A protocol to differentiate the chondrogenic ATDC5 cell-line for the collection of chondrocyte-derived extracellular vesicles.

Skeletal growth and fracture healing rely on the mineralization of cartilage in a process called endochondral ossification. Chondrocytes firstly synthesize and then modify cartilage by the release of a wide range of particles into their extracellular space. Extracellular vesicles (EVs) are one type of such particles, but their roles in endochondral ossification are yet to be fully understood. It remains a challenge to obtain representative populations of chondrocyte-derived EVs, owing to difficulties both in preserving the function of primary chondrocytes in culture and in applying the serum-free conditions required for EV production. Here, we used the ATDC5 cell-line to recover chondrocyte-derived EVs from early- and late-differentiation stages, representing chondrocytes before and during cartilage mineralization. After screening different culture conditions, our data indicate that a serum-free Opti-MEM-based culture medium preserves chondrocyte identity and function, matrix mineralization and cell viability. We subsequently scaled-up production and isolated EVs from conditioned medium by size-exclusion chromatography. The obtained chondrocyte-derived EVs had typical ultrastructure and expression of classical EV markers, at quantities suitable for downstream experiments. Importantly, chondrocyte-derived EVs from late-differentiation stages had elevated levels of alkaline phosphatase activity. Hence, we established a method to obtain functional chondrocyte-derived EVs before and during cartilage mineralization that may aid the further understanding of their roles in endochondral bone growth and fracture healing.

Targeting the myeloid microenvironment in neuroblastoma.

Myeloid cells (granulocytes and monocytes/macrophages) play an important role in neuroblastoma. By inducing a complex immunosuppressive network, myeloid cells pose a challenge for the adaptive immune system to eliminate tumor cells, especially in high-risk neuroblastoma. This review first summarizes the pro- and anti-tumorigenic functions of myeloid cells, including granulocytes, monocytes, macrophages, and myeloid-derived suppressor cells (MDSC) during the development and progression of neuroblastoma. Secondly, we discuss how myeloid cells are engaged in the current treatment regimen and explore novel strategies to target these cells in neuroblastoma. These strategies include: (1) engaging myeloid cells as effector cells, (2) ablating myeloid cells or blocking the recruitment of myeloid cells to the tumor microenvironment and (3) reprogramming myeloid cells. Here we describe that despite their immunosuppressive traits, tumor-associated myeloid cells can still be engaged as effector cells, which is clear in anti-GD2 immunotherapy. However, their full potential is not yet reached, and myeloid cell engagement can be enhanced, for example by targeting the CD47/SIRPα axis. Though depletion of myeloid cells or blocking myeloid cell infiltration has been proven effective, this strategy also depletes possible effector cells for immunotherapy from the tumor microenvironment. Therefore, reprogramming of suppressive myeloid cells might be the optimal strategy, which reverses immunosuppressive traits, preserves myeloid cells as effectors of immunotherapy, and subsequently reactivates tumor-infiltrating T cells.

Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin.

Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 µl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.