Evaluating Nanoparticles Penetration by a New Microfluidic Hydrogel-Based Approach.

Reliable predictions for the route and accumulation of nanotherapeutics in vivo are limited by the huge gap between the 2D in vitro assays used for drug screening and the 3D physiological in vivo environment. While developing a standard 3D in vitro model for screening nanotherapeutics remains challenging, multi-cellular tumor spheroids (MCTS) are a promising in vitro model for such screening. Here, we present a straightforward and flexible 3D-model microsystem made out of agarose-based micro-wells, which enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescent imaging, including live high-resolution optical microscopy, can be done in situ without manipulating spheroids.

Integrated platform for culture, observation, and parallelized electroporation of spheroids.

Reversible electroporation is a method to introduce molecules into cells by increasing the permeability of their membranes, thanks to the application of pulsed electric fields. One of its main biomedical applications is electro-chemotherapy, where electroporation is used to deliver anticancer drugs into tumor tissues. To improve our understanding of the electroporation effect on tissues and select efficient treatments, in vitro tumor models are needed. Cell spheroids are relevant models as they can reproduce tumor microenvironment and cell-cell interactions better than 2D cell cultures. Various methods offering a relatively simple workflow are now available for their production. However, electroporation protocols usually require handling steps that may damage spheroids and result in random spacing, inducing variations in electric field distribution around spheroids and non-reproducible electroporation conditions. In addition, only a few microsystems allow the production and electroporation of spheroids, and the spheroids produced lack reproducibility in size and location. To overcome these issues, we developed a unique device enabling culture, monitoring, and electroporation of hundreds of regular spheroids in parallel, with a design ensuring that all spheroids are submitted to the same electric field conditions. It is comprised of a microfluidic chamber encompassing a micro-structured agarose gel, allowing easy medium exchange while avoiding spheroid handling. It also enables optical imaging of spheroids in situ, thanks to transparent electrodes. In this paper, we describe the fabrication and characterization of the developed microsystem and demonstrate its applicability to electroporation of a network of spheroids. We present a first successful application as an anticancer drug testing platform, by evaluating the bleomycin effect on HT29 colorectal cancer cell spheroids. This work opens new perspectives in the development of in vitro assays for the preclinical evaluation of electroporation-based treatment.

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

Correction for 'Quantifying nanotherapeutic penetration using a hydrogel-based microsystem as a new 3D in vitro platform' by Saba Goodarzi et al., Lab Chip, 2021, 21, 2495-2510, DOI: 10.1039/D1LC00192B.

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

The huge gap between 2D in vitro assays used for drug screening and the in vivo 3D physiological environment hampered reliable predictions for the route and accumulation of nanotherapeutics in vivo. For such nanotherapeutics, multi-cellular tumour spheroids (MCTS) are emerging as a good alternative in vitro model. However, the classical approaches to produce MCTS suffer from low yield, slow process, difficulties in MCTS manipulation and compatibility with high-magnification fluorescence optical microscopy. On the other hand, spheroid-on-chip set-ups developed so far require a practical knowledge of microfluidics difficult to transfer to a cell biology laboratory. We present here a simple yet highly flexible 3D model microsystem consisting of agarose-based microwells. Fully compatible with the multi-well plate format conventionally used in cell biology, our simple process enables the formation of hundreds of reproducible spheroids in a single pipetting. Immunostaining and fluorescence imaging including live high-resolution optical microscopy can be performed in situ, with no manipulation of spheroids. As a proof of principle of the relevance of such an in vitro platform for nanotherapeutic evaluation, this study investigates the kinetics and localisation of nanoparticles within colorectal cancer MCTS cells (HCT-116). The nanoparticles chosen are sub-5 nm ultrasmall nanoparticles made of polysiloxane and gadolinium chelates that can be visualized in MRI (AGuIX®, currently implicated in clinical trials as effective radiosensitizers for radiotherapy) and confocal microscopy after addition of Cy5.5. 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 MCTS. The nanoparticles are first found in both extracellular and intracellular space of MCTS. While the extracellular part is washed away after a few days, we evidenced intracellular localisation of AGuIX®, mainly within the lysosomal compartment, but also occasionally within mitochondria. Hence, our agarose-based microsystem appears as a promising 3D in vitro user-friendly platform for investigation of nanotherapeutic transport, ahead of in vivo studies.

