> DISCOVERING 3D CELL CULTURE THROUGH THE MICROCHANNELS

3D as the microenvironnement

> WHY USING MICROCHANNELS DEVICES?

 

Cells in the body are physically confined by neighboring cells, tissues, and the extracellular matrix. Then, the microenvironment surrounding a cell significantly influences cell function through both biochemical and biophysical parameters.
More and more, microfluidic cell culture shows its capacity to allow new insights into cellular function because it enables precise control over multiple factors among multiple cell types in a single in vitro device. For example, it will facilitate the establishment and the control of biochemical gradients, and provide improved access for imaging. Furthermore, in contrast to most conventional platforms, which are limited to two-dimensional studies, microfluidic systems may integrate scaffolds that enable cell culture studies in three dimensions in a more physiologically relevant model. Moreover, using microfluidic devices, such as microchannels, enable a lower samples and expensive reagents consumption.
Although animal models have vastly improved our understanding of cell biology, complementary in vitro systems have the potential to offer valuable quantitative insights into how biophysical properties influence cell biology, with or without pathophysiology context.

> HOW ARE MICROCHANNELS DEVICES MADE?

 

Elastomeric micromolding techniques were developed by Bell Labs in the 1970s, and first applied to microfluidics and cell biology in the 1980’s. Nowadays, Polydimethylsiloxane (PDMS) is the most common microfluidic substrate in use in academic laboratories due to its bio-compatibility, reasonable cost, easy use, and optical transparency.
With PDMS, it becomes easy to replicate microchannel structures by molding. Once molded, the chip has to be inverted on a glass substrate to enclose the channels.
The surface of native PDMS is hydrophobic and must therefore be specifically treated prior to cell culture to facilitate attachment and growth. A number of different methods have been developed to reduce hydrophobicity and coat PDMS surfaces for enhanced cell attachment such as oxygen plasma.
Once activated, the extracellular matrix can be added to create the most suitable environment to grow your cells.

1. Cell migration studies

 

Cell migration is a key component for many biological processes including embryonic development, immune responses, wound healing, organ regeneration, and cancer cell metastasis, thus making it an exciting and crucial field of study.
Several processes are involved in cell migration, such as chemotaxis, haptotaxis, transmigration, chemoinvasion… Traditional 2D cell migration assays do not recapitulate the complex topography found in the body. Microchannel cell culture offers an alternative to traditional methods to study cell migration and its mechanism at single cell resolution or at a collective scale, in a more physiologically relevant way.

Microchannels fabricated for the study of cell migration during physical confinement are typically straight, ranging in cross-sectional area from ∼20 μm² to more than 1,000 μm² (reviewed in Paul et al., 2016). Cells adopt distinct signaling strategies to modulate cell migration in different physical microenvironments, to confined (3D) or unconfined environments (2D). Migration speed in microchannels is often greater than the one observed in 2D environments.

 

2. Morphology and plasticity related studies

 

The bioengineering microchannels have been used to explore how confinement and mechanical forces influence the biomolecular properties of cells. In particular, how physical confinement modulates intracellular molecules responsible for cell morphology, adhesion, contractility, and gene expression. Microchannels’ geometries allow testing of cells’ response to anisotropic physical gradients or cues. Moreover, the geometry of the microchannel influences cell signaling and mechanosensing pathways to modulate cell decision making. For example, it has been shown that cytoskeletal architecture, cellular adhesion, gene expression, and activation of mechanotransduction pathways are mechanism dependent processes (review in Paul et al., 2016).
Some confined geometry devices, such as small size channels or constrictions, also demonstrate the capacity of the cell to distort and cross such issues in a normal or pathologic context. Malboubi et al., were also using a confined environment to study the deformability of cancer cells. Such experiments were also done on other cell types such as red blood cells.
In the future, elucidating the precise effects of physical forces on cell behavior will be crucial, to have a better understanding of mechanotransduction pathways for instance.

 

3. Experimental models

 

> Example n°1: Angiogenesis and metastasis assay

 

