Artificial zona pellucida for in vivo culture of nude blastomeres

An artificial zona pellucida is provided for implantation in the peritoneal cavities of small laboratory animals. The assembly includes a hydrogel cartridge for containing one or more nude isolated blastomeres which can be cultured therein to an implant stage in the peritoneal cavity of a small animal such as a mouse. The combination is particularly advantageous for culturing of nude isolated blastomeres of a species different from the host small laboratory animal, such as blastomeres of domestic animals.

FIELD OF INVENTION 
The field of this invention is mammalian embryo culture, and, in 
particular, the culturing of separated embryo cells to a stage at which 
they can be implanted. 
BACKGROUND OF INVENTION 
Although it is known that individual cells (blastomeres) of a developing 
mammalian embryo prior to the blastocyst implant stage are capable of 
developing into totipotent embryos, manipulation of embryos in the 2-, 4-, 
or 8-cell morulae stage has met with limited experimental success and has 
not developed into a commercial practice. Moore, et al. (1968) reported on 
a series of experiments with rabbit ova in which individual blastomeres 
were cultured in their own ova membrane (the zona pellucida). The 
culturing was compared with that of nude blastomeres without zona, and 
separated blastomeres inserted in the host zona. The culturing of the 
blastomeres was carried out in the fallopian tubes of the recipient does. 
No single blastomere devoid of zona survived, but survival was obtained 
with some of the blastomeres separated from the 2-, 4-, and 8-cell ova 
enclosed in their own zona, and the surviving blastomeres developed into 
normal rabbits. Separation of the blastomeres at the 2-cell stage gave a 
higher rate of survival (30%) than those separated at the 4- and 8-cell 
eggs (19% and 11%). Single blastomeres of 4-cell ova inserted in host zona 
showed limited development, undergoing one or more cleavages. 
Isolated blastomeres and blastomere clusters prepared by microsurgery from 
pre-implantation embryos require some form of protection when the zona 
pellucida is removed or its integrity compromised (Moore, et al., 1968; 
Willadsen, 1979). Agar has been used to seal or encapsulate ruptured zona 
pellucida containing blastomeres (Willadsen, 1979). In the experiments 
reported by Willadsen, 2-celled sheep embryo were manipulated by severing 
the zona pellucida, removing and separating the blastomeres, and 
re-inserting a single blastomere into an evacuated zona. The 
zona-containing blastomeres were transferred to an agar solution held in 
the tip of a pipette until the solution hardened. A tiny solid cylinder of 
the agar containing the zona-encased blastomere was ejected. The small 
cylinders were encapsulated in larger solid cylinders of agar. The 
cylinders were transferred to ewe oviducts for incubation. After reaching 
the late morulae or early blastocyst stage, the agar cylinders were 
recovered, the agar was removed, and the embryos were implanted. Some live 
offspring were produced. The reduced embryo survival rate compared to 
standard embryo transfer was attributed to the difficulty of manipulating 
the embryos in the agar, which required the use of hypodermic needles, and 
other procedures. 
The embedding of embryos in agar is referred to in the literature as the 
agar "chip" technique. The micromanipulations involved in the agar chip 
technique were summarized by Willadsen in 1982 in a treatise on "Mammalian 
Egg Transfer". After referring to the disappointing results in experiments 
on the developmental potential of isolated blastmeres, Willadsen concluded 
"with near certainty that the central problem in all instances arose from 
the lack of adequate methods for culture of the micromanipulated embryos 
in vitro in line with the inability of the embryonic cells to survive in 
vivo after the zona pellucida had been ruptured or removed". He further 
concluded that "none of the methods currently used is entirely 
satisfactory for the culture of early embryos, i.e., cleavage stages which 
are most dependent on the zona pellucida for their survival in vivo." 
With reference to agar embedding, Willadsen (1982) had reported that 
"single blastomeres may be embedded without a zona pellucida, but this is 
not advisable, because it makes it very difficult to release the embryos 
at a later stage". Willadsen recommended that isolated blastomeres be 
contained in their own zona or in host zona, and that agar chip embedding 
be used only for such zona-contained blastomeres. 
Egestone et al. (1985) reported on experiments in which 1-cell bovine 
embryos were embedded in agar and successfully cultured to the blastocyst 
stage in ewe oviducts. A tendency of the chips to dissolve in the oviduct 
fluid was observed. Boland (1984) reported on experiments using the rabbit 
oviduct as a screening tool for the viability of mammalian eggs embedded 
in agar chips. He concluded that the rabbit oviducts were unsuitable for 
such screening "because of the high rate of degradation of agar chips". 
More recently, Adaniya, et al. (1987) reported on the coating of rabbit 
embryos ready for implantation with sodium alginate. Following 
implantation in the uterus, the rate of degradation of the sodium alginate 
was observed. It was found that after four hours only 38% of the capsules 
remained, and after six hours none of the capsules were recoverable. 
