http://humrep.oxfordjournals.org/content/16/9/1965.full
本篇詳列cryoloop冷凍之使用方法
其cryoprotectant 濃度略高於人類使用之抗凍劑
其balance timing 亦較短
本篇發表於2004年是目前發展成熟的cryotop方法的前身
Cryoloop vitrification yields superior survival of Rhesus monkey blastocysts
- R.R. Yeoman1,4,
- B. Gerami-Naini3,
- S. Mitalipov3,
- K.D. Nusser3,
- A.A. Widmann-Browning3 and
- D.P. Wolf1,2,3
+ Author Affiliations
- Received January 11, 2001.
- Accepted June 8, 2001.
Abstract
BACKGROUND: Vitrification using the cryoloop procedure was evaluated
for preservation of non-human primate blastocysts by comparing survival results
from two different cryoprotectant mixtures with prior results from controlled
rate cooling. METHODS: Rhesus monkey blastocysts were produced by
intracytoplasmic sperm injection of mature oocytes from cycling females
stimulated with recombinant human hormones. Morphologically well-formed
blastocysts were divided between Procedure A (2.8 mol/l dimethylsulphoxide and
3.6 mol/l ethylene glycol with 0.65 mol/l sucrose and 25 μmol/l Ficoll in
TALP-HEPES with 20% fetal bovine serum (TH20)) and Procedure B (3.4 mol/l
glycerol and 4.5 mol/l ethylene glycol in TH20). After >48 h in liquid
nitrogen, the removal of cryoprotectants was accomplished in the presence of a
3-step series of decreasing sucrose concentrations in TH20. Surviving embryos
were co-cultured on buffalo rat liver cells. RESULTS: Of 16 blastocysts
vitrified via Procedure A, 38% survived with minimal lysis and only one hatched
in culture; in contrast, of 33 blastocysts vitrified by Procedure B, 85%
survived and 71% hatched. Of 22 blastocysts cryopreserved by conventional slow
cooling, 36% survived and 6% hatched. Transfer into three recipients, each with
two embryos vitrified with Procedure B, resulted in a successful twin-term
pregnancy. CONCLUSION: Modified cryoloop vitrification with a final solution of
3.4 mol/l glycerol and 4.5 mol/l ethylene glycol is a promising procedure for
preserving Rhesus monkey blastocysts that is simple, rapid, and inexpensive.
Key words
Introduction
Embryo cryopreservation is a vital aspect of assisted reproduction
technologies and reliable procedures for zygotes and cleaving embryos have been
developed and routinely applied in humans and non-human primates (Kuzan and Quinn, 1988; Wolf et
al.1989). With the ability to culture embryos to the blastocyst stage
(Zhang et al.1994; Gardner and Lane, 1997), the low
temperature storage of these advanced embryos becomes a necessity. While
in-vitro developed blastocysts may have greater implantation potential, their
survival rate after conventional cryostorage in glycerol has been disappointing,
to the extent that it diminishes this advantage (Hartshorne et
al.1991; Kaufman et al.1995). Alternative
cryopreservation techniques include vitrification or the rapid cooling of a
liquid to achieve a glass-like solid state (Rall and Fahy, 1985). In
this approach, damaging ice crystal formation is minimized and water removal is
enhanced by short-term exposure to high concentrations of cryoprotectants.
Moreover, rapid cooling rates are obtained by minimizing the mass of the embryo
suspension and the container. This has been optimized with a small nylon loop
adapted from protein crystallography (Teng, 1990). The loop has a
volume of <1 μl and no thermo-insulating layer which yields very high cooling
rates and the successful vitrification of rodent, bovine and human blastocysts
(Lane et al.1999a, b). Because of the limited success with
conventional slow freezing of Rhesus monkey blastocysts, we investigated the use
of the cryoloop to cryopreserve Rhesus embryos.