Cancer is one of the leading causes of death around the world. It has been reported that more than 90% of human deaths diagnosed with cancer are attributed to metastasis. Cancer is considered a localized disease in its early stages; however, in the process of metastasis, cancer cells of a typical solid tumor must loosen their adhesion to neighboring cells, escape from the tissue of origin, invade other tissues by degrading the tissues’ extracellular matrix until reaching a blood or lymphatic vessel, to enter circulation, and proliferate in the new environment in which they ultimately reside. Cancer cell migration and associated subjects, as drug testing, are a topic of interest in the field of oncology. To control microenvironment in such topic is essential, and microchannel devices allow it.
Irimia et al. used microchannel devices to observe directly and quantify cancer migration at single cell level, with very high spatial and temporal resolution. They concluded that cancer cells have a higher motility than non-tumoral cells in conditions similar to the in vivo ones.
Furthermore, the channel features are also a point of interest. An essential aspect of micro-fabrication is that it supports the construction of channels of different shapes and coated with different extracellular matrix proteins. For example, cell plasticity can be challenged by reducing the size of the channels during migration. Dolega et al. demonstrated that epithelial cell behaviour can be modulated in microchannels by mechanical constraint (i.e. by adjusting the diameter of the microchannel). Using constriction, Byun et al., found that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential.
Those examples show that microchannels can be used to study morphology, phenotype, and migration potential of tumor cells. However, they can also be used as new diagnostic and therapeutic approaches in oncology (reviewed in Wu et al., 2017).

 

> Example n°2 : Immune cell migration assay

 

Cell migration is a hallmark of some immune cells functions, and represents a multi-factorial process difficult to study in their natural environment. Micro-fabrication offers the possibility to develop robust bottom-up approaches that allow the evaluation of a specific contribution of individual physical and biochemical extracellular cues to cell migration. Microchannels therefore represent a valuable controlled experimental system from which hypotheses to be later tested in vivo can be generated.
Matthieu Piel’s lab, one of our partners, largely investigates cell polarization and pathway signaling, during immune cells migration, such as Dendritic cells (DC), thanks to micro-fabricated devices (reviewed in Vargas et al., 2015). For instance, they used microchannels to characterize organelle location, protein and calcium dynamics, as the impact of extracellular cues, during DC migration.

 

> Example n°3 : Vascularization models

 

Vascularization is also a topic of interest in current biomedical research. Vascularization is involved in several processes such as angiogenesis, organ-on-a-chip models, and hematologic studies. In order to better mimic the physiological conditions, those microfluidic networks generally include a controlled flow rate, and levels of shear stress have been designed.
For instance, organ-on-a-chip technologies integrate several well-understood microfluidic components into a single in vitro device, allowing researchers to recapitulate more closely in vivo functions (both normal and disease states): gut, lung-, blood vessel-, cancer-, and kidney-on-a-chip (reviewed in Sackman et al., 2014). Sophisticated in vitro assays with which drugs could be tested, increasing the predictability of a new drug before animal testing (possibly even replacing animal trials) and human clinical trials.
Microchannels can mimic the sizes and structure of the capillaries inside living tissues, where endothelial cells are successfully cultured into, or were being used to perfused and supplied metabolites into hydrogel constructions.
Blood presents a complex inner structure, comprising a wide variety of cells of different sizes and elastic properties, which are immersed in the slightly viscoelastic blood plasma. The understanding of complex systems benefits from a simplified model that focuses on their basic ingredients, facilitating the identification of the relevant mechanisms responsible for the macroscopic behaviour of the fluid.
Alterations in the biophysical properties, such as cell adhesion, cell aggregation, and cell deformability, of blood cells contribute to the pathophysiology of disease states, ultimately leading to compromise of microvascular flow in vital organs. In infectious contexts, microfluidic channels were shown to be efficient to study the behavior of P. falciparum-infected erythrocytes under capillary-like conditions. Shelby et al., demonstrated that contrary to normal erythrocyte, late-stage infected RBCs cause blockage in capillaries. Thanks to their PDMS microstructure, they dissected the contributions of decreased cell deformability and membrane rigidity on movement through capillaries of precise dimensions.

 

> Example n°4 : Neuronal system models

 

Neurons are often cultured in vitro on a flat, open, and rigid substrate, a platform that does not reflect well the native microenvironment of the brain. Once again, 3D confined environments were demonstrated to be efficient to better represent the in vivo conditions.Microfluidic devices form the basis of unique experimental approaches that visualize the development of neural structure at a micro-scale and aid the guidance of neurite growth in an axonal isolation compartment. Claverol-Tinturé et al., thanks to a related microchannel device, studied the activity-dependent neuronal development and characterized the information processing in neuronal networks. Their device includes inlets, as microwells, for cell body confinement, and microchannels capable of guiding neuritis for network topology specification. Bae et al., also utilized microfluidics technology to monitor the differentiation and migration of neural cells derived from human embryonic stems cells. These experimental tools offer advantages for the visualizing of neural structures development within a micro-scale device, which allows the guidance of neurite growth into an axonal isolation compartment.
More complex models could also be elaborated, such as 3D Human Blood-Brain-Barrier-on-a-Chip, to dissect the neuroinflammatory response in an in vitro relevant model for instance.

> CONCLUSION: SIZE DOES MATTER!

