, and high
frequency of immortalization with serial passage in
culture. These techniques can be used, with modification,
to immortalize rat embryo fibroblasts as well.
Furthermore, variations of these techniques can be
applied to other murine cell types (e.g., astrocytes or
keratinocytes) for the study of cell-type specific pathways
(see Section V).
The genetics of immortalization in murine cells are
now largely understood [Fig. 1, reviewed in Sharpless
and DePinho (1999)]. As opposed to human cells, the
spontaneous immortalization of cultured rodent cells
occurs with high frequency resulting from a stochastic
genetic event. This difference in frequency is attributable
to the additional requirement in human cells for
telomerase expression as detailed in the previous
article. In murine cells, the act of cell culture, with a
few exceptions, potently induces both antiproliferative
) of the Ink4a/Arf
which regulate the Rb and p53 pathways, respectively
(Fig. 1). Immortalization of murine cells requires circumventing
at least p19ARF
-p53 or, in some cell types,
both of the pathways regulated by the products of the
Ink4a/Arf locus (for examples, see Bachoo et al.
Kamijo et al.
, 1997; Randle et al.
, 2001). This can be done
in one of three ways: spontaneous, stochastic genetic deletion through serial culture; the use of immortalizing
oncogenes; or the use of genetically defined mice.
This article deals predominantly with the former
method, the latter two are described in Section V.
|FIGURE 1 The genetics of senescence. In murine cells, the act of
culture [("culture shock" (Sherr and DePinho, 2000)] induces both
products of the Ink4a/Arf locus, p16INK4a inhibits cyclin-dependent
kinases 4 and 6, leading to Rb hyphophorylation and growth arrest.
p19ARF stabilizes p53 by inhibiting its mdm2-mediated degradation;
p53 also induces growth arrest. In MEFs, the p19ARF-p53 axis dominates
the growth phenotype (Kamijo et al., 1997; Sharpless et al.,
2001), although both p16INK4a and p19ARF contribute to senescence in
other cell types (Bachoo et al., 2002; Randle et al., 2001).
-Sterile 10-cm dishes (Falcon Cat. No. 353003
-Sterile 6-cm dishes (Falcon Cat. No. 353002
-Sterile phosphate-buffered saline (PBS, GIBCO Cat.
-100 and 70% ethanol
-0.25% trypsin-EDTA (GIBCO Cat. No. 25200-056
-DMEM (with glucose and L-glutamine, GIBCO Cat.
-Heat-inactivated fetal calf serum (FCS, Sigma Cat. No. F-2442
; to heat inactivate, thaw and then place in
water bath at 56°C for 30min)
-100× penicillin/streptomycin (P/S 10,000 units/ml,
GIBCO Cat. No. 15140-122
-Optional: β-mercapoethanol (β-ME, Sigma Cat. No. M-7522
-Liquid nitrogen-safe cryotybes (Corning Cat. No. 2028
-Dimethyl Sulfoxide (DMSO, Mallinkrodt AR Cat. No. 4948
III. INSTRUMENTATION (ALL
- 6 ¾in. Mayo scissors (Fisher Cat. No. 13-804-8
- Large forceps (Fisher Cat. No. 13-812-36
- Curved fine scissors (Fisher Cat. No. 08-951-10
- Two curved small forceps (Fisher Cat. No. 08-953D
- Two 5 ½ in. Kelly clamps (Fisher Cat. No. 08-907
- 9-in. cotton-plugged Pasteur pipettes (Fisher Cat. No. 13-678-8B
- Razor blades (place in 100% ethanol in 15-m dish prior
A. Murine Embryo Fibroblast Production
- The embryo dissection need not be performed in
the hood but rather can be done on a clean bench
covered with a diaper. Use sterile reagents (e.g., autoclave
or flame dissecting equipment).
- Sacrifice the pregnant mother at approximately
noon on postcoital day 13.5 (see Section VI) through
CO2 inhalation. If the embryos are of potentially different
genotypes (e.g., the progeny of a heterozygous
intercross), regenotype the mother at this stage.
