Proc. Natl. Acad. Sci.
USA
Vol. 77, No. 6, pp. 3455-3459, June 1980
Cell Biology
Culture of hormone-dependent functional epithelial cells
from rat thyroids
(thyroglobulin/iodide transport/hormones/differentiation/aging)
F. S. AMBESI-IMPIOMBATO*, L. A. M. PARKSt, AND H. G.
*Centro di Endocrinologia ed Oncologia Sperimentale del C.N.R.c/o Istituto di Patologia Generale, II Facolta' di Medicina, Universita' di Napoli, Naples, Italy;
and tLaboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205
Communicated by Rita Levi-Montalcini, March 4,1980
ABSTRACT Primary cultures of rat thyroid cells were made
in medium supplemented with 0.1-0.5% calf serum and con-
taining six hormones or growth factors: insulin, thyrotropin,
transferrin, hydrocortisone, somatostatin, and glycyl-L-histi-
dyl-L-lysine acetate. The FRTL strain was purified by successive
colonial isolations and was found to maintain highly differen-
tiated features (secretion into the culture medium of physio-
logical amounts of thyroglobulin and concentration of iodide
by 100-fold). The FRTL strain has been observed for more than
3 years in continuous culture. It has maintained the same bio-
chemical and morphological characteristics that typified the
primary cultures of thyroid follicular cells immediately after
their enzymatic release from the rat thyroid. Thyroid epithelial
cells that were grown under more conventional cell culture
conditions failed to retain these specialized characteristics. We
show that maintenance in vitro of these specialized functions
of rat thyroid follicular cells is dependent on low serum con-
centrations and supplementation with hormones in the primary
cultures. Our observations indicate that this culture strategem
may be applicable to the general problem of maintenance of
differentiated characteristics in cultures of other epithelial
cells.
In spite of many advances in cell culture there has been rela-
tively little success in culturing differentiated epithelial cells.
The majority of cultures that have been adapted to grow in vitro
and have been cloned are mesenchymal cells or cells derived
from tumors in which it may be difficult to distinguish between
the epithelial and fibroblast-like phenotypes. Recently, how-
ever, the number of amniote epithelial cells grown and studied
in vitro has been increasing as a consequence of a better defi-
nition of the cellular requirements (1-6) and the availability
of improved growth media. Most cultured cells, especially those
of the fibroblast type, require the addition of relatively high
amounts of serum (5-20%) for growth. The addition of serum
is considered necessary to provide the cells with unknown
"serum factors" needed for cell attachment to the substrate and
for cell division.
The pioneering work of Sato (7), which has validated the
hypothesis that for many cultured cells serum serves as a source
of hormones (many of which probably are still undefined), has
made possible new precision in regulating the in vitro envi-
ronment. This idea, which has proven successful with estab-
lished cell lines (8) and tumor-derived cell lines, has served as
the basis for our approach to the study of normal thyroid cell
strains derived from primary culture. It has allowed us to grow
highly differentiated thyroid follicular cell strains from the rat
in continuous culture. When traditional culture techniques were
used, strains of epithelial thyroid follicle cells no longer ex-
pressed important aspects of thyroid differentiation (production
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3455
COONtf
of thyroglobulin and concentration of iodide), whereas cell
strains maintained continuously in medium supplemented with
0.1-0.5% calf serum and hormones did retain these thyroid-
specific functions.
MATERIALS AND METHODS
Trypsinization and Primary Cultures. The cell dissociation
procedures used were similar to those now in standard use for
the isolation of primary cell lines (9, 10). Thyroid glands were
excised from 5- to 6-week-old Fischer rats that had been killed
by CO2 asphyxiation. Usually, the glands from three to six rats
were pooled and used for the primary cultures. All the proce-
dures were performed under sterile conditions. The glands were
freed from adherent connective tissue, cut into small pieces, and
washed in Ca2+- and Mg2+-free Hanks' salt solution (11) by
centrifugation at approximately 500 X g. Enzymatic digestion
was carried out by adding to the washed pellet 3 ml of colla-
genase (CLSPA, Worthington), 20 units per ml trypsin (1:300,
ICN Pharmaceuticals), 0.75 mg per ml heat-inactivated di-
alyzed chicken serum (GIBCO), 2% in Ca2+- and Mg2+-free
Hanks' salt solution (CTC solution) (1).
