SEGURAM

Seguram
ReadyCell Communications Department
21 February 2024
ProgrammeCIEN 2019 (CDTI)
Budget505.321 Euros
Duration2019-2023 (48 months)
ConsortiumGrupo Uvesa (project leader), Idukern, Betelgeux, Incarlopsa, Biolan, Delafruit and ReadyCell

As part of the collaborative consortium SEGURAM, our focus lied within the realm of innovative strategies aimed at enhancing productivity, quality, and safety within the meat industry, while concurrently reducing reliance on antibiotics. 

Development of a gut-on-a-chip  

ReadyCell has designed and developed a microfluidic gut-on-a-chip device that mimics the key properties of the intestine to study nutrient bioavailability and product safety, aligning with the goals of the SEGURAM project framework.  

The prototype, based on PMMA (polymethyl methacrylate), integrates two independent channels separated by a PET membrane. In the upper channel, Caco-2 cells are cultured to form a monolayer on the membrane, while the lower channel is used to introduce culture medium and collect basal fluid. The prototype, optimized with 5 subunits per device and standardized size, is compatible with commonly used microscope slides (76×26 mm) and therefore, with various laboratory equipment. Additionally, the high transparency of the device allows for microscopic assays to be conducted without manipulation and disassembly.  

The evaluation of cell growth and epithelial barrier formation using microscopy techniques highlighted the adhesion and formation of a cell monolayer on the PET membrane. Lucifer Yellow (LY) permeability assay was conducted to assess barrier integrity. Results confirmed that Caco-2 cells successfully formed a well-developed epithelial barrier by the fifth day of culture, establishing this day as optimal for conducting ingredient bioavailability assays. The acceptance criteria set for this gut-on-a-chip model were Papp < 10·10-6 cm/s and flow < 0.5%. 

Concurrently, efforts included adapting ReadyCell’s Shipping Medium, originally designed for transporting our Caco-2 models in 2D plate formats, for using in the new organ-on-a-chip models and aiming to transport prototypes at room temperature.  

Bioavailability assays in gut-on-a-chip 

ReadyCell aimed to validate gut-on-a-chip devices’ use in nutrient bioavailability research. Attempts were made to correlate in vitro nutrient bioavailability assay results using feed extracts from in vivo studies with analysis of samples collected (feed and feces) from poultry farm studies. An acid-free extraction methodology was implemented to analyze nutrients and ginger components in animal feed samples provided by some consortium partners, confirming minerals and gingerol detection studies without compromising the integrity of the intestinal barrier. 

Difficulties in detecting the components of interest through analytical techniques in the in vitro samples hindered correlating the mineral absorption and gingerol impact observed in vitro with the in vivo patterns. Despite these analytical challenges, experimental procedures for gut-on-a-chip bioavailability assays were developed and optimized to minimize variability sources. Gut-on-a-chip device selection streamlined in vivo intestinal environment simulation compared to 2D plate systems and proved useful for bioavailability and efficacy assays. Yet, continued research is essential to enhance biological relevance and validate the system.

