Overview
Our lab has developed a microfluidics-assisted technique, the Cellular Capsule Technology (CCT) that enables to encapsulate cells and grow organoids or tumor models within a shell of porous hydrogel.
The CCT is based on a co-extrusion micro-device fabricated with a 3D printer. The working principle consists in injecting an alginate solution and the cell suspension in concentric capillaries to generate a composite liquid jet. Exploiting the Rayleigh-Plateau instability that fragments the jet into microdroplets, we produce spherical capsules upon alginate crosslinking in a calcium bath. Inhibiting the Rayleigh-Plateau instability leads to the formation of tubular capsules.
Quantitative imaging of the growth of the organoids using state-of-the art optical sectioning microscopy and analyzing the cellular fate using custom optofluidic devices allow us to investigate both fundamental mechanotransduction issues in stem cell biology and self-organization processes involved in tissue engineering, and to explore biomedical applications, in particular in oncology and neurodegenerative diseases.
Part of our work is now transferred to industry through the creation of a start-up by two post-doctoral fellows.
Mechanotransduction & Self-organization
Cells self-organize in a spatio-temporal and context-dependent manner, leading to fascinating supracellular assemblies such as blood vessels, intestinal crypts, or embryos. Cells also sense mechanical cues from their surroundings and respond by altering their behavior or fate. Cell self-organization directs the architecture of a healthy or pathological tissue. Cell mechanotransduction is now recognized as an important regulator of many physiologic and pathologic processes. Our capsule technology allows us i/ to investigate the mechanisms of cell self-organization by using the capsules as micro-compartments or scaffolds for larger scale assembly, ii/ to study the mechanisms of mechanotransduction in 3D by using the elastic capsules as micro-dynamometers.
Mechanotransduction
Multicellular tumor spheroids under mechanical stress
We have shown that colon carcinoma cells encapsulated in alginate capsules exhibit enhanced motility in post-confluent stages of spheroid growth. Cells located at the periphery of pre-compressed spheroids are also more prone escape the spheroid upon implantation in a collagen matrix after capsule dissolution, indicating that compressive forces trigger a switch towards an invasive phenotype (collaboration: Danijela Vignjevic (PI), Institut Curie, Paris). The growth response (kinetics, cell density and proliferation mapping) was quantitatively modelled using an hybrid numerical approach based on a DCM (Deformable Cell Model) combined with a CBM (Center Based Model) (collaboration: Dirk Drasdo (PI) and Paul Van Liedekerke, INRIA BANG, Paris).
References: Alessandri et al., PNAS, 2013; Van Liedekerke et al., PLOS Comput. Biol., 2019
Fundings: ANR CapCell, Fondation Pierre-Gilles de Gennes, PhysiCancer Inserm
Contact: Pierre Nassoy
Illustrations: actin staining at the surface of a spheroid (left), simulated spheroid under compression (right)
Mechanosensing mechanisms of adipose stem cells
We are investigating the influence of internally generated or externally applied mechanical stress on adipose growth. More specifically, we wish to decipher the molecular basis of adipose precursor cells mechanotransduction (collaboration: Eric Honoré – IPMC, Sophia Antipolis Nice).
Fundings: ANR MecanoAdipo, Défi MecanoBiologie
Contact: Gaëlle Recher & Pierre Nassoy
Illustrations: working hypothesis about differenciation of Adpiose Precursor Cell into Hypertrophic Adipocyte (left), pre-adipocytes encapsulated into alginate shell before and after confluency (lipid droplets in yellow and nuclei in blue)
Self-organisation
Guided self-organization of functional blood vessels
We have developed a strategy to engineer perfusable contractile mature vessels by directing the self-organization of endothelial and smooth muscle cells in hollow tubular capsules internally coated with extracellular matrix (collaboration: Andreas Bikfalvi (PI) and Laetitia Andrique (post-doc)- LAMC, Bordeaux).
References: Patent: Andrique et al., 2017 WO 2018/162857 Al & Andrique, Recher et al., (in Press) Science Advances
Fundings: ANR VascTubes
Contact: Gaëlle Recher
Illustrations: self-organisation of smooth muscle cells and enfothelial cells within the alginate shell after one day of culture (left), calcium imaging of a similar tube (before and after application of a vasocompressive drug) (right), (credits Laetitia Andrique)
Spontaneous oscillations and rheological properties of tissue cysts
Hollow vesicular tissues of various sizes and shapes arise in biological organs such as ears, guts, hearts, brains and even entire organisms. We have developed an in-vitro experimental setup to generate encapsulated hollow tissue spheroids (cysts). We monitored their growth and spontaneous oscillations driven by between osmotic swelling and intermittent epithelium rupture. We have created a minimal theoretical framework to explain the timescales and amplitudes of the inflation-deflation cycles both in free and confined growth conditions. We propose that hydraulically-gated oscillations may be a general mechanism for organ size regulation (collaboration: L. Mahadevan (PI) and Teresa Ruiz (post-doc)- Harvard University).
