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  MaTissE  
Magnetic Approaches to Tissue Mechanics and Engineering

ERC consolidator grant #648779

PI: Claire Wilhelm

Cell therapies and medically oriented nanotechnologies are currently among promising biotechnologies. One attractive approach is to associate magnetic nanoparticles with cells in order to supply them with sufficient magnetization to be detectable by MRI,  manipulated by magnetic forces, or treated with therapeutic hyperthermia.
In MaTisse, we explored nanoparticles internalization in (human stem) cells. We investigated how intracellular magnetic nanoparticles allow applications of tissue engineering mediated by cellular magnetic force to mimic the most closely multicellular organisations found in the living. The aim is to confine (stem) cells in three dimensions at the millimetric scale by using home-designed miniaturized magnetic devices, in order to create cellular patterns for stem cell differentiation and tissue engineering. In parallel, we monitored the becoming of the nanoparticles after their internalization, and evidenced not only a massive biodegradation, but also a possible re-crystallisation of biogenic magnetic nanoparticles by the cells themselves.


project table of content

MAGNETIC TISSUE ENGINEERING

Magnetic tissue stretcher

We have built a magnetic tissue stretcher which provides a versatile tissue stimulator: frequency, longitudinal extension, speed of displacement and force amplitude can be varied at will. A 750 µm diameter soft iron tip magnetized either by a permanent magnet is first used to form a cell aggregate. A second identical tip is then approached. With the permanent magnet, the aggregate is rapidly deformed into a rectangular shape, and stretched when the second tip is drawn away. 
tissue stretcher
Such stretching and compression capacity is particularly suited to cardiac and skeletal muscle geometry. For cardiac stimulation, a cyclic stretching was imposed, and resulted in almost total differentiation of embryonic mouse stem cells to cardiomyocytes.

Magnetic molding of multicellular aggregates (spheroids)

Current limitations in standard techniques for spheroid formation persist and limit their applicability, like low degree of sphericity, poor control of spheroid size and long maturation times. We implemented and reported a fast and highly reproducible spheroid generation technique to address these limitations based on the labeling of cells with magnetic nanoparticles and their subsequent aggregation by means of an external magnet.

Scale-up of magnetic spheroid formation and stimulation

A scalable Ni-based magnetic micropattern was next developed for spheroid formation and stimulation. It provided a unique all-in-one solution to both create hundreds of spheroids, for instance made of embryoïd stem cells (embryoïd bodies) with no direct contact and no supporting matrix, in a high-throughput manner, and to stimulate them mechanically in situ (with electromagnet), in an on/off cyclical manner that mimics cardiac muscle contraction. It was sufficient to guide almost all embryoïd bodies towards the cardiac lineage commitment, a feat that was not reached with other approaches of embryoïd bodies differentiation.

Magnetic cells aligner

Another technique based on remote magnetic cell actuation was developed to align magnetically labeled cells (in this case cardiomyocytes) in hydrogel. Remarkably, the hydrogel viscosity, before gel transition, allowed multilayer cell formation of aligned cell structures, at centimeter scale. The key stage then consisted in exploiting gel transition to stabilize the pattern and create aligned, functional and viable tissues. Importantly, this strategy was implemented without the need for external mechanical cues (no molds, frames nor posts). Instead, magnetic actuation provided high precision and accuracy for spatial and temporal pattern control.

Magnetic tissue rheometer

Finally, cells magnetization allowed developing a unique all-in-one rheometer by first forming a multicellular aggregates of controlled cylindrical shape, and then applying a precise magnetic compression at the tissue scale, to retrieve the rheological properties of the construct.


INTRACELLULAR NANO-FATE

Massive intracellular degradation of nanoparticles

With the growing medical use of magnetic nanoparticles, in particular for cell therapies, it is crucial to study their long-term intracellular fate within living tissues. We provided multiscale quantitative methods to study intracellular iron oxide nanoparticle degradation, showing an unexpected near-total nanoparticle degradation during long-term maturation of a stem cell model tissue. In brief, we developed stem cell spheroids as new biological tools to monitor intracellular nanoparticle degradation, and we managed to perform single spheroid magnetism in situ as a fingerprint of nanoscale transformations. Remarkably, and unexpectedly, the nanoparticles were found to be more than 90% purged inside the tissue in the first ten days of tissue maturation, barely affecting cellular iron homeostasis. The same massive degradation was recapitulated at the single endosome level, using a unique approach based on single purified endosome nano-magnetophoresis. It was also evidenced in situ, inside cells, in operando thanks to an original magnetic sensor. These results evidencing for the first time a total breakdown of nanoparticles by endosomes in stem cells composing a model tissue bodes well for their safety in medical applications, especially regenerative medicine.

