J. Cell S,i. 15, 419-427 (1974) 419

Printed in Great Britain

RELATIONSHIP BETWEEN FREEZING RATE,

ULTRASTRUCTURE AND RECOVERY IN A

HUMAN DIPLOID CELL LINE

D. J. BEADLESchool of Biological Sciences,Thames Polytechnic, London, S.E. 18, England

AND L. W. HARRISMicrobiological Research Establishment,Porton, Salisbury, Wiltshire, England

SUMMARY

The relationship between freezing rate, ultrastructure and recovery in a human diploidcell line has been studied by freezing cells at rates that are known to give high and low recoveriesand examining them immediately after thawing. Some correlation was found betweenstructural damage and recovery. The main types of damage observed were loss of cytoplasmand nucleoplasm, indicating disruption of cellular membranes, and swelling of subcellularorganelles due to osmotic changes during the freeze-thaw cycle. No simple correlation wasfound between freezing rate and structural damage. In the absence of a cryoprotectant bothrapid and slow freezing produced similar types and amounts of damage resulting in lowrecovery. In the presence of 10 % dimethylsulphoxide, however, slowly frozen cells showedfew signs of damage and recovery was high. DMSO had no such protective effect on rapidlyfrozen cells.

INTRODUCTION

Mazur (1965), Mazur, Leibo & Chu (1972) and Meryman (1966) have demon-strated that slow freezing gives maximal survival of mammalian cells provided thata cryoprotective agent and rapid thawing are used. It could be supposed, therefore,that this type of freezing would cause minimal structural and functional damageto the cells and indeed Trump, Young, Arnold & Stowell (1965) found that relativelyslow rates of freezing and rapid rates of thawing tended to minimize cytologicalterations in slices of mouse liver. However, in a more recent study Bank & Mazur(1972) found little correlation between structural alterations due to freezing andthawing and changes in the level of survival. It seems that more information isneeded before correlations between freezing rates, structural damage and recoverycan be made. The present study is an attempt to relate structural changes in a humandiploid cell line to freezing rate, presence of a cryoprotectant and recovery. Thishas been done by selecting freezing rates commonly used in laboratories which areknown to give high and low recoveries and examining the cells immediately afterrapid thawing.

420 D. J. Beadle and L. W. Harris

MATERIALS AND METHODS

The human diploid cell, MRC-5, was derived from foetal lung tissue and was grown inEagle's minimum essential medium supplemented with 10% foetal calf serum. Cells werefrozen in borosilicate glass ampoules which contained 1 ml of growth medium and 3 x 1 0 ' cellswith or without 10% dimethylsulphoxide (DMSO) as a cryoprotectant. The chosen freezingrates were 17 cC/min and approximately 100 °C/min and a BF4 Biological Freezer System(Linde Division of Union Carbide) was used to ensure a steady drop to —196 °C. The100 °C/min cooling rate is the nominal value for ampoules plunged directly into liquidnitrogen. Thawing was carried out at 200—300 °C/min by taking the ampoules straight fromliquid nitrogen and plunging them into a waterbath at 37 °C for 1 min. A complete analysisof all these cooling and warming rates has been published elsewhere (Harris & Griffiths,1974a, b).

Immediately after thawing the cells were spun into a pellet which was fixed for 30 min atroom temperature in 2 5 % glutaraldehyde buffered at pH 7 2 with cacodylate buffer. Afterfixing the pellet was washed overnight in buffer and postfixed for 2 h at 4 °C in 1 % osmiumtetroxide buffered at pH 7-1 with veronal acetate. The pellet was then broken into a numberof smaller pellets and each of these was dehydrated in a series of ethanols prior to embeddingin Araldite. Sections were cut on an LKB Ultratome III and stained with uranyl acetateand lead citrate. Gold and silver sections were examined on an AEI EM6B electron microscope.

Survival of cells from freeze/thaw cycles is given as a recovery index (RI). This was measuredby adding triplicate-o-25 ml amounts of the resuscitated cell suspension to 10 ml of growthmedium in 100-ml culture vessels and incubating for 24 h. Identical aliquots of control cellsuspensions were treated in an identical manner. After 24 h incubation cells were harvestedand counted in a haemocytometer and the number of viable cells (not stained by trypanblue) grown from the frozen and thawed samples was expressed as a proportion of the numbergrown from the unfrozen control cell suspension (RI = 100 when the number of cellsgrown from the resuscitated cell sample equals that from the control sample). This measure-ment was used because MRC-5 cells are incapable of growing from low inoculum levels thusrendering the plating efficiency method unsuitable (Harris & Griffiths, 1974a).

