Comsol Drug Delivery Simulation

Screening Applications

Anurag Mathur1,2, Peter Loskill1,2, Kaifeng Shao1, Nathaniel Huebsch4,5, SoonGweon Hong1, Sivan G. Marcus1, Natalie Marks1, Mohammad Mandegar4,5, Bruce R. Conklin4,5, Luke P. Lee1,3

& Kevin E. Healy1,2

Berkeley, California 94720, USA, 4Gladstone Institute of Cardiovascular Disease, San Francisco, California 94158, USA, Drug discovery and development are hampered by high failure rates attributed to the reliance on non-human animal models employed during safety and efficacy testing. A fundamental problem in this inefficient process is that non-human animal models cannot adequately represent human biology. Thus, there is an urgent need for high-content in vitro systems that can better predict drug-induced toxicity.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Systems that predict cardiotoxicity are of uppermost significance, as approximately one third of safety-based pharmaceutical withdrawals are due to cardiotoxicty. Here, we present a cardiac microphysiological system (MPS) with the attributes required for an ideal in vitro system to predict cardiotoxicity: i) cells with a human genetic background; ii) physiologically relevant tissue structure (e.g. aligned cells); iii) computationally predictable perfusion mimicking human vasculature; and, iv) multiple modes of analysis (e.g. biological, electrophysiological, and physiological). Our MPS is able to keep human induced pluripotent stem cell derived cardiac tissue viable and functional over multiple weeks. Pharmacological studies using the cardiac MPS show half maximal inhibitory/effective concentration values (IC50/EC50) that are more consistent with the data on tissue scale references compared to cellular scale studies. We anticipate the widespread adoption of MPSs for drug screening and disease modeling.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

T

he current drug development process is inefficient and expensive. The average time to develop and launch a new drug is 10–15 years, and the average cost is approximately 5 billion USD1,2. Preclinical development and clinical studies account for 63% and 32% of this cost, respectively3. A significant limitation in the restricted predictability of preclinical screening is that it relies heavily on animal models and animal derived cell lines. Due to inter-species differences in ion channels, biological pathways, and pharmacokinetic properties4 animal models fail to fully recapitulate human physiology, and thus in many cases do not faithfully predict human cardiotoxicity.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Simple high throughput in vitro assays play a crucial role in assessing the safety of cardiac drugs by monitoring, for instance, currents through ‘hERG’ ion channels due to their role in causing life-threating arrhythmias5. Yet, the heavy reliance on simplified model cell lines (e.g., HEK293, CHO) overexpressing hERG ion channels is risky.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

These assays produce false positives (Verapamil) and false negatives (Alfuzosin)6, putting human lives at risk and resulting in a waste of valuable resources during clinical drug development. To mimic the anisotropic structure of the cardiac ventricles, Grosberg et al.7 employed mouse embryonic stem cell derived cardiomyocytes (CMs) to make a 2D anisotropic cardiac muscle tissue by microfabrication of fibronectin layers. While they successfully showed that stimulated CMs cultured in serum aligned in the 2D structure and generated contractile force, the model is not scalable to generate a 3D thick volume tissue. Using the same cell line but a different approach, Boudou et al.8 created freestanding 3D tissue constructs and examined the effect of various drugs. Besides issues arising from the lack of biomimicry, the applicability of both systems for drug testing are limited due to the use of animal cells. With the discovery of patient-specific human induced pluripotent stem cells (hiPSCs)9,10, it is now possible to engineer human in vitro systems amenable for drug screening. Advances in generating stem cell derived human cardiomyocytes offer an unprecedented opportunity to create systems, which are better predictors of drug toxicity. In recent years, considerable progress has been

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made on the use of static 2D dish culture with human stem cell derived CMs11–14. However, the predictability of such systems is still limited, as they do not recapitulate the human in vivo structure and organization of the myocardium. To address this other groups intro- duced multiple types of 3D tissue constructs based on different meth- ods and cell types (hESC, hiPSCs)8,15–18. However, all of them are restricted to the use of a static bath to deliver the nutrients and drugs, hence do not provide microcirculation like the vasculature, which includes the capability to continuously exchange the nutrients, con- stant exposure of the tissue to fresh drug in the plasma, and the convective-diffusive oxygen/nutrient transport.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

