Certain applications (12) may require prolonged maintenance of patterned co-cultures. To test whether our technology is suitable for these applications, we maintained co-cultures for seven days, finding patterns were extremely stable; even after one week in culture, the original boundary between the two cell populations was clearly visible (Figure 2E-H). This was true for homotypic (Figure 2H) as well as heterotypic co-cultures (Figure 2F). We quantified the extent of pattern disruption for a homotypic co-culture (Supplementary Figure S4). Pattern disruption is significantly less than what was observed using microcontact printing with the same combination of cell types (22). This limited cell-cell mixing behavior is potentially useful for multi-day experiments where the extent of cell-cell contact between the two cell types could change signaling properties and hence cellular function. Future work will include more detailed characterization of the pre-patterned surfaces to elucidate the mechanism responsible for the increased stability of patterns generated using the present method.
Depending on the specific application, it may also be desirable to generate co-cultures in wells of different sizes. Therefore, we assessed the compatibility of our method with different well sizes. We attempted to pattern co-cultures in 6-, 12-, 24-, 48-, and 96-well plates. We focused on generating robust patterns at the first cell seeding stage, because we found in our 24-well plate system that this step was the most critical for successful patterning. We were able to successfully pattern cell localization in 6-, 12-, 24-, and 48-well plates (Figure 3A-H). For the 96-well plate size, successful cell patterns could be generated (Figure 3I and J) using a higher concentration of BSA and a higher angle of tilting to make sure the two fronts formed by the meniscus of BSA solution did not touch. We verified that the higher BSA concentration in the pre-patterning step did not negatively affect the outcome of co-culture in 96-well plates (Supplementary Figure S5). We are confident our method can produce reliable patterning in wells of 96-well plates and larger.
It should be noted that with decreasing well size, we found that the contour of the pattern became more curved. In 6-well plates, approximately 50% of the interface was straight; however, in smaller wells the BSA solution generated a curved interface (Figure 3G). The curvature of the interface results from meniscus formation when BSA solution is placed in a well. In the case of 6-well plates, the contribution of the meniscus to the final shape is negligible. However, in smaller wells the meniscus effect is so large that the two fronts of BSA solution join at the top of the well, resulting in an oval-shaped interface as opposed to a semicircle. We expect that supplementing the BSA solution with a cell culture–compatible surfactant (23) or changing the tilt angle (as used in the 96-well plate case) will enable the generation of straighter interfaces.
Among the most important requirements for assays designed for high-throughput applications are consistency and reproducibility. For example, it is important that patterning in all of the wells of an individual plate in parallel is successful. To better characterize the reliability of our patterning, we measured the reproducibility of interface generation at the pre-patterning and cell patterning steps. Analysis of the reproducibility of our protein pre-patterning step (Supplementary Figure S6A) shows that by tilting the plate we were able to ensure reproducible BSA adsorption in all wells of the plate. Furthermore, we verified that the tilting of the plate does not result in uneven protein adsorption in different locations of the pre-patterned surface (Supplementary Figure S6B). We next attempted simultaneous patterning of cells in all wells of a 24-well plate using ARPE-19 cells to characterize the consistency of the cell patterning step. We were able to successfully generate cell patterns in all 24 wells, demonstrating the consistency of our method.
Reproducible patterning is also particularly important in high-throughput imaging applications where low variation in the positioning of the interface between the two cell types allows the interface region to be captured in a single image obtained at the same position from well to well, preventing the need for cumbersome post-acquisition image analysis. To test whether our method produced sufficiently reproducible patterning for high-content imaging, we assessed the spatial reproducibility of our pattern. Specifically, we quantified the well-to-well variation in the location of the interface of the patterned cell populations. For each well, we measured the y-coordinates of the interface at set values of x-coordinates. Figure 4A shows the results of this analysis in coordinates converted into distance from the bottom/ right corner of the screen. Deviations in the position of interface in different wells were minimal with the relative standard deviation in different locations ranging from 0.6% to 9.1%, corresponding to a displacement of less than 100 μm at the center point of the interface. This deviation easily allows for capturing the interface in the same image frame location in each well at a 4× magnification. To demonstrate this, we overlaid images of the interface with the closest representation of the average localization against two images representing the localization of interfaces with the most deviation from the average (Figure 4B). All three interfaces are easily captured with the same image frame location using automated microscopy. This is significant because existing patterning methods often have an imperfect success rate and positioning the pattern in the same location in every well requires careful alignment of the stamp or stencil within a well.
To demonstrate the utility of generating a defined interface between the two cell types, a co-culture of wild type MDCK and MDCK cells overexpressing GFP-N-cadherin (Supplementary Figure S7A) was studied. At the pattern interface, GFP-N-cadherin MDCK cells are surrounded on one side with wild type cells and on the other side by self-like GFP-N-cadherin cells. Interestingly, we noticed that in these interface cells GFP-N-cadherin was preferentially enriched on the side of the cells in contact with other GFP-N-cadherin overexpressing cells. We confirmed this asymmetric localization of GFP-N-cadherin by quantifying GFP signal intensity at the membrane on either side of the cells at the interface (Supplementary Figure S7A). The planar polarization of the GFP-tagged protein is consistent with homotypic inter-cellular interactions of N-cadherin (24) between neighboring cells. This specific model system could be useful for screening for molecules involved in this intercellular signaling process.
We present for the first time a method for medium-throughput generation of 2-D patterned co-cultures. The simplicity and reliability of our method, combined with its compatibility with high-content microscopy, make it a potentially useful tool for generating a more in vivo–like cell culture platform for screening applications.
We acknowledge R. Sodi and SI Ontario for technical assistance. This work was funded by a Natural Science and Engineering Research Council of Canada (NSERC) Discovery grant and a Connaught Early Researcher Award to A.M. and an NSERC graduate scholarship and CIHR Training Program in Regenerative Medicine Fellowship to S.J. The authors have no conflict of interests to declare. S.J. and K.L. designed the project, conducted experiments, analyzed the data and wrote the manuscript, A.M. designed the project, analyzed the data, and wrote the manuscript.
The authors declare no competing interests.
Address correspondence to Alison P. McGuigan, Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada. E-mail: [email protected]
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