In this thesis, cell micropatterning method in microchannel was created. Firstly, the strategy of surface modification method combining MPC polymer and PL was demonstrated. Secondly, cell micropatterning using photochemical reaction was developed. Thirdly, cell micropatterning was realized in microchannel.

In chapter 2, glass surface was synthesized by PL and MPC polymer. MPC polymer is known as resistant barrier for biological samples and PL is utilized in selective patterning of biological samples. By combining two compounds, chemicals with different cell adhesiveness surface was realized and estimated. Two different kinds of surface modification methods were performed and the property of MPC polymer-modified surface was confirmed. In 2.2, firstly, MPC polymer was synthesized with PL1 (5-Amino-4-Methyl-2-nitroactophenone) and grafted on activated APTS glass surface (APTS-DSC). The contact angles showed that the surface remained in hydrophobic (45°) even though hydrophilic photolabille MPC polymer (PL1-MPC) was introduced. This result showed that the photolabile MPC polymer was not introduced on the surface, since activity of the hydroxyl groups in photolabile MPC polymer was too low to react with APTS-DSC surface. In 2.3, I altered the surface modification to step-by-step synthesis method. After silanization the glass surface using APTS, photolabile linker and MPC polymer were modified in order. I chose the commercialized Fmoc protected PL (PL2) and coupled to APTS surface and then Fmoc was deprotected. To this surface (APTS-PL), MPC polymer was grafted and the result of contact angle indicated that hydrophilic surface was formed (12°). Furthermore, photocleavage of the MPC polymer was evaluated by UV (365 nm, 150 mW/cm2) illumination in order to make cell adhesive surface. The contact angle increased up to 3 min of UV exposure and no significance change was confirmed after 3 min. The optimum UV irradiation time was determined to 3 min and adapted to next experiments. Additional surface characterization tools such as XPS and AFM was also used to analyze the modification and elimination of the MPC polymer on the glass surface. The results were in agreement with my expectation. The developed surface modification methods and photocleavage conditions of MPC polymer were applied in micropatterning of biological molecules in next chapter.

In chapter 3, micropatterning of the biological samples using photochemical reaction was verified on glass surface. In 3.2, reduction of the non-specific adsorption on MPC polymer modified surface was demonstrated. The amount of adsorbed proteins (BSA and fibronectin) and cells (MC-3T3 El) were evaluated and compared to other modified surface. To the MPC polymer grafted surface, 0.22 μg/cm2 of BSA, 0.05 μg/cm2 of fibronectin was adhered. Compared to APTS and APTS-PL surface, the amounts of adsorbed proteins were reduced to 10-14 %. However, after UV illumination, adsorbed proteins (2.12 μg/cm2 of BSA, 0.43 μg/cm2 of fibronectin) were recovered to almost same amounts as those of APTS (2.28 μg/cm2 of BSA, 0.52 μg/cm2 of fibronectin), APTS-PL surface (2.28 μg/cm2 of BSA, 0.36 μg/cm2 of fibronectin). In the case of attachment rate of the cells, there was no cell had observed on MPC polymer modified surface. In contrast, on UV irradiated surface, 212 cells were attached in square millimeter, which showed that cell adhesiveness was recoved by photochemical reaction. In relation to UV exposed surface, cell attachment rate and cell proliferation rate were compared with ECM proteins such as fibronectin, poly (lysine)-matrigel coated surface. The results indicated that no significant changes were observed in 3 different surfaces.

In 3.3, firstly, micropatterning of proteins and cells were demonstrated by UV exposure through the photomask. After selective removal of MPC polymer, FITC-BSA was attached on UV irradiated area and fluorescence was observed (200 μm wide stripes). For MC-3T3 E 1 cell patterning, two different sizes of photomasks (200 μm, 100 μm wide stripes) were used and localized on UV irradiated region. In addition, photomask having 40 μm wide stripes was used and ECs were patterned on UV illuminated region as same size as photomask. To verity single cell level patterning, round shaped photomask with 30 mm spots were used, which resulted in single cell was attached on one spot of pattern. Secondly, isotropic tendency of the localized MC-3T3 El cells on pattern size was investigated. Three different conditions were used, which were without a photomask, 200 μm and 70 μm wide stripes of photomask. The cells were placed by each different conditions and a part of adhered cells (140 μm × 100 μm) were observed. No isotropic direction was seen in the case of without a photomask and 200 μm wide stripes, except in 70 μm wide stripes.

