Zygote 13 (November), pp. 277–282. C© 2005 Cambridge University Pressdoi:10.1017/S0967199405003278 Printed in the United Kingdom

Effect of cryopreservation on fusion efficiency and in vitrodevelopment into blastocysts of bovine cell lines used insomatic cell cloning

O. Hayes1, LL. Rodrıguez2, A. Gonzalez1, V. Falcon3, A. Aguilar1 and F.O. Castro2

Division of Animal Biotechnology and Division of Chemistry and Physics, Centro de Ingenierıa Genetica y Biotecnologıa,La Habana 10600, Cuba, and Faculty of Veterinary Medicine, Universidad de Concepcion, Chillan, Chile

Date submitted: 21.03.05. Date accepted: 29.04.05

Summary

The outcome of the process of cloning by nuclear transfer depends on multiple factors that affect itsefficiency. Donor cells should be carefully selected for their use in somatic nuclear transfer, and theprotocols used for keeping frozen cell banks are of cardinal importance. Here we studied the effect oftwo protocols for freezing donor cells on fusion rate and development into blastocysts. Our hypothesisis that freezing affects cell membranes in a way that interferes with the fusion process upon cloning butwithout hampering normal cell development in vitro. We found that freezing cell lines without controllingthe cooling rate gives lower yields in the fusion step and in the final development into blastocysts,compared with cells frozen with a controlled cooling rate of approximately 1 ◦C/min. Transmissionelectron microscopy of the cells subjected to different freezing procedures showed major damage tothe cells frozen with a non-controlled protocol. We conclude that freezing of donor cells for cloning isa critical step in the procedure and should be monitored carefully using a method that allows for astep-wise, controlled cooling rate.

Keywords: Bovine, Cell fusion, Freezing, Nuclear transfer

Introduction

In somatic cell cloning it is of cardinal importance tohave well-characterized, cryopreserved cell lines thatare useful for nuclear transfer. While many studieshave focused on the importance of nuclear donor celltype (Oback & Wells, 2002), cell cycle stage (Campbellet al., 1996a), genetic background (Wakayama &Yanagimachi, 1999), epigenetic differences (Kikyo &Wolfee, 2000) and culture conditions upon cloning

All correspondence to: F.O. Castro, Department of AnimalSciences, Faculty of Veterinary Medicine, University ofConcepcion, Chillan, Chile. Tel: 56-42-208741. Fax: 56-42-270212. e-mail: fidcastro@udec.cl1 Division of Animal Biotechnology, Centre for GeneticEngineering and Biotechnology, PO Box 6162, Havana 10600,Cuba.2 Department of Animal Sciences, Faculty of VeterinaryMedicine, University of Concepcion, Chillan, Chile.3 Division of Chemistry and Physics, Centre for GeneticEngineering and Biotechnology, PO Box 6162, Havana 10600,Cuba.

results (Vajta et al., 2002), relatively little attention hasbeen paid to the effect of the freezing procedure oncell fusion and subsequent embryonic developmentafter nuclear transfer. The importance of well-characterized working and master banks should notbe underestimated; instead it should be an asset fora cloning laboratory. Different methods for freezingsomatic cells are described in several excellent manuals(Celis, 1998; Freshney, 2000). Factors such as the typeof cryoprotectant and the rate of cooling have themost dramatic impact on the survival of cells afterfreezing and thawing. Our hypothesis is that thecryopreservation protocol might affect the quality ofthe membrane and therefore the fusion process duringcloning.

Materials and methods

All reagents used were purchased from SigmaChemical (St Louis, MO) unless otherwise indicated.

278 O. Hayes et al.

Cell lines

Skin fibroblasts were obtained from adult cattle ofa local breed (Siboney) by ear punching. After theremoval of hair and rinsing in 70% ethanol, tissuewas washed in Dulbecco phosphate-buffered saline(DPBS), minced thoroughly with small scissors andplaced on plastic Petri dishes, allowed to dry slightlyto increase its attachment to the surface of the plastic,and the dishes carefully filled with DMEM:F12 (1:1)medium supplemented with 10% fetal calf serum(FCS), 10 mg/l insulin, 10 ng/ml human recombinantepidermal growth factor, 1 mM sodium pyruvate,1% (v/v) non-essential amino acids and 10 µg/mlpenicillin–streptomycin. After confluence, the cellswere dissociated using 0.08% trypsin and 1 mM EDTAin DPBS and pelleted at 300 g for 10 min.

