Minoru Seki

Hydrodynamic filtration for on-chip particle concentration and classification utilizing microfluidics

We propose here a new method for continuous concentration and classification of particles in microfluidic devices, named hydrodynamic filtration. When a particle is flowing in a microchannel, the center position of the particle cannot be present in a certain distance from sidewalls, which is equal to the particle radius. The proposed method utilizes this fact, and is performed using a microchannel having multiple side branch channels. By withdrawing a small amount of liquid repeatedly from the main stream through the side channels, particles are concentrated and aligned onto the sidewalls. Then the concentrated and aligned particles can be collected according to size through other side channels (selection channels) in the downstream of the microchannel. Therefore, continuous introduction of a particle suspension into the microchannel enables both particle concentration and classification at the same time. In this method, the flow profile inside a precisely fabricated microchannel determines the size limit of the filtered substances. So the filtration can be performed even when the channel widths are much larger than the particle size, without the problem of channel clogging. In this study, concentrations of polymer microspheres with diameters of 1-3 microm were increased 20-50-fold, and they were collected independently according to size. In addition, selective enrichment of leukocytes from blood was successfully performed.

Continuous particle separation in a microchannel having asymmetrically arranged multiple branches

A new method for continuous size separation and collection of particles in microfabricated devices, asymmetric pinched flow fractionation (AsPFF), has been proposed and demonstrated. This method improves the separation scheme of pinched flow fractionation (PFF), which utilizes a laminar flow profile inside a microchannel. In this study, multiple branch channels with different channel dimensions were arranged at the end of the pinched segment, so that the flow rate distributions to each branch channel were varied, and a large part of the liquid was forced to go through one branch channel (drain channel). In the proposed channel system, the flow profile inside the microchannel was asymmetrically amplified, enabling the separation of one-order smaller particles compared with PFF. After introducing the method, we examined the effect of the asymmetric amplification by controlling the outlet of the drain channel. Also, a mixture of 1.0 approximately 5.0 microm particles was separated, and erythrocytes were successfully separated from blood. The results indicate that the AsPFF method could be applied to the separation of much smaller-size particles, since more precise separation can be achieved simply by changing the geometries of branch channels.

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane‐glass hybrid microchip

We report on the development of a hybrid polydimethylsiloxane (PDMS)‐glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS‐glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS‐glass hybrid microchip with 500 bp target of λDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS‐glass microchip. The chip could be considered as an inexpensive single‐use apparatus compared to glass or silicon‐made microchips for the same purpose.

Controlled formation of heterotypic hepatic micro-organoids in anisotropic hydrogel microfibers for long-term preservation of liver-specific functions

We have developed a hydrogel-based cell cultivation platform for forming 3D restiform hepatic micro-organoids consisting of primary rat hepatocytes and feeder cells (Swiss 3T3 cells). Sodium alginate solutions containing hepatocytes/3T3 cells were continuously introduced into a microfluidic channel to produce cell-incorporating anisotropic Ba-alginate hydrogel microfibers, where hepatocytes at the center were closely sandwiched by 3T3 cells. Hydrogel fiber-based cultivation under high oxygen tension enabled the formation of heterotypic micro-organoids with a length of up to 1 mm and a diameter of ∼50 μm, mimicking the hepatic cord structures found in the liver, while maintaining a high hepatocyte viability (∼80%) over 30 days. Long-term observation of up to 90 days revealed a significant enhancement of hepatic functions because of heterotypic and homotypic cell-cell interactions, including albumin secretion and urea synthesis as well as expression of hepatocyte-specific genes, compared with conventional monolayer culture and single cultivation in the hydrogel fibers. The encapsulated hepatic constructs were recovered as scaffold-free micro-organoids by enzymatically digesting the hydrogel matrices using alginate lyase. This technique for creating heterotypic micro-organoids with precisely ordered multiple cell types will be useful for the development of a new liver tissue engineering approach and may be applicable to the fabrication of extracorporeal bioartificial liver (BAL) devices and assessment tools for drug development and testing.

Microfluidic synthesis of chemically and physically anisotropic hydrogel microfibers for guided cell growth and networking

Hydrogel materials with microscale heterogeneity are of great interest in the effort to spatially control cellular microenvironments in tissue engineering applications. Here we present a microfluidic system to continuously synthesize chemically and physically anisotropic Ca–alginate hydrogel microfibers enabling the guidance of cell proliferation to form linear cell colonies and intracellular networks. The microfluidic gelation process involves 2 critical steps to obtain alginate microfibers using axisymmetric microchannels with uniform depth: introduction of a buffer solution between the sodium alginate (NaA) and CaCl2 solutions to modulate the gelation speed, and use of a thickener to balance the viscosities of the solutions. We synthesized hydrogel fibers with diameters of ∼7 to 200 μm, maintaining the anisotropy in the cross-section, and examined factors affecting the fiber diameter and uniformity. Moreover, parallel alginate flows with and without propylene glycol alginate (PGA) enabled the formation of sandwich-type solid-soft-solid hydrogel fibers, which were used to guide the direction of growth of cells inoculated in the soft-core, with the help of outer polycation membranes made of poly-L-lysine. We demonstrated the formation of linear colonies of 3T3 and HeLa cells inside the anisotropic fiber and observed elongated nuclei along the fiber direction. In addition, the heterogeneous morphology of the fiber was utilized to guide neurite elongation and generate cellular networks by using neuron-like PC12 cells. The hydrogel fibers reported here can be used as an innovative tool for investigating cell and tissue morphogenesis in heterogeneous microenvironments, and for creating tissue models with precise control of cellular alignment and elongation.

