On-chip culture system for observation of isolated individual cells

Abstract

To investigate the properties of isolated single cells with their environment, we developed the differential analysis method for single cells using an on-chip microculture system. The advantages of the system are, (i). continuous cultivation of a series of isolated single cells or a group of cells under contamination free conditions, (ii). continuous observation and comparison of those cells with 0.2 microm spatial resolution by a phase-contrast/fluorescent microscopy system with digital image processing. The core of the system is an n x n (n = 20-50) array of chambers, where each is 20-70 microm in diameter and 5-30 microm deep holes etched into a biotin-coated 0.17 mm thick glass slide. The biotin-coated glass slide is covered with the streptavidin coated cellulose semipermeable membrane, which is fixed on the surface of the glass slide by streptavidin-biotin attachment, separating those holes from the nutrient medium circulating through a 'cover chamber' above. A single cell or group of cells can thus be isolated from environment perfused with the same medium, and the medium in each chamber can be changed within the diffusion time (<1/30 s). In addition, the microchamber volumes of specific cells or cell groups can be controlled by the sizes of the chambers. By using this system we found that the length of isolated Escherichia coli increased at 0.06 microm min(-1) between cell divisions regardless of the chamber volume, and that the cell concentration reached 10(12) cells ml(-1) under contamination free conditions. The system is thus particularly useful for one cell level analysis because the direct descendants of single cells can be cultured and compared in the isolated microchambers, and the physical properties of the cells in each microchamber can be continuously observed and compared.

Noise-driven growth rate gain in clonal cellular populations

“…The important differences are (i) the microchannels are directly created on a glass coverslip, not on PDMS; and (ii) the channel region is covered by a semipermeable membrane (SI Appendix, Fig. S1) via biotin-streptavidin bonding by chemically decorating the surface of a microfabricated glass coverslip with biotin and membrane with streptavidin (23). The narrow and shallow growth channels can harbor 25 ∼ 40 cells at a time.…”

Sex allocation pattern of the diatomCyclotella meneghiniana

“…We designed an on-chip single-cell microfluidic system [16,17] for tracking differentiation processes at the single-cell level. Cells were randomly seeded on arrays of 100 Â 100 mm microchambers fabricated on a glass slide (figure 1).…”

“…Each microchamber was approximately 100 Â 100 Â 20 mm. After the sample cells were placed in the array, the array was sealed with a semipermeable membrane (molecular weight cut-off, 25 000; Spectrum Laboratories, Irving, TX) using an avidin-biotin attachment to prevent the cells from escaping [17]. Microchambers comprising various cell numbers were prepared using stochasticity for the process of cell application and sealing with the membrane.…”

Optimal Lineage Principle for Age-Structured Populations

“…The basic configurations of the microfluidics device used in this study are the same as those reported in the previous studies (Wakamoto et al 2005;Inoue et al 2001;Wakamoto et al 2001), which use the microstructures fabricated on a glass slide, the sealing method of semi-permeable membrane via streptavidin-biotin bonding, and the medium perfusion unit to control the culture conditions around the cells. The microstructure used in this study was an array of 2000(l) × 3(w) × 1(d)-μm micro-grooves fabricated on a 0.17-mm-thick coverslip (MATSUNAMI NEO Micro Cover Glass, 24 × 60 mm, thickness No.…”