Proc. NatL Acad. Sci.
USA
Vol. 79, pp. 4795-4799, August 1982
Medical Sciences
Human amniotic fluid cells grown in a hormone-supplemented
medium: Suitability for prenatal diagnosis
(primary culture/growth-promoting factors/low-serum medium/chromosome analysis)
HSIAO-CHEN CHANG*, OLIVER W. JONES*, AND HIDEO MASUIt
*Department of Medicine, M-013, and tDepartment of Biology, Q-058, University of California at San Diego, La Jolla, California 92093
Communicated by Morris Enton Friedkin, May 3, 1982
ABSTRACT A new supplemented medium has been devel-
oped to improve human amniotic fluid cell growth and to reduce
the dependence on exogenously added serum. The medium con-
sists of a mixture of Ham's F12 medium and Dulbecco's modified
Eagle's medium supplemented with Hepes, antibiotics, and 10
growth-promoting factors at 4% fetal bovine serum. Good clonal
growth is achieved consistently in 8-13 days and is associated with
large numbers of metaphase cells. Primary clones may be ana-
lyzed directly, thereby reducing difficulty with interpretation of
chromosomal mosaicism. This medium could also be used for cul-
tivation of fetal solid tissues and peripheral blood cultures of
lymphocytes.
Numerous hormones have been detected in human amniotic
fluid-such as human growth hormone, human follicle-stimu-
lating hormone, human luteinizing hormone, human chorionic
gonadotropin, thyroid hormones, insulin, glucagon, testoster-
one, progesterone, estradiol, and cortisone, among others. The
source and entry of some hormones, their concentrations, and
their possible physiological role in pregnancy have been re-
viewed by Dawood (1).
Most prenatal genetic diagnostic studies rely on amniocen-
tesis and the subsequent growth of human amniotic fluid cells
(HAFC). Despite various techniques used in HAFC cultures,
fetal bovine serum (FB serum) is perhaps one of the most im-
portant determinants in achievement of cell growth in vitro. A
level of 15-30% FB serum is most often added to HAFC cul-
tures in a wide range of chemically defined nutrient media
(2-5). The high concentration of the serum may increase the
chance of mycoplasma contamination, which is known to cause
structural aberrations in chromosomes prepared from HAFC
cultures (6). It may also exhibit an inhibitory effect on cell
growth (7).
The studies described here were -undertaken to determine
whether a partial supplement of some pregnancy-related hor-
mones, originally present in amniotic fluid, to a nutrient me-
dium would lower serum requirements of HAFC. In addition,
experiments were designed to determine if a supplement of
growth additives would minimize vast variations in growth stim-
ulation properties in commercially prepared serum used for tis-
sue culture purposes. By a modification of serum-free culture
systems, a medium supplemented with 10 growth-promoting
factors at 4% FB serum consistently resulted in good clonal
growth in 8-13 days. In conjunction with the use of chamber/
slides, chromosomal analysis from primary culture in situ could
be performed in a relatively short time.
The publication costs ofthis article were defrayed in part by page charge
payment. This article must therefore be hereby marked "advertise-
ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
4795
MATERIALS AND METHODS
Culture Medium. Coon's modification of Ham's F12 me-
dium (Coon's F12 medium) was routinely used in our prenatal
diagnostic laboratory; therefore, it was included as a control
medium for various growth assays (8).
Unless otherwise stated, the basic culture medium [serum-
free (SF) medium] was a 1:1 mixture of Dulbecco-Vogt mod-
ified Eagle's medium (DVME medium) and Ham's F12 medium
(F12 medium) supplemented with 15 mM Hepes and 1.2 g of
NaHCO3, 40 mg of penicillin, 8 mg of ampicillin, and 90 mg of
streptomycin per liter.