OBSERVATIONS

Assessment of damage

In order to assess damage to frozen/thawed cells electron micrographs of untreatedMRC-5 cells were used as a standard for comparison. These cells were scrapedfrom the bottom of a Roux bottle with a rubber policeman and were then treatedfor electron microscopy in an identical manner to the experimental cells. A typicalcell is shown in Fig. 1. The nucleus is large and lobular and contains granularnucleoplasm. A single nucleolus is present and clumps of heterochromatin occuradjacent to a regular nuclear membrane. The cytoplasm is granular and containsmany polysomes and microfilaments. The rough endoplasmic reticulum is abundant,forming a complex anastomosis that is often dilated. The mitochondria are cylindricalwith regular, transverse cristae. Although classical Golgi bodies are occasionallyseen the Golgi apparatus is generally poorly defined. Numerous microvillar processesare present at the cell surface. In order to assess damage between 400 and 500 cellswere examined after each freeze/thaw regime and compared structurally to the controlcells. Only cells showing severe and obvious damage were scored as damaged. It wasnot considered possible to determine whether cells varying slightly from the controlwere in fact damaged.

Freezing rate, ultrastructure and recovery 421

Cells frozen in the absence of DM SO (RI = 001)

The majority of cells frozen without a cryoprotectant at either of the chosenrates showed extreme structural damage (Fig. 2). Of those frozen at 1-7 °C/min85%, and at 100 °C/min 99%, of the cells examined showed severe damage. Themost obvious sign of this was almost complete loss of cytoplasm and nucleoplasmindicating a disruption of both the cell and nuclear membranes. Where the plasmamembrane was still intact it appeared to be very irregular but often it had beencompletely disrupted leaving a loosely packed group of vesicles. The nuclear envelopenormally remained intact but there was a separation of its constituent membranessometimes resulting in gross swellings and the appearance of vesicular structuresin the resulting space. A small amount of chromatin remained attached to the nuclearenvelope and the nucleolus was normally intact.

The cytoplasmic organelles were distorted often beyond recognition. Mitochondriacould be recognized because of their double membrane and occasional cristae whichwere reduced in number and the matrix was not evident. The ribosomal endoplasmicreticulum was swollen to form large vesicles and could only be identified by thepresence of ribosomes on the membranes. Most of the other organelles had swolleninto large, empty vesicles which could not be identified. They were presumablyderived from the Golgi, lysosomes and smooth endoplasmic reticulum. A few cellswere found that did not exhibit such extremes of damage.

Cells frozen in the presence 0/10% DM SO

Of the cells frozen at 1-7 °C/min, 89% showed no structural damage (Fig. 3) andthe recovery index at this rate was 0-90, whereas 62 % of the cells frozen at 100 °C/minexhibited severe structural damage (Fig. 4) and the recovery index was only 0-06.In the undamaged cells the plasma membrane was regular in outline althoughoccasional discontinuities were seen. The cytoplasm was granular and there wasno evidence of loss of cytoplasmic contents. The nuclei were irregular in shape butthe nuclear envelope was intact and there were no indications of separation of its2 membranes. The nucleoplasm was granular and clumps of heterochromatin wereobvious close to the nuclear envelope. The endoplasmic reticulum was extensivebut occasionally slightly swollen. The mitochondria were elongate and unswollenwith numerous cristae. Golgi regions were present but classical Golgi structureswere lacking. A large number of membrane-bound vesicles that were morphologicallysimilar to lysosomes were present and many of these appeared to be autolytic. Large,empty vacuoles were often present in the peripheral cytoplasm. Many of these cellshad cytoplasmic blebs on their surface and these contained intact microtubules(Fig. 5). Bundles of microfilaments were often seen in the cytoplasm although theydid not have any obvious orientation. Damaged cells had structural characteristicssimilar to those described for cells frozen in the absence of a cryoprotectant.

