Periodic Reporting for period 1 - BioMag (A companion magnetic sensor for in operando detection of magnetic biosynthesis in cancer and neurodegenerative models)
Période du rapport: 2022-10-01 au 2024-03-31
Iron plays a vital role in essential metabolic processes across all life forms, including oxygen transport, enzymatic functions such as DNA synthesis and repair, and overall organ growth and survival. In humans, sophisticated mechanisms regulate iron homeostasis, controlling its storage, transport, and export. Recently, the involvement of iron in diseases has garnered attention, notably in ferroptosis, a form of cell death triggered by iron-mediated oxidative damage. While ferroptosis holds promise for cancer therapy, it poses risks for neurodegenerative diseases like Alzheimer's and Parkinson's. Additionally, iron's magnetic properties, particularly in its iron oxide form, have propelled it into the forefront of nanomedicine, offering novel approaches to diagnostics and therapeutics. We recently demonstrated that human stem cells are able to produce magnetic nanoparticles. Despite its significance for understanding the presence of biogenic magnetic iron in human cells, especially in the human brain, the value of producing biogenic magnetic nanoparticles remain limited by yield, detection, and environment of the producer cells. Addressing this gap, the BioMag Proof of Concept (PoC) project aims to develop a multifunction bioreactor that can all in one mature stem cells into spheroids or organoids (e.g. cerebral), while generating biogenic magnetic nanoparticles post administration of non-magnetic iron salts, and enabling real-time magnetic measurement of the onset of such intracellular magnetic iron biogenesis. The long term goal was thus to achieve a sensitive magnetic sensor for in situ measurement coupled with cell bioreactors for 3D culture, overall facilitating long-term culture and optimized biomagnetic detection.
Several approaches have been taken, in parallel.
1) The initial proof of biosynthesis of magnetic nanoparticles by human mesenchymal stem cells (MSC) was obtained upon internalization of chemically-synthesized magnetic nanoparticles, their degradation, and the use of the iron ions released over this degradation for the biological production of new magnetic nanoparticles. A first aim was to obtain this biosynthesis from iron salts (non-magnetic), and this way be able to track the synthesis of the biological material only. To do so, we decoded the cellular response following exposure to iron II vs. III ions, continuously or in pulses, with or without the addition of trisodium citrate, and at different concentrations. The incorporation of iron into cells, cell viability, and the potential triggering of magnetic biosynthesis were analyzed. A preliminary kinetics of iron incorporation and magnetic particle biosynthesis was obtained during continuous exposure to iron quinate at 34 µM in human and mouse MSCs. The results indicate an increase in internalized iron over time and the appearance of a detectable magnetic moment after 14 days of culture, when cells contain more than 1.6 pgFe. However, the magnetic moments remain low, around 10-12 emu/cell after 36 days of incubation but provide initial evidence of magnetic biosynthesis from iron ions. These results have been published in Nanoscale 2023, 15, 10097–10109, as Biomineralization of magnetic nanoparticles in stem cells, by A Fromain, A Van de Walle, G Curé, C Péchoux, A Serrano, Y Lalatonne, A Espinosa and C Wilhelm.
2) Since the biosynthesis obtained from iron salts remains much weaker than that obtained from a precursor in nanoparticle form, we went back to monitoring, over time, the biotransformations of magnetic nanoparticles in human cells. The aim is to deepen our understanding of the mechanisms leading to the biosynthesis of magnetic nanoparticles. This monitoring was carried out using a magnetic sensor enabling in situ measurements and time tracking on living cells. The impact of the spatial conformation of the cells (2D vs. 3D), of components present in the culture medium, and of the addition of iron chelators (e.g. citrate) was assessed. Results indicate that the spatial conformation and metabolic activity of the cells during labeling play an important role in the amplitude and timing of biosynthesis. An article on these studies is currently being finalized.
