工藤 信樹, 木下 勇人

Nobuki KUDO, Yuto KINOSHITA

北海道大学大学院情報科学研究科

Graduate School of Information Science and Technology, Hokkaido University

キーワード : sonoporation, microbubbles, cell damage, scaffold stiffness, gel scaffold

目的：In vivoソノポレーションに向けた基礎研究として，硬さが異なる足場上に単層培養した細胞のソノポレーションにより生じる細胞膜損傷率を検討した．方法：コラーゲンゲル，10％，30％アクリルアミドゲル，およびカバーガラスを用いて作成した4種類の細胞培養足場を単層細胞の培養に用いた．原子間力顕微鏡を用いて測定したヤング率はゲル足場では0.09‐8.6 kPa，生細胞では4.5 kPaであった．波数3，100および10,000波でピーク正圧/負圧が8.0/-1.3 MPaのパルス超音波を微小気泡が付着した細胞に1回のみ照射した．結果：Propidium iodideを用いて細胞膜損傷を蛍光顕微鏡により可視化した．波数3波の超音波パルスでは有意な変化は見られなかったが，波数100および10,000波のパルスではヤング率の増加に伴い損傷が増加する明確な傾向が確認された．結論：実験結果より，接着細胞の下層にある足場層の硬さをソノポレーション条件の重要なパラメータとして考慮に入れる必要があること，またin vivoソノポレーションに対する最適照射条件は生体組織の物理的特性を考慮して決定すべきであることが示唆された．

Purpose: As basic studies to realize in vivo sonoporation, rates of cell membrane damage during sonoporation were evaluated using monolayer cells cultured on scaffolds withdifferent degrees of stiffness. Methods: Four types of scaffolds, constructed using collagen gel, 10 and 30% acrylamide gels, and a coverslip, were used for cultivation of monolayer cells. Young’s moduli measured using an atomic force microscope were in the range 0.09-8.6 kPa for the gel scaffolds, whereas Young’s modulus for living cells was 4.5 kPa. Cells with attached microbubbles were exposed to one-shot pulsed ultrasound of 8.0/-1.3 MPa in peak positive/negative pressures with durations of 3, 100, and 10,000 cycles. Results: Cell membrane damage was visualized by fluorescence microscopy using propidium iodide. The 3-cycle ultrasound pulse had no significant effect; however, the rates of damage caused by 100-cycle and 10,000-cycle pulses showed a strong tendency for higher rates of damage with a higher Young’s modulus. Conclusion: The experimental results indicate that the stiffness of the underlying layer of adherent cells should be considered as an essential parameter of the sonoporation condition and that the optimum exposure conditions for in vivo sonoporation should be determined with consideration of the physical properties of underlying tissues.