NIH 3T3 mouse embryonic fibroblast cells were initiated from a cell line isolated in

1962 at the New York University School of Medicine Department of Pathology. 3T3
refers to the cell transfer and inoculation protocol for the line, and means “3-day
transfer, inoculum 3 x 105 cells.” Using this protocol, the immortal cell line begins to
thrive and stabilize in cell culture after about 20-30 generations of in vitro growth.
George Todaro and Howard Green, the scientists who first cultured this cell line,
obtained the cells from desegregated NIH Swiss mouse embryo fibroblasts. The cell
line has since become a standard fibroblast cell line.

NIH3T3 Characteristics

As one of the most commonly utilized cell lines, the NIH3T3 cell line has been
incorporated in studies for a range of mechanistic and cell based assays, including
protein functional analysis. The morphology of the cells are adherent, fibroblastic and
are considered to be among the relatively easy to grow cell lines.

The NIH 3T3 cells doubling time is 20-26 hours. Cell lines have a set of conditions for
which they grow best. This includes a specific serum to provide additional growth
factors, growth media, antibiotics to prevent bacterial growth and CO2 at a percentage
for which the cells thrive (i.e. 5% CO2). Some cell lines require supplements such as
non-essential amino acids (NEAA) or glutamine. Mammalian cells are grown in
incubators set to 37°C.

Expression

Lysophosphatidylcholine (lyso-PC) induces AP-1 activity and c-jun N-terminal kinase

activity (JNK1) by a protein kinase C-independent pathway.

NIH3T3 Cytogenetics

3T3 mouse cells are hypertriploid. The modal chromosome number is 68, which
occurs in 30% of cells. Higher ploidies occur at a much lower rate of 2.4%.

NIH 3T3 Cell Migration

Cell interactions with a surface influences behaviors such as proliferation, migration

and differentiation. In order to study the behavior of a cell in relation to cell migration,
the scrape or wound healing assay can be incorporated. Although there are many
variations of the assay, the classic scrape assay is performed by making a scratch or
wound in the monolayer of cultured cells. Using photographic data, cell migration into
the area of the wound can be quantified using images captured during the experiment.

The scrape assay is an older technology with many flaws incorporated during the
formation of the wound (i.e. inconsistent pressure applied to well bottom, cell
confluency at wound site, etc). To combat these inconsistencies, there are now
chemical and automated ways to create the wound for migration studies. Also, there
are cartridge style assays in which cells exhibit migration through a membrane in
pursuit of fresh culture medium.
From other references
Origin of 3T3 Cells

In 1961 a young medical student, George Todaro, asked to spend a term working in
my laboratory at New York University Medical School. His stay was extended by a
summer, then by several more terms and finally by a postdoctoral stay of several
years.

We began cultivation of mouse embryo fibroblasts with a view to establishing an

immortalized cell line suitable as a target for viral transformation. At that time, it was
believed that mammalian cells became immortalized in culture only rarely and that it
was impossible to predict when such an event might occur or under what conditions.
In order to avoid haphazard conditions of cultivation, I thought it necessary to keep
both the inoculation density and the transfer interval constant during repeated
subcultivation, because those two variables might influence the ability of the cells to
become immortalized; in addition, knowledge of the correct conditions might make it
possible to develop immortalized cell lines reproducibly.

We settled on inoculation densities of 3, 6, or 12 x 105 cells per 20 cm2 dish and a

transfer interval of 3 or 6 days. After a period of declining growth rate that lasted for
10-20 cell generations, during which the doubling time of the murine fibroblasts
increased to as much as 100 hours, we were pleasantly surprised to find that the
growth rate began to increase and in 9 of 11 cultures carried under several conditions,
there evolved cell lines with doubling times of 15-24 hours (Todaro and Greenl, 1963).
The ease of evolution of murine fibroblasts into immortalized lines has since been
repeated in many laboratories but fibroblasts of some species, especially the human,
immortalize extremely rarely. The reason for this important difference is still being
studied.

