Periodic Reporting for period 1 - MIGMIPS (Micro-guts for the study of translocation of microplastic)
Période du rapport: 2021-05-01 au 2023-04-30
Plastic waste dumped into the environment is in constant progression, due to the increasing use of plastic and the low percentage of recycling (9%). Between 4.8 and 12.7 millions of cubic meters of plastic end in the oceans every year, and get degraded by the effect of multiple factors (UV, mechanical abrasion by wind or waves, bacterial digestion) into particles with sizes under 5 mm, called microplastics, or under 1 μm, called nanoplastics. Micro/nanoplastics (MNplastics) have already been detected in the oceans, surface water, drinking water, as well as in phytoplanctons, molluscs and fishes which are usually eaten by humans. MNplastics could be easily ingested and inhaled by animals and humans and have been found in human stool, whereas polystyrene microspheres were accumulating in internal organs of mice. Several studies showed that non-plastic nanoparticles could easily pass through the biological barriers (lung, gut, skin) and accumulate in organs. Studying the toxicity on internal organs of real environmental MNplastics, instead of simplistic model particles, is urgent. To achieve this goal, an important prerequisite is to identify which MNplastics are able to pass through the biological barriers, enter the blood circulation and reach the whole body.
In this project I focused on the gut barrier, which is the entry door to all the MNplastics found in food or drinking waters. In order to study these translocations, in-vivo experiments with animals, mostly mice, should be reduced due to ethical and practical reasons: results are long to obtain, are time and resource consuming and the physiology of the gut varies a lot between species, leading to results which are not applicable to humans. Characterizing and detecting heterogeneous MNplastics accumulated in biological tissues is still a huge challenge, and only limited information could be obtained after the sacrifice of the animals. For all these reasons, a lot of experiments testing the toxicity and translocation of nanoparticles are done in-vitro with a better control of the experimental conditions, for example “Transwell” systems, where an epithelium (first layer of the gut barrier) is cultured on a rigid, porous and plastic membrane. However, it has been shown that these Transwells are too simplistic compared to the real gut barrier. More complex in-vitro systems are then necessary. Such tools, called microphysiological systems (MPS) or organs-on-chip, have already proved to give more reliable results compared to more simplistic classical in-vitro systems. However, to our knowledge, such systems has never been developed to specifically study the translocation of environmental MNplastics through the gut.
Research questions and objectives:
In this project, called MIGMIPS (Micro Gut for MIPlastic Studies), I proposed to measure which quantity of MNplastics are able to pass through the gut, using advanced MPS and detection techniques, with the following specific objectives:
- developing a MPS of the gut barrier including a reconstructed 3D epithelium, on a thin extracellular matrix, while remaining easy-to-use and to manipulate, and compatible with both open-surface sensing and microfluidics,
- optimizing detection and characterization techniques of MNplastics in culture medium, using Raman spectroscopy, electronic and optical microscopy,
- combining the two previous tools to measure the translocation rate through the gut barrier in function of the MNplastics characteristics (size, material, shape), with a first validation step using model fluorescent particles and confocal microscopy,
At the end of the project, we developed a new method for fabricating scaffolds with large pores, using high-resolution 3D printing, allowing the creation of nets with fibres of 200 nanometers and pores of 1 to 10 micrometers. We succeeded to print these nets on frames which could be secured on classical cell culture well plate, or inserted inside 3D printed microfluidic devices in order to perform several actions: seeding of gut cells on the nets, culture of this epithelium, characterization by optical microscopy. After seeding and culture, gut cells formed a tight epithelium reminiscent to the gut epithelium. We also optimized the purification of micro and nanoparticules from cell culture medium to de-ionized water using filtration and dialysis. We then concentrated and evaporated 6μl of NPs-containing water on super-hydrophobic surfaces, on which we could count the particles by fluorescence and image with electronic microscopy and Raman spectroscopy the larger particles. The study of the translocation of MNplastics through the created gut model did not start before the end of the project, but will be implemented in the next months as the main beneficiary of this grant, Dr. Bastien Venzac, got a permanent position in his host laboratory.
In this project I focused on the gut barrier, which is the entry door to all the MNplastics found in food or drinking waters. In order to study these translocations, in-vivo experiments with animals, mostly mice, should be reduced due to ethical and practical reasons: results are long to obtain, are time and resource consuming and the physiology of the gut varies a lot between species, leading to results which are not applicable to humans. Characterizing and detecting heterogeneous MNplastics accumulated in biological tissues is still a huge challenge, and only limited information could be obtained after the sacrifice of the animals. For all these reasons, a lot of experiments testing the toxicity and translocation of nanoparticles are done in-vitro with a better control of the experimental conditions, for example “Transwell” systems, where an epithelium (first layer of the gut barrier) is cultured on a rigid, porous and plastic membrane. However, it has been shown that these Transwells are too simplistic compared to the real gut barrier. More complex in-vitro systems are then necessary. Such tools, called microphysiological systems (MPS) or organs-on-chip, have already proved to give more reliable results compared to more simplistic classical in-vitro systems. However, to our knowledge, such systems has never been developed to specifically study the translocation of environmental MNplastics through the gut.
