A Cardiac Microphysiological System for Studying Ca2+ Propagation via Non-genetic Optical Stimulation

Summary

Using light to control cardiac cells and tissue enables non-contact stimulation, thereby preserving the natural state and function of the cells, making it a valuable approach for both basic research and therapeutic applications.

Abstract

In vitro cardiac microphysiological models are highly reliable for scientific research, drug development, and medical applications. Although widely accepted by the scientific community, these systems are still limited in longevity due to the absence of non-invasive stimulation techniques. Phototransducers provide an efficient stimulation method, offering a wireless approach with high temporal and spatial resolution while minimizing invasiveness in stimulation processes. In this manuscript, we present a fully optical method for stimulating and detecting the activity of an in vitro cardiac microphysiological model. Specifically, we fabricated engineered laminar anisotropic tissues by seeding human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) generated in a 3D bioreactor suspension culture. We employed a phototransducer, an amphiphilic azobenzene derivative, named Ziapin2, for stimulation and a Ca²⁺ dye (X-Rhod 1) for monitoring the system's response. The results demonstrate that Ziapin2 can photomodulate Ca²⁺ responses in the employed system without compromising tissue integrity, viability, or behavior. Furthermore, we showed that the light-based stimulation approach offers a similar resolution compared to electrical stimulation, the current gold standard. Overall, this protocol opens promising perspectives for the application of Ziapin2 and material-based photostimulation in cardiac research.

Introduction

The use of light for stimulating living cells and tissues is emerging as a significant game-changer in biomedical research, offering touchless stimulation capabilities with precise temporal and spatial resolution^1,2,3,4,5,6. One of the leading techniques used to make cells sensitive to light is optogenetics, which involves genetically modifying cells to express light-sensitive ion channels or pumps^7,8. This approach has demonstrated impressive effectiveness in regulating cells within living tissue; however, its reliance on viral gene transfer has hindered its widespread adoption in research and clinical applications.

To overcome this limitation, organic and inorganic materials have been used as light-sensitive transducers to develop non-genetic, material-based light-mediated stimulation techniques^9,10. Organic nanostructured phototransducers^{11,12,13,14,15} have recently demonstrated remarkable success in triggering cellular responses across diverse applications, including neurons, cardiomyocytes, and skeletal muscle cells.

Herein, we propose Ziapin2^16,17,18, an azobenzene derivative, for investigating Ca²⁺ propagation in engineered laminar cardiac tissues. The amphiphilic structure of the molecule allows for precise targeting of the cell plasma membrane, while the azobenzene core enables light-induced isomerization, leading to its conformational change^16,17,18. In cardiac cells, this trans-to-cis isomerization alters the plasma membrane thickness, inducing a cascade of effects that generates an action potential, which in turn triggers the excitation-contraction process^19,20,21.

Additionally, we describe the fabrication process of an engineered platform for the anisotropic growth of cardiac tissue²² and detail the experimental setup used for optically triggering and monitoring its activity, with a particular focus on acquiring Ca²⁺ dynamics within the tissue^23,24. Finally, we compare the acquired signals with those obtained through electrical stimulation, which is considered the reference standard. Overall, this protocol highlights the application of a novel light-responsive transducer in advancing our understanding of cardiac cellular behavior, especially in the context of engineered tissues.

Protocol

The human pluripotent stem cell (hiPSC) culture used is a wild-type human male iPSC line that harbors a doxycycline (Dox)-inducible CRISPR/Cas9 system, created by introducing CAGrtTA::TetO-Cas9 into the AAVS1 locus (Addgene: #73500). The study was conducted in accordance with protocols approved by the Boston Children's Hospital Institutional Review Board. Informed consent was obtained from patients prior to their participation in the study. The generation of hiPSC-derived cardiomyocytes (hiPSC-CMs) was induced as previously described^25,26. The protocol will be briefly summarized in the following section:

1. Generation and preparation of human induced pluripotent stem cell-derived cardiomyocytes

1. Wash hiPSCs being cultured in T75 flasks once with PBS. Detach the cells by incubating with the chelating agent Versene for 10-15 min and seed into bioreactor vessels pretreated with 1% solution of non-ionic surfactant at a density of 50 million IPSCs/vessel in 100 mL of E8 medium supplemented with 10 µM ROCK inhibitor Y-27632.
2. Set the following bioreactor parameters: agitation 60 rpm, temperature 37 °C, pH 7, and overlay gassing (O₂ and CO₂) at 3 standard L/h.
3. After 1 day in culture, confirm that the embryoid body (EB) diameter has reached 100-300 µm and treat the cells with basic RPMI medium (RPMI 1640 medium supplemented with B27 minus Insulin), containing 7 µM CHIR99021 for 24 h, followed by media change to RPMI for another 24 h. Add basic RPMI medium containing 5 µM IWR-1-endo for another 48 h to start the differentiation process towards the cardiac lineage. After 48 h, change the medium back to basic RPMI.
4. On day 7 of differentiation, culture cells in a basic RPMI medium supplemented with 1:1,000 (v/v) human insulin. Refresh the media every 2 days or every day with a 50% medium change if oxygen consumption reaches more than 30%.
5. On day 15, enzymatically dissociate the bioreactor cells for 3 h using a Collagenase II solution containing HBSS, Collagenase II (200 units/mL), HEPES (10 mM), ROCK inhibitor Y-27632 (10 µM), and N-benzyl-p-toluenesulfonamide (BTS, 30 µM). To do this, first wash the differentiated hiPSC-CM EBs 2x with prewarmed HBSS and incubate them in Collagenase II solution (200 µl volume of EBs / 12 ml of Collagenase II solution) at 37 °C in 5% CO₂ until single cells appear from full EB dissociation.
6. Stop the dissociation reaction by adding an equal volume of blocking buffer (RPMI-1640 without B27 and DNase II, with 6 µL of DNase II per mL of RPMI-1640) to the single-cell suspension. Count the differentiated hiPSC-CMs, centrifuge (200 × g, 5 min), and prepare for downstream applications by freezing the cells at -80 °C in isopropanol for 24 h and moving them to liquid nitrogen tanks till seeding is performed.

2. Engineered laminar tissue fabrication

1. Adhere two layers of laboratory tape, one white and one blue, to a 1 mm-thick clear, scratch- and UV-resistant acrylic sheet. Using a CO₂ laser engraver, cut the acrylic sheet in circles (20 mm diameter to fit the 12 MW) and the chip pattern (3 x 7.5 mm squares, three per chip) on the tape as designed in vector graphics software (e.g., CorelDraw); acrylic cutting parameters: 100 power, 20 speed, and 1,000 PPI; tape cutting parameters: 8 power, 6 speed, and 1,000 PPI. Remove the two layers of tape inside the innermost line using tweezers, soak the chips in pure bleach for 30 min to 1 h to remove thick lines and dark spots from cutting, leaving a sharp line, and rinse the chips in a beaker with running deionized (DI) water overnight or for at least 3 h.
   NOTE: Do not bleach for longer than 2 h, as this will etch the adhesives on the sidewalls, preventing gelatin from adhering properly.
2. Sonicate the chips for 10 min and the polydimethylsiloxane (PDMS, Sylgard 184) stamps with line groove features (25 µm ridge width, 4 µm groove width, and 5 µm groove depth) for 30 min in clean 70% ethanol. Transfer the chips and stamps to a clean area under a hood and allow them to dry under airflow for ~1-2 h.
3. Weigh 1 g of gelatin from porcine skin (gel strength ~175 g Bloom, Type A) into a 50 mL tube. Add 5 mL of phosphate-buffered saline (PBS) and mix thoroughly to ensure immediate dissolution of the gelatin. Place the tube in a 65 °C water bath for 30 minutes to complete the dissolution process. Similarly, weigh 1 g of microbial transglutaminase (MTG) into a separate 50 mL tube. Add 12.5 mL of PBS, mix thoroughly, and place the tube in a 37 °C water bath for 30 minutes to ensure full dissolution of the MTG.
4. Sonicate the gelatin tube for 15 min, then return it to the 65 °C water bath before use. Place the MTG tube in a desiccator with the cap slightly loosened and turn on the vacuum slowly. Observe the bubbles being removed from the MTG solution under vacuum. After degassing, return the MTG tube to the 37 °C water bath.
   NOTE: Monitor the process and adjust the vacuum to avoid vigorous boiling.
5. Cover a grid sheet with clean parafilm, place the chips on the grid, and keep the stamp nearby, ready for use. Add 5 mL of MTG to the 5 mL of gelatin solution, pipetting carefully to avoid bubbles; then, quickly pipette the gelatin onto the chip, using enough to cover the chip area (approximately 0.5 mL each). Then, place the line-patterned PDMS²² (line width 25 µm, height 5 µm, spacing 4 µm) stamp on top and apply a 200 g weight to ensure the gelatin is patterned parallel to the tissue's longitudinal axis. Once all chips are molded, cover them with a glass jar to avoid environmental disturbance and let them crosslink overnight.
   NOTE: Use the mixed solution gelatine-MTG within 5 min; otherwise, the target pattern will be altered due to a higher crosslinked state of the gelatine.
6. Transfer the chip and PDMS stamp sandwich to a new P150 dish filled with PBS and hydrate the gelatin for 30 min to 1 h to facilitate the separation of the PDMS stamp from the chip. Remove any excess unmolded gelatin around the chip and transfer the clean chip to a new P150 dish filled with PBS. Store the PDMS stamps in 70% ethanol.
7. Sterilize the chips by soaking them in ethanol for 10 min under the hood.
   NOTE: Do not exceed 10 min in ethanol, as longer exposure may deform the gelatin.
8. Transfer the chips to PBS, soak for 10 min, and rinse 3x. Prepare coating solutions by mixing 20 µg/mL fibronectin with 1:100 diluted basement membrane matrix in culturing media (approximately 0.5 mL per well). Coat the chips for 2 h in the incubator at 37 °C and 5% CO₂ or at 4 °C overnight.
9. Thaw and seed cells (8 × 10⁵ hiPSC-CMs per cm²) in RPMI medium with Y-27632 (10 µM), then replace it with RPMI without Y-27632 after 24 hours.
10. Three days after cell seeding, remove the white tape using tweezers.