Protection of a magnetic or silver core by gold or polymeric shell as a shield to degradation

Beyond its obvious nanosafety implications, the impact of the cellular environment on nanomaterials also raises concerns as to their therapeutic applicability, for either the tissue engineering field (long-term stimulation of engineered tissues), or for cancer therapies (serial treatments). It can thus be beneficial to protect iron oxide nanoparticles to massive intracellular dissolution. We have showed that a gold shell can prevent the intracellular biodegradation of a magnetic or a silver core and thereby maintain their physical magnetic and photothermal potential. Besides, it demonstrates that not only magnetic metrics, but also thermal ones can be quantitative mirrors of the intracellular status. Finally, a finely tuned polymeric coating can also partially prevent the degradation of a magnetic core.

biodegradation

Magnetic intracellular biosynthesis

Finally, we have demonstrated that the iron freed by the magnetic nanoparticles intracellular degradation could be used by the cells to re-synthesize biogenic magnetic nanoparticles, first evidence of a magnetic biosynthesis in human cells.


PEOPLE HIRED BY THE PROJECT
Claire Wilhelm, PI

Gaëtant Mary, phD student 2016-2019
Anouchka Plan Sangnier, phD student 2019 (4 months)
Alexandre Fromain, phD student 2018-2021
Ana Espinosa, postdoctorant 2015-2016
Alberto Curcio, postdoctorant 2017-2019
Sophie Richard, postdoctorant 2016-2017
Aurore Van de Walle, postdoctorant 2016-2020
Efrain Jose Perez, postdoctorant 2019-2021


COLLABORATORS

MSC, UMR7057 : Amanda Silva Brun, Florence Gazeau, Myriam Reffay, Nathalie Luciani, Jean-Claude Bacri, Cyprien Gay
PHENIX, UMR8234 : Christine Ménager, Nébéwia Griffete, Ali Abou-Hassan
LVTS, U1148 : Laurence Motte, Yoann Lalatonne
B2A, UMR 8256: Onnik Agbulut
LIOAD, UMR_S 791: Catherine Le Visage
IIT, Genova, Italy : Teresa Pellegrino


PUBLICATIONS
                                    

Massive Intracellular Remodeling of CuS Nanomaterials Produces Nontoxic Bioengineered Structures with Preserved Photothermal Potential. Curcio A, Van de Walle A, Benassai E, Serrano A, Luciani N, Manshian BB, Sargsian A, Soenen S, Espinosa A, Abou-Hassan A*, Wilhelm C*. In revision (2021)

All-in-one rheology of multicellular aggregates. Mary G, Mazuel F, Nier V, Fage F, Nagle I, Devaud L, Bacri J-C, Asnacios S, Asnacios A, Gay C, Marcq P, Wilhelm C*, Reffay M*. under review (2021)

Magnetic molding of tumor spheroids: Emerging model for cancer screening. Perez JE, Nagle, I, Wilhelm C. Biofabrication 13, 015018 (2020)

Transient cell stiffening triggered by magnetic nanoparticle exposure Perez JE, Fage F, Pereira D, Abou-Hassan A, Asnacios S, Asnacios A, Wilhelm C. Journal of Nanobiotechnology In press (2021)

High‐Throughput Differentiation of Embryonic Stem Cells into Cardiomyocytes with a Microfabricated Magnetic Pattern and Cyclic Stimulation. Mary G, Van de Walle A, Perez JE, Ukai T, Maekawa T, Luciani N, Wilhelm C. Advanced Functional Materials 12002541 (2020)

Transformation Cycle of Magnetosomes in Human Stem Cells: From Degradation to Biosynthesis of Magnetic Nanoparticles Anew. Curcio A, Van de Walle A, Serrano A, Preveral S, Péchoux C, Pignol D, Menguy N, Lefèvre CT, Espinosa A, Wilhelm C. ACS nano 14, 1406-1417 (2020)

Ever-Evolving Identity of Magnetic Nanoparticles within Human Cells, the Interplay of Endosomal Confinement, Degradation, Storage, and Neo-Crystallization. Van de Walle A, Kolosnjaj-Tabi J, Lalatonne Y, Wilhelm C. Accounts of chemical research 53, 2212-2224 (2020)

