Bioengineered three-dimensional physiological model of colonic longitudinal smooth muscle in vitro

Abstract

Background: The objective of this study was to develop a physiological model of longitudinal smooth muscle tissue from isolated longitudinal smooth muscle cells arranged in the longitudinal axis.

Methods: Longitudinal smooth muscle cells from rabbit sigmoid colon were isolated and expanded in culture. Cells were seeded at high densities onto laminin-coated Sylgard surfaces with defined wavy microtopographies. A highly aligned cell sheet was formed, to which addition of fibrin resulted in delamination.

Results: (1) Acetylcholine (ACh) induced a dose-dependent, rapid, and sustained force generation. (2) Absence of extracellular calcium attenuated the magnitude and sustainability of ACh-induced force by 50% and 60%, respectively. (3) Vasoactive intestinal peptide also attenuated the magnitude and sustainability of ACh-induced force by 40% and 60%, respectively. These data were similar to force generated by longitudinal tissue. (4) Bioengineered constructs also maintained smooth muscle phenotype and calcium-dependence characteristics.

Summary: This is a novel physiologically relevant in vitro three-dimensional model of colonic longitudinal smooth muscle tissue. Bioengineered three-dimensional longitudinal smooth muscle presents the ability to generate force, and respond to contractile agonists and relaxant peptides similar to native longitudinal tissue. This model has potential applications to investigate the underlying pathophysiology of dysfunctional colonic motility. It also presents as a readily implantable band-aid colonic longitudinal muscle tissue.

FIG. 1.

Process schematic for bioengineering rabbit longitudinal smooth muscle cell (RLSMC) sheet. Replicating molds with defined wavy surfaces were patterned with Epo-Tek using thermal expansion, oxidation, and UV curing. Poly(dimethylsiloxane) (PDMS)-based wavy culture plates were cured at room temperature for at least 2 days. Circles (35 mm) were cut out from the cured PDMS and secured with more PDMS in a standard 35 mm tissue dish. The plates were sterilized and prepped for culture. Laminin is deposited onto the plate, and silk sutures are pinned down. LSMCs were cultured separately and seeded onto the plate, and allowed to align along the axis of the waves on the plate. Once alignment was microscopically confirmed (approximately 4 days postcell seeding), a fibrin hydrogel was added to promote delamination and subsequent aligned construct formation. Once alignment was microscopically confirmed (approximately 4 days postcell seeding), a fibrin hydrogel was added to promote delamination and subsequent aligned construct formation.

FIG. 2.

Engineering highly aligned LSMC sheets. (A) 16 × optical image of the PDMS mold fixed onto a 35 mm culture plate, with the wavy patterns seen as dark lines. (B) The wavy plate was functionalized with laminin, and silk sutures were pinned down 12 mm apart. The growth medium was added to the plate and left to sterilize under UV light before cells were plated onto them. (C) 10 × inverted microscope images show that cells were unaligned 1.5 h after plating them, and show almost complete alignment on day 3. Highly aligned cell sheet formation can be seen on day 5. (D) Addition of the fibrin gel on day 5 promoted delamination (indicated by the black arrow), and the aligned cells contracted to form a highly longitudinally aligned construct 7 days after initial cell seeding, attached to the silk sutures.

FIG. 3.

Immunofluorescence on isolated RLSMC. (A) α-Smooth muscle actin, a smooth muscle marker, shows stained stress fibers and smooth muscle actin. (B) Smooth-muscle-specific heavy Caldesmon stains positive. (C) Interstitial cells of cajal (ICC) marker, c-Kit, shows no positive areas, indicating the absence of ICC cells in the longitudinal smooth muscle culture. (D) Negative control of the rabbit secondary antibody indicates weak background staining like (C). Scale bar = 50 μm.

FIG. 4.

Passive tension and signal-to-noise ratio (SNR). (A) A trace of passive tension obtained from the bath medium without tissue. To determine the degree of system noise in the measurement, the bioengineered tissue was attached to the measurement system with no external stretch. (B) Active tension tracing. The SNR of the system, representing the degree of active tension is the ratio of mean signal strength to the standard deviation of the noise. SNR = ∼250 units.

FIG. 5.

Force generation in response to 1 μM acetylcholine (ACh) in (A) bioengineered tissue and (B) native longitudinal tissue. RLSMC sheets generated rapid-rising contractions of 94.4 ± 13.244 μN (n = 7) in response to 1 μM ACh in regular calcium media. Contractions were sustained over a 12-min period. Native longitudinal tissue also generated rapid rising contractions of 53.067 ± 5.014 (n = 6). Addition of ACh is indicated by the arrow at 180 s. Representative tracings for bioengineered constructs and native tissue have been chosen.

FIG. 6.

Dose–response to ACh in bioengineered tissue and native longitudinal tissue. Rabbit longitudinal tissue responded to ACh in a dose-dependent manner. Doses tested were between 1 nM and 1 μM. Bioengineered sheets from isolated LSMCs retained this dose-dependence property.

FIG. 7.

Response to ACh in zero calcium in bioengineered tissue and rabbit longitudinal tissue. Native longitudinal tissue generated only 16.57 ± 2.095 μN (n = 6) in response to 1 μM ACh in the absence of extracellular calcium. This force was also sustained only for 2.5 min, versus the 12 min of force sustenance in regular calcium. Bioengineered tissue also showed force attenuated in magnitude by 50% and a reduction in time by 60%.

FIG. 8.

Inhibition of force generation by preincubation with vasoactive intestinal peptide (VIP) in bioengineered tissue and rabbit longitudinal tissue. On treatment with VIP, both the native longitudinal tissue and the bioengineered tissue relaxed as indicated by the drop in baseline force. On subsequent stimulation with 1 μM ACh, force generated was lowered by 40% in magnitude. Force was also sustained only for 5 min, versus the 12 min of force sustenance in control ACh responses.

FIG. 9.

Comparison of force generated by ACh in bioengineered tissue and native longitudinal tissue. Force generation in response to ACh was significantly attenuated in magnitude and time by preincubation with VIP or in the absence of extracellular calcium. The accompanying table summarizes the mean values of force generated by the native tissue as well as the bioengineered tissue in response to 1 μM ACh under different conditions of treatment (*p < 0.05, **p < 0.001).