Assessment of culture substrate functionalization in a stretchable cardiac culture platform
Board Location: #120
Discipline: Technology and Engineering
Subcategory: biomedical
Session: 3
Genevieve Vega - University of Texas at San Antonio
Co-Author(s): Claudia E. Varela, Boston University, Boston, MA
Myocardial infarction (MI) occurs when blood flow to a heart muscle region is disrupted leading to cardiomyocyte (CM) death. This results in permanent impairment of heart function. While it is known that altered biomechanics in the non-infarcted muscle contribute to remodeling after MI, the exact mechanisms are unknown [1]. To study how aberrant mechanical forces impact CM biology, we adapted an in vitro platform to enable pathological loading (over-stretching) of human induced pluripotent stem cell-derived (iPSC) CMs. However, how different ECM coatings or CM alignment cues influence the system has not been investigated. Here, we investigate how a surface coating of matrigel and fibronectin, as well as microcontact printing of fibronectin, impact CM attachment and alignment in our in vitro platform. Devices were casted from polydimethylsiloxane (PDMS), plasma treated, assembled, and baked overnight. Matrigel (12.5 µL/mL) or fibronectin (10 µg/mL) solutions were deposited or micropatterned with 20 µm fibronectin lines spaced 20 µm apart on the PDMS devices. After incubation, iPSC-derived CMs [2] were seeded and cultured for at least 3 days, exposed to 4-5 h of cyclic stretch using a custom platform and a 3D printed stretch adapter, and fixed for immunohistological assessment. Cell alignment and attachment of stretched and micropatterned samples were quantified using ImageJ. Our results demonstrate that different ECM coatings, such as matrigel and fibronectin yield comparable cell attachment and alignment, regardless of exposure to cyclic stretch. This suggests both ECM coatings are suitable for the in vitro platform. Additionally, fibronectin micropatterning appeared to direct the alignment of CMs as indicated by the alignment of sarcomeres along the fibronectin lines. These findings will aid in optimizing our in vitro culture model so it can be utilized to study the role of mechanical forces in CM biology in the infarcted heart, potentially enabling the development of post-MI remodeling interventions.
References: 1.- Calcagno, D. M., Taghdiri, N., Ninh, V. K., Mesfin, J. M., Toomu, A., Sehgal, R., Lee, J., Liang, Y., Duran, J. M., Adler, E., Christman, K. L., Zhang, K., Sheikh, F., Fu, Z., & King, K. R. (2022). Single-cell and spatial transcriptomics of the infarcted heart define the dynamic onset of the border zone in response to mechanical destabilization. Nature Cardiovascular Research, 1(11), 1039–1055.
2.- Lian, X., Zhang, J., Azarin, S. M., Zhu, K., Hazeltine, L. B., Bao, X., Hsiao, C., Kamp, T. J., & Palecek, S. P. (2012). Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nature Protocols, 8(1), 162–175.
Funder Acknowledgement(s): This work is supported by NSF Award # 1647837 - Nanosystems Engineering Center for Directed Multiscale Assembly of Cellular Metamaterials with Nanoscale Precision: CELL-MET, REU Supplement. A special thanks to Dr. Chen, Dr. Varela, and Xining Gao for their guidance and endless support throughout this program.
Faculty Advisor: Christopher Chen M.D. Ph.D/ Claudia E. Varela Ph.D., claudiav@bu.edu
Role: I differentiated iPSCs into cardiomyocytes. I also fabricated PDMS devices used for the experiments by bonding and baking components. Then I incubated the devices with the corresponding ECM coatings and performed microcontact printing of the fibronectin pattern. I seeded the devices with the cells I maintained in culture, assisted with sample imaging, and analyzed the acquired images for cell attachment and alignment using ImageJ. Additionally, I enhanced the original stretch adapter design used for cyclic stretching of samples for improved functionality and throughput.