The SF medium plus 10 growth-promoting factors was
termed H medium (supplemented medium), and the derivation
is shown in the next section. The growth-promoting factors
added were: transferrin, 5 gg/ml; selenium, 20 nM; insulin,
10 pug/ml; triiodothyronine, 0.1 nM; glucagon, 1 gg/ml; fibro-
blast growth factor, 10 ng/ml; hydrocortisone, 1 nM; testos-
terone, 1 nM; estradiol, 1 nM; and progesterone, 1 nM. All 10
growth-promoting factors were added to the SF medium just
prior to seeding HAFC. In some experiments, twice the amount
of each growth-promoting factor was added to the SF medium;
this was defined as H-1 medium (supplemented medium). The
added factors in H-1 medium were transferrin, 10 gg/ml; se-
lenium, 40 nM; insulin, 20 pug/ml; triiodothyronine, 0.2 nM;
glucagon, 2 pug/ml; fibroblast growth factor, 20 ng/ml; and hy-
drocortisone, testosterone, estradiol, and progesterone at 2 nM
each.
Growth-Promoting Factors in Subcultures. Because these
experiments required many duplicate samples, it was not prac-
tical to use primary cultures; therefore, the subcultures from
trypsin-treated primary cultures (19-24 wk of gestation) were
grown with multiple well plates (Linbro, 76-033-05, Flow Lab-
oratories, Hamden, CT).
The trypsin-treated subcultures were plated with 2.5% FB
serum in SF medium overnight. After three washes, the cells
were then treated with various growth-promoting factors in SF
medium. The final cell numbers were then measured by a Coul-
ter Counter on day 3.
Primary Cultures. HAFC from 19-24 wk of gestation were
from women undergoing elective therapeutic abortions and
were cultured in 35-mm plates (Linbro FB-6TC, Flow Labo-
ratories). The amount of amniotic fluid used to initiate these
primary cultures was expressed in volume (8 ml per plate for
each treatment) because sample fluids at different gestational
ages contained variable amounts of viable and nonviable cells.
HAFC were centrifuged at 700 rpm for 8 min (EC clinical
tabletop centrifuge). After removal of the supernatant fluids,
Abbreviations: HAFC, human amniotic fluid cell(s); FB serum, fetal
bovine serum; DVME medium, Dulbecco-Vogt modified Eagle's me-
dium; SF medium, serum-free medium.
4796 Medical Sciences: Chang et al. Proc. Natl. Acad. Sci. USA 79 (1982)

the cell suspensions made from the cell pellets were seeded in CA) for 1 min and stained with Giemsa by a modification of
culture vessels as indicated. There was one change of nutrient Seabright (9).
media after 5-7 days. H medium or H-1 medium (or both) plus Materials. Bovine insulin, human transferrin, glucagon,
FB serum at 1%, 4%, 8%, and 20% were used for the cultures. triiodothyronine, hydrocortisone, estradiol, progesterone, and
The primary cultures of 10-13 days were evaluated and cell testosterone were purchased from Sigma. Sodium selanite and
numbers were determined by using a Coulter Counter. Colony- phytohemagglutinin were from Difco. Human fibronectin was
forming assays were performed with the cultures fixed with 4% from Collaborative Research (Waltham, MA).
formaldehyde and. stained with 5% Giemsa.
Chromosome Analysis in Situ. Amniotic fluid (6-10 ml) from RESULTS
clinical patients at 16-18 wk ofgestation were cultured for chro- Secondary Growth in a Supplemented SF Medium. Primary
mosome analysis. For multiple cultures, the uncentrifuged, and cultures of HAFC were first established in Coon's F12 medium
centrifuged amniotic fluid samples were placed directly in with 20% FB serum for 10-18 days. Then, 10 growth-promoting
growth chamber/slides (Lab-Tek 4801, Miles). H-1 medium factors were added individually or in combination, and growth
plus 4% FB serum was added, and the cultures were observed response of trypsin-subcultured cells was measured after a cul-
for cell growth. There was a change of the nutrient media at 5-7 ture period of 3 days in SF medium (Table 1). Each individual
days and every other day thereafter. Good clonal. growth was factor stimulated growth by 20-76%. However, with combi-
achieved reproducibly in 8-13 days. nations of 2 to 10 factors, the growth response increased
Cell harvest and chromosome preparation were performed 48-213%. Therefore, the 10 growth-promoting factors close to
in situ on the chamber/slides. After 3-3.5 hr in colcemid their optimal concentrations (data not shown) were simulta-
(0.05-0.1 ug/ml, GIBCO), most of the medium was removed neously added to SF medium-termed H and H-i media-and
by gentle suction and replaced with 3.0 ml of hypotonic solu- used to study HAFC primary growth.