We hypothesized that combining the genetic background of human cells with appropriate biophysical tissue architecture and ‘‘tis- sue-like’’ drug gradients would recapitulate a minimal organoid of the human myocardium sufficiently to allow accurate prediction of the cardiotoxicity of drugs. To test this hypothesis, we have developed a microphysiological system (MPS) that: i) uses 3D confinement with biomimetic dimensions to facilitate a self-organization of hiPSC derived cardiomyocytes into an aligned 3D m-tissue; and, ii) mimics the shear flow protection of the endothelial barrier. We applied this system to test the cardiac response with four model drugs. Our results suggest that this hiPSC-derived cardiac-MPS could significantly improve the ability to prognosticate on drug efficacy and toxicity in vitro, reducing both the cost and duration of bringing a new drug candidate to market.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Results

To mimic a minimal organoid structure of the heart we have developed a cardiac microphysiological system (MPS) (Fig. 1 a–b) based on three major concepts: a) aligned three-dimensional tissue structure, inspired by the human myocardium by recapitulating the geometry of the perimysial collagen fiber spacing in the human heart (width 100–200 mm)19; b) microcirculation mimicking the in vivo transport, which includes continuous exchange of nutrients, con- stant exposure of the tissue to fresh drug compounds, and removal of metabolic waste products; and, c) shear flow protection of the tissue and controlled diffusive transport to the tissue.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

The major components of the device are a central cell chamber, two adjacent media channels, and arrays of connecting microchan- nels. The media channels (width 30–40 mm) recapitulate the va- sculature and allow for a precise and computationally predictable delivery of nutrients and drugs in microfluidic perfusion chambers.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

The cell chamber is connected to the media channels by 2 mm wide microchannels, which mimic endothelial barriers separating the car- diac microtissue from perfusion medium, protecting the tissue itself from shear forces, but allowing nutrient delivery via diffusion from the ‘vascular’ channels. The narrow cross-section of these micro- channels creates a total fluidic resistance into the cell culture area 10 times greater than through the media channel, and a convection/ diffusion ratio (Peclet number) of 693 in the flow channel and <0 in the barrier channels. Thus, the transport from the media channels to the cell chamber is purely diffusive (Fig. 1c), featuring diffusion properties characteristic of the endothelial barrier for humans in vivo. The diffusive transport was verified by performing fluorescence recovery after photobleaching (FRAP) experiments on the MPS (Supplementary Movie 1), yielding a characteristic diffusion time of 2.5 s for 4 kDa FITC-dextran (Fig. 1d).

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

To create a robust and reproducible hiPSC derived cardiac micro- tissue in the cell chamber, we relied on a consistent, well-character- ized, high yield, and high-efficiency cardiomyocyte differentiation protocol (Supplementary Fig. 1). This was achieved by modifying a hiPSC-CM differentiation protocol introduced by Lian et al.20 Using optimized procedures for cell singularization and device loading, hiPSC derived CMs (hiPSC-CMs) on days 15–20 of the CM differ- entiation were introduced into the MPS at a high packing density while keeping pressure and stresses low (Supplementary Movie 2).

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Cells were loaded at this stage to achieve robust CM commitment without excessive buildup of extracellular matrix (ECM). Additionally, this early injection time point promoted the formation the cardiac tissue without use of either exogenous ECM or synthetic matrices.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

The hiPSC-CMs formed a 3D cardiac tissue in serum-free media within 24 hours of loading, which started to beat spontaneously with robust and homogenous beating at physiological beat rates of 55– 80 bpm without any external stimulation (Fig. 2a, Supplementary Movie 3). Within 7 days of formation, the beating became aligned in an uniaxial manner and consisted of multiple cell layers, as

Microscopy

(Fig. 2b). Computational motion-tracking of the beating velocity indicated the beating was aligned along the long axis of the MPS (Fig. 2 c– h). Aligned 3D cell structure and consistent physiological beating are crucial prerequisites for a cardiac MPS, since functionality is dictated by structural alignment of the cells. Drug response, for instance, is strongly dependent on the initial spontaneous beat rate of the tissue21 and the electromechanical activity of the heart is governed by the intracellular connections and 3D alignment of the cardiac tissue22,23.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

