Long-Term Electrical and Mechanical Function Monitoring of a Human-on-a-Chip System

Abstract

The goal of human-on-a-chip systems is to capture multi-organ complexity and predict the human response to compounds within physiologically relevant platforms. The generation and characterization of such systems is currently a focal point of research given the long-standing inadequacies of conventional techniques for predicting human outcome. Functional systems can measure and quantify key cellular mechanisms that correlate with the physiological status of a tissue, and can be used to evaluate therapeutic challenges utilizing many of the same endpoints used in animal experiments or clinical trials. Culturing multiple organ compartments in a platform creates a more physiologic environment (organ-organ communication). Here is reported a human 4-organ system composed of heart, liver, skeletal muscle and nervous system modules that maintains cellular viability and function over 28 days in serum-free conditions using a pumpless system. The integration of non-invasive electrical evaluation of neurons and cardiac cells and mechanical determination of cardiac and skeletal muscle contraction allows the monitoring of cellular function especially for chronic toxicity studies in vitro. The 28 day period is the minimum timeframe for animal studies to evaluate repeat dose toxicity. This technology could be a relevant alternative to animal testing by monitoring multi-organ function upon long term chemical exposure.

Keywords: 28-day; electrical function; mechanical function; multi-organ system; serum-free.

Figure 1.. Non-invasive technology to monitor cellular function in the 4-Organ system.

Schema of the microfluidic platform and the interface used to measure the mechanical and electrical functional activity. Electrical signals were recorded from the MEA chips (cMEA) utilizing a MultiChannel Systems MEA amplifier via a printed circuit board and an elastomeric connector (right). Cardiac and skeletal muscle contractile function was measured under stimulation with inserted electrodes from the cantilever chips (CL) using a laser-deflection based apparatus and a detector (left).

Figure 2.. Diagram of the pumpless microfluidic system with representative non-invasive mechanical and electrical functional readouts of the 4-Organ co-culture.

An acrylic housing holds PDMS gaskets that define the microfluidic pathway and the organ compartments along the path. Cells were cultured on their respective surfaces and compartments for 28 days. Representative images of hepatocytes on coverslip (compartment A (250 μm scale)), cardiomyocytes on cantilever (compartment B (250 μm scale)), and pattern on cMEA chips (compartment C (1000 μm scale)), skeletal muscle myotubes on cantilever chips (compartment D (250 μm scale)) and motoneurons pattern on cMEA chips (compartment E (1000 μm scale)) show each cell type in a different location in the system. Medium exchange was performed daily through both reservoirs, and the supernatant was used to quantify hepatic function (A). The contractile machinery of cardiomyocytes and myotubes was challenged on the cantilever chips using a laser-deflection based apparatus that recorded cantilever movement and a wave amplitude (B and D). The electrical signal of cardiomyocytes and motoneurons was recorded from the cMEAs connected to an amplifier via a printed circuit board and an elastomeric connector, translating current changes detected on the electrodes into field potential waveforms (C and E).

Figure 3.. Computational fluid dynamic simulation of the pumpless 4-Organ microfluidic system.

A transient model for gravity driven flow on the rocking system was developed onCFD-ACE+ (ESI Group, Paris, France) The shear stress profiles in the different organ chambers are shown as contour plots (spatially) and temporal plots over one flow cycle. The shear stresses for the liver chamber are computed over the entire chamber whereas only at the areas where the cells are patterned (center square) for the other organ chambers.

Figure 4.. Long-term hepatic characterization of the 4-Organ system under serum-free and flow conditions using non-invasive measurements.

Human primary hepatocytes were co-culture in the 4-Organs system (chamber A) under a serum-free medium (HSL3) and flow for up to 28 days. Weekly morphology images of hepatocytes in the system demonstrate good morphology (top). Hepatic urea (p<0.001) and albumin (p<0.05) daily production in the 4-Organ system is plotted as weekly averages along with a range (grey fraction) of previous reported literature values using human primary hepatocytes (urea^[–29] and albumin^{[, –30]}) (A-B). Hepatic cytochrome p450 1A1 (p>0.05) and 3A4 (p>0.05) activities in the 4-Organ system were evaluated at the endpoint (C-D). All functions were compared to hepatocytes in mono-culture (static or flow), confirming that hepatic function in the 4-Organ system is preserved compared to the mono-culture conditions. (100 μm scale).

Figure 5.. Long-term cardiac electrical characterization of the 4-Organ system under serum-free and flow conditions using non-invasive measurements.

Human iPSc derived cardiomyocytes pattern on custom MEA chips (chamber C) were co-culture in the 4-Organs system under a serum-free medium (HSL3) and flow for up to 28 days. Weekly morphology images of pattern cardiomyocytes on cMEA chip in the system demonstrate good morphology and stable pattern (top). Cardiac electrical parameters studied under spontaneous or stimulated conditions showed stable function over 28 days; beat frequency (p>0.05) (A), conduction velocity (spontaneous p>0.05, stimulated p>0.05) (B) and QT-interval (p>0.05), except for the mISI (p<0.001) (D). (100 μm scale).

Figure 6.. Long-term cardiac contractile characterization of the 4-Organ system under serum-free and flow conditions using non-invasive measurements.

Human iPSc derived cardiomyocytes on cantilever chips (chamber B) were co-culture in the 4-Organs system under a serum-free medium (HSL3) and flow for up to 28 days. Weekly morphology images of cardiomyocytes on cantilever chips in the system demonstrate good morphology (top). Contractile parameters studied under stimulated conditions showed stable cardiac function over 28 days; contractile force (p>0.05) (A), and time to peak (p<0.05) (B). (100 μm scale).

Figure 7.. Long-term skeletal muscle contractile characterization of the 4-Organ system under serum-free and flow conditions using non-invasive measurements.

Human skeletal muscle myotubes on cantilever chips (chamber D) were co-culture in the 4-Organs system under a serum-free medium (HSL3) and flow for up to 28 days. Weekly morphology images of myotubes on cantilever chips in the system demonstrate good morphology (top). Contractile parameters studied under stimulated conditions showed stable myotubes function over 28 days; contractile force (p>0.05) (A), and time to peak (p>0.05) (B). (100 μm scale).

Figure 8.. Long-term neuronal electrical characterization of the 4-Organ system under serum-free and flow conditions using non-invasive measurements.

Human motoneurons pattern on custom MEA chips (chamber E) were co-culture in the 4-Organs system under a serum-free medium (HSL3) and flow for up to 28 days. To improve cell stability on the neuronal MEA patterns, the incorporation of shield barriers at the two microfluidic entrances of chamber E were simulated and later experimentally tested with successful results at protecting the patterns, and thus enabling the electrical recording up to 28 days (A). Weekly morphology images of pattern motoneurons on cMEA chip in the system demonstrate good morphology and stable pattern (B). Neuronal electrical parameters studied under spontaneous conditions showed activity over 28 days; weekly average of spike wave forms (C), spike amplitude (p=0.3) (D) and spike rate (p=0.3) (E). (100 μm scale).