Exosome Production and Regenerative Potential is Influenced by Mechanical Stimulus and Cell Origin
Authors: Mullen Michael , Bahney Chelsea , Ravuri Sudheer , Huard Johnny , Ehrhart Nicole 
Published: April 2020

INTRODUCTIONExosomes are nanosized membrane bound vesicles (30–150 nm) secreted by cells. Exosomes contain nucleic acids and proteins used to signal and influence the cells around them. It has been shown that mesenchymal stem cells (MSCs) influence tissue regeneration and healing through their paracrine secretion of pro‐regenerative factors. Many of these factors are contained within exosomes suggesting that exosome suspensions may be an effective cell‐free therapy for tissue regeneration and repair. Previously, our group has found that mechanical stimulus can be used to rescue muscle derived stem cells from an aged mouse model (progeria mice), indicating that physical environment influences MSC function. With this knowledge, we hypothesized that myoblasts subjected to mechanical stimulation would improve exosome quantity and function. Given the fact that several studies have also shown muscle to bone cross talk improves fracture repair, we further hypothesized that optimized myoblast exosomes could improve osteoblast cell function.METHODSC2C12 myoblasts were cultured using the FlexCell FX‐5000TT bioreactor. Two exercise regimens were programmed using biaxial or uniaxial strain: 1) low intensity regimen (LIR) (0–15% strain at 0.5 Hz for 24 hours) 2) high intensity interval regimen (HIIR) (12–22% strain at 1 Hz for 10 minutes followed by 50 minutes of rest repeated for 24 hours). Unexercised, control cells were cultured in parallel. MC3T3‐E1 osteoblasts were cultured for 24 hours for exosome collection. Exosomes were isolated using Invitrogen Total Exosome Isolation Reagent. System Bioscience’s ExoELISA‐ULTRA CD81 Kit and a BCA assay were used to quantify exosome number and protein concentration. 20 ug of exosomes (~5e9 exosomes) were used for treatment in proliferation and differentiation assays. Proliferation was assessed using PrestoBlue (Thermo Fisher). Myogenic differentiation was evaluated using image analysis (15 images per group).ResultsAll exercise regimens resulted in increased exosome concentrations in myoblasts. Biaxial strain and HIIR resulted in the greatest exosome production. Further, we found increased functionality of exosomes from exercised cells resulting in greater proliferation and differentiation of myoblasts. Interestingly, myoblast exosomes also significantly increased proliferation in osteoblast cells. However, in contrast, exosomes isolated from osteoblasts significantly decreased proliferation when treated on myoblasts but significantly increased proliferation when treated back upon themselves.DISCUSSION and CONCLUSIONOur results indicate that mechanical stimulus and cell origin directly influence exosome production. We found that exercise can be optimized to improve the production and regenerative capacity of myoblast exosomes. Further, we found a positive effect of myoblast exosomes on osteoblasts, but the converse, osteoblast exosomes on myoblasts, had a negative effect on cell proliferation. Coupling this with findings to indicate exosomes remain stable at −80° C for at least 4 months, we see exosomes as a potential cell‐free therapy for tissue regeneration following musculoskeletal traumas.Support or Funding InformationBorgen Family Foundation(A) Myoblast Cells secrete more exosomes when exercised (flex) compared to non‐exercised (static) cells. (* = p < 0.05, n = 8) (B) Exercise protocol attenuates exosome production. (**** = p < 0.0001, n = 4) (C–F) Exosomes from exercised cells increase myogenic differentiation. (* = p < 0.05, *** = p < 0.001, n = 4)Figure 1(A) Exosomes from exercised cells increase proliferation. (* = p < 0.05, *** = p < 0.001, n = 4) (B) Exosomes effect on proliferation is dependent on cell origin. (*** = p < 0.001, n = 3)Figure 2