c5011 hollow fiber bioreactor  (FiberCell Systems)

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mesenchymal stem cell-derived exosome subpopulations remained consistent for 28 culture days, displaying therapeutic effects in a silicosis mouse model

doi: 10.3389/fcell.2025.1550447

Figure Lengend Snippet: Monitoring of culture parameters during exosome production in a hollow fiber bioreactor demonstrates the feasibility and consistency of maintaining cellular homeostasis for long-term exosome preparation. (A) Schematic diagram illustrating the entire workflow of EV production by hUC-MSCs in the hollow fiber bioreactor system. The hollow fiber cell culture system requires a 4-day preparation period, followed by cell seeding, wherein PBS circulates for 2 days, followed by complete cell culture medium MSC-XF circulation for 2 days. On Day 0, hUC-MSCs are seeded into the hollow fiber cartridges. After a 3-day cell seeding period, MSC-EV culture medium replaces the inner and outer circulating MSC-XF medium. From Day 4 to Day 28, a 25-day EV production period begins, with 2 mL of culture medium extracted daily from the external circulation for monitoring glucose concentration and pH. Every 2 days, 20 mL of EV-rich cell-conditioned medium is collected from the internal circulation and frozen for future EV isolation and analysis. On Day 28, new parent hUC-MSCs are seeded into their respective hollow fiber cartridges, with both inner and outer circulating media being MSC-XF, and on Day 30, the circulating media are changed to MSC-EV culture medium. From Day 31 to Day 60, a 30-day EV production period begins, with monitoring and sample collection conducted in the same manner as in the first production cycle. (B) Daily monitoring of glucose concentration in the circulating culture medium to ensure continuous glucose consumption by the cells. Two data points per day represent glucose supplementation by adding fresh cell culture medium. pH measurement using circulating culture medium samples ensures that cells do not undergo metabolic stress. Red represents hUC-MSC-0214 cells, and blue represents hUC-MSC-1103 cells. (C) Comparison of glucose consumption rates before and after media change. Black represents hUC-MSC-0214 cells and red represents hUC-MSC-1103 cells. (D) Nanoparticle concentration of EV-rich fluid harvested from the internal circulation at different time points. Black represents hUC-MSC-0214 cells and red represents hUC-MSC-1103 cells. (E) Number of particles secreted per single cell harvested during different production cycles. Black represents hUC-MSC-0214 cells and red represents hUC-MSC-1103 cells.

Article Snippet: The Fiber Cell hollow fiber bioreactor system (FiberCell Systems, C2011), which has an internal circulating volume of 500 mL and an extra-capillary space (ECS) volume of 20 mL, was prepared according to the manufacturer’s instructions.

Techniques: Cell Culture, Concentration Assay, Isolation, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mesenchymal stem cell-derived exosome subpopulations remained consistent for 28 culture days, displaying therapeutic effects in a silicosis mouse model

