February 1, 2010 (Vol. 30, No. 3)
Deepa Patke Ph.D.
Anis H. Khimani Ph.D.
Renee M. Howell Ph.D.
Mark Manak Ph.D.
Preserving Fundamental Characteristics for Immune Monitoring and Drug Development
The advent of new and precise high-throughput screening technologies has opened up a vast resource for understanding the molecular mechanisms underlying disease, genetic disorders, and immune dysfunction. It is estimated that each year 100 or more cellular targets of disease are discovered, making it critical to develop and optimize appropriate screening and evaluation approaches to accelerate the development of successful drug therapies or potent vaccines.
The most promising small molecules or substances identified as lead compounds during screening then undergo extensive optimization and characterization processes in ex vivo/in vitro studies. During this phase of drug development, toxicity, target reactivity, and various properties of lead compounds are studied.
Advanced molecular and cellular techniques are used to measure specific biological effects and responses to drug candidates. While biochemical assays have been routinely used to measure compound activity and metabolism, these assays do not provide a clear picture of the physiological reactions of the compounds. This has led to the emergence of cell-based assays that closely mimic physiological reactions that occur in the context of the in vivo microenvironment.
Peripheral blood mononuclear cells (PBMCs) have been a popular model system that serves as a circulatory mirror of the in vivo physiological and metabolic activity of cells. Among the plethora of available primary cells, PBMCs and its individual subsets have enabled a broad spectrum of applications from in vitro cell-based assays to the monitoring of ex vivo changes before and after treatment. The applications span across basic discovery stages to preclinical and clinical stages, including direct monitoring of immune responses to therapeutic and vaccine strategies (Figure 1).
Consistency, reproducibility, and robustness of test systems are of primary importance when large amounts of data are collected from multisite longitudinal or cross-sectional studies designed for drug development. Traditionally, fresh PBMCs isolated from whole blood or leukopaks have supported most studies. Although these conventional protocols have their benefits, they do pose challenges and limitations from the perspective of consistency and continuity in sourcing of starting material and lot-to-lot variation, exacerbating variability in data points collected for a particular study.
Numerous reports in the literature have argued the benefits of using cryopreserved PBMCs in order to facilitate and improve upon several aspects of a study where longitudinal and/or cross-sectional data is routinely collected. Several researchers have demonstrated that cryopreserved cells are comparable to their fresh counterparts in terms of function, proliferation, and differentiation.
The results from our study specifically demonstrate that the ability to isolate and retain large batches of PBMCs from a single donor visit via apheresis methods, coupled with the use of optimized processing, cryopreservation, and recovery conditions, leads to well-preserved characteristics of cryopreserved PBMCs.
In this article, we present a snapshot of studies conducted at SeraCare Life Sciences on the cryopreservation and characterization of frozen PBMCs and on companion assays for evaluation of cellular function and for support of a number of other preclinical and clinical applications.
PBMCs were isolated from leukopaks collected from three healthy donors with differences in gender, demographics, and age, using a chemical-free procedure developed by SeraCare Life Sciences. These cells were processed and cryopreserved using stringently controlled methods and stored under liquid nitrogen vapor. Three cryopreserved vials from each donor were randomly selected and thawed using a standard protocol where adequate care was taken to prevent osmotic shock to the cells. The vials were tested for their percent recovery and viability, and proliferative and functional capacity.
Recovery and viability were measured using a standard protocol. For the proliferation assay, thawed PBMCs were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and incubated with or without phytohemagglutinin (PHA) for 72 hours according to a standard protocol. Cell divisions were measured at 0, 24, 48, and 72 hour time points. Briefly, cells were harvested at each time point and surface-stained with CD45 PerCP.
Events were acquired using the Beckman Coulter Quanta MPL Flow Cytometer by gating on the CD45 positive lymphocyte population that had transformed into the blast phase in response to PHA. The extent of cell division within this population was measured by calculating the division index (DI) based on the number of cell divisions identified by individual peaks of successive CFSE dilutions obtained at 72 hours in the presence of PHA. Cells incubated in the absence of PHA were used as controls.
By using our optimized protocol for thawing of cryopreserved PBMCs, each vial of PBMCs yielded close to 100% recovery of cells and greater than 90% viability with an overall average of 92% viability for all vials of PBMCs tested.
Figure 2 represents proliferation data with division indices from a single thawed vial of PBMCs selected from each of the three batches of cryopreserved donor PBMCs, at 72 hours post incubation in the presence or absence of PHA. Results demonstrate the robust response of cryopreserved PBMCs to PHA regardless of the inherent differences in donor characteristics. All of the cryopreserved PBMCs tested show more than three distinct cell division populations at 72 hours and a DI greater than 0.9.
Functional characteristics of the cryopreserved PBMCs were tested in an IFN-γ ELISpot assay using the SeraCare Life Sciences kit and protocol. PBMCs from three randomly selected vials from each lot were thawed and incubated in the presence of CMV and CEF peptide pools and PHA as a positive control for 18 hours at 37ºC in a CO2 incubator. Culture medium alone without any antigens was used as a negative control.
Results shown in Figure 3 represent the IFN-γ ELISpot responses of one of the PBMC lots to CMV and CEF antigenic peptide pools as compared to the negative control. The response to PHA was strong with more than 1,000 spot-forming cells per 200,000 PBMCs. Data from these experiments provides substantial evidence on the robustness of cryopreserved PBMCs with respect to fundamental properties of PBMCs such as viability, proliferative capacity, and functionality.
The use of standardized conditions for cryopreservation using specific media and well-monitored storage conditions in conjunction with the use of optimized thawing protocols leads to optimal preservation of fundamental characteristics of PBMCs. This enables adherence to critical requirements important in the design and execution of large-scale longitudinal or cross-sectional studies throughout drug development.
In addition, the availability of well-characterized cryopreserved PBMCs enables ex vivo testing of leads under varying conditions. The large batch sizes contribute to consistency and reproducibility of results and provide sufficient cells that can be used as controls pertinent to a particular study. Cyropreserved PBMCs and companion assays such as ELISpot and other cell-based assays comprise a unique toolbox to facilitate progress in preclinical and clinical stages of drug development.
Deepa Patke, Ph.D., is project scientist, Anis H. Khimani, Ph.D., is senior product manager, Christine Marking is research associate III, Renee M. Howell, Ph.D., is director of product development, and Mark Manak, Ph.D. ([email protected]), is chief scientist at SeraCare Life Sciences. Web: www.seracare.com.