Volumetric imaging of human mesenchymal stem cells (hMSCs) for non-destructive quantification of 3D cell culture growth

Abstract

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. The use of spherical microcarriers, however, precludes in-process visualization and monitoring of cell number, morphology, and culture health. The development of novel expansion methods also motivates the advancement of analytical methods used to characterize these microcarrier cultures. A robust optical imaging and image-analysis assay to non-destructively quantify cell number and cell volume was developed. This method preserves 3D cell morphology and does not require membrane lysing, cellular detachment, or exogenous labeling. Complex cellular networks formed in microcarrier aggregates were imaged and analyzed in toto. Direct cell enumeration of large aggregates was performed in toto for the first time. This assay was successfully applied to monitor cellular growth of mesenchymal stem cells 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 presented study motivates the development of on-line optical imaging and image analysis systems for robust, automated, and non-destructive monitoring of bioreactor-microcarrier cell cultures.

Comparison of polystyrene and hydrogel microcarriers for optical imaging of adherent cells

“…The software accepts refractive index (n) inputs for the immersion medium and particle, particle size, illumination wavelength, and illumination polarization. The polystyrene particles were modeled as a sphere with 160 µm diameter and n equal to 1.59 [54], and the gelMA microcarriers were spheres with 120 µm diameter and n equal to 1.35 [47]. Cells were simulated to have a diameter of 10 µm and n equal to 1.45 [55], [56].…”

“…Passage 4 ihMSCs were first expanded in low-density monolayer cell culture in CCM to obtain the required cell numbers. As previously described elsewhere [47], the ihMSCs [52] were cultured in a rotating wall vessel (RWV) bioreactor (RCCS-8DQ bioreactor, Synthecon) at 24 RPM on custom 120 ± 6.2 µm diameter gelMA microcarriers at 1000 cells/cm 2 [46]. Specimens were suspended in 1 mM concentration of CellTracker Green (CTG) (Thermo Scientific) for 45 minutes, then fixed with 4% PFA, and stored in PBS.…”

“…We have previously demonstrated the advantages of gelMA microcarriers for in toto imaging-based quantitative monitoring of cell density and morphology of microcarrier-based cell cultures [47]. The combination of volumetric optical imaging, via planar illumination methods, and water-based hydrogel microcarriers provides the ability to rapidly and non-invasively observe the entire 3D microcarrier culture surface area, as is routinely performed for monolayer cultures [47], [48]. Light-sheet fluorescence microscopy (LSFM) is a fast and photo-gentle camera-based imaging method that utilizes fluorescence contrast for image formation.…”

“…Unfortunately, LSFM requires exogenous labels for fluorescence contrast, or higher laser power for autofluorescence imaging, both of which can be toxic for live organisms [49]. Light-sheet tomography, however, presents a powerful imaging modality based on label-free elastic scattering contrast for rapid and direct visualization of individual cell morphology on microcarriers and thus enables robust non-invasive and non-destructive monitoring of cell culture critical quality attributes [47]. Since elastic scattering contrast relies on refractive-index mismatches in the sample, highly scattering microcarriers are expected to preclude the visualization of cells on their surface.…”

Scalable manufacture of therapeutic mesenchymal stromal cell products on customizable microcarriers in vertical wheel bioreactors that improve direct visualization, product harvest, and cost

“…This group previously described an approach to fabricate customizable, degradable gelatin methacryloyl microcarriers (GelMA-M) using a step-emulsification microfluidic device [ 42 ]. The microcarriers exhibited improved optical characteristics as compared to their polystyrene counterparts [ 43 ] and simplified cell harvest by complete digestion of the microcarriers. When cultured on GelMA-M in vertical wheel bioreactors (VWB) at 100 mL scale, the ihMSCs retained in vitro trilineage differentiation potential and immune modulatory capacity [ 42 ].…”