doi: 10.3389/fcell.2025.1550447

Figure Lengend Snippet: Fine separation of exosomes achieved using tangential flow filtration combined with strong anion exchange chromatography. (A) Schematic diagram illustrating the process of fine separation of exosomes, with clarification using a 0.65 μm filter, sterilization using a 0.8/0.2 μm filter, tangential flow filtration using a 300 kDa membrane, and strong anion exchange chromatography column using CIMmultus™ EV. (B) Particle size distribution of exosome concentrates harvested from different exosome separation methods as detected by TRPS. Blue represents the original solution of mixed exosomes from Hollow Fiber hUC-MSC-0214 Day 4–40 before separation, red represents exosome concentrates after tangential flow filtration combined with strong anion exchange chromatography, and green represents nanoparticle concentrates after enrichment by Exodus. (C) Detection of exosome marker proteins in nanoparticle-enriched fluid after separation by TFF + BIA Strong AEX and Exodus using Wes Protein Simple. “M” denotes Marker, “1” represents mixed original sample 1 of hUC-MSC-0214, “2” represents exosome-enriched fluid after separation by TFF + BIA-EV, “3” represents mixed original sample 2 of hUC-MSC-0214, and “4” represents nanoparticle-enriched fluid after separation by Exodus. Exosome-specific positively expressed proteins include CD9, CD63, CD81, and TSG101. (D) Electron microscopy images showing typical exosome structures of enriched particles in the mixed original sample of hUC-MSC-0214, exosome-enriched fluid after separation by TFF + BIA-EV, and nanoparticle-enriched fluid after separation by Exodus at resolutions of 500 nm, 200 nm, and 100 nm. (E) Particle size distribution of exosomes harvested from hUC-MSC-0214 under different culture media and conditions, with all detected samples being nanoparticle-enriched fluid after Exodus enrichment. “2D XF” represents a mixture of cell supernatants from MSC-XF culture Day 1–3, “2D EV” represents a mixture of cell supernatants from Day 4 to 10, “3D XF” represents a mixture of cell supernatants from Hollow Fiber bioreactor Day 1–3, and “3D EV” represents a mixture of cell supernatants from Day 4 to 40. (F) Particle size distribution of exosomes harvested from hUC-MSC-0214 at different time stages in the Hollow Fiber bioreactor, with all detected samples being nanoparticle-enriched fluid after Exodus enrichment. “3D EV 5–15” represents a mixture of cell supernatants from Day 5 to 15, “3D EV 15–25” represents a mixture of cell supernatants from Day 15 to 25, “3D EV 25–35” represents a mixture of cell supernatants from Day 25 to 35, and “3D EV 35–45” represents a mixture of cell supernatants from Day 35 to 45.

Article Snippet: The Fiber Cell hollow fiber bioreactor system (FiberCell Systems, C2011), which has an internal circulating volume of 500 mL and an extra-capillary space (ECS) volume of 20 mL, was prepared according to the manufacturer’s instructions.

Techniques: Filtration, Chromatography, Clarification Assay, Membrane, Marker, Electron Microscopy

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mesenchymal stem cell-derived exosome subpopulations remained consistent for 28 culture days, displaying therapeutic effects in a silicosis mouse model

doi: 10.3389/fcell.2025.1550447

Figure Lengend Snippet: Monitoring of culture parameters during exosome production in a hollow fiber bioreactor demonstrates the feasibility and consistency of maintaining cellular homeostasis for long-term exosome preparation. (A) Schematic diagram illustrating the entire workflow of EV production by hUC-MSCs in the hollow fiber bioreactor system. The hollow fiber cell culture system requires a 4-day preparation period, followed by cell seeding, wherein PBS circulates for 2 days, followed by complete cell culture medium MSC-XF circulation for 2 days. On Day 0, hUC-MSCs are seeded into the hollow fiber cartridges. After a 3-day cell seeding period, MSC-EV culture medium replaces the inner and outer circulating MSC-XF medium. From Day 4 to Day 28, a 25-day EV production period begins, with 2 mL of culture medium extracted daily from the external circulation for monitoring glucose concentration and pH. Every 2 days, 20 mL of EV-rich cell-conditioned medium is collected from the internal circulation and frozen for future EV isolation and analysis. On Day 28, new parent hUC-MSCs are seeded into their respective hollow fiber cartridges, with both inner and outer circulating media being MSC-XF, and on Day 30, the circulating media are changed to MSC-EV culture medium. From Day 31 to Day 60, a 30-day EV production period begins, with monitoring and sample collection conducted in the same manner as in the first production cycle. (B) Daily monitoring of glucose concentration in the circulating culture medium to ensure continuous glucose consumption by the cells. Two data points per day represent glucose supplementation by adding fresh cell culture medium. pH measurement using circulating culture medium samples ensures that cells do not undergo metabolic stress. Red represents hUC-MSC-0214 cells, and blue represents hUC-MSC-1103 cells. (C) Comparison of glucose consumption rates before and after media change. Black represents hUC-MSC-0214 cells and red represents hUC-MSC-1103 cells. (D) Nanoparticle concentration of EV-rich fluid harvested from the internal circulation at different time points. Black represents hUC-MSC-0214 cells and red represents hUC-MSC-1103 cells. (E) Number of particles secreted per single cell harvested during different production cycles. Black represents hUC-MSC-0214 cells and red represents hUC-MSC-1103 cells.

