细胞力学仿体制备及其弹性特性定量表征

细胞力学是生物力学的前沿领域，也是组织工程学的重要组成部分， 它可以了解细胞与外界环境信号的传递机制，包括细胞膜信号转导、细胞 内信号通路等，这对于组织工程学中细胞生长、分化、再生等具有重要意 义。由于细胞不同阶段、不同状态其力学特性在不断变化。为了校验仪器 在测量细胞状态时的准确性，亟需制备细胞仿体来标定细胞力学测量仪器。 近年来，基于甲基丙烯酰基明胶（Methacrylate Gelatin，GelMA）微球 作为细胞仿体的研究在生物医学工程上引起了广泛的关注，它可以用于递 送药物、打印生物支架、培养干细胞等等。研究指出它不仅具有良好的生 物相容性，且可以模拟细胞的力学特性。 本文制备可用于模拟细胞的 GelMA 微球和可诱导细胞仿体发生形变的微流控芯片，并对其力学特性进 行表征。通过搅拌乳化的方法制备得到 GelMA 微球，该微球与细胞粒径大 小和力学性能相似。通过光学显微镜、扫描电子显微镜（ Scanning Electronic Microscopy，SEM）等方法表征微球的形貌。用粒径测量仪测量 微球的粒径大小，测得 GelMA 微球的粒径大小区间为 0.8~100 μm。然后对 制得的 GelMA 水凝胶进行力学性能表征，通过动态机械分析仪测试得到的 冻融循环后的 GelMA 水凝胶弹性模量最低可达 12 kPa。满足模拟细胞的粒 径大小和弹性模量的要求。在制得细胞仿体后，制备声表面波芯片诱导细 胞仿体发生形变。通过分析 3D 打印和微纳加工的优缺点，选用微纳加工的 方法对声表面波芯片进行制备。以声表面波为激励源，超声频率为 39 MHz， 700 mv。在输入功率为 694.35 mW 的激励下，细胞仿体可快速排成一列， 从而实现单粒流过，为超声挤压细胞奠定基础。挤压实验将参数调制到 76.1 MHz，200 mv，同时循环数调至 76100。在输入功率为 1.54 W，持续 时间为 0.01 s 的激励下，细胞在杜氏磷酸盐缓冲溶液中被挤压发生形变，18 μm的微球在横向产生 3 μm的挤压变形。 通过对 GelMA 微球的粒径和力学性能测试，验证 GelMA 微球模拟细 胞仿体的可行性。并通过制备不同类型的声表面波芯片，对细胞仿体进行 操控和测量，该微球的制备有望用于校准细胞力学测量仪器，为细胞力学 研究奠定坚实的基础。

[1] 张毅奕,高宇欣,陶祖莱.细胞粘附强度与细胞活力的关系[J].医用生物力学,2000.
[2] SATCHER R. L, DEWEY C. F. Theoretical estimates of mechanical properties of theendothelial cell cytoskeleton. Biophysical Journal[J], 1996, 71(1): 109–118.
[3] WANG N, NARUSE K, FREDBERG JJ, et al. Mechanical behavior in living cellsconsistent with the tensegrity model[J]. Proceedings of the National Academy ofSciences 2001, 98: 7765– 7770.
[4] HAGA H, SASAKI S, KAWABATA K, et al. Elasticity mapping of living fibroblastsby AFM and immunofluorescence observation of the cytoskeleton[J]. Ultramicroscopy2000, 82: 253–258.
[5] ANANTHAKRISHNAN R, EHRLICHER A. The forces behind cell movement[J].International Journal of Biological