Finally, two different types of cells were patterned on the same glass surface. After placing 1(st) cell type (MC-3T3 El) cells, UV was illuminated to 900 μm distance from 1(st) patterned cells, then 2(nd) type cells (ECs) were seeded and ECs were introduced on 2(nd) UV exposed area. I succeeded in positioning two different types of cells on the same glass surface by photochemical reaction.

In 3.4, the stability of micro-patterned cells was demonstrated by culturing of MC-3T3 El cell patterns (width 100 μm stripe) for 5 weeks and ECs cell patterns (width 40 μm stripe) for 3 weeks on each glass surface. They maintained the micropatterns in safe for long-term culture. These results indicated that MPC was chemically bonded in safe to glass surface and not affected by synthesized and secreted proteins from the patterned cells during the cultures.

In chapter 4, cell micropatterning using photochemical reaction was adopted in microchannel. In 4.2, cell micropatterning conditions were set up and realized on the glass surface in chapter 2, 3. These conditions should be re-tested whether it can be applied in microchannel directly or not. Firstly, I evaluated the UV transparent on microchip which was made by quartz plate. The model system was demonstrated by inserting quartz plate with the same thickness as microchip between MPC polymer modified glass surface and photomask. In results, cell micropatterning was successful and no significance difference was found on micro-patterned cells. This result was in agreement with former results in chapter 3.3. Secondly, the rate of cell adherence on UV illumination time was confirmed. UV irradiation time was varied to 1, 3, and 5 min, and the cell suspension was introduced into microchannel. After 2 h of incubation, 35 cells, 266 cells, and 246 cells were immobilized on each UV exposed surface. After 3 min, the cell attachment rate reached a plateau; no significant difference was occurred. These are in good accord with the results in chapter 2.3.2. Thirdly, the concentration of MC-3T3 El cell suspension to introduce in microchannel was optimized. The concentration of the suspension was varied to and cell culturing in microchannel was observed. When the concentration of cell suspension was higher than 1 × 107 cells/mL, cells could not spread out onto the micochannel surface, because of the aggregation of cells. In the flow condition of medium, most of the cells were flusheded out. It was clearly proved that optimum concentration of cell suspension was 5~8 × 106 cells/mL.

In 4.3, based on re-estimated experimental conditions in chapter 4.2, cell micropatterning was performed in microchannel. The MC-3T3 El cells were adhered to photochemically micro-patterned region, after 2 hr of incubation, the micro-patterned cells were found to be flowed out under flow condition. The cell aggregation is supposed to make cells out of the microchnnel. To reduce the risk, cell attachment rate should be also confirmed before introducing the cell suspension. Cells are likely to make aggregation when they are seeded on non-biofouling surface. When cell densitiy was reduced to 4 × 106 cells/mL according to cell adhesive area, cells were patterned in safe on UV exposed area after 24 h of incubation. The cell adhesive area is also important point to be considered for cell micropatterning in microchannel. Finally, ECs and MC-3T3 El cells were placed in same microchannel using photochemical reaction. The Distance of cells was regulated by 200 μm.

In 4.4, the stability of micro-patterned ECs (width 200 μm stripes) was observed in microchannel. In the flow system, patterned cells were maintained the patterns in safe for 14 days of culture. MPC polymer was stable enought to inhibit the cell migration out of the patterns.

Finally, in chapter 5, conclusions and future perspectives are described.