In parallel, we are investigating the mechanical properties of these closed spherical epithelia (cysts) using all-optical methods in the framework of an extended finite elasticity model (collaboration: Martine Ben Amar (PI)- LPS, Paris, and Loïc Le Goff (PI) – Institut Fresnel, Marseille)
References: Ruiz-Herrero et al., 2017, Development
Fundings: ANR MecaTiss
Contact: Pierre Nassoy
Illustrations: encapsulated cyst (color code as a function of depth) (left, credits: Basile Gurchenkov), cartoon with minimal physical ingredients for oscillations (right)
Self-assembled architecture of tumor models
We are investigating the dynamic organization of stromal and tumor cells in spherical capsules as realistic tumor mimics. This approach is pursued for solid tumor (colon carcinoma in collaboration with Danijela Vignjevic (PI) and Fabien Bertillot (PhD student), Institut Curie, Paris) and for liquid tumors (follicular lymphoma in collaboration with Laurence Bresson-Bépoldin (PI), Simon Latour (PhD student), Valérie Lemorvan (IE)- Institut Bergonié, Bordeaux, and Pr. Karin Tarte -Faculté de médecine, Rennes).
References: Patent: Bresson-Bépoldin et al., 2017 WO 2018/185439 Al
Fundings: ANR Invaders, SIRIC BRIO, Fondation ARC, La Ligue Contre le Cancer
Contact: Pierre Nassoy & Gaëlle Recher
Illustrations: Fibroblasts spreading onto the surface of a tumor spheroid (left, credits Fabien Bertillot), B cells mixed with lymphoid stroma progenitors (right, credits: Simon Latour).
Biomedical applications
The cellular capsules technology is used to produce micro-tumors and micro-tissues. For applications in oncology, 3D tumor spheroids have been shown to exhibit higher resistance to chemotherapy treatment than 2D cultures and thus better mimic in vivo tumor response. For applications in tissue-engineering, our general approach consists in encapsulating stem cells that are further differentiated in capsulo to produce ‘balls’ of specific tissue. Cell therapy for neurodegenerative diseases is a natural direction to go.
Engineered vascularised tissues from spherical and tubular capsules, multiscale assembly with resorbable scafold
Organogenesis knowledge is crucial to personalised regenerative medicine but also in the field of fundamental developmental biology. Centimetre-sized engineered tissues/organs would not only provide a platform to study a realistic organ homoeostasis and function, but also, would be an excellent candidate for grafts as an alternative to tissue transplants. The bottleneck is that growing such a big cell architecture requires oxygen and nutrient supply as well as a cocktail of growth factors that can be provided uniquely by a micro and meso vascularisation. The overarching aim of this project is to develop a framework to engineer multiscale scaffold-free and vascularised tissues and to investigate the vascular network establishment and functioning.
This project relies on the hypothesis that combining ontological principles of developmental biology (differentiation and maturation of different cell types has to be concomitant to ensure the harmonious development of the niche ) with tissue engineering approaches (hydrogels, microfluidics, microfabrication, encapsulation) will permit to shape and grow a structured and physio-mimetic tissue. Practically, the strategy is to take advantage of self-organising properties of cells when grown together with directed assembly which consists in positioning preassembled elementary bricks.
Reference: Andrique et al., 2019
Funding: ANR JCJC MUSCOVADO
Contact: Gaëlle Recher
Illustrations: prevascularised liver spheroid (left), assembly of prevascularised spheroids and vesseloid in a hydrogel mould (right)
(credits: Naveen Vijayan Mekhileri & Gaëlle Recher)
Dopaminergic neuronal organoids for Parkinson disease cell therapy
Controlled differentiation of induced pluripotent stem cells capsules leads to the formation of micro-ball composed of dopaminergic post-mitotic neurons. These neuronal organoids (or BrainCaps) can then be implanted into rodent brain that exhibit Parkinson symptoms. (collaboration: Erwan Bézard (PI), IMN – Institut des Maladies Neurodégénératives, Bordeaux)
Reference: Feyeux et al., 2016
Fundings: Idex, CNRS pré-maturation, ANR Parkington
Contact: Pierre Nassoy
Illustrations: neuronal network grown within a capsules (left), axons spreading within the brain after engraftment (right)
(credits: Kevin Alessandri, Maxime Feyeux & Frédéric Naudet)
Realsitic tumor model for chemotherapy assays
Co-encapsulation of B lymphocytes and stromal cells was shown to enhance the survival and proliferation of B cells. The resulting spheroids provide a unique in vitro 3D model for liquid tumors and allow to perform high throughput drug screening assays. (collaboration: Laurence Bresson-Bépoldin(PI), Institut Bergonié, Bordeaux)
Reference: Bresson-Bépoldin et al., 2017
Contact: Gaëlle Recher & Pierre Nassoy
Illustrations: example of capsules filled with tumor cells and stromal cells (left, credits: Simon Latour), prototypic curve to illustrate the improvement of 3D culture system over 2D
Instrumental & analytical developments
There is growing evidence that 2D cell cultures fail to recapitulate the architecture and function of living tissues. Our microfluidic technique allows us to produce 3D cell-based assays in a controlled manner. Visualizing their growth and internal organization over days or weeks with subcellular resolution and reduced phototoxicity requires i/ to use or develop advanced optical sectioning microscopy, ii/ to design dedicated culture and observation environments, iii/ to perform quantitative 3D+time image analysis (using either our home-made microscopes or the Bordeaux Imaging Center facility ones). The Cellular Capsules Technology generates capsules at ultra-high rate (5000.sec-1). To exploit this massive production and to perform on-line cell fate monitoring for high throughput assays, we need to develop an optofluidic platform that integrates i/ fast label-free optical methods based on extinction measurements and light scattering and ii/ microfluidic cell sorting devices.
