Development of a Multi‐Organ Microphysiological (MPS) Model to Study Age‐Related Comorbidities

Figures

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  FIGURE 1. Development of the multi‐organ system. (A) Layout of the pumpless microfluidic device developed for multi‐organ chips showing the position of each cell type on the device. Medium reservoir 1 serves as the inlet for fresh medium while reservoir 2 serves as the outlet for spent medium. (B) Representative cantilever and MEA readouts. (C) Close‐up image of MEA electrodes with patterned cardiac cells. (D) Graphic showing cantilever mechanism. (E) Representative phase images of each cell type cultured onto cantilevers (SKM and Cardiac), MEAs (cardiac), and coverslips (hepatocytes) in a multiorgan system. Scale bar: 100 µm (unless mentioned otherwise).

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  FIGURE 2. Development and characterization of the sarcopenic phenotype. (A) Timeline for induction of sarcopenia in skeletal muscle using TNF‐α, (B) Representative phase images for changes in myotube width for control and sarcopenic conditions, (C) Comparison of skeletal muscle width over a period of 28 days after culture. Data were normalized to the healthy condition in the same experimental batch on the corresponding days (n = 6 coverslips from 3 experiments). (D) Representative images for comparative ROS accumulation in control and sarcopenic muscle (n = 6 coverslips from 3 experiments). (E) Comparative ROS concentration in control and sarcopenic conditions (n = 6 coverslips from 3 experiments). Data were normalized to the healthy condition in the same experimental batch. (F) A significant increase in skeletal muscle contraction force was observed on days 14, 17, 21, and 28 after culture (n = 9 systems from 3 experiments). Data were normalized to the healthy condition in the same experimental batch on the corresponding days. Data represents: Mean +/‐ SEM. Student's t‐Test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 100 µm.

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  FIGURE 3. Development and characterization of cardiomyopathy phenotype. (A) Timeline for induction of hypertrophic cardiomyopathy in cardiac cells using Phenylephrine (PE). (B) Increased troponin levels for hypertrophic cardiomyocytes with representative images for cardiac troponin T (cTNT) staining (n = 120 images from 12 coverslips, p‐value = 9.99533e(‐06). (C) Representative phase images for an increase in cell width for the HCM condition, the graph indicates a significant increase in cardiomyocyte width for HCM cells (n = 6 coverslips from 3 experiments, p‐value= 0.001595). (D) Both healthy and HCM cells were tested on cantilevers (n = 9 systems from 3 experiments) to measure cardiomyocytes' contraction force and beat frequency. A highly significant increase in cardiac force was noted for HCM cells on days 5, 7, 9, and 14, with p‐values 3.03834e(‐11), 1.77653e(‐07), 7.04072e(‐06), and 1.68674e(‐07), respectively. (E) No significant difference from the control in the beat frequency. All HCM data were normalized to healthy conditions in the same batch on corresponding days, and Student's t‐test was performed on all data. Data represents: Mean +/‐SEM. **** p < 0.0001. Scale bar: 100 µm.

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  FIGURE 4. Analysis of sarcopenia phenotype in the multi‐organ system. (A) Representative phase images for skeletal myofibers in cantilevers on day 14. (B) The skeletal myofibers were tested on cantilever systems to measure muscle contraction force. The skeletal muscle force for the diseased (sarcopenic) was significantly smaller than the healthy skeletal muscle (n = 15 systems from 5 batches). The force was normalized to healthy muscle force, and a Student's t‐test was performed. There was a significant decrease on all four days, 5, 7, 9, and 14, with p‐values 1.3058e(‐09), 9.1154e(‐06), 0.00014, and 3.46e(‐06), respectively. Data represents: Mean +/‐SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 100µm.

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  FIGURE 5. Analysis of the hypertrophic cardiomyopathy phenotype in the multi‐organ system. All data were normalized to the healthy condition within the same batch on corresponding days (n = 15 systems from 5 batches). Data represents: Mean +/‐SEM, and a Student's t‐test was performed. (A) Increases in cardiomyocyte contraction force for HCM on all four testing days (day 5 p = 0.0316, day 7 p = 0.0032, day 9 p = 0.0204, and day 14 p = 0.0004). (B) Reduced cardiac beat frequency on day 5 (p = 0.0407). (C) An initial increase was observed for conduction velocity that fell off with time. (D) An upward trend was observed for QT interval with a significant increase on days 5 and 14 (p = 0.0210, 0.0012, respectively). (E) Representative phase images of cardiac cells on cantilevers on day 14. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Scale bar: 100µm.

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  FIGURE 6. Analysis of hepatocyte function in the multi‐organ systems (n = 15 systems from 5 batches). (A) Representative phase image for hepatocytes on day 14 indicating good cell coverage. (B) A non‐significant decrease in the normalized CYP3A4 activity was observed in diseased systems, indicating a potential disease burden on hepatocytes. The CYP3A4 data were normalized to hepatocyte MTT viability from the same coverslip and further normalized to the healthy systems within the same experimental batch. (C) Relative albumin production shows no significant difference between healthy and diseased groups. (D) Results obtained for urea production showed no significant difference. Data represents: Mean +/‐SEM. Scale bar: 100µm.

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  FIGURE 7. Alamar blue was used as a cell viability test on day 14 after disassembly (n = 15 systems from 5 batches). No significant difference in cell viability was observed for all three cell types: hepatocytes (A), cardiomyocytes (B), skeletal muscle (C). Data represents: Mean +/‐SEM.