A novel technique for positioning multiple cell types by liquid handling

Abstract

The spatial control of cells on a surface and the patterning of multiple cell types is an important tool for fundamental biological research and tissue engineering applications. A novel technique is described for the controlled seeding of multiple cell types at specific locations on a surface without requiring the use of specialized equipment or materials. Small-volume, quasi-hemispherical drops of cell solution are deposited onto a cell culture surface immersed under barrier oil, which serves to contain the drop and prevents evaporation of the cell culture medium during the time necessary for cells to attach to the cell culture surface. Subsequent flooding with an aqueous cell-compatible buffer displaces the barrier oil, allowing the cells to grow freely across the surface. This technique offers a simple and easily implemented solution for defining the initial position of cultured cells. The coculture of multiple cell types may be carried out by incorporating different cell types in each drop. A suitable drop volume was found to be 1 l dispensed with a standard 0.5-10 l pipette. The drop formed resulted in a footprint diameter of approximately 2 mm. Mineral oil and silicone oil do not compromise the viability of cultured cells when used in this technique. Moreover, a surface with heparin-immobilized FGF2 is shown to retain its bioactivity following drying of the substrate and contact with mineral oil.

“One Cell−One Well”: A New Approach to Inkjet Printing Single Cell Microarrays

“…The use of dual liquid phase inkjet printing has also been reported, in the form of aqueous biphasic systems, where each of the layers is loaded with a high concentration of a non-volatile component that forces the two solutions to remain separate . Moreover, the combination of arrays of macrodroplets (≥1 μL) of cell cultures submerged in mineral oil has also been recently reported . However, the combination of inkjet printing with water-in-oil cell culturing has not been reported, but offers a practical means of overcoming evaporation-related challenges of living cell microarray fabrication, and to miniaturize sample size further into the nanoliter/subnanoliter range of volumes.…”

“…In a recent article, 37 the use of mineral oil and silicone oil as a protective layer for standing aqueous droplets was reported. In this approach, macroscopic droplets (1 μL) of cell culture were deposited onto a surface that had been submerged in various hydrophobic liquid media, and was shown to protect the droplets from evaporation.…”

“…30 Moreover, the combination of arrays of macrodroplets (g1 μL) of cell cultures submerged in mineral oil has also been recently reported. 37 However, the combination of inkjet printing with waterin-oil cell culturing has not been reported, but offers a practical means of overcoming evaporation-related challenges of living cell microarray fabrication, and to miniaturize sample size further into the nanoliter/subnanoliter range of volumes. Because of the sample volumes involved, inkjet is particularly well-suited as a dispensing technique for delivering single living cells to predetermined locations.…”

Coalescence-assisted generation of single nanoliter droplets with predefined composition

“…We used mineral oil due to its low toxicity and prevalence in digital microfluidic applications. [31][32][33][34] The addition of 1% oleic acid, a biocompatible, naturally occurring fatty acid, 35 lowered surface tension by reorientation of molecules at the water/oil interface, 36 which reduced droplet adherence to channel walls at low droplet velocities. The lack of surfactant makes the droplets amenable to coalescence and allows the manipulation of larger droplet volumes as well as overcoming biocompatibility issues.…”

Digital Microfluidic Cell Culture

“…Nonetheless, the use of immiscible fillers is promising because some fluorinated liquids and silicone oils are biocompatible with endothelial cells and are used in vitreoretinal surgery (33). In addition, silicone oil has been used in cell (34) and embryo (35) culture systems to prevent evaporation and preserve sterility. Accordingly, several groups (26,27,36,37) have used silicone oil for cell-based applications in DMF without reported toxicity (although no specific studies have been done to verify these effects).…”