Hiroko Kikuchi

Culture of bone-marrow-derived cells in microfabricated pit arrays

Tissue cells cannot survive without the attachment to extracellular matrix (ECM). Furthermore, the geometry of ECM is known to play an essential role in the regulation for these cells to proliferate and differentiate so that tissues with normal morphologies can be formed or maintained. We have introduced microfabricated surface structures coated with ECM protein collagen into cell culture studies to examine a possibility of 3D patterning of ECM.

Microchannel array flow analyzer for measurement of whole blood rheology and flow characteristics of leukocytes activated by bacterial stimulation

Microgrooves (width 6, 7, and 8 micrometer, each with length 20, 30, and 40 micrometers, respectively; depth 4.5 micrometers; number 4704 in parallel of one size per chip; chip dimensions 12 multiplied by 12 mm) photofabricated in the surface of a single-crystal silicon substrate were converted to leak-proof microchannels by tightly covering them with an optically flat glass plate. Using the microchannels as a model of physiological capillaries, total flow rate of heparinized whole blood taken from healthy subjects was determined under a constant suction of 20 cmH2O, while flow behavior of blood cells through individual channels was microscopically observed. The apparent viscosity (ratio to that of saline) of whole blood was obtained as 4.7 plus or minus 0.5, 3.7 plus or minus 0.3, and 3.4 plus or minus 0.2 (mean plus or minus SD, n equals 4) for 6, 7, and 8 micrometer width channels, respectively. Normal leukocytes passed, showing a round shape, through the channels much more slowly then erythrocytes, but caused no appreciable interference with passage of erythrocytes. Meanwhile, cells exposed to the chemotactic peptide FMLP (1 - 10 nM) and bacterial cells (Escherichia coli K 12; 6 multiplied by 106/ml) slowed further greatly, showing very irregular shapes, and eventually blocked the channels. Such a response of leukocytes took place immediately after the exposure to FMLP, but it appeared gradually with time after the exposure to the cells.

Microchannel arrays with improved accessibility and use for cell studies and emulsification

Arrays of microgrooves (groove width; 2, 3, 4, 5, 6, 7, 8, 10, 12, and 14 micrometer, groove interval; width x3, x10, and x20, one size and interval per chip) each connecting a center well and a side edge of a silicon substrate were created by photolithography and anisotropic wet etching. A penetrating hole was made by sand blast at the substrate center for the access to the center well. By tightly covering the substrate surface with a glass plate, the microgroove arrays were converted to microchannel arrays having one ends open at the side edges of the substrate. These microchannel arrays were used for cell trapping for microinjection and also used for emulsification. Poplar (Populus alba) protoplasts were used for the test of cell trapping. Cells showed a very large variation in size and irregularity in shape, and, furthermore, the protoplast preparation contained a number of cell membrane fragments and chloroplasts. Despite the cell size and shape variations and obstruction by the admixtures, many cells could be trapped by aspiration at the channel ends because of their openness to the outside free space and also their large multiplicity in parallel. The free space outside the side of the substrate allowed a free manipulation of a glass micropipette under microscopic observation using transmitted illumination. The microscopic observation direction nearly perpendicular to the movement directions of the micropipette further allowed the movement of the pipette tip nearly always in focus. These led to an easy pointing and puncturing. In addition, the cell trapping points in a line made successive approach to adjacent cells easier. Soybean oil containing 1.5 wt% polyoxyethylene(20)sorbitan monoolete as a surfactant was forced to flow into physiological saline filling the outside of the substrate through the microchannels. Regularly sized oil particles were created by this process with a variation coefficient (S.D./mean) 16% of their diameter. This variation, which is larger than those (minimum 1 - 2%) obtained in our previous trials using our previous microchannel arrays, appeared to be attributable to an irregularity of the channel ends due to microchipping by saw cutting. As an advantage over the previous ones, the present microchannel arrays allowed an easy collection of the created oil particles and also an easy change of the composition of the suspending fluid during the process. The substrate side surface is thus indicated to be useful for interfacing structures or devices microfabricated in the main substrate surface, which may be covered with a glass plate, with conventional or hand-operated tools or processes outside the substrate.

Measurement of oxyradicals from leukocytes lodged in a microchannel array

The oxyradical production by leukocytes is crucial for killing invading bacteria, while it has been widely discussed as a tissue injuring factor. Despite such importance of the event, it is still unclear, probably because of lack of simple and reliable measurement methods, to what extent it varies among different subjects and either to what extent it is affected by environmental factors including diet. The present paper describes use of microfabricated channel arrays, that have been developed for use in studies of blood rheology, in the oxyradical measurement by chemiluminescence.

Changes in stiffness of yeast cells during cell cycle by passability through micromachined channel arrays

Possibility of cell sorting by cellular deformability was examined using yeast cells and previously described microchannel arrays. Cells harvested at every hour during incubation were washed and suspended in sorbitol solution at a concentration of O.D. 0.3. An aliquot of each suspension was caused to flow through the microchannel concentration of O.D. 0.3. An aliquot of each suspension was caused to flow through the microchannel arrays by applying 20 cmH2O suction. Cells at two hours of incubation could not enter into the microchannels, while cells at 4-5 hours of incubation could enter into the microchannels despite their larger size due to budding than the preceding ones and some few cells were observed to pass through 8 micrometers width microchannels. The number of cells that could enter into the microchannels decreased at 7-8 hours and re-increased at 9- 10 hours, but the synchronism in this second cycle appeared to decrease. Protoplasts prepared by treatment with zymolyase from cells at 4-5 hours of incubation showed no appreciable resistance to the microchannel passage.