Biomimetic Culture Strategies for the Clinical Expansion of Mesenchymal Stromal Cells.

Mesenchymal stromal/stem cells (MSCs) typically require significant ex vivo expansion to achieve the high cell numbers required for research and clinical applications. However, conventional MSC culture on planar (2D) plastic surfaces has been shown to induce MSC senescence and decrease cell functionality over long-term proliferation, and usually, it has a high labor requirement, a high usage of reagents, and therefore, a high cost. In this Review, we describe current MSC-based therapeutic strategies and outline the important factors that need to be considered when developing next-generation cell expansion platforms. To retain the functional value of expanded MSCs, ex vivo culture systems should ideally recapitulate the components of the native stem cell microenvironment, which include soluble cues, resident cells, and the extracellular matrix substrate. We review the interplay between these stem cell niche components and their biological roles in governing MSC phenotype and functionality. We discuss current biomimetic strategies of incorporating biochemical and biophysical cues in MSC culture platforms to grow clinically relevant cell numbers while preserving cell potency and stemness. This Review summarizes the current state of MSC expansion technologies and the challenges that still need to be overcome for MSC clinical applications to be feasible and sustainable.

Assessment of chimeric antigen receptor T cytotoxicity by droplet microfluidics in vitro.

Chimeric antigen receptor T (CAR-T) cells are cytotoxic T cells engineered to specifically kill cancer cells expressing specific target receptor(s). Prior CAR-T efficacy tests include CAR expression analysis by qPCR or ELISA, in vitro measurement of interferon-γ (IFNγ) or interleukin-2 (IL-2), and xenograft models. However, the in vitro measurements did not reflect CAR-T cytotoxicity, whereas xenograft models are low throughput and costly. Here, we presented a robust in vitro droplet microfluidic assay for CAR-T cytotoxicity assessment. This method not only enabled assessment of CAR-T cytotoxic activity under different fluid viscosity conditions, but also facilitated measurement of CAR-T expansion and dissection of mechanism of action via phenotype analysis in vitro. Furthermore, our data suggested that label-free cytotoxicity analysis is feasible by acquiring data before and after treatment. Hence, this study presented a novel in vitro method for assessment of cellular cytotoxicity that could potentially be applied to any cytotoxicity experiment with varying solvent composition.

Cisplatin prevents breast cancer metastasis through blocking early EMT and retards cancer growth together with paclitaxel.

Cancer growth is usually accompanied by metastasis which kills most cancer patients. Here we aim to study the effect of cisplatin at different doses on breast cancer growth and metastasis. Methods: We used cisplatin to treat breast cancer cells, then detected the migration of cells and the changes of epithelial-mesenchymal transition (EMT) markers by migration assay, Western blot, and immunofluorescent staining. Next, we analyzed the changes of RNA expression of genes by RNA-seq and confirmed the binding of activating transcription factor 3 (ATF3) to cytoskeleton related genes by ChIP-seq. Thereafter, we combined cisplatin and paclitaxel in a neoadjuvant setting to treat xenograft mouse models. Furthermore, we analyzed the association of disease prognosis with cytoskeletal genes and ATF3 by clinical data analysis. Results: When administered at a higher dose (6 mg/kg), cisplatin inhibits both cancer growth and metastasis, yet with strong side effects, whereas a lower dose (2 mg/kg) cisplatin blocks cancer metastasis without obvious killing effects. Cisplatin inhibits cancer metastasis through blocking early steps of EMT. It antagonizes transforming growth factor beta (TGFß) signaling through suppressing transcription of many genes involved in cytoskeleton reorganization and filopodia formation which occur early in EMT and are responsible for cancer metastasis. Mechanistically, TGFß and fibronectin-1 (FN1) constitute a positive reciprocal regulation loop that is critical for activating TGFß/SMAD3 signaling, which is repressed by cisplatin induced expression of ATF3. Furthermore, neoadjuvant administration of cisplatin at 2 mg/kg in conjunction with paclitaxel inhibits cancer growth and blocks metastasis without causing obvious side effects by inhibiting colonization of cancer cells in the target organs. Conclusion: Thus, cisplatin prevents breast cancer metastasis through blocking early EMT, and the combination of cisplatin and paclitaxel represents a promising therapy for killing breast cancer and blocking tumor metastasis.