Fresh Versus Cryopreserved Leukopaks

Leukopaks, or leukapheresis products, are incredibly useful as a tool for drug discovery screening or as a starting material for ex vivo cell & gene therapy workflows. However, researchers often ask: should I order my leukopak fresh or cryopreserved?

The answer depends on what research is being executed, associated costs, and degree of convenience in scheduling.

Fresh Leukopaks

Leukopaks are traditionally shipped fresh at ambient temperatures to avoid the costs associated with cryopreservation media as well as the inevitable stress on the cells during the freezing and thawing cycles. This is also the most representative configuration for the patient starting material used in autologous cell therapies. While viabilities can vary cell type-to-cell type, a 90% viability can be sustained from fresh leukopaks for the first 24 to 36 hours. After this point, PBMCs begin to be affected by neutrophil degranulation. Supplementation of the leukopak with autologous plasma can extend viability for potentially up to 72 hours.

Users of fresh leukopaks must coordinate their laboratory schedules to receive the material and process immediately for cellular isolation, activation, and expansion. The benefits of fresh leukopaks, therefore, are only truly realized when collection centers are located within close proximity to research locations. Further, collection sites must accommodate donor schedules to perform leukapheresis early enough in the day to be convenient to the donor and arrive within hours of operation at the researcher’s site for true same-day use. An excellent compromise exists using next-day, early morning delivery services by courier – thus allowing full donor schedule flexibility and maximizing the laboratory time for downstream use. BioIVT’s donor centers are strategically located near biotechnology hubs around the world to provide researchers with fresh LEUKOMAX® leukopaks in either same-day or next-day delivery schedules, depending on location.

Chain-of-custody is especially critical with time-sensitive material such as fresh leukopaks. Ideally, researchers would be provided information on time of collection, when the leukopak has been picked up by the courier, when it was delivered to the researcher’s site, and who at the researcher site received the material. BioIVT offers digital chain-of-custody tracking for all same-day courier fresh LEUKOMAX® leukopak orders.

Cryopreserved Leukopaks

The process of freezing leukopaks is facilitated by a cocktail consisting on an isotonic electrolytic solution, a cryoprotectant, and a protein source, and then gradually reducing the temperature of the cells and solution to less than -150°C. BioIVT’s LEUKOMAX® leukopaks are frozen with serum-free CryoStor® Freeze Media at 200mL for full leukopaks, 100mL for half leukopaks, and 50mL for quarter leukopaks. The lack of serum enables easier shipment globally, as it contains one less component with required source documentation such as CITES permits. Cryopreservation of early passage primary cells allows them to be used months or years later with little to no loss of functionality and pluripotency.

Proper cryopreservation presents challenges at both the freezing and thawing stages. The process of freezing leaves cells susceptible to damage on two fronts: the formation of intracellular ice crystals and the effects of increased solute concentration (solution effects). Successful cryopreservation protocols like those used at BioIVT balance these two factors to result in minimal cell death. The appearance of intracellular ice crystals can be minimized by slow cooling the cells at a rate of -1°C per minute, using either a purpose-built slow cooling freezer rack or a rate-adjustable freezer. Slow cooling allows ice crystals to form in the extracellular space first, thus increasing the extracellular solute concentration and drawing water out of the cells to prevent formation of excessive ice crystals within the cells. Cellular dehydration, if unchecked, can in turn cause substantial damage to cells during the cryopreservation process due to the increased concentration of salt and other solutes. This can be alleviated by the addition of membrane-permeable cryoprotectants, the most common of which are dimethyl sulfoxide (DMSO) and glycerol. These function by reducing the salt concentration at a given temperature so that solution effects due to critical salt concentration occur at a lower temperature (ideally one low enough to prevent any lethal biochemical reactions due to high salt concentration).¹ BioIVT’s LEUKOMAX® leukopaks are frozen using this method.