The tissue was incubated for 20 min at 37°C in a vigorously
shaking waterbath. Usually, two 20-min incubations and some
vigorous pipetting were sufficient to reduce the whole tissue
mass to a cell suspension. After a 2-min settling period (to allow
large fragments to settle), the supernatants were collected,
combined, washed in complete medium, and distributed into
10-cm Falcon plastic tissue culture dishes at 104 to 105 cells per
dish.
Passage of Cells. Secondary cultures were made by trans-
ferring individual epithelial colonies with cloning cylinders.
When uniform cell suspensions were desired, the CTC made
2 mM in ethylene glycol bis(f3-aminoethyl ether)-N,N,-
N',N'-tetraacetic acid (Sigma) which was added as a 0.2 M
solution in 1 M NaOH/Ca2+- and Mg2+-free Hanks' saline,
1:1.
Culture Medium. The culture medium used was a modifi-
cation (12) of Ham's F-12M (13) supplemented with 0.1-0.5%
calf serum (GIBCO). Fetal calf serum was found to be unsuit-
able for these cells. The culture medium and serum supplement
were terminally sterilized by filtration through 0.22-,um pre-
washed Millipore filters. Just before use, this medium was
supplemented with a 60- or 100-fold concentrate of the sterile
hormone mixture. The complete supplemented medium has
been designated mF12+6H to indicate that it is a modified
F-12M formulation supplemented with the mixture of six
hormones (Table 1).
Abbreviations: mF12, modified F-12 medium (see Table 1); mF12+
6H, mF12 supplemented with 0.5% calf serum and six hormones.
t To whom reprint requests should be addressed.
3456 Cell Biology: Ambesi-Impiombato et al. Proc. Natl. Acad. Sci. USA 77 (1980)
Table 1. Composition of the modified F-12 medium (mF12) Radioimmunoassay for Thyroglobulin. Thyroglobulin was
Concentration Concentration determined by P. R. Larsen by a double-antibody immu-
mg/ mg/ noprecipitation radioimmunoassay technique. Details of this
Component liter mM Component liter mM method and of 'the specificity of the antibodies have been
L-Arginine. Calcium published (14).
HC1 4422.0 2.0 pantothe-
L-Histidine. nate 0.5 0.001 RESULTS
HCI 42.0 0.2 Niacinamide 0.04 0.0003 Most of the culture regimens and media tested produced either
L-Isoleucine 7.8 0.06 Linoleic acid 0.09 0.0003 nondividing cultures or dividing cultures of nonfunctional
L-Leucine 26.2 0.2 Pyridoxine-HCl 0.06 0.0003 epithelial or fibroblast-like cells. Two of the culture strategies
L-Lysine. Thiamine.HCI 0.285 0.0008 consistently yielded epithelial cell strains but with opposite
HC1 73.0 0.4 Riboflavin 0.04 0.0003 functional properties. The FRT cell strain was isolated in me-
Glycine 16.0 0.2 Folic acid 1.0 0.002 dium supplemented with 5% serum and dibutyryl cyclic AMP
L-Methionine 9.0 0.06 Vitamin B-12 1.0 0.0007 and then was "conditioned" by culture on thyroid fibroblasts
L-Phenylala- Thioctic acid 0.2 0.0009 for 24 hr. The FRT cells were able to produce thyroglobulin in
nine 10.0 0.06 myo-Inositol 36.0 0.2 primary cultures but after adaptation to growth in uncondi-
L-Serine 21.0 0.2 Ascorbic acid 45.0 0.250 tioned medium they lost the ability to concentrate iodide or to
L-Threonine 23.8 0.2 Choline-HCl 13.8 0.1 produce thyroglobulin. The FRTL strain was isolated in me-
L-Trypto- Thymidine 0.7 0.003 dium supplemented with 0.5% calf serum and purified hor-
phan 4.0 0.02 Hypoxanthine 4.0 0.030 mones. FRTL cells appear to represent differentiated thyroid
L-Tyrosine 11.0 0.06
follicle cells growing in culture. Because our success or failure
L-Valine 0.2 NaCl 7530.0 130.0
L-Cystine
23.4

0.2 KCI 305.0 4.0 in producing functional cell cultures was determined by the
interaction between primary cells and the different media,
48.0

L-Asparagine. Na2HPO4-
HCO 30.0 0.2 7H20 250.0 0.9 these are described in detail below.
