(278d) Micropatterned Hydrogels to Promote Alignment in Co-Cultures Systems for an in Vitro Neuromuscular Model
In vitro models are of immeasurable value to researchers and have become the gold standard in most tissue engineering and drug discovery applications. However, many of the currently used models do not fully capture the complex cell-cell interactions and 3D environment of multicellular organisms. The phenotype of cells can be vastly influenced by chemical and mechanical interactions within their microenvironment, therefore, it is critical to mimic native tissue environment when designing an in vitro models. .
Primary cardiac muscle cells, cardiomyocytes (CMs), seeded on and inside of hydrogels, often lose their propensity to spontaneously contract, a visual cue of endogenous CM phenotyping, after only a few days . However, the beating characteristics and phenotype of CM can be maintained in vitro for longer periods of time when CMs are subjected to external mechanical  or electrical stimuli . Additionally, the presence of cardiac fibroblast in CM co-culture systems has been shown to prolong CM beating . We hypothesize that in an in vitro model, the intrinsic cardiac neurons that help modulate heart rate  also may play a role in maintaining CM phenotype.
To test this hypothesis, we aim to construct a 3D co-culture system by encapsulating cardiac neurons and CMs in a methacrylated gelatin (GelMA) hydrogel. To align both cell types and mimic the architecture of native heart tissue, we have designed a two-step photolithography technique to encapsulate multiple cell types in a micropatterned GelMA hydrogel. This novel technique can be employed to study the interactions between the nervous system and the myocardium in a biomimetic environment.
Encapsulated cells are aligned in the 3D hydrogel constructs through a multi-step photolithography technique modified from a single step technique developed by Nikkhah et al . GelMA hydrogels are synthesized by photocrosslinking of pregel solutions using free radical chemistry. GelMA is produced by reacting methacrylic anhydride to the anime groups in gelatin as previous reported by Nichol et al . Crosslinking is mediated via exposing the pregel solution: 10% (w/v) GelMA solutions containing 0.1% (w/v) free radical initiator (Irgacure 2959) to UV light (300-400nm, intensity 6.9mW/cm2) for up to 75 seconds.
As a proof of concept, fluorescent beads were used as analogs for trypsinized cells. To pattern these beads, first a solution of a pregel-bead mixture is exposed to UV light though a photomask to form patterned lines of crosslinked GelMA (200 um widths with 200 um separation). Next, in order to create a contiguous hydrogel with distinctly aligned beads, the uncrosslinked suspension is washed away and a second pregel-bead solution is crosslinked in between partially patterned hydrogel by irradiating the entire sample. Hydrogels are imaged using fluorescent microscopy and the degree of gel segmentation in 3D is analyzed via intensity profiles of z-stacks taken at 10x on a Zeiss Axio Observer with NIH ImageJ software.
Results and Discussion:
We have successfully fabricated patterned hydrogels of two alternating type of fluorescent beads. Analysis of patterned hydrogels demonstrates two distinct patterns inside 3D GelMA hydrogels showed only a small percentage of leakage between line sections. However, this method is relatively efficient for spatially containing two individual cell suspensions. We have further plans to validate this process for a number of different line segment and spacing measurements.
Utilizing this technique, muscle cells will be patterned prior to neurons for our in vitroneuromuscular model. Future work will combine the neurons found in the heart and CMs into our patterned hydrogel to investigate if their interactions have an effect on neurite sprouting and growth as well as CM phenotype.
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