Journal: The EMBO Journal

Article Title: Photoreceptor nanotubes mediate the in vivo exchange of intracellular material

doi: 10.15252/embj.2020107264

Figure Lengend Snippet: A, B Examples of live Nrl::GFP and C57BL/6J retinal cultures showing actin + (SirActin) and GFP + (A) and tubulin + (ViaFluor) and MitoTracker Green + (MTG) protrusions (B) connecting photoreceptors. DIC, differential interference contrast microscopy. C Quantification of photoreceptor protrusions in Nrl::GFP retinal cultures (culture wells, n = 8, images, n = 24, protrusions, n = 868). Data are presented as mean ± SEM. D Images from FRAP experiments carried out on protrusion connected and isolated Nrl::GFP cultures pre‐ and post‐photobleaching. Area of photobleaching (white dashed circles). Analysed regions (yellow, orange and red dashed circles). Photoreceptor FRAP results shown from multiple experiments (isolated cells, n = 14; connected to recipient, n = 21; connected donor, n = 21). All data presented as percentage, mean ± SEM and fitted to the curve in a non‐linear regression with two‐phase association. E Time lapse imaging showing movement of MitoTracker Green + puncta in photoreceptors connected by a protrusion. White arrows point to MTG + puncta inside of the protrusion during the time lapse imaging. Data information: n.s. not statistically significant, **** P ≤ 0.0001 and *** P < 0.001; unpaired t ‐test and extra‐sum‐of‐squares F ‐test (Y0 constrained to 0.0). Scale bars: 5 μm. Source data are available online for this figure.

Article Snippet: To live‐image actin or microtubules, cultures were supplemented overnight with SiR‐Actin (100 μM) (CY‐SC001, Cytoskeleton Inc. Denver, CO, USA) or ViaFluor ® (50 μM) (70062, Biotium, Fremont, CA, USA).

Techniques: Microscopy, Isolation, Imaging

Journal: Scientific Reports

Article Title: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

doi: 10.1038/s41598-020-67039-w

Figure Lengend Snippet: Secreted Factors following Perfusion of Activated PBMCs through MSC-Bioreactor Circuits. PBMCs activated with PHA (5 ug/mL) and supplemented with IL-2 (100 ng/mL) were perfused through bioreactor circuits containing 0 M or 9 M (n = 3) and medium was recovered. Both ( A ) factor concentration and ( B ) extracellular vesicle characteristics were modulated with MSC treatment.

Article Snippet: Viafluor labeled PBMCs acquired from AllCells (Quincy, Massachusetts) were resuspended at 2.7 ×10 6 cells per mL in supplemented RPMI 1640 media then stimulated with 5 ug/mL PHA and 100 ng/mL IL-2 (all Sigma-Aldrich).

Techniques: Concentration Assay

Journal: Scientific Reports

Article Title: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

doi: 10.1038/s41598-020-67039-w

Figure Lengend Snippet: Prevention of lymphocyte activation in MSC bioreactors. PBMCs were labeled with ViaFluor, stimulated (PHA/IL-2) and perfused for 5-days via circuits containing microreactors seeded with 0, or 9 ×10 6 MSCs per device. MSC treatment was shown to inhibit T-cell proliferation as demonstrated by ViaFluor labelling (A) . This was observed for four PBMC donors (n = 4 donors, n = 3/donor) (B) . Percent decrease in PBMC proliferation with MSC (9 M) is indicated. Perfusion via MSC-seeded microreactors (9 M) exhibited changes in CD4, CD8, and CD19 + lymphocytes when compared to acellular controls (0 M) (n = 2 donors, n = 3/donor) (C) . Graphs show average values for each cell dose group + standard deviations for two donors. A student’s t-test was performed on each set. **p ≤ 0.01 *p ≤ 0.05.

Article Snippet: Viafluor labeled PBMCs acquired from AllCells (Quincy, Massachusetts) were resuspended at 2.7 ×10 6 cells per mL in supplemented RPMI 1640 media then stimulated with 5 ug/mL PHA and 100 ng/mL IL-2 (all Sigma-Aldrich).

Techniques: Activation Assay, Labeling

Journal: Scientific Reports

Article Title: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

doi: 10.1038/s41598-020-67039-w

Figure Lengend Snippet: MSC bioreactor reprogramming of T cell cytokine responses. Stimulated (PHA/IL-2) PBMCs perfused in a bioreactor (+/− MSC) for 5-days were plated on a 6-well tissue culture dish for two additional days ( A ). Lymphocyte secreted factors (in the supernatant) were assayed using multiplex cytokine panel. Th1/Th2 specific cytokines are shown as a percentage change with MSC (9 M) treatment relative to acellular (0 M MSC) (n = 1 donor, 3 runs/donor) ( B ).

Article Snippet: Viafluor labeled PBMCs acquired from AllCells (Quincy, Massachusetts) were resuspended at 2.7 ×10 6 cells per mL in supplemented RPMI 1640 media then stimulated with 5 ug/mL PHA and 100 ng/mL IL-2 (all Sigma-Aldrich).

