Journal: Journal of visualized experiments : JoVE

Article Title: Isolation of Primary Mouse Lung Endothelial Cells

doi: 10.3791/63253

Figure Lengend Snippet: Materials

Article Snippet: T-75 cell culture flask with vent cap, CellBIND treated , Corning Life Sciences , 3290 , .

Techniques: Control, Recombinant, Sterility, Suction Filtration, Cell Culture

Journal: Bioactive Materials

Article Title: Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

doi: 10.1016/j.bioactmat.2022.07.004

Figure Lengend Snippet: hMSC expansion and metabolism on Synthemax II microcarriers in PBS Vertical-Wheel Bioreactors at 25, 40, and 64 RPM. (A) Process flow for hMSC expansion on Synthemax II microcarrers in PBS Vertical-Wheel Bioreactors; (B) hMSC growth kinetics during expansion and EV collection stages; (C) Glucose and Lactate metabolism: (i) Glucose concentration kinetics; (ii) Lactate concentration kinetics. (iii) Glucose consumed normalized to cell number; (iv) Lactate produced normalized to cell number; (v) Lactate production to glucose consumption ratios. (D) Glutamine metabolism: (i) Glutamine concentration kinetics. The extracellular vesicle (EV) collection medium is formulated with Glutamax. Therefore, the glutamine concentration at day 5 is 0. (ii) Glutamic acid concentration kinetics; (iii) Ammonia concentration kinetics.

Article Snippet: In parallel to bioreactors, Corning CellBIND T-25 cm 2 , T-75 cm 2 , and T-175 cm 2 Rectangular Canted Neck Cell Culture Flasks with Vent Cap were inoculated with hMSCs at 1250 cells/cm 2 and grown in standard culture conditions.

Techniques: Concentration Assay, Produced

Journal: Bioactive Materials

Article Title: Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

doi: 10.1016/j.bioactmat.2022.07.004

Figure Lengend Snippet: Metabolic and extracellular vesicle (EV) biogenesis pathways and reactive oxygen species (ROS) analysis for hMSCs in PBS Vertical-Wheel Bioreactors at 25, 40, and 64 RPM. Various genes were measured by RT-qPCR on the mRNAs isolated from the harvested cells at the end of EV collection (n = 6, results were from two independent bioreactor runs), relative to 2D control. (A) Glycolytic genes; (B) Autophagy genes; Flow cytometry histograms for (C) Total ROS; (D) Mitochondrial ROS; and (E) Autophagosomes. Red line: negative control; blue line: 2D culture control; orange line: 25 RPM bioreactor culture; light green: 40 RPM, and dark green: 64 RPM. (F) ESCRT-dependent EV biogenesis markers; (G) ESCRT-independent EV biogenesis markers. * indicates p < 0.05; ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: In parallel to bioreactors, Corning CellBIND T-25 cm 2 , T-75 cm 2 , and T-175 cm 2 Rectangular Canted Neck Cell Culture Flasks with Vent Cap were inoculated with hMSCs at 1250 cells/cm 2 and grown in standard culture conditions.

Techniques: Quantitative RT-PCR, Isolation, Control, Flow Cytometry, Negative Control

Journal: Bioactive Materials

Article Title: Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

doi: 10.1016/j.bioactmat.2022.07.004

Figure Lengend Snippet: Characterizations of extracellular vesicles (EVs) secreted by 0.1L bioreactor-expanded hMSCs. (A) Western Blot of exosomal marker expression, including HSC70, TSG101, CD81, Calnexin (negative marker); HRS, Syntenoin-1, CD81, and CD63. (B) Comparison of EV yield for the number of EVs per mL spent medium (n = 3), (C) Number of EVs normalized to the cell number (n = 3). (D) EV size and distribution determined based on TEM images (n = 50–80). (E) TEM images of EVs for various groups. Scale bars: 100 nm. (F) miRNA expression in the isolated EVs determined by RT-qPCR (n = 6). The value shows ΔΔCt. Results were combined from two independent bioreactor runs. *p < 0.05; **p < 0.01, ***p < 0.001.

Article Snippet: In parallel to bioreactors, Corning CellBIND T-25 cm 2 , T-75 cm 2 , and T-175 cm 2 Rectangular Canted Neck Cell Culture Flasks with Vent Cap were inoculated with hMSCs at 1250 cells/cm 2 and grown in standard culture conditions.

