Production of retroviral vectors in continuous high cell density culture.

Retroviral vectors derived from murine leukemia virus (MLV) are used in somatic gene therapy applications e.g. for genetic modification of hematopoietic stem cells. Recently, we reported on the establishment of a suspension viral packaging cell line (VPC) for the production of MLV vectors. Human embryonic kidney 293-F (HEK293-F) cells were genetically modified for this purpose using transposon vector technology. Here, we demonstrate the establishment of a continuous high cell density (HCD) process using this cell line. First, we compared different media regarding the maximum achievable viable cell concentration (VCC) in small scale. Next, we transferred this process to a stirred tank bioreactor before we applied intensification strategies. Specifically, we established a perfusion process using an alternating tangential flow filtration system. Here, VCCs up to 27.4E + 06 cells/mL and MLV vector titers up to 8.6E + 06 transducing units/mL were achieved. Finally, we established a continuous HCD process using a tubular membrane for cell retention and continuous viral vector harvesting. Here, the space-time yield was 18-fold higher compared to the respective batch cultivations. Overall, our results clearly demonstrate the feasibility of HCD cultivations for high yield production of viral vectors, especially when combined with continuous viral vector harvesting. KEY POINTS: • A continuous high cell density process for MLV vector production was established • The tubular cell retention membrane allowed for continuous vector harvesting • The established process had a 18-fold higher space time yield compared to a batch.

Novel suspension retroviral packaging cells generated by transposition using transposase encoding mRNA advance vector yields and enable production in bioreactors.

To date, the establishment of high-titer stable viral packaging cells (VPCs) at large scale for gene therapeutic applications is very time- and cost-intensive. Here we report the establishment of three human suspension 293-F-derived ecotropic MLV-based VPCs. The classic stable transfection of an EGFP-expressing transfer vector resulted in a polyclonal VPC pool that facilitated cultivation in shake flasks of 100 mL volumes and yielded high functional titers of more than 1 × 106 transducing units/mL (TU/mL). When the transfer vector was flanked by transposon terminal inverted repeats (TIRs) and upon co-transfection of a plasmid encoding for the transposase, productivities could be slightly elevated to more than 3 × 106 TU/mL. In contrast and using mRNA encoding for the transposase, as a proof of concept, productivities were drastically improved by more than ten-fold exceeding 5 × 107 TU/mL. In addition, these VPC pools were generated within only 3 weeks. The production volume was successfully scaled up to 500 mL employing a stirred-tank bioreactor (STR). We anticipate that the stable transposition of transfer vectors employing transposase transcripts will be of utility for the future establishment of high-yield VPCs producing pseudotype vector particles with a broader host tropism on a large scale.

Simple assessment of viability in 2D and 3D cell microarrays using single step digital imaging.

Simple and rapid imaging and analysis of 2D and 3D cell culture compatible with miniaturized arrays of nanoliter droplets are essential for high-throughput screening and personalized medicine applications. In this study, we have developed a simple one-step, cost-effective and sensitive colorimetric method for the analysis of cell viability in 2D and 3D cell cultures on a nanoliter droplet microarray. The method utilizes a flatbed document scanner that detects a color change in response to cell metabolism in nanoliter droplets with high sensitivity in a single step without the need for expensive specialized equipment. This new nanoliter-based method is faster and more sensitive than equivalent methods using multi-well plate assays. The method detects quantifiable signal from as few as 10 cells and requires only 5 min. This is 2.5 to 10-fold more sensitive and 12 times faster than the same assay in multi-well plates. The method is simple, affordable, fast and sensitive. It can be used for various applications including high-throughput cell-based and biochemical screenings.