A granulosa cell line was established from muralgranulosa cells collected by aspirating one ovarianantral follicle (3–10 mm in diameter) from the samecow using an ultrasound-guided transvaginal probe(Pieterse et al., 1988). After the removal of unattachedclumps of cells or explants, attached cells were furthercultured until confluence at 39 ◦C in an atmosphere of5% CO2 and subcultured at intervals of 5–7 days.

Fetal fibroblasts were obtained from a 55-day-old bovine female fetus, collected by cesarean hys-terectomy, eviscerated, minced and then subjectedto the same treatment as for skin fibroblasts. Afterconfluence, the cells were dissociated using 0.1%trypsin and 1 mM EDTA in DPBS and pelleted at 300 gfor 10 min.

Freezing protocols

After trypsinization, the cells were counted, pelletedby centrifugation, the supernatant discarded andthe pellet resuspended in ice-cold freezing medium(10% DMSO, 90% fetal calf serum) to yield a finalconcentration of 2.5–3 × 106 cells/ml. The cells weredistributed in freezing vials and frozen by one of thefollowing two methods:

Method 1 (discontinuous gradient): Cells were kept onice for 1 h, after which time the vials were wrappedin a cloth and placed at − 20 ◦C for another hourand finally stored at − 80 ◦C for at least 4 h beforeplunging them into liquid nitrogen.

Method 2 (continuous gradient): Cells were placed in aCryo Freezing Container (Nalge Nunc International,Hereford, UK) and frozen according to themanufacturer’s instructions. This cryofreezing con-tainer provides the critical, repeatable − 1 ◦C/mincooling rate required for successful cell preservationand recovery. Briefly, the container filled withisopropyl alcohol was kept at − 20 ◦C for at least3 h before use. After this, the cryovials were placedin the inner rack and the container was transferred to

the bottom of a − 80 ◦C mechanical freezer and leftundisturbed for a minimum of 4 h. The vials werethen plunged into liquid nitrogen.

Cells frozen by both methods were thawed and usedin transmission electron microscopy and in somaticnuclear transfer. Cells subcultured without freezingwere used in nuclear transfer as a control. For fusionexperiments, cells were used between passages 5 to 8and the experiments were replicated five times. Cellswere thawed by direct immersion of the cryovial in awater bath set to 37 ◦C. The vials were held in waterbriefly until the contents melted completely and thencarefully pipetted to the appropriate tissue cultureflask. The cells, either frozen/thawed or fresh, wereplaced under a transmission electron microscope toanalyse the integrity of the cell membrane, or they wereused in nuclear transfer experiments.

Transmission electron microscopy

Cells were seeded in 60 mm Petri dishes (GreinerBioOne, Frickenhausen, Germany) at a density thatallowed the dish to become around 80% full andleft overnight in culture. The next day the cells weretrypsinized mildly, by using 0.08% trypsin insteadof 0.25% as is normally used in our laboratory. Therationale for this is that mild trypsinization avoidsexcessive damage to cell membranes. The cells werecollected by centrifugation and washed three timesin PBS before fixation for 1 h at 4 ◦C in 1% (v/v)glutaraldehyde and 4% (v/v) paraformaldehyde. Afterthis, cells were rinsed in 0.1 M sodium cacodylate(pH 7.4), postfixed for 1 h at 4 ◦C in 1% osmiumtetroxide, and dehydrated in increasing concentrationsof ethanol. The embedding was done as previouslydescribed, with minor modifications (Spurr, 1969).Briefly, ultrathin sections (40–50 nm) made with anultramicrotome (NOVA, LKB, Stockholm, Sweden)were placed on 400 mesh grids, stained with saturateduranyl acetate and lead citrate, and examined using aJEOL/JEM 2000 EX transmission electron microscope(JEOL, Tokyo, Japan). To avoid sample bias, 2000microphotographs were analysed in this study.

