T-Cell Culture Protocols

T-cells, which belong to a family of immune cells called lymphocytes, are generated during an adaptive immune response. T-cells originate from bone marrow stem cells and mature in the thymus. There are several different types of T-cells, each with a different function. T-cells are distinguished from each other by the attached T-cell antigen receptor. A subset of T-cells carries the CD4 marker and helps to induce an immune response.

Sources of T-Cells

• T-cells can be obtained from various sources, including the spleen, lymph nodes, and thymus, but peripheral blood is the most readily available source of T-cells for culture purposes. Blood samples are thoroughly screened for infections and other contaminants and carefully handled under a safety cabinet. T-cells are cultured for several reasons, including toxicity measurements and to study immunological vaccine responses.

Isolation

• T-cells are commonly separated from whole blood samples by density-gradient separation, as T-cells are less dense than red blood cells such as erythrocytes and therefore extracted by centrifugation. An appropriate neutral salt solution is added to the blood sample, followed by a Ficoll isopaque solution. Ficoll isopaque has a density greater than T-cells but less than erythrocytes and during centrifugation forms a middle layer between both cells, allowing T-cells to be removed from the top. The T-cells are further centrifuged, the supernatant is removed, and a small aliquot is counted to determine the cell viability. The cells are either used directly for experiments or cryopreserved for future use.

Cryopreservation

• Cryopreservation reagents are prepared in advance to avoid T-cell degradation at room temperature. If not possible, cells are kept on ice or refrigerated. Reagents consist of cell culture media and 10-13 percent human serum albumin (HSA) protein, as well as cell culture media and 10 percent Dimethyl Sulphoxide (DMSO). DMSO minimizes dehydration, pH changes, and electrolyte concentration changes associated with freezing. T-cells are re-suspended in the reagent-containing cell culture media and HSA in a cryotube. The reagent-containing DMSO is slowly added to the cells and gently swirled. The cells are then transferred to a freezing vessel containing isopropanol to ensure a gradual temperature decrease and stored.

Thawing

• When T-cells are thawed, a cryotube is removed from storage and incubated at 37 degrees Celsius. Subsequently, the appropriate thawing medium is slowly added to the cells until twice the volume is reached, transferred to a separate tube, and centrifuged. Benzonase is sometimes added to the thawing medium to prevent the clumping of dead T-cells. The centrifuged pellet is responded in cell culture media and the cell viability is assessed. The T-cells are then ready to use for respective experiments.