SUMMARY OF INVENTION 
The present invention provides an artificial zone pellucida which can be 
used for culturing of isolated mammalian blastomeres, or blastomere 
clusters, without the use of any natural zona pellucida. The nude 
blastomeres are inserted directly in the artificial zona pellucida, which 
comprises a hollow cartridge of a size suitable for implantation in the 
peritoneal cavity or fallopian tube of a host mammal. The cartridge 
provides an enclosed incubation chamber having a chamber-enclosing wall 
formed of a cross-linked microporous hydrogel which is essentially 
non-biodegradeable. At least one viable nude mammalian blastomere is 
removably contained in the incubation chamber, and an aqueous culture 
medium is contained in the chamber in contact with the blastomere and the 
inside of the hydrogel wall. After implantation, transfer of substances 
from the fallopian or peritoneal fluid can take place through the hydrogel 
wall, thereby promoting the development of the blastomere to the 
blastocyst stage, ready for recovery and implantation. 
For convenient insertion and removal of the blastomeres, the artificial 
zona pellucida cartridges can be provided with removable end plugs. If it 
is desired to culture more than one blastomere in the same cartridge, 
dividers may be provided within the cartridge separating the interior of 
the cartridge into a series of separate compartments. The dividers may 
also be made removable for convenient insertion and recovery of the 
embryos. 
A preferred hydrogel material is a cross-linked methacrylate polymer, such 
as the hydrogel known as HEMA. HEMA consists primarily of polymerized 
2-hydroxyethyl methacrylate. HEMA polymers may be cross-linked with glycol 
dimethacrylates, such as ethylene glycol dimethacrylate or tetraethylene 
glycol dimethacrylate. For purpose of the present invention, the 
cross-linking should not be so extensive that it prevents the hydrogel 
from functioning as a microporous membrane. In general, the amount of 
cross-linker employed should not be over 2.5 mole percent of the hydrogel.

DETAILED DESCRIPTION 
The artificial zona pellucida assembly of this invention can be employed 
for a number of different micromanipulations and culturings of mammalian 
embryos. In a preferred application, the assembly is employed for in vivo 
culturing of nude blastomeres, or clusters of nude blastomeres. For 
example, mammalian embryos in the morulae stage prior to blastulation can 
be separated by microsurgical techniques into individual blastomeres. Use 
of blastomeres derived from 2-, 4-, or 8-cell embryos can provide the 
means for the use of chimaeric production of multiple viable, totipotent 
embryos from a single fertilized ova. The techniques for separation and 
recovery of the blastomeres can be the same as previously employed. (See, 
for example, Willadsen, 1984.) Instead of individual nude blastomeres, 
clusters of two or four blastomeres can be used. For example, a 4-cell 
embryo can be separated into two-cell clusters. 
For purpose of the present invention, the assembly comprising the 
artificial zona pellucida includes a hollow cartridge of a size suitable 
for implantation in a peritoneal cavity or fallopian tube of a host 
mammal. Where small mammals are to be employed, such as mice or rabbits, 
as may be preferred, the dimensions of the cartridge should be quite 
small, that is, the length may be from 1 to 3 cm and the exterior diameter 
from 3 to 5 mm. To achieve effective culture of the nude blastomere to the 
blastocyst stage, it is important that the body of the cartridge be formed 
from a microporous hydrogel. 
Polymers of alkyl acrylates are known to form hydrogels which can be 
cross-linked to provide microporous structures. A preferred monomer for 
forming the hydrogel is 2-hydroxyethyl methacrylate (HEMA). HEMA can be 
polymerized in the presence of glycol dimethacrylate monomers which 
function as cross-linkers. Preferred cross-linkers are ethylene glycol 
dimethacrylate or tetraethylene glycol dimethacrylate. A limited degree of 
cross-linking is desirable. The swollen or hydrated membranes of 
cross-linked pHEMA should function essentially as pore-type membranes. 
They should also be essentially non-biodegradeable under the conditions of 
use. Solute transport should take place through water-filled regions of 
the hydrogel, which acts as the pores. At high cross-linking levels, the 
hydrogel membrane begins to function as a partition-type membrane. The 
solute interacts with the membrane matrix and diffusion is retarded. See 
Wisniewski, et al. (1976). 
Data presented by Wisniewski indicated that cross-linked HEMA membranes up 
to about 2.5 mole % cross-linker can function essentially as microporous 
or pore-type membranes. For the purpose of this invention, it is therefore 
preferred that the hydrogel contain not over 2.5 mole percent of 
cross-linker, such as from 0.1 to 2.5 mole %. A preferred cross-linker 
range is from 0.2 to 1.0 mole percent in the hydrogel. Such exhibit high 
diffusivities as shown by Lee, et al., 1978). An optimum formulation is 
from 0.3 to 0.5 mole % of a glycol dimethacrylate cross-linker. 
Procedures for preparing the cross-linked polymerized hydrogels are known. 
A mixture of the HEMA monomer and the cross-linker is prepared in the 
desired proportions using a suitable solvent such as ethylene glycol, or a 
mixture of ethylene glycol and water. Initiator solutions are also 
prepared, such as an aqueous solution of ammonium persulfate, and an 
aqueous solution of sodium metabisulfite. These reactants are combined 
under pressure to minimize the entrapment of bubbles in the hydrogel. A 
suitable pressure casting procedure using two hypodermic syringes is 
described in Pinchuk et al. (1981), and this procedure is also described 
in the following experimental examples. 