Nuclear transfer

For nuclear transfer, the cells were seeded in 60 mmplastic dishes, as described above for electron micro-scopy, left to reach confluence and serum-starved(0.5%) for 3–5 days. Mild trypsin treatment was appliedand only fast-detaching cells were used for nucleartransfer. The rationale for using only fast-detachingcells was based on our observation that in monolayercultures of primary fibroblasts the pure fibroblastsdetach faster than other contaminating cell populationssuch as epithelial cells (data not published). The cloningprocedures were essentially those described by Wells

Freezing affects efficiency of cloning 279

Table 1 Effect of different freezing protocols on fusion rate and development into blastocysts of cloned bovine embryos

Oocytes or embryos

Cell line Freezing method n Fused (%) Cultured Cleaved (%) Blastocysts (%)

Adult fibroblasts Discontinuous 1202 308 (25.62)a 277 87 (31.40)a 0a

Adult granulosa Discontinuous 915 249 (27.21)a 249 86 (34.53)a 2 (0.80)a

Adult fibroblasts Continuous 1411 771 (54.64)b 588 458 (77.89)b 69 (11.73)b

Fetal fibroblasts Discontinuous 552 172 (31.15)a 172 60 (34.88)a 4 (2.32)a

Fetal fibroblasts Continuous 1136 690 (60.73)b 690 437 (63.33)b 86 (12.46)b

Adult fibroblasts Not frozen 340 218 (64.11)b 218 166 (76.14)b 44 (20.18)b,c

Adult granulosa Not frozen 283 176 (62.19)b ND ND ND

Different superscripts within a column indicate a significant difference. p < 0.05.

et al. (1999) with certain modifications introduced inour laboratory (Rodrıguez et al., 2004). Enucleationwas performed 18 h after the onset of maturation,without Hoechst staining and ultraviolet confirmation.Electrical fusion and initiation of activation wereconducted at the same time by means of a singlepulse (1800 V/cm, 30 µs, 2 times) in a 0.3 M mannitolfusion medium. Reconstructed couplets were furtheractivated by brief incubation (5 min) in 7% absoluteethanol diluted in TCM 199 supplemented with 25 mMHepes and 5% FCS at room temperature. After this, thecouplets were kept for 5 h in cycloheximide (10 µg/ml)and cytochalasin B (5 µg/ml) in bicarbonate-bufferedTCM 199 in a CO2 atmosphere to achieve completeactivation, after which the fusion rate was evaluatedand the fused couplets were placed in 400 µl of a CR1aamedium supplemented with 2% FCS, 0.3% essentiallyfatty-acid-free bovine serum albumin (BSA) and 50 µMbeta-mercaptoethanol in 4-well dishes in a 5% CO2atmosphere at 39 ◦C and complete humidity for theirculture until the blastocyst stage.

Statistics

Percentages of fused, cleaved embryos and blastocystswithin the two treatment and control groups werecompared by chi-square and Kruskal–Wallis tests.

Results and discussion

We have evaluated two methods for cryopreservationof nuclear donor cells. The first method we termed thediscontinuous method, since there is no continuouscontrol over the cooling rate, and it does not useany device to provide controlled freezing, making itsimple and inexpensive while yielding excellent resultsin terms of survival after thawing and subsequentsubculture of most cell lines. It has been successfullyused in our laboratory for routine cell freezing (F.O.Castro, unpublished data). The second, or continuous,

method implies the use of a cooling device for stepwisedecrease of the temperature. The two methods yieldedsimilar results in terms of cell survival after thawingand subculture for routine cell work in the laboratory.Given its simplicity and low cost, we decided to extendthe discontinuous method for the freezing of nucleardonor cells for cloning experiments. However, thefusion efficiency after nuclear transfer of two frozensomatic adult cell lines and one fetal cell line usingthe discontinuous method was low for all three celllines assayed (Table 1). Attempts to increase the fusionefficiency by changing key parameters such as number,intensity and duration of the electrical pulses were notsuccessful (data not shown).

On the other hand, we found that cells isolated fromadult skin or granulosa and subcultured continuouslyin vitro for 5 to 8 passages but never exposed to freezing,fused almost 2 times better than their counterpartsfrozen by the discontinuous method (Table 1). Thisphenomenon was independent of the cell type and ageat the moment of isolation, since essentially the samefusion figures were obtained for fetal fibroblasts usingthe discontinuous method of freezing.

