Bioprocessing Considerations towards the Manufacturing of Therapeutic Skeletal and Smooth Muscle Cells

Abstract

Tissue engineering approaches within the muscle context represent a promising emerging field to address the current therapeutic challenges related with multiple pathological conditions affecting the muscle compartments, either skeletal muscle or smooth muscle, responsible for involuntary and voluntary contraction, respectively. In this review, several features and parameters involved in the bioprocessing of muscle cells are addressed. The cell isolation process is depicted, depending on the type of tissue (smooth or skeletal muscle), followed by the description of the challenges involving the use of adult donor tissue and the strategies to overcome the hurdles of reaching relevant cell numbers towards a clinical application. Specifically, the use of stem/progenitor cells is highlighted as a source for smooth and skeletal muscle cells towards the development of a cellular product able to maintain the target cell's identity and functionality. Moreover, taking into account the need for a robust and cost-effective bioprocess for cell manufacturing, the combination of muscle cells with biomaterials and the need for scale-up envisioning clinical applications are also approached.

Keywords: cell manufacturing; skeletal muscle cells; smooth muscle cells; tissue engineering.

Figure 1

Schematic representation and comparison of isolation methods for primary SkMCs and SMCs: enzymatic digestion and explant-based approaches. Enzymatic-based methods for SkM and SM processing involve tissue sample collection, followed by tissue mincing, digestion with proteases (e.g., collagenase and/or dispase enzymes), filtering through a cell strainer, resulting in a cell suspension that is usually plated on coated surfaces for SkMCs (such as Matrigel) and on plastic surfaces for SMCs. Another cell isolation method is the explant-based protocol, which is mainly applied for SMCs, while for SkMCs, it is highly limited and has been mainly purposed towards tissue and disease modelling. The explant technique involves fragmentation of the tissue sample into approximately 1–2 mm diameter explants, followed by cell adhesion, migration and proliferation from the explants onto the plastic surface. Each isolation approach is compared in different categories: scale-up is easier when using enzymatic approaches, while explant-based can facilitate good manufacturing practice (GMP) compliance and simpler protocol optimization. As enzymatic approaches comprise the selection of enzyme(s) composition, concentration and digestion duration, there is a need for a balance between milder digestion protocols and insufficient cell retrieval in contrast to more harsh protocols that can result in higher cell numbers, but with limited viability. Although the resulting cell yield from both explant and enzyme-based methods is described as limited, enzymatic methods can be advantageous when having a reduced amount of starting sample. Adapted from [17,56,57]. GMP: good manufacturing practice; SkMCs: skeletal muscle cells; SMCs: smooth muscle cells.

Figure 2

Considerations on the key points for SMC and SkMC manufacturing. From selection of cell source to culture setup, with potential combination of biomaterials. In terms of cell source, MSCs and iPSCs present broader availability and higher expansion potential, though with a limited maturation phenotype of the target cell type. Due to their low immunogenic profile, MSCs can be used in an allogeneic therapeutic setting, in contrast to primary isolated muscle cells. Concerning culture systems, a balance between complexity (e.g., cocultures that need to accommodate more than one cell type) and feasibility must be considered, with mechanical stimulus representing a critical aspect in muscle tissue engineering. Scaffold design and selection should take into account multiple characteristics such as its composition and stiffness, and if it targets in vitro/ex vivo use only, or if it is intended to be used as a cell delivery vehicle in vivo or as an architectural implanted scaffold. Adapted from [155,156,157]. ECs: endothelial cells; iPSCs: induced pluripotent stem cells; MSCs: mesenchymal stromal cells; SkMCs: skeletal muscle cells; SMCs: smooth muscle cells.