Cryopreservation Of Immune Cells

Patrick J. Stiff, Loyola University Medical Center, Maywood, Illinois, USA

Copyright © 1998 Elsevier Ltd. All Rights Reserved.

Primitive experiments in cryopreservation date to the mid-eighteenth century but it was not until 1949 that Polge first used glycerol to successfully cryopreserve both animal and human spermatozoa at -79°C. After the demonstration of glycerol's effect on the low-temperature storage of spermatozoa, early cryopreservation studies focused on the use of glycerol and then dimethylsulfoxide (DMSO) to cryopreserve murine bone marrow for syngeneic bone marrow transplantation. The goal was to circumvent the lethal effects of radiation on the bone marrow, which were first seen as a result of the atomic bombs that had been dropped on Japan a few years earlier. These initial studies demonstrated the efficacy of these agents in the cryopreservation of marrow cells frozen at a constant rate of 1°C min-1 to -79°C, with storage for periods up to 6 months. Viability was determined by the demonstration of viable cells post-thaw by dye exclusion studies and the successful engraftment of the bone marrow when injected into syngeneic animals after lethal irradiation. This technology now forms the basis of numerous successful autologous bone marrow transplant programs worldwide, with clinical successes not only in the leukemias and lymphomas but also in solid tumors.

The use of cryopreserved lymphocytes for in vitro studies is also now commonplace. The initial studies were reported in 1964 by Ashwood-Smith, who cryopreserved murine lymphocytes using 15% DMSO. Cells were held in liquid nitrogen (-196°C) for 3 months and then assayed for viability, both by using phase microscopy and by the demonstration of splenomegaly after they were injected into irradiated hybrid mice. With the need for standardization of cytochemical staining, surface marker analysis and in vitro stimulation studies of lymphocytes, cryopreservation subsequently became the easy way to store cells for comparative studies. It is now easy to follow and compare changes over time or after therapy in individual patients using cryopreserved cells, with all assays done at the same time. While cryopreservation is used mainly for in vitro testing of lymphocytes, with the advent of recombinant cytokine therapy, cryopreservation may permit the storage of large numbers of either resting or 'stimulated' lymphocytes treated ex vivo, either after or before cryopreservation, in an attempt to augment the in vivo immune response to tumor cells. When infused later at a time of minimal residual disease, they might decrease relapses when used as consolidation therapy following conventional or transplant therapies.

Cryoprotectants and the freezing process

The initial use of glycerol for the freezing of spermatozoa was a 'chance observation' in 1949; it and other cryoprotectants work by stabilizing cell membranes and intracellular contents during the freezing and thawing processes. Glycerol and related compounds and DMSO are intracellular cryoprotectants in that they diffuse readily into the cell when added prior to cryopreservation, while hydroxyethyl starch (HES), dextran and polyvinylpyrrolidone (PVP) act as extracellular cryoprotectants by stabilizing the cell membrane during the freezing and thawing process. Lethal damage to cells can occur at various points in the freezing-thawing process, with the majority of damage occurring during the phase change from liquid to solid, and during the thawing and manipulation of the previously frozen cells. When cells in suspension are cooled, extracellular ice crystals form, raising the intracellular solute concentration. This osmotic change, if it occurs in the absence of a cryo-protectant, can lead to their death with cell membrane lysis. While rapid cooling decreases water shifts, it causes cell death due to intracellular ice formation. By diffusing into the cell, intracellular cryoprotectants decrease the solute shifts that occur during the freeze-thaw cycle, thereby decreasing the potential damage caused by high intracellular solute concentrations. This permits the use of slow cooling rates (1-3°C min-1), which in turn decreases the risk of damage caused by intracellular ice formation when faster rates are used.

The phase change from liquid to solid, with the liberation of the heat of fusion that occurs during the freezing process, can also be damaging to the cells. Without the use of a controlled-rate freezing apparatus, the temperature can actually rise several degrees (heat of fusion), and does not begin to fall again until the entire solution has been frozen. A rapid transit during this transition at l°Cmin~' appears to be optimal for the preservation of marrow stem cells, but seems to be less critical for other cells, including lymphocytes and platelets, especially when small ali-quots with a greater surface area available for the dispersion of this heat arc available.

While little damage is felt to occur during storage at low temperatures, provided that there are no significant temperature fluctuations, significant damage can occur during the thawing of the cryopreserved cells. During the thawing process, cells swell because of the reversal of the water shifts that occur during freezing. Intracellular cryoprotectants that diffuse out of the cells rapidly, extracellular cryoprotectants and proteins, including albumin, appear to decrease the damage to cell membranes that occurs during this swelling. While some controversy exists as to the best way to thaw frozen cells, in general most cells thaw rapidly and, once thawed, should be diluted slowly, 1:1 over 3-10 min, and then more rapidly to minimize further swelling. When used for transplantation purposes the thawed cells are infused rapidly, unfil-tered and undiluted.

