Isolated immune cell populations support a wide variety of research and cell therapy applications. Because purification and preliminary characterization of immune cell subsets can be time-consuming and costly, many researchers prefer to purchase isolated immune cells rather than raw materials.
The quality of the starting cellular material directly impacts the downstream efficacy of the final product. This is particularly pertinent when working with cryopreserved starting cellular materials. Certain cell types are more sensitive than others to the cryopreservation process, which should be optimized to preserve maximum cell viability. This includes eliminating any granulocyte contamination as it is detrimental to the quality of mononuclear cells (MNCs) from apheresis and bone marrow-derived products.
THE IMPACTS OF GRANULOCYTE CONTAMINATION
Unfortunately, researchers often fail to consider two of the most critical factors impacting cryopreserved cell quality:
- The amount of granulocyte contamination present.
- The length of storage time in which isolated cells are exposed to their presence.
Granulocyte contamination has multiple impacts on starting material quality. Unlike most cells, granulocytes are poorly cryopreserved in standard freezing media, and release DNA and lysosomal enzymes upon thawing, causing clumping and damage to other cells (1).
Generally, density gradient centrifugation is used to separate the peripheral blood MNCs and immune cell precursors from the heavier cellular components, including granulocytes and red blood cells. Over time, however, granulocytes can become activated, and degranulation is accompanied by a loss in granulocyte cell density, resulting in less efficient separation from the MNC population by density gradient procedures and increased risk of co-purification with white blood cells (2).
There are two intertwined processes at work here. Purification efficiency dictates the overall percentage of granulocyte contamination and the risk of immune cell loss and damage. Storage time is equally important. The longer immune cells are exposed to granulocytes prior to processing, the greater the risk that granulocyte activation will impact immune cell viability and function.
A study has shown that delays between blood collection and processing of more than 8 hours at room temperature or hypothermic temperature, significantly affect the performance of white blood cells in immunological assays (2). The overall immune cell integrity is compromised with the NK cells showing a decreased ability to degranulate and secrete cytokines (3) and the response of dendritic cells and monocytes to Toll-like receptors being depressed (4). There is also a direct relationship between adverse events during infusion of autologous peripheral blood stem cells for transplant and the number of granulocytes in the leukapheresis product (5).
The function of T cells, which are used for several different cell therapy treatment strategies, can be severely impacted by activated granulocytes. Granulocyte contamination and activation due to longer blood storage periods reduces the relative number of T cells present in white blood cell populations and inhibits T cell proliferation (2). Contaminating granulocytes have also been reported to be associated with cellular integrity loss of regulatory T cells (6).
HEMACARE BEST PRACTICES MITIGATE GRANULOCYTE CONTAMINATION
The good news is that proper precautions can mitigate all of these various impacts. At HemaCare, we leverage years of cell isolation and processing expertise to safeguard the viability and functionality of our isolated immune cell products.
Because the time interval between blood collection and processing is a critical parameter for optimal white blood cell function, HemaCare isolated immune cell products are processed, purified, and cryopreserved on-site on the same day they are collected. This avoids lengthy storage times as well as granulocyte contamination and activation.
Same-day cell isolation and cryopreservation eliminate the need for multiple freeze-thaw cycles, preserving cell quality and stability. Granulocyte populations are removed from the outset using optimized techniques such as density gradient centrifugation for MNC purification or immunomagnetic cell separation to isolate targeted cell types. In addition, our customers can choose between positive or negative cell selection to suit their anticipated application.
HemaCare cell isolation and processing service offers:
- Optimized same-day cell isolation and cryopreservation protocols
- Pure isolated cellular populations free of granulocyte contamination
- Stringent quality standards
- Rigorous QC testing
- Guaranteed cell counts
- Significant savings when isolating cells from full leukopaks
In summary, cryopreserved leukopaks can be necessary as starting materials for process development and research programs where multiple cell types and populations are being examined. However, without optimal quality oversight, the presence of granulocytes can negatively affect the stability and functionality of target cells. For this reason, many researchers and cell therapy developers are now making the switch to isolated immune cell starting materials as an alternative to raw materials.
Let HemaCare supply the quality and expertise you need while you focus on your project–visit our website today to learn more about our isolated immune cell products!
- Bakken AM. Cryopreserving human peripheral blood progenitor cells. Curr Stem Cell Res Ther. 1(1):47-54. Jan 2006.
- McKenna K. C., et al. Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function. J Immunol Methods. 341(1-2):68-75. Feb 2009.
- Naranbhai V., et al. Impact of blood processing variations on natural killer cell frequency, activation, chemokine receptor expression and function. J Immunol Methods. 7; 366(1-2):28-35. Mar 2011.
- Meier A., et al. Rapid loss of dendritic cell and monocyte responses to TLR ligands following venipuncture. J Immunol Methods. 31; 339(2):132-40. Dec 2008.
- Cordoba R., et al. The occurrence of adverse events during the infusion of autologous peripheral blood stem cells is related to the number of granulocytes in the leukapheresis product. Bone Marrow Transplantation. 40; 1063–1067. 2007.
- Agashe C., et al. Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells. J Immunol Methods. 449; 23-27. Oct 2017.