Steps to Improving Cryopreservation Outcome: Understanding key factors influencing efficacy

by John M. Baust, Kristi K. Snyder, Aby J. Mathew, Robert G. Van Buskirk and John G. Baust

Introduction

Cryopreservation is often viewed as a standard procedure in the cell culture laboratory. Until recently, however, hypothermic storage (4 to 8 C) has been used almost exclusively in the organ transplant field. In this regard the “gold standard” solutions (media) utilized were developed several decades ago. Recently, a growing awareness and concern has arisen in groups, such as stem cell therapy companies, pertaining to the quality and efficacy of the biopreservation processes utilized to develop a cellular-based product. Within both industry and academia groups recognize the critical role shipping and storage processes play in the distribution of living products in support of therapeutic intent. Traditional cryopreservation can often result in a 50 to 70% cell loss a problem that is not often appreciated and often erroneously perceived as not critical to cell system quality.⁽¹⁾ A loss of this magnitude of stem or dendritic cells under either hypothermic storage or cryopreservation conditions translates to a loss in the value of the product, as well as to potential uncertainty in therapeutic dose. In addition, the potential of launching an immune response in the patient as a consequence of cellular debris from the poorly preserved cells becomes a concern. Furthermore, poor preservation can result in viable cells that have little to no cell-specific function.⁽²⁾ Thus, it is important that researchers become more aware of the critical factors necessary to support successful preservation.

Pitfall #1 freeze slowly/thaw quickly

One of the most talked about cryopreservation steps, yet least understood in a general sense, are freeze/thaw rates. As a rule of thumb, successful cryopreservation can be achieved by applying slow cooling and rapid warming. Studies dating back to the 1970’s and early 1980’s described the importance of cooling rate through cell survival data which generated inverse U-shaped curves as one varied cooling rates from very slow to very fast.^(3,4) For many nucleated mammalian cells, a rate of 1 C/min was found to most often yield the best survival. Today, one of the most common freezing procedures applied is the 2-step method to bring cells slowly to a low subfreezing temperature prior to storage in liquid nitrogen. Typically this procedure involves placing samples at 20 C for 2 hours then 80 C for 2 hours followed by plunging in LN₂. While popular due to simplicity, this is the least effective of the cooling methods. Other more effective procedures for cooling cell systems available to researchers include programmable controlled rate cooling devices (active) and thermally buffered containers with samples centered in, but isolated from, the alcohol “ring”. These methods provide for more linear rates of sample cooling. Another often-overlooked aspect of the freezing procedure is the ice nucleation step (seeding). It is very important that ice nucleation begin at temperatures just below 0 C (2 C to 5 C). This provides a uniform cooling rate that supports water efflux from the cells, thereby minimizing the probability of lethal intracellular ice formation. Sample seeding can be achieved by a programmed “burst” or spray of LN₂ in a controlled rate cooler by abrupt agitation of the sample (tapping or flicking the vial) or application of a cold source to the surface of the sample vial.

Figure 1. Comparative Viability of Cryopreserved Human Cells at Various Time Intervals Following Thawing. Cryopreserved cell systems typically exhibit high viability levels immediately following thawing but over the course of 24-48 hours continue to die yielding much lower actual survival. The continuous death process is referred to as delayed-onset cell death and is commonly observed in many cell types [i.e. renal (MDCK), endothelial (CAEC), smooth muscle (CASMC), fibroblasts (NHDF) and peripheral blood mononuclear cells (PBMC)]. All cells were cryopreserved in serum supplemented media containing 5-10% DMSO. Click to enlarge.

Thawing is relatively simple compared to freezing. Essentially, one should thaw samples as quickly as possible, but samples should not be allowed to warm much beyond the point of ice dissipation (~5 to 10 C) within the sample prior to dilution. When samples are thawed, the warmer the medium in which the cells are contained, the more extensive the cell damage or death that can occur due to the toxic nature of cryoprotective agents such as dimethyl sulfoxide (DMSO). Further, it has been determined that rapid warming is the best way to thaw cells in order to reduce cellular damage due to re-crystallization phenomena (coalescence and re-growth of ice crystals). Rapid warming (typically in a 37 C water bath) reduces further ice damage to samples. Keeping the samples cool to the touch until diluted helps assure higher quality and more consistent post-thaw results.

Pitfall #2 timing assessments

One of the most common errors associated with the development of preservation strategies is linked to the timing utilized to assess cell viability. Typically, cryopreservation viability assessment is conducted immediately post-thaw (within an hour). While numerous assays exist to assess viability, the most commonly utilized is the Trypan Blue dye exclusion assay. Regardless of the assay employed, viability assessment immediately post-thaw provides for elevated, or false positive, indications. This problem relates to the recently discovered phenomena of delayed onset cell death (DOCD), which is characterized by continued cell death many hours to days following re-warming.⁽⁵⁾ DOCD has been shown to be a direct result of the activation of both apoptotic and necrotic cell death cascades during and following preservation.⁽⁶⁾ As a result, many subpopulations of cells are present immediately post-thaw that appear viable but succumb to preservation-based stress many hours later. Due to the contribution of apoptosis and necrosis, a careful selection of the assessment timing interval is important. Considering the timing of apoptotic and necrotic cell death processes coupled with cell recovery, cell cycle, and cell attachment processes and assessment of cell viability should be performed at 24-hours post-thaw. Viability assessment 2 days post-thaw also often masks true survival but in comparison to 24 hour assessment provides for an indication of re-growth of the surviving subpopulation.