While effective at low temperatures, intracellular cryoprotectants such as DMSO are toxic to cells at room temperatures or higher. For optimal use, DMSO should be added at 4°C just prior to freezing. In addition, for in vitro work it should be diluted or washed to a concentration of <1% as soon as possible post thaw. A recent analysis of the mechanism of action of DMSO by Arakawa suggests a reason for the disparity of the effects of DMSO at different temperatures. At low temperatures, DMSO is excluded from the hydration cover of proteins which leads to their stabilization during the freezing process; however, at room temperatures DMSO interacts in a hydrophobic way with these same proteins, leading to denaturation or destabilization. Exposure at concentrations >1% for as little as 30-60 min is sufficient to cause irreversible cell membrane damage.

Cryopreservation techniques Hematopoietic stem cells

There are currently two methods of hematopoietic stem cell cryopreservation in use worldwide. The most commonly employed method is based on the animal studies of the 1950s. After bone marrow is harvested in the operating room, it is brought to the cryopreservation laboratory where, using a cell sorter or density gradient separation, the majority of the red cells are removed. After adjusting the cell count to 60-70 million ml"', using tissue culture media admixed with 20% autologous plasma, an equal volume of media/plasma solution containing 20% DMSO is slowly admixed with the cells at 4°C; 100 ml aliquots are then placed into polyolefin freezing bags, sealed, placed into aluminum freezing frames and then frozen using a programmable rate-controlled freezer, at a constant freezing rate of

1°C min"' to -80-120°C, at which time the bags are placed into the liquid or vapor phase of liquid nitrogen. At the time of reinfusion, the cells are thawed at the bedside and infused rapidly over 5-15 min. Rapid reinfusion is required because this method does not successfully cryopreserve granulocytes, which lyse rapidly after thawing and release nucleo-proteins which cause clumping and a gel formation if left at room temperature for even 30 min.

A newer method, which has also been proven to be effective for both marrow and blood stem cells, uses a combination of both DMSO, an intracellular cryoprotectant, and low molecular weight HF.S, an extracellular cryoprotectant. This combination successfully cryopreserves both stem cells and terminally differentiated granulocytes, permitting the cryopreservation of large volumes of bone marrow in a single bag. In addition, this method does not require the use of a rate-controlled freezing process, nor does it require storage at liquid nitrogen temperatures (although recent data suggest that cells preserved in DMSO alone can also be frozen without a rate-controlled freezer or stored for short periods at -80°C). After harvesting, the majority of the red cells are removed as described above. The cells are brought to a final volume of 300 ml and are admixed with an equal volume of a cryoprotectant solution containing 10% DMSO, 12% HES and 8% human albumin in lieu of autologous plasma; 300 ml aliquots are placed into freezing bags, which are then placed into aluminum freezing frames. The marrow is frozen and stored simply by placement in a -80°C freezer, where it is kept until it is autotransfused. Reinfusion is done by infusing the 300 ml aliquots over 30 min each. Successful engraftment has been seen for cells stored at -80°C for periods of 2 years.

The newest source of stem cells being utilized for transplantation is cord blood, obtained at birth and used primarily for unrelated allogeneic transplantation. Cord blood banks are being developed in many countries, using standard cryopreservation techniques; successfully thawed, these cells are used as a transplant source primarily for children who have neither a related sibling donor nor an unrelated donor available through national and international computer registries. While standard cryopreservation techniques are used, many programs concentrate the stem cells (HES sedimentation) prior to cryopreservation to maximize freezer space. This type of transplant will likely increase as there appears to be a lower incidence of graft versus host disease. Furthermore, techniques have been developed to expand hematopoietic stem cells ex vivo, making these grafts available to adults.

Lymphocytes

While post-thaw recovery of cryopreserved lymphocytes appears to be optimal when the cells are frozen in 7.5-10% DMSO using a rate-controlled freezing process at 1°C min-1, many routinely freeze lymphocytes by simple placement into a -80°C freezer, or even by simple placement in insulated containers into the vapor phase of liquid nitrogen. Alternatively, lymphocytes can be held at room temperature for several days if nutrient media are properly buffered at pH 7.0 with HEPES buffer, and care is taken to avoid bacterial contamination. The cryopreservation process using rate-controlled freezing is similar to that for bone marrow; however, after freezing to -30°C at 1°C min 1 the rate is increased to 5°C min 1 to -80°C, at which time the cells are transferred to the vapor phase of liquid nitrogen. Alternatively, good results can be seen if the cells are put in a styro-foam box and placed in either a -80°C freezer for a minimum of 2 h before transfer to the vapor phase of liquid nitrogen or left in the mechanical freezer, or directly in the vapor phase of liquid nitrogen for 30 min and then transferred to the liquid phase of liquid nitrogen. Thawing and processing is done in a similar manner to marrow cells, with dilutions performed slowly over 5-10 min using a buffered, protein-containing solution. To wash the cells free of the residual DMSO, they are centrifuged at 150g for 10 min and then gently resuspended with the same media. Cells can also be frozen in tissue culture wells for later use, e.g lymphocytotoxicity assays.

