Decellularized Apium graveolens Scaffold for Cell Culture and Guided Alignment of C2C12 Murine Myoblast

Abstract

The development of in vitro meat from validated tissue engineering techniques has emerged as a more sustainable and ethical method of meat production. To date, the expansion of cellular agriculture is believed to be dependent on four pillars: animal free scaffolds, serum free media, bioreactors and cell lines or stem cells. In 2014, decellularized plant tissue emerged as an animal free scaffold for three-dimensional cell culture. Despite the fact that plant parenchyma provides a relatively porous and biocompatible substrate with stiffness values similar to those of muscle tissue, it lacks a number of physical, biochemical, and topographical cues necessary to recapitulate the microenvironment sensed by cells. A crucial characteristic of skeletal muscle tissue is the aligned arrangement of myofibers. Yet, a great deal of knowledge has come from in vitro studies where cells appear randomly scattered. This is turn has created a discrepancy between in vivo and in vitro studies due to widely supported observation that spatial orientation greatly influences gene expression. As part of this thesis, microchannels were developed by longitudinally cutting the vascular bundle of celery (Apium graveolens). Based on the guided alignment of cells on synthetic microchannels, I hypothesize that myoblast will not only align parallel to the vascular bundle but fuse into aligned myotubes. Results show that following 10 days in culture, the normalized orientation of F-actin filaments was determined to be 1.2 ± 2.0°. Subsequent to myoblast alignment, differentiation for 5 days led to the formation of myotubes with a normalized orientation of 8.6 ± 23.8°. Granted that alignment is a crucial characteristic of skeletal muscle tissue, it constitutes only one parameter. Fully recapitulating the microenvironment will need to extend beyond topographical cues, as plant vascularization is partially hydrophobic, approximately 30x stiffer than muscle tissue, and lacks the biochemical cues of mammalian extracellular matrix.