Mechanical Control of Cell Proliferation Increases Resistance to Chemotherapeutic Agents.

While many cellular mechanisms leading to chemotherapeutic resistance have been identified, there is an increasing realization that tumor-stroma interactions also play an important role. In particular, mechanical alterations are inherent to solid cancer progression and profoundly impact cell physiology. Here, we explore the influence of compressive stress on the efficacy of chemotherapeutics in pancreatic cancer spheroids. We find that increased compressive stress leads to decreased drug efficacy. Theoretical modeling and experiments suggest that mechanical stress decreases cell proliferation which in turn reduces the efficacy of chemotherapeutics that target proliferating cells. Our work highlights a mechanical form of drug resistance and suggests new strategies for therapy.

High-Frequency Mechanical Properties of Tumors Measured by Brillouin Light Scattering.

The structure of tumors can be recapitulated as an elastic frame formed by the connected cytoskeletons of the cells invaded by interstitial and intracellular fluids. The low-frequency mechanics of this poroelastic system, dictated by the elastic skeleton only, control tumor growth, penetration of therapeutic agents, and invasiveness. The high-frequency mechanical properties containing the additional contribution of the internal fluids have also been posited to participate in tumor progression and drug resistance, but they remain largely unexplored. Here we use Brillouin light scattering to produce label-free images of tumor microtissues based on the high-frequency viscoelastic modulus as a contrast mechanism. In this regime, we demonstrate that the modulus discriminates between tissues with altered tumorigenic properties. Our micrometric maps also reveal that the modulus is heterogeneously altered across the tissue by drug therapy, revealing a lag of efficacy in the core of the tumor. Exploiting high-frequency poromechanics should advance present theories based on viscoelasticity and lead to integrated descriptions of tumor response to drugs.

Drug delivery to tumours using a novel 5-FU derivative encapsulated into lipid nanocapsules.

In this work, a novel lipophilic 5-fluorouracil (5-FU) derivative was synthesised and encapsulated into lipid nanocapsules (LNC). 5-FU was modified with lauric acid to give a lipophilic mono-lauroyl-derivative (5-FU-C12, MW of about 342 g/mol, yield of reaction 70%). 5-FU-C12 obtained was efficiently encapsulated into LNC (encapsulation efficiency above 90%) without altering the physico-chemical characteristics of LNC. The encapsulation of 5-FU-C12 led to an increased stability of the drug when in contact with plasma being the drug detectable until 3 h following incubation. Cytotoxicity assay carried out using MTS on 2D cell culture showed that 5-FU-C12-loaded LNC had an enhanced cytotoxic effect on glioma (9L) and human colorectal (HTC-116) cancer cell line in comparison with 5-FU or 5-FU-C12. Then, HCT-116 tumour spheroids were cultivated and the reduction of spheroid volume was measured following treatment with drug-loaded LNC and drugs alone. Similar reduction on spheroids volume was observed following the treatment with drug-loaded LNC, 5-FU-C12 and 5-FU alone, while blank LNC displayed a reduction in cell viability only at high concentration. Globally, our data suggest that the encapsulation increased the activity of the 5-FU-C12. However, in-depth evaluations of LNC permeability into spheroids are needed to disclose the potential of these nanosystems for cancer treatment.

Collective regulation of cell motility using an accurate density-sensing system.

