Team:UniGE-Geneva/Demonstrate

Unige iGEM 2019

Demonstrate

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Measurement of multiple activities in different cell types in co-culture

We encapsulated mammary breast cancer cells (BC) in red fluorescent capsules, and liver tumor cells (LC) in unmarked capsules (Figure 1). The cells had been genetically modified to allow the detection of two distinct proliferative states via the expression of distinct fluorescent reporter proteins. After co-cultivating the encapsulated cells, we were able to measure the proportion of cells in each proliferative state, in either of the two cell types (Figure 2). This validates the ability of our method to allow individual measurement of several biological parameters in a co-culture of several cell types.

Figure 1: Co-culture of FUCCI-BC cells in far-red capsules (blue) with FUCCI-LC cells in unlabeled capsules (non-fluorescent) in FBS 15%. Cell segmentation profile used to quantify the different fluorescent intensities following different combinations is shown at the right.

Figure 2: Quantification of two cellular proliferation states in BC cells and LC cells in co-culture.

Multiple biological activities and interactions between cell types

Co-culture of BC and LC also represents basic inter-tissue communication that would be occuring in vivo because the presence of liver cells enabled metabolism of the pro-drug tamoxifen, and the active metabolite of this drug was then able to exert anti-cancer effects on BC. To test this possibility, we also did a co-culture of BC cells and LC cells treated with cytotoxic concentrations of Tamoxifen (Figure 3). We were able to discriminate the cytotoxic activity (Figure 4) from the cytostatic activity (Figure 5) by using two different fluorescent reporters in two differently labeled capsules, and the LC cells were in a third labeled capsule. This experiment showed that when BC cells are co-cultured with LC cells, Tamoxifen is more cytotoxic when compared to BC cell cultured alone. This demonstrates the ability of our novel co-culture method to reproduce a physiological interaction between two tissues, as observed in the clinic.

Figure 3: Co-culture of zipGFP-BC cells in far-red capsules (blue) with FUCCI-BC cells in unlabeled capsules (non-fluorescent) with LC cells in green capsules (green).

Figure 4: Caspase-dependent apoptotic activity in BC cells treated with Tamoxifen and co-cultured or not with LC cells.

Figure 5: Proliferation states of BC cells treated with Tamoxifen and co-cultured or not with LC cells.

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.

Sakaue-Sawano A, Yo M, Komatsu N, Hiratsuka T, Kogure T, Hoshida T, et al. Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle. Mol Cell. 02 2017;68(3):626-640.e5.