A versatile method for dynamically controlled patterning of small populations of epithelial cells on substrates via non-contact piezoelectric inkjet printing

Abstract

Intercellular interactions play a central role at the tissue and whole organism level modulating key cellular functions in normal and disease states. Studies of cell-cell communications are challenging due to ensemble averaging effects brought about by intrinsic heterogeneity in cellular function which requires such studies to be conducted with small populations of cells. Most of the current methods for producing and studying such small cell populations are complex to implement and require skilled personnel limiting their widespread utility in biomedical research labs. We present a simple and rapid method to produce small populations with varying size of epithelial cells (10–50 cells/population) with high-throughput (~ 1 population/second) on flat surfaces via patterning of extracellular matrix (ECM) proteins and random seeding of cells. We demonstrate that despite inherent limitations of non-contact, drop-on-demand piezoelectric inkjet printing for protein patterning, varying mixtures of ECM proteins can be deposited with high reproducibility and level of control on glass substrates using a set of dynamically adjustable optimized deposition parameters. We demonstrate high consistency for the number of cells per population (~1 cell standard error of mean), the population’s size (~0.2 coefficient of variation) and shape, as well as accurate spatial placement of and distance between colonies of a panel of metaplastic and dysplastic esophageal epithelial cells with differing adhesion and motility characteristics. The number of cells per colony, colony size and shape can be varied by dynamically varying the amount of ECM proteins deposited per spatial location and the number of spatial locations on the substrate. The method is applicable to a broad range of biological and biomedical studies including cell-cell communications, cellular microenvironment, migration, and stimulus response.

Simultaneous Multiparameter Cellular Energy Metabolism Profiling of Small Populations of Cells

“…( a ) Process flow. First, to facilitate preferential adhesion to specific areas on the substrate, a solution of the extracellular matrix (ECM) protein fibronectin is deposited using a non-contact piezoelectric printing approach 30 ; Second, cells suspended in culture medium are deposited and allowed to gravity-settle on the substrate; Third, the substrate is washed with PBS 3 times to remove the weakly adhered cells from the interstitial areas. ( b ) Fluorescence emission micrograph of cell colonies stained with JC-1 after 2 hours of incubation following deposition.…”

“…Forty to fifty pL of the fibronectin solution were deposited per single location on the glass substrate in a pattern matching the lid array geometry. The deposition was performed by a non-contact piezoelectric dispensing robot (Rainmaker au301, Aurigin Technology Inc., Phoenix, AZ) 30 . Each location was deposited with two spots, and each spot was made up of two drops.…”

“…It has been shown that the average number of cells per spot for the 20 × 20 µm and 40 × 40 µm geometries was 1.3, and 3.1, respectively. Our group has recently reported a non-contact method for the generation of small (<100 cells/population) populations of epithelial cells with high consistency 30 . Microcontact printing for developing arrays of cell adhesion regions, and cell adhesion protein for cell adhesion, are the methods of choice to achieve such cell cluster patterning.…”

“…Building on the above mentioned technologies of cell adhesion proteins 26 – 28 , 30 and microchamber-based design 21 – 24 , we report a new technology that allows simultaneous quantification of multiple extracellular and intracellular physiological parameters of small (<100 cells) populations of cells. The technology is based on the creation of an isolated microenvironment for the cell population, for measuring transmembrane fluxes of rapidly diffusing small molecules involved in the bioenergetic machinery such as oxygen and protons.…”

Inkjet Printing of Drug-Loaded Mesoporous Silica Nanoparticles—A Platform for Drug Development

“…Hepatocytes have, for instance, been printed to create “liver-on-chip” models [ 73 ]. Printing of epithelial cells has been conducted and the method has also been suggested as applicable for biological and biomedical studies to gain more understanding about e.g., cell–cell communication and stimulus response [ 74 ].…”