Materials and methods
All procedures were performed in accordance with the NIH Guide for
Care and Use of Laboratory Animals and approved by the Institutional Animal Use
and Care Committee of the Oregon Regional Primate Research Center, Oregon Health
Science University. The procedures for follicular stimulation, fertilization,
and embryo culture have been reviewed recently (Ouhibi et
al.2000). Twenty adult Rhesus macaques were entered into a follicular
stimulation protocol starting 1 to 4 days after menses (Zelinski-Wooten et
al., 1995). Cycling females received twice daily injections of recombinant
human FSH (rFSH; 30 IU i.m., Ares Advanced Technology, Norwell, MA, USA) and
once daily injections of Antide® (a gonadotrophin-releasing hormone
antagonist; 0.5 mg/kg s.c., Ares Advanced Technology) for 8 consecutive days. On
the last two days of rFSH/Antide stimulation, animals also received twice daily
injections of recombinant human LH, 30 IU i.m. (Ares Advanced Technology). On
the last day of hormonal stimulation, ovarian morphology was evaluated by
ultrasonography (Ultrasound Unit, ATL, Bothell, WA, USA) and animals with
follicles ≥3 mm indiameter received an injection of recombinant human chorionic
gonadotrophin, 1000 IU i.m. (Ares Advanced Technology). Cumulus–oocyte complexes
were collected laparoscopically 29–32 h later and held in HEPES-buffered TALP
(modified Tyrode's solution with albumin, lactate and pyruvate) medium (Bavister et al.1983) containing 3
mg fatty acid free, fraction V bovine serum albumin per ml (TH3) (Sigma, St
Louis, MO, USA). After 1 min exposure to hyaluronidase (1 mg/ml) (Sigma),
intracytoplasmic sperm injection was performed under embryo-tested mineral oil
(Sigma) in a chamber (Falcon 1009, Becton-Dickinson, Franklin Lakes, NJ, USA)
containing two drops: a sperm drop consisting of 4 μl of 10%
polyvinylpyrrolidone (Irvine Scientific, Santa Ana, CA, USA) in TH3 and 1 μl of
sperm suspension (3×106/ml) and an oocyte drop, 20 μl of TH3, into
which mature oocytes were placed. Individual spermatozoa were injected using a 7
μm outer diameter micropipette (Humagen, Charlettesville, VA, USA) using
micromanipulators (Narishige, Tokyo, Japan) on an inverted microscope equipped
with Hoffman optics (IX70, Olympus, Melville, NY, USA). Injected oocytes were
placed in co-culture with buffalo rat liver (BRL) cells (American Type Culture
Collection, Manassas, VA, USA) in CMRL (Connaught Medical Research Laboratories)
medium (Life Technologies, Rockville, MD, USA) containing 10% fetal bovine serum
(HyClone, Logan, UT, USA), 10 mmol/l l-glutamine, 5 mmol/l
sodium pyruvate, 1 mmol/l sodium lactate, 100 units/ml of penicillin, and 100
μg/ml streptomycin (Sigma) at 37°C in 5% CO2 (Zhang et
al.1994). Embryos were transferred to fresh plates of BRL cells every
other day to reduce nitrogen accumulation. Blastocysts, defined as the expansion
of the embryo after compaction and cavitation to include both a discernable
trophectoderm and inner cell mass, were distributed evenly among the low
temperature storage groups.
Blastocyst preservation
Conventional slow cooling involved exposure of embryos at room
temperature for 10 minutes to 0.68 mol/l (5%, volume/volume) glycerol (Sigma) in
TH3 followed by 10 min in 1.22 mol/l (9% volume/volume) glycerol (Ménézo et al.1992). Samples were
loaded into cryovials (Nalgene, Rochester, NY, USA) with 1 ml of the final
solution, cooled at –3°C/min to –8°C for seeding, and further cooled at
–0.3°C/min to –30°C before plunging into liquid nitrogen for storage. For
recovery, vials were warmed until liquefied in a 37°C water bath and recovered
embryos were then incubated sequentially in 0.5 mol/l and 0.2 mol/l sucrose
(Sigma) at room temperature for 10 min each before being placed in co-culture
with BRL cells in CMRL media.
