Process Summary

The Shiekh Lab currently uses Matrigel to coat ePlate wells before experiments. Their hiPSC derived cardiomyocytes are reseeded in these wells and their electrical and contratile properties are observed in the ACEA Biosciences RTCA CardioECR machine in response to various compounds. However, the cells experience long recovery times post reseeding onto the expensive Matrigel coating.

Based on literature, fibronectin (10 ug/mL), laminin (15ug/mL), and laminin E8 fragments (15ug/mL) were chosen as adhesive substrate coatings to test in the wells for the hiPSC derived cardiomyocytes. These adhesives were compared against Matrigel to evaluate if they are a more, less, or equally effective alternative.

The expriments showed that the fibronectin coating was equally as effective as Matrigel when looking at cell recovery time and signal consistency. Fibronectin was more effective than Matrigel when it came to signal strength, suggesting greater cell viability and/or adhesion. In addition to these positives, it is 98% less expensive than Matrigel, freeing up funds for other aspects of the research that Dr. Sheikh is conducting.

From our literature search we found papers that showed a decrease in recovery time when reseeding primary rat cardiomyocytes onto a micropatterned surface. We wanted to apply this idea to hiPSC derived cardiomyocytes.

Furthermore, cardiomyocytes cultured in vitro form disorganized monolayers or 3D structures where cells form more connections (most of which are not intercalated disc gap junctions) than in vivo. These cardiomyocytes also do not form aligned sarcomeres, among other discrepencies to their in vivo counterparts. In addition to this, hiPSC derived cardiomyocytes are missing some of the features of human adult cardiomyocytes including t-tubules and multiple nuclei. All of these discrepancies introduce some error to physiological modeling of disease and screening compounds on cardiomyocytes. Literature has shown that some of these errors, such as the unaligned sarcomeres and many connections, can also be corrected through micropatterning.

Two different designs were chosen for the micropatterning of our wells. Pattern 1 consists of a 30 micron lane that can fit two cells per lane width with a 13 micron space between lanes. Pattern 2 consists of a 17 micron lane that can fit one cell per lane width with a 13 micron space between lanes. The 13 micron spacing allows for a few connections to occur across lanes and thus create a synchronously beating monolayer. This is necessary for this project as the ACEA Biosciences RTCA CardioECR takes an aggregate measurement across all the impedance electrodes. Thus, asynchronous beating would introduce varying amounts of noise into the signal.

A stamping protocol was developed through testing and validation with guidance from Jeniffer Stowe of the Cardiac Mechanics Research Group. The current stamping protocol developed by our group includes coating PDMS stamps with 10ug/mL of fibronectin in PBS, incubation, removal of excess fibronectin, stamping with tweezers, incubation, and removal of stamps with tweezers. The stamping was done onto glass slides to mimic the glass surface of the E-Plate as well as onto BSA (1% BSA in PBS) coated glass slides to mimic the E-Plate wells with a backfill to increase the efficacy of micropatterning. Fibronectin transfer in patterns 1 and 2 were validated with a Coomassie stain.

The ACEA Biosciences RTCA CardioECR consumable E-Plates are marketed as single use and are very expensive. Their cost limits the progression of experiments in the lab as well as the resources of the lab. In literature, these exact E-Plates have been washed with a 0.25% EDTA Trypsin wash and reused without issue [1]. We decided to incorporate this into our project in order to reduce costs at the laboratory and reduce waste.