Bioprocess development for the cultivation of human T-lymphocytes

The ex vivo expansion of human T-lymphocytes is of increasing importance in medical applications, especially for the adoptive immunotherapy of viral i nfection and malignant diseases of immunosuppressed patients. In these the rapies, the cells of the patient (dendritic cells or lymphocytes for exampl e) are isolated from the blood and subsequently cultivated, expanded, and/o r genetically modified er vivo [1,2], Afterwards, these expanded or modifie d cells are transplanted back into the patient (Fig. 1). For example, patie nts with leukaemia are very susceptible to otherwise harmless infections li ke cytomegalovirus (CMV) after chemo- and radiotherapy or bone marrow trans plantation. The use of CMV specific cytotoxic T-lymphocytes can reduce the risk of such infections. These lymphocytes are cultivated together with ant igen presenting cells, leading to the proliferation of specific T-lymphocyt es which act against CMV. Peptides or proteins of the viral capsid can act as such antigens. For one medical application 2(.)10(9) specific cells are necessary. This am ount of cells can not be isolated from donated blood. Usually there are > 1 00 specific T-lymphocytes in one donation. In some cases (e.g. CMV) up to 1 0(5) specific cells can be found, but there is still a demand for cell expa nsion before therapy. It is very difficult to produce such an amount of cel ls with conventional cultivation techniques like tissue culture flasks or c ulture bags. There is also a permanent risk of contamination with microorga nisms. Therefore biotechnological methods are necessary which unable the cu ltivation of such an amount of cells under controlled conditions with a rep roducible course of events. A controlled change of culture medium, the proc ess control of pH, pO(2), and temperature, the easy sampling during cultiva tion and collection of cells after cultivation is of increasing importance. To produce clinical relevant numbers of specific T-lymphocytes. 70 tissue culture flasks (T75) would be needed. With regard to easy sampling during c ultivation and isolation of the cells before transplantation, we endeavour to produce cells in high density culture systems. The production of a relev ant number of cells is possible in one culture system with cell retention. There are different systems available, for example fed-batch processes or p erfusion systems with cell retention by filtration, or continuously centrif ugation. The aim of this investigation was to optimize the conditions for the select ive activation and cultivation of human T-lymphocytes in tissue culture fla sks starting with mononuclear cells (MNC). Afterwards a transfer of the opt imized culture conditions to a high density culture system is presented.