Speaker
Description
Introduction: Fecal incontinence has a high impact on patient quality of life. Available treatments based on surgical and non-surgical approaches range from change in diet, to bowel training or sacral nerve stimulation, none of which represent a long-term solution. Novel therapies are emerging that aim to regenerate the sphincter muscle and, therefore, restore continence. These approaches usually consist of administering a suspension of previously expanded cells to the damaged tissue. This strategy often results in a reduced cell viability due to the harsh step of cell harvesting from the culture platform in which cells were expanded, as well as the unnatural way the unattached adherent cells are delivered as a suspension.
Methodology: Here, we propose a new strategy for the treatment of fecal incontinence, by means of a two-step process. First, skeletal muscle cells (SkMCs) are expanded under static and planar culture conditions until relevant cell numbers are reached. The expanded SkMCs are then combined in bioreactors with implantable, biocompatible and biodegradable polymeric microcarriers, prepared by thermally induced phase separation (TIPS). Different bioreactor culture scenarios were tested: (1) SkMCs from different commercially available sources vs from primary muscle samples from different patients, (2) vertical wheel bioreactor (VWBR) vs spinner flask, (3) xeno(geneic)-free vs non-xeno-free conditions, (4) culture time, (5) agitation scheme. These parameters were optimized to maximize cell adhesion efficiency. Cell viability (calcein and DAPI staining) and distribution throughout the microcarriers, presence of the CD56 myogenic marker (flow cytometry), cell differentiation potential (desmin staining to assess myotube formation) and cell migration from the microcarriers were also assessed.
Results: The optimized adhesion process allowed us to obtain a 70-80% efficient SkMC adhesion onto the TIPS microcarriers. This was achieved by applying an intermittent agitation scheme to patient-derived SkMCs adhered to the microcarriers in VWBR, under xeno-free conditions, after 24h. SkMCs maintained their myogenic features (expression of CD56 marker) after the expansion phase in planar systems under static conditions, as well as after adhesion and culture in the microcarriers. SkMCs were able to migrate from the microcarriers and differentiate into multinucleated myotubes, as well as maintaining high cell viability throughout the process.
Conclusion(s): By optimizing the choice of bioreactor, as well as its operating conditions, we were able to obtain a high percentage of viable SkMCs adhered to TIPS microcarriers, with relevant muscle regeneration potential. Additionally, by performing the entire cell adhesion process under xeno-free conditions, we avoid the use of fetal bovine serum, which addresses regulatory issues. The xeno-free conditions established for the cell-microcarrier combination, associated to the single-use feature of the bioreactor selected, make this process more amenable to GMP compliance. The use of implantable microcarriers should also greatly increase the likelihood of success of the proposed cell-based therapy, as it avoids the drawbacks associated with harvesting of SkMCs and their subsequent delivery in an unattached state to the damaged tissue.
The AMELIE project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874807.
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