L-Proline 70.0 0.6 KH2PO4 68.0 0.5 Primary cell suspensions were made by serial treatment with
L-Alanine 18.0 0.2 MgSO4.7H20 104.0 0.4 collagenase and trypsin of three to five thyroid glands pooled
L-Aspartic MgCl246H20 106.0 0.4 from 6-week-old littermates of the NIH Fischer 344 inbred
acid 26.0 0.2 CaC12.2H20 165.0 1.1 strain of rats. After washing by centrifugation, the primary cell
L-Glutamic CuS04-5H20 0.002 0.000008 suspensions were counted and distributed into tissue culture
acid 30.0 0.2 ZnSO4.7H20 0.144 0.0005 dishes at different densities in the experimental media.
FeSO4-7H20 0.8 0.0028 Medium with 2-10% Serum Supplement Only. Both the
Sodium
pyruvate 220.0 2.0 Glucose 2000.0 11.0 epithelial and fibroblast-like cells from primary suspensions of
Putrescine- NaHCO3 2500.0 29.7 rat thyroid glands attached efficiently in mF12 supplemented
2HCI 0.3 0.001 Phenol red 1.25 0.0035 with 2-10% fetal calf serum. However, within 1 week the epi-
Glutamine 292.0 2.0 thelial cells were usually found to have lysed selectively leaving
Biotin 0.07 0.003 only the fibroblast-like cells in the cultures. Supplementation
of mF12 with the same concentrations of calf serum yielded
Hormone Supplement for Rat Thyroid Cells. The final cultures in which the epithelial component (initially >80% of
hormone mixture was arrived at by testing various hormones the attached cell population) survived but did not divide.
or factors at physiological or nearly physiological concentra- Cultures at high serum concentrations (2-10%) without other
tions. This approach led to the total replacement of the serum additives were eventually overgrown by fibroblast-like cells in
requirements for already established cell lines such as GH3 cells all cases.
(8). The final concentrations of the hormones used were: insulin Epithelial cells grew in medium that was supplemented with
(Sigma), 10 ,ug/ml; hydrocortisone (Sigma), 10 nM; transferrin 5% calf serum and dibutyryl cyclic AMP (10 ttM) after it was
(Sigma), 5 ,ug/ml; glycyl-L-histidyl-L-lysine acetate (Calbio- conditioned by thyroid fibroblasts. This regimen allowed the
chem), 10 ng/ml; somatostatin (Calbiochem), 10 ng/ml; and epithelial cells to divide and apparently restricted division of
thyrotropin (Pituitary Hormone Distribution Program, National the fibroblast-like cells. These epithelial cells were later adapted
Institute of Arthritis, Metabolism and Digestive Diseases), 10 to grow in medium containing 5% serum without prior condi-
milliunits/ml. These hormones were made up as a single 60- tioning or added dibutyryl cyclic AMP. These cells were found
to 100-fold concentrated stock solution in Ca2+- and Mg2+-free not to produce detectable amounts of thyroglobulin or to con-
Hanks' saline, divided into aliquots, and frozen rapidly in liquid centrate iodide (strain FRT in Tables 2 and 3), except when they
nitrogen (thyrotropin preparations may lose activity unless they were cocultivated as primary or secondary cultures with thyroid
are frozen rapidly). After freezing, the stock solutions were fibroblasts.
stored at -80'C. No antibiotics were added. The medium was Medium Without Serum or Hormones. When primary rat
renewed twice weekly. thyroid cells were cultured in mF12 with no serum or hormone
Iodide Uptake Assay. We adapted the method described supplement, cell attachment was exceedingly rare. Within 3-4
by Tong (9) for suspensions of freshly isolated cells. Between days, floating vesicles or follicles made up of initially undisso-
0.5 and 1.0 X 106 cpm of 125I per dish was added to the growth ciated or reaggregated cells could still be found. However, no
medium in the presence of 1 gM cold iodide and 3 mM 1- cell division or colony growth was ever observed.
