Browsing by Author "Matsubara, Mie"

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In situ laser micropatterning of proteins for dynamically arranging living cells
(2013)
Okano, Kazunori
;
Matsui, Ai
;
Maezawa, Yasuyo
;
Hee, Ping-Yu
;
Matsubara, Mie
This study shows the modification of the surface of polymer-layered glass substrates to form biofunctional micropatterns through femtosecond laser ablation in an aqueous solution. Domains of micrometer size on a substrate can be selectively converted from proteinphobic (resistant to protein adsorption) to proteinphilic, allowing patterning of protein features under physiological aqueous conditions. When femtosecond laser pulses (800 nm, 1 kHz, 200-500 nJ per pulse) were focused on and scanned on the substrate, which was glass covered with the proteinphobic polymer 2-methacryloyloxyethylphosphorylcholine (MPC), the surface became proteinphilic. Surface analysis by X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) reveals that the laser ablates the MPC polymer. Extracellular matrix (ECM) proteins were bound to the laser-ablated surface by physisorption. Since femtosecond laser ablation is induced under physiological aqueous conditions, this approach can form micropatterns of functional ECM proteins with minimal damage. This method was applied to pattern collagen, laminin, and gelatin on the substrate. Removal of an ECM protein from the substrate followed by replacement with another ECM protein was achieved on demand at a specific location and time by the same laser ablation method. Living cells adhered to the fabricated domains where ECM proteins were arranged. The modification of patterning during cell culture was used to control cell migration and form arrays of different cells.
In situ observation of cell-detachment process initiated by femtosecond laser-induced stress wave
(2010-10-01)
Maezawa, Yasuyo
;
Hosokawa, Yoichiroh
;
Okano, Kazunori
;
Matsubara, Mie
;
Masuhara, Hiroshi
When a stress wave generated by focusing a femtosecond laser is loaded on an animal cell adhered on a substrate, the cell is detached from the substrate. There are two possible mechanisms for the cell detachment: (a) The cell is detached from a scaffold coated on a glass plate, and (b) the cell is detached from the glass plate with the scaffold. In this work, we have studied the cell-detachment mechanism by visualizing the scaffold with a fluorescence probe of quantum dots. When the cell was detached from the substrate, fluorescence from the scaffold simultaneously disappeared from the glass plate, although the scaffold was not irradiated by the laser. This indicates that detachment due to the stress wave is attributed to mechanism (a). On the other hand, when the cell was detached from the substrate by a trypsin treatment, the fluorescence from the scaffold remained, suggesting mechanism (b). By comparing both results, it is considered that physiological damage of the cell membrane during the detachment process by femtosecond laser-induced stress wave is less than that due to the trypsin treatment.
In situ observation of cell-detachment process initiated by femtosecond laser-induced stress wave
(2010-10-01)
Maezawa, Yasuyo
;
Hosokawa, Yoichiroh
;
Okano, Kazunori
;
Matsubara, Mie
;
Masuhara, Hiroshi
When a stress wave generated by focusing a femtosecond laser is loaded on an animal cell adhered on a substrate, the cell is detached from the substrate. There are two possible mechanisms for the cell detachment: (a) The cell is detached from a scaffold coated on a glass plate, and (b) the cell is detached from the glass plate with the scaffold. In this work, we have studied the cell-detachment mechanism by visualizing the scaffold with a fluorescence probe of quantum dots. When the cell was detached from the substrate, fluorescence from the scaffold simultaneously disappeared from the glass plate, although the scaffold was not irradiated by the laser. This indicates that detachment due to the stress wave is attributed to mechanism (a). On the other hand, when the cell was detached from the substrate by a trypsin treatment, the fluorescence from the scaffold remained, suggesting mechanism (b). By comparing both results, it is considered that physiological damage of the cell membrane during the detachment process by femtosecond laser-induced stress wave is less than that due to the trypsin treatment.
Induction of Cell-Cell Connections by Using in situ Laser Lithography on a Perfluoroalkyl-Coated Cultivation Platform
(2011-03-21)
Okano, Kazunori
;
Yu, David
;
Matsui, Ai
;
Maezawa, Yasuyo
;
Hosokawa, Yoichiroh
This article describes a novel laser-directed microfabrication method carried out in aqueous solution for the organization of cell networks on a platform. A femtosecond (fs) laser was applied to a platform culturing PC12, HeLa, or normal human astrocyte (NHA) cells to manipulate them and to facilitate mutual connections. By applying an fs-laser-induced impulsive force, cells were detached from their original location on the plate, and translocated onto microfabricated cell-adhesive domains that were surrounded with a cell-repellent perfluoroalkyl (R(f)) polymer. Then the fs-laser pulse-train was applied to the Rf polymer surface to modify the cell-repellent surface, and to make cell-adhesive channels of several mm in width between each cell-adhesive domain. PC12 cells elongated along the channels and made contact with others cells. HeLa and NHA cells also migrated along the channels and connected to the other cells. Surface analysis by X-ray photoelectron spectroscopy (XPS) and atomic force microscopy (AFM) confirmed that the Rf polymer was partially decomposed. The method presented here could contribute not only to the study of developing networks of neuronal, glial, and capillary cells, but also to the quantitative analysis of nerve function.
Laser-Assisted Control of Protein Adsorption for Dynamically Arranging Viable Cells
(2013)
Okano, Kazunori
;
Matsui, Ai
;
Maezawa, Yasuyo
;
Matsubara, Mie
;
Hosokawa, Yoichiroh
Polymers and proteins covering glass surface were ablated by femtosecond laser pulses under physiological aqueous condition. The prepared micropatterns of proteins were confirmed by fluorescence imaging and their cell adhesion characteristics was demonstrated.
Morphological evaluation of cell differentiation after the isolation of single cells by a femtosecond laser-induced impulsive force
(2011-02-01)
Maezawa, Yasuyo
;
Okano, Kazunori
;
Matsubara, Mie
;
Masuhara, Hiroshi
;
Hosokawa, Yoichiroh
When nerve growth factor (NGF) is interacted with PC12 cells derived from rat pheochromocytoma, they are partially differentiated into neuron-like cells with neurites. In this work, PC12 cells differentiated by NGF were selectively isolated using a localized impulsive force in a mu m-scale area, which was generated by focusing an infrared femtosecond laser into a cell culture medium. In order to evaluate the ability of the isolation method, differentiated and undifferentiated cells were isolated and their morphological changes after the isolation were compared. In both cases, their neurites were once contracted and some of them gradually regenerated day by day. When differentiated cells were isolated, the percentage of differentiated cells with regenerated neurites, 6 h after the isolation, was about 3.3 times higher than that when undifferentiated ones were isolated. This result was compared with a control trypsin experiment. In the comparison, it was indicated that the same degree of cell function was maintained when the present isolation method was used.