We were surprised a second time to discover that although immortalization occurred

under most of the culture conditions we used, the properties of the resulting cell lines
depended on the inoculation density and the transfer interval. The line that emerged
from cells regularly inoculated with the highest inoculation density and subcultured
every 3 days (3T12) grew to the highest saturation density (about 350,000 per cm2).
Cells regularly subcultured with half this inoculation density gave rise to a line (3T6)
with a somewhat lower saturation density than 3T12. But the most interesting line
resulted from serial subcultivation using the smallest inoculum. This line (3T3) grew
as vigorously as the others as long as the cells were sparse, but arrested its growth
sharply and entered a stable resting state when the cells became confluent at a
saturation density of 50,000 cells/cm2, only one sixth that of secondary cultures of
strains of mouse fibroblasts. When transferred with dilution, the 3T3 cells resumed
exponential growth, and again reached saturation density at 50,000 cells/cm2. These
experiments showed that murine fibroblasts maintained at low population density
during the period of their immortalization evolved into a cell line which, when allowed
to become confluent, entered a reversibly resting state at a very low population
density.

This property, which could be described as highly developed density-dependent

inhibition of growth, was unprecedented among previously established cell lines, most
of which could grow to saturation densities 10-30 fold higher than 3T3. The exceptional
behavior of 3T3 was evidently due to culture conditions ensuring the absence of
selection for variant cells with the ability to continue growing at high density; without
these conditions, cells of the emerging 3T3 phenotype would have been selectively
eliminated.

The 3T3 line, unlike nearly all immortalized cell lines known previously, or others that
we developed in the same experiments, did not give rise to tumors when injected into
mice. Yet 3T3 cells were not normal either, since they had undergone many
chromosomal alterations and become aneuploid. The behavior of 3T3 cells drew a
clear distinction between the ability to grow indefinitely (immortalization) and the ability
to form tumors (oncogenic transformation) (see GENES 2000: 29.2 Tumor cells are
immortalized and transformed). There is now a great deal of evidence that telomere
shortening, which can lead to chromosomal rearrangements, is an important cause of
finite lifetime in cultured cells and must be overcome before the cells become
immortalized (see Great Experiment: Immortalizing Human Cells with Telomerase,
and GENES 2000: 29.22 Telomere shortening causes cell senescence).

The arrest of growth of 3T3 cells at low saturation density was not simply the result of
exhaustion of some metabolite from the culture medium or the production of inhibitors.
When cells that had entered the resting state were dissociated with trypsin and
replated with dilution in the very same medium that was harvested from the resting
culture, the cells had no difficulty in resuming multiplication. Similarly, if the monolayer
was wounded by removal of 3T3 cells (Todaro et al., 1967), the wound was quickly
repaired by cells that migrated into the wound and proliferated until the bare area was
covered with cells ( Figure 1).

An important determinant of the saturation density was the serum concentration of the
medium. But the property of arresting its growth at such a low density in the standard
medium containing 10% serum made the 3T3 line well-suited for use as a target of
transformation by oncogenic viruses such as polyoma and SV40 (Todaro and Green,
1964; Todaro and Green, 1964). The transformants could easily be detected by
release of the cells from growth inhibition and the formation of dense, multilayered
colonies that could readily be scored ( Figure 2). The transformants, when injected
into mice, gave rise to tumors.

While work on transformation of 3T3 cells by DNA viruses continued in numerous

laboratories, this cell line was relatively insensitive to the RNA viruses of the murine
sarcoma group. Using the same protocol that had led to the development of the 3T3
line, it was possible to evolve new 3T3-like cell lines from fibroblasts of Balb-c and NIH
Swiss mice (Aaronson and Todaro, 1968; Jainchill et al., 1969). These lines were less
sensitive to density-dependent inhibition, but were more sensitive to transformation by
murine sarcoma viruses than the original 3T3, which was of random-bred Swiss origin.

When cellular oncogenes were discovered, a suitable system for cell-based assay was
lacking. NIH 3T3, which was highly susceptible to transfection (Smotkin et al., 1975),
was found to be the best line for transformation by cellular oncogenes (Shih et al.,
1979) and the use of this cell line for scoring transformation promoted vigorous
development of the field (see Great Experiment: The Discovery of Oncogenes in
Human Tumors) (Krontiris and Cooper, 1981; Perucho et al., 1981; Weinberg, 1982).