- Place the mother on a dissection stand and clean
by spraying generously with 70% ethanol.
- Using Mayo scissors and large forceps, carefully
open the abdomen, locate the embryos, and remove
the bicornuate uterus with embryos. The embryos will
appear as ~l-cm "beads on a string," in general there
will be 6-12 embryos in a normal litter. If a mating
produces an embryo with a genotype associated
with developmental lethality, it is often possible to see
partially resorbed embryos at this time.
- Place uterus plus embryos in sterile PBS in 10-cm
dish. Using fine forceps and/or scissors, gently dissect
each balloon-like embryo with fetal membranes from
the thicker uterus and place individually into a 6-cm
dish with sterile PBS. If there are a large (>12) number of embryos, place the 6-cm dishes on ice. If the fetal
membranes rupture during dissection, separate the
embryos from the placenta and membranes and put in
a 6-cm dish in PBS. Some investigators use a dissecting
microscope for this step, although we do not find
- Perform the rest of the dissection in a tissue
- Dissect away placenta and fetal membranes. Of
note, the placenta is partly of maternal origin and can
therefore contaminate genotyping if the mother's
genotype differs from those of the embryos. To remove
placenta, use a pair of fine forceps. Grasp umbilical
vessels from the placenta to the embryo with one set
of forceps and grasp the placenta with the other and
pull in opposite directions. Pulling the placenta
directly can cause the abdominal viscera to herniate
and detach. The fetal membranes may still be wrapped
around the embryo after placental removal, but these
can be gently pulled away from the embryo after placental
detachment. The fetal membranes are of embryonic
origin and can be used for genotyping, although
we prefer to use nonadherent cells obtained on day 2
- Place the entire embryo without membranes
or placenta in a 10-cm dish with 1 ml 0.25%
trypsin-EDTA (this is preferable to the 0.05%
trypsin-EDTA commonly used to passage cells).
- Hold a razor blade in each of two Kelly clamps
and flame after dipping in 100% ethanol. Briefly cool
razor blades in sterile PBS in a 10-cm dish and then
use to mince the embryos. The pieces of tissue need
not be too fine; chopping approximately 30 times per
embryo is generally adequate. Change razor blades
between embryos if the embryos are of differing
- Allow minced tissue to sit in trypsin-EDTA for
10min at 37°C, 5% CO2.
- Add 2ml of growth media (DMEM + 10% FCS
+ pen/strep with 50µM β-ME; the β-ME is optional)
and disaggregate by repeated pipetting: 10 times with
a 5-ml pipette and 10 times with a plugged Pasteur
pipette. After disaggregation, there should be no large
(<1mm in largest dimension) chunks of tissue
- Add 8 ml of growth media and swirl plates for
even seeding. We grow cells at this stage in an incubator
dedicated for primary cells in the rare event of
- The 10-cm dish should be 100% confluent after
attachment of MEFs overnight. If subconfluent, this
generally indicates poor disaggregation of the embryo, improper media, or bacterial contamination (see
- Gently remove the media and save nonadherent
cells for genotyping (see later). Take care as the cells
are poorly adherent at this stage and large chunks of
the monolayer can detach with poor handling.
- Add 10 ml of fresh growth media.
- If genotyping is necessary (i.e., the embryos are
of different genetic composition), this can be done on
DNA prepared from nonadherent cells collected on
day 2. This is done by pelleting these cells in 15-cm
conical tubes (1000rpm × 5 min), detergent lysis, and
precipitation of DNA. The exact method used depends
on the manner of genotyping (e.g., polymerase chain
reaction vs Southern blot). One can easily obtain >100
µg of good-quality DNA from these pellets if needed.
We prepare using a commercially available DNA
extraction kit (Promega #Al125).
B. Thawing MEFs
- The culture should be superconfluent on day 3
and ready to freeze down or passage for further experiments.
