Isochoric supercooled preservation and revival of human cardiac microtissues

Figures

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  Fig. 1. Cardiac microphysiological system and isochoric supercooling protocol. a A glass-PDMS microfluidic device featuring two independent MPS. b Details of the microfluidic features including media inlet (IN) and outlet (OUT) as well as the cell loading port (cell in), cell chamber with anchor pillars, media channels, and connecting fenestration channels. c Cardiac microtissue after cellular self-assembly within the cell chamber. d Example fluorescence traces of a genetically encoded calcium channel reporter (GCaMP) showing spontaneous and externally triggered (1.25 Hz) beating activity before cryopreservation as well as disturbed and restored tissue function after cryopreservation. The width of the calcium transient at 30% and 80% peak amplitude was measured as a proxy for action potential duration (APD30 and APD80, respectively). e Isochoric supercooling enables the preservation of biological matter in a metastable ice-free condition at temperatures below the freezing point of water/physiological saline. Isochoric conditions are achieved by confining the preservation solution in a high-rigidity container totally absent of bulk gas phase and denying it access to the atmospheric…

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  Fig. 2. Recovery rate and comparison of key cardiac functionality parameters pre- and post- isochoric supercooling. Statistical difference between groups (p < 0.05) is marked by horizontal lines with asterisks. All raw data points are shown within violin plots. a Total recovery rate of preserved tissues as a function of preservation time. Gray bars indicate tissues that presented some activity post-isochoric supercooling, but were not coherent over the entire tissue (i.e., only part of the microtissue recovered). Scatter markers represent the weighted means of individual experiments, and marker diameter reflects the number of tissues (total sample sizes, i.e., number of independent cardiac tissues, for each condition: 24 h: n = 39; 48 h: n = 36; 72 h: n = 17). Error bars mark the standard error of the mean (SEM) across experimental groups, weighted by the total number of tissues evaluated in each group. These error bars reflect the SEM of the total surviving tissue population, i.e., the combined population of fully recovered and partially recovered tissues. b Confocal microscopy image of cardiac microtissue before and after 24 h supercooled preservation (α-actinin in red and nuclei in…