When fetal fibroblasts that had been frozen by eithermethod were used in nuclear transfer experiments, thesame pattern of fusion as seen for adult cells wasobserved.

In nuclear transfer experiments using fetal fibroblastsfrozen and thawed by either the continuous or discon-tinuous method, we also found the former to be moreefficient. In these sets of experiments, no attempts tofuse non-frozen fetal fibroblasts were conducted. Allthree cell lines isolates described here – adult or fetalfibroblasts and granulosa – have been subcultured rou-tinely in our laboratory for more than 20 passageswithout any appreciable loss of viability. These findingssuggest the hypothesis that the cell membranes suf-fered damage during freezing that does not com-promise cell viability but which is incompatible withefficient fusion after nuclear transfer, and prompted usto search for another freezing method.

280 O. Hayes et al.

The occurrence of this phenomenon could beexplained if damaged membranes are rebuilt afterfreezing-thawing, during or after cell doubling.However, since we thawed and seeded the frozen cellsin such a concentration as to fill about 85–90% of theculture area after the cell expansion that normallyoccurs upon attachment of the cells to the plasticsurface, we expected that the vast majority of the cellswill not divide but instead will fill the culture vessel andenter into quiescence by cell-to-cell contact inhibitionor serum deprivation (Campbell et al., 1996a, b; Oback &Wells, 2002). Therefore most of the cells used fornuclear transfer will probably not have rebuilt theirmembranes, which in turn could affect the cell fusionrates observed.

Freezing protocols with a stepwise, controlleddecrease in temperature have been suggested asalternatives for efficient and quick freezing that aresuitable for laboratories where a programmable cellfreezing device is not available (Kuleshova et al., 2002).We decided to freeze two of our cell lines (adultskin and fetal fibroblasts) using the Cryo FreezingContainer. This cryofreezing container guarantees acritical, repeatable 1 ◦C/min cooling rate. As shownin Table 1, the figures for fusion efficiency using thismethod of cell freezing were higher than for thediscontinuous method and very similar to those fornon-frozen cells.

To study the effect of the freezing method on in vitrodevelopment into the blastocyst stage, we comparedthe two methods with non-frozen cells in terms ofcleavage and blastocyst rates upon nuclear transfer. Wefound that in the three cell lines assayed the freezingmethod significantly affected not only the efficiencyof fusion but also the cleavage and blastocyst rate,regardless of the age of the cells (adult or fetal; p <

0.05). The figures from groups pooled according tothe method of freezing were: cleavage 33.60 ± 1.91and 70.61 ± 7.28 for discontinuous and continuousfreezing, respectively, and 1.04 ± 0.96 and 12.09 ± 0.36for blastocysts. Interestingly, adult fibroblasts frozen bythe continuous method were comparable to non-frozencells in terms of cleavage, but yielded significantlyfewer blastocysts (p < 0.05). This finding might indicatethat cell freezing per se has a negative effect onblastocyst production upon nuclear transfer in bovine.There is a very limited amount of data published onthis subject with which to compare our findings, andthe impact of freezing protocols on the outcome ofsomatic cell cloning by nuclear transfer has previouslybeen overlooked. Kishi et al. (2003) also found that thecleavage rates of frozen-thawed cells were significantlylower than those of fresh cells in bovine cloningexperiments using mammary epithelial cells. Thereason for this impairment on in vitro developmentis not clear and could be related to structural

damage to the donor cells by the cryopreservationprotocol.

Despite the relatively low rate of development toblastocyst observed in our experiments, we do notthink that our findings related to the effect of freezingmethod on blastocyst development are the result of asub-optimal nuclear transfer system. This is supportedby the fact that reasonably high rates of blastocysts (upto 35%) have been produced in our laboratory usingother fresh donor cell types and oocytes producedboth in vitro or in vivo by ovum pick-up (data notshown).Nevertheless, our findings indicate that thedamage to the membrane upon freezing affects keyparameters of the nuclear transfer procedure, such asfusion efficiency, cleavage and blastocyst rate, and thatthis damage does not preclude normal development ofcells after in vitro passages.