本論文は「Cell patterning in microchannel using photochemical reaction(光化学反応を利用した微小空間における細胞パターニングの研究)」と題し、高機能マイクロ細胞実験システムのための細胞マイクロパターニング法の開発に関する研究結果をまとめたものである。

第1章では、近年のμ-TASやLab-on-a-chipといわれる類似的研究の歴史的背景とその意義をまとめ、マイクロ化学システムの有用性を示した。また微小空間で細胞を操作する有用性や開発されている細胞操作デバイスについてまとめた。しかし、細胞生物学研究の最前線における現状と高度な要求(単一細胞分析、擬似組織空間、細胞と細胞の相互作用分析)に答えるためにはマイクロメートルサイズの細胞接着領域の制御が必要である。従来のマイクロ化学チップ技術では細胞の数と配置の制御が不可能だったので、達成されていない。この高度な要求に対し、マイクロチップ内の細胞マイクロパターン培養技術を着想した。マイクロ化学チップは狭い閉じた空間であるため、光を使って、細胞接着領域、非接着領域をコントロールすることを考案した。そして、微小空間における細胞マイクロパターニング法の開発を本研究の目的にした。

第2章では、細胞非接着性である2-メタクリロイルオキシエチルホスホリルコリン(MPC)ポリマーと光反応を利用した表面修飾法を開発した。マイクロチップ内の狭い閉じた空間へ、細胞接着領域を構築するためには、光分解で選択的に細胞接着領域と非接着領域を構築することが有効である。しかし、光分解リンカーを持つ細胞非接着性化合物はないため、新たな化合物が必要である。そこで、細胞非接着性化合物MPCポリマーに光分解リンカーを組み込むことを着想した。ガラス表面にシラン化剤、光分解リンカー、MPC ポリマーを順に修飾することに成功した。接触角、XPS(X-ray photoelectron spectroscopy)、AFM(atomic force microscopy)などの表面分析ツールを利用してMPC ポリマーのガラス表面への修飾と光分解反応によるMPCポリマーの剥離を確認し、光パターニングの基礎を実現した。

第3章では、2章で創製した修飾法を利用して、ガラス基板上への細胞パターニングを実証した。まず、MPCポリマー表面への細胞接着タンパク質と細胞の非特異吸着量を評価した。MPCポリマー導入表面への細胞接着タンパク質の吸着量は、シラン化剤表面、光分解リンカー表面の吸着量と比べて10分の1まで減少した。一方、UV360 nm照射表面の場合は、光分解リンカー表面と同等の吸着量を示した。同様に、細胞接着率もMPCポリマー表面には細胞の接着が抑制されるのに対し、UV照射表面には細胞の接着率が光分解リンカー表面と同等の細胞接着率が確認された。このような表面とフォトマスクを利用してタンパク質と細胞のマイクロパターニングを実現した。直径30マイクロメートルサイズの丸型フォトマスクを利用して、単一細胞のパターニングにも成功した。また、同一ガラス表面への2週間の細胞パターニングを実現した。単一細胞レベルでの異種細胞の空間配置できる初めての技術である。最後に、細胞パターンは1日目のパターンを35日に渡って維持しながら生存していることが検証された。

第4章では、マイクロチャネル内の細胞マイクロパターニング法を実現した。まず、基礎実験としてマイクロチップ基板材質のUV透過性を検証した。次にマイクロチャネル内導入する細胞懸濁液濃度を細胞接着表面に関して最適化した。次に細胞マイクロパターニングをマイクロチャネル内で実現し、細胞パターンの2週間の安定性を確認した。また、マイクロチャネル内への2種類の細胞マイクロパターニングを世界ではじめて実証した。本研究で開発した技術は、細胞間の情報伝達など細胞生化学の新しい研究ツールとして期待出来る。

第5章では、第2章から第4章までに開発した光化学反応を利用した微小空間における細胞パターニング法の意義についてまとめ、展望を示した。

以上のように本論文では、表面修飾の分子の設計からマイクロデバイスへの展開までに成功した。これらの研究成果は、マイクロ化学チップ研究の発展のみならず細胞生物学研究に貢献できる重要な知見である。

よって本論文は博士(工学)の学位請求論文として合格と認められる。