It was found that the nude blastomeres could be loaded into the artificial 
zona pellucida tube using only sterile normal saline as the medium. This 
evidences that transfer of substances from the peritoneal or fallopian 
tube fluid, takes place and promotes blastomere development. It is 
preferred to use a starting medium which contains nutrients known to 
promote the growth of embryos or blastomeres. Whitten's medium (Whitten et 
al., 1968) supplemented with bovine serum albumin (BSA) can be used. Other 
suitable media for culturing embryos or blastomeres are described by 
Fisher (1987), Camous et al. (1984), and Brinster (1984). These include 
(suggest describing one or two suitable media. 
The body cavity, referred to as the peritoneal cavity, is a suitable site 
for implantation of the artificial zona pellucida assembly in the host 
animal. For small laboratory animals, such as mice, hamsters, rabbits, 
etc., this is a more convenient site than the fallopian tubes. For 
implants in larger animals, such as sheep (ewes), the fallopian tubes can 
be used, and may be employed in ligated form. Means may be provided 
surgically for convenient insertion and removal in fallopian tubes. The 
fluid present in the peritoneal cavity of female mammals is similar to 
that found in its fallopian tubes (Bouckerchart, et al., 1986, and Bryans, 
et al., 1954). The peritoneal cavity of a female laboratory animal is 
preferred, such as a female mouse, it has been found that the peritoneal 
cavity of male laboratory animals can also be employed, although with 
somewhat less effective embryo development. 
ILLUSTRATED EMBODIMENTS 
Several embodiment of the artificial zona pellucida assemblies of this 
invention are shown in the accompanying drawings. FIG. 1 represents a 
tubular hydrogel cartridge which is provided with end closures in the form 
of removable end plugs. The hydrogel cartridge is prepared from a 
cross-linked hydrogel as described above. Illustrative dimensions of the 
hydrogel cartridge shown in FIG. 1 include a chamber length of 1 cm, an 
outside diameter of 3.5 mm, an inside diameter of 1.7 mm, and a wall 
thickness of 0.9 mm. The wall thickness is given as 0.9 mm., but the wall 
thickness, as well as the other dimensions, may vary. Wall thicknesses 
within the range from 0.5 to 3 mm can be used. The end plugs 11 and 12 may 
be formed from a solid biocompatible polymer such as a silicone polymer. 
Alternatively, the end plugs may be formed of cross-linked HEMA, but 
transfer through the end plugs from the peritoneal or fallopian fluid is 
not required. In FIG. 2, the artificial zona pellucida of FIG. 1 is shown 
in assembled condition, containing a nude blastomere 13 immersed in a 
suitable aqueous medium 14. 
In FIG. 3, there is shown, a multiple compartment artificial zona pellucida 
assembly 15. In this embodiment, the ends of the hydrogel cartridge are 
slightly enlarged to receive the removable end plugs 16 and 17. Within the 
cartridge there is provided three spaced removable dividers 18, 19 and 20. 
These dividers may be formed of cross-linked HEMA, or a non-permeable 
biocompatible plastic may be employed as a silicone polymer. The dividers 
separate the cartridge chamber into four compartments, and within each 
compartment there is provided a nude blastomere. This assembly permits the 
simultaneous culturing of four blastomers 21-24. Suitable aqueous media 25 
will be employed within the compartments as previously described. 
For culturing the artificial zona pellucida assemblies, such as the 
assembly of FIG. 2 or FIG. 3, the peritoneal cavity of a mouse may be 
used. A mid-line incision may be made in the abdomen of the mouse, the 
assembly inserted, and the incision sutured. The inserted artificial zona 
pellucida assembly 15 will normally rest in the lower portion of the 
peritoneal cavity C of the mouse M, as illustrated in FIG. 4. Instead of a 
host mouse, other laboratory animals may be employed, including rabbits, 
guinea pigs, hamsters, etc. While contained in the laboratory animal, such 
as the preferred mouse, the zona pellucida assembly may be shipped to a 
detination. Within shipping times of 2 to 4 days, such as by air 
transport, the nude blastomeres can continue to develop to the blastocyst 
stage, and will be ready for implantation when the host mouse or other 
laboratory animal reaches its destination. 
The artificial zona pellucida of this invention may also be employed as an 
ova fertilization assembly 26, as illustrated in FIG. 5. The cConstruction 
of the hydrogel chamber and the end plugs is as previously described. A 
divider 27 is positioned centrally in the compartment. The divider may be 
formed from pHEMA or a silicone polymer, and is provided centrally with an 
opening of sufficient size to permit sperm to pass therethrough. Ova 
culture medium is provided in the compartments. In the left-hand 
compartment 28 viable sperm is placed and in the right-hand compartment 29 
an unfertilized ova 30. To facilitate capacitance of the sperm and thereby 
obtain fertilization, the assembly may be placed in the vagina of a host 
animal. After fertilization occurs, the ova may be incubated in the 
cartridge until it is ready for implanting. Preferably on fertilization, 
the assembly is removed from the vagina and inserted in the peritoneal 
cavity or fallopian tube of the host animal. For embryo development, 
vaginal fluid is not as effective as peritoneal or fallopian fluid. 