tion. A hypotonic treatment of 0.075 M KCI/0;6% sodium ci- Primary Growth in a Supplemented Medium. HAFC were
trate (2:1) was carried out at 370C for 35 min. At the end of the cultured in FB serum at concentrations of 1%, 4%, 8%, and 20%
treatment, 1.5 ml ofthe Carnoy's fixative (methanoVacetic acid,
3:1) was added directly to the slide-without removing the
hypotonic solution-for 15 min at room temperature. After A B C
three more complete changes of the fixatives (10-15 min each
time), the chamber walls were removed. The slides were then
dried briefly on a hot plate and were left overnight in an oven
(55°C) or left to air-dry for 2-5 days. For G-banding, the slides
were treated with 0.05% trypsin (Irvine Scientific, Santa Ana,
Table 1. Growth response of trypsinwsubcultured HAFC cultures 0 F
in a SF medium* ..:, 0
=
"
Am
% of
Additiont growth
None 100
Selenium 120
Insulin 161
Triiodothyronine 126
Fibroblast growth factor 159 G H
Glucagon 169
Hydrocortisone 176
Progesterone 144
Estradiol 140 S I/
Testosterone 159
Transferrin 169
Above + selenium 148
Above + insulin 181
Above + triiodothyronine 190.
Above + glucagon 219.
Above + fibroblast growth factor 256 0.
Above + hydrocortisone 267
Above + estradiol 230 _. _.
Above + progesterone 289
Above + testosterone 313
FIG. 1. Primary cultures of HAFC in a supplemented medium. All
The concentrations of the added factor(s) were: transferrin, 5 ,ug/ cells were cultured with DVME medium plus F12 medium (1: 1) except
ml; selenium, 20 nM; insulin,. 10 ,ug/ml; triiodothyronine,.0.1 nM; glu- in plate J, where Coon's modified Ham's F12 medium (Coon's F12 me-
cagon, 1 ,ug/ml;.fibroblast growth factor, 10 ng/ml; hydrocortisone, 1 dium) was used as a control. Percentages indicate FB serum concen-
nM; testosterone, 1 nM; estradiol, 1 nM; progesterone, 1 nM. The num- trations in each plate. Levels of added 10 factors (see text) varied in
bers represent the averages of duplicate.35-mm plates. H and H-1 media at a specified serum concentration. Plates A, B, and
* Primary culture of HAFC had been first, grown with 20% FB serum C were at 1% in SF, H, and H-1 media, respectively; plates D, E, and
in Coon's F12 medium, and subcultures were obtainedby using 0.05% F were at 4% in.SF, H,.and H-1 media,-respectively; plates G,.H, and
trypsin/0.02% EDTA. The growth response to various factors was I were at 8% in. SF, H, and H-1 media, respectively; plates J, K, and
measured with the subcultured cells in a SF medium after 3 days of L. were at 20% in Coon's F12, H, and SF media, respectively. HAFC
growth. seeded from 8 ml of amniotic fluid per plate were grown for 13 days,
t Above, in each instance where appropriate, refers to the line im- fixed with 4% formaldehyde in phosphate-buffered saline, and stained
mediately preceding. with 5% Giemsa.
 Medied Sciences: Chang et d Proc. NatL Acad. Sci. USA 79 (1982) 4797

6
I). The further stimulation was generally exhibited with growth
at 8% FB serum in H-1 medium (plate I). These cells normally
reached confluence 8-10 days after seeding. Cells with 20%
FB serum in H medium consistently demonstrated the most
5F rapid growth rate (plate K).
In a similar experiment, total cell count was obtained after
/ a growth period of 10-13 days. Primary growth response of
HAFG-cell number times 105 per 35-mm tissue culture
4 plate-was studied as a function of FB serum concentrations
/ in SF, H, and H-1 media (Fig. 2). With serum alone in the SF
x medium, the growth of the cells was maximal at 4% FB serum
and the plateau remained between 8% and 20% FB serum.