Article Snippet: We established a 28-day biomanufacturing workflow using a Hollow Fiber 3D bioreactor integrated with the RoosterBio exosome-harvesting system.

Techniques: Cell Culture, Concentration Assay, Isolation, Comparison

Journal: Frontiers in Cell and Developmental Biology

Article Title: Mesenchymal stem cell-derived exosome subpopulations remained consistent for 28 culture days, displaying therapeutic effects in a silicosis mouse model

doi: 10.3389/fcell.2025.1550447

Figure Lengend Snippet: Fine separation of exosomes achieved using tangential flow filtration combined with strong anion exchange chromatography. (A) Schematic diagram illustrating the process of fine separation of exosomes, with clarification using a 0.65 μm filter, sterilization using a 0.8/0.2 μm filter, tangential flow filtration using a 300 kDa membrane, and strong anion exchange chromatography column using CIMmultus™ EV. (B) Particle size distribution of exosome concentrates harvested from different exosome separation methods as detected by TRPS. Blue represents the original solution of mixed exosomes from Hollow Fiber hUC-MSC-0214 Day 4–40 before separation, red represents exosome concentrates after tangential flow filtration combined with strong anion exchange chromatography, and green represents nanoparticle concentrates after enrichment by Exodus. (C) Detection of exosome marker proteins in nanoparticle-enriched fluid after separation by TFF + BIA Strong AEX and Exodus using Wes Protein Simple. “M” denotes Marker, “1” represents mixed original sample 1 of hUC-MSC-0214, “2” represents exosome-enriched fluid after separation by TFF + BIA-EV, “3” represents mixed original sample 2 of hUC-MSC-0214, and “4” represents nanoparticle-enriched fluid after separation by Exodus. Exosome-specific positively expressed proteins include CD9, CD63, CD81, and TSG101. (D) Electron microscopy images showing typical exosome structures of enriched particles in the mixed original sample of hUC-MSC-0214, exosome-enriched fluid after separation by TFF + BIA-EV, and nanoparticle-enriched fluid after separation by Exodus at resolutions of 500 nm, 200 nm, and 100 nm. (E) Particle size distribution of exosomes harvested from hUC-MSC-0214 under different culture media and conditions, with all detected samples being nanoparticle-enriched fluid after Exodus enrichment. “2D XF” represents a mixture of cell supernatants from MSC-XF culture Day 1–3, “2D EV” represents a mixture of cell supernatants from Day 4 to 10, “3D XF” represents a mixture of cell supernatants from Hollow Fiber bioreactor Day 1–3, and “3D EV” represents a mixture of cell supernatants from Day 4 to 40. (F) Particle size distribution of exosomes harvested from hUC-MSC-0214 at different time stages in the Hollow Fiber bioreactor, with all detected samples being nanoparticle-enriched fluid after Exodus enrichment. “3D EV 5–15” represents a mixture of cell supernatants from Day 5 to 15, “3D EV 15–25” represents a mixture of cell supernatants from Day 15 to 25, “3D EV 25–35” represents a mixture of cell supernatants from Day 25 to 35, and “3D EV 35–45” represents a mixture of cell supernatants from Day 35 to 45.

Article Snippet: We established a 28-day biomanufacturing workflow using a Hollow Fiber 3D bioreactor integrated with the RoosterBio exosome-harvesting system.

Techniques: Filtration, Chromatography, Clarification Assay, Membrane, Marker, Electron Microscopy