Genome scale analyses include
Single cell and Single nuclei RNAseq on cell lines and organoids
Bulk and single nuclei ATACseq
Many cell types are found in cerebral organoids made from DO mouse ES cell lines
Plate based assays include
high content analysis (PE Opera)
RTCA impedance assays (ACEAbio)
microelectrode array (Axion Maestro)
Fl, Lum, hTRF etc. (PE Envision)
Astrocyte swelling kinetics, RTCA assay.
We often develop custom assays for our projects but for a recent project involving genetics of tyrosine kinase inhibitor induced cardiotoxicity we also built our own instrumentation. We needed to conduct beating assays for over 700K wells of cardiomyocytes derived from over 200 ESC lines. No commercially available instrument or assay met the throughput and cost constraints of the project so we designed and built a custom imager to meet our needs.
Fully automated high throughput beating cell imager
Using three instruments, our current screening capability is 10K wells per day, scalable to over 100K wells per day
Scans a 384 well plate in 20 minutes, capturing 20 seconds of beating per well
Real-time analysis of image data
Completely non-invasive and label-free assay
Uses conventional low-cost plasticware
Can measure beating in very small regions of the well
Uses mouse or human ES/IPS cell lines "off the shelf" without modification or purification, i.e., does not require purified cardiomyocytes
The video below shows a day 9 embryoid body in a single well of a 384 well plate. The motion is digitized by the imager, depicted as black dots in the panel on the right. The analysis software analyzes the motion and automatically places up to four ROI, (small red circles) at points of optimal beating.
Only the motion inside an ROI is quantified and graphed as shown below. This graph represents beating for ROI #1 in the video above. Each beat consists of two peaks, one for contraction and one for relaxation. A built-in peakfinding routine then catalogs the beats to calculate the beat-to-beat cycle time. The mean and coefficient of variation for beat score and beat cycle are also automatically calculated.
Contractile properties of cardiomyocytes are reflected in the Beat Scores (Y-axis). Arrhythmic responses result in altered mean and CV for beat cycle times. Contractile and arrhythmic activity are measured simultaneously.
Embroid bodies exhibit a high degree of variability and heterogeneity of cells and the location and size of cardiomyocyte patches are unpredictable. In our assay, very small beating patches are scored for beating, and any cells/activity outside of the ROI is ignored by the assay. This assay does not require synchronized beating across the entirety of the well surface. In fact, wells with multiple independent beating clusters can serve as in-well replicates.
Cisapride: lengthening beat cycle and fibrillation-like beating with increasing dose
Blebbistatin: inhibition of contractility but not rhythmicity
Sunitinib: effect on contractility is resolved from effect on beat cycle, and shows different potencies in different genetic backgrounds