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Human Reproduction vol.9
no.
10
pp.
1927-1931,
1994
Established cell lines and their conditioned media
support bovine embryo development during in-vitro
culture
M.W.Myers, J.R.Broussard1, Y.Menezo2,
S.G.Prough, J.Blackwell, R.A.Godke3 and
J.K.Thibodeaux4
In Vitro Fertilization Laboratory, Tulsa Center for Fertility and
Women's Health, 1145 South Utica, Suite 1209, Tulsa, OK 74104,
Departments of 'Dairy and 3Animal Science, Louisiana State
University, Baton Rouge, LA 70803, USA and 2INSA, Laboratorie
de Biologie, Villeurbanne
69621,
France
4To whom correspondence should be addressed
These experiments were conducted to evaluate the ability of
different somatic-cell monolayers or conditioned medium
from somatic cells for supporting bovine embryo development
in vitro. In the first experiment, bovine embryos (2- to 4-cells)
were allocated randomly to a control (medium 199 with 10%
fetal bovine serum and antibiotics) group or co-cultured with
bovine oviduct epithelial (BOEC), buffalo rat liver (BRL),
Madin Darby bovine kidney (MDBK) or African green
monkey kidney (Vero) cells. In the second experiment, bovine
embryos (1-cell) were allocated randomly to the following
groups: control medium or conditioned medium from BOEC,
BRL,
MDBK and Vero monolayers. In both experiments,
development to the blastocyst stage was assessed after 8 days
of incubation at 39°C and 5% CO2. In Experiment 1, co-
culture improved development to the blastocyst stage
compared with control medium alone, and the highest
development was observed after co-culture with BOEC. In
Experiment 2, conditioned medium enhanced development
to morulae and blastocysts compared with the control
medium; however, no differences were detected among
different cell supports. These results indicate that both co-
culture and conditioned medium from different cell
monolayers supported development to the blastocyst stage at
a higher efficiency than control medium alone.
Key words: bovine embryos/co-culture/in-vitro fertilization
Introduction
Embryos of the domestic farm animal species often become
arrested in development during in-vitro culture. The
developmental arrest of embryos observed in vitro is thought to
be a result of inadequate culture
systems.
Improvements in culture
systems for early-stage embryos are imperative for enhancing
embryo viability, and thus improving pregnancy rates.
Investigators have used various somatic helper cells to overcome,
to some degree, the in-vitro induced cell blocks (see reviews by
Rexroad, 1989; Bongso et al., 1990; Thibodeaux and Godke,
© Oxford University Press
1992).
Co-culture systems have been used with farm animal
embryos to increase the rate of blastocyst formation during
extended culture in
vitro.
In addition, one research group (Voelkel
and Hu, 1992) has indicated that co-culturing bovine embryos
with buffalo rat liver (BRL) cells would increase the survival
of embryos following cryopreservation.
Although co-culture systems have been shown to enhance
embryonic development and increase post-thaw survival, their
use to culture human embryos in assisted reproductive technology
programmes has been reported only recently (Bongso
et
al., 1989;
Wiemer et al., 1989a,b; Menezo et al., 1990). Many human
assisted reproduction programmes have used somatic-cell co-
culture to enhance embryo development and increase the quality
of embryos available for replacement. Human embryos have been
co-cultured with various helper cells including bovine uterine
fibroblast (Wiemer et al., 1989a,b), bovine oviduct cells (Wiemer
etal., 1993, 1994), human ampullary cells (Bongso etal., 1989,
1990,
1991, 1992; Yeungefa/., 1992), granulosa cells (Mansour
et al., 1992; Freeman et al., 1993; Plachot etal, 1993) and
established cell lines such as African green monkey kidney (Vero)
cells (Menezo etal, 1990, 1992a,b) and Madin Darby bovine
kidney (MDBK) cells (Mitropoulou et al, 1993).
Established cell lines offer an attractive co-culture system for
human assisted reproduction programmes by providing a
continuous supply of cells that are easily cultured in vitro, that
readily survive cryopreservation and are readily screened for
pathological organisms (Gardner and Lane, 1993). However,
some researchers have suggested that reproductive tract cells may
be more successful than established cell lines for supporting
embryo development during co-culture. To date, a comparison
of these different somatic-cell supports has not been made in a
controlled experiment.
The objective of this study was to compare directly the
development rates of bovine embryos following co-culture with
different somatic-cell monolayers that have been used to culture
human embryos. A second objective was to compare the
development rates of bovine embryos following culture in
conditioned medium from different cell monolayers.