Synthesis of Polymeric Microspheres with Narrow Size Distributions Employing Microchannel Emulsification

Polymeric divinylbenzene microspheres (MS) of narrow size distributions were prepared by microchannel (MC) emulsification and subsequent polymerization. Using two types of MC plates, large MS with 9.2 μm diameter on average and 5.7% coefficient of variation, as well as small MS with 3.4 μm diameter on average and 7.4% coefficient of variation were prepared. The MS diameter can be controlled by the size of the MC. This method is advantageous for synthesizing polymeric MS of narrow size distributions because of its simplicity.

Taxol (paclitaxel) production using free and immobilized cells of Taxus cuspidata

The production characteristics for Taxol (paclitaxel) using free and immobilized cells of Taxus cuspidata were investigated in a perfusion culture bioreactor. Although the cell growth was inhibited by higher dilution rates, the specific production rate of Taxol was increased by perfusion compared with that using batch operation. Perfusion cultures using a nylon-mesh cell separator for free suspension cells showed similar production profiles to those obtained using immobilized cells. Continuous Taxol production was successfully obtained at an approximate specific production rate of 0.3 mg/g DCW (dry cell weight) per day for up to 40 days. (c) 1997 John Wiley & Sons, Inc.

Effect of temperature and its shift on growth and anthocyanin production in suspension cultures of strawberry cells

Abstract Temperature effect on growth and anthocyanin production in the cultured strawberry, Fragaria ananassa cv Shikinari, was investigated at a temperature range of 15–35°C. Cultivation temperatures have a pronounced effect on cell growth, anthocyanin content and production. Maximum growth was found at 30°C, however, the lower temperature induced an increased level of anthocyanin content within the examined temperatures. As a result, the maximum anthocyanin production was obtained at 20°C. The culture condition was changed step-wise by a temperature shift after 3 days from 30 to 20°C for the rest of the cultivation. In this process, anthocyanin production of 270 mg/l on day 9 was increased 1.8, 3 and 4-fold over that of cultures at 20, 25 and 30°C, respectively.

Key role for transketolase activity in erythritol production by Trichosporonoides megachiliensis SN-G42

Erythritol is an important sugar alcohol industrially produced only by fermentation. The highly osmophilic yeast-like fungi, Trichosporonoides megachiliensis SN-G42, enables commercial production of erythritol with a high conversion from glucose to erythritol of more than 47%. However, the microbial production pathway of erythritol remains unclear. In the present study, the activities of enzymes in the pentose phosphate pathway of Trichosporonoides megachiliensis SN-G42 used for industrial erythritol production were measured under various culture conditions to examine the production mechanism and the key-enzymes. As a result, the various enzyme activities of this organism are revealed in the pentose phosphate pathway, i.e., those of hexokinase, glucose-6-phosphate dehydrogenase, gluconate dehydrogenase, transketolase, transaldolase, and erythrose reductase. In the cultures in which erythritol was produced after completion of cell growth, the enzyme activities of the pentose phosphate pathway were higher than those of the TCA cycle. In particular, transketolase activity was correlated with erythritol productivity under various production cultures with different agitation speeds and thiamine concentrations. These results suggest that erythritol may be produced mainly through the pentose phosphate pathway. In addition, the high activity of transketolase is required to produce abundant intermediates, which results in high erythritol productivity. As such, transketolase appears to be a key-enzyme for erythritol production in the organism studied.

Cultivation of yeast and plant cells entrapped in the low-viscous liquid-core of an alginate membrane capsule prepared using polyethylene glycol.

A liquid-core alginate-membrane capsule was prepared by a novel method using polyethylene glycol as a thickener and the cells of Saccharomyces cerevisiae were encapsulated in its core and cultured. After 24 h of cultivation, the cell concentration in the capsule core-liquid reached 222 microg/mm3 on a dry weight basis, which was 1.4 times as large as that in the core of double-layered alginate beads, i.e., alginate-coated alginate-gel beads. The diameter increase of the capsule prepared by the proposed method using immobilized cell growth was suppressed compared to those using the double-layer method and simple alginate-gel bead entrapment, most likely because of the mobility of the entrapped cells in the capsule. We also confirmed that this encapsulation method is applicable for the cultivation of cultured cells of the plant Fragaria ananassa. Additionally, the time-course of the changes in thickener concentration in the liquid-core of the capsule was measured after encapsulation, and revealed the residual thickener, i.e., polyethylene glycol, was able to leak through the alginate shell membrane. This results in low-viscosity of the core liquid enabling good mass-transfer performance, whereas xanthan gum as a thickener could not leak through.