3) Moreover, the magnetic sensor has been coupled with an automatized microfluidic setup allowing the formation of droplets that can be collected and perfused through the magnetic sensor at a given speed rate. This way, magnetic measurements can be obtained in series, from droplets containing cells or solutions.
4) Concerning the design and fabrication of 3D cell bioreactors, it was successful, and allowed the maturation of stem cells into spheroids within. Besides, a different option was selected than a blade to gently mix the cells within (still confidential).
5) Finally, magnetic spheroids were produced from primary neurons and glial cells to set-up the mini-brain model, published in Advanced Science 10, 2302411 (2023) as Surface Tension and Neuronal Sorting in Magnetically Engineered Brain‐Like Tissue, by JE Perez, A Jan, C Villard, and C Wilhelm.
1) The initial proof of biosynthesis of magnetic nanoparticles by human mesenchymal stem cells (MSC) was obtained upon internalization of chemically-synthesized magnetic nanoparticles, their degradation, and the use of the iron ions released over this degradation for the biological production of new magnetic nanoparticles. A first aim was to obtain this biosynthesis from iron salts (non-magnetic), and this way be able to track the synthesis of the biological material only. To do so, we decoded the cellular response following exposure to iron II vs. III ions, continuously or in pulses, with or without the addition of trisodium citrate, and at different concentrations. The incorporation of iron into cells, cell viability, and the potential triggering of magnetic biosynthesis were analyzed. A preliminary kinetics of iron incorporation and magnetic particle biosynthesis was obtained during continuous exposure to iron quinate at 34 µM in human and mouse MSCs. The results indicate an increase in internalized iron over time and the appearance of a detectable magnetic moment after 14 days of culture, when cells contain more than 1.6 pgFe. However, the magnetic moments remain low, around 10-12 emu/cell after 36 days of incubation but provide initial evidence of magnetic biosynthesis from iron ions. These results have been published in Nanoscale 2023, 15, 10097–10109, as Biomineralization of magnetic nanoparticles in stem cells, by A Fromain, A Van de Walle, G Curé, C Péchoux, A Serrano, Y Lalatonne, A Espinosa and C Wilhelm.
2) Since the biosynthesis obtained from iron salts remains much weaker than that obtained from a precursor in nanoparticle form, we went back to monitoring, over time, the biotransformations of magnetic nanoparticles in human cells. The aim is to deepen our understanding of the mechanisms leading to the biosynthesis of magnetic nanoparticles. This monitoring was carried out using a magnetic sensor enabling in situ measurements and time tracking on living cells. The impact of the spatial conformation of the cells (2D vs. 3D), of components present in the culture medium, and of the addition of iron chelators (e.g. citrate) was assessed. Results indicate that the spatial conformation and metabolic activity of the cells during labeling play an important role in the amplitude and timing of biosynthesis. An article on these studies is currently being finalized.
3) Moreover, the magnetic sensor has been coupled with an automatized microfluidic setup allowing the formation of droplets that can be collected and perfused through the magnetic sensor at a given speed rate. This way, magnetic measurements can be obtained in series, from droplets containing cells or solutions.
4) Concerning the design and fabrication of 3D cell bioreactors, it was successful, and allowed the maturation of stem cells into spheroids within. Besides, a different option was selected than a blade to gently mix the cells within (still confidential).
5) Finally, magnetic spheroids were produced from primary neurons and glial cells to set-up the mini-brain model, published in Advanced Science 10, 2302411 (2023) as Surface Tension and Neuronal Sorting in Magnetically Engineered Brain‐Like Tissue, by JE Perez, A Jan, C Villard, and C Wilhelm.
Besides basics advancements in the understanding of human stem cells magnetic biomineralization, the project main results in terms of demonstration, access to markets, commercialization is the development of the novel 3D cell culture bioreactor, with patent filed in 2023. Not only this new device allowed spheroids formation and maturation, but also it can be used for extracellular vesicles production from the stem cells within. Another patent was filed in 2024, deriving from these results.