Cryopreservation is routinely utilized in a diverse range of areas including cell therapy, tissue engineering, and cell banking. In addition to direct clinical applications, the use of cryopreserved cells, such as hepatocytes, LNCaP, keratinocytes, CHO cells, and others, plays a critical role in drug discovery and in vitro tox testing.
While the use of cryopreserved cells is commonplace, the quality and utility of cryopreserved cells continue to have significant challenges. As the use of in vitro cell systems continues to rise, more attention has been directed toward developing new approaches to assure effective cryopreservation of cellular systems (maintaining a high degree of cell viability and function). Most cell cryopreservation techniques use a mixture of cell culture media, animal sera, and DMSO as a freezing solution combined with slow cooling rates to prevent ice crystal damage and other physically related effects associated with the freezing process.
However, the rapid recovery of viable, functional cells able to quickly attach and grow in culture is often a problem following cryopreservation. Cells that appear to be viable immediately upon thawing are often observed floating or poorly attached after 24 hours incubation. These cells usually become necrotic or apoptotic and, as a result, recover slowly, if at all.
Additionally, these traditional freezing solutions usually contain an animal-derived protein and/or serum component, which can contribute to variability in cell survival, increased contamination risks, and added cost.
Beyond Freeze Rupture
While control of ice formation within a cell is important and fully avoidable with standard preservation protocols, it represents only part of the story. During the freeze-thaw process, a host of detrimental molecular events occur, these include alterations in the cellular genome, proteome, plasma membrane, and mitochondria.
Recent reports have shown that the ultimate post-preservation result is often the activation of the apoptotic cell death cascade. This activation continues many hours after thawing (cryopreservation-induced delayed-onset cell death, or CIDOCD) and is responsible for many of those “floating cells” observed a day or two following thawing. To avoid this problem a new understanding of preservation dependent on novel approaches to cryopreservation has been developed to reduce cell stress thereby improving overall recovery.
Cell Molecular Response
Over the past six years the field of cryopreservation has undergone a paradigm shift from that with a primary focus on ice formation to one of understanding and controlling molecular-based cellular responses to low-temperature exposure. In fact, this shift continues as molecular-based responses to thermal stress continue to be uncovered.
One new approach that has emerged as beneficial in influencing outcome is that of cellular process control with a focus on reducing stressor buildup and stress response activation during and following (1–18 hours) the freeze-thaw process, yielding significant improvement in culture quality.
Influence of Post-thaw Culture
While preservation media changes have helped reduce cell death through modulation of the stress response, a level of CIDOCD remains and compromises many cell systems. CIDOCD is hypothesized to be a result, in part, of death-pathway activation due to stresses experienced by a cell during post-thaw culture. Cell culture process components such as media type, culture conditions (O2/CO2 concentration), and culture substrate can provide additional stressors if not properly managed.
Cells are in a biochemically fragile state following thawing due to damage to membranes and proteins, high intracellular free-radical levels, depleted energy stores, and cytoskeletal alterations. Thus, it is believed that many routine processes that divert cellular energy resources away from repair/recovery mechanisms, such as the establishment of cell adherence, can lead to stress-response amplification and increased cell death.
This tutorial demonstrates that by providing novel culture substrates, such as the Corning (www.corning.com) CellBIND® Surface, which is designed to help cellular-adhesion processes, a further reduction in cryopreservation-associated cell stress response is achieved.
Various events associated with the process of cryopreservation are often viewed as independent steps (isolation process vs. freeze vs. thaw vs. culture), when in fact they are intimately linked. As such, we explored the effect of coupling cryopreservation media with advanced culture substrates on cryopreservation outcome.
We specifically investigated the use of the CellBIND Surface combined with the protein-free BioLife CryoStor™ CS5 freezing solution (BioLife Solutions; www.biosolutions.com) for the cryopreservation and post-thaw culture of LNCaP cells, a human prostate cancer cell line that is difficult to preserve and maintain due to its slow growth and poor attachment properties.
The CellBIND Surface is created using a patented process that improves the cell attachment properties of polystyrene culture vessels. This surface has been shown to improve the recovery and attachment of LNCaP cells following cryogenic storage.
The CryoStor CS5 cryopreservation solution provides improved cell survival and recovery during and following cryogenic storage. It contains 5% DMSO and is based on a fully defined, cGMP manufactured protein-free formulation originally developed for the hypothermic (4°C) storage of tissues and organs.
