Get Cross-Network PBMC Processing Standard Operating Procedure - Hanc

Because erythrocytes and granulocytes are much more fragile to freezing and thawing, PBMC isolation is a common prerequisite to cryopreservation of blood cells; and the most common method for PBMC isolation is density gradient centrifugation using Ficoll-, a high molecular weight carbohydrate solution.

The frequencies of cell types within the lymphocyte population include 70 – 85% CD3+ T cells (45 – 70% of PBMC), 5 – 20% B cells (up to 15% of PBMC), and 5 – 20% NK cells (up to 15% of PBMC). The CD3+ compartment is composed of CD4 (25 – 60% of PBMC) and CD8 T cells (5 – 30% of PBMC), in a roughly 2:1 ratio.

Peripheral blood mononuclear cell (PBMC) isolation is commonly done via density gradient centrifugation over Ficoll-, a labor-intensive procedure that requires skilled technicians and can contribute to sample variability.

PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells.

The most common PBMC isolation method involves using a density gradient medium (e.g. Ficoll™ or Lymphoprep™) and centrifugation. This method takes advantage of the differences in density between the cells in blood and the density gradient medium.

This chapter deals with T lymphocytes derived from peripheral blood mononuclear cells (PBMCs). T cells comprise, approximately, 45–70 % of PBMCs in human peripheral blood, with a count in healthy subjects in the range of 1 million cells/mL.

The PBMC isolation protocol involves directly separating lymphocytes from whole blood. Whole blood is the blood that flows through the human body — the raw fluid with no components removed or separated. This is composed primarily of plasma, platelets, red blood cells, and PBMCs.

To obtain T cells for CAR-T cell production, blood is collected and leukapheresis is performed to isolate and extract peripheral mononuclear blood cells (PBMCs, e.g., leukocytes and lymphocytes). The T cells in this PMBC fraction are then separated out using cell processors or centrifugation-based cell separators.