Sep
28
Written by:
Emily Sherman
9/28/2010 7:24 AM
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Cryopreserved PBMCs: Quality Standards
A Path to Preserving Viability, Recovery, and Functionality
Vials of cryopreserved PBMCs
yielded 80-90% viability and
recovery post thaw.
Peripheral bloodmononuclear cells (PBMCs) are a popular, ex vivo model systemthat serve as a principal cellularmodel ormarkers of immune activity in a preclinical study or clinical trial follow ups.
Therefore, the applications using PBMCs in immune monitoring and immunotoxicity studies span through preclinical and clinical stages within the drug discovery and development paradigm(Figure 1). Consistency, reproducibility, and robustness are key when large amounts of data are collected from multi-site longitudinal or cross sectional studies designed for drug development.
Traditionally, fresh PBMCs isolated from whole blood or leukopaks are used inmost studies. Although the conventional methods have their merits, reproducibility and lot-to-lot consistency are compromised due to lack of continuity in procurement of starting material from the same source resulting in variability in data points collected for a particular study.
Published literature have described the benefits of cryopreserved PBMCs as part of several aspects of a study where longitudinal and/or cross sectional data are routinely collected. The studies have demonstrated that cryopreserved cells are comparable to fresh cells in function, proliferation, and differentiation.
Our studies specifically demonstrate that isolation and retention of large PBMC lots from a single donor via apheresis methods—combined with optimized processing, cryopreservation, and recovery conditions—results in well preserved characteristics of the cryopreserved PBMCs. Vials of cryopreserved PBMCs yielded 80-90% viability and recovery post thaw. In this article, we present selected studies conducted at SeraCare Life Sciences (www.seracare.com) on cryopreservation and characterization of frozen PBMCs.
By using our optimized protocol for thawing of cryopreserved PBMCs, each vial of PBMCs yielded 80-90% viability and recovery post thaw (data not shown).
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.
PBMCs were processed and cryopreserved using stringently controlled methods developed by SeraCare and stored under vapor-phase liquid nitrogen. Four (4) 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. Recovery, viability, and functional activity were then tested.
Recovery and viability were measured using a standard protocol developed at SeraCare.
Functional activity was assessed by testing the cell’s ability to proliferate in response to phytohemagglutinin (PHA) and to produce IFN-γ after specific antigen stimulation (Table 1a and b, respectively).
Proliferation response was measured by MTT colorimetric method. Three (3) vials of PBMCs from each donor were thawed and 50,000 cells were cultured in presence or absence of PHA for 96 hours according to a protocol developed by SeraCare.
Briefly, the MTT assay is a colorimetric assay that monitors the reduction of yellow 3-(4,5-dimethylthiazol- 2yl)-2,5-diphenyl tetrazolium (MTT) by mitochondrial succinate dehydrogenase. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, colored (dark purple) formazan product. The cells are then solubilized with an organic solvent (eg., isopropanol) and the released, solubilized formazan reagent is measured spectrophotometrically.
Data were analyzed and results were represented as a proliferation index (PI)—a ratio of absorbance readings of PHA-stimulated PBMCs and unstimulated PBMCs (Table 1a). Although we observed variability among different donors, tested PBMCs showed high proliferation capability.
Indeed, all of the cryopreserved PBMCs tested show PI values greater than five (5). Results demonstrated the robust response of cryopreserved PBMCs to PHA, regardless of the inherent differences in donor characteristics.
Functional characteristics of the cryopreserved PBMCs were also tested in an IFN-γ ELISpot assay using the SeraCare kit and protocol. Three randomly selected vials of PBMCs from one donor were thawed and incubated in the presence of CMV and CEF peptide pools for 18 hours at 37ºC in a CO2 incubator. PHA and medium alone were used as positive and negative control, respectively.
Results shown in Table 1b represent the IFN-γ ELISpot responses of one PBMC lot to CMV and CEF antigenic peptide pools, as compared to the negative control. The response to PHA was strong with an average of 828 ± 41 spot forming cells (SFCs) per 200,000 PBMCs .
Conclusions
The data presented here support the recoverability of cryopreserved PBMCs with respect to viability, proliferative capacity, and functionality. Standardized conditions such as specific media (SeraCare CryoMedia™), cryopreservation protocol, and well monitored storage conditions, along with the use of optimized thawing protocols, resulted in optimal preservation of fundamental characteristics of PBMCs. Optimization of each step within the process, followed by implementation of quality standards and related documentation within SeraCare’s Quality Management System(QMS), resulted in lot to lot consistency and functional performance of the cryopreserved cells.
About the Authors
Anis H. Khimani, Ph.D. (pictured) is Senior Product Manager; Tiziana diPucchio, Ph.D. is Principal Scientist (Immunology); Mark Manak, Ph.D. is Chief Scientist; and Kent Treichler is Director of Quality at SeraCare Life Sciences.
Address correspondence to ktreichler@seracare.com
Figure 1. PBMC Acquisition, Prep/Preservation, Recovery & Assays
Within the Drug Development Paradigm
Table 1. PBMC Quality Assessment by Measuring Functional Activity
Article Source: http://www.pharma-ii.com/enewsletter/pdf/Quality_Matters-Cryopreserved_PBMCs.pdf