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Cytokines, crucial in immune modulation, impact disease progression when their secretion is dysregulated. Existing methods for profiling cytokine secretion suffer from time‐consuming and labor‐intensive processes and often fail to capture the dynamic nature of immune responses. Here, iSECRETE, an integrated platform that enables synchronous cell activation, wash‐free, and target‐responsive protein detection for single‐cell IFN‐γ cytokine secretion analysis within 30 min at room temperature is presented. By incorporating a DNA proximity assay (DPA) into a multifunctional microfluidic system, one‐pot homogenous cytokine signal amplification, with a limit of detection of ≈50 secreted molecules per cell is achieved. iSECRETE can robustly handle various sample types that are shown. Two distinct immune activation assay modalities are demonstrated on iSECRETE. Finally, the detection of single‐cell IFN‐γ secretion as an activation hallmark of chimeric antigen receptor T cells within 6 h of exposure to cancer targets is shown. iSECRETE represents the fastest single‐cell sample‐to‐result cytokine secretion assay to date, providing a powerful tool for advancing the understanding of biological phenotypes, functions, and pathways under in vivo‐like conditions. [ABSTRACT FROM AUTHOR]

Regenerative therapies, including the transplantation of spinal cord progenitor cells (SCPCs) derived from induced pluripotent stem cells (iPSCs), are promising treatment strategies for spinal cord injuries. However, the risk of tumorigenicity from residual iPSCs advocates an unmet need for rapid SCPCs safety profiling. Herein, a rapid (≈3000 cells min‐1) electrical‐based microfluidic biophysical cytometer is reported to detect low‐abundance iPSCs from SCPCs at single‐cell resolution. Based on multifrequency impedance measurements (0.3 to 12 MHz), biophysical features including cell size, deformability, membrane, and nucleus dielectric properties are simultaneously quantified as a cell is hydrodynamically stretched at a cross junction under continuous flow. A supervised uniform manifold approximation and projection (UMAP) model is further developed for impedance‐based quantification of undifferentiated iPSCs with high sensitivity (≈1% spiked iPSCs) and shows good correlations with SCPCs differentiation outcomes using two iPSC lines. Cell membrane opacity (day 1) is also identified as a novel early intrinsic predictive biomarker that exhibits a strong correlation with SCPC differentiation efficiency (day 10). Overall, it is envisioned that this label‐free and optic‐free platform technology can be further developed as a versatile cost‐effective process analytical tool to monitor or assess stem cell quality and safety in regenerative medicine. [ABSTRACT FROM AUTHOR]

Mesenchymal stromal cells (MSCs) are promising candidates for cartilage repair therapy due to their self-renewal, chondrogenic, and immunomodulatory capacities. It is widely recognized that a shift from fetal bovine serum (FBS)-containing medium toward a fully chemically defined serum-free (SF) medium would be necessary for clinical applications of MSCs to eliminate issues such as xeno-contamination and batch-to-batch variation. However, there is a notable gap in the literature regarding the evaluation of the chondrogenic ability of SF-expanded MSCs (SF-MSCs). In this study, we compared the in vivo regeneration effect of FBS-MSCs and SF-MSCs in a rat osteochondral defect model and found poor cartilage repair outcomes for SF-MSCs. Consequently, a comparative analysis of FBS-MSCs and SF-MSCs expanded using two SF media, MesenCult™-ACF (ACF), and Custom StemPro™ MSC SFM XenoFree (XF) was conducted in vitro. Our results show that SF-expanded MSCs constitute variations in morphology, surface markers, senescence status, differentiation capacity, and senescence/apoptosis status. Highly proliferative MSCs supported by SF medium do not always correlate to their chondrogenic and cartilage repair ability. Prior determination of the SF medium's ability to support the chondrogenic ability of expanded MSCs is therefore crucial when choosing an SF medium to manufacture MSCs for clinical application in cartilage repair. [ABSTRACT FROM AUTHOR]