A simple and Rapid Method for Generating Patterned Co-Cultures with Stable Interfaces

Abstract

In native tissues, different cell types are organized into defined structures and architectures that are critical for correct tissue function. In vitro cellular patterning methods enable control over the spatial organization of cells, permitting, to some extent, the reproduction of native tissue structures and the generation of a more "in vivo-like" culture platform. While this is advantageous for applications such as drug screening, existing patterning methods are time-consuming, labor-intensive, and low-throughput. Here, we describe a novel medium-throughput patterning strategy for generating spatially controlled co-cultures of two cell types based on differential deposition of BSA solution in a tilted plate. Our method allows generation of homotypic and heterotypic co-cultures that are stable for at least seven days in culture. The reproducibility and consistency of this patterning technique, together with its low cost and ease of use, make it a promising cell culture platform for medium- to high-throughput screening using high-content imaging.

Micropatterning Strategies to Engineer Controlled Cell and Tissue Architecture in Vitro

“…Furthermore, due to the largely manual and complex nature of the patterning process, these methods have not been adapted for use with 96-well plates in many cases, which hinders high-throughput quantification. To combat these issues, our lab recently developed two extremely inexpensive and easily implemented techniques based on the use of parafilm inserts (26) and liquid tilting (56), as shown in Figure 4. The parafilm insert method is a stencil-based approach that avoids the challenges associated with soft-lithography based stencils by generating parafilm inserts that are manually cut and pressure-stuck to a tissue culture surface, shielding' the underlying area from cell attachment.…”

“…Top, schematic illustrating the parafilm insert method (26); bottom, representation of the plate tilting approach (56). …”

“…Note that patternability can be affected substantially by this choice and, in the particular case of the plate tilting method (56) described earlier, different BSA concentrations result in different pattern curvatures within the well. …”

A Triple Co-Culture Method to Investigate the Effect of Macrophages and Fibroblasts on Myoblast Proliferation and Migration

“…Diffusion rates of soluble factors in larger culture volumes (as required in the overflow chamber) are also a consideration; if the rate of diffusion is too low, signalling factors may not be able to reach their targets timeously (13,19). Secondly, there are methods that allow direct interactions between cells (i.e., micropatterning); this is usually achieved by spatially controlling the position of the adhering cell populations within a culture dish (such as that reported by Javaherian et al, 2013, or the co-culture method presented in this paper) (Figure 1A iii and iv) (20). Co-culturing by way of micropatterning involves the creation of a distinct population of cells within a culture dish by selectively controlling the attachment of cells (Figure 1A iii).…”

“…Co-culturing by way of micropatterning involves the creation of a distinct population of cells within a culture dish by selectively controlling the attachment of cells (Figure 1A iii). This can be achieved by creating a nonadhesive area via a physical barrier (using stamps, as seen in (21)) or a chemical barrier (e.g., bovine serum albumin (BSA) (20)). The first population of cells will adhere to the area surrounding the barrier; once the barrier is removed (either by physical removal or by coating the nonadhesive area with an adhesive matrix factor (20)), the second cell population binds in the remaining space (22).…”