The adoption of cell-based therapies into the clinic will require tremendous large-scale expansion to satisfy future demand, and bioreactor-microcarrier cultures are best suited to meet this challenge. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed using fluorescence and elastic scattering contrast. This assay was successfully applied to monitor cellular growth of induced pluripotent derived mesenchymal stem cells (iH-MSCs) attached to spherical hydrogel microcarriers over time. Elastic scattering and fluorescence lightsheet microscopy were used to quantify cell volume and cell number at varying spatial scales. The fluorescence-based assay is used as ground truth data to correlate the elastic scattering-based assay data.
Fig 1e Fixed passage 4 day 3 ih-MSC on a gelMA microcarrier
ih-MSCs were cultured in a rotating wall vessel (RWV) bioreactor (RCCS-8DQ bioreactor (Synthecon, Houston, TX) fitted with 10 mL high aspect ratio vessels on custom-fabricated 120 ± 6.2 μm diameter gelMA microcarriers. For this purpose, approximately 110,000 gelMA microcarriers with a combined growth area of 50 cm2 and 5×104 cells (1000 cells/cm2) were incubated in 10 mL of CCM in the RWV bioreactor at 24 revolutions per minute. Half of the media was replaced with fresh CCM every 2 days. iH-MSCs fixed with 4% paraformaldehyde and fluorescently labeled with CellTracker Green (green), DRAQ-5 (blue), and DiI (gold) in the cytoplasm, nuclei, and plasma membrane, respectively. Label-free elastic scattering (white) and trasmitted light (grey) are also used to visualize the sample.
Passage 4 Day 3 single cell imaged with elastic scattering contrast and illuminated with 638 nm