FIG. 6 illustrates a further use of the assembly. With the construction of 
FIG. 1, the fertilized ova 31 is placed within the hydrogel cartridge 32 
and is surrounded with an aqueous ova culture medium 33. The assembly is 
then placed in the peritoneal cavity or fallopian tube of the host animal 
and retained therein until the ova develops to the implant stage. 
The method of carrying out this invention and the results obtainable 
thereby are further illustrated by the following experimental examples. 
EXPERIMENTAL EXAMPLES 
Materials and Methods 
Construction of Hydrogel Chambers 
The hydrogel chambers were made from the mixture of HEMA monomer and 
tetraethylene glycol dimethacrylate (TGD) as the cross-linker. Ethylene 
glycol (DG) was used as a solvent. The formulation of reactants was that 
described by Lee et al. (1978, Table 1) for a ratio of 10 ml HEMA to 0.1 
ml TGD. The mole % cross-linker in the hydrogel is approximately 0.4%. 
Before casting the chambers, 3 stock solutions were prepared and placed in 
glass test tubes: Solution A--a mixture of 10 ml HEMA, 0.1 ml TGD, 3.0 ml 
EG, and 2.0 ml distilled water; solution B--initiator, 1.0 ml of ammonium 
persulfate, 40 g per 100 ml distilled water or 1.75 M; solution 
B--co-initiator, 1.0 ml of sodium metabisulfite, 15 g per 100 ml distilled 
water or 0.79 M. Each stock solution was purged with nitrogen and the 
reactants were mixed and polymerized under pressure, as described by 
Pinchuk et al. (1981) for the casting of ureteral anastomotic nipples. 
The hydrogel chambers were case at room temperature within the barrel of 71 
mm long, 3.5 mm ID, 0.5 ml insulin syringes (No. 8471 single use, 
plastipak LO-dose U-100; Becton Dickinson and Co., Rutherford, N.J.). The 
needle and its hub were removed to free the needed connector of the 
syringe. The plunger was withdrawn from the syringe and the rubber gasket 
at the tip of the plunger was removed and inverted so that the hollow 
cavity that previously attached the gasket to the plunger was directed 
toward the barrel of the syringe. The inverted gasket was then reinserted 
in the barrel and aligned with the 50-unit mark of the syringe. The 
syringe with the gasket in position was hand-held vertically with the 
needle connector directed upwardly. The reactants, 1.51 ml of Solution A, 
0.1 ml o Solution B, and 0.1 ml of Solution C were then mixed within a 3 
ml polyethylene syringe just before loading the casting insulin syringes. 
The syringe containing the polymerizing mixture was fitted with a 11/3 
inch (38.1 mm), 18-gauge needle, which was passed through the connector to 
fill the barrel of the insulin syringe with 0.5 ml of the polymerizing 
mixture. Immediately after filling the casting syringe, a 5.6 cm long, 0.9 
mm OD stainless steel rod, inserted into a 5.6-cm long, 1.7 mm OD Teflon 
tubing (Cole-Parmer International, Chicago, Ill.) was passed through the 
needle connector and the mixture of reactants in the barrel of the insulin 
syringe to the rubber gasket in order to form a hollow chamber in the 
polymerizing gel. The Teflon tubing with the inserted steel rod was held 
in the center of the barrel by the needle connector and the hollow cavity 
of the inverted rubber gasket. A 6.0-cm long, 16 mm OD plexiglass rod, 
drilled through with a 7/32-inch bit (5.56 mm), was used to brace the 
barrel of the insulin syringe and prevent breakage during pressurized 
polymerization. A 4-cm long, 3.5 mm OD stainless steel rod was inserted 
into the flanged end of the barrel of the syringe and used to apply 
pressure to the gasket and polymerizing mixture. The braced syringe was 
then placed between size 9 neoprene stoppers attached to the jaws of a 
pipe claimp. Pressure was applied for 15 minutes by closing the jaws of 
the clamp until all visible air bubbles were displaced from the 
polymerizing gel. No attempt was made to measure the pressure applied with 
the clamp. After this 15-minute period of perssurized polymerization, the 
cast hydrogel was removed from the syringe and the centrally located 
Teflon tubing with the rod was withdrawn from the cast hydrogel which now 
was a hydrogel tube of approximately 5 cm long, with a 1.7 mm lumen and 
0.9 mm thick wall. The pHEMA hydrogel tubes, in batches of 10 tubes, were 
then placed for 96 hours in a 100 ml glass beaker containing 95% ethanol 
which was changed every 12 hours to remove nonpolymerized HEMA. After 
ethanol washing, the pHEMA tubes were placed in a beaker containing 500 ml 
of distilled water, and heated at a slow boil for 4 hours. The distilled 
water was changed and the procedure repeated 12 times. After boiling, the 
pHEMA tubes were cut into 1 cm-long segments with a razor blade under 
observation with a stereoscopic microscope at 10X. Segments containing 
visible flaws were discarded. Solid plugs, 2 mm long were made from 0.085 
inch (2.16 mm) OD Silastic tubing (Dow Corning Corp., Medical Products 
Midland, Me.) filled with Silastic adhesive and used to close the ends of 
the pHEMA tube to form a chamber, as illustrated in FIGS. 1 and 2. Each 
1-cm segment of hydrogel tube and the 2 solid plugs to close the ends of 
the chamber were placed into a 2 ml glass ampulle (Wheaton Scientific, 
Millville, N.J.) containing distilled water. The ampulles were then 
autoclaved for 40 minutes at 120.degree. C., fire-sealed, and stored at 
room temperature until used. 