Rapid cooling is a less common method for cryopreservation and relies on vitrification instead of dehydration of cells. Vitrification is carried out using high concentrations of non-membrane-permeable cryoprotectants that dehydrate the cell and flash freeze the remaining water with minimal crystallization, thereby minimizing damage from ice nucleation. Solution effects do not come into play because of the speed with which the cells are frozen. Hydroxy-ethyl-starch (HES), polyvinyl pyrrolidone (PVP) and Polyethylene oxide (PEO) are three common non-penetrating cryopreservation agents.¹

Cell death during the thawing process also occurs on two fronts: solvent concentration and ice recrystallization. The concentration of solvents used to prevent solution effects are cytotoxic at normal biological temperatures and must therefore be added immediately prior to freezing and removed just after thawing or the recovered cell viability will be low. A second major cause of cell death during the cryopreservation and thawing process is ice recrystallization during thawing process; current research is investigating the use of ice recrystallization inhibitors such as polyvinyl alcohol or antifreeze glycoproteins to enhance the recovery rate of cryopreserved cells.² Cryopreservation in liquid nitrogen has been shown to yield similar cell viability to cells cultured ex vivo if proper protocols are followed. ³

Cryopreservation of leukapheresis products offers researchers access to consistent and well-characterized specimens that can reduce inter- and intra-experiment variability. Freezing is also especially beneficial when having to transport leukopaks over a distance that can take over 24 hours to reach. This approach not only simplifies logistics but also gives researchers a level of flexibility in their laboratory scheduling to choose when they will process their leukopak. This is especially useful for allogeneic therapies, where the convenience of “off-the-shelf” generation should extend upstream to the starting material. Of course, this approach necessitates a well-developed standard operating procedure (SOP) for thawing cryopreserved leukopaks. You can access the workflow developed and used by BioIVT here.

Additionally, ordering cryopreserved leukopaks from a supplier reduces the time between collection and cryopreservation upon receipt. This helps to preserve cell functionality and viability downstream.

Cell Type-Specific Considerations

Although the basic protocol for cryopreservation of primary mammalian cells is similar among cell types, there are certain cell type-specific challenges that must be overcome for successful cryopreservation. These include the length of time in ex vivo culture as well as the addition of stimulants to promote growth of the cell type of interest as well as the type of medium used for cryopreservation (and addition of any factors to improve viability during the freezing process).

T cells

T cell immunotherapies are an emerging field of cancer research. Such therapy can take the form of autologous tumor-reactive T cells, engineered T cell receptors (TCR), or engineered chimeric antigen receptor (CAR) tumor reactive T cells. With the former, tumor-reactive CD8+ T cells are isolated from a patient and expanded ex vivo and then returned to the patient, often in concert with the cytokine IL-2. Research has shown that adoptive cell therapies work best when the transferred cells contain both mature cytotoxic T lymphocytes ready to kill cancer cells and a population of largely undifferentiated, multipotent cells that can continue to produce effector cells over time.⁴ In long term culture, though, T cells begin to lose their multipotency and show signs of replicative senescence. It thus appears that a strategy involving rapid expansion of T cells followed by cryopreservation would best suit ex vivo T cell therapy.

A study of isolated murine CD8+ T-cells determined that cryopreserved and ex-vivo cultured murine T cells showed similar surface marker expression (CD44, CD62-L, CD69 and CD25), pro-inflammatory cytokine secretion patterns, and 3D scanning abilities. Both cryopreserved and fresh T cells were able to efficiently differentiate into effector T cells, as measured by surface marker expression. In addition, both cryopreserved and fresh cells were able to infiltrate and reduce tumor size by similar amounts.⁵

