1Dept of Physics, MIT - Massachusetts Institute of Technology, 2Department of Electrical Engineering and Computer Science, MIT - Massachusetts Institute of Technology
This article is a part ofJoVE General. If you think this article would be useful for your research, please recommend JoVE to your institution's librarian.Recommend JoVE to Your Librarian
Current Access Through Your IP Address
Current Access Through Your Registered Email Address
Mittal, N., Flavin, S., Voldman, J. Patterning of Embryonic Stem Cells Using the Bio Flip Chip. J. Vis. Exp. (8), e318, doi:10.3791/318 (2007).
Cell-cell interactions consisting of diffusible signaling and cell-cell contact (juxtacrine signaling) are important in numerous biological processes such as tumor growth, stem cell differentiation, and stem cell self-renewal. A number of methods currently exist to modulate cell signaling in vitro. One method of modulating the total amount of diffusible signaling is to vary the cell seeding density during culture. Due to the random nature of cell seeding, this results in considerable variation in the actual cell-cell spacing and amount of cell-cell contact, and cannot prescribe the local environment. A more specific approach for modulating cell signaling is to use molecular inhibitors or genetic approaches to knock down specific signaling proteins, but both of these methods are best suited to manipulating small numbers of molecules. Here, we demonstrate a new approach to modulating cell-cell signaling that modulates the local environment of a cluster of cells by placing different numbers of cells at desired locations on a substrate. This method provides a complementary way to control the local diffusible and juxtacrine signaling between cells. Our method makes use of the Bio Flip Chip (BFC), a microfabricated silicone chip containing hundreds-to-thousands of microwells, each sized to hold either a single cell or small numbers of cells. We load the chip with cells simply by pipetting them onto the array of wells and washing unloaded cells off the array. The chip is then flipped onto a substrate, whereby the cells fall out of the wells and onto the substrate, maintaining their patterning. After the cells have attached, the chip can be removed (or left on). This approach to cell patterning is unique in that it: 1) doesn't alter the chemistry of the substrate, thus allowing cells to proliferate and migrate; 2) allows patterning onto any substrate, including tissue-culture polystyrene, glass, matrigel, and even feeder cell layers; and 3) is compatible with traditional microcontact printing, allowing the creation of extracellular matrix islands with cells placed inside those islands. In this video, we demonstrate the patterning of mouse embryonic stem cells onto tissue-culture polystyrene using the BFC.
Making a BFC
BFCs are made by molding polydimethylsiloxane (PDMS) over a 4" Si master wafer.
Making a master wafer
Preparing the BFC for use
Patterning cells with a BFC
Although the protocol and video shown here describe using the BFC 1 to pattern mouse embryonic stem cells (mESCs), BFCs can be used to pattern practically any cell type of interest. The only change one may need to make is to alter the size of the microwells. In our experience, to use the BFC to pattern single cells of another cell type, a microwell diameter and height equal to 10 microns greater than the unattached cell diameter works best.
BFCs can also be used to pattern cells onto other substrates, including other cells, without significant changes in the protocol. Patterning onto an existing cell layer is one advantage of the BFC approach over traditional cell patterning approaches since certain celltypes, including mESCs and human ESCs, require support or feeder cells to maintain their proper phenotype in culture.
One application of the BFC that we are pursuing is to use the BFC to investigate signaling between mouse embryonic stem cells. By seeding single cells in square lattices with different grid spacings, we modulate the rate at which signaling molecules are exchanged by cells. By making larger wells (with a diameter of 40 microns), we can also collect larger numbers (~2-10) cells at a given location, locally altering cell density. In addition, we can place a number of small wells in close proximity to each other, resulting in a substrate pattern where local cell density is high by cells are not in contact. In this fashion, we can independently modulate diffusible and juxtacrine signaling between cells. Having cells in a grid also makes it much easier to track cells over several days. We believe that the ease of cell patterning with the BFC will encourage other researchers to use it in their studies.
This work was supported by NIH grant EB007278.
|PDMS||Dow Corning||Sylgard 184|
|Silicon master mould|
|Tissue culture dishes||Falcon BD||35-3001||35 mm|
|3/4" binder clips|
|New Item||MicroChem Corp.||SU8-2050|
|BSA||7.5% Bovine Serum Albumin|
1. Rosenthal A., Macdonald A., Voldman J. Cell Patterning Chip for Controlling the Stem Cell Microenvironment. Biomaterials 28, 3208-3216 (2007).