The capacity of living cells to sense their population density and to migrate accordingly is essential for the regulation of many physiological processes. However, the mechanisms used to achieve such functions are poorly known. Here, based on the analysis of multiple trajectories of vegetative Dictyostelium discoideum cells, we investigate such a system extensively. We show that the cells secrete a high-molecular-weight quorum-sensing factor (QSF) in their medium. This extracellular signal induces, in turn, a reduction of the cell movements, in particular, through the downregulation of a mode of motility with high persistence time. This response appears independent of cAMP and involves a G-protein-dependent pathway. Using a mathematical analysis of the cells' response function, we evidence a negative feedback on the QSF secretion, which unveils a powerful generic mechanism for the cells to detect when they exceed a density threshold. Altogether, our results provide a comprehensive and dynamical view of this system enabling cells in a scattered population to adapt their motion to their neighbours without physical contact.

In-depth phenotypic characterization of multicellular tumor spheroids: Effects of 5-Fluorouracil.

MultiCellular Tumor Spheroids (MCTS), which mimic the 3-Dimensional (3D) organization of a tumor, are considered as better models than conventional cultures in 2-Dimensions (2D) to study cancer cell biology and to evaluate the response to chemotherapeutic drugs. A real time and quantitative follow-up of MCTS with simple and robust readouts to evaluate drug efficacy is still missing. Here, we evaluate the chemotherapeutic drug 5-Fluorouracil (5-FU) response on the growth and integrity of MCTS two days after treatment of MCTS and for three colorectal carcinoma cell lines with different cohesive properties (HT29, HCT116 and SW480). We found different sensitivity to 5-FU for the three CRC cell lines, ranging from high (SW480), intermediate (HCT116) and low (HT29) and the same hierarchy of CRC cell lines sensitivity is conserved in 2D. We also evidence that 5-FU has a strong impact on spheroid cohesion, with the apparition of a number of single detaching cells from the spheroid in a 5-FU dose- and cell line-dependent manner. We propose an innovative methodology for the chemosensitivity evaluation in 3D MCTS that recapitulates and regionalizes the 5-FU-induced changes within MCTS over time. These robust phenotypic read-outs could be easily scalable for high-throughput drug screening that may include different types of cancer cells to take into account tumor heterogeneity and resistance to treatment.

Magnetophoretic manipulation in microsystem using carbonyl iron-polydimethylsiloxane microstructures.

This paper reports the use of a recent composite material, noted hereafter i-PDMS, made of carbonyl iron microparticles mixed in a PolyDiMethylSiloxane (PDMS) matrix, for magnetophoretic functions such as capture and separation of magnetic species. We demonstrated that this composite which combine the advantages of both components, can locally generate high gradients of magnetic field when placed between two permanent magnets. After evaluating the magnetic susceptibility of the material as a function of the doping ratio, we investigated the molding resolution offered by i-PDMS to obtain microstructures of various sizes and shapes. Then, we implemented 500 µm i-PDMS microstructures in a microfluidic channel and studied the influence of flow rate on the deviation and trapping of superparamagnetic beads flowing at the neighborhood of the composite material. We characterized the attraction of the magnetic composite by measuring the distance from the i-PDMS microstructure, at which the beads are either deviated or captured. Finally, we demonstrated the interest of i-PDMS to perform magnetophoretic functions in microsystems for biological applications by performing capture of magnetically labeled cells.

A tapered channel microfluidic device for comprehensive cell adhesion analysis, using measurements of detachment kinetics and shear stress-dependent motion.

