A study of bioprocess designs for development of the myogenic secretome as a source of bioactive extracellular vesicles

The human skeletal muscle myoblasts (HSMM) secretome decreases cytokine-induced inflammation in chondrocytes, indicating potential as therapy for arthritis. The lack of an efficient scalable workflow for the human skeletal myoblast secretome limits its development as a source of therapeutic biologics. Therefore, the principal objective of this study is to investigate approaches that could support manufacture of a bioactive human skeletal myoblast secretome at therapeutically relevant yields. An upstream process for myogenic secreted EVs and proteins was optimised over 7-days. Analytics involved quantification of secretome yield and extracellular vesicle (EV) characterisation. Internalisation of myogenic EVs by human chondrocytes was investigated to examine bioactivity. Effects of EVs on matrix metalloproteinase-13 and nuclear factor-κB gene expression and EV ability to limit degradation of collagen II in tumour necrosis factor-α treated chondrocytes was also assessed. Potential mechanisms of action of HSMM secretome proteins were investigated by predicting their interactions with proteins involved in arthritis pathology using NetworkX, and the STRING database. Myogenic secretome yields increased 2-fold on 75cm2 and 175cm2 surface areas compared to 25cm2 surface areas. The myogenic secretome yield on poly (lactic-co-glycolic) acid microcarriers was 3-fold greater than that produced on a 2D surface. The yield of EVs from 175cm2 and 75cm2 surface areas following SEC isolation were 4-fold and 2-fold greater respectively, compared to those achieved using precipitation. Particles within exosome size ranges (30-150 nm) were isolated at higher concentrations than microvesicle particles. Chondrocyte internalisation of 2D and 3D myogenic EVs possibly via phagocytosis, displayed heterogeneous EV distribution. The myogenic secretome may enhance collagen II expression and reduce NF-kB expression in chondrocytes. Protein - protein interaction (PPI) networks between secretome proteins and arthritis proteins were complex. Clustering using k-means demonstrated high proportions of highly weighted PPI scores, and key proteins with potential therapeutic bioactivities were identified using betweenness centrality. Taken together, this study is a basis for upstream and downstream processing of the human myogenic secretome demonstrating how this novel medicine modality may be applied as a therapeutic for arthritis.

Citation

Rageh, A. (2025) A study of bioprocess designs for development of the myogenic secretome as a source of bioactive extracellular vesicles. University of Wolverhampton. https://wlv.openrepository.com/handle/2436/626060

Publisher

University of Wolverhampton

URI

https://wlv.openrepository.com/handle/2436/626060

Type

Thesis or dissertation

Language

en

Description

This thesis is submitted in partial fulfilment at the University of Wolverhampton for the degree of Doctor of Philosophy in Cell Biology.

Collections

Theses and Dissertations