 

Microfluidic methods have demonstrated advantages over traditional methods. More and more, the importance of physical microenvironments in regulating cell signaling and motility has gained increasing recognition. Microchannels technologies enhance the capabilities to deeper investigate biology, and medical research, offering new possibilities to solve in vitrotechnical issues such as gradient establishment.

This mini-review focused on main microchannels applications. Other research fields, such as dental surgery, also investigate this micro-confinement technology to better understand some biological processes.

> REFERENCES

 

Bae, J., Lee, N., Choi, W., Lee, S., Ko, J.J., Han, B.S., Lee, S.C., Jeon, N.L., and Song, J. (2016). Use of Microfluidic Technology to Monitor the Differentiation and Migration of Human ESC-Derived Neural Cells. pp. 223-235.

Balzer, E.M., Tong, Z., Paul, C.D., Hung, W.-C., Stroka, K.M., Boggs, A.E., Martin, S.S., and Konstantopoulos, K. (2012). Physical confinement alters tumor cell adhesion and migration phenotypes. FASEB J. 26, 4045-4056.

Bauer, M., Su, G., Beebe, D.J., and Friedl, A. (2010). 3D microchannel co-culture: method and biological validation. Integr. Biol. 2, 371

Bissell, M.J., and LaBarge, M.A. (2005). Context, tissue plasticity, and cancer. Cancer Cell 7, 17-23.

Cavallo, F., Huang, Y., Dent, E.W., Williams, J.C., and Lagally, M.G. (2014). Neurite guidance and three-dimensional confinement via compliant semiconductor scaffolds. ACS Nano 8, 12219-12227.

Choi, J.S., Piao, Y., and Seo, T.S. (2014). Circumferential alignment of vascular smooth muscle cells in a circular microfluidic channel. Biomaterials 35, 63-70.

Claverol-Tinturé, E., Ghirardi, M., Fiumara, F., Rosell, X., and Cabestany, J. (2005). Multielectrode arrays with elastomeric microstructured overlays for extracellular recordings from patterned neurons. J. Neural Eng. 2, L1-7.

Dolega, M.E., Wagh, J., Gerbaud, S., Kermarrec, F., Alcaraz, J.-P., Martin, D.K., Gidrol, X., and Picollet-D’hahan, N. (2014). Facile Bench-Top Fabrication of Enclosed Circular Microchannels Provides 3D Confined Structure for Growth of Prostate Epithelial Cells. PLoS One 9, e99416.

Fernandez, M.-I., Heuzé, M.L., Martinez-Cingolani, C., Volpe, E., Donnadieu, M.-H., Piel, M., Homey, B., Lennon-Dumenil, A.-M., and Soumelis, V. (2011). The human cytokine TSLP triggers a cell-autonomous dendritic cell migration in confined environments. Blood 118, 3862-3869.

Halldorsson, S., Lucumi, E., Gómez-Sjöberg, R., and Fleming, R.M.T. (2015). Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens. Bioelectron. 63, 218-231.

Herland, A., van der Meer, A.D., FitzGerald, E.A., Park, T.-E., Sleeboom, J.J.F., and Ingber, D.E. (2016). Distinct Contributions of Astrocytes and Pericytes to Neuroinflammation Identified in a 3D Human Blood-Brain Barrier on a Chip. PLoS One 11, e0150360.

Heuzé, M.L., Collin, O., Terriac, E., Lennon-Duménil, A.-M., and Piel, M. (2011). Cell Migration in Confinement: A Micro-Channel-Based Assay. In Methods in Molecular Biology (Clifton, N.J.), pp. 415-434.

Hümmer, D., Kurth, F., Naredi-Rainer, N., and Dittrich, P.S. (2016). Single cells in confined volumes: microchambers and microdroplets. Lab Chip 16, 447-458.

Hung, W.-C., Chen, S.-H., Paul, C.D., Stroka, K.M., Lo, Y.-C., Yang, J.T., and Konstantopoulos, K. (2013). Distinct signaling mechanisms regulate migration in unconfined versus confined spaces. J. Cell Biol. 202, 807-824.

Hwang, H., Park, J., Shin, C., Do, Y., and Cho, Y.-K. (2013). Three dimensional multicellular co-cultures and anti-cancer drug assays in rapid prototyped multilevel microfluidic devices. Biomed. Microdevices 15, 627-634.

Irimia, D., and Toner, M. (2009). Spontaneous migration of cancer cells under conditions of mechanical confinement. Integr. Biol. 1, 506.