Research questions and objectives:
In this project, called MIGMIPS (Micro Gut for MIPlastic Studies), I proposed to measure which quantity of MNplastics are able to pass through the gut, using advanced MPS and detection techniques, with the following specific objectives:
- developing a MPS of the gut barrier including a reconstructed 3D epithelium, on a thin extracellular matrix, while remaining easy-to-use and to manipulate, and compatible with both open-surface sensing and microfluidics,
- optimizing detection and characterization techniques of MNplastics in culture medium, using Raman spectroscopy, electronic and optical microscopy,
- combining the two previous tools to measure the translocation rate through the gut barrier in function of the MNplastics characteristics (size, material, shape), with a first validation step using model fluorescent particles and confocal microscopy,
At the end of the project, we developed a new method for fabricating scaffolds with large pores, using high-resolution 3D printing, allowing the creation of nets with fibres of 200 nanometers and pores of 1 to 10 micrometers. We succeeded to print these nets on frames which could be secured on classical cell culture well plate, or inserted inside 3D printed microfluidic devices in order to perform several actions: seeding of gut cells on the nets, culture of this epithelium, characterization by optical microscopy. After seeding and culture, gut cells formed a tight epithelium reminiscent to the gut epithelium. We also optimized the purification of micro and nanoparticules from cell culture medium to de-ionized water using filtration and dialysis. We then concentrated and evaporated 6μl of NPs-containing water on super-hydrophobic surfaces, on which we could count the particles by fluorescence and image with electronic microscopy and Raman spectroscopy the larger particles. The study of the translocation of MNplastics through the created gut model did not start before the end of the project, but will be implemented in the next months as the main beneficiary of this grant, Dr. Bastien Venzac, got a permanent position in his host laboratory.
In this project, called MIGMIPS (Micro Gut for MIcroPlastic Studies), we proposed to measure the translocation rate of MNplastics through the gut, using an advanced microphysiological system (MPS) and detection techniques that had to be developed in a first time.
- Gut microphysiological system:
In order to allow the translocation of MNplastics through the gut barrier model, gut cells have to be cultured on a porous membrane with large pores, which can be shaped in 3D like the folded inner layer of the gut. We developed a new method for fabricating such scaffolds, using high-resolution 3D printing, allowing the creation of nets with fibres of 200 nanometers and pores of 1 to 10 micrometers. We succeeded to print these nets on frames which could be secured on classical cell culture well plate, or inserted inside 3D printed microfluidic devices in order to perform several actions: seeding of gut cells on the nets, culture of this epithelium, characterization by optical microscopy.
After seeding and culture, gut cells formed a tight epithelium reminiscent to the gut epithelium.
- Detection and characterization of MNplastics
In parallel, we worked on the detection and characterization of microplastics below 5μm of diameter, which turned out to be extremely difficult to detect and characterize, while most of the techniques used in the microplastics field only work for larger microplastics. In particular, we optimized the purification of micro and nanoparticules from cell culture medium to de-ionized water using filtration and dialysis. We then concentrated and evaporated 6μl of NPs-containing water on super-hydrophobic surfaces, on which we could count the particles by fluorescence and image with electronic microscopy and Raman spectroscopy the larger particles.
- Gut microphysiological system:
In order to allow the translocation of MNplastics through the gut barrier model, gut cells have to be cultured on a porous membrane with large pores, which can be shaped in 3D like the folded inner layer of the gut. We developed a new method for fabricating such scaffolds, using high-resolution 3D printing, allowing the creation of nets with fibres of 200 nanometers and pores of 1 to 10 micrometers. We succeeded to print these nets on frames which could be secured on classical cell culture well plate, or inserted inside 3D printed microfluidic devices in order to perform several actions: seeding of gut cells on the nets, culture of this epithelium, characterization by optical microscopy.
After seeding and culture, gut cells formed a tight epithelium reminiscent to the gut epithelium.
- Detection and characterization of MNplastics
In parallel, we worked on the detection and characterization of microplastics below 5μm of diameter, which turned out to be extremely difficult to detect and characterize, while most of the techniques used in the microplastics field only work for larger microplastics. In particular, we optimized the purification of micro and nanoparticules from cell culture medium to de-ionized water using filtration and dialysis. We then concentrated and evaporated 6μl of NPs-containing water on super-hydrophobic surfaces, on which we could count the particles by fluorescence and image with electronic microscopy and Raman spectroscopy the larger particles.
Both the MPS and the MNplastics detection are beyond the state of the art. Unfortunately, the study of the translocation of MNplastics through the gut barrier using these results did not start during the project duration due to delays in the development of the gut MPS. However, as the main beneficiary of this fellowship (Bastien Venzac) got a permanent position in his host laboratory, this project will go on during the next years until significant results on the behaviour of MNplastics could be obtained.