To ascertain this, we conducted an electronmicroscopy study of cells subjected to both freezingmethods, as well as of control non-frozen cells. Ourfindings point to a relatively higher degree of damageto the cell membrane in the group subjected todiscontinuous freezing, regardless of the origin ofthe donor cell (granulosa or fibroblasts; Fig. 1c, f)compared with cells frozen by the continuous method(Figure 1b, e) or non-frozen cells (Fig. 1a, d). The mostcommon feature found in damaged cells was the lossof the outer cellular membrane (arrows on Fig. 1c, f),as well as vacuolization (V on Fig. 1f). No similardamage was found in cells frozen by the continuousmethod, or in the control, non-frozen cells. We didnot find obvious damage to intracellular organellesother than some vacuolization in cells subjected to thediscontinuous method of freezing. Nuclear structureswere not affected by either method of freezing (Fig. 1and data not shown), probably indicating that the lowerfusion rate of cells frozen by the discontinuous methodwas due to cell membrane damage rather than otherstructural damage. We did not conducted an in-depthstudy of cytoskeletal injury in these cells; however, wecan not rule out the possibility of certain damage atthis level. A previous paper has pointed to damage tothe spectrin-based membrane skeleton as a key factoraffecting cell fusion in erythrocytes by preventing therounding up process of the cell membrane (Sowers,1995).

The lack of any blastocyst development inthe discontinuously frozen adult fibroblasts wasunexpected and we do not have a clear explanation forit. It does not seem to be a consequence of extra stressresulting from the electrical pulses during fusion, sinceno special efforts were made to re-fuse non-fused cells.Most likely, damage to the cell cytoskeleton couldaccount for deficient reorganization of cell structuresthat in turn led to poor embryonic development. Infavour of this hypothesis is the fact that the percentage

Freezing affects efficiency of cloning 281

Figure 1 Transmission electron micrographs of ultrathinsections of fibroblasts (a–c) or granulosa cells (d–f) used incloning experiments. Cells were subcultured and not frozen(a, d), frozen by a continuous protocol (b, e), or frozen by adiscontinuous protocol (c, f). Arrows in (e) and (f) indicate lackof continuity of the cell membrane. V, vacuoles. Magnification×20 000.

of morulae obtained after nuclear transfer fromcells frozen by the discontinuous method was 4.2(13/308) but no blastocysts were produced. Essentiallythe same figures were found for adult granulosacells, where 5.6% of morulae (14/249) and 0.8% ofblastocysts (2/249) were obtained. A marked degreeof blastomere fragmentation was documented forthese cells. We hypothesize that only a few cells frozen

by this method maintain a strictly intact membranecapable of fusing with the oocyte membrane in away that allows proper blastomere assembly anddevelopment without fragmentation, thus making theevent of blastocyst formation an exception.

From the results described above, it seems thatthe freezing method affects the structure of the cellmembrane and this, in turn, affects the efficiency offusion after nuclear transfer and in vitro developmentto the blastocyst stage. It appears that fusion requiresthe existence of an absolutely intact membrane, whilethis is not necessarily the case for cell subculture.Scientifically demonstrated data on the effect ofcytoskeletal damage on nuclear transfer are notavailable in the literature, thus making any conclusiondifficult to draw.

The process of cell fusion is controlled by biologicallyrelevant membrane factors among which the roundingof the membrane, the cell shape and the formation ofpores are of major influence (Sowers, 1989; Wu et al.,1994; Fuhr et al., 1996; Baumann & Sowers, 1996). Wecan not rule out the possibility of integral damage tothe membrane skeleton in our discontinuous freezingprotocol. The lack of membrane integrity found hereat the electron microscopy level can lead to a loss ofintimate physical contact at fusion, which is a criticalstep for the electrically disrupted bilayers of adjacentmembranes to fuse together instead of fusing withother portions of the same disrupted membrane. Ifthis occurs, there are not enough blunt ends of thedisrupted membranes available for resealing the pores,and the communication between the karyoplast andthe cytoplast thus becomes irreversible (Tesarik et al.,2000).

We are not aware of similar work on the effectof the freezing method on cell fusion and the invitro development of cloned embryos. Based on ourfindings, we suggest a method of cell freezing with astepwise controlled cooling rate. Whether this findingis true for other cell lines remains to be tested.

Acknowledgements

We are indebted to Jesus Seona and Nilda Reyes forphotographic work.

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