Techniques: Multiplex Assay

Journal: Scientific Reports

Article Title: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

doi: 10.1038/s41598-020-67039-w

Figure Lengend Snippet: Dose and duration effects of ex vivo MSC perfusion on human lymphocytes ( A ) Stimulated (PHA/IL-2) PBMCs were perfused for either 24 hours, 72 hours or for 5 days through circuits containing microreactors seeded with either 0, 3, or 9 ×10 6 MSCs per device (0 M, 3 M, 9 M) (n = 2 donors, n ≥ 3/donor) (5 day historical only has one donor). The 24- and 72-hour perfusion groups were first placed into static culture for 24 hours prior to perfusion initiation. Each group was perfused for the designated time and then placed into static culture until collection on Day 5. Relative to 0 M control MSC treatment was shown to inhibit lymphocyte proliferation in all conditions ( B ), with a trend correlating with MSC dose response. CD8 + T cell proliferation was also inhibited by perfusion ( C ) while B-cell proliferation increased ( E ) in a dose and duration dependent manner for each subpopulation. A student’s t-test was performed on each set. ****p ≤ 0.0001 ***p ≤ 0.001 **p ≤ 0.01 *p ≤ 0.05. n.s. = not significant. Graphs show average values for each cell dose + standard deviation. ( F ) Culture media samples were collected at Day 5 and analyzed via multiplex. Measurement of percent change was calculated by determining the output of any condition relative to the 0 M control. The absolute value of the percent change was then charted into columns according to MSC dose and perfusion duration. Comparative analysis of intensities were calculated within each row with darker colors representing larger values. Red blocks indicate decreases in percent change while green blocks indicate increases. Of all conditions, the 9 M MSC 24-hour perfusion group showed the largest changes in analyte values (n = 1 donor).

Article Snippet: Viafluor labeled PBMCs acquired from AllCells (Quincy, Massachusetts) were resuspended at 2.7 ×10 6 cells per mL in supplemented RPMI 1640 media then stimulated with 5 ug/mL PHA and 100 ng/mL IL-2 (all Sigma-Aldrich).

Techniques: Ex Vivo, Control, Standard Deviation, Multiplex Assay

Journal: Scientific Reports

Article Title: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

doi: 10.1038/s41598-020-67039-w

Figure Lengend Snippet: Transfer of MSC bioreactor/PBMC perfusate alter primary human monocyte differentiation. MSC reprogrammed PBMCs (PHA/IL-2 activated) perfusate/supernatant from a 5-day bioreactor (with −/+ MSC) was added on monocytes cultured on a cell-repellent tissue culture dish for two days ( A ). After two days, monocytes were stained using CD14 and CD16 antibodies and dot blots are shown ( B ) and percentage change in monocyte subsets with and without MSC-reprogrammed PBMC is shown (n = 2 donors, n = 3/donor) ( C ). The levels of TNFα and IL-10 in the supernatant of monocytes after two-days of addition of PBMCs perfusate from circuits with or without MSCs is plotted as percentage change with MSC addition (n = 2 donors, n = 3/donor) ( D ).

Article Snippet: Viafluor labeled PBMCs acquired from AllCells (Quincy, Massachusetts) were resuspended at 2.7 ×10 6 cells per mL in supplemented RPMI 1640 media then stimulated with 5 ug/mL PHA and 100 ng/mL IL-2 (all Sigma-Aldrich).

Techniques: Cell Culture, Staining

Journal: Scientific Reports

Article Title: Mesenchymal Stromal Cell Bioreactor for Ex Vivo Reprogramming of Human Immune Cells

doi: 10.1038/s41598-020-67039-w

Figure Lengend Snippet: Role of monocytes in MSC bioreactor immune reprogramming. PHA/IL-2 stimulated PBMCs were cultured in static tissue culture flasks for 4-days. On day 2 and day 4, monocytes (~2.8 M/ bioreactor) were added into PBMC flask (in the monocyte addition group in gray). At day 4, after addition of monocytes PBMCs were perfused in bioreactor for 24 hours via circuits seeded with 0, or 9 ×10 6 MSCs per device ( A ). Following 24-hours of perfusion, the cells were assayed via flow cytometry ( B ) and supernatant was assayed for TNFα and IL-10 levels both without and with monocyte addition ( C ). Overall, the addition of monocytes to the PBMCs enhanced the MSC immunomodulatory effects as shown by changes in lymphocyte population, TNFα and IL-10 levels ( D ). A student’s t-test was performed on each set (**p ≤ 0.01 *p ≤ 0.05). Graphs show average values + standard deviation (n = 1 donors, n = 3/donor).

Article Snippet: Viafluor labeled PBMCs acquired from AllCells (Quincy, Massachusetts) were resuspended at 2.7 ×10 6 cells per mL in supplemented RPMI 1640 media then stimulated with 5 ug/mL PHA and 100 ng/mL IL-2 (all Sigma-Aldrich).

Techniques: Cell Culture, Flow Cytometry, Standard Deviation