Enhancing the functionality and applicability of engineered skeletal muscle tissue

Gapinske, Lauren

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https://hdl.handle.net/2142/116067 Copy

Description

Title

Enhancing the functionality and applicability of engineered skeletal muscle tissue

Author(s)

Gapinske, Lauren

Issue Date

2022-07-12

Director of Research (if dissertation) or Advisor (if thesis)

Bashir, Rashid

Doctoral Committee Chair(s)

Bashir, Rashid

Committee Member(s)

• Kong, Hyunjoon
• Gillette, Martha
• Saif, Taher

Department of Study

Bioengineering

Discipline

Bioengineering

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Skeletal muscle
• biohybrid machine
• biobot
• tissue engineering
• connexin
• cryopreservation

Abstract

Skeletal muscle represents the most abundant tissue type in the body, amounting to 40% of the body’s weight. Skeletal muscle is essential for movement, respiration, glucose disposal, and thermoregulation. Although skeletal muscle possesses an incredible ability to regenerate in response to small-scale injuries, this self-repair capacity becomes overwhelmed with aging, genetic myopathies, and large muscle loss. Loss of skeletal muscle leads to poor outcomes, including metabolic disease, physical disability, and even death. Engineered in vitro skeletal muscle tissue therefore has important implications in regenerative medicine, as well as the development of high-fidelity models which recapitulate muscle development, genetic diseases, regeneration, and drug response. Furthermore, the inherent regenerative potential, adaptability, scalability, and energy efficiency of skeletal muscle has led to its more recent application as a bioactuator for the development of biohybrid machines. With recent advancements in the development of brain organoids, there is an additional interest in the development of skeletal muscle bioactuators to serve as a functional output for the study of brain development and disorders. The aim of this body of work is to advance not only the functionality of engineered skeletal muscle tissues, but their utility for new applications and availability to other research groups. The utility of skeletal muscle tissues for any application hinges upon its ability to produce force. Previously, the functional lifespan of these muscle tissues in culture was limited to only a few weeks in culture, due to the degradation of the tissue’s ECM and eventual rupture. Here, we present the use of the protease inhibitor E-64 as a means to prevent tissue degeneration and rupture, thereby extending the functional lifespan of the muscle up to 200 days. In addition to extending the lifespan of the tissues in culture, we also enable batch fabrication, storage, and shipment of these tissues through the optimization of a protocol for their long-term cryopreservation. We found that by freezing tissues prior to myogenic differentiation, we not only preserve cell viability, but increase the force generation of differentiated muscle. Following up on these surprising results, we performed studies to determine the mechanism by which cryopreservation leads to a functional improvement in engineered skeletal muscle. We found that cryopreservation alters the microstructure of the tissue by increasing pore size of the ECM and decreasing elastic modulus, which leads to increased expression of genes related to cell migration, cell-matrix adhesion, ECM secretion, and protease activity. Specifically, cryopreservation leads to the upregulation of many ECM proteins including laminin, fibronectin, and several types of collagens, as well as integrins and matrix metalloproteinases. We believe that these freezing-induced changes to the ECM structure and composition improve the tissue microenvironment for the efficient migration and fusion of myoblasts into differentiated myotubes, as evidenced by the associated upregulation of late-stage myogenic markers, in addition to the observed increase in force generation of mature tissue. Another long-term goal of this body of research is to develop engineered neuromuscular tissues capable of large-scale force generation and locomotion of an engineered “biobot.” Here, we transfect C2C12 skeletal myoblasts with three connexin gap junction isoforms (37, 40, and 43) in an attempt to electrically couple the muscle tissue and reduce the required level of motor neuron innervation for maximal force generation. We assess the impact of connexin expression on myogenic differentiation and force generation of the muscle, and compare the relative force generation of the tissues under localized point optical stimulation vs global stimulation in order to assess connexin gap junction functionality. We found that connexin 37 not only leads to the development of larger myotubes capable of greater force generation than wild type, Cx40, and Cx43 C2C12s, but also maintains greater stability of Cx37 RNA and protein expression throughout differentiation and exhibits a greater degree of apparent electrical coupling of the tissue. In future studies, we will use this cell line in the development of a neuromuscular bioactuator to hopefully enable greater force generation under motor neuron stimulation of the muscle tissue. This would enable the development of smarter biomachines which sense and process their environment through integration with an engineered nervous system.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Lauren Gapinske

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