Stable, Covalent Attachment of Laminin to Microposts Improves the Contractility of Mouse Neonatal Cardiomyocytes

The mechanical output of contracting cardiomyocytes, the muscle cells of the heart, relates to healthy and disease states of the heart. Culturing cardiomyocytes on arrays of elastomeric microposts can enable inexpensive and high-throughput studies of heart disease at the single-cell level. However, cardiomyocytes weakly adhere to these microposts, which limits the possibility of using biomechanical assays of single cardiomyocytes to study heart disease. We hypothesized that a stable covalent attachment of laminin to the surface of microposts improves cardiomyocyte contractility. We cultured cells on polydimethylsiloxane microposts with laminin covalently bonded with the organosilanes 3-glycidoxypropyltrimethoxysilane and 3-aminopropyltriethoxysilane with glutaraldehyde. We measured displacement of microposts induced by the contractility of mouse neonatal cardiomyocytes, which attach better than mature cardiomyocytes to substrates. We observed time-dependent changes in contractile parameters such as micropost deformation, contractility rates, contraction and relaxation speeds, and the times of contractions. These parameters were affected by the density of laminin on microposts and by the stability of laminin binding to micropost surfaces. Organosilane-mediated binding resulted in higher laminin surface density and laminin binding stability. 3-glycidoxypropyltrimethoxysilane provided the highest laminin density but did not provide stable protein binding with time. Higher surface protein binding stability and strength were observed with 3-aminopropyltriethoxysilane with glutaraldehyde. In cultured cardiomyocytes, contractility rate, contraction speeds, and contraction time increased with higher laminin stability. Given these variations in contractile function, we conclude that binding of laminin to microposts via 3-aminopropyltriethoxysilane with glutaraldehyde improves contractility observed by an increase in beating rate and contraction speed as it occurs during the postnatal maturation of cardiomyocytes. This approach is promising for future studies to mimic in vivo tissue environments.


Fig. S1
Covalent attachment of laminin to the surface of PDMS using organosilanes. A) Strategy to create a layer of laminin on PDMS surfaces. PDMS is treated with plasma to oxidize the surface, and incubated in a solution of organosilane with a functionality (F) that will bind laminin. B) Two types of organosilanes were used: 3-glycidoxypropyltrimethoxysilane (GPTMS) and 3aminopropyltriethoxysilane (APTES). C) Binding mechanisms of organosilane functionalities (glycidoxy group for GPTMS and amine group for APTES) to amines within laminin. Glycidoxy binds directly to amines. Glutaraldehyde is used to crosslink amines between laminin and APTES.

Fig. S2
Calculation of post deflection from the movement induced by the contraction of attached neoCMs. A) Calculation of micropost deflection. 1) Representative frame of videos acquired with bright-field microscopy. 2) Thresholding of an 8-bit frame with dark background to isolate the tops of posts. 3) Only the tops of the posts can be visualized after background subtraction. 4) Post deflection as a function of time (δt) was calculated by tracking the variations in post positions. Pixels were converted to µm with a microscope calibration slide, and frames were converted based on the camera frame rate.

Fig. S3
Molecular representation of the specific dyes we used for the colorimetric assays that probe for the presence of amines and amine binding chemical functions on the surface. A) Toluidine blue O contains an amine group that binds amine-reactive species, such as the glycidoxy groups on GPTMS-functionalized surfaces and the aldehyde groups on APTES-functionalize surfaces after incubation with glutaraldehyde ( Fig. S1). B) In solution, ninhydrin is yellow; binding to amines generates a purple compound.

Fig. S6
Time-dependent contact angle analysis of PDMS surfaces functionalized with GPTMS and APTES after being stored in water or methanol. A) Representative images of a 50-µL droplet of water in contact with tested PDMS surfaces at different times after surface functionalization. B) Mean surface contact angle at the day of functionalization (day 0) and after two days of storage in water or methanol (day 2). Ten measurements were taken at different surface locations of each sample. Three surfaces were used for each surface type. Error bars represent standard error of the mean. *p < 0.002. Intensity was measured for 53 microposts for each type of functionalization. One-way analysis of variance, p < 0.001 and p < 0.001 calculated by two-sided Student's t-test between any of the presented pairs of samples. Error bars represent standard error of the mean. C) Ratio between average fluorescence intensities of microposts sonicated for 1 h in phosphate-buffered saline (IS) and not sonicated (I0) after incubation in fluorescent gelatin with plasma-treated, GPTMS-, and APTES-functionalized surfaces. IS/I0 semi-quantifies the amount of loss of surface protein.
No loss is expected when IS/I0 = 1, and complete loss occurs when IS/I0 = 0. Intensity was measured for more than 50 microposts. Oneway analysis of variance, p < 0.001. Between any pair of samples, p < 0.03 by two-sided Student's t-test.

Table S1
Summary of results from quantitative characterization of surface properties related to cardiomyocyte binding to PDMS surfaces functionalized with laminin via physisorption or covalent attachment with the organosilanes GPTMS and APTES. Mean and standard error of the mean are presented.