Stimulation of CHO-­‐K1 Cell Growth by Mitoplicator™

A single stimulation with nanosecond pulsed electrical fields

will increase cell proliferation of CHO-­‐K1 cell line in vitro.

INTRODUCTION

CHO cells are a most widely used cell line in R&D, scale up and manufacturing of (therapeutic) biologicals. Here we provide proof of concept, that a single treatment using the ECPR technology can stimulate the proliferation rate of the CHO-­‐K1 cell line.

RESULTS

CHO-­‐K1 cells were trypsinized, seeded, allowed to adhere and then treated in the Mitoplicator™ instrument (figure 1) for 5 min. Paired control cultures were kept on a heated plate aside the instrument and underwent all handlings in parallel with treated cultures. Controls and treated monolayers were incubated, after which cultures were trypsinized, collected and counted using Burker-­‐Turk counting chambers.

As shown in figure 2, treated CHO-­‐K1 cells proliferate faster than control cultures. After 2 days of incubation cell counts for treated cells showed (125 ± 5; n = 10) percent growth over controls and after three days of culture the difference increased to (141 ± 4; n = 15) percent growth

Treated & control cells displayed identical morphologies under the light microscope.

CONCLUSION

ECPR technology can accelerate the growth rate of the CHO-­‐K1 in vitro and thereby shows the potential to improve the cycle time between seeding and harvesting of cells for biomolecule production. We are now aiming at expanding this principle to larger scale cultures, including fermentor-­‐scale.

METHODS

Cells. CHO-­‐K1 (ATCC CCL-­‐61) were cultured following ATCC guidelines1 in F-­‐12 medium (Gibco, 21765-­‐037) with 10% FBS (Gibco 10500-­‐064) and 1% sodium pyruvate (Gibco, 11360-­‐039). Cells were passaged twice weekly (1000 cells/cm2) and while harvested 50-­‐80% confluent. 3ml of trypsinized, resuspended CHO-­‐K1 cells were seeded (at 625 or 1250 cells/cm2) in 10ml tubes with a 6cm2 optically clear surface. Cells were allowed to adhere overnight prior to stimulation.

Stimulation. Prior to Mitoplicator treatment, tubes were coded. The handling before, during & after Accelerator treatment was as previously described2 and in accordance with the CHO-­‐K1 ATCC culture guidelines. Mitoplicator treatment conditions were 5 minutes at 50Hz. Following treatment, tube lids were opened and tubes placed in the incubator.

Cell counting. Monolayers were trypsinized, collected and cells were gently resuspended in medium containing 10% FBS. The number of cells was counted using a Burker-­‐Turk counting chamber. Cell counting was done blind and in duplicate. The average of duplicates was recorded. Codes were broken after cell count results were recorded.

Data handling. Six independent experiments were executed serially. Each experiment included triplicates or quadruplicates of control and treatment. Within each experiment, controls and treated cultures were ranked by cell count result and paired in rank order. The percentage growth over controls was derived for the ranking-­‐matched pairs. The clusters in figure 2 reflect the cell counts of the replicates from the six independent experiments.

ABOUT MITOPLICATOR™ & ECPR TECHNOLOGY

The Enhanced Cell Proliferation Reactor (ECPR) technology was discovered, validated & developed by the Vabrema founders since 2009 and in close collaboration with the Eindhoven Technical University (TU/e).

Today, ECPR technology is available for R&D purposes as the Mitoplicator™ instrument and for fermenter-­‐scale cell or protein production (assay/engineering project).

Proof of principles for ECPR technology includes mammalian & human cell lines, primary cells & isolates, stem cells, plant seedlings, algae. Vabrema actively pursues an R&D plan, aiming at widening ECPR applicability.