If cells are less than 100% confluent by day 3,
freeze down on day 4.
- To freeze, wash cells once with sterile PBS and
add 1 ml of 0.25% trypsin-EDTA. Cells will detach in
5-10min and then add to a 15-ml conical tube with
10 ml of growth media.
- Spin cells at 1000rpm for 5 min.
- Resuspend cell pellet in 3 ml of ice-cold freezing
media (90% FCS + 10% DMSO) and put in three welllabeled,
chilled cryovials. We label with date prepared,
the line number, and the passage number upon
thawing. The lines are numbered as the mother's
number first, followed by the embryo number (e.g.,
115-7 would be the seventh embryo of mother 115).
The cells will be passage 2 upon thawing if not passaged
further before freezing.
- Keep cells on ice for 10-15 minutes and then
put in a -80°C freezer in a sealed styrofoam block
overnight. Alternatively, the cells can be placed in a
cryo-freezing container (Nalgene Cat. No. 5100-001)
and then placed in a -80°C freezer overnight.
- The day after freezing, cells should be moved to
a liquid nitrogen tank for long-term storage.
C. Immortalizing MEFs
- Keep cells on dry ice until absolutely ready to
- Place the vial of cells in a 37°C water bath and
watch carefully. As soon as the cells begin to thaw,
pour the entire pellet into a 15-ml conical tube.
- Add 10 ml of growth media dropwise over about
30s with gentle mixing while adding. Mix well after
all 10ml of media is added.
- Spin cells down (1000rpm for 5min), resuspend
in 3 ml media, and plate in a 10-cm dish. One vial of
frozen cells can go into one 10-cm dish; add another
7 ml media.
- The cells should be 90-100% confluent the day
after thawing. If confluence is less with large numbers
of floating (dead) cells noted, this suggests improper
freezing, improper storage (e.g., freezer malfunction),
or improper thawing (see Section VI).
This can be done using a rigorously defined passaging
protocol (e.g., 3T3 or 3T9 assay) or, more simply,
by serial replating of cells. The advantage of the passaging
protocol is that one learns about the growth
kinetics and immortalization frequency of the cell (see
Section V). The immortalization of wild-type MEFs
requires a stochastic genetic event: generally p53 loss
or, less frequently, loss of p19ARF
(Kamijo et al.
Other immortalizing events, such as mdm2 amplification
(Olson et al.
, 1993), have also been noted, but these
are considerably rarer. In our experience, this genetic
event is most likely to be loss of p
53 (~60-70% of
immortalized lines) o r p19ARF
(~20-30%) regardless of
growth conditions (passage at high or low density).
Therefore, serial passaging without counting cells at
every passage is an appropriate way to derive immortalized
cells. In fact, the "3T3 cell" has become a
nondescript term referring to immortal murine
embryo fibroblasts obtained through serial passaging,
but few laboratories except those interested in growth
and senescence do rigorous 3T3 assays to obtain
D. Immortalization by Serial Passage
E. 3T9 Assay
- Split MEFs 1:3 twice per week during the rapid
growth phase of the culture (the first 6-10 passages).
- As cells enter senescence, split 1:2 or merely
replate (aim to keep cells near 100% confluency on
the day of passaging). If the cultures are seeded
too sparsely, this will decrease the immortalization
- Between passages 10 and 20 (4 to 9 weeks in
culture), immortalized lines will begin to overgrow the
culture. These cells are smaller and spindle shaped,
initially appearing as small nests of cells obvious
on the day of passaging. These cells can be subcloned,
but are used more frequently as pooled immortalized
This assay was originally described by Todaro and
Green (1963). The "3" refers to passaging every 3 days,
and the "9" refers to 9 x 105 cells plated per passage.
3T3 (3 x 105 cells) and 3T12 (1.2 x 10 6 cells) assays can
also be done to study growth and immortalization at
varying densities. In our experience, immortalization
frequency is highest for the 3T3 assay (and therefore
this assay has lent its name to all immortalized lines of
murine fibroblasts), whereas 3T9 is more useful for
measuring immortalization frequency (Sharpless et al.