LC
3F When the factor levels were doubled in H-1 medium, there
generally were higher cell numbers than those found in cul-
-a
tures grown in H medium. Nevertheless, these slight differ-
LU ences in growth rate in H and H-1 media were not statistically
2 /1 significant.
The addition of human fibronectin-a pretreatment at room
temperature for 45 min-greatly enhanced growth of the cells
I T in H-1 medium at 1% FB serum (Fig. 3). Two samples from
1-
different patients (plates A, B, and C and plates D, E, and F)
I I were grown at 1% in SF medium (plates A and D), H medium
(plates B and E), and H-1 medium (plates C and F), and the
I I I I sII I U I latter exhibited a much-preferred growth condition. The total
0 2 4 6 8 10 12 14 16 18 20
FETAL BOVINE SERUM, %
FIG. 2. Response of primary culture of HAFC in a supplemented
medium. The growth-cell number times 10' per 35-mm tissue culture *A
plate-was a function of FB serum concentrations at 1%, 4%, 8%, and
20%. All cells were grown in SF medium (v), H medium (e), or H-1 * e
medium (0). See text for identification of the added 10 factors. Values
given were the mean SEM of 14 separate determinants except those
±
.... .~~~~~~~~~~~~~
cells treated with 4% FB serum in SF, H, and H-1 media, where only
6 separate determinants of each were run. ..I
0 E/ FI
in H and H-I media. Growth stimulation by the media is shown Ar9

in Fig. 1. In all cases, there was an increased number of clones ..wo~~~~.

* 4A4I#
when compared with serum alone (plates A, D, G, J, L) versus
serum plus 10 factors (plates B, C, E, F, H, I, K). The 2-fold
increase of the added factors resulted in additional growth in
these cells (compare paired plates B and C, E and F, and H and
G .A
WI 1996.

FIG. 4. Response of primary cultures of HAFC to deletion of a fac-

FIG. 3. Primary culture of HAFC grown in supplemented medium
at 1% FB serum. Cells shown were from two separate patients (A, B,
and C and D, E, and F), and plates A and D, B and E, and (and F were
in SF, H, and H-i media, respectively. See text for identification of the
added 10 factors. To improve attachment of the cells, these plastic
plates had been coated withhuman fibronectin (10 yg per 35-mm plate)
before seeding of the cells. HAFC growth, fixation, and staining as in
Fig. 1.
tor from a supplemented medium. Plates A, B, and C were 20% FB
serum in Coon's F12, H, and SF media, respectively. Plate D was 4%
FB serum in SF medium. The remainder of the plates were all at 4%
FB serum in H-1 medium, with or withoutthedeletion of a factor. Plate
E, without transferrin; plate F, without selenium; plate G, a control
with all 10 factors; plate H, without insulin; plate I, without triio-
dothyronine; plate J, without fibroblast growth factor, plate K, without
glucagon; plate L, without four steroids. See text for identification of
the added 10 factors. HAFC growth, fixation, and staining as in
Fig. 1.
4798 Medical Sciences: Chang et aL
cells from those primary cultures, ranging between 0.9-5.2
X 1i0 cells per ml, proved to be enough to make chromosome
preparations in 10 of 15 samples tested.
The growth condition of 4% FB serum in H and H-1 media
(see Figs. 1 and 2) was chosen for the deletion experiment (Fig.
4). Plates A, B, and C were at 20% FB serum in Coon's F12,
H, and SF media, respectively. The remainder of the plates
were at 4% FB serum in H-1 medium (plate G) or one factor
at a time was deleted in H-1 medium (plates E, F, H, I, J, K,
and L). This proved that the simultaneous addition of the 10
factors to SF medium was better for the cell growth than any
of the combinations of the 9 factors; thus, each factor is impor-
tant for growth stimulation.
Clinical Application of Chromosome Analysis in Situ from
Primary Culture. As little as 2 ml ofthe HAFC was seeded into
a growth chamber/slide.-From each 1-ml volume of amniotic
fluid there were 3.0-8.8 clones cultured on the glass slide. By
using the chamber/slide, the preserved mitotic cells remained
fixed in the original clones. Cell loss was minimal; thus, the
number of metaphases was increased to 10-12% in certain
clones. Fig. 5A demonstrates a culture harvested for chromo-
some preparation in situ after 11 days in culture. At the edge
of one clone, a high percentage of mitotic cells can be seen by
phase microscopy. After a brief treatment with trypsin and
Giemsa, a G-banded metaphase spread shows a normal female
(46,XX) (Fig. 5B). This medium also supported good cytogenetic
analysis when coverslips were used in culturing HAFC.