Assessments at 24-hours post-thaw allow for a manifestation of most all the cell stress cascades and the peak of apoptotic (8-12 hours) and necrotic (4-8 hours) activities. An example of the profile of cell death time course can be seen in numerous cell systems when samples are cryopreserved under a standard protocol and viability is assessed at 0 and 24-hours post-thaw (Figure 1). By comparing the data points, a 50% overall difference in cell viability with the 24-hour time interval is noted. This reduction has been reported in numerous cell systems in both in vitro and in vivo studies. This temporal-based dynamic change in cell viability often means the difference between a successful outcome and system failure. In the case of cell therapy, the reduction in cell numbers affects both clinical outcome and dosing parameters. As such, proper temporal assessment of preservation outcome is critical.

Pitfall #3 assay selection

Coupled with the issue of assessment timing is the issue of assay selection. Often following a cryopreservation procedure, assays measuring cellular membrane integrity such as Trypan Blue or Calcein-AM are utilized to evaluate sample viability. Inherent in these assays are their inabilities to distinguish between subpopulations of cells which are viable or undergoing the early stages of apoptotic or necrotic cell death, where in both cases cell membranes are still intact at that time. This results in the frequent determination of dying populations of cells as “viable”. Even at later time points of 24-48 hours post-thaw, these assays typically yield elevated survival numbers. In order to gain a more accurate assessment of post-thaw viability, assays that measure parameters beyond membrane integrity should be utilized. Assays that measure metabolic activity (Alamar Blue, MTT, etc.), cell death type, etc. offer effective alternatives to Trypan Blue (Figure 2).⁽⁷⁾ Another common assessment is the Colony Forming Unit (CFU) assay, which is useful as a downstream assay to monitor reproductive capacity of the surviving cell population. While a useful tool, the CFU assay is often misapplied due to pre-selection of the viable population and adjustment for cell yields. In many cases, this pre-selection step results in elevated CFU values, thereby making comparative assessment between cryopreserved samples and conditions difficult. The issue of CFU assay pre-selection is most simply overcome by straight plating of post-thaw samples based on the sample pre-freeze values without any adjustment for post-thaw yields or viability. By eliminating the post-thaw cell number adjustment, sample and condition comparison becomes much more straightforward. Coupled with the previously discussed parameter of timing, proper assessment regimes can be developed which can be applied to accurately determine population viability.

Figure 2. Comparison of Post-Thaw Cell Survival Utilizing Several Different Viability Assays. Due to the contribution of the time dependent, molecular-driven cell death processes associated with cryopreservation, standard assay systems tend to provide highly variable viability measures. This variability between assays is often greatest immediately following thawing but reduces over time as populations stabilize. These data show the variability in survival assays of corneal endothelial cells following cryopreservation and between assays (including TrypanBlue = blue; Annexin V/PI = yellow; and alamarBlue = maroon), assessment time (1 and 24 hours post-thaw), and preservation medium (culture media supplemented with FCS and 5% DMSO or CryoStor CS2 a serum-free solution containing 2% DMSO). Click to enlarge.

Pitfall #4 cell viability does not imply cell function

It is often assumed in areas of stem cell research, drug discovery screening, bioreactor seeding, etc. that, if cells are viable subsequent to cryopreservation, they are also equally functional. In many cells, such as hepatocytes, stem cells, keratinocytes, etc., the post-thaw surviving cells lose cell-specific functions as a consequence of preservation-related stress.(sup)(2,8,9) Thus, one pitfall in preservation is to limit one’s assay set to those that merely examine viability. Cell specific function must also be analyzed simultaneously with the viability assays. Interestingly, studies have shown that the effect of preservation on cell function is not only specific to cell type, such as endothelial cells vs. smooth muscle cells, but also influences individual biochemical pathways within a given cell. For example, in hepatocytes it has been shown that cryopreservation has a differential effect on albumin synthesis and urea secretion⁽²⁾. In the area of hypothermic storage, the cardiomyocyte system serves as a model for the effect of preservation on function. In a comparative study it was demonstrated that following storage in solutions such as Viaspan there was a high degree of cell viability but a substantial loss of contractile function, but in other solutions, such as HypoThermosol-FRS, there was maintenance of both a high level of survival and function.⁽¹⁰⁾

Pitfall #5 genetic selection

One concept that has been virtually unstudied in the preservation sciences is the possibility of genetic selection in cryopreserved cell lines. No preservation process preserves 100% of all the cells. Indeed, there is often at least a 30-50% or more cell loss with preservation. This loss implies that the 50-70% of cells that survive may do so given the differential activation/deactivation of various stress pathways thereby making those cells more resistant to the rigors of preservation. Thus, successive preservation episodes, such as commonly utilized in master cell banks, may apply a selection pressure on those subpopulations better able to survive extreme cold and other related stresses. Along these lines, we have begun a series of studies utilizing both cDNA microarray and proteonomic analysis of surviving cell populations following preservation to shed light on this issue. Preliminary data from cDNA array and SELDI-TOF analysis indicate that there are significant alterations/ activations of many stress response pathways following low temperature exposure. An interesting observation from these studies noted changes in post-preservation yields depending on the preservation medium utilized. Higher levels of survival may significantly reduce the negative effects of the preservation process not only on cell survival and function but on cell genomics and proteonomics as well.

Summary

Stem cell therapy and other processes within the field of Regenerative/Reparative Medicine make it even more critical to improve the overall quality of laboratory biopreservation protocols. Utilization of new technologies when coupled with attention to the aforementioned issues can lead to more successful preservation, thereby providing greater utility to the various cell-based industries.

About the authors

John M. Baust, Kristi K. Snyder and Robert G. Van Buskirk are with Cell Preservation Services Inc., Owego, NY. Aby J. Mathew and John G. Baust are with BioLife Solutions Inc., Owego, NY. More information about cryopreservation methods and technologies is available from the authors at their respective firms.

References

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