2001). As stated earlier, serial replating of murine
fibroblasts at almost any density will eventually yield
immortalized lines as long as the cells are not seeded
A. Immortalization through the Use of
Oncogenes or Genetic Background
- For the first passage, wash cells growing in a
10-cm dish (usually passage 2 at this stage) with PBS,
trypsinize (0.05% trypsin-EDTA is adequate for this
purpose), dilute into 10ml of growth media, and
- Spin down and resuspend cells at a concentration
of 9 × 105 cells/ml. Seed 1 ml of cells (9 × 105 cells) into
a 6-cm dish and add 2.5 ml of media. Swirl the cells to
ensure uniform seeding. Label the dish with the MEF
line number and the passage number.
- For subsequent passages, trypsinize and recount
cells every 3 days in a manner identical to steps 1 and
2. Record the number of cells counted before each
passage. Growth curves and immortalization frequencies
can be determined from analysis of these data as
described in Section V.
- Immortalized lines will emerge between passages
10 and 20; virtually all lines proliferating after 20
passages will be immortal. If necessary, these lines can
be subcloned by limiting dilution, but this is not generally
done. The majority of immortalized lines will
have lost either p53 or p19ARF function (Kamijo et al.,
1997), although various other genetic events can
increase or decrease the frequency of immortalization
(Frank et al., 2000; Jacobs et al., 1999; Kamijo et al., 1999;
Sharpless et al., 2001).
A problem with immortalization by serial passage
is that the nature of the immortalizing genetic event is
not known, although predominantly p53 or p19ARF
inactivated as MEFs escape senescence. As the behavior of p19ARF-deficient cells may be quite different from
p53-deficient cells, however, the stochastic inactivation
of these pathways may produce significant line-to-line
variability and therefore be undesirable. For example, p53
null MEFs are more aneuploid and more resistant
to DNA damage than p19ARF
null MEFs (Kamijo et al.
1997; Pomerantz et al.
, 1998; Serrano et al.
, 1996; Stott et al.
, 1998). To assure that all lines will evade senescence
via a similar mechanism, cells can either be
immortalized through the use of an immortalizing
oncogene or by using cells derived from animals of a
genetic background that resists senescence.
Classically, the most commonly used oncoprotein to
immortalize cells is the SV40 large T antigen (Colby
and Shenk, 1982; Jat and Sharp, 1986; Todaro and
Green, 1966). This molecule inactivates the p53 and Rb
pathway, and the majority of murine cell types can be
immortalized by transfection or retroviral transduction
of TAg. A disadvantage of TAg, however, is that
cells expressing this molecule are unstable and are
prone to clonal in vitro
evolution. Furthermore, as Rb
pocket proteins are required for the differentiation of
many cells types (Dannenberg et al.
, 2000), TAg generally
impairs or precludes the study of differentiation.
Alternatively, MEFs and several other murine cell
types can be immortalized with a dominant-negative
form of p53 [e.g., p53-DD (Bowman et al.
, 1996)], which
preserves the Rb pathway, although these cells are still
more prone to aneuploidy than p19ARF
The most elegant method of obtaining immortal cell
lines is by deriving cells from animals resistant to
senescence. The most commonly used strains for this
purpose are Ink4a/Arf
-deficient (Serrano et al.
-deficient (Kamijo et al.
, 1997), or p53
(Donehower et al.
, 1992) mice. These strains are widely
available and can be obtained from the mouse
models of the human cancer consortium (MMHCC
http: / / web. ncifcrf, gov / researchresources / mmhcc/).
For studies of genetically defined animals, the genetic
background of interest can be crossed two generations
to mice of these backgrounds and then MEFs (or other
cell types) derived as described earlier, which will be
immortal in most cases if derived from p53- or p19ARF
embryos. This method can be employed to
derive immortal cells from difficult genetic backgrounds
that undergo premature senescence in culture
(Frank et al.