Kondo, Y., Yada, Y., Haga, T., Takayama, Y., Isomura, T., Jimbo, Y., Fukayama, O., Hoshino, T., and Mabuchi, K. (2017). Temporal relation between neural activity and neurite pruning on a numerical model and a microchannel device with micro electrode array. Biochem. Biophys. Res. Commun. 486, 539-544.

Lázaro, G.R., Hernández-Machado, A., and Pagonabarraga, I. (2014b). Rheology of red blood cells under flow in highly confined microchannels: I. effect of elasticity. Soft Matter 10, 7195-7206.

Lázaro, G.R., Hernández-Machado, A., and Pagonabarraga, I. (2014a). Rheology of red blood cells under flow in highly confined microchannels. II. Effect of focusing and confinement. Soft Matter 10, 7207-7217.

Lee, M.K., Rich, M.H., Baek, K., Lee, J., and Kong, H. (2015). Bioinspired Tuning of Hydrogel Permeability-Rigidity Dependency for 3D Cell Culture. Sci. Rep. 5, 8948.

Mak, M., Reinhart-King, C.A., and Erickson, D. (2013). Elucidating mechanical transition effects of invading cancer cells with a subnucleus-scaled microfluidic serial dimensional modulation device. Lab Chip 13, 340-348.

Malboubi, M., Jayo, A., Parsons, M., and Charras, G. (2015). An open access microfluidic device for the study of the physical limits of cancer cell deformation during migration in confined environments. Microelectron. Eng. 144, 42-45.

Molino, D., Quignard, S., Gruget, C., Pincet, F., Chen, Y., Piel, M., and Fattaccioli, J. (2016). On-Chip Quantitative Measurement of Mechanical Stresses During Cell Migration with Emulsion Droplets. Sci. Rep. 6, 29113.

Novo, P., Dell’Aica, M., Janasek, D., and Zahedi, R.P. (2016). High spatial and temporal resolution cell manipulation techniques in microchannels. Analyst 141, 1888-1905.

Park, T.H., and Shuler, M.L. (2003). Integration of cell culture and microfabrication technology. Biotechnol. Prog. 19, 243-253.

Paul, C.D., Hung, W.-C., Wirtz, D., and Konstantopoulos, K. Engineered Models of Confined Cell Migration.

Qin, Y., Yang, X., Zhang, J., and Cao, X. (2017). Developing a non-fouling hybrid microfluidic device for applications in circulating tumour cell detections. Colloids Surfaces B Biointerfaces 151, 39-46.

Raab, M., Gentili, M., de Belly, H., Thiam, H.-R., Vargas, P., Jimenez, A.J., Lautenschlaeger, F., Voituriez, R., Lennon-Dumenil, A.-M., Manel, N., et al. (2016). ESCRT III repairs nuclear envelope ruptures during cell migration to limit DNA damage and cell death. Science (80-. ). 352, 359-362.

Rusanov, A.L., Luzgina, N.G., Barreto, G.E., and Aliev, G. (2016). Role of Microfluidics in Blood-Brain Barrier Permeability Cell Culture Modeling: Relevance to CNS Disorders. CNS Neurol. Disord. Drug Targets 15, 301-309.

Sackmann, E.K., Fulton, A.L., and Beebe, D.J. (2014). The present and future role of microfluidics in biomedical research. Nature 507, 181-189.

Scianna, M., and Preziosi, L. (2013). Modeling the influence of nucleus elasticity on cell invasion in fiber networks and microchannels. J. Theor. Biol. 317, 394-406.

Shelby, J.P., White, J., Ganesan, K., Rathod, P.K., and Chiu, D.T. (2003). A microfluidic model for single-cell capillary obstruction by Plasmodium falciparum-infected erythrocytes. Proc. Natl. Acad. Sci. U. S. A. 100, 14618-14622.

Thiam, H.-R., Vargas, P., Carpi, N., Crespo, C.L., Raab, M., Terriac, E., King, M.C., Jacobelli, J., Alberts, A.S., Stradal, T., et al. (2016). Perinuclear Arp2/3-driven actin polymerization enables nuclear deformation to facilitate cell migration through complex environments. Nat. Commun. 7, 10997.

Vargas, P., Chabaud, M., Thiam, H.-R., Lankar, D., Piel, M., and Lennon-Dumenil, A.-M. (2016). Study of dendritic cell migration using micro-fabrication. J. Immunol. Methods 432, 30-34.

Wu, J., Chen, Q., and Lin, J.-M. (2017). Microfluidic technologies in cell isolation and analysis for biomedical applications. Analyst 142, 421-441.

Zhao, Y., Xu, D., and Tan, W. (2017). Aptamer-functionalized nano/micro-materials for clinical diagnosis: isolation, release and bioanalysis of circulating tumor cells. Integr. Biol. 9, 188-205.