Animals 
Twenty-three uniparous, crossbred rabbit does ranging from 1.0 to 1.5 years 
of age were used as embryo donors or recipients for Experiments 1 and 2. 
Does were individually caged for at least 21 days before assignment to the 
experiments, fed commercial rabbit food, and maintained during the 
experimental period in a room with controlled temperature (20.degree. to 
22.degree. C.) and light (12 hours' light/12 hours' dark). Two crossbred, 
mature rabbit bucks of known fertility were individually caged, maintained 
in the same room, and fed, as described for the does. 
Fifteen mature, cycling female and 10 mature male Balb/c mice were used as 
intermediate recipients for the 2 experiments of this study. Mice were 
caged by sex in groups of 4 to 5 females or 2 males per cage, fed 
commercial mouse food, and maintained in a room with controlled 
temperature (20.degree. to 22.degree. C.) and light (14 hours light/10 
hours dark). For the female mice intermediate recipients, the stage of the 
estrous cycle was determined by vaginal smears and only females which were 
late in the afternoon of the day of estrus (D1) were used. 
EXPERIMENT 1 
Transfer of Rabbit Embryos Cultured in pHEMA Hydrogel Chambers in the 
Peritoneal Cavity of Intermediate Mouse Recipients. 
Embryo Recovery and Culture 
Ten does were randomly selected from the 23 does to serve as embryo donors 
and 10 does were selected to serve as recipients. To induce 
superovulation, each donor doe was given a subcutaenous injection of 0.5 
mg of follicle-stimulating hormone (FSH-P, Burns Biotec, Omaha, Nebr.) 
every 12 hours for 72 hours. Donor does were mated twice to each of the 2 
bucks, 24 hours after the last FHS injection. Each of the 10 mated donors 
was then randomly paired with a recipient. To synchronize donors and 
recipients, each unmated recipient was induced to ovulate by a single 
intramuscular injection of 50 IU of human chorionic gonadotropin (hCG, 
Fort Dodge Laboratories, Ft. Dodge, Iowa), given 14 hours after the fourth 
mating of the corresponding paired donor. 
To recover 1-cell rabbit embryos, does were anesthetized with Halothane 
(Fort Dodge Laboratories, Ft. Dodge, Iowa) 18 hours after the 4th mating 
and each oviduct was flushed from the uterotubal junction with 3 ml of 
0.9% (or 0.154 M sodium chloride) sterile saline solution. After flushing 
and recovery, embryos were examined with an inverted microscope at 100 
.times.while still in the collecting dish and flushing fluid. Oocytes that 
had spermatozoa in the perivitelline space or embryos that had extruded 
the second polar body were considered to be 1-cell embryos. The 1-cell 
embryos from each doe were washed 3 times by trasnfer between 10.times.35 
mm culture dishes (Lux, Miles Laboratories, Inc., Naperville, Ill.) 
containing sterile 0.9% saline solution before loading in the pHEMA 
hydrogel chambers. 
Three pHEMA hydrogel chambers were prepared for each doe before embryo 
collection. The sealed tip of each glass ampulle was broken and the tube 
and plugs were withdrawn with sterile forceps and placed in 10.times.35 mm 
culture dishes containing sterile saline solution. With the aid of 
forceps, one of the solid plugs was inserted into one end of each of the 3 
tubes and the tubes with the uninserted plugs were then incubated in a 
dish containing sterile saline solution for at least 110 minutes at 
37.degree. C., in an incubator with 5% co.sub.2 in humidified air, before 
loading the chamber with embryos. 
The 1-cell embryos recovered from each donor doe were subdivided into 3 
groups of equal numbers of embryos. The embryos from each group were 
transferred into the lumen of a saline solution-filled pHEMA tube, while 
the tube was immersed in sale. (The saline was used instead of an embryo 
media to confirm tranport from the peritoneal fluid through the hydrogel.) 
