Studying functionality and antigen-specificity of resident kidney T cells derived from a kidney biopsy is hampered by the lack of sufficient numbers of T cells obtained by the standard method of enzymatic tissue dissociation. Enzymatic dissociation of kidney tissue was compared to a novel method of whole kidney tissue culture allowing T cells to migrate into the medium in the presence of exogenous IL-2 and IL-15. T cell numbers were quantified and phenotype of resident T cells (CD69+CD103+/-), TCR Vβ repertoire and functional characteristics were analyzed with multi-parameter flow cytometry. Renal tissue culture for four weeks in the presence of exogenous IL-2 and IL-15 yielded significantly higher numbers of T cells (1.3 × 104/mm3) when compared to cultures without exogenous cytokines (71/mm3) or direct isolation by enzymatic dissociation (662/mm3 T cells, p < 0.05). The proportion of T cells with a resident phenotype did not change in the tissue culture; percentages amounted to 87.2% and 85.1%, respectively. In addition, frequencies of CD4+, CD8+, CD4-CD8-, T cells and MAIT T cells remained similar. For both CD4+ and CD8+, T cells had a more differentiated memory phenotype after tissue culture, but the distribution of TCR Vβ families did not change. In addition, the predominant Th1 cytokine secretion profile and poly-functionality of resident kidney T cell remained intact. T cell proliferation potential was not affected, excluding exhaustion and enrichment of BKV- and CMV-reactive resident T cells was observed. In conclusion, the kidney tissue culture method yields significantly increased numbers of resident T cells without major effects on composition and functionality.