Micropatterning Alginate Substrates for in vitro Cardiovascular Muscle on a Chip

Abstract

Soft hydrogels such as alginate are ideal substrates for building muscle in vitro because they have structural and mechanical properties close to the in vivo extracellular matrix (ECM) network. However, hydrogels are generally not amenable to protein adhesion and patterning. Moreover, muscle structures and their underlying ECM are highly anisotropic, and it is imperative that in vitro models recapitulate the structural anisotropy in reconstructed tissues for in vivo relevance due to the tight coupling between sturcture and function in these systems. We present two techniques to create chemical and structural heterogeneities within soft alginate substrates and employ them to engineer anisotropic muscle monolayers: (i) microcontact printing lines of extracellular matrix proteins on flat alginate substrates to guide cellular processes with chemical cues, and (ii) micromolding of alginate surface into grooves and ridges to guide cellular processes with topographical cues. Neonatal rat ventricular myocytes as well as human umbilical artery vascular smooth muscle cells successfully attach to both these micropatterned substrates leading to subsequent formation of anisotropic striated and smooth muscle tissues. Muscular thin film cantilevers cut from these constructs are then employed for functional characterization of engineered muscular tissues. Thus, micropatterned alginate is an ideal substrate for in vitro models of muscle tissue because it facilitates recapitulation of the anisotropic architecture of muscle, mimics the mechanical properties of the ECM microenvironment, and is amenable to evaluation of functional contractile properties.

Keywords: Biomedical Applications; Hydrogels; Microcontact Printing; Surface Modification; Tissue Engineering.

Figure 1

Mechanical characterization of 5% w/v of alginate. (A) Example photograph of 12 test samples punched out for mechanical testing. (B) Stress-strain curve collected for alginate substrates ionically crosslinked with 10mM, 60mM, and 100mM CaCl₂. The straight line fits reveal a strong linear correlation implying elastic behavior. (C) Young’s moduli for 10, 60, and 100mM CaCl₂ conditions calculated from the corresponding slope of the linear fits were determined to be 4.77 ± 0.08 kPa, 56.98 ± 0.86 kPa and 60.60 ± 0.64 kPa respectively (Mean ± SEM, N=3 samples).

Figure 2

Micropatterned alginate thin films fabricated using (A, B) microcontact printing and (C, D) micromolding. (A, C i) A layer of APTES is deposited on the glass coverslip (A, C ii–iii) A flat or patterned calcium-loaded agar stamp is applied on a drop of alginate (A, C iv) The thin film of hydrogel is submerged in a solution of streptavidin mixed with the reagents EDC and Sulfo-NHS and then washed and dried (A, C v) Biotinylated fibronectin is applied either by microcontact printing with a stamp of PDMS or by simple submersion (A, C vi) Samples are ready for being cut for contractility assay. (B i) Fluorescent imaging of immunostained 2D fibronectin pattern on flat alginate films (B ii) Section profile of fluorescence level. (D i) 3D reconstruction of AFM imaging of the topography of micromolded films (D ii) Height profile along a section perpendicular to the features.

Figure 3

Tissues generated from (A, B, C) cardiac myocytes and (D, E, F) vascular smooth muscle cells on (A, D) isotropic fibronectin, (B, E) microcontact printed, and (C, F) micromolded alginate substrates. (A–C i) Phase contrast pictures of cardiac tissues for each condition (A–C ii) Immunofluorescence composite images where actin is red, nuclei are blue and α-actinin is green and (A–C iii) zoomed-in grayscale images for α-actinin in white squares. (D–F i) Phase contrast pictures of vascular smooth muscle tissue for each condition and (D–F ii) Immunofluorescence composite images where actin is white and nuclei are blue.

Figure 4

Orientational order parameter for (A) sarcomeric alignment in cardiac tissue and (B) F-actin alignment in vascular smooth muscle tissue cultured on isotropic alginate surface, microcontact printed alginate surface and micromolded alginate surface. (Mean ± SD, N=6 coverslips with at least 3 fields of view of 160µmX160µm per coverslip for each condition, * = statistically different from isotropic tissue, p < 0.05, OOP: Orientational Order Parameter, Iso: Isotropic fibronectin on flat alginate substrate, µCP: Microcontact printed lines of fibronectin on flat alginate, µM: Micromolded alginate substrate with a uniform coating of fibronectin).

Figure 5

Results from cardiac muscular thin films constructed from micropatterned alginate substrates. (A) Schematic of the assay: cantilevers are cut and peeled out of the alginate film and exposed to external electrical field stimulation (B) Optical photograph of the alginate coverslip after peeling the cut regions showing a high number of MTFs from a single experiment. Scale bar represents 5mm. (C) Tracking of the horizontal projection of the 2 representative films during one contraction cycle at 2Hz stimulation frequency (D) Representative time trace of stress generated by one MTF at different pacing frequencies. Average diastolic, peak systolic and twitch stresses generated by (E) microcontact printed and (F) micromolded alginate cardiac MTFs. (Mean ± SEM, N = 7 MTFs in (E) and N = 6 MTFs in (F), spont = spontaneous contraction).

Figure 6

Results from vascular smooth muscle thin films constructed from micropatterned alginate substrates. (A) Optical photograph of the alginate coverslip after peeling the cut regions and peeling the MTFs. Scale bar represents 1mm. (B) Tracking of the horizontal projection of the 2 representative films during one experiment; no treatment at 0 minutes, after stimulation with endothelin-1 at 30 minutes and after exposure to HA-1077 at 60 minutes. Scale bar represents 1mm. (C) Averaged time traces of stress generated by engineered vascular smooth muscle on microcontact printed (red circles, N=7) and micromolded (black squares, N=21) alginate MTFs. Untreated samples are stimulated by 100nM endothelin-1 at 10 minutes and treated with 100µM HA-1077 at 40 minutes. The calculation of contraction stress generated in response to vasoconstrictor treatment, basal tone revealed by vasodilator treatment and the residual stress are indicated in the panel. Average contraction stress, basal tone and residual stresses recorded on (D) microcontact printed and (E) micromolded alginate vascular smooth muscle MTFs (Mean ± SEM, N = 7 MTFs in (D) and N = 21 MTFs in (E)).