Materials and Methods
LNCaP cells were obtained from ATCC and cultured under standard conditions in RPMI 1640 (Invitrogen; www.invitrogen.com) with 10% FBS (Invitrogen). For the cryopreservation protocol, LNCaP cell suspensions were split into aliquots of five million cells and pelleted. The supernatant was decanted, and cell pellets were resuspended in each of the three freeze media—culture media + 5% DMSO; 90% serum + 10% DMSO; CryoStor CS5.
Samples were cooled at -1°C/min to -80°C in an isopropanol freezing container in an -80°C freezer, held overnight, and transferred into to the vapor phase of liquid nitrogen storage. Vials were stored in LN2 for an average 7–14 days prior to thawing.
Following storage, samples were quickly thawed in a 37°C water bath, diluted in culture media, and seeded into flasks at 1 x 106 cells/flask with 5 mL of growth media. For each condition, four T-25 flasks (tissue culture-treated, TCT; Corning) and the CellBIND Surface were utilized—one for visual assessment and three for quantitative analysis of cell survival.
In addition, samples were plated into 96-well plates (TCT and CellBIND Surface) to assess various culture configurations. Survival analysis was conducted immediately post-thaw as well as following 24 hr post-thaw culture. Quantitative determination of cell number was performed on T-25 samples via hemocytometer counts. Samples were prepared for analysis by rinsing with PBS, trypsinization of adherent cells, collection, and staining with trypan blue. Cell viability for samples seeded in 96-well plates was determined using the metabolic activity indicator alamarBlue™. Additionally, micrographs were taken of each condition following 24 hours culture. Each study was performed in triplicate and repeated three times at each study site.
Results and Discussion
To test the overall effects of cryopreservation media and post-thaw culture configuration, LNCaP samples were cryopreserved in traditional preservation solutions or CryoStor CS5 and plated on either the CellBIND Surface or a TCT surface.
Analysis of cell survival immediately post-thaw revealed no significant difference in cell viability regardless of the preservation media (Table). This observation was expected and consistent with previous reports given that the timing of assessment does not account for CIDOCD. As observed in numerous other cell systems, such as hepatocytes, it is only after 24 hours culture post-thaw that a true level of survival is reached (Figure 1). When samples were allowed to recover in culture for 24 hours and then assessed for survival, significant differences were seen in the effectiveness of both the preservation media and the culture conditions (Figure 2).
Overall, the combination of CryoStor CS5 and the CellBIND Surface resulted in a significant increase in the survival and attachment of LNCaP cells over traditional solutions (media/DMSO or Serum/DMSO) and TCT surface. This observation was made in both the T-25 flask (Figure 2a) and 96-well plate (Figure 2b) culture configurations. The CellBIND Surface alone had a positive impact on the recovery of LNCaP cells (increase of 28% over TCT using standard freeze medium).
Further, the use of CryoStor in place of media and DMSO significantly increased, by 45%, the recovery of LnCAP cells on the TCT surface (p<0.001). The use of serum/DMSO as the cryopreservation media had a slight positive effect on overall survival but remained significantly below that of CryoStor in all conditions.
On average, the combined CryoStor/ CellBIND surface approach yielded a 58% increase in cell survival over traditional freeze media and standard tissue culture treated surfaces (p=0.0013). Importantly, when cultures were visualized by light microscopy, the CryoStor CS5/CellBIND surface approach showed an increase in attached cell number and improved cell morphology relative to traditional media/DMSO and tissue culture ware approaches (Figure 3). Analysis of cell membrane integrity using the fluorescent stain Calcein-AM corroborated the light microcopy data showing that the surviving cells were more numerous and retained a high degree of membrane integrity (Figure 4).
Through this study we tested the hypothesis that both the freezing media formulation and post-thaw culture surface might significantly impact cryopreservation outcome. It was further determined that by combining the use of novel cryopreservation media and cell culture surfaces, one can positively impact the overall level of cell survival and attachment.
Specifically, the CryoStor/CellBIND Surface approach resulted in a significant increase in LNCaP cell viability following cryopreservation and provided for the retention of a more native cellular attachment morphology. This is important for numerous reasons including the fact that these two factors have been correlated directly with overall functionality and usability of cryopreserved cellular systems.
The CryoStor/CellBIND Surface combination approach now offers researchers a leg up in this area by reducing much of the time and effort presently required thereby increasing productivity and cost-effectiveness of in vitro screening processes.