Generation of static and dynamic patterned co-cultures using microfabricated parylene-C stencils

Abstract

Many biological processes, such as stem cell differentiation, wound healing and development, involve dynamic interactions between cells and their microenvironment. The ability to control these dynamic processes in vitro would be potentially useful to fabricate tissue engineering constructs, study biological processes, and direct stem cell differentiation. In this paper, we used a parylene-C microstencil to develop two methods of creating patterned co-cultures using either static or dynamic conditions. In the static case, embryonic stem (ES) cells were co-cultured with fibroblasts or hepatocytes by using the reversible sealing of the stencil on the substrate. In the dynamic case, ES cells were co-cultured with NIH-3T3 fibroblasts and AML12 hepatocytes sequentially by engineering the surface properties of the stencil. In this approach, the top surface of the parylene-C stencil was initially treated with hyaluronic acid (HA) to reduce non-specific cell adhesion. The stencil was then sealed on a substrate and seeded with ES cells which adhered to the underlying substrate through the holes in the membrane. To switch the surface properties of the parylene-C stencils to cell adhesive, collagen was deposited on the parylene-C surfaces. Subsequently, a second cell type was seeded on the parylene-C stencils to form a patterned co-culture. This group of cells was removed by peeling off the parylene-C stencils, which enabled the patterning of a third cell type. Although the static patterned co-culture approach has been demonstrated previously with a variety of methods, layer-by-layer modification of microfabricated parylene-C stencils enables dynamic patterning of multiple cell types in sequence. Thus, this method is a promising approach to engineering the complexity of cell-cell interactions in tissue culture in a spatially and temporally regulated manner.

Applications of Microscale Technologies for Regenerative Dentistry

“…It is well-known that tooth development is the result of the continued reciprocal interaction between epithelial and mesenchymal cells in distinct, but local, environments (Kapadia et al, 2007;Salazar-Ciudad, 2008). Such conditions can be recreated in a controlled manner by the use of microscale techniques to isolate, seed, and study single cells or collections of cells (Khademhosseini et al, 2005;Rosenthal et al, 2007;Wright et al, 2007Wright et al, , 2008. Thus, microscale techniques may provide new tools for the exploration of tooth development.…”

“…Fabrica tion of micro-bioreactor arrays (Figallo et al, 2007) that provide myriad functionalities to monitor and control cell growth are technologies that will likely advance the field. Also, reproducible cellular patterning (Rosenthal et al, 2007;Wright et al, 2008), patterned co-cultures (Wright et al, 2007), and control of the microenvironment over large areas permit arrays of cell constructs to be assessed in a high-throughput manner (Moeller et al, 2008). Such techniques permit the assessment of a variety of growth factors, biomaterials (Anderson et al, 2005), and substrate and cellular interactions, alone or in combination (Khademhosseini et al, 2005) (Fig.…”

A microfluidic array with cellular valving for single cell co-culture

“…1 Similarly, heterotypic cellular interfaces can be produced by selectively masking surfaces using microfluidic poly(dimethylsiloxane) (PDMS) stencils 2 or Parylene C stencils. 3 Sequential cell patterning can also be achieved using photoresponsive polymer films 4 or by using electrochemically active surfaces to modulate ligand presentation. 5 More recently, cells have been metabolically decorated with DNA sequences and patterned as adjacent co-cultures by hybridization with complementary surface-tethered DNA sequences 6 or directly to cells likewise encoded with the complementary sequence.…”

A novel technique for positioning multiple cell types by liquid handling

“…This may be achieved by depositing cell adhesive or repellent molecules on the surface [4][5][6][7] or by using a removable cell-seeding stencil to prevent contact with the cell culture substrate. 8 The former generally restricts cell evolution to the attachmentpermissive region, while the latter may allow cells to evolve freely over the cell culture surface. Physical barriers, such as cloning rings, are able to confine a cell to a specific location on a substrate.…”

“…9,10 When using these techniques, the seeding of more than one cell type may require multiple processing steps, where the attachment of one cell type is completed before the next can be introduced. 3,5,8 More complex methods for accurate cell seeding make use of ink-jet printing and microfluidic devices to deposit cells at the desired positions. 11,12 These methods are suitable for implementing a high level of automation and they allow excellent spatial resolution.…”