Evaluation of cryopreserved cells

Depending on the cryoprotectant used to preserve hematopoietic stem cells, post-thaw recovery of nucleated cells varies from 50 to 90%. Unfraction-ated marrow cells cryopreserved in DMSO alone are associated with a lower recovery because terminally differentiated granulocytes are not successfully preserved, lyse and release nucleoproteins, causing clumping which entraps additional cells. This problem is eliminated if this cell population is removed prior to cryopreservation, or the DMSO/HES cryoprotectant is used, which successfully preserves these cells. Post-thaw recovery of committed stem cells such as CFU-GM and BFU-E also appears to be higher using the DMSO/HES method, ranging from 80 to 90% of prefreeze assays. However, post-trans-plant engraftment times appear similar for the two methods, with time post-transplant for a granulocyte recovery >500 cells pi"1 and platelets >20 000 cells ph1 of 17-22 days. Engraftment times appear to be shorter (5-7 days) for blood stem cell transplants, largely due to the increased number of cells that are mobilized, collected and cryopreserved.

Cryopreserved lymphocytes have many uses, including histocompatibility studies, functional assays to compare differences over time or therapy given in vivo for determination of cell surface markers. Having a reproducible source of cells, and the ability to improve quality control by assaying all target cells at the same time under the same conditions, is of significant value. Total recovery of viable lymphocytes, cryopreserved as described above, is in the range of 70-80%, and as early as 1962 Pcgg demonstrated that thawed lymphocytes could be used in stimulation assays, with little difference compared with fresh controls.

A variety of lymphocyte assays have been performed on cryopreserved lymphocytes. The relative number of B and T cells, the number of E (sheep erythrocyte) and EAC (erythrocyte-antibody-complement) rosettes, and the response to the mitogens PHA, PWM and Con A are not affected by cryopreservation but their recovery appears to be optimal when programmed freezing methods are used. The handling of the cells prior to cryopreservation appears to be important: specifically, the use of ammonium chloride is toxic to T cells when used prior to cryopreservation; it can, however, be safely used post thaw for separation studies.

Cryopreservation with DMSO appears to decrease the number and intensity of CD4+, CD7% CD 13* and CD33' cells assayed by flow cytometry, but appears to have no effect on B-lineage cells or on CDS' cells. Multiple studies have demonstrated a decrease in natural killer (NK) cells, i.e. cytotoxicity against K562, as well as antibody-dependent cellular cytotoxicity (ADCC) and mixed lymphocyte responses for cryopreserved lymphocytes. NK cell recovery is 50-70% compared with that of fresh cells; however, this value significantly improves when the thawed cells are incubated for 18 hours prior to assay. Lym-phokine-activated killer (LAK) cell activity may also be reduced following cryopreservation. Activity levels vary widely, ranging from 33 to 87%, depending on the effector:target ratios and the target cell line used for the assay. Cytotoxicity, however, improves upon incubation with recombinant interleukin 2 (rIL-2) for several days after thawing, and, if stimulated both before and after cryopreservation, activity levels may exceed that for fresh mononuclear cell populations due to loss of macrophage function during cryopreservation. The cause for the lowered NK and LAK activity appears to be a lowering of the binding to target cells early after thawing which recovers upon incubation for 5-24 h. Thymocyte recovery is also poor following standard cryopreservation using DMSO but preliminary studies suggest a benefit to thymocyte recovery if both DMSO and HES are used together as cryoprotcct-ant agents.

With the advent of many recombinant cytokines, the ability to cryopreserve both bone marrow and lymphocytes will attain even more significance. Clinical trials in which cryopreserved marrow is incubated for 24 h prior to the reinfusion into patients as part of an autologous transplant are under way.

See also: Antibody-dependent cellular cytotoxicity; Hematopoietic stem cell transplantation; Cell separation techniques; Flow cytometry; Hybridomas, B cell; Hybridomas, T cell; Leukocyte culture; Lympho-kine-activated killer (LAK) cells; Rosetting techniques; Tissue typing; Viability, methods of assessing leukocyte.

Further reading

Arakawa T, Carpenter JF, Kita Y and Crowe JH (1990) The basis for toxicity of certain cryoprotectants: a hypothesis. Cryobiology 27: 401-415.

Gorin NC (1986) Collection, manipulation and freezing of hematopoietic stem cells. Clinics in Hematology 15: 19-48.

Lorentzen DF (1990) Cell preservation. In: Zacharv AA and Teresi GA (eds) (1990) ASH1 Laboratory Manual, 2nd edn. American Society of Histocompatibility and Immunogenetics (ASHI), Kansas, USA.

Rubinstein F, Dobrila L, Rosenfeld RE et al (1995) Processing and cryopreservation of placental/umbilical cord blood for unrelated bone marrow reconstitution. Proceedings of the National Academy of Sciences of the USA 92: 10119-10122.

Stiff PJ, Koester AR, Weidner MK, Dvorak K and Fisher RI (1987) Autologous bone marrow transplantation using unfractionated cells cryopreserved in dimethyl-sulfoxide and hydroxyethyl starch without controlled-rate freezing. Blood 70: 974-978.

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