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Cytomatrix |
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In vitro generation of functional human T lymphocytes in an artificial three dimensional matrix (Cellfoam) |
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T.P.H. Meyer, M. Poznansky*, D.T. Scadden*, M. Pykett, M. Rosenzweig;
Cytomatrix, Woburn, MA, 01801 and Massachusetts General Hospital*, Boston, MA, 02114 |
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The in vitro generation of T lymphocytes has an absolute dependency on thymic elements as well as a three dimensional structure. We have developed a culture system that incorporates an inert, three dimensional matrix, termed Cellfoam (CF), that together with adherent thymic cells facilitates the in vitro differentiation of human hematopoietic progenitors into mature T cells. Thymus tissue from 4-6 week old BALB/c mice was cultured in CF until a confluent murine stromal cell network was established. Freshly isolated human bone marrow CD34+ (or AC133+) cells were then co-cultured on the mouse thymus in the absence of cytokines until developing T cells were observed to migrate out of the CF (about 2 weeks). Flow cytometry revealed that those non adherent cells included CD4+/8+ double positive, as well as CD4+/8- and CD4-/8+ single positive cells. Molecular analysis (RT-PCR) of T cells generated in vitro after 14 days revealed elevated levels of RAG-2 expression and TRECs, both associated with and indicative of successful T cell receptor rearrangement during T cell maturation. To specifically test the function of these T cells we performed in vitro antigen stimulation. Incubation with various antigens (cmv, measles, candida) or the mitogen Con. A resulted in elevated levels of the cytokines IL-2 and INF-g as determined by intracellular staining and flow cytometry. After 6h stimulation, intracellular IL-2 could be detected in 7-10% of the generated CD4+ T cells. INF-g was detected in both CD4+ and CD8+ cells. Using both phenotypic and functional evaluation of these cells, we have demonstrated that human T cells can be effectively generated from bone marrow progenitor cells in the context of a three dimensional microenvironment. |
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