methyl-2-mercaptoimidazole (to inhibit organification of io- Medium with Hormones Alone or Low Serum Alone. In
dide). Cells were incubated for 2 hr. At the end of the incuba- the primary cultures, cell attachment was clearly enhanced in
tion, the medium was removed and its radioactivity was de- the presence of the added hormones. Cell division and colony
termined. Cells were then scraped with a rubber policeman, growth were evident among the epithelial cells immediately
pelletted in pre-weighted tubes, and assayed for radioactivity. after plating. In contrast, fibroblast-like cells were rarely ob-
The C/M ratio (i.e., cpm/pl of medium:cpm/mg of cell pellet) served to divide. Our attempts to transfer, by trypsinization,
was calculated. the primary epithelial cells grown in medium with hormone
 Cell Biology: -Ambesi-Impiombato et al.
Table 2. Thyroglobulin production by cultured cells
Thyroglobulin
Cells ng/ml* pg/cell/day
FRT <2.5 - (with no calf serum) together with several controls are shown
Thyroid fibroblasts <2.5
Primary cultures of
thyroid epithelial cells
on fibroblast feeder layer 20-25 2.5
FRTL 7000 14
* Sensitivity limit of the method was 2.5 ng/ml.
supplement but without serum failed, and no cell attachment
was seen in secondary cultures. The epithelial cells grown under
these conditions were extremely sensitive to even the mild
trypsin/collagenase treatment used. The low plating efficiency
and the low number of cells present in the primary cultures did
not leave enough cells to start successful secondary cultures.
When primary cell suspensions were cultured in growth
medium supplemented with 0.1-0.5% calf serum alone, cell
survival was greatly improved over that in media without any
serum supplement but the epithelial and nonepithelial cells that
did attach did not divide as judged by repeated observations
of the same groups of cells over periods of up to 3 weeks.
Medium with Low Serum and Hormones: Hormonal Re-
quirements. Cell attachment was frequent and mitotic figures
were regularly noted among the epithelial cells in medium to
which both low levels of calf serum and hormones were added.
Starting with the hormone cocktail of Hayashi and Sato (8)
individual hormones were deleted or added until evidence of
sustained cell division was obtained. Neither fibroblast growth
factor (15) nor epithelial growth factor (16) produced any de-
tectable effect. The final concentrations of the six hormones in
the medium are given in Materials and Methods. Primary cells
released from the gland by trypsin and collagenase and later
passages from these cultures attached and grew continuously
when these six hormones were present in mF12 supplemented
with 0.5% calf serum (mF12+6H).
Primary thyroid cultures and strain FRTL were absolutely
dependent upon added thyrotropin and insulin for growth and
survival. At low population densities (<50 cells/mm2) of FRTL
cells, both transferrin and somatostatin were required; at higher
population densities (>100 cells/mm2), however, it was hard
to show an unambiguous requirement for either. In some ex-
periments they appeared to be required; in others, slow growth
would continue without them for months or until cells were
transferred by trypsinization, at which time the hormone-
deficient cells were found to be most vulnerable. Experiments
attempting to demonstrate a requirement for glycylhistidyl-
lysine and hydrocortisone gave variable results. About half of
the low-density cultures showed poor growth when either was
deleted. At higher cell densities (>50 cells/mm2), however, it
was difficult or impossible to show requirement for either (with
or without added 0.5% calf serum). In spite of this, sparse cul-
tures and sparse regions of more dense cultures as well as pri-
mary cultures grew best when all six hormones were included
Table 3. Iodide uptake by cultured cells
C/M
Cells ratio
FRT 1.2
FRT fibroblasts 1.5
FRL* 1.3
FRTL 100.0
*
Epithelial cell line from Fischer rat liver.
Proc. Natl. Acad. Sci. USA 77 (1980) 3457
and we have chosen to use the full mF12+6H throughout the
continuous passage of FRTL cells.