Experiments had been conducted simultaneously in four
combinations of culturing cells in two different media and two
culture vessels. Table 2 briefly summarizes HAFC growth with
H-1 medium at 4% FB serum in a chamber/slide and with
Coon's F12 medium at 20% FB serum in a culture dish in a
conventional method. Clearly, chromosome analysis is obtained
sooner with cells cultured with the experimental method. Fur-
thermore, primary cultures of fetal skin fibroblasts from solid
tissues of six abortuses were used for karyotyping purposes be-
tween 12 and 16 days after culture initiation with H-1 medium
at 4% FB serum. Successful results of 20 peripheral blood cul-
tures, including one sample of Ficoll/Paque-isolated lympho-
cytes, were also obtained by using this medium.
To exclude the possibility that maternal cell growth was not
,D!*
.a-,¶ . , a' .
,S1
j n, t .
% ~~ i ~ i! ~ U
* "%.
* * ,74
4 .. . q / ,,,t
.o
Proc. Natl. Acad. Sci. USA 79 (1982)
stimulated by the hormone-supplemented medium, several
guidelines were carefully followed (10). Three or more multiple
cultures for each sample were used for chromosome analysis;
mitoses were selected from clonal cells by the in situ method.
Parallel experiments of karyotype analysis in 35 amniotic fluid
samples were performed with H-1 medium at 4% FB serum and
Coon's F12 medium at 20% FB serum in culture plates. An
identical result was obtained in confirming 17 of 46,XY karyo-
types and 18 of 46,XX karyotypes. Fluorescent polymorphisms
were compared between maternal blood lymphocytes and
HAFC when necessary.
DISCUSSION
Kaback and Leonard described studies to determine whether
specific media with increased serum concentration or selected
hormones would enhance the rate of HAFC culture growth (11).
They found that neither insulin nor fetuin nor cortisone had
any stimulatory effect on cell growth. Gospodorawicz et al. (12)
reported that fibroblast growth factor at 100 ng/ml in the pres-
ence of 20% FB serum induces significant mitogenic effects on
primary culture growth of human and bovine amniotic fluid
cells. However, epidermal growth factor at 100 ng/ml had no
statistically significant effect on cell growth. Since then, fibro-
blast growth factor has been widely tested along with standard
20% FB serum to promote faster-growing HAFC cultures (13,
14). Cartilage growth factor isolated from bovine scapular car-
tilage has been shown to increase cell numbers by 70% in pri-
mary cultures of 14-16 days in a small blind clinical trial (15).
Some investigators have attempted other modifications of
tissue culture media. For example, Reidy and Chen used 5%
FB serum in MCDB 104 to culture cells (16). They provided
no data about how many colonies per flask and how much fluid
they used to initiate cultures. These investigators also used a
combination of 5% FB serum plus 15% amniotic fluid in
McCoy's 5A medium for growing HAFC (17). Nevertheless,
three colonies per flask seem to be a low yield in cell growth.
Another system that used maternal serum in HAFC cultures
has been reported by Young et al. (18).
Taken together, it is apparent that there is a need to develop
further a culture system that would enhance HAFC growth at
f
s. U..
i. I-M0.
j
',
_-5
*f t P?
vs w
rxa-M-44C w
isW
.
A,
41 FIG. 5. Growth of primary culture
_v- v
t : of HAFC in a supplemented medium
41 in a growth chamber/slide. Fresh am-
niotic fluid (not centrifuged) in a 2-ml
0%*-
a portion was directly seeded with an
equal amount of H-1 medium at 4% FB
serum. A culture was harvested for in
situ chromosome preparation after 11
t
days in culture. (A) At the edge of an
active clone showing many preserved
metaphase cells. (x 160.) (B) A G-
banded metaphase spread after tryp-
B 4
sin treatment and Giemsa staining.
(x 750.)
 Medical Sciences: Chang et al.
Table 2. HAFC grown in chamber/slide with H-1 medium at 4%
FB serum and in culture dishes with Coon's F12 medium at 20%
FB serum*
Experimental Standard
Variable method method
Culture medium H-1 medium Coon's F12 medium
+ 4% FB serum + 20% FB serum
Culture vessel Chamber/slide Culture dish
Amniotic fluid used
for seeding 2-5 mlt 5-15 mlt
Chromosome Individual clones of Mixed primary clones
analysis primary cultures and subcultures
in situ in most cases
Mitotic index Higher Lower
Diagnostic results 8-13 days 10-16 days
Medium used for Solid tissuest Solid tissuest
other cells Peripheral blood lym-
phocyte culture§
* H-1 medium refers to 4% FB serum plus 10 factors in a 1:1 mixture
of DVME medium/F12 medium (see Materials and Methods); Coon's
F12 medium refers to 20% FB serum in Coon's modified Ham's F12
medium as a positive control.
t HAFC (20 samples) were collected from pregnant women with ges-
tation at 16-18 wk.
t Fetal skin fibroblasts were grown from solid tissues of six abortuses.
§ Peripheral blood (20 samples) was used for short-term lymphocyte
cultures in H-1 medium; RPMI 1640 medium at 15% FB serum was
run for comparison.
low serum concentration. The results shown here reflect one
approach to this goal. In the culture system described, there
is a shortened period of time required to achieve good clonal
growth and increased metaphase cells cultured from amniotic
fluid. The primary growth of HAFC at 4% FB serum in H-1
medium is equivalent to those cultured at 20% FB serum in
Coon's F12 medium (19). Growth uniformity among clinical
patients is observed in these HAFC cultures (20). It is also fea-
sible to analyze individual primary clones in situ to reduce pos-
sible misinterpretation of chromosome mosaicism (21, 22).
Although this supplemented medium at 4% FB serum has
been developed mainly for primary cultures, it can also be used
for HAFC secondary cultures, peripheral blood cultures, and
fetal solid tissue. Primary growth can be further stimulated by
raising serum to 5-20% in this medium for those samples of low
cell numbers or early gestation (or both) (Figs. 1 and 2). When-
ever there are 5-7 active growing clones with 200-1,000 cells
per clone, these cultures could be processed for chromosome
analysis in growth chamber/slides. For secondary culture, it is
suggested that 5-20% FB serum be used in H-1 medium. Be-
cause cells grown in low serum or SF medium generally tend
to be more sensitive to trypsin/Pronase, subcultures with this
treatment should be handled with care.
The replacement of DVME medium and F12 medium in the
supplemented medium with Ham's F10 medium, Ham's F12
medium, Coon's F12 medium, McCoy's 5A medium, RPMI
1640 medium, Ham's MCDB 104 medium, DVME medium,
Eagle's minimal essential medium, minimal essential a-me-
dium, or medium 199 also promotes rapid multiplication of
HAFC (ref. 19; unpublished data). Therefore, the best com-
bination for the supplemented medium could be found among
these media. For example, minimal essential a-medium that
contains deoxyribonucleosides and ribonucleosides could be a
possible alternative to be one of the basic culture media (23).
The presence of many pregnancy hormones in commercial
serum is presumably responsible for its growth-promoting ac-
tivity in tissue culture systems (24). Barnes and Sato also have
Proc. Natl. Acad. Sci. USA 79 (1982) 4799
indicated that the primary role of serum is to provide hormones
and that serum can be replaced by a group of hormones in a
SF medium (see ref. 25). Our results strongly support these
observations. Other hormones have been tested for growth-
promoting activity-including growth hormone, luteinizing
hormone, luteinizing-hormone release factors, chorionic
gonadotropin, placenta lactogens, reverse triiodothyronine,
and prostaglandins. All have shown some activities in enhancing
growth of amniotic fluid cells (unpublished data). Perhaps a
substitute of triiodothyronine with reverse triiodothyronine is
required for further improvement of the supplemented me-
dium (26).
