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 approac....

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:

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