A closed culture system for the ex vivo transduction and expansion of human T lymphocytes.

A phase I clinical trial is currently being performed at our institution, with the aim of evaluating the feasibility and toxicity related to the administration of herpes simplex thymidine kinase gene-expressing human primary T lymphocytes following allogeneic hematopoietic stem cell transplantation. The need for safe and standardized preparation conditions for gene-modified cells is crucial. We describe the closed culture system used in the current trial for ex vivo retroviral-mediated gene transfer and transduced cell selection. Cell handling is performed in closed systems using a sterile connection device that avoids opening the culture system. Cell numbers during the production process increased from 93 +/- 16 on day 0 to 440 +/- 92 x 10(6) on day 12 (7.2 +/- 1.4-fold increase) (n = 11). Transduction efficiency before and after G418 resistance-based selection was 13.5 +/- 3.8% and 90.0 +/- 1.4%, respectively. Safety and efficacy testing included a search for replication-competent retrovirus, endotoxins, Mycoplasma, and bacterial contamination (n = 0/9), PCR-DNA, % CD3+ cells (91 +/- 2%), and viability after thawing (82 +/- 3%). Effective working time from day 0 to day 12 is approximately 20 h. The closed system we developed allows for safe and reproducible ex vivo preparation of gene-modified primary T lymphocytes for clinical use.