Growth curves for relatively dense populations of FRTL with
each of the six hormones deleted one at a time from mF12+6H
in Fig. 1. Growth was greatly reduced in media lacking thy-
rotropin or insulin, whereas at these densities growth in media
lacking any one of the other hormones continued like the con-
trols. After these cells became confluent they were passaged at
5 X 105/60-mm plate, and the cells lacking insulin, thyrotropin,
or somatostatin died, as ultimately also did those lacking
transferrin. The remaining two, lacking glycylhistidyllysine
or hydrocortisone, still continued to grow at a reduced rate.
We believe that the low-level calf serum supplement is re-
quired mostly for cell attachment both for primary cultures and
for subsequent passages. When the serum supplement was
deleted after the cells had attached and spread (as in Fig. 1),
the presence of the hormones alone was sufficient to maintain
the culture dividing for periods of 2-3 months or until the
culture was again subcultured by treatment with trypsin and
collagenase. At this time, the low serum supplement had to be
present for cell attachment to occur.
Growth and Morphology of FRTL Cells and Colonies in
mF12+6H. The FRTL strain was obtained after three succes-
sive colonial subculturings in mF12+6H. It is difficult to pre-
pare perfect single-cell suspensions from these cultures by using
enzymatic digestion alone; more extreme measures such as
vigorous pipetting or chelation yield better single-cell suspen-
sions, but cell survival suffers. Repeated selection of colonies,
even when one cannot be sure that the colony originated from
. 7 ~~~~~~~~~~~9
6~~~~~~~~~~~~~~
106
0
3 7 11 15 19 23 27 31
Time, days
FIG. 1. Growth of densely plated FRTL cells in mF12+6H
lacking calf serum and one hormone of the six-hormone supplement.
FRTL cells were plated at densities of 2 X 105 cells/60-mm petri plate
in different media. At this density, cells plated in mF12 with other
hormone supplements but lacking thyrotropin or insulin showed
greatly reduced growth (2, 3). Cells grown in media singly deficient
for the other hormones showed growth patterns similar to those of
the controls (lines 1 and 9). FRTL cells plated in mF12 containing
0.5% calf serum but no hormone supplement died (8). When FRTL
cells were passaged into mF12 alone, no attachment or division oc-
curred (not shown). Curves: 1, lacking calf serum; 2, lacking serum
and thyrotropin; 3, lacking serum and insulin; 4, lacking serum and
transferrin; 5, lacking serum and somatostatin; 6, lacking serum and
glycylhistidylysine; 7, lacking serum and hydrocortisone; 8, lacking
all six hormones; 9, complete mF12+6H.
3458 Cell Biology: Ambesi-Impiombato et al.
a single cell, is probably the best method for achieving homo-
geneity in cell strains of this kind that have very low plating
efficiencies.
The FRTL strain grows with a population doubling time of
5-7 days in mF12+6H at 370C. We calculate conservatively
that the FRTL strain has undergone more than 175 population
doublings since its initiation in July 1976. These cells have re-
mained diploid. Late in 1978, 25 metaphases were scanned; 23
of them had 42 chromosomes and 2 had 41 (and probably
represented broken metaphases); all the chromosomes appeared
normal in shape as judged by the quinacrine mustard fluo-
rescent banding technique. These results were duplicated in
the fall of 1979.
The morphological characteristics of the primary cells are
unchanged in the FRTL strain after more than 3 years of con-
tinuous culture. By phase-contrast microscopy, individual cells
are small with a small nucleus containing one or two nucleoli.
The FRTL cells are round and smaller than most cultured cells
but, because they do not adhere broadly to the plastic support
(due to the low serum concentration), the cytoplasm is often
stretched in various directions, particularly immediately after
isolation. The cytoplasm of such isolated cells sometimes outlides
large lumen-like structures (Fig. 2). Colonies often develop
follicular lumina at their center, which tend to grow or to fuse
into larger "follicles" in time (Fig. 3). At high cell densities and
after several weeks in the same culture dish, the cells tend to
grow on top of one another to form three-dimensional structures
(often without a discernible lumen) rather than to expand in
a monolayer to fill the empty spaces on the dish.
