Graduate Student Carnegie Mellon University Pittsburgh, Pennsylvania, United States
Introduction:: Extracellular vesicles (EVs) are naturally-occurring membrane bound nanoparticles that carry biomolecules and facilitate communication between cells. Secreted by cells and found in a wide range of biofluids, a growing area of research aims to utilize the strengths of EVs as a low immunogenomic lipid-based carrier of biomolecules between cells. However, the scalability of EV isolation processes remains as a time and resource intensive process. Milk-derived extracellular vesicles (milk-EVs) are of interest because they are an abundant source of various bioactive molecules including proteins, lipids, and nucleic acids. We attenuated a three-step centrifugation process to extract the milk-EVs from a simplified pasteurization process for A2/A2 milk (breed: Holstein Friesian, Henry Farms, Knox, PA). We investigated the ability of A2/A2 milk-EVs to aid in myoblast migration in C2C12 cells in a scratch wound assay. We found that A2 milk-EV treated cells show faster migration (p< 0.01) to the injury site compared to untreated cells over a 12-hour treatment.
Materials and Methods:: Pasteurized, non-homogenized milk was collected from genetically-tested A2/A2 cows(GeneSeek, Neogen Corp). Upon milking from source, sample was refrigerated at 4°C, funneled into a pasteurization unit(AD Process Systems, Stanford,WI) where it was heated at 60-70°C for 30 minutes and cooled down to 4°C prior bottling. Post-process, milk was reduced with a single round of centrifugation at 3,000-5,000rpm at 4°C. Milk-EVs were extracted via two rounds of centrifugation at 15,000-20,000rpm at 4°C for 1-6 hours. The relative centrifugation time was extrapolated based on the k-factors from the centrifuge rotor (ThermoFisher F28/50). The actual centrifugation time is equivalent to the maximum rotational speed from an exponential curvefit based on the extrapolated k-factor and the ratio of the centrifugation radius.
C2C12 cells(primary mouse myoblasts, source: ATCC) were cultured in growth medium(Dulbecco's Modified Eagle Medium (DMEM)‐high glucose,1% penicillin‐streptomycin,1% L‐glutamine,10% heat‐inactivated fetal bovine serum) for 7 days. Myoblasts were seeded(density:150,000 cells/mL) on polydimethylsiloxane(PDMS)-coated coverslips with a top layer of fibronectin (concentration:50 ug/mL). At 80% confluency, a scraper was used to make a scratch wound (dimensions:~1mmx5mm). Cells were treated with milk-EVs(100 ug/mL, ThermoFisher 23227 bicinchoninic acid assay). Cells were imaged at 0,6 and 12 hours post-treatment. At 6-hour treatment, treated cells with stained milk-EVs(1:200 Vybrant DiD) were washed with a stripping buffer(500 uM sodium chloride, 0.5% acetic acid in deionized water, pH of 3), fixed with 4% formaldehyde and incubated in a staining solution(1:200 DAPI, 1:300 phalloidin, in phosphate buffered saline) for 30 minutes prior imaging.
Results, Conclusions, and Discussions:: Both control- and milk-EV treatment groups showed an overall decreasing wound area over a 12-hour observation time. The addition of milk-EV treatment demonstrates a significant effect on wound closure (F = 6.92, p = 0.01, two-way ANOVA). The rate of wound closure over time is significantly increased by milk-EV treatment (p< 0.01, paired t-test). At 12 hours after the scratch was made, over 41% of the wound had closed with milk-EV treatment compared to 31% wound closure in the control group. The proposed uptake mechanism suggests the initial activation of the membrane surface from calcium-gated channels resulting in capillary pressure range of 1400-3000 Pa. EV contact with FcRn receptors triggers internalization of EV particles via membrane fusion resulting in 50-100nm pore sizes. Applying Darcy’s Law with these parameters, membrane permeability of C2C12 cells estimates the passive internalization of milk-EVs (particle size: 90-200nm). A 2D-random walk time-scale model estimates optimal EV uptake within 2-3 hours of treatment with majority of EVs internalized after 6 hours.
In this study, we demonstrate that treatment with isolated milk-EVs extracted using a facile three-step centrifugation can promote cell migration into a wound area. This preliminary work can hopefully encourage more research in this field. With improved understanding, future studies can be developed on A2/A2 sourced milk-EVs and its potential applications in the development of new therapies, non-invasive biomarkers or enhanced nutritional products.
Dairy farming provides jobs and improves food security for many rural communities. Maintaining this economy can be challenging due to issues such as competition, high production costs and environmental issues. As plant-based alternatives to conventional milk have become more mainstream, milk consumption in the United States has decreased. Many studies have shown that specialized grazing from a specific A2/A2 genetic cow strain can curb the discomfort and digestive issues from drinking conventional milk. A2/A2 milk can be an inexpensive and viable alternative for consumers based on their individual dietary needs and preferences. Furthermore, the simplification of dairy processing can promote sustainable farming practices that can mitigate greenhouse gas emissions and address social concerns with animal welfare regarding the use of antibiotics and hormones. At a macroscale, these parallel efforts to optimize farming practices and discover new contributions of milk in health and medicine can aid in facilitating a collective and equitable social transformation for dairy farming.
Acknowledgements (Optional): Preliminary experiments for the extraction of extracellular vesicles were conducted as an independent project in Jiang Lab (Principal Investigator: Shaoyi Jiang, PhD), Department of Biomedical Engineering, Cornell University. In vitro testing was conducted as a class project for Tissue Engineering (Professor: Adam Feinberg, PhD), Department of Biomedical Engineering at Carnegie Mellon University. Subsequent modeling for uptake mechanism were conducted as a class project for Biologically Inspired Microsystems Engineering (Professor: Mingming Wu, PhD), Department of Biological and Environmental Engineering, Cornell University. A special thanks to Wenchao Gu, PhD for sparking interest in this field. We would also like to acknowledge Misti West, our teaching assistants and the members of the Sherman and Joyce Bowie Scott Hall – Collaborative Laboratory space at Carnegie Mellon University for access to workspace and equipment. Many thanks to the owners of Henry Farms for providing us with raw milk.
