本年度の実験では，ファイバ状hiPS由来心筋組織の収縮力計測法確立のために下記の2つの実験を実行した．
(1) 電気刺激を用いたファイバ状心筋組織の成熟化評価
本年度は，二重同軸マイクロ流体デバイスによって生成されたファイバ状のhiPS由来心筋組織に対し，電気刺激を定期的に与えることによって心筋組織の成熟化を達成した．1 Hzの双極パルス電気刺激を3-5日間印加することにより，ファイバ状心筋組織の拍動伝播速度が上昇した．また免疫蛍光染色によりαアクチニンを染色することで，筋組織の収縮発生を担うサルコメア構造の周期間隔が増大した．これらの結果より，電気刺激の印加によりファイバ状心筋組織の成熟化が促進され，次年度以降に実施予定である薬物試験への適用可能性が期待できる．

(2) 画像処理を用いた心筋組織の収縮力計算
本研究で形成した心筋組織はファイバ形状であり，心筋組織の拍動はファイバの屈曲という形で容易に光学観察が可能である．この屈曲量とハイドロゲルの機械特性から有限要素法ソフトウェアのCOMSOLにより，数値計算により心筋組織の収縮力を見積もる手法を確立した．具体的には，自律的な拍動をするファイバ形状心筋組織を動画で撮影し，画像の自動追尾解析により時系列的な拍動強度（屈曲角度）を算出した．得られた屈曲角度の差分から数値計算を用いて心筋組織の収縮力を得た．これにより，投薬の前後での相対的な心筋組織の収縮力の差分が，ファイバ形状の心筋組織を用いることで求められることを示した．
In this year, the following two experiments were performed to establish a method for measuring the contractility of microfiber-shaped hiPS-derived cardiac tissues.
(1) Maturation of microfiber-shaped cardiac tissue using electrical stimulation
We achieved maturation of cardiac tissue by applying electrical stimulation to microfiber-shaped hiPS-derived cardiac tissue produced by a double coaxial microfluidic device. One-hertz bipolar pulsed electrical stimulation for 3-5 days increased the beating velocity of the microfiber-shaped cardiac tissue. The rate of propagation of the microfiber-shaped cardiac tissue was also increased with the electrical stimulus. Immunofluorescent staining for α-actinin increased the periodic spacing of sarcomere structures that generate contraction in cardiac tissue. These results showed that the microfiber-shaped cardiac tissue was matured by the bipolar pulsed electrical stimulation. Our matured microfiber-shaped cardiac tissue could apply to drug testing, which will be conducted in the next year.

(2) Calculation of contractility of myocardial tissue using image processing
The cardiac tissue formed in this project is fiber-shaped, enabling that the beating of the cardiac tissue can be easily observed optically in the form of fiber bending. We established a method to estimate the contractile force of the cardiac tissue numerically using COMSOL, a finite element method software, based on the amount of bending and the mechanical properties of the hydrogel. Specifically, a video was taken of microfiber-shaped cardiac tissue beating autonomously, and the beating intensity (bending angle) was calculated by automatic tracking analysis of the video. The numerical calculation was used to obtain the contraction force of the myocardial tissue from the differences in the flexion angles obtained. The difference in the relative contractility of the cardiac tissue before and after drug administration could be obtained by using our microfiber-shaped microfiber-shaped.