Thyroglobulin Production. The FRTL cells were found, by
radioimmunoassay, to secrete thyroglobulin into the growth
medium. Tests were made first after 4 months and again at least
once a year for the 3 years of their continuous culture. The su-
pernatant medium (6 ml/2-4 X 106 cells per 100-mm plate)
was removed for radioimmunoassay 3 days after the last com-
plete medium exchange. Thyroglobulin was found in this me-
dium at a concentration of 7.0.ug/ml (Table 2). Control media
from other cell strains including nonfunctional thyroid epi-
FIG. 2. Phase-contrast photomicrograph of isolated FRTL cells
in primary culture. The large vacuolated structures are typical of
primary cultures of thyroid cells and may represent attempts to
produce follicle-like structures. The irregular shape of some of the
cells when not part of a colony (bottom) is probably due to incomplete
attachment to the plastic support and occurs only in low serum con-
centrations. Bar indicates lOm.
100
Proc. Natl. Acad. Sci. USA 77 (1980)
FIG. 3. Phase-contrast photomicrograph of a portion of a large
colony of FRTL cells at the third passage. The tendency to form
three-dimensional follicle-like structures that increase in size with
time can be noted even in the absence of nonepithelial cells. Bar in-
dicates 100,um.
thelial cells (strain FRT that were grown in medium with 5%
serum but without hormones), thyroid fibroblasts, and liver and
kidney cells all showed no thyroglobulin production.
Iodide Concentration. The C/M ratio was determined for
several cell lines (Table 3). Fibroblast cells and nonfunctional
thyroid epithelial cells, as well as cells of nonthyroid origin, all
had a C/M close to 1.0, indicating that the equilibrium position
reached by the radioactive iodide added to the medium was
limited by a simple diffusion process. FRTL cells were able to
concentrate iodide to 100 times the external concentration.
DISCUSSION
Our observations indicate that the low-serum hormone-sup-
plement approach to cell culture may be generally applicable
to the culture of other specialized epithelial cells from kidney,
liver, uterus, and lung. The obvious special advantage that
thyroid offers is that one specific tropic hormone for it is already
well known.
Perhaps one of the most valuable advantages of the low-
serum strategy is that, from the first few days of primary cul-
ture, the epithelial cells, not the fibroblasts, are selectively fa-
vored in medium with 0.5% calf serum. The well-known pro-
gressive selection in favor of fibroblast-like cells, which are
better adapted to growth in conventional culture conditions
(17), is thus avoided.
As our results show, it may not be easy to make unambiguous
decisions as to whether or not a growth factor is required. In
low-cell-density experiments the degree of dissociation of the
cells was found to be critical. In plates containing incompletely
dissociated clusters of 25-50 cells, division may continue slowly
in these regions and after many weeks even restore or exceed
the original inoculum (as seen with curves 2 and 3 in Fig. 1). We
found it difficult or impossible to establish an absolute re-
quirement for glycylhistidyllysine and hydrocortisone at rel-
atively high cell densities. With inocula >100 cells/mm2 even
the requirements for thyrotropin and insulin and especially
those for transferrin and somatostatin were slow to be expressed.
Our results clearly show that the physiology of the cells may be
different under different conditions of cell density.
After more than 3 years of continuous culture, FRTL cells
 Cell Biology: Ambesi-Impiombato et al.
still retain a principal differentiated characteristic of follicle
cells: dependence on thyrotropin for growth. When a graded
series of thyrotropin concentrations were tried, we found that
cell growth and the persistence of the morphological and dif-
ferentiated characteristics were dose dependent. Although an
effect could be seen at 0.5 milliunit of thyrotropin per ml, a
dramatic stimulation of growth and maintenance of normal
morphology was present at 5.0 and 10.0 milliunits/ml; con-
centrations of 30, 50, or 100 milliunits/ml showed little further
improvement. When these experiments were repeated with a
more highly purified preparation of thyrotropin the findings
were identical. This indicates that the growth-stimulating effect
was due to the thyrotropin and not to possible contaminants
present in the less pure preparations that have been used rou-
tinely as supplements in our cultures. Because of the sharp effect
of thyrotropin on division in these cells it is possible that thy-
rotropin deprivation might prove helpful in obtaining synch-
rony of DNA synthesis or cell division.