Techniques: Western Blot, Marker, Expressing, Comparison, Isolation, Quantitative RT-PCR

Journal: Bioactive Materials

Article Title: Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

doi: 10.1016/j.bioactmat.2022.07.004

Figure Lengend Snippet: Bioreactor-expanded hMSCs and the secreted EV protein cargo analysis by proteomics. (A) Venn diagram of the differentially expressed proteins (DEPs) from hMSCs of bioreactor culture vs. 2D culture. (B) GO annotation of the hMSC DEPs (558 as in A) for the enriched pathways. (C) Venn diagram of the DEPs for hMSC-EVs. (D) GO annotation of the hMSC-EV DEPs (616 as in C) for the enriched pathways. (E) GO annotation of the DEPs (863 as in C) for the Bioreactor only subset of hMSC-EV protein cargo.

Article Snippet: In parallel to bioreactors, Corning CellBIND T-25 cm 2 , T-75 cm 2 , and T-175 cm 2 Rectangular Canted Neck Cell Culture Flasks with Vent Cap were inoculated with hMSCs at 1250 cells/cm 2 and grown in standard culture conditions.

Techniques:

Journal: Bioactive Materials

Article Title: Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

doi: 10.1016/j.bioactmat.2022.07.004

Figure Lengend Snippet: hMSC expansion and metabolism on Synthemax II microcarriers in 0.5L PBS Vertical-Wheel Bioreactors. (A) Process flow for hMSC expansion on Synthemax II microcarrers in PBS Vertical-Wheel Bioreactors; (B) hMSC numbers during expansion and EV collection stages; (C) Glucose and Lactate metabolism: (i) Glucose concentration kinetics; (ii) Lactate concentration kinetics. (iii) Glucose consumed normalized to cell number; (iv) Lactate produced normalized to cell number; (v) Lactate production to glucose consumption ratios. (D) Glutamine metabolism: (i) Glutamine concentration kinetics. The EV collection medium is formulated with Glutamax. (ii) Glutamic acid concentration kinetics; (iii) Ammonia concentration kinetics.

Article Snippet: In parallel to bioreactors, Corning CellBIND T-25 cm 2 , T-75 cm 2 , and T-175 cm 2 Rectangular Canted Neck Cell Culture Flasks with Vent Cap were inoculated with hMSCs at 1250 cells/cm 2 and grown in standard culture conditions.

Techniques: Concentration Assay, Produced

Journal: Bioactive Materials

Article Title: Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

doi: 10.1016/j.bioactmat.2022.07.004

Figure Lengend Snippet: Metabolic and extracellular vesicle biogenesis pathways for hMSCs in 0.5L PBS Vertical-Wheel Bioreactors. Various genes were measured by RT-qPCR on the mRNAs isolated from the harvested cells at the end of EV collection (n = 6, results were from two independent bioreactor runs), relative to 0.1L bioreactor control. (A) Glycolytic genes; (B) Autophagy genes; (C) ESCRT-dependent EV biogenesis markers; (D) ESCRT-independent EV biogenesis markers. * indicates p < 0.05; ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: In parallel to bioreactors, Corning CellBIND T-25 cm 2 , T-75 cm 2 , and T-175 cm 2 Rectangular Canted Neck Cell Culture Flasks with Vent Cap were inoculated with hMSCs at 1250 cells/cm 2 and grown in standard culture conditions.

Techniques: Quantitative RT-PCR, Isolation, Control

Journal: Bioactive Materials

Article Title: Upscaling human mesenchymal stromal cell production in a novel vertical-wheel bioreactor enhances extracellular vesicle secretion and cargo profile

doi: 10.1016/j.bioactmat.2022.07.004

Figure Lengend Snippet: Characterizations of extracellular vesicle secreted by 0.5 L bioreactor-expanded hMSCs. (A) Comparison of EV production yield as the number of EVs normalized to the cell number. (B) Protein content of isolated EVs normalized to EV number. (C) Western Blot of exosomal marker expression. (D) miRNA expression in the isolated EVs determined by RT-qPCR (n = 6, results were from two independent bioreactor runs). (E) In vitro wound healing assay for the human fibroblasts that received the 2D and bioreactor hMSC-EVs. Scale bar: 100 μm. VWB: vertical-wheel bioreactor. * indicates p < 0.05; ** indicates p < 0.01, *** indicates p < 0.001.

Article Snippet: In parallel to bioreactors, Corning CellBIND T-25 cm 2 , T-75 cm 2 , and T-175 cm 2 Rectangular Canted Neck Cell Culture Flasks with Vent Cap were inoculated with hMSCs at 1250 cells/cm 2 and grown in standard culture conditions.

Techniques: Comparison, Isolation, Western Blot, Marker, Expressing, Quantitative RT-PCR, In Vitro, Wound Healing Assay