3. Synthesis and application of the phototransducer

NOTE: Ziapin2 was synthesized according to a previously published procedure^16,18 and was administered to hiPSC-CMs directly in the culturing medium.

1. Add 25 µM of Ziapin2 to the cell culture and incubate at 37 °C and 5% CO₂ for 7 min.
2. Gently wash out excess molecules and rinse with fresh culturing medium.

4. Viability assay

NOTE: Alamar Blue is a resazurin-based assay that can permeate cells and act as a redox indicator to monitor cell viability. Resazurin dissolves in physiological buffers, resulting in a deep blue solution that is added directly to cells in culture. Viable cells with active metabolism reduce resazurin to resofurin, which is pink and fluorescent.

1. Plate cells in a 96-well plate previously coated with 20 µg/mL fibronectin and 1:100 diluted basement membrane matrix in culture media. Mix by shaking and then aseptically add Alamar Blue to each well in an amount equal to 10% of the well volume.
2. Incubate cultures with Alamar Blue for 4 h.
3. Treat cells with the phototransducer and vehicle (DMSO) as previously described. Apply light stimulation (1 Hz pulsed light for 1 min) through an optical fiber (cyan, 470 nm) to assess the effect of Ziapin2 internalization and light exposure on hiPSC-CM viability.
4. Read fluorescence with a plate reader at excitation 560 nm and emission 590 nm.

5. Assessment of engineered laminar cardiac tissue anisotropy

NOTE: This protocol outlines a systematic approach for assessing the anisotropy of engineered laminar cardiac tissue using immunostaining, confocal microscopy, and nuclei analysis²⁷.

1. Wash the substrate with PBS several times before fixation with 4% (v/v) paraformaldehyde containing 0.05% (v/v) Triton X-100 in PBS for 10 min.
2. Block non-specific binding by incubating samples with 5% (w/v) bovine serum albumin (BSA) in PBS for 30 min.
3. Incubate samples with Hoechst (1:500) at room temperature for 1 h.
4. Wash with PBS before mounting onto microscope slides with an anti-fade agent. Allow slides to dry overnight and store at 4 °C until imaging.
5. Image samples using a spinning disk confocal microscope at 20x magnification. Determine the orientation of the cells using OrientationJ Measure²⁸, a FiJi plugin.

6. Optical mapping recordings

NOTE: Optical mapping was performed after 5 days in culture on hiPSC-CMs seeded on gelatin-molded tissue chips.

1. To follow this protocol, ensure that the optical mapping apparatus consists of a modified tandem-lens microscope equipped with a high-speed camera and the excitation light source, a 200 mW Mercury lamp. Place a dichroic mirror in front of the designated Ca²⁺ imaging camera to ensure that the preparation is exposed to excitation light and to collect the emitted fluorescence coming from the sample. 
2. Incubate the sample with 2 µM X-Rhod 1 added to the culturing medium for 30 min at 37 °C.
   NOTE: Expose the sample to light only during image acquisition to avoid photobleaching of the dye.
3. Incubate the phototrasducer as previously described in section 3.
4. Wash with fresh culturing medium and transfer the chips to phenol red-free RPMI 1640 medium supplemented with B27 minus Insulin.
5. Place the tissue chips in a temperature-controlled dish and start recordings at physiological temperature (37 °C), acquiring images at a frame rate of 2.5 frames/s.
6. For optical pacing, apply the optical point stimulation at one end of the tissue using an LED light source (465/25 nm) allowing Ziapin2 stimulation. Pace the tissues at 0.5 or 1 Hz frequency via a temporally regulated optical fiber positioned 1 mm away from the tissue. For electrical pacing, apply a point stimulation using a bipolar platinum electrode positioned at the center of the most distal end of the rectangular tissue. This placement ensures that Ca²⁺ wave propagation originates from the midpoint of the tissue's width, which corresponds to the shorter side of the rectangle. The stimulation should be applied at a frequency of 0.5 Hz, with an amplitude of 10 V and a pulse duration of 100 ms.
7. After the recordings, apply a spatial filter with a 3 x 3 pixel size to improve the signal-to-noise ratio.
8. Determine the Ca²⁺ wave conduction velocity at each pixel for every pulse, calculating the rate of change along both the x- and y-directions. To do this, measure the spatial gradient of the Ca²⁺ signal intensity in these directions and relate it to the time delay of the signal's propagation. By combining these directional change rates, quantify how quickly the wave travels across the cellular field in both dimensions.

