Fig 1.
LSM and hydrogel microcarriers enable direct cell enumeration (DRAQ-5) and volume (CTG) quantification in toto.
Representative 2D LSM max. intensity projections of ih-MSCs attached to gelMA microcarriers at passage 4 day 3 (P4 D3), passage 4 day 7 (P4 D7), passage 7 day 3 (P7 D3), and passage 7 day 7 (P7 D7) using a-d) CTG fluorescence and e-h) elastic scattering contrast. i-l) DRAQ-5 labeled nuclei were used to estimate the m) normalized frequency of counted ih-MSCs attached to single gelMA microcarriers at P4 D3 (n = 30), P4 D7 (n = 20), P7 D3 (n = 23), and P7 D7 (n = 26). A zero-truncated Poisson distribution is fit over the sampled data (red line). Scale bar = 25 μm.
Fig 2.
The optical sectioning capabilities of LSM permits visualization into the hydrogel microcarrier.
a) 2D max. intensity and orthogonal projection of P7 D3 microcarrier with a single CTG-expressing cell. 2D max. intensity projections using the middle 1/3 of the volume to view the interior of the microcarrier using b) DRAQ-5, c) CTG, d) DiI plasma membrane stain, and e) elastic scattering contrast. Owing to the optical sectioning capabilities of LSM and refractive index matching of the microcarriers, biological features such as cell infiltration into the microcarrier can be visualized (white arrow). Scale bar = 25 μm.
Fig 3.
Large aggregates can be imaged and analyzed in toto using fluorescence and label-free LSM.
2D maximum fluorescence intensity Z-projections of ih-MSCs labeled with a) CTG, b) DiI, and c) DRAQ-5. (d) Elastic scattering also allows for visualization of cells within large aggregates. e) Small scatterers in agarose and cell debris can be segmented out based on intensity and/or size using Imaris. f) CTG + DRAQ-5 merge and g) elastic scattering + DRAQ-5 merge at higher resolution. Scale bar = 400 μm.
Fig 4.
Optical imaging and semi-automated image analysis enable rapid characterization of microcarrier-expanded cell growth in toto.
a) The overall average CPM at each sampled timepoint for all microcarriers sampled including aggregates from the DRAQ-5 data. b) The average single cell volume quantified by the fluorescence-based and the elastic scattering-based methods. Data are presented as mean with standard deviation (error bars). The DRAQ-5 labeled cell number versus the total cell volume quantified from the CellTracker Green fluorescence for all c) passage 4 and d) passage 7 samples. The DRAQ-5 labeled cell number versus the total cell volume quantified by the ELIAS method for all e) passage 4 and f) passage 7 samples. The linear trends and R2 values are shown.