Results

Figure. Prototype of the PMMA gut-on-a-chip device. A) Upper part of the device; includes the upper channel and reservoirs. B) Lower part of the device; includes the lower channel. C) Gut-on-a-chip device assembled. D) Gut-on-a-chip device in use.
Figure. Prototype of the PMMA gut-on-a-chip device. A) Upper part of the device; includes the upper channel and reservoirs. B) Lower part of the device; includes the lower channel. C) Gut-on-a-chip device assembled. D) Gut-on-a-chip device in use.
Figure. PMMA gut-on-a-chip device seeded with Caco-2 cells. Phase-contrast microscopy images over a period of 18 days. Scale bar: 100 µm.
Figure. PMMA gut-on-a-chip device seeded with Caco-2 cells. Phase-contrast microscopy images over a period of 18 days. Scale bar: 100 µm.
Figure. Immunostaining of Caco-2 cells in PMMA gut-on-a-chip devices after 18 days of culture. Nucleus marker (DAPI) is showed in blue and actin cytoskeleton (Actin-stain™ 488 phalloidin) is showed in green. A: Upper channel seeded with Caco-2 cells. Scale bar: 1000 µm. B: Amplification of the red rectangle in image A. Scale bar: 200 µm. C: Amplification of the orange rectangle in image A. Scale bar: 200 µm.
Figure. Immunostaining of Caco-2 cells in PMMA gut-on-a-chip devices after 18 days of culture. Nucleus marker (DAPI) is showed in blue and actin cytoskeleton (Actin-stain™ 488 phalloidin) is showed in green. A: Upper channel seeded with Caco-2 cells. Scale bar: 1000 µm. B: Amplification of the red rectangle in image A. Scale bar: 200 µm. C: Amplification of the orange rectangle in image A. Scale bar: 200 µm.
Figure. Immunostaining of Caco-2 cells in PMMA gut-on-a-chip devices after 18 days of culture. Nucleus marker (DAPI) is showed in blue and actin cytoskeleton (Actin-stain™ 488 phalloidin) is showed in green. A: Upper channel seeded with Caco-2 cells. Scale bar: 1000 µm. B: Amplification of the red rectangle in image A. Scale bar: 200 µm. C: Amplification of the orange rectangle in image A. Scale bar: 200 µm.
Figure. Immunostaining of Caco-2 cells in PMMA gut-on-a-chip devices after 18 days of culture. Nucleus marker (DAPI) is showed in blue and actin cytoskeleton (Actin-stain™ 488 phalloidin) is showed in green. A: Upper channel seeded with Caco-2 cells. Scale bar: 1000 µm. B: Amplification of the red rectangle in image A. Scale bar: 200 µm. C: Amplification of the orange rectangle in image A. Scale bar: 200 µm.
Figure. Immunostaining of Caco-2 cells in PMMA gut-on-a-chip devices after 18 days of culture. Nucleus marker (DAPI) is showed in blue and actin cytoskeleton (Actin-stain™ 488 phalloidin) is showed in green. A: Upper channel seeded with Caco-2 cells. Scale bar: 1000 µm. B: Amplification of the red rectangle in image A. Scale bar: 200 µm. C: Amplification of the orange rectangle in image A. Scale bar: 200 µm.
Figure. Immunostaining of Caco-2 cells in PMMA gut-on-a-chip devices after 18 days of culture. Nucleus marker (DAPI) is showed in blue and actin cytoskeleton (Actin-stain™ 488 phalloidin) is showed in green. A: Upper channel seeded with Caco-2 cells. Scale bar: 1000 µm. B: Amplification of the red rectangle in image A. Scale bar: 200 µm. C: Amplification of the orange rectangle in image A. Scale bar: 200 µm.
Figure. Evolution of paracellular permeability for Lucifer yellow (LY) in gut-on-a-chip devices at different days of culture. The graph shows the apparent Permeability (Papp) and flux (%) over time.
Figure. Evolution of paracellular permeability for Lucifer yellow (LY) in gut-on-a-chip devices at different days of culture. The graph shows the apparent Permeability (Papp) and flux (%) over time.
Figure. Evolution of paracellular permeability for Lucifer yellow (LY) in gut-on-a-chip devices at different days of culture. The graph shows the apparent Permeability (Papp) and flux (%) over time.
Figure. Evolution of paracellular permeability for Lucifer yellow (LY) in gut-on-a-chip devices at different days of culture. The graph shows the apparent Permeability (Papp) and flux (%) over time.
Figure. Evolution of paracellular permeability for Lucifer yellow (LY) in gut-on-a-chip devices at different days of culture. The graph shows the apparent Permeability (Papp) and flux (%) over time.
Figure. Evolution of paracellular permeability for Lucifer yellow (LY) in gut-on-a-chip devices at different days of culture. The graph shows the apparent Permeability (Papp) and flux (%) over time.

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ReadyCell

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