A Combined Western and Bead-Based Multiplex Platform to Characterize Extracellular Vesicles.

In regenerative medicine, extracellular vesicles (EVs) are considered as a promising cell-free approach. EVs are lipid bilayer-enclosed vesicles secreted by cells and are key players in intercellular communication. EV-based therapeutic approaches have unique advantages over the use of cell-based therapies, such as a high biological, but low immunogenic and tumorigenic potential. To analyze the purity and biochemical composition of EV preparations, the International Society for Extracellular Vesicles (ISEV) has prepared guidelines recommending the analysis of multiple (EV) markers, as well as proteins coisolated/recovered with EVs. Traditional methods for EV characterization, such as Western blotting, require a relatively high EV sample/protein input for the analysis of one protein. We here evaluate a combined Western and bead-based multiplex platform, called DigiWest, for its ability to detect simultaneously multiple EV markers in an EV-containing sample with inherent low protein input. DigiWest analysis was performed on EVs from various sources and species, including mesenchymal stromal cells, notochordal cells, and milk, from human, pig, and dog. The study established a panel of nine antibodies that can be used as cross-species for the detection of general EV markers and coisolates in accordance with the ISEV guidelines. This optimized panel facilitates the parallel evaluation of EV-containing samples, allowing for a comprehensive characterization and assessment of their purity. The total protein input for marker analysis with DigiWest was 1 µg for all nine antibodies, compared with ∼10 µg protein input required for traditional Western blotting for one antibody. These findings demonstrate the potential of the DigiWest technique for characterizing various types of EVs in the regenerative medicine field.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.