Materials and methods
In-vitro maturation
Bovine oocytes used for in-vitro maturation and in-vitro
fertilization (IVF) were obtained from two abattoir sources;
however, embryos derived from each source were allocated
randomly across replicates and treatment groups for each
experiment. For the first source, ovaries were collected and
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M.W.Myers et al.
transported to the laboratory in sterile saline (0.9% NaCl) at
26-30°C within 7 h of collection. Ovaries were washed with
warm saline and oocytes were aspirated from follicles (2-8 mm)
with an 18 gauge needle and a 10 ml syringe. The recovered
cumulus-intact oocytes were washed twice with maturation
medium and matured for 22-24 h at 39°C and 5% CO2 in
humidified air as described previously (Thibodeaux etal., 1993;
Broussard et al., 1994). The maturation medium was medium
199 with Earle's salts and 25 mM HEPES (Gibco, Grand Island,
NY, USA) supplemented with 10% fetal bovine serum (FBS;
Hyclone Laboratories, Logan, UT, USA), antibiotics (100 IU
penicillin and 100 /ig streptomycin/ml) and hormones [0.01 IU
purified bovine follicle-stimulating hormone (FSH) and luteinizing
hormone (LH)/ml; Nobl Laboratories, Sioux Center, IA, USA].
Oocytes were matured in four-well tissue culture plates (Nunclon,
VWR Scientific, Bridgeport, NJ, USA) containing 500 jtl of
maturation medium and 500 fd of light mineral oil (Sigma
Chemical Co., St Louis, MO, USA) that had been equilibrated
previously (39°C and 5% CO2 in humidified air) overnight.
For the second source, cumulus-intact oocytes were recovered,
washed with maturation medium as described above and
transferred into 1.5 ml culture tubes (Nunclon) containing 500
li\ of maturation medium and layered with 500 fd of light mineral
oil.
The culture tubes containing the oocytes were placed in a
pre-equilibrated battery-powered incubator (Mini-Tube of
America, Inc., Madison, WI, USA) and transported to the IVF
laboratory via an express courier. The oocytes were matured
during transit and arrived at the IVF laboratory within 18-20 h.
In-vitro fertilization
After a maturation interval of 22-24 h, oocytes were pooled
and washed twice in IVF medium. Groups of 25-30 oocytes
were transferred into 50 fi\ drops of IVF medium covered with
light mineral oil. The IVF medium consisted of modified Tyrode-
lactate medium that contained 10 mM Na lactate (Sigma), 0.25
mM pyruvate (Sigma) and 6 mg/ml fatty acid-free bovine serum
albumin (BSA), as described previously (Voelkel and Hu,
1992).
This medium was modified to include
15
/tg/ml of heparin
(Sigma) and antibiotics (100 IU penicillin and 100 fig
streptomycin/ml).
Frozen-thawed semen from the same dairy bull (Select Sires,
Plain City, OH, USA) was thawed in a 37°C water bath, layered
directly on a 90 and
45%
discontinuous Percoll (Sigma) gradient
(Keefer et al., 1993) and centrifuged at 700 g for 30 min. After
centrifugation, the top layers of solution were removed and the
sperm pellet was washed once by centrifugation (200 g for 6 min)
in IVF medium. After the final centrifugation, the sperm pellet
was resuspended in IVF medium to the desired volume containing
0.75 xlO6 spermatozoa/ml. A 50
/*1
aliquot of the sperm-cell
suspension was added to the 50
fi\
droplet containing the oocytes
and co-incubated for 18 h at 39°C and 5% CO2 in humidified
air.
Experiment 1
The objective of this experiment was to evaluate the efficiency
of different somatic-cell support systems for embryo development
during co-culture. Embryos (n = 718) were allocated randomly
to the following groups: control medium alone consisting of
medium 199 with Earle's salts supplemented with 10% FBS and
antibiotics (control; n = 134) or co-cultured with bovine oviduct
epithelial cells (BOEC; n = 148), BRL cells (n = 145), MDBK
cells (n = 138) or Vero cells (n = 153). BOECs were prepared
as described previously (Thibodeaux et al., 1993) and used
following the first or second passage. Established cell lines (BRL,
MDBK, Vero) were purchased from American Type Culture
Collection (Rockville, MD, USA) and cultured for one to three
passages post-thaw prior to seeding in co-culture plates. All cells
except MDBK were maintained in control medium during
incubation. In our laboratory, MDBK cells exhibit a higher
growth rate and higher cell viability when incubated in Ham's
F-12 medium (Gibco) with 10% FBS and antibiotics. However,
all cell preparations were seeded in four-well plates 48 h prior
to embryo co-culture in control medium, so that 60% of the wells
were confluent at the time embryos were placed with cells.
