An efficient cell count method using a lattice molded on indents of a culture dish

Cell count is an important task for obtaining biological and medical information. In this paper, a novel cell count method is presented for improving the efficiency of the procedure as well as reducing microbial contamination compared to the conventional cell count method using a hemocytometer. The proposed method involves a lattice array consisting of a 50 μm × 50 μm square with lines of 2-μm width and 1.4-μm depth on a surface indented from a culture dish bottom. This configuration enables observation of cells at the same focus as the lattice. Therefore, an instant cell count during incubation is possible without the tedious, error-prone preparation steps such as harvesting and loading required in conventional methods. In addition, cells can be preserved with minimal contact with the external environment. These advantages become magnified with a periodic long-term cell count. A polystyrene culture dish, 35 mm in diameter, was fabricated by injection molding using a nickel mold, wherein indents of 3 mm × 3 mm in area and 1 mm in height were electroplated based on microfabrication technology. For easy separation from the nickel mold, the four sides of the indents in the mold are inclined at 54.74° via anisotropic silicon etching. The usefulness of the suggested method was verified using adhering HeLa (cervical carcinoma cell) cells and floating Jurkat cells. Both were placed in culture dishes and cultivated for 3 days in a carbon dioxide incubator (5% CO₂, 95% air) at 37 °C, and then successfully observed through the divided lattice using an inverted microscope. The dish was also assessed with a hemocytometer by counting HEK 293T (human embryonic kidney) cells and yeast cells.