Only the three most relevant seeding densities are shown for each pattern size. Thus, Poisson’s distribution provides a correlation between the dimensions of the stencil, the seeding densities, and the droplet size to the end pattern. physical, chemical, or combination techniques have been developed to Rabbit polyclonal to ZNF238 create orderly patterns of mammalian or bacterial cells.20 Physical D-AP5 approaches include inkjet cell printing21,22 and cell localization using optical,23,24 laser-based,25,26 magnetic,27 acoustic,28,29 or dielectrophoretic30 forces. While these techniques physically trap/propel cells at desired coordinates and offer unique features for specific applications, their level of complexities limits their wide-scale adaptation for cell micropatterning. On the other hand, chemical techniques are used to manipulate surface characteristics, such as the electrostatic charge, extracellular matrix (ECM) deposition, etc., making the surface amenable to mammalian or bacterial cell attachment.31,32 To immobilize individual or clusters of adherent cells that readily attach to surfaces through ECM binding, a combination of physical and chemical micropatterning techniques are commonly utilized to control multiple parameters such as spatial arrangement, underlying ECM substrate, cell spread area, cell-cell contact, and morphology of cells.33C35 Among all the available techniques, currently there are two strategies commonly utilized to achieve cell/ECM patterning: (a) microcontact printing (CP) and (b) microstencil patterning (SP). Microcontact printing is an established single cell analysis (SCA) technique used to create a selective extracellular matrix (ECM) environment for cells to adhere and grow.36 Proteins are stamped onto the culture surfaces, which are subsequently treated again to be selectively hospitable to cell adhesion. 37 Many different substrates and proteins can be used as ink for micro-contact stamps, and once created, stamps can be cleaned, reused, and maintained over time.38,39 Alternatively, the microstencil printing is accomplished through a stencil membrane containing through-holes of the desired pattern geometry.40C42 The membrane is placed in close contact with an untreated substrate (e.g., polystyrene Petri dish), and the open surface (through membrane perforations) is chemically or ionically modified to support cell adhesion. Though, the activated surfaces last for a relatively shorter duration and membrane stencils do not have a similar lifespan as microcontact printing stamps.43 While both methods have merits for specific applications, the microstencil approach has been eschewed in favor of CP primarily due to the delicacy of stencils and the complexity of processes involved in fabricating them. Following surface modification, multiple factors limit the production of clean and reproducible cellular patterns for high-throughput single cell analyses including (1) requirement of a large number of cells even though the pattern area is relatively small, (2) lack of control over cell clustering within individual patterns, (3) inability to customize spatial distribution of cells to obtain specific cell density within each pattern, and (4) inability to pattern cells without requiring an underlying protein substrate. Although cell clumping can be minimized by passing the cells through a filtering sieve to obtain a homogeneous single cell suspension, the addition of cells several orders of magnitude in excess than the number D-AP5 of available adhesive patterns exacerbates cell clustering. The requirement of excess cells results as the existing procedures involve filling of the entire patterned Petri dish with culture media to obtain uniform patterns. Attempts have been made to circumvent this need by placing a small D-AP5 droplet of cell suspension specifically on the patterned region.9 However, when placed inside the CO2 incubator for attachment, the droplet experiences evaporation induced convective flows, a phenomenon well known and characterized for the formation of coffee stains.44,45 In case of saline droplets, these flows are governed by the forces generated due to temperature, concentration, and density D-AP5 gradients as a result of uneven evaporation. Additional factors such as aspect ratio, wettability, substrate temperature, humidity, and surface roughness also influence intra-droplet convective currents.46.