Vitrification with the cryoloop involved exposure to a solution of 1.4
mol/l (10%, volume/volume) dimethylsulphoxide (DMSO) (Sigma) and 1.8 mol/l (10%,
volume/volume) ethylene glycol (Sigma) in HEPES-buffered TALP medium containing
20% fetal bovine serum (TH20) for 2 min before transfer to a solution of 2.8
mol/l (20%, volume/volume) DMSO, 3.6 mol/l (20%, volume/volume) ethylene glycol,
25 μmol/l (10 mg/ml) Ficoll (400 000 molecular wt., Sigma), and 0.65 mol/l
sucrose in TH20 for ~25 s during which time the blastocysts were placed onto a
film of this solution within the loop. The cryoloop consisted of a 20 μm nylon
filament in a 0.7–1.0 mm diameter loop mounted on a stainless steel tube fixed
to the inside of a 1.8 ml cryovial cap (Hampton Research, Laguna Niguel, CA,
USA) (Figure 1). The loop was plunged immediately into liquid nitrogen within
the cryovial which was then fixed onto a cane in liquid nitrogen for storage
(Procedure A; Lane et al.1999a). Warming was performed by placing the
loop in a solution of 0.25 mol/l sucrose in TH20 and allowing the blastocysts to
fall to the bottom of the dish for a 2 min exposure. Further cryoprotectant
removal occurred during 3 min in 0.125 mol/l sucrose in TH20 prior to rinsing
twice in media for 5 min before co-culture on BRL cells in CMRL media as above.
Figure 1.
A pictorial sequence of vitrification with
cryoloops is displayed. (1) Embryos were exposed in sequence to
the cryoprotectant solutions placed into four wells of the plate. Blastocysts
are transferred through these sequential solutions using a glass pipette.
(2) A film on the loop is made by dipping into the last
cryoprotectant solution. (3) Embryos are then added to the film
within the loop using a pulled glass transfer pipette. (4) The
cryoloop is plunged into liquid nitrogen and screwed into place using the
magnetic holding rod. The cryoloop consisted of a 20 μm nylon filament in a
0.7–1.0 mm diameter loop mounted on a stainless steel tube fixed to the inside
of a 1.8 ml cryovial cap (Hampton Research, Laguna Niguel, CA, USA).
Alternatively, Procedure B (Agca et al.1998)
involved incubation for 3 min in 1.36 mol/l (10%) glycerol in TH20 and then 3
min in combined 1.36 mol/l (10%) glycerol and 2.7 mol/l (20%) ethylene glycol in
TH20. During the last 20 s of this last step, the loop was dipped into a
cryoprotectant solution of 3.4 mol/l (25%) glycerol plus 4.5 mol/l (25%)
ethylene glycol in TH20. At the completion of the last step, the blastocysts
were rinsed quickly in the cryoprotectant solution, placed into the loop and
plunged into liquid nitrogen within 25 s. Warming and recovery of the
blastocysts was performed as with Procedure A except that the initial incubation
occurred in a 0.5 mol/l sucrose solution for 3 min followed by 0.25 mol/l
sucrose and 0.125 mol/l sucrose for 3 min each before the blastocysts were
rinsed and then cultured. Whenever sufficient blastocysts were available,
vitrifications involving the two alternative solutions were carried out in
parallel with blastocysts of equal quality and maturity evenly distributed.
After 4 h in culture, survival was based on clarity of blastomeres and
blastocoel re-expansion. Further development was recorded when applicable with
attachment assessed at 24 h after hatching and outgrowth assessed after 48 h.
Embryo transfers were conducted on multiparous female recipients 4–5 days after
the mid-cycle oestradiol peak. Pregnancies were detected by measuring hormone
concentrations and subsequent fetal development was monitored periodically by
ultrasonography.
Data analysis
Statistical significance of survival and further growth after warming
among treatment groups was compared using a χ2 analysis (SigmaStat,
Jandel Scientific, San Rafael, CA, USA).
Results
The developmental stages of the 71 blastocysts available for
cryopreservation varied: 31% were early blastocysts, 15% blastocysts, 42%
expanded blastocysts, and 10% were hatched. The distribution of developmental
stages within the three treatments did not differ significantly from this
overall distribution (P = NS, χ2-test). Cryoloop
vitrification of 16 blastocysts with Procedure A resulted in 38% of the embryos
surviving after warming (Figure 2). Only one blastocyst developed further with
co-culture on BRL cells and hatched. With our conventional slow freezing method,
36% of 22 blastocysts survived with one hatching under similar culture
conditions (Figure 2). In contrast, vitrification of 33 blastocysts after
processing with Procedure B resulted in 28 surviving (85%) at 4 h, 25 expanding
(77%), and 23 hatching (71%) (Figure 2). Twelve of these blastocysts were
cultured further and attached with outgrowth of cells (Figure 3). Cracked zonae
were occasionally seen with the slow freezing protocol but were not observed
with either vitrification procedure. Three transfers, each composed of two
expanded blastocysts from vitrification Procedure B, were made into recipients
4–5 days after the estimated time of ovulation. A twin pregnancy with two live
births has resulted.