422 D. J. Beadle and L. W. Harris

DISCUSSION

Bank & Mazur (1972), studying the effects of different freezing and thawingregimes on Chinese hamster cells, found no obvious relationship between structuralalterations and changes in survival. The observations presented above, however,suggest that there is a good correlation between structural damage and recovery inMRC-5 human diploid lung cells where recovery is either very high or very low butthat at intermediate recovery levels the relationship is less clear. For example, cellsfrozen at 100 °C/min with 10% DMSO have an RI of 0-06 whereas only 62% ofthe cells exhibit severe structural damage. Hence at least 30 % of the cells that failto recover appear to be undamaged and work is now in progress to determine whythis is so. The most common form of damage seen was loss of contents from membrane-bound structures. This was particularly evident in the case of the cytoplasm andnucleoplasm in damaged cells indicating disruption to both the plasma membraneand the nuclear envelope during the freezing and thawing regime. Mazur et al.(1970) have stressed the importance of the plasma membrane in freezing damage andLevitt & Dear (1970) have suggested that freezing could damage cell membranesby bringing about the formation of 'holes' leading to an efflux of cell solution.Such 'holes' might be formed by ice crystals penetrating the membrane duringintracellular freezing or by tension on the membrane due to cell collapse duringextracellular freezing. Either process could account for the loss of cytoplasmicmaterial observed during this study. Another common form of cytoplasmic alterationin these cells was swelling in mitochondria and other subcellular organelles. Suchswelling is almost certainly osmotically induced (Farrant, Walter & Armstrong,1967).

Although there appears to be a correlation between structural damage and recoveryin these cells there is no such simple relationship between freezing rate and damage.Both of the chosen rates resulted in similar types and amounts of damage resultingin low recovery on thawing. The presence of a cryoprotectant, DMSO, protectedcells from the consequences of slow freezing resulting in good recovery on thawing,a protection not imparted to cells frozen at the more rapid rate. DMSO is generallythought to protect cells from freezing damage through its colligative properties(Nash, 1966; Ashwood-Smith, 1971). The presence of DMSO in the mediumlowers the concentration of electrolytes in equilibrium with ice at any temperaturebelow freezing so that the cells are not subjected to excessively high concentrationsof solutes until the temperature is so low that any damage produced by the increasedelectrolyte levels is reduced sufficiently to be tolerated by the cells. The protectiveaction of DMSO depends upon its ability to penetrate cells and in this respect itwould be of interest to investigate the ultrastructure of cells protected by a non-penetrating cryoprotectant such as poly-vinylpyrrolidone.

Since DMSO protected only at the slower freezing rate it is likely that the majorcause of damage at this rate is due to solute concentration effects whereas at thehigher rate some other factor might be involved that is unaffected by the presenceof DMSO. Mazur (1966) and Mazur et al. (1970, 1972) have suggested that the

Freezing rate, ultrastructure and recovery 423

major cause of damage at rapid cooling rates is the formation of intracellular icecrystals; and that solution effects such as exposure to concentrated solutes, dehydration,osmotic shrinkage and pH changes cause the damage at the slower rates. However,Farrant (1965), using freezing methods that caused ice formation in smooth musclefound little evidence of damage due to the formed ice. Whether or not the damagecaused in MRC-5 cells frozen at 100 °C/min is due to ice formation or to some otherfactor is not known at present but as the type of damage seen in cells frozen at the2 different rates appears to be similar it is possible that the causes of damage inboth cases are similar but the more rapid freezing rate may prevent the DMSO fromexerting its protective effect.

The present study has been concerned with comparing populations of cells frozenat different rates and not with the effect of a particular rate on individual cells. Forthis reason it provides no information as to why such a range of structural alterationsshould be seen in a single ampoule. In this respect it is important to determine towhat extent these variations are due to physical parameters such as the spatialpositions of cells within the ampoule and to what extent the physiological conditionof the cell itself is involved. The next phase of this work will be concerned withattempting to answer these questions.

We would like to thank Dr J. B. Griffiths for useful discussion and advice during thiswork and to thank Mr F. Morsley for photographic assistance and Mrs B. Bates for typingthe manuscript.

REFERENCES

ASHWOOD-SMITH, M. J. (1971). Radioprotective and cryoprotective properties of DMSO.In Dimethyl Sulphoxide (ed. S. W. lacob, E. E. Rosenbaum & D. C. Wood), pp. 147-188.New York: Marcel Dekker.

BANK, H. & MAZUR, P. (1972). Relation between ultrastructure and viability of frozen-thawedChinese hamster tissue culture cells. Expl Cell Res. 71, 441-454.

FARRANT, J. (1965). Mechanism of cell damage during freezing and thawing and its prevention.Nature, Lond. 205, 1284-1287.

