MyoBio: An Automated Bioreactor System Technology for Standardized Perfusion-Decellularization of Whole Skeletal Muscle.

Paul Ritter,Raymund Horch,Elke Kretzschmar,Aijia Cai,Kristin Hartmann,Oliver Friedrich,Benjamin Fries,Gerhard Prols,Andreas Arkudas,Maren Fiedler,Barbara Reischl,Christian Lesko,Mena Michael,Haitham Salti,Friedrich Paulsen,Michael Haug

doi:10.1109/tbme.2022.3142317

Abstract

Decellularizing solid organs is a promising top-down process to produce acellular bio-scaffolds for 'de novo' regrowth or application as tissue 'patches' that compensate, e.g., large volumetric muscle loss in reconstructive surgery. Therefore, generating standardized acellular muscle scaffolds marks a pressing area of need. Although animal muscle decellularization protocols were established, those are mostly manually performed and lack defined bioreactor environments and metrologies to assess decellularization quality in real-time. To close this gap, we engineered an automated bioreactor system to provide chemical decellularization solutions to immersed whole rat gastrocnemius medialis muscle through perfusion of the main feeding arteries. Perfusion control is adjustable according to decellularization quality feedback. This was assessed both from (i) ex situ assessment of sarcomeres/nuclei through multiphoton fluorescence and label-free Second Harmonic Generation microscopy and DNA quantification, along with (ii) in situ within the bioreactor environment assessment of the sample's passive mechanical elasticity. We find DNA and sarcomere-free constructs after 72 h of 0.1% SDS perfusion-decellularization. Furthermore, passive elasticity can be implemented as additional online decellularization quality measure, noting a threefold elasticity decrease in acellular constructs. Our MyoBio represents a novel and useful automated bioreactor environment for standardized and controlled generation of acellular whole muscle scaffolds as a valuable source for regenerative medicine.

Highlights

IntroductionThis article has been accepted for publication in a future issue of this journal, but has not been fully edited
The washing time was optimized via residence time experiments to 21 min which is three times the time that is needed to displace 95% of the total volume to ensure the full removal of SDS and cellular debris in the bioreactor vessel
Our engineered MyoBio bioreactor system represents a resourceful advancement towards the automated production of skeletal muscle scaffolds for recellularization or reconstructive surgery

Summary

Introduction

This article has been accepted for publication in a future issue of this journal, but has not been fully edited. Content may change prior to final publication. More than 10% of all reconstructive surgeries eventually fail due to graft infection [4], and are accompanied by the risk of donor site morbidity [2]. To overcome such limitations of autologous transpositions [1], [2], [5], new methods in tissue engineering aim to find sustainable alternatives for heterologous transplantation approaches [6]–[8]. The topdown approach to obtain bio-scaffolds from previously intact organs reflects a promising method as it conserves the vascular network and the specific tissue-inherent architecture which is usually challenging to achieve in bio-printing and has not yet been convincingly demonstrated in bottom-up processes [15]– [17]

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