The MPS system is highly scalable and presents the opportunity to model human genetic disease via the use of patient-derived or gen- ome-edited hiPSC-CMs in a rapid and cost effective manner. For example, we used hiPSC-CMs derived from a genetically engineered hiPSC line expressing a GCaMP6 reporter to monitor Ca21 cur- rents21, which provides the ability to collect electrophysiological data on a high-content basis using simple optical microscopy, as shown in Fig. 3 and Supplementary Movie 4.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Figure 1 | The cardiac microphysiological system (MPS). (a) Schematic of the MPS nutrient channels (red) and cell-loading channel (green). (b) Scanning electron micrograph of the MPS showing in inset the 2 mm endothelial-like barriers connecting the nutrient channel and the cell channel. Red rectangular box shows the weir that enables efficient and consistent loading of singularized high density hiPSC-CMs. (c) Schematic of the cardiac MPS showing nutrient inlet and outlet ports connected to tubes. (d) Simulated velocity profile of flow in the MPS, inset shows the magnified view. Note the lack of convection within the diffusive barriers and predominant convective flow through the nutrient channels. Thus, mass transport to the tissue is exclusively diffusive. (e) Diffusion dynamics in the microphysiological system. Normalized fluorescence recovery of 4 kDa FITC–dextran (0.2 mg/mL). Insets are confocal microscopy images corresponding to the FRAP experiment. Finitial is the time regime that corresponds to the initial fluorescence before bleaching; F0 is the fluorescence measurement immediately after photobleaching; Ft . 0 corresponds to the recovery of fluorescence after photobleaching; Ft ? 0 corresponds to maximal recovery of fluorescence at the end of the experiment. Scale bar: 10 mm.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

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To validate the drug response of the cardiac MPS we tested two pharmacological agents, Isoproterenol (b-adrenergic agonist) and E-4031 (hERG blocker), and two clinically used drugs with known clinical effects, Verapamil (multi-ion channel blocker) and Metoprolol (b-adrenergic antagonist) (Table 1). We chose these compounds since they represent four different important classes of pharmacological agents with well-characterized clinical responses. To characterize drug responsiveness, we used beat-rate as a metric. Due to the controlled dimensions and the alignment of the engineered cardiac tissue formed in our MPS, we obtain consistent and spontaneous beat rates on the order of 55–80 beats per min, allowing us to make sensitive, repro- ducible measurements regarding how drug treatment affects beat rate.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Furthermore, the pharmacological agents tested in this study have a known effect on the beat rate. Hence, mechanical motion is an import- ant method to analyze behavior in a non-invasive manner without the use of any synthetic dyes or complex electrophysiological measurements.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Our results demonstrate good concordance with clinical observa- tions. Administration of Isoproterenol increased the beat rate with an EC50 of 315 nM (Fig. 4a and Supplementary Movie 5 and 6). This

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Figure 2 | Characterization of the 3D cardiac tissue in the microphysiological system (MPS). (a) Optical microscopy image of a cardiac tissue in the MPS. (b) Confocal fluorescence microscopy imaging of the microtissue in the MPS reveals an aligned multiple cell layer thick structure. Inset shows a magnified view of the sarcomeric alpha actinin and DAPI staining. (c, d) Heat map of the time-averaged beating motion and corresponding average beating kinetics in the MPS, (e, f) along the channel axis, and (g, h) perpendicular to the channel axis.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

Figure 3 | Cardiac tissue derived from a genetically engineered hiPS cell line expressing a GCaMP6 reporter in the MPS. (a) Frames from a fluorescence movie (GFP channel) showing the switching from dim to bright during activity of Ca21 channels. (b) Time-course of the normalized fluorescence intensity (green) and the beating motion (red) obtained by computational analysis of the movie. This combination allows high throughput analysis of mechanical and electrophysiological properties.

Figure: System Model & Simulation Flow for Comsol Drug Delivery Simulation

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