Figure 2.
The percentages of warmed Rhesus monkey blastocysts
that survived, expanded, hatched and attached are displayed after low
temperature storage in liquid nitrogen. `Slow' is the group that was frozen
slowly by programmed freezing. `Vit.1' is the group vitrified using the cryoloop
with solutions and sequence of Procedure A.. `Vit.2' is the group vitrified
using the cryoloop with the solutions and sequence of Procedure B. The results
with Vit.2 were significantly improved versus Slow and Vit.1 (P <
0.05). The numbers in parentheses denote the number of blastocysts preserved.
Figure 3.
Micrographs of Rhesus monkey blastocyst after
storage in liquid nitrogen. (A) A thawed blastocyst frozen in
glycerol-based medium via a slow controlled rate cooling protocol. Note the
presence of degenerating cells and cracked zona. (B) A
surviving blastocyst vitrified by cryoloop Procedure A with notable cellular
granularity. (C) Two blastocysts from cryoloop Procedure B
which expanded and hatched. Note the presence of an empty zona pellucida.
(D) A hatched blastocyst vitrified by the modified cryoloop
protocol and maintained in culture for 3 days. Bars designate scale.
Discussion
The current study presents a superior method to preserve primate
blastocysts by vitrification with a nylon loop. The cryoprotectant system with a
final concentration of 3.4 mol/l glycerol and 4.5 mol/l ethylene glycol in TH20
protected embryos sufficiently such that with warming, blastocysts re-expanded
and developed at high rates.
Successful vitrification was first achieved using 8-cell stage mouse
embryos in 0.25 ml straws (Rall and Fahy, 1985). With sufficiently
rapid warming, a high survival rate was obtained, presumably by avoiding
damaging ice crystal reformation which occurs with slower warming rates (Rall, 1987). More recently, vitrified mouse
blastocysts cryopreserved using cryoloops and open-pulled straws have yielded
high pregnancy rates upon transfer (Nowshari and Brem, 1998; Cseh et al.1999; Lane et al.1999a; Kong et al.2000). Similarly, high
pregnancy rates (21–39%) following vitrification of domestic livestock
blastocysts have been reported (Ishimori et al.1993; Agca et al.1998; Vajta et al.1998).
Cryopreservation of human embryos by conventional slow freezing
methods has yielded numerous pregnancies and full-term births (Ménézo et al.1992). However,
embryo survival post-thaw is generally <50%. With a vitrification method
using an electron microscope grid as a support (Martino et
al.1996) and ethylene glycol and sucrose as cryoprotectants, 48 of 93
blastocysts survived warming and five of 20 patients became pregnant (Choi et al.2000). Additionally, a
case study has reported a pregnancy from two blastocysts frozen in straws using
ethylene glycol and DMSO as cryoprotectants (Yokota et al.2000).
Vitrification using nylon loops allows for a very small volume of
liquid to encase the blastocyst and eliminates the thermo-insulating effect of a
pipette or an electron microscope grid resulting in extremely high rates of
cooling. Additionally, fracture damage observed in the zona pellucida, which is
caused by mechanical stresses produced from non-uniform volume changes of the
suspending medium (Rall and Meyer, 1989; Kasai et
al.1996; Van den Abbeel and Van Steirteghem, 2000),
was not seen in our vitrification groups. Cryoprotectant toxicity and osmotic
swelling during water re-entry may have reduced survival of monkey blastocysts
vitrified by Procedure A since blastomere darkening was frequently observed
immediately after warming. These outcomes may relate to species-specific
membrane and microtubule fragility. Vitrification Procedure B utilized
cryoprotectant formulations and procedures developed in the bovine (Agca et al.1998; Donnay et al.1998), a species
known to be sensitive to chilling injury (Martino et
al.1996). The longer exposure and higher cryoprotectant concentrations
employed in Procedure B suggest that more extensive permeation is desirable. Of
course, superimposed on this conclusion is the knowledge that embryos have
different permeabilities to various cryoprotectants (Kaidi et
al.1999; Paynter et al.1999). Additionally,
cryoprotectant removal in a higher sucrose concentration may have slowed water
diffusion reducing damage related to osmotic shock. Thus, a promising procedure
for vitrifying Rhesus monkey blastocysts has been developed that is simple,
rapid, and inexpensive and may also have clinical applications.
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