7. Data export and handling

1. Extract and analyze the data with the referenced software.
2. Apply a spatial filter of 3 x 3 pixels to improve the signal-to-noise ratio.
3. Extract the Ca²⁺ transients (CaT) traces as the mean of a specific manually selected region of interest (ROI).
4. Measure the CaT parameters in a central region of interest (ROI) of 50 x 50 pixels (≈ 25 mm²).
5. For each CaT, analyze CaT amplitude, rise time (t_peak), time of maximum decay slope (Max Decay Slope), and time to 90% transient decay (Decay Time₉₀).

8. Statistical analysis

1. Assess the normality of the distribution using an appropriate statistical test, such as a normality test, to determine whether parametric or non-parametric methods should be applied.
2. Evaluate statistical significance between two conditions using the Student's t-test or Mann-Whitney U-test for continuous or categorical data, respectively. When comparing more than two groups, use one-way ANOVA or the Kruskal-Wallis test depending on data assumptions, followed by appropriate post-hoc tests to identify significant pairwise differences.

Results

A multistep process was developed and implemented for the fabrication of engineered laminar cardiac tissue using a combination of laser patterning, gelatin molding, and cell seeding techniques. Originally established by McCain et al.²² and Lee et al.²⁴, this technique was re-implemented, following their protocols to construct the engineered laminar microtissues. The process integrates precise laser-based patterning for structural guidance, gelatin as a scaffold material, an...

Discussion

This approach provides a robust platform for advancing cardiac research, providing insights into the complex dynamics of cardiac tissue opening up new possibilities for long term in vitro cardiac mechanistic studies that could potentially lead to new therapeutic strategies. To ensure the success of this methodology, it is crucial to reproduce a microphysiological environment that closely mimics in vivo conditions of the human heart. Therefore, careful attention must be given to designing and aligning th...

Materials

Name	Company	Catalog Number	Comments
alamarBlue Cell Viability Reagent	Thermo Fisher Scientific	DAL1025	Cell Viability Assay
B-27 Supplement, minus insulin	Thermo Fisher Scientific	A1895601	For cell culture
Bovine Serum Albumin	Sigma-Aldrich	A9056-50G	For cell staining
BrainVision Analyzer software	Brain Products	https://www.brainproducts.com/downloads/analyzer/	Data export and handling
BTS	Sigma	203895-5MG
CHIR99021	Stem Cell Technologies	72054
Clear Scratch- and UV-Resistant Acrylic Sheet, 12" x 12" x 0.01 inch	McMaster Carr	4076N11	Tissue chip fabrication
Collagenase Type II	Worthington	CLS-2 / LS004176
DNase II	VWR	89346-540
Essential 8 Medium	Thermo Fisher Scientific	A1517001	For cell culture
Fibronectin	VWR	47743-654	Coating
Gelatin from porcine skin gel strength 175 Type A	Sigma-Aldrich	G2625-100G	Tissue chip fabrication
Geltrex LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix	Thermo Fisher Scientific	A1413302	Coating
HBSS	Thermo Fisher	14175-095
HEPES (1 M)	Thermo Fisher Scientific	15630080
Hoechst 33342	Life technologies	H1399	For cell staining
Insulin solution human	Sigma Aldrich	I9278-5ML
IWR-1-endo	Stem Cell Technologies	72564
Paraformadehyde 16% Aqueous Solution (PFA)	VWR	100503-917	For cell staining
PBS, sterile, 500 mL	Thermo Fisher Scientific	10010049	Tissue chip fabrication
phosphate buffered saline	Thermo Fisher Scientific	10010049
Pluronic F-127 (20% Solution in DMSO)	Thermo Fisher Scientific	P3000MP	Non-ionic surfactant
ROCK inhibitor Y-27632	Stem Cell Technologies	72304
RPMI 1640 Medium, GlutaMAX Supplement	Thermo Fisher Scientific	61870127	For cell culture
RPMI 1640 Medium, no phenol red	Thermo Fisher Scientific	11835030	Optical mapping
Versene Solution	Thermo Fisher Scientific	15040066	chelating agent
VWR General-Purpose Laboratory Labeling Tape	VWR	89098-058	Tissue chip fabrication
X-Rhod-1 AM	Thermo Fisher Scientific	X14210	Optical mapping