Team:UniGE-Geneva/Design

Unige iGEM 2019

Design

Monolayer VS 3D cell culture

To make a cell culture method with high physiological relevance, we first evaluated existing methods, identified our goals on how the process could be improved, and then designed experiments to develop this novel drug testing approach.

Currently, the most popular drug testing method uses monolayer cell culture. However, monolayers do not reflect the spatial organization of 3D tissues of human organs in vivo (3D).

By preventing the attachment of cells to a solid support, they associate into multicellular 3D structures called spheroids, which mimic the organization of cells in 3D tissues.

We therefore choose 3D cell culture as a core feature of our new cell culture method.

3D cell co-culture vs Encapsulated 3D cell co-culture

The human body is composed of many different organs, comprised of tissues that communicate with each other by releasing soluble factors that circulate in the blood. Incorporating inter-organ communication into new co-culture methods is therefore important because our aim is to simulate in vivo conditions as closely as possible. For example, ovaries produces the hormone estrogen, which can travel via the blood to the breast where it stimulates mammary epithelial cell proliferation.

Current cell culture methods completely overlook these inter-organ communication stimuli, which can be involved in essential physiological pathways. Existing cell co-culture methods mostly employ two different cell types which are simply mixed together. This is problematic because certain cell-cell contacts never exist in normal physiology (e.g. brain-liver cell direct contacts), and could lead to artefactual results in a cell co-culture drug testing experiment.

The laboratory of Prof. Aurélien Roux developed a microfluidics-based technology that allows the encapsulation of cells inside an alginate capsule, which are then further incubated to generate encapsulated cell spheroids. We envisaged the encapsulation of different cell types in separate capsules, so direct physical contact would not occur between different cell types, even after extended co-culture together.

Find out more about encapsulation technique.

Encapsulated cell culture provides additional advantages like the control of the size of the spheroids. Standard 3D cell culture can result in spheroid overgrowth, leading to large spheroids with a necrotic center because their size is not compatible with an optimal diffusion of oxygen and nutrients. We produced capsules that were 200 μm in diameter to prevent cell spheroid overgrowth, and physilogically normal tissue perfusion rates of oxygen and nutrients. Moreover, the permeability of the alginate capsule allows free passage of all soluble factors that are smaller than 100 kDa, which is the majority of drugs and biologics except for intact antibodies. This opens the opportunity to use alginate encapsulated 3D co-cultures to recapitulate in vivo soluble molecule interactions between different tissues.

Fluorescently-labeled capsules
Encapsulated 3D cell co-culture represents a very attractive tool to study physiological communication between different tissues. However, as for most cell co-culture methods, a major challenge is the identification of different cell types once they are mixed. To more precisely study individual biological processes it would be advantageous to know in which cell type they are occuring. On the other hand, a bulk analysis of different co-cultured cell populations may produce misleading or irrelevant results due to the combination of results from completely disparate cell types.

The Alginate molecule contains a carboxylic acid group that can react with amine groups. By using amine-containing fluorophores, we covalently modified alginate to make it fluorescent. By testing different fluorophores, we generated a palette of many color-encoded capsules. The encapsulation of specific cell types in different fluorescent capsules allows their identification by fluorescence microscopy in an encapsulated 3D co-culture. Instead of current cell co-culture methods that mix no more than two cell types, this strategy can lead to make a cell co-culture with up to five different color-encoded encapsulated cell types (unstained, blue, green, orange, far-red) according to fluorescence spectra characteristics and fluorescence microscopy limitations. Overcoming some of these limitations may allow the incorporation of more than five different cell types per co-culture.

Fluorescent reporter cells

To study biological processes in a co-culture, we should be able to measure biological activities on individual cell types. A system of fluorescent reporters can be used to monitor specific biological activities. Fluorescent intensity can depend on the expression levels of the reporter, or on its maturation or stability. Expression of the reporter can be under the control of a response element regulated by a specific biological activity (hormonal response, xenobiotic response,...). Maturation or stability of the reporter can be controlled by processes like proliferation or apoptosis.

Each reporter system can use a different colored fluorescent protein, allowing the identification of the reported biological activity by using a reporter color-code. We can use lentiviral transduction to stably insert a reporter construct in the genome of a cell type. It allows the genera:on of stable reporter cell lines that we can encapsulate to follow separately several biological actvities simultaneously.

Fluosphera

The combination of both color-encoded capsules and color-encoded reporters allows the simultaneous measurement of up to 16 individual combinations of several cell types reporting the same or different biological activities.

To make it possible, image acquisition of 96-well plates containing the encapsulated 3D cell co-culture is performed with a high-content screening confocal microscope. This kind of microscope is less common in academic laboratories, but represents a very conventional instrument used by many pharmaceutical industry laboratories, which is where our invention is likely to make the most impact in the drug discovery process.

After image acquisition, a segmentation analysis is performed to quantify fluorescence intensities of the different reporters while defining which capsule they belong to. This allows the simultaneous quantification of the levels of several biological activities in several individual co-cultured cell types.

References

Alessandri K, Feyeux M, Gurchenkov B, Delgado C, Trushko A, Krause K-H, et al. A 3D printed microfluidic device for production of functionalized hydrogel microcapsules for culture and differentiation of human Neuronal Stem Cells (hNSC). Lab Chip. 26 2016;16(9):1593‑604.

Andersen T, Auk-Emblem P, Dornish M. 3D Cell Culture in Alginate Hydrogels. Microarrays (Basel). 24 mars 2015;4(2):133‑61.

de Médina P, Favre G, Poirot M. Multiple targeting by the antitumor drug tamoxifen: a structure-activity study. Curr Med Chem Anticancer Agents. nov 2004;4(6):491‑508.

Langhans SA, Three-Dimensional in Vitro Cell Culture Models in Drug Discovery and Drug Repositioning. Front. Pharmacol. (2018). 9:6. doi: 10.3389/fphar.2018.00006