Journal: Journal of Extracellular Vesicles
Article Title: GMP‐Compliant Process for the Manufacturing of an Extracellular Vesicles‐Enriched Secretome Product Derived From Cardiovascular Progenitor Cells Suitable for a Phase I Clinical Trial
doi: 10.1002/jev2.70145
Figure Lengend Snippet: In vitro functional assays and immunogenicity assay. (a) Proliferation assay. HUVEC were plated and cultured in typical HUVEC media (unstressed, Control +) and in protein‐depleted media (stress condition). Stressed HUVEC were untreated (Control –) or treated with 50 µL of final product or 50 µL of PBS 1X (vehicle control). HUVEC proliferation was quantified by measuring BrdU uptake. Values were double normalised (baseline [negative control] subtracted, and normalised to the positive control). Data in the figure represent a single, representative experiment (average +/– SD; n = 5 technical replicates). (b) Cardiomyocyte survival assay. This test is a staurosporine‐induced cardiotoxicity assay. Cardiomyocytes were exposed to NucSpot Live 650 dye without staurosporine (Control +), with staurosporine alone (Control –) or with stauroporine and increasing concentrations of the final product. At the 24‐h time‐point, cell counts were determined by Incucyte. Values were double normalised (baseline [negative control] subtracted, and normalised to the positive control). Data in the figure represent a single, representative experiment (average +/– SD; n = 3 technical replicates). (c) Scratch wound healing assay. After establishing a monolayer of HUVEC, a scratch was induced using the Wound Maker according to the manufacturer's directions. Following the scratch, HUVEC were cultured in typical HUVEC media (unstressed, Control +) and in protein depleted (stress condition) media. Stressed HUVEC were untreated (Control –) or treated with final product. Plates were imaged by Incucyte at T0 and every 2 h up to 18‐h. Representative images taken on the Incucyte depicting individual wells are shown immediately after the scratch (T0) and 18 h post‐treatment (T18). Data in the figure represent a single, representative experiment. (d) Scratch wound healing assay. The results of HUVEC scratch wound healing assay described above were determined using the Scratch Wound Healing Module. Normalised wound confluence results at the 18‐h timepoint were double normalised (baseline [negative control] subtracted, and normalised to positive control). Data in the figure represent a single, representative experiment (average +/– SD; n = 5 technical replicates). (e) NK cell degranulation. The percent of human NK cells expressing CD107a was measured by flow cytometry. Human NK cells were cultured in the presence of human K562 cells (positive control), PMA/ionomycin 1X (second positive control) or 50 µL of PBS 1X (vehicle control) or 50 µL of final product. In presence of final product, there is a dramatic absence of expression of CD107a compared with the positive controls. Data in the figure represent a single, representative experiment (average +/– SD; n = 3 technical replicates). (f) PBMC alloreactive activation. The levels of multiple cytokines, IFN‐γ, and IL‐2 were measured by flow cytometry. Human PBMC were cultured in the presence of PMA/ionomycin 1X (positive control), 50 µL of PBS 1X (vehicle control) or 50 µL of the final product. Data in the figure represent a single, representative experiment.
Article Snippet: After 18 h, cells were harvested, washed and stained with CD16‐PE, CD56‐PE, CD8‐APC Vio770, CD4 Viogreen specific antibodies and 7‐AAD (Miltenyi Biotec), and the expression of CD107a was analysed on CD16 + CD56 + human NK cells using a MQ10 Flow Cytometer (Miltenyi Biotec).
