A new agarose-based microsystem to investigate cell response to prolonged confinement

Prunet, Audrey; Lefort, Sylvain; Delanoë-Ayari, Hélène; Laperrousaz, Bastien; Simon, Gilles; Barentin, Catherine; Saci, Souhila; Argoul, Françoise; Guyot, Boris; Rieu, Jean-Paul; Gobert, Stéphanie; Maguer-Satta, Véronique; Rivière, Charlotte

doi:10.1039/d0lc00732c

Cited by 13 publications

(19 citation statements)

References 85 publications

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“…In the present study, the standard agarose used for the preparation of the microsystems provides a cell-repellent surface, with a stiffness in the 150 kPa range. 65,66 In future studies, the mechanical properties of the agarose gels will be adjusted using different concentrations and types of agarose. Elastic hydrogels as soft as 1 kPa can be obtained using low concentrations of ultra-low agarose, 65 matching the physiological range of stiffness.…”

Section: Discussionmentioning

confidence: 99%

“…65,66 In future studies, the mechanical properties of the agarose gels will be adjusted using different concentrations and types of agarose. Elastic hydrogels as soft as 1 kPa can be obtained using low concentrations of ultra-low agarose, 65 matching the physiological range of stiffness. It now calls for dedicated studies to assess how nanoparticle penetration and therapeutic efficacy is affected by the size of the 3D cell assembly, the presence of an extracellular matrix of different stiffness and composition, and the presence of associated tumour cells.…”

Section: Discussionmentioning

confidence: 99%

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Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform

et al. 2021

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform

et al. 2021

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“…The stiffness of PDMS is much higher than the stiffness encountered by immune cells in their physiological environments. Therefore, a soft confiner made using agarose gel was proposed more recently (Prunet et al 2020). This has the advantage that it has a stiffness close to the physiological context.…”

Section: Durotaxismentioning

confidence: 99%

Characterization of immune cell migration using microfabrication

Vesperini

Montalvo

et al. 2021

Biophys Rev

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The immune system provides our defense against pathogens and aberrant cells, including tumorigenic and infected cells. Motility is one of the fundamental characteristics that enable immune cells to find invading pathogens, control tissue damage, and eliminate primary developing tumors, even in the absence of external treatments. These processes are termed “immune surveillance.” Migration disorders of immune cells are related to autoimmune diseases, chronic inflammation, and tumor evasion. It is therefore essential to characterize immune cell motility in different physiologically and pathologically relevant scenarios to understand the regulatory mechanisms of functionality of immune responses. This review is focused on immune cell migration, to define the underlying mechanisms and the corresponding investigative approaches. We highlight the challenges that immune cells encounter in vivo, and the microfabrication methods to mimic particular aspects of their microenvironment. We discuss the advantages and disadvantages of the proposed tools, and provide information on how to access them. Furthermore, we summarize the directional cues that regulate individual immune cell migration, and discuss the behavior of immune cells in a complex environment composed of multiple directional cues.

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“…In future studies, the mechanical properties of the agarose gels can be adjusted using different concentrations and type of agarose. Elastic hydrogels as soft as 1 kPa can be obtained using low concentration of ultra-low agarose 61 , matching the physiological range of stiffness. It now calls for dedicated studies to assess how nanoparticles penetration and therapeutic efficiency is affected by the size of the 3D-cell assembly, the presence of an extracellular matrix of different stiffness and composition, and the presence of associated tumour cells 63 .…”

Section: Conclusion and Outlooksmentioning

confidence: 99%

“…Combined with optical and digital clearing 64 , our approach opens up the possibility to resolve tumour heterogeneity, at the single cell-level, in a physiological context. In the present study, the standard agarose used for the preparation of the microsystems provides a cell-repellent surface, with a stiffness in the 150 kPa range 65,66 . In future studies, the mechanical properties of the agarose gels will be adjusted using different concentrations and type of agarose.…”

Section: Conclusion and Outlooksmentioning

confidence: 99%

Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3Din vitroplatform

Goodarzi

Prunet

Rossetti

et al. 2021

Preprint

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Despite progress in therapeutic strategies and understanding of cancer cell biology, there is a large attrition of promising therapeutics into the clinic. One predominant reason is the huge gap between 2D in-vitro assays used for drug screening, and the in-vivo 3D-physiological environment. This is particularly important for a specific category of emerging therapeutics: nanoparticles. The lack of physiological context hampered reliable predictions for the route and accumulation of those nanoparticles in-vivo. For such nanotherapeutics, Multi-Cellular Tumour Spheroids (MCTS) is emerging as a good alternative in-vitro model. However, the classical approaches to produce MCTS suffer from low yield, poor reproducibility and slow process, while spheroid-on-chip set-ups developed so far require a microfluidic practical knowledge difficult to transfer to a cell biology laboratory.We present here a simple yet highly flexible 3D-model microsystem consisting of agarose-based micro-wells. Fully compatible with the multi-well plates format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. It is compatible with live high-resolution optical microscopy and provides a user-friendly platform for in-situ immunostaining.As a proof-of-principle of the relevance of such in-vitro platform for the evaluation of nanoparticles, the aim of this study was to analyse the kinetic and localization of nanoparticles within colorectal cancer cells (HCT-116) MCTS. The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI and confocal microscopy (AGuIX®, currently implicated in clinical trials as effective radiosensitizers for radiotherapy). We show that the amount of AGuIX® nanoparticles within cells is largely different in 2D and 3D. Using our flexible agarose-based microsystems, we are able to resolve spatially and temporally the penetration and distribution of AGuIX® nanoparticles within tumour spheroids. The nanoparticles are first found in both extracellular and intracellular space of spheroids, within lysosomes compartment. While the extracellular part is washed away after few days, we evidenced trafficking of AGuIX® nanoparticles that are also found within mitochondria. Our agarose-based microsystem appears hence as a promising 3D in-vitro platform for investigation of nanotherapeutics transport, ahead of in-vivo studies.

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A new agarose-based microsystem to investigate cell response to prolonged confinement

Abstract: A unique tool to analyze the role of long-term effects of mechanical confinement in normal and pathological conditions.

Cited by 13 publications

References 85 publications

Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform

Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform

Characterization of immune cell migration using microfabrication

Quantifying nanotherapeutics penetration using hydrogel based microsystem as a new 3Din vitroplatform

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