, 2000; Jacobs et al.
, 1999). This strategy can
also be employed to obtain cells of nonfibroblast lineages.
For example, immortal melanocytes (Chin et al.
1997), skin keratinocytes (unpublished observations),
glia (Bachoo et al.
, 2002), lymphocytes (unpublished
observations; Randle et al.
, 2001), and macrophages
(Randle et al.
, 2001) have been derived successfully
- or Ink4a/Arf
-deficient mice using standard culture methods for these cell types. To immortalize
with high efficiency, cells must be homozygous null for
, or Ink4a/Arf;
therefore, the principal disadvantage
of this approach is the extra time needed to
backcross to these defined genetic backgrounds.
B. Data Analysis of 3T9 or 3T3 Assay
These data can be plotted as cell number per
passage or population doublings (PDs) per passage
(Fig. 2). PDs for any given passage = log2(Nf
), where Nf
= number of cells counted at
the end of the passage and No
= number of cells seeded
at the beginning of the passage (i.e., 9 × 105
for a 3T9
assay). For the purpose of immortalization frequency,
"senescence" occurs if less than 9 × 105
cells are recovered
for two consecutive passages. The immortalization
frequency = 1-number of senescent lines/total
number of lines analyzed. Measured in this way, the
immortalization frequency of wild-type MEFs can
vary significantly depending on culture conditions,
method of embryo preparation, and so on and therefore
is only meaningful when compared to proper littermate
control embryos analyzed concurrently.
|FIGURE 2 The 3T9 assay can be used to quantify both growth and immortalization frequency. Data from
a 3T9 (or 3T3) assay can be graphed in either of two ways: (a) as cell number per passage or (b) as population
doublings (PDs) per passage (PDs defined in text). The same data are graphed by either method showing
a senescent line, an immortalized line, or a line lacking Ink4a/Arf. The p19ARF-dependent slow growth period
seen in wild-type MEFs between passages 5-15 is called "senescence," although in actuality it represents a
- Embryo dates. By convention, the day a coital plug
is detected is day 0.5 for timed matings, and embryos should be made at midday 13 days later. While it is difficult
to be sure of correct plugging dates at the time
of dissection, in general day 13.5 embryos have
paddle-like front paws, whereas day 14.5 embryos
have more fully formed individual digits. Embryos
that are too large and well developed or too small
suggest an incorrect date of plugging. Cells of different
embryonic ages do differ in several in vitro growth
properties; therefore, littermate controls are always
preferable in MEF experiments. In our experience,
MEFs from embryos older than 13.5 grow less well and
immortalize less frequently than 13.5 embryos.
- MEFs are not confluent the day after dissection. In
general, cells from a single embryo should cover a
10-cm plate fully the morning after plating. Poor coverage
of the dish can result from inadequate embryo
disaggregation, tissue mincing with razor blades that
were not cooled properly after flaming, improper
media, or bacterial contamination. In particular, one
should be vigilant for bacterial contamination as this
can be difficult to note given that there is copious
debris in the culture 1 day after plating.
- MEFs are not confluent the day after thawing. This
results most often from freezer or liquid nitrogen tank
malfunction, but can also be due to improper technique
when the cells were frozen, malfunction of the
cryo-freezing container, or improper thawing. DMSO
is toxic to these primary cells so it is important to
resuspend the frozen cell pellet well in growth media,
spin the cells down, and then respsund in fresh growth
media prior to plating. Thawed vials of cells should not be left in freezing media for a significant period of
time prior to replating.
- Cells grow poorly and/or fail to immortalize. This can
result from poor growth media (e.g., the fetal calf
serum is too old), the use of embryos significantly later
than E13.5, or occult pathogen contamination.
Embryos of certain genetic backgrounds grow poorly
in culture ("premature senescence"), which can
sometimes be obviated by backcrossing to Ink4a/Arf-, p19ARF-, or p53-deficient animals (Frank et al., 2000;
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