Studies with human peripheral blood mononuclear cells have shown similar results with regard to the T cell compartment. A paper using human samples from HIV+ and HIV- donors determined that cellular proliferation and the proportion of CD4+ and CD8+ T cells was unchanged after storage in liquid nitrogen. When the cells were examined in more detail, however, fresh and cryopreserved T cells exhibited some differences in population dynamics. For example, the researchers found that certain cell surface markers – particularly CD45RO and CD62-L – appeared to be easily damaged during the cryopreservation process and thus skewed the analysis of post-thaw T cell populations. Likewise, T cell subsets correlating to activated and memory and regulatory T cells were underrepresented after thawing. Despite these slight changes in population distribution, T cell functionality was comparable between fresh and cryopreserved cells.³

PBMCs

The PBMC population is made up of leukocytes (T cells, B cells, NK cells) and monocytes (which can differentiate into macrophages or dendritic cells). In contrast to T cells, which are generally isolated and grown in culture for a short time prior to freezing, PBMCs obtained via leukapheresis are processed and frozen as soon as possible after acquisition.

Several studies have investigated the impact of cryopreservation on PBMCs in both healthy and diseased donors. In a 2007 study, researchers found that while the type of anticoagulant, PBMC isolation protocol, time to cryopreservation and cell shipping method all affected post-thaw viability, the largest decrease in viability, cell recovery and cell functionality was correlated with length of time between sample collection and cryopreservation. In all cases, as expected, a fresh sample yielded more cells than a cryopreserved sample; however, it was shown that with prompt processing (less than 8 hours between collection and freezing) recovery of >94% was possible.⁶

Depending on the source and freezing protocol used, thawed PBMCs showed slight alteration in secretion of some cytokines including IFN-γ, IL-6, IL-10, IL-12 and IL-13.^7,8 Despite these background levels of cytokine expression, antigen-specific cytokine responses were still present and sometimes amplified, indicating that cellular functionality is retained following cryopreservation. PBMCs stored in liquid nitrogen have shown minimal loss of viability and function even after 15 months of storage.³

Final Thoughts

The tradeoffs between fresh and cryopreserved leukopaks are a balance between convenience, cost, and logistics. Researchers should prioritize their chosen cell type or cell types of interest and follow protocols that preserves both viability and function therein. Researchers must also consider the configurations of their final desired application when choosing what leukopak format to order – mimicking real-world conditions as closely as possible to the end-use case.

For more information around BioIVT’s leukopak screening, scheduling, and cancellation policies, please visit our Leukapheresis Collections page. To purchase fresh or cryopreserved leukopaks, please visit our LEUKOMAX® leukopak product page.

Sources

1. McGann, LE (1978). “Differing actions of penetrating and nonpenetrating cryoprotective agents.” Cryobiology 15 (4): 382-90.
2. Deller RC, Pessin JE, Vatish M, Mitchell DA and Gibson MI (2016). “Enhanced non-vitreous cryopreservation of immortalized and primary cells by ice growth inhibiting polymers.” Biomaterials Science (advance epub.).
3. Weinberg A, Song L-Y, Wilkening C et al (2009). “Optimization and Limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T cell characterization.” Clinical and Vaccine Immunology 16(8): 1176-86.
4. Crompton JG, Sukumar M and Restifo NP (2013). “Uncoupling T-cell expansion from effector differentiation in cell based immunotherapy.” Immunological Reviews 257 (1): 264-76.
5. Nino JLG, Kwan RYK, Weninger W and Biro W (2016). “Antigen specific T cells fully conserve antitumor function following cryopreservation.” Immunology and Cell Biology 94: 411-18.
6. Bull M, Lee D, Stucky J, Chiu YL, Rubin A, Horton H, McElrath MJ (2007). “Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials. J Immunol Methods 322(1-2):57-69.
7. Axelsson S, Faresjö M, Hedman M, Ludvigsson J, Casas R (2008). “Cryopreserved peripheral blood mononuclear cells are suitable for the assessment of immunological markers in type 1 diabetic children.” Cryobiology 57(3):201-8.
8. Mallone R et al (2011). “Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society” Clinical and Experimental Immunology 153(1): 33-49.