The open end of the tube was then sealed with the second Silastic plug and 
the sealed chambers were examined at 20.times.with a stereoscopic 
microscope to verify the number of embryos and the integrity of the 
chamber. The 3 chambers containing the embryos from each donor were then 
randomly assigned to one of the following treatment groups: Group 1: in 
vitro controls cultured for 72 hours in a 10.times.35 mm culture dish 
containing 3 ml of 0.9% sterile saline solution in an incubator at 
37.degree. C. with 5% CO.sub.2 in humidified air; Group 2: cultured in 
vivo for 72 hours in the peritoneal cavity of an adult female Balb/c mouse 
on D1 of the cycle; Group 3: cultured in vivo for 72 hours in the 
peritoneal cavit of an adult male Balb/c mouse. The embryo-loaded chambers 
assigned to Groups 2 and 3 were surgically implanted in anesthetized 
(Metofane, Pitman-Moore, Washington Crossing, N.J.) female or male mice 
through a 1-cm long incision in the ventral abdominal wall. After 72 hours 
of either in vitro or in vivo culture, the chambers were recovered and 
examined at 25 to 100.times.with an inverted microscope to determine the 
stage of embryonic development. Embryos were classified as follows: 
Embryos that did not cleave or had fragmented blastomeres were considered 
degenerated; embryos that cleaved beyond the 1-cell stage, had 
recognizable, intact blastomeres, but did not reach the morula stage, were 
considered retarded; morulae were embryos that cleaved beyond the 16 cell 
stage but had not yet developed a blastocoele, while blastocysts were 
embryos with a clear, defined blastocoele. The number of degenerated or 
retarded embryos and the number of morulae and glastocysts obtained from 
each donor were recorded. 
Embryo Transfer. The morulae and blastocysts obtained after in vivo culture 
in the pHEMA chambers were transferred to the paired recipient. Recipients 
were anesthetized with Halothane, as described for the donor does. The 
ventral area of the abdominal wall was clipped free of hair, disinfected, 
draped, and the uterine horns were exposed through a 6-cm long midventral 
incision. For each paired recipient, the left or right uterine horn was 
alternated as to receive embryos cultured in vivo in either male or female 
mouse. Each horn was punctured with the eye of a No. 22 suture needle and 
the embryos from treatment groups 2 or 3 were transferred to the lumen of 
the assigned horn using a 5 .mu.l Wiretrol pipet (Fisher Scientific, 
Springfield, N.J.). The abdominal incision was sutured and each doe was 
fitted with an Elizabethan plastic collar until recovery from surgery. 
Twenty-one days after transfer, recipient does were laparotomized. Before 
laparotomy, each doe was sedated by an intramuscular injection of 1 mg per 
kg of body weight of Acepromazine (Ceva Laboratories, Inc., Overland Park, 
Kans.). The midventral abdominal area was disinfected and then infiltrated 
with 10 mg/kg body weight of a 2% solution of Lidocaine (Astra 
Pharmaceutical Products, Inc., Worcester, Mass.). The uterine horns were 
exposed and the number of fetuses within each horn was recorded. Fetuses 
from embryos cultured in male mice were marked in utero by an injection of 
1 .mu. of India ink deposited, as a subcutaneous drop, in the rump area of 
each fetus. At parturition, which occurred 6 to 8 days after laparotomy, 
the number of offspring derived from each of the 2 treatment groups was 
recorded. Bunnies were observed daily for general health until weaning. 
Experiment 2 
Blastomere Isolation and Culture in Compartmentalized pHEMA Hydrogel 
Chambers in the Peritoneal Cavity of Female Mouse Recipients. 
Four-cell rabbit embryos were recovered from the 3 remaining does. 
Superovulation was induced and the embryos recovered, as described for 
Experiment 1, except that the oviducts were flushed 32 hours after the 4th 
mating with Whitten s medium (WM, Whitten and Biggers, 1968) supplemented 
with 1 mg bovine serum albumin (BSA, Fraction V, Sigma Chemical Co., St. 
Louis, Mo.) per ml of medium. The flushing medium was filtered through a 
0.2 .mu.m filter. 
To isolate blastomeres, the 4-cell embryos recovered from each doe were 
placed in 10.times.35 mm culture dishes pretreated with Prosil 28 (PRC 
Inc., Gainesville, Fla.) to decrease adhesion of the blastomeres to the 
culture dish. The embryos were washed as a group 3 times at room 
temperature in filtered WV-BSA medium and then incubated for 15 minutes at 
27.degree. C. with 5% CO.sub.2 in humidified air in Ca.sup.+ and MG.sup.++ 
-free, modified Dulbecco's phosphate buffered saline solution (DPBS, 
Dulbecco and Bogt, 1954), supplemented with 0.02% (or 0.68 mM) EDTA and 
0.25 M glycerol. Next the embryos were incubated for 15 minutes in 
modified DPBS, supplemented with 0.50 M glycerol and then transferred to 
Prosil 28 treated culture dishes containing modified DPBS supplemented 
with 1.0 M glycerol to remove the zona pellucida. The zona was removed 
under observation with inverted microscope at 100.times.with a hand-held 
microknife made from a piece of razor blade. The zona-free blastomere 
clusters were then transferred and incubated 2 times for 15-minute 
periods, first in a culture dish containing modified DPBS supplemented 
with 0.50 M glycerol and then in modified DPBS supplemented with 0.25 M 
glycerol. To separate the blastomeres, each blastomere cluster was 
transferred to a culture dish contianing modified DPBS only and then 
subjected, during observation with a stereoscopic microscope at 20.times., 
to repeated aspiration into and expulsion from a 50 .mu.m ID silicone 
coated capillary tube (Polymicro Technologies, Phoenix, Ariz.) attached to 
a 5 .mu.l Wiretrol pipet. The isolated blastomeres were then washed 5 
times by transfer between dishes containing WM-BSA medium. Only those 
embryos from each of the 3 donor does that yielded 4 intact blastomeres 
were used in this experiment. Th 4 blastomeres isolated from each embryo, 
hence a monozygotic group, were placed within individual compartments of a 
sterile pHEMA hydrogel chamber, under observation with a stereoscopic 
microscope at 20.times.. These chambers were made from HEMA, as described 
for Experiment 1, except that the length of each chamber was increased to 
2 cm in length. One of the open ends was sealed with a plug and the lumen 
of the chamber was partitioned into 4 compartments, as illustrated in FIG. 