FARRANT, J., WALTER, C. A. & ARMSTRONG, J. A. (1967). Preservation of structure andfunction of an organised tissue after freezing and thawing. Proc. R. Soc. B 168, 293-310.

HARRIS, L. W. & GRIFFITHS, J. B. (1974a). An assessment of methods used for measuringthe recovery of mammalian cells from freezing and thawing. Cryobiology (in Press).

HARRIS, L. W. & GRIFFITHS, J. B. (19746). The effect of cooling on cellular changes during thefreeze-thaw cycle. Cryobiology (in Press).

LEVITT, J. & DEAR, J. (1970). The role of membrane proteins in freezing injury and resistance.In The Frozen Cell (ed. G. E. W. Wolstenholme & M. O'Connor), pp. 149-174. CibaFdn Symp. London: Churchill.

MAZUR, P. (1965). Causes of injury in frozen and thawed cells. Fedn Proc. Fedn Am. Socsexp. Biol. 24, Suppl. 15, 5175-5182.

MAZUR, P. (1966). Physical and chemical basis of injury in single-celled micro-organismssubjected to freezing and thawing. In Cryobiology (ed. H. T. Meryman), pp. 213-315.New York and London: Academic Press.

MAZUR, R. P., LEIBO, S. P., FARRANT, J., CHU, E. H. Y., HANNA, JR. , M. G. & SMITH, L. H.

(1970). Interactions of cooling rate, warming rate and protective additive on the survivalof frozen mammalian cells. In The Frozen Cell(ed. G. E. W. Wolstenholme & M. O'Connor),pp. 69—88. Ciba Fdn Symp. London: Churchill.

MAZUR, P., LEIBO, S. P. & CHU, E. H. Y. (1972). A two factor hypothesis of freezing injury.Evidence from Chinese hamster tissue culture cells. Expl Cell Res. 71, 345-355.

424 D. J. Beadle and L. W. Harris

MERYMAN, H. T. (1966). Review of biological freezing. In Cryobiology (ed. H. T. Meryman),pp. 1-114. New York and London: Academic Press.

NASH T. (1966). Chemical constitution and physical properties of compounds able to protectliving cells against damage due to freezing and thawing. In Cryobiology (ed. H. T. Meryman),pp. 179-211. New York and London: Academic Press.

TRUMP, B. F., YOUNG, D. E., ARNOLD, E. A. & STOWELL, R. E. (1965). The effects offreezing and thawing on the structure, chemical constitution and function of cytoplasmicstructures. Fedn Proc. Fedn Am. Socs exp. Biol. 24, Suppl. 15, 5144-5172.

{Received 25 May 1973 -Revised 22 November 1973)

ABBREVIATIONS ON PLATES

er, endoplasmic reticulum mv, microvillig, Golgi region n, nucleus/, lysosome ne, nuclear envelopem, mitochondria

Fig. 1. Control cell. The cytoplasm and nucleoplasm are granular. The nucleus islobular with a regular nuclear envelope. Mitochondria are numerous and dilatedelements of the endoplasmic reticulum can be seen, x 25 000.Fig. 2. MRC-5 cell frozen at 17 °C/min without DMSO. This cell shows loss ofcytoplasm and nucleoplasm. The nuclear envelope is distorted and the cytoplasmicorganelles all show signs of swelling. Mitochondria and swollen endoplasmic reticulumare present, x 17500.

Freezing rate, ultrastructure and recovery 425

426 D. J. Beadle and L. W. Harris

Fig. 3. MRC-5 cell frozen at 1-7 °C/min with 10% DMSO added. This cell hasvery granular cytoplasm and nucleoplasm. The nucleus has heterochromatic clumpsand the nuclear envelope is intact. The cytoplasmic organelles all appear normal.A number of lysosomes are present and the endoplasmic reticulum is extensive andattached to the nuclear envelope. The plasma membrane is intact, x 8750.

Fig. 4. MRC-5 c e ' l frozen at 100 °C/min with 10 % DMSO added showing extremedamage. The plasma membrane has been completely lost and the endoplasmicreticulum has swollen grossly where it is attached to the nuclear envelope, x 17500.

Fig. 5. MRC-5 cell frozen at 17 °C/min with 10% DMSO added showing thepresence of microtubules (arrows) in the cytoplasmic extensions, x 35000.

Freezing rate, tdtrastructure and recovery