The use of T cells is increasing both in healthcare and in research yet the preservation methodologies for longer periods of times are yet to be optimized. In order to overcome these issues, we have optimized a protocol in sample handling and preservation of T cells in order to perform a successful donor homologous co-culture with DCs and preserve these cells for subsequent testing. This method will help in saving time and effort as well as the ease of use for experiments requiring use of T cells in mono or co-cultures. Handling and preservation of T cells using our methodology showed stability and viability of these cells in co-cultures. Data showed viability of >93% before and after liquid nitrogen preservation. Moreover, preserved cells had no unspecific activation which can be seen in unchanged expression of the T cell activation marker CD25. T cell proliferation profile showed that preserved T cells used in DC-T cell co-cultures (LPS stimulated DCs) had the ability to interact and proliferate indicating potency of these cells. This provides evidence of the efficiency of our handling and preservation methodology in maintaining cell viability and stability. Preserving donor T cells would facilitate reuse of these cells in donor homologous co-cultures reducing inconvenience of multiple donations of fresh blood and provides accessibility of the same population of T cells for experiments that requires repetition, commercial availability of the cells or for preservation of cells for clinical therapies such as chimeric antigen receptor T cells.