3, by the insertion of 3, 1.7 mm OD.times.2 mm thick pHEMA discs, while in 
a 10.times.35 mm culture dish containing WM-BSA medium. The WM-BSA in the 
compartments served as the initial blastomere culture medium. These discs 
were made from HEMA hydrogel polymerized at room temperature within a 1.7 
ID Teflon tubing without applying pressure. The first blastomere was 
placed in the chamber and one of the pHEMA discs was inserted in the lumen 
of the chamber and positioned at approximately 2.5 mm from the sealed end 
of the chamber. This procedure was repeated until each of the 4 
blastomeres was loaded into the compartmentalized chamber, together with 
the WM-BSA medium. The chamber was then sealed with the remaining plub, as 
shown in FIG. 3. The chambers containing 4 isolated blastomeres were 
surgically implanted in the peritoneal cavity of a female Balb/c mouse on 
D1 and incubated in vivo for 72 hours as described for Experiment 1. 
Because of the number of monozygotic groups obtained, 4 chambers were 
implanted in the peritoneal cavity of each of 5 female mice. At the end of 
the 72-hour period of incubation, the compartmentalized chambers were 
recovered and the isolated blastomeres were examined for development with 
an inverted microscope at 25 to 100.times.. 
Statistical Analysis 
End points for stage of development of embryos cultured in vivo and for the 
results of transfer of morulae and blastocysts to recipient does between 
groups 2 and 3 of Experiment 1 were compared by Chi-square analysis, with 
1 degree of freedom and Yates correction (Steel and Torrie, 1960). 
Significance was established at P.ltoreq.0.05. Data for embryos cultured 
in vitro (control group 1 of Experiment 1) were not statistically compared 
with those of groups 2 and 3 due to the 0 values obtained. 
RESULTS 
Experiment 1 
A total of 357, 1-cell embryos recovered from 10 donor does was used in 
this experiment (Table 1). all of the 238 embryos that were placed in the 
pHEMA chambers and cultured in vivo in the peritoneal cavity of male or 
female mice were recovered at the end of the 72 hours of in vivo culture, 
as reported in Table 1. All of the 119 embryos incubated in vitro in pHEMA 
chambers had degenerated during the 72 hours of culture. In comparison, 
only 10.1% cultured in male mice and 8.4% of the embryos cultured in 
female mice degenerated during the in vivo culture period. These 
differences were not significant (P&gt;0.05, Table 1). More (P&lt;0.0005 Table 
1) of the embryos cultured in female mice developed to blastocysts 
(68/119) than those cultured in male mice (25/119). The transfer of 188 
morulae and blastocysts (Table 1) recovered from pHEMA chambers implanted 
in the peritoneal cavities of male and female mice resulted in 23 (12.2%) 
live offspring, as shown in Table 2. Fewer (P&lt;0.005) offsprings were born 
from the transfer of embryos cultured in male mice (3/97, Table 2) than 
those cultured in female mice (20/81). Survival to term was not influenced 
(P&gt;0.05) by the horn to which embryos were transferred. The 23 bunnies 
developed in an apparently normal fashion and were released for adoption 
after weaning. 
Experiment 2 
A total of 43, 4-cell embryos was collected from 3 donor does and eighty 
intact blastomeres were isolated from 20 of these 4-cell embryos. All of 
the blastomeres that were cultured in vivo were recovered from the 
compartmentalized pHEMA chambers after the 72 hours of culture. Of these 
80 isolated blastomeres, 16 (20%) were retarded or degenerated, 21 (26%) 
developed to the morula and 43 (54%) to the blastocyst stages, as shown in 
Table 3. Eleven (44 blastomeres) of the 20 originally cultured monozygotic 
groups developed, apparently in synchrony, to become either morulae or 
blastocysts. Including the 4 retarded blastomeres that cleaved but did not 
develop to the morula or blastocyst stages, 85% (68/80, Table 3) of the 
isolated blastomeres survived the isolation procedure and cleaved, while 
cultured in vivo in the compartmentalized pHEMA chambers. However, these 
de novo formed morulae and blastocysts were fragile and fragmented during 
withdrawal from the chamber. 
DISCUSSION 
In the present study, all of the embryos and blastomeres cultured within 
pHEMA chambers were recovered following the 72 hours period of culture. 