Following the 18 h insemination interval, oocytes were washed
twice in control medium to remove sperm cells, then cultured
in 500 /il of the control medium in four-well plates for an
additional 36 h. Subsequently, cleaved embryos (>2-cell stage)
were freed of cumulus cells and washed through three changes
of fresh control medium. Embryos were then allocated randomly
to one of the five treatment groups and incubated in 500 fi\ of
medium under light mineral oil in four-well plates
(
= 20
embryos/well) at 39CC and 5% CO2 in humidified air. The
culture medium was not replaced during the incubation period
and embryo development to the blastocyst or expanded blastocyst
stages was assessed on day 8 of incubation (day 0 =
insemination).
Experiment 2
The objective of this experiment was to evaluate the efficiency
of conditioned medium from different somatic-cell monolayers
to maintain embryo development during in-vitro culture. Cells
were prepared and maintained as outlined in Experiment 1.
Conditioned medium was prepared from each somatic-cell
monolayer (BOEC, BRL, MDBK, Vero) following a 48 h
conditioning period in 500 fA of medium in four-well plates at
39°C and 5% CO2 in humidified air. After the conditioning
period, the medium was harvested, centrifuged and stored
(—20°C) as described previously (Eyestone et al., 1991) until
12 h before allocation of embryos to the respective treatment
groups. Prior to the experiment, conditioned medium was thawed
and 50 fil drops of medium were placed under light mineral oil
and equilibrated overnight at 39°C and 5% CO2 in humidified
air.
After the 18 h fertilization interval, zygotes (1-cell) were placed
into 15 ml conical tubes and cumulus cells removed by vortexing.
Zygotes (n = 922) were subsequently washed through two
washes of
control
medium to ensure the removal of
cumulus
cells,
then allocated randomly to treatment groups (20-25
zygotes/droplet) and incubated at 39 °C and 5% CO2 in
humidified air. As in Experiment 1, the culture medium was not
replenished during the incubation period. Morula or blastocyst
stage development was assessed on day 7 of
culture.
In addition,
development to the blastocyst or expanded blastocyst stages was
assessed on day 8 of culture (day 0 = insemination).
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Co-culture of embryos with different cell supports
Control BOECBRLMDBKVero
Fig. 1. Development in vitro of bovine embryos (2- to 4-cells) to
the blastocyst stage following co-culture on different somatic-cell
monolayers until day 8 after in-vitro insemination (day 0 = day of
insemination). Control, culture medium alone; BOEC, bovine
oviduct epithelial cells; BRL, buffalo rat liver cells; MDBK, Madin
Darby bovine kidney cells; Vero, African green monkey kidney
cells.
Groups with different labels (a, b, c) were significantly
different (/> < 0.05).
Control BOECBRLMDBKVero
Fig. 2. Development in vitro of bovine embryos (2- to 4-cells) to
the expanded blastocyst stage following co-culture on different
somatic-cell monolayers until day 8 after in-vitro insemination
(day 0 = day of insemination). Groups with different labels (a, b)
were significantly different (P < 0.05). See legend to Figure 1 for
abbreviations.
Statistical analysis
Development of embryos to morula and blastocyst stages was
evaluated by x2 analysis. A value of P < 0.05 was considered
statistically significant.
Results
Experiment 1
The percentage of embryos developing to blastocyst stage on day
8 of incubation was improved with co-culture compared with
control medium alone (Figure 1). Those embryos co-cultured with
BOEC had the highest developmental rate to blastocyst compared
with all other treatment groups. The rate of development to the
expanded blastocyst stage was also improved significantly when
co-cultured with BOEC, BRL, MDBK and Vero compared with
those cultured in control medium alone (Figure 2). There was,
however, no difference among co-culture groups in the proportion
of embryos developing to the expanded blastocyst stage.
Experiment 2
There was no difference in embryos developing to the 2-cell stage
or greater among treatment groups (Table I). The rate of
development to morula and blastocyst stages was enhanced when
incubated in conditioned medium from the different cell
monolayer cultures compared with control medium alone.