We have developed a method for studying cellular adhesion by using a custom-designed microfluidic device with parallel non-connected tapered channels. The design enables investigation of cellular responses to a large range of shear stress (ratio of 25) with a single input flow-rate. For each shear stress, a large number of cells are analyzed (500-1500 cells), providing statistically relevant data within a single experiment. Besides adhesion strength measurements, the microsystem presented in this paper enables in-depth analysis of cell detachment kinetics by real-time videomicroscopy. It offers the possibility to analyze adhesion-associated processes, such as migration or cell shape change, within the same experiment. To show the versatility of our device, we examined quantitatively cell adhesion by analyzing kinetics, adhesive strength and migration behaviour or cell shape modifications of the unicellular model cell organism Dictyostelium discoideum at 21 °C and of the human breast cancer cell line MDA-MB-231 at 37 °C. For both cell types, we found that the threshold stresses, which are necessary to detach the cells, follow lognormal distributions, and that the detachment process follows first order kinetics. In addition, for particular conditions' cells are found to exhibit similar adhesion threshold stresses, but very different detachment kinetics, revealing the importance of dynamics analysis to fully describe cell adhesion. With its rapid implementation and potential for parallel sample processing, such microsystem offers a highly controllable platform for exploring cell adhesion characteristics in a large set of environmental conditions and cell types, and could have wide applications across cell biology, tissue engineering, and cell screening.

A quorum-sensing factor in vegetative Dictyostelium discoideum cells revealed by quantitative migration analysis.

BACKGROUND: Many cells communicate through the production of diffusible signaling molecules that accumulate and once a critical concentration has been reached, can activate or repress a number of target genes in a process termed quorum sensing (QS). In the social amoeba Dictyostelium discoideum, QS plays an important role during development. However little is known about its effect on cell migration especially in the growth phase. METHODS AND FINDINGS: To investigate the role of cell density on cell migration in the growth phase, we use multisite timelapse microscopy and automated cell tracking. This analysis reveals a high heterogeneity within a given cell population, and the necessity to use large data sets to draw reliable conclusions on cell motion. In average, motion is persistent for short periods of time (t ≤ 5 min), but normal diffusive behavior is recovered over longer time periods. The persistence times are positively correlated with the migrated distances. Interestingly, the migrated distance decreases as well with cell density. The adaptation of cell migration to cell density highlights the role of a secreted quorum sensing factor (QSF) on cell migration. Using a simple model describing the balance between the rate of QSF generation and the rate of QSF dilution, we were able to gather all experimental results into a single master curve, showing a sharp cell transition between high and low motile behaviors with increasing QSF. CONCLUSION: This study unambiguously demonstrates the central role played by QSF on amoeboid motion in the growth phase.

The MRI assessment of intraurethrally--delivered muscle precursor cells using anionic magnetic nanoparticles.

Autografting of cultured myogenic precursor cells (MPC) is a therapeutic strategy for muscle disorders, including striated urethral sphincter insufficiency. Implantation of myofibers with their satellite cells into the urethra is a recently described method of MPC transfer aimed at generating a new sphincter in incontinent patients. In this study, we magnetically labeled muscle implants with dextran-free anionic iron oxide nanoparticles (AMNP). The aim was to evaluate the biocompatibility of the labeling procedure and its utility for non-invasive MRI follow-up of cell therapy in a female pig model. After adsorption of AMNP to the implant surface, various cell types, including MPC, were magnetically labeled within the implants. Magnetic labeling did not affect cell proliferation or differentiation. Autograft detection in vivo by 0.3-T MRI was possible for up to 1 month. Ex vivo, Perl's, anti-desmin and anti-myosin heavy chain staining confirmed the co-localization of AMNP and regenerated myofibers. AMNP labeling was thus useful for locating myofiber implant autografts in vivo and for ex vivo monitoring of the biology of this cell transfer method.

Aortic aneurysms in a rat model: in vivo MR imaging of endovascular cell therapy.

