Christian Freudigmann (Reutlingen, DE), Ellena Fuhrmann (Reutlingen, DE), Verena Singer (Reutlingen, DE), Jessica Pfannstiel (Reutlingen, DE), Dr. Hanna Hartmann (Reutlingen, DE)
Abstract text (incl. figure legends and references)
Introduction: A simple monitoring of cell viability is essential, to assess suitability of matrix materials for 3D cell culture and potential stress that cells are exposed.
Objectives: Establishment of the LDH assay as a quantitative tool for the evaluation of cell viability in 3D cultures.
Materials & methods: In this study NIH/3T3 cells were embedded into gels of two widely used bioinks, namely alginate and alginate/NFC and the activity of LDH released by cells with damaged cell membrane was measured using a commercial kit. To determine LDH activity of viable cells with intact cell membrane, a detergent-based lysis protocol was developed by using live /dead staining as readout. Release kinetics of LDH and its stability in solution under various temperatures were investigated as well as the limit of quantification of the assay. As a proof-of-concept, cell viability was determined after exposing them to different amounts of shear stress, comparing three mixing methods for gel embedding.
Results: After embedding cells into the hydrogels, LDH activity of dead cells with damaged cell membrane was successfully detected in the supernatant. By using 0.4 % Triton X-100, a lysis protocol to quantify viable cells was successfully developed. The maximum amount of actively released LDH was detected after 8 h, being stable for at least 24 h at 4 °C and 37 °C in cell culture medium or lysis buffer, respectively. The limit of quantification was 0.25 Mio dead cells/ml for alginate and 0.5 Mio cells/ml for alginate/NFC. Regardless of the mixing method used, cells in alginate/NFC showed a viability of about 80 %, whereas cells in alginate showed a viability of 30-50 %, depending on the mixing method used.
Conclusion: The LDH assay has been successfully established for assessing the viability of cells in bioinks and is a helpful tool for cell analysis in 3D-bioprinting.
This work received financial support from the Federal Ministry of Education and Research (13XP5071C).