3D Magnetic Alignment of Cardiac Cells in Hydrogels. Richard S, Silva A, Mary G, Ragot H, Perez JE, Menager C, Gazeau F, Boucenna I, Agbulut O, Wilhelm C. ACS Applied Bio Materials, 3, 6802-6810 (2020)

Endosomal confinement of gold nanospheres, nanorods and nanoraspberries governs their photothermal identity and is beneficial for cancer cells therapy. Plan Sangnier A, Van de Walle A, Motte L, Guenin E, Lalatonne Y*, Wilhelm C*. Advanced Biosystems 4, 1900284 (2020)

Magnetic nanoparticles in regenerative medicine: what of their fate and impact in stem cells? Van de Walle A, Perez JE, Abou-Hassan A, Hemadi M, Luciani N, Wilhelm C. Materials Today Nano 100084 (2020)

Real-time in situ magnetic measurement of the intracellular biodegradation of iron oxide nanoparticles in a stem cell-spheroid tissue model. Van de Walle A, Fromain A, Plan Sangnier A, Curcio A, Lenglet L, Motte L, Lalatonne Y, Wilhelm C. Nano Research 13, 467-476 (2020) 

Biosynthesis of magnetic nanoparticles from nano-degradation products revealed in human stem cells. Van de Walle A, Plan Sangnier A, Abou-Hassan A, Curcio A, Hémadi M, Menguy N, Lalatonne Y, Luciani N, Wilhelm C.  PNAS 116, 4044-4053 (2019)

Impact of magnetic nanoparticle surface coating on their long-term intracellular biodegradation in stem cells. Plan Sangnier A, Van de Walle A, Le Borgne R, Motte L, Lalatonne Y, Wilhelm C. Nanoscale, 11, 16488 (2019)

The biological fate of magnetic protein-specific molecularly imprinted polymers: toxicity and degradation. Boitard C, Curcio A, Rollet A-L, Wilhelm C, Ménager C, Griffete N. ACS Applied Materials & Interfaces, 11, 35556-35565 (2019)

Role of growth factors and oxygen to limit hypertrophy and impact of high magnetic nanoparticles dose during stem cells chondrogenesis. Van de Walle A, Faissal W, Wilhelm C*, Luciani N*. Computational and Structural Biotechnology Journal 16, 532-542 (2018)

Intracellular Biodegradation of Ag Nanoparticles, Storage in Ferritin, and Protection by Au Shell for Enhanced Photothermal Therapy. Espinosa A, Curcio A, Cabana S, Radtke G, Bugnet M, Kolosnjaj-Tabi J, Péchoux C, Alvarez-Lorenzo C, Botton GA, Silva AKA, Abou-Hassan A*, Wilhelm C*. ACS nano 12, 6523–6535 (2018)

A 3D magnetic tissue stretcher for remote mechanical control of embryonic stem cell differentiation. Du V, Luciani N, Richard S, Mary G, Gay C, Mazuel F, Reffay M, Menasché P, Agbulut O, Wilhelm C. Nature Communications, 8, 400 (2017)

Magneto-Thermal Metrics Can Mirror the Long-Term Intracellular Fate of Magneto-Plasmonic Nanohybrids and Reveal the Remarkable Shielding Effect of Gold. Mazuel F, Espinosa A, Radtke G, Bugnet M, Neveu S, Lalatonne Y, Botton GA, Abou-Hassan A*, Wilhelm C*. Advanced Functional Materials, 27, 1605997 (2017)

3D magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration. Van de Walle A, Wilhelm C*, Luciani N*. JoVE, 122, e55221 (2017)

Forced and Self Rotation of Magnetic Nanorods Assembly at the Cell Membrane: a Bio-Magnetic Torsion Pendulum. Mazuel F, Mathieu S, Di Corato R, Bacri J-C, Meylheuc T, Pellegrino T, Reffay M, Wilhelm C. Small, 13, 1701274 (2017)

Successful chondrogenesis within scaffolds, using magnetic stem cell confinement and bioreactor maturation. Luciani N, Du V, Gazeau F, Richert A, Letourneur D, Le Visage C, Wilhelm C.  Acta Biomaterialia, 37, 101-110 (2016)

Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single Endosome and Tissue Levels. Mazuel F, Espinosa A, Luciani N, Reffay M, Le Borgne R, Motte L, Desboeufs K, Michel A, Pellegrino T, Lalatonne Y, Wilhelm C. ACS Nano, 10, 7627- 38 (2016)