PURPOSE: To prospectively evaluate in rats whether magnetic cell labeling can be used to noninvasively assess the technical success of endovascular cell therapy for abdominal aortic aneurysms (AAAs). MATERIALS AND METHODS: The study was approved by an institutional animal care and use committee. Vascular smooth muscle cells (VSMCs) labeled with iron oxide nanoparticles were seeded endovascularly in already formed AAAs. T2*-weighted gradient-echo and T2-weighted spin-echo magnetic resonance (MR) imaging was performed in vivo at 1.5 T before and 30 minutes after the injection of iron-loaded VSMCs (14 rats), nonlabeled VSMCs (three rats), or iron-free particles (three rats). Ten rats were euthanized shortly after the injection (day 0). Of the 10 remaining rats, which were seeded with iron-loaded cells, three were imaged on day 7 after cell delivery; three, on day 14; and four, on day 28; then they were euthanized. Ex vivo high-field-strength MR imaging of two AAAs was performed 28 days after cell delivery. Histologic examination of cross sections of all AAAs was performed. Statistical evaluations were performed with a nonparametric Wilcoxon correlation test. RESULTS: Magnetic cell labeling did not alter the capability of VSMCs to stabilize the diameter of the aneurysms. T2*-weighted gradient-echo images showed areas of hypointense signal within the aortic wall immediately and up to 1 month after cell therapy. The mean signal intensity decreased significantly after cell delivery (from 2362 +/- 244 [standard deviation] before to 434 +/- 275 after delivery, P < .001). Areas of hypointense signal and iron-loaded VSMCs were colocalized in the area of aortic wall reconstruction at both high-field-strength MR imaging and histologic analysis. CONCLUSION: MR imaging with magnetic cell labeling can be used to document endovascular cell delivery in already formed AAAs in rats.

Magnetic targeting of nanometric magnetic fluid loaded liposomes to specific brain intravascular areas: a dynamic imaging study in mice.

PURPOSE: To prospectively determine, by using dynamic imaging, whether a magnet placed over a specific area of the mouse brain could target systemically administered rhodamine-labeled magnetic fluid-loaded liposomes (MFLs) to that brain region. MATERIALS AND METHODS: Experiments were performed with a French Ministry of Agriculture permit and regional ethics committee authorization. In seven anesthetized C57BL/6 mice, a closed cranial window was implanted above the left parieto-occipital cortex. A laser-scanning confocal fluorescence microscope (LSCFM) was used to track the intravenously injected rhodamine-labeled MFLs within this cortical area, through the cranial window. The MFLs were video monitored for 2 minutes every 15 minutes for 1 hour after injection. A magnet was placed on the cranial window implanted in four mice, while no magnet was placed in three (control) mice. After dynamic in vivo imaging, static in vivo imaging was performed with a different LSCFM. Ex vivo fluorescence histologic analysis was then performed. Paired Student t testing was used to compare the cerebral blood flow and two-dimensional flow values before and 1 hour after MFL injection. For image analysis, intergroup comparisons were performed by using an independent t test. RESULTS: In vivo video monitoring through the window revealed that the rhodamine-labeled MFLs accumulated in the mouse brain microvasculature exposed to the magnet-first within superficial brain venules and then within intracerebral venules-with no significant change in blood flow (P > .05). MFLs accumulated neither in the arterioles of the mice with a magnet nor in the arterioles of the control mice. Static in vivo imaging findings confirmed the microvascular localization of the rhodamine-labeled MFLs, and histologic findings specified their accumulation on the side of the magnet only. CONCLUSION: Real-time in vivo imaging of rhodamine-labeled MFLs in the mouse brain cortex revealed that these nanosystems can be magnetically targeted, through microvessels, to selected brain areas.

Intraurethral transfer of satellite cells by myofiber implants results in the formation of innervated myotubes exerting tonic contractions.

