to BioTechniques free email alert service to receive content updates.
A simple and rapid method for generating patterned co-cultures with stable interfaces
Sahar Javaherian#1, Katherine J. Li#1, and Alison P. McGuigan1,2
Full Text (PDF)
Supplementary Material

Figure 2.  Patterned co-cultures. (Click to enlarge)

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.

Figure 3.  Patterning cells in wells of different sizes. (Click to enlarge)

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.

Figure 4.  Reproducibility of patterning. (Click to enlarge)

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.

Competing interests

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]

1.) Herzog, E.L., and R. Bucala. 2010. Fibrocytes in health and disease. Exp. Hematol. 38:548-556.

2.) Nguyen, D.X., P.D. Bos, and J. Massague. 2009. Metastasis: from dissemination to organ-specific colonization. Nat. Rev. Cancer 9:274-284.

3.) Whitesides, G.M., E. Ostuni, S. Takayama, X. Jiang, and D.E. Ingber. 2001. Soft lithography in biology and biochemistry. Annu. Rev. Biomed. Eng. 3:335-373.

4.) Bhatia, S.N., M.L. Yarmush, and M. Toner. 1997. Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts. J. Biomed. Mater. Res. 34:189-199.

5.) Jinno, S., H.C. Moeller, C.L. Chen, B. Rajalingam, B.G. Chung, M.R. Dokmeci, and A. Khademhosseini. 2008. Microfabricated multilayer parylene- C stencils for the generation of patterned dynamic co-cultures. J. Biomed. Mater. Res. A 86:278-288.

6.) Wallace, C.S., and G.A. Truskey. 2010. Direct-contact co-culture between smooth muscle and endothelial cells inhibits TNF-alpha-mediated endothelial cell activation. Am. J. Physiol. Heart Circ. Physiol. 299:H338-H346.

7.) Bhatia, S.N., U.J. Balis, M.L. Yarmush, and M. Toner. 1998. Probing heterotypic cell interactions: hepatocyte function in microfabricated co-cultures. J. Biomater. Sci. Polym. Ed. 9:1137-1160.

8.) Bhatia, S.N., U.J. Balis, M.L. Yarmush, and M. Toner. 1999. Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J. 13:1883-1900.

9.) Truskey, G.A. 2010. Endothelial Cell Vascular Smooth Muscle Cell Co-Culture Assay For High Throughput Screening Assays For Discovery of Anti-Angiogenesis Agents and Other Therapeutic Molecules. Int J High Throughput Screen. 2010:171-181.

10.) Shearer, M.C., and J.W. Fawcett. 2001. The astrocyte/meningeal cell interface--a barrier to successful nerve regeneration?. Cell Tissue Res. 305:267-273.

11.) Cho, C.H., J. Park, A.W. Tilles, F. Berthiaume, M. Toner, and M.L. Yarmush. 2010. Layered patterning of hepatocytes in co-culture systems using microfabricated stencils. Biotechniques 48:47-52.

12.) Paz, A.C., S. Javaherian, and A.P. McGuigan. 2012. Micropatterning Co-cultures of Epithelial Cells on Filter Insert Substrates. Journal of Epithelial Biology & Pharmacology:77-85.

13.) Khademhosseini, A., K.Y. Suh, J.M. Yang, G. Eng, J. Yeh, S. Levenberg, and R. Langer. 2004. Layer-by-layer deposition of hyaluronic acid and poly-L-lysine for patterned cell co-cultures. Biomaterials 25:3583-3592.

14.) Javaherian, S., K.A. O'Donnell, and A.P. McGuigan. 2011. A fast and accessible methodology for micro-patterning cells on standard culture substrates using Parafilm inserts. PLoS ONE 6:e20909.

15.) Peerani, R., C. Bauwens, E. Kumacheva, and P.W. Zandstra. 2009. Patterning mouse and human embryonic stem cells using micro-contact printing. Methods Mol. Biol. 482:21-33.

16.) Fernandes, T.G., M.M. Diogo, D.S. Clark, J.S. Dordick, and J.M. Cabral. 2009. High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research. Trends Biotechnol. 27:342-349.

17.) Lee, M.Y., R.A. Kumar, S.M. Sukumaran, M.G. Hogg, D.S. Clark, and J.S. Dordick. 2008. Three-dimensional cellular microarray for high-throughput toxicology assays. Proc. Natl. Acad. Sci. USA 105:59-63.

18.) Chung, S., R. Sudo, V. Vickerman, I.K. Zervantonakis, and R.D. Kamm. 2010. Microfluidic platforms for studies of angiogenesis, cell migration, and cell-cell interactions. Sixth International Biol.-Fluid Mechanics Symposium and Workshop March 28-30, 2008 Pasadena, California. Ann. Biomed. Eng. 38:1164-1177.

19.) Tumarkin, E., L. Tzadu, E. Csaszar, M. Seo, H. Zhang, A. Lee, R. Peerani, K. Purpura. 2011. High-throughput combinatorial cell co-culture using microfluidics. Integr Biol (Camb) 3:653-662.

20.) Jeyachandran, Y.L., E. Mielczarski, B. Rai, and J.A. Mielczarski. 2009. Quantitative and qualitative evaluation of adsorption/desorption of bovine serum albumin on hydrophilic and hydrophobic surfaces. Langmuir 25:11614-11620.

21.) Kowalczynska, H.M., M. Nowak-Wyrzykowska, A.A. Szczepankiewicz, J. Dobkowski, M. Dyda, J. Kaminski, and R. Kolos. 2011. Albumin adsorption on unmodified and sulfonated polystyrene surfaces, in relation to cell-substratum adhesion. Colloids Surf. B Biointerfaces 84:536-544.

22.) Javaherian, S., N. Anesiadis, R. Mahadevan, and A.P. McGuigan. 2013. Design principles for generating robust gene expression patterns in dynamic engineered tissues. Integr Biol (Camb) 5:578-589.

23.) Srigunapalan, S., I.A. Eydelnant, C.A. Simmons, and A.R. Wheeler. 2012. A digital microfluidic platform for primary cell culture and analysis. Lab Chip 12:369-375.

24.) Derycke, L.D., and M.E. Bracke. 2004. N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling. Int. J. Dev. Biol. 48:463-476.

  1    2    3