We consider that the maintenance of a strict dependence on
thyrotropin is strong evidence for the normality and the dif-
ferentiated character of FRTL cell cultures. Recently it was
reported (18) that thyrotropin is not a growth factor for human
thyroid cells in culture. This conclusion was based on the finding
that conventional mass cultures of human thyroid tumors
showed a dose-related decrease of [3H]thymidine incorporation
in a 24-hr interval after administration of increasing doses of
bovine or human thyrotropin preparations. The functional
status of these comparatively short-term cultures was not re-
ported. The data appear to fall short of establishing a role for
thyrotropin in cultures of normal differentiated thyroid follicle
cells. Our cultures of functional rat thyroid epithelial cells re-
quired thyrotropin for sustained cell division and long-term
survival at all cell densities.
We have observed no evidence of senescence in vitro of these
FRTL cells or among other epithelial cell strains derived from
rat liver (BRL) (2). The FRTL cells have undergone at least 175
population doublings without a discernible crisis or change in
karyotype, growth rate, or level of differentiated function.
FRTL cells did not form tumors when 2 X 106 cells were in-
jected into each of five Fischer rats that were subsequently
monitored for more than a year. The cellular senescence phe-
nomenon reported by Hayflick (19)-in which cultures of
human lung and skin fibroblasts ceased to divide after 50-100
population doublings in vitro- does not seem to apply to epi-
thelial cells of the rat. One of the considerations that has in-
hibited investigators from using apparently normal primary
cell strains as opposed to transformed cell lines for biochemical
studies has been the fear that these cell strains would suddenly
become "senescent" and stop growing, making it difficult to
complete long-term studies. Our FRTL culture has been
maintained free of heteroploidy or of any of the characteristics
of the transformed state without evidence of senescence. To
avoid a circular argument, we must deny that remaining ap-
parently unchanged for 200 cell generations is in itself evidence
of transformation or of becoming an established cell line.
During most of the 3 years of continuous culture of strain
FRTL, the cells were harvested and replated at 106/100-mm
plate every 2-3 weeks. The cells from one dish were not pooled
with others before transfer, so that any change, such as a
spontaneous transformation, that might have occurred in one
plate would be detected before it had spread to all the plates
being carried. Periodically, samples of several lineages were
frozen and stored in liquid nitrogen. Twice a year, chromosome
Proc. Natl. Acad. Sci. USA 77 (1980) 3459
counts were made and quinacrine mustard banded preparations
were scanned for obvious anomalies. During the second and
third years of FRTL serial passages, two spontaneous trans-
formation events were detected and segregated from the main
lineage; during the third year, a translocation was found and
eliminated by return to a previously frozen sample. One of the
spontaneous transformations gave a fully functional trans-
formed subline with a population doubling time of about 1 day
and a modal chromosome number of 67. The second appeared
fibroblast-like and had lost both differentiated functions. The
translocation strain had nearly the same population doubling
time, morphology, and differentiated function as the main
lineage FRTL. With relatively simple precautions like passage
without pooling and with periodic chromosome counts, it should
be possible to propagate nontransformed epithelial strains for
at least 200 population doublings and perhaps without limit.
We anticipate that the ability to culture and colonially purify
apparently normal differentiated thyroid cells in vitro will
prove useful in a wide range of studies in cellular genetics, gene
regulation, pharmacology, and aging, apart from the obvious
applications in studies of thyroid endocrine physiology and
biochemistry. In particular, with the availability of diploid
hormone-dependent endocrine cells in vitro, novel approaches
to the study of the interactions between cells and hormones or
hormone-like substances will become possible. This will cer-
tainly lead to a more detailed understanding of the physiological
interactions between organs and hopefully will lead to a better
understanding of the integration among cell types and tissues
within an organ.
Note Added in Proof. Low serum conditions are critical during early
passages when selection of the more differentiated cells occurs.
However, once isolated, a substrain of FRTL was grown for >1 yr in
mF12+6H plus 5% serum, had a doubling time of 30-40 hr, and re-
mained diploid and differentiated.
We acknowledge the excellent technical assistance of Ann Rahe and
we thank Dr. L. D. Kohn for the gift of purified thyrotropin, Dr. R.
Larsen for the thyroglobulin radioimmunoassay, and Dr. G. H. Sato
for helpful discussions and advice.
1.
Coon, H. G. (1966) Proc. Natl. Acad. Sci. USA 55,66-73.
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