Label-free thick & live tissue imaging with Optical Coherence Tomography
Full-field Optical Coherence Tomography – principle
Development of a remote scanning method to increase the size of the effective field of view, to enable the scan of thick samples without the need of moving the stage
Implementation of machine learning approaches for smart acquisition
Reference: Recher et al., 2020
Contact: Amaury Badon
Illustration: Imaging capsules with wide-field microscopy and full-field optical coherence tomography.
(Credits: Amaury Badon)
Optimization of the Cellular Capsules Technology
The initial set-up relied on a co-extrusion chip mounted with glass capillaries and glue. We have now made the fabrication of the chip more accessible by using a stereolithography 3D printer and by adapting the design of the microfluidic circuitery. In addition, to reduce the occurrence of droplet coalescence, we have implemented a ring-shaped electrode that favors droplet splay and improve capsule monodispersity.
Reference: Alessandri et al., 2016
Contact: Pierre Nassoy
Illustrations: new hybrid chip combining 3D-printed body and glass nozzle (left), schematics of the co-extrusion principle (right, credit: Basile Gurchenkov)
Implementation of light-sheet microscopy dedicated to long term stress-free spheroid imaging.
We have set up a simple custom-made light sheet microscope to perform long term imaging in depth and with reduced phototoxicity. Due to the symmetry of the samples, dual-sided illumination and sample rotation are not necessary. We managed to avoid embedding the sample in agarose carrots. Instead, the capsules lie on an agarose mattress in constraint-free conditions. Angle scan allows to reduce stripe and shadowing effects.
Contact: Gaëlle Recher & Pierre Nassoy
Illustrations: beam path for generating the light-sheet (top left), chamber and sample holder for stress-free imaging (bottom left), exploded view of a stack acquired by scanning through a spheroid (right) (credits: Dan Strehle)
Design of a versatile culture and microscopy chamber for live imaging: the Universlide
Besides the technology used to produce the capsules, the environment of the sample is an important aspect that needs to be optimized to perform proper visualization of the encapsulated micro-tissues. We have designed and validated a multi-usage observation chamber for live 3D imaging. The Universlide is assembled from 3D printed parts and compatible with all commercial microscopes. The chamber is applicable for medium/high throughput screening and automatized multi-position image acquisition.
References: Recher et al., 2016; Alessandri et al., 2017
Contact: Gaëlle Recher
Illustrations: exploded view of the different parts of the chamber (left), pictures array of individual capsules imaged within the chamber’s wells (right)
Calcium imaging and analysis
We developp in collaboration with Pierre Bon (permanent researcher, with Laurent Cognet) a calcium-analysis method that is automated and non-biaised. The software sequentially process mathematical operations to provide the variations calcium signalling (or any other fluorescence intensity fluctuations) within a single cell. We are currently improving the bêta-version and making proofs-of-principle on different biological systems.
Contact: Gaëlle Recher
Illustrations: recorded calcium signalling
Implementation of a light absorption analyser for cell viability and differentiation fast monitoring
While 3D optical microscopy techniques allow us to visualize and monitor with great details the fate and internal cellular architecture of encapsulated organoids, they are intrinsically slow. To perform fast and automated analytical assay, we need to design image-free methods and to integrate the detection module into a microfluidic chip. In the first step towards building such an optofluidic platform, we develop an analyser based on light extinction measurements. This approach does not require cell labelling and allows to monitor cell viability and cell differentiation with good statistics.
Contact: Pierre Nassoy
Illustrations: 3D-printed mold for PDMS casting, microfluidics cell-sorter (left), capsule flowed through a capillary (right)
The BiOf lab has developed the Cellular Capsule Technology, which enables the alginate-encapsulation of multicellular spheroids. Our optofluidic devices allow producing efficiently stem cell spheroids, tissue cysts (cystic 3D cell cultures), and various 3D tumor models, including liquid tumor organoids. The experience of our academic research team, based in Bordeaux, France, in self-assembled 3D cell culture and tissue engineering has led us to explore cell mechanics and cellular mechanotransduction, design novel 3D cell-based assays using fluorescence light-sheet microscopy and investigate novel therapeutic strategies, such as the use of dopaminergic neuronal organoids as a new parkinson’s disease cell therapy.