PURPOSE: We investigated a new method of muscle precursor cell transfer in the urethra for the treatment of urinary incontinence, consisting of implanting myofibers with their satellite cells. MATERIALS AND METHODS: In preliminary experiments to test the regenerative capacities of satellite cells histological analysis was performed on days 7 and 30 after the implantation of myofiber cores in the urethra of 6 female pigs. In the main experiments 11 pigs underwent baseline urodynamics, followed by endoscopic destruction of the striated urethral sphincter located around the distal urethra (day 0). On day 30 circular myofiber strips in 7 experimental cases and adipocytes in 4 controls were implanted in the proximal urethra. Seven days later (day 37) 1 case was sacrificed to verify satellite cell activation. On day 60 urodynamics were performed without and with curarization. Urethral cryosections were immunostained for desmin (activated satellite cells), fast myosin heavy chain/bungarotoxin (myotubes/acetylcholine receptors), neurofilament/vesicular acetylcholine transporter (nerve endings) and CD45/CD68 (inflammatory response). RESULTS: Preliminary histological studies revealed a myogenic process consisting of myofiber degeneration and satellite cell activation (day 7), followed by myotube formation replacing parental myofibers (day 30). In the main experiments endoscopic injury abolished striated urethral sphincter activity. Implantation of myofiber strips generated a pressure peak that decreased after curarization (mean+/-SEM 71.5+/-17.8 vs 33.5+/-14.8 cm H2O, p=0.031) and reappeared 60 minutes later, revealing that this action was tonic and under neural control. Nerve endings connected to the acetylcholine receptors of myotubes were observed on day 60. An inflammatory response was observed only on day 7 in the myofiber implantation group. Adipocyte implantation resulted in no significant intraurethral pressure changes. CONCLUSIONS: Urethral implantation of myofibers regenerates as myotubes that exert tonic activity under neural control. This has potential clinical value as a means to create an additional striated urethral sphincter.

Hybrid gadolinium oxide nanoparticles: multimodal contrast agents for in vivo imaging.

Luminescent hybrid nanoparticles with a paramagnetic Gd2O3 core were applied as contrast agents for both in vivo fluorescence and magnetic resonance imaging. These hybrid particles were obtained by encapsulating Gd2O3 cores within a polysiloxane shell which carries organic fluorophores and carboxylated PEG covalently tethered to the inorganic network. Longitudinal proton relaxivities of these particles are higher than the positive contrast agents like Gd-DOTA which are commonly used for clinical magnetic resonance imaging. Moreover these particles can be followed up by fluorescence imaging. This study revealed that these particles suited for dual modality imaging freely circulate in the blood vessels without undesirable accumulation in lungs and liver.

Iron oxide nanoparticle-labeled rat smooth muscle cells: cardiac MR imaging for cell graft monitoring and quantitation.

PURPOSE: To perform a quantitative analysis of anionic maghemite nanoparticle-labeled cells in vitro and determine the effect of labeling on signal intensity at magnetic resonance (MR) imaging. MATERIALS AND METHODS: The study was approved by the institutional animal care and use committee at Hôpital Bichat. In vitro cell proliferation, iron content per cell, and MR signal intensity of cells were measured in agarose phantoms for 0-14 days of culture after labeling of rat smooth muscle cells with anionic maghemite nanoparticles. Next, iron oxide-labeled smooth muscle cells were injected into healthy hearts and hearts with ischemic injury in seven live Fisher rats. Ex vivo MR imaging experiments in excised hearts 2 and 48 hours after injection were performed with a 1.5-T medical imaging system by using T2-weighted gradient-echo and spin-echo sequences. Histologic sections were obtained after MR imaging. Correlation analyses between division factor of iron load and cell amplification factor and between 1/T2 and number of labeled cells or number of days in culture were performed by using linear regression. RESULTS: Viability of smooth muscle cells was not affected by magnetic labeling. Transmission electron micrographs of cells revealed the presence of iron oxide nanoparticles in vesicles up to day 14 of culture. Intracellular iron concentration decreased in parallel with cell division (r2 = 0.99) and was correlated with MR signal intensity (r2 = 0.95). T2*-weighted MR images of excised rat hearts showed hypointense signal in myocardium at 2 and 48 hours after local injection of labeled cells. Subsequent histologic staining evidenced iron oxide nanoparticles within cells and confirmed the presence of the original cells at 2 and 48 hours after implantation. CONCLUSION: Magnetic labeling of smooth muscle cells with anionic maghemite nanoparticles allows detection of cells with MR imaging after local transplantation in the heart.