A human neural stem cell line, HK532, isolated from a fetal cortex and conditionally immortalized (US Patent No. 7544511) was further genetically modified by exposing the cells in culture to replication-incompetent recombinant lentivirus expressing EGFP under the human ubiquitin C promoter. The resulting cells were washed free of the transducing virus, further propagated for several passages, and stored frozen to create working stocks. This cell line had been used previously to over-express another potentially therapeutic protein, human IGF-1, using similar method19 (link). One day prior to the transplantation surgery, the frozen cells were thawed, washed several times, concentrated to the target cell density of 50 k cells/µL in a proprietary cell suspension medium, and transported to the surgery site overnight by a commercial carrier. Once received, the cell viability was checked by trypan blue exclusion method and found to be 88% viable. The cells were stored on ice until the time of injection and used without further manipulation20 (link).
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