The development of 1-cell rabbit embryos to the morulae and blastocyst 
stages within saline-filled pHEMA chambers implanted in the peritoneal 
cavities of mice, as well as the birth of live offspring resulting from 
the transfer of these embryos, demonstrate that the pHEMA hydrogel 
chambers permit the passage of essential factors from the peritoneal 
cavity into the lumen of the chamber, such that embryonic development 
could occur. These results also confirm that the peritoneal cavity of mice 
can support the development of 1-cell rabbit embryos to the blastocyst 
stage (see Briones, et al., 1954.) 
The percentage of 1-cell rabbit embryos that developed to the morula and 
blastocyst stages during culture for 72 hours in the peritoneal cavity of 
female mice is comparable to that reported for 2- and 4-cell embryos 
cultured within the ligated oviducts of estrous does (Adams, 1973). The 
development of embryos cultured in male mice was retarded when compared to 
the development of embryos cultured in female mice in our study or when 
compared to the rate of development in ligated oviducts (Adams, 1973). 
This suggests that the peritoneal cavity of the male mouse is a less 
favorable environment for embryonic development than that of the female 
mouse. 
The percentage of offspring born from the transfer of morulae or 
blastocysts cultured from the 1-cell stage in the peritoneal cavity of 
female mice (20/91, 22%) appears to be greater than that resulting from 
embryos cultured in vitro for 72 hours (7.0%, Adams, 1970, 14%, Maurer, 
1978) and compares with the percentages resulting from the transfer of 2 
to 4-cell embryos cultured in the ligated oviduct of estrous does (17%, 
Adams, 1973) or obtained after transfer of noncultured embryos to 
asynchronous recipients (27%, Yang, et al., 1986). 
The fewer offspring born from the transfer of 1-cell rabbit embryos 
cultured in the peritoneal cavity of male mice (3/97, 3%) could be the 
result of the transfer of more morulae than blastocysts (72 morulae and 25 
blastocysts, Table 2) than for the female mice (23 morulae and 68 
blastocysts). 
The number of isolated blastomeres that developed during in vivo culture to 
morulae and blastocysts, while contained in the compartmentalized pHEMA 
chambers, as well as the recovery at the end of the incubation period of 
all of the resulting product of each of the isolated blastomeres, attest 
to the protective nature of the pHEMA chamber. The development of 
apparently normal blastocysts and the lack of trophoblastic vesicles in 
the blastocysts derived from isolated blastomeres suggests complete 
development while in the pHEMA chamber. 
In summary, the results of this study demonstrate that pHEMA hydrogel can 
be cast into sealable and easily retrievable chambers for the in vivo 
culture of embryos. 
TABLE 1 
______________________________________ 
Number of Embryos 
Treat- Re- 
ment Cultured Degenerated 
tarded.sup.a 
Morula 
Blastocyst 
______________________________________ 
In Vitro 
119 119 0 0 0 
Controls 
Male 119 12 10 72* 25 
Mouse 
Female 119 10 18 23 68* 
Mouse 
______________________________________ 
.sup.a Embryos that cleaved beyond the 1cell but did not advance to the 
morula stage. 
*Significantly (P &lt; 0.0005) different from the corresponding in vivo 
treatment group. 
TABLE 2 
__________________________________________________________________________ 
Sex of 
Recipient 
intermediate 
Number (stage)* 
Uterine 
Fetuses 
Offspring 
number 
mouse recipient 
transferred 
horn day 25** 
born alive 
__________________________________________________________________________ 
1 male 7 (M), 3 (B) 
left 0 -- 
female 1 (M), 9 (B) 
right 
0 -- 
2 male 8 (M), 4 (B) 
right 
0 -- 
female 11 (B) left 3 3 
3 male 6 (M), 2 (B) 
left 0 -- 
female 9 (B) right 
3 3 
4 male 10 (M), 1 (B) 
right 
0 -- 
female 13 (B) left 4 4 
5 male 7 (M), 1 (B) 
left 0 -- 
female 7 (M) right 
0 -- 
6 male 7 (M), 2 (B) 
right 
0 -- 
female 1 (M), 7 (B) 
left 2 2 
7 male 11 (M), 2 (B) 
left 1 1 
female 3 (M), 9 (B) 
right 
4 4 
8 male 5 (M), 5 (B) 
right 
2 2 
female 9 (B) left 4 4 
9 male 7 (M), 1 (B) 
left 0 -- 
female 6 (M) right 
0 -- 
10 male 4 (M), 4 (B) 
right 
0 -- 
female 5 (M), 1 (B) 
left 0 -- 
Totals 
male 97 (72M), (25B) 
-- 3 3 
female 91 (23M), (68B) 
-- 20 20 
__________________________________________________________________________ 
*Stage: 
(M), morula; 
(B), blastocyst. 
**Determined by laparotomy on day 25 of gestation. 
TABLE 3 
______________________________________ 
Blastomeres isolated and cultured 
80 
Of these . . . 
Degenerated 12 
Retarded.sup.a 4 
Morulae 21 
Blastocysts 43 
Total recovered after culture 
80 
Total developing to the morula or blastocysts stages 
64 [11] 
______________________________________ 
.sup.a Blastomeres that cleaved but did not advance to the morula or 
blastocyst stages. 
[ ] Brackets indicate monozygotic groups. 
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