Techniques: In Vitro, Functional Assay, Immunopeptidomics, Proliferation Assay, Cell Culture, Control, Negative Control, Positive Control, Clonogenic Cell Survival Assay, Wound Healing Assay, Expressing, Flow Cytometry, Activation Assay
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![In vitro functional assays and immunogenicity assay. (a) Proliferation assay. HUVEC were plated and cultured in typical HUVEC media (unstressed, Control +) and in protein‐depleted media (stress condition). Stressed HUVEC were untreated (Control –) or treated with 50 µL of final product or 50 µL of PBS 1X (vehicle control). HUVEC proliferation was quantified by measuring BrdU uptake. Values were double normalised (baseline [negative control] subtracted, and normalised to the positive control). Data in the figure represent a single, representative experiment (average +/– SD; n = 5 technical replicates). (b) Cardiomyocyte survival assay. This test is a staurosporine‐induced cardiotoxicity assay. Cardiomyocytes were exposed to NucSpot Live 650 dye without staurosporine (Control +), with staurosporine alone (Control –) or with stauroporine and increasing concentrations of the final product. At the 24‐h time‐point, cell counts were determined by Incucyte. Values were double normalised (baseline [negative control] subtracted, and normalised to the positive control). Data in the figure represent a single, representative experiment (average +/– SD; n = 3 technical replicates). (c) Scratch wound healing assay. After establishing a monolayer of HUVEC, a scratch was induced using the Wound Maker according to the manufacturer's directions. Following the scratch, HUVEC were cultured in typical HUVEC media (unstressed, Control +) and in protein depleted (stress condition) media. Stressed HUVEC were untreated (Control –) or treated with final product. Plates were imaged by Incucyte at T0 and every 2 h up to 18‐h. Representative images taken on the Incucyte depicting individual wells are shown immediately after the scratch (T0) and 18 h post‐treatment (T18). Data in the figure represent a single, representative experiment. (d) Scratch wound healing assay. The results of HUVEC scratch wound healing assay described above were determined using the Scratch Wound Healing Module. Normalised wound confluence results at the 18‐h timepoint were double normalised (baseline [negative control] subtracted, and normalised to positive control). Data in the figure represent a single, representative experiment (average +/– SD; n = 5 technical replicates). (e) NK cell degranulation. The percent of human NK cells expressing <t>CD107a</t> was measured by flow cytometry. Human NK cells were cultured in the presence of human K562 cells (positive control), PMA/ionomycin 1X (second positive control) or 50 µL of PBS 1X (vehicle control) or 50 µL of final product. In presence of final product, there is a dramatic absence of expression of CD107a compared with the positive controls. Data in the figure represent a single, representative experiment (average +/– SD; n = 3 technical replicates). (f) PBMC alloreactive activation. The levels of multiple cytokines, IFN‐γ, and IL‐2 were measured by flow cytometry. Human PBMC were cultured in the presence of PMA/ionomycin 1X (positive control), 50 µL of PBS 1X (vehicle control) or 50 µL of the final product. Data in the figure represent a single, representative experiment.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5392/pmc12365392/pmc12365392__JEV2-14-e70145-g001.jpg)
![DNA/LION vaccine induces durable T cell responses in rhesus macaques (A and B) Spike-specific T cell responses were measured in PBMCs over time by flow cytometry in (A) study 1, as IFN-γ + T cells, and (B) study 2, as cytokine + (IFN-γ, TNF-α single- and/or double-positive) T cells. Scatterplots with median line and minimum and maximum values are shown. The pie chart depicts the frequency of spike-specific IFN-γ, TNF-α single- and/or double-positive T cells. (C) Comparison of CD4 + and CD8 + IFN-γ + spike-specific T cell responses induced by DNA/LION (study 1, group 1) and by DNA(IM) prime followed by DNA/LION boost (study 1, group 2) and a historical study examining spike DNA vaccination by EP alone ( n = 4) or co-immunized with adjuvanted spike protein ( n = 4). (D) Frequency of spike-specific cytokine + T cells CD4 + and CD8 + T cells at the day of V4 and 2 and 3 weeks later (study 1). Bars indicate mean values. p values from generalized estimating equation (GEE) and are defined as ≤0.05, ∗; ≤0.01, ∗∗; ≤0.001, ∗∗∗; ≤0.0001,∗∗∗∗. (E) Frequency of cytokine + spike-specific CD8 + T CM (CD28 + CD95 + ) and T EM (CD28 − CD95 + CCR7 − ) after V4. Scatterplots with mean values are shown. (F) Proportion of cytokine + CD8 + T cells expressing EOMES and/or T-bet (mean of 8 animals). (G) Manually gated spike-specific cytokine + <t>CD107a</t> + CD8 + T CM and T EM cells (V4 [blue], V4w2 [red], V4w3 [green]) were overlaid in histograms depicting expression of EOMES, T-bet, GzmB, and Ki67 (mean fluorescent intensity [MFI] as mean of 8 animals at each time point). Gray dotted line marks the border between the negative and positive population.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4941/pmc11994941/pmc11994941__gr2.jpg)
