We report an unordered 2-D array of eukaryotic cells completely embedded in a 3-D matrix. Every cell is located at the same distance from the gel surface, which ensures uniformity of growth conditions and ease of observation characteristic of a 2-D culture. Each cell is firmly immobilized, and each has a unique address in the array. The cells can be rapidly screened, individually monitored during extended time periods, and cultured with the formation of spheroid microcolonies characteristic of a 3-D culture. Individual microcolonies can be extracted from the gel and further propagated, thus enabling isolation of pure cell clones from rather dense cell populations and rapid drug-free generation of stable cell lines.
There is ample evidence that 3-D cell cultures better mimic the in vivo conditions of a multicellular organism than do 2-D cultures, in which cells adhere to glass or plastic surfaces (1-4). Often, the responses of cells in a 2-D culture to various cues are quite dissimilar from those of the cells in vivo or in a 3-D culture (5-8). This is partly because the physiology of a cell is determined, in addition to the genome, by the microenvironment, including mechanical properties of the extracellular matrix (ECM) and physical and chemical anisotropies (2, 9-11), which are quite different in 3-D and 2-D cultures (7, 12, 13). Therefore, 3-D cultures are preferable over 2-D for the use in a number of fields, such as studies on stem cell differentiation, tissue morphogenesis, cancer biology, cell-virus interactions, and cell-based drug screening and toxicology assays (1, 14, 15).
Still, 2-D cultures cultures have the advantage of presenting every cell in the same plane. This ensures that all the cells are cultured under identical conditions, including gas exchange, nutrition supply and waste removal, and permits the cells to be easily monitored, screened, and collected for further use. On the contrary, in a 3-D culture, cells located at different distances from the surface encounter different physiological conditions (3, 8). A variety of sophisticated and expensive systems have been developed to minimize such chemical gradients (3). Another problem is the difficulty of using conventional microscopes for monitoring cells immersed at different depths in a highly scattering medium (7). Both the problems were partially overcome by “on-top” cultures, in which cells are cultured in a liquid medium while being attached to a surface of a gel, while mimicking to some extent the conditions of a 3-D culture (16, 17). However, in such a format, cells and their progeny are not securely immobilized and may occasionally migrate into the surrounding solution. Also, before they become attached to the gel surface, cells tend to aggregate with one another and may unevenly spread over the gel surface (17).
Here we report unordered 2-D arrays of eukaryotic cells that combine the advantages of the microenvironment of a 3-D culture with the uniformity of conditions and ease of observation characteristic of a 2-D culture. This planar 3-D culture may be of use in a number of research and applied fields requiring observation, manipulation, and proliferation of a large number of individual eukaryotic cells under strictly controlled conditions.Materials and methods
Adherent cells—HeLa (CCL-2; ATCC, Manassas, VA, USA), HEK-293 (CRL-1573), H1299 (CRL-5803), and SC-1 (CRL-1404)—were grown to 90%–100% confluence in 25 cm2 flasks and dispersed according to the ATCC protocol (www.lgcstandards-atcc.org) followed by the addition of 10 mL Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (PAA Laboratories, Pasching, Austria), 4 mM glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin. Suspension cells DT40 (CRL-2111) were grown in DMEM supplemented with 8% FBS, 2% chicken serum (cat. no. C5405; Sigma-Aldrich, St. Louis, MO, USA), 2 mM glutamine, 50 µM 2-mercaptoethanol, 50 U/mL penicillin, and 50 µg/mL streptomycin until the density of 106 cell/mL, whereas cells K-562 (CCL-243) were grown in RPMI-1640 medium supplemented with 10% FBS, 4 mM glutamine, 50 U/mL penicillin, and 50 µg/mL streptomycin until the density of 0.5 × 106 cells/mL.
For transfecting HEK-293 cells, 0.3 g GFP-encoding plasmid pEGFP-C3 (Clontech Laboratories, Mountain View, CA, USA) and 1 L Unifectin-56 (Unifect Group, Moscow, Russia) were mixed with 225 L serum-free DMEM, incubated for 20 min at 22°C, and added to a well of a 12-well plate containing 1-day-old 50%–70% confluent cell layer under 0.9 mL of the complete growth medium, and the cells were grown for one more day before use.
Polyacrylamide (PAA) gels were cast in 14-mm-diameter, 0.4-mm-deep wells, then washed and dried as reported (18). Merged gels were prepared as follows. Cells were pelleted in a centrifuge for 5 min at 200× g (or 300× g for DT40), resuspended in Dulbecco's phosphate-buffered solution (DPBS; 8 mM Na2HPO4/1.5 mM KH2PO4, pH 7.5, 138 mM NaCl, 2.7 mM KCl, 0.9 mM CaCl2, 0.5 mM MgCl2) to the desired concentration, and incubated for 2 min at 30°C. The cell suspension was mixed (1:1, v/v) with a cooled to 30°C molten 1% agarose (Type IX, Ultra-low Gelling Temperature, cat. no. A5030; Sigma-Adrich) in DPBS, and 70 L mixture were poured into a well containing dry PAA gel film attached to its bottom, with simultaneous sliding a coverslip over the well. In case of inverted merged gels, the PAA gel was attached to the coverslip. Where indicated, the slide was spun for 1 min at 100× g, 25°C in centrifuge 5804R (Eppendorf, Hamburg, Germany), by placing the slide with the agarose layer facing up on the tube adapter of a bucket rotor (cat. no. A-4–44; Eppendorf). The agarose was then solidified at 4°C for 20 min.