We thank Dr. G. Sato for his gift of fibroblast growth factor, members
of the Department of Reproductive Medicine at University of Califor-
nia, San Diego, for providing amniotic fluid samples, and Ms. Kathy
Logan for manuscript preparation. H-c.C. was supported by National
Research Service Award 5 F32 AM 05887-02 from the National Institute
of Arthritis, Metabolism, and Digestive Diseases. Support was also
provided in part by State of California Department of Health Services
Grant 81-00012. This study was presented in part at the Joint Meeting
of the Western Society for Clinical Research at Carmel, California, in
February 1981.
1. Dawood, M. Y. (1977) Am. J. Obstet. Gynecol. 128, 576-583.
2. Chang, H. C., Jones, 0. W., Bradshaw, C., Sarkar, S. & Por-
reco, R. P. (1981) In Vitro 17, 81-90.
3. Felix, J. S., Doherty, R. A., Davis, H. T. & Ridge, S. C. (1974)
Pediatr. Res. 8, 870-874.
4. Nadler, H. L. & Gerbie, A. B. (1970) N. Engl. J. Med. 282,
596-599.
5. Steele, M. W. & Breg, W. R. (1966) Lancet i, 383-385.
6. Schneider, E. L., Stanbridge, E. J., Epstein, C. J., Golbus, M.,
Abbo-Halsbasch, G. & Rodgers, G. (1974) Science 184, 477-480.
7. Elmore, E. & Swift, M. (1977) In Vitro 13, 837-842.
8. Coons, H. G. & Weiss, M. C. (1969) Proc. Natl. Acad. Sci. USA
62,852-859.
9. Seabright, M. (1971) Lancet ii, 971-972.
10. Peakman, D. C., Moreton, M. F. & Robinson, A. (1977)J. Med.
Genet. 14, 37-39.
11. Kaback, M. M. & Leonard, C. 0. (1972) in Antenatal Diagnosis,
ed. Dorfman, A. (Univ. Chicago Press, Chicago), pp. 81-94.
12. Gospodarowicz, D., Moran, J. S. & Owashi, N. D. (1977)J. Clin.
Endocrinol. Metab. 44, 651-659.
13. Chettur, L., Christensen, E. & Philip, J. (1978) Clin. Genet. 14,
223-228.
14. Porreco, R. P., Bradshaw, C., Sarkar, S. & Jones, 0. W. (1980)
Obstet. Gynecol 55, 55-59.
15. Golbus, M. S., Djalal, M., Klagsbrun, M., Kaback, M. M., Lev-
enson, R. M. & Epstein, C. J. (1980) Am. J. Med. Genet. 6,
107-111.
16. Reidy, J. A. & Chen, A. T. L. (1980) In Vitro 16, 213 (abstr.).
17. Reidy, J. A. & Chen, A. T. L. (1980) N. Engl. J. Med. 303,
704-705.
18. Young, S. R., Hixson, E. T. & Wade, R. V. (1979) Am. J. Hum.
Genet. 31, 86A (abstr.).
19. Chang, H. C. & Jones, 0. W. (1982) in Growth of Cells in Hor-
monally Defined Medium, Cold Spring Harbor Conferences on
Cell Proliferation, eds. Sato, G. H., Pardee, A. B. & Sirbasku,
D. A. (Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY), Vol. 9, in press.
20. Chang, H. C. & Jones, 0. W. (1981) Karyogram 7, 54.
21. Hecht, F., Peakman, D. C., Kaiser-McCaw, B. & Robinson, A.
(1981) Am. J. Med. Genet. 10, 51-54.
22. Hsu, L. Y. F., Yahr, F., Kim, H. J., Kerenyi, T. & Hirschhorn,
K. (1978) Mt. Sinai J. Med. 45, 135-149.
23. Stanners, C. P., Eliceiri, G. L. & Green, H. (1971) Nature (Lon-
don) New Biol 230, 52-54.
24. Esber, H. J., Payne, I. J. & Bogden, A. E. (1973)J. Natl. Cancer
Inst. 50, 559-562.
25. Barnes, D. & Sato, G. (1980) Cell 22, 649-655.
26. Chopra, I. J. & Crandall, B. F. (1975) N. Engl. J. Med. 293,
740-743.