Conversely, there was no difference in development to the morula
or blastocyst stages among conditioned media treatment groups.
Discussion
This study demonstrated that established cell lines could enhance
viability of IVF-derived bovine embryos during co-culture over
the control medium, although to a lesser extent when compared
with that of oviduct epithelial cells. However, co-incubation of
embryos in medium conditioned by oviduct cells resulted in
similar developmental rates, although these were higher than in
the control culture medium.
Development of bovine embryos to the blastocyst stage was
greater following co-culture with oviduct cells (BOEC) than with
BRL,
MDBK or Vero cells. Development to the blastocyst stage
was not different among established cell lines. Also, development
Table I. Effect of somatic-cell conditioned media on the development of bovine zygotes to the blastocyst stage during in-vitro culture
Treatment groups"Number culturedNumber cleavedbNumber of morulacNumber of blastocystsd
Control
BOEC
BRL
MDBK
Vero
182
186
183
187
184
104 (57.1)
114 (62.6)
116 (63.4)
111 (59.4)
120 (65.2)
29
(15.9)c
69
(37.1)f
70
(38.3)f
56 (30.0/
64 (34.8/
5
(2.8)e
27 (14.5/
23 (12.6/
17 (9.1/
27 (14.7/
Values in parentheses are percentages.
"Control, control medium alone; BOEC, bovine oviduct epithelial cells; BRL, buffalo rat liver cells; MDBK, Mardin Darby bovine kidney cells; Vero,
African green monkey kidney cells.
bEmbryos developing to the 2-cell or greater stage.
cEmbryos developing to the morula stage or greater on day 8 after in-vitro fertilization.
dEmbryos developing to the blastocyst stage or greater on day 8 after in-vitro fertilization.
c'fDifferent superscripts within columns differ significantly (P < 0.01).
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M.W.Myers et at.
to the expanded blastocyst stage was similar among all co-culture
groups. Previously, Voelkel and Hu (1992) reported that bovine
IVF embryos co-cultured with BRL cells had similar development
rates to those embryos co-cultured with BOECs. In contrast, those
embryos co-cultured with BRL cells had a higher post-thaw
survival rate as compared with those embryos co-cultured with
oviduct cells. Trounson et al. (1994) also noted similar
developmental rates of bovine embryos into morulae and
blastocysts following co-culture with oviduct, BRL and granulosa
cells or culture with synthetic oviduct fluid (SOF) medium.
However, the number of cells per embryo was higher following
co-culture with oviduct and granulosa cells compared with those
embryos co-cultured with BRL cells or cultured in SOF medium.
Voelkel and Hu (1992) also reported that bovine embryos co-
cultured with BRL cells required a lower oxygen concentration
(5%) for development in vitro, whereas oviduct-cell cultures
required a 20% oxygen concentration for embryo development.
The oxygen concentration in a recent report from Trounson et
al. (1994) and from our study was 20% for all embryo co-culture
groups. The BRL co-culture system also apparently requires that
embryos be transferred to a fresh cell population during the
incubation period (Voelkel and Hu, 1992). In this study, embryos
were left undisturbed during the incubation period. Collectively,
differences in incubation conditions among studies may account
for the differences reported following co-culture with oviduct and
BRL cells.
Conditioned medium may have some advantages over that of
monolayer co-culture in that no additional cells are required for
the culture system and the medium may be stored frozen,
eliminating the need for preparing cells fresh for each subsequent
embryo group (Eyestone et al., 1991). Eyestone et al. (1991)
have reported similar developmental rates for bovine morulae
and blastocysts following incubation with fresh or frozen-thawed
oviductal-cell conditioned medium. The frozen-thawed
conditioned medium culture system would allow the same batch
of medium to be used with sequential groups of embryos. This
would potentially reduce the variation in embryo development
due to variations often noted in monolayer culture systems.
Several groups have reported that conditioned medium from
oviduct, cumulus or granulosa cells enhances the rate of blastocyst
formation in bovine embryos during extended in-vitro culture
(Eyestone and First, 1989; Kobayashi etal., 1992; Harper and
Brackett, 1993), while others have noted no difference in
development over that in control medium alone. However,
conditioned media apparently may not support embryo
development in all species (Bongso et al., 1990).
Conditioned medium from all the somatic-cell monolayers in
this study supported the development of embryos to the blastocyst
stage at a similar rate. It is interesting to note that co-culturing
embryos with oviduct cells resulted in a higher blastocyst
formation than with BRL, MDBK and Vero cells (Experiment
1),
but this finding was not detected when embryos were
incubated in conditioned medium from the same cell monolayers
(Experiment 2). In our study, the proportion of embryos forming
blastocysts was lower following culture with oviduct-cell
conditioned medium (Experiment 2) when compared with direct
co-culture on oviduct-cell monolayers (Experiment 1). The exact
mechanism(s) for enhancement of the development potential of
embryos by somatic-cell co-culture systems remains unclear. It
has been proposed that these cells remove toxic components of
the culture medium, reduce metabolic by-products, reduce oxygen
tension, secrete embryotrophic substances or possibly there is
a combination of one or more of these actions (Rexroad, 1989;
Bavister et al., 1992). Co-culture on somatic cells may allow
for an extended period of detoxification of the medium or
maintenance of reduced oxygen tension, as opposed to a brief
conditioning period (48 h) of the medium prior to embryo
exposure. Such a mechanism may explain the differences in
development between monolayer co-culture and culture in
conditioned medium in this study. Furthermore, the production
of one or more growth factors (platelet-derived growth factor)
by oviduct cells (Thibodeaux et al., 1993) may also have been
reduced during the brief medium-conditioning period, which
could account for the lowered blastocyst formation in the
conditioned medium in Experiment 2.
Although several different types of somatic cell are capable
of supporting the development of embryos to the blastocyst stage,
the viability
as
judged by the ability to establish viable pregnancies
warrants further evaluation. In this study, embryos co-cultured
with MDBK cells resulted in similar blastocyst formation rates
as those co-cultured with BRL and Vero cells; however, the
quality of the blastocyst formed in the MDBK cell co-culture
group was below that of the other co-culture groups evaluated
(data not
shown).
The poorer quality blastocysts from the MDBK
cell co-culture group reflect the fact that MDBK cells are known
to exhibit higher growth rates in Ham's F-12 medium than in
the standard bovine embryo culture medium (medium 199). In
fact, different environmental requirements for growing and
developing both somatic-cell populations and embryos may
explain the lack of a beneficial effect of co-culture with some
somatic cells (Bavister et al., 1992). Previously, different culture
media used to maintain somatic cells in vitro have been shown
to have profound effects on the growth patterns of bovine uterine
and oviduct cells (Thibodeaux et al., 1992).
Improved in-vitro development of human embryos has been
observed following co-culture with oviduct cells from human
(Bongso etal., 1989, 1990, 1991, 1992; Yeung etal., 1992)
or bovine uterine and oviduct cell (Wiemer et al., 1989a,b, 1993,
1994) origin. Also, other somatic-cell types, such as Vero
(Menezo et al., 1990, 1992a,b) or granulosa (Freeman et al.,
1993;
Plachot et al., 1993) cells, have been shown to improve
human embryo development in
vitro.
Although the actual success
of somatic-cell co-culture experiments in human assisted
reproduction programmes remains unclear, largely due to the
number of embryos replaced (Trounson et al., 1994), this
methodology certainly has merit and further evaluation is needed.
Recently, Wiemer et al. (1994) reported the beneficial effects
of co-culture for those patients with consistently poor quality
embryos and in patients with poor prognosis for pregnancy
following IVF. Zona thickness can also be diminished following
co-culture, and that may enhance zona hatching (Wiemer et al.,
1994).
Perhaps the most beneficial effects of co-culture in human
assisted reproduction programmes may arise from the 'rescue'
of poor quality embryos, or increasing the implantation rates
following replacement (Wiemer et al., 1993).
In summary, this study indicates that different established cell
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Co-culture of embryos with different cell supports
lines which are commercially available are capable of supporting
bovine embryo development to the blastocyst stage during in-
vitro culture. Although co-culture with oviduct cells did result
in a greater proportion of blastocysts, development to expanded
blastocysts was similar between all the somatic-cell co-culture
groups evaluated in this study. Conditioned medium from
different cell monolayers also supported embryo development
to the blastocyst stage, but not to the same extent as the somatic-
cell monolayers. Collectively, these findings may provide useful
information to human assisted reproduction programmes for
developing the optimum culture system to increase the quality
of human embryos available for replacement.
Acknowledgements
The authors are grateful to Select Sires, Inc. (Plain City, OH, USA)
for donating the semen used in the IVF procedures. This research was
supported by a grant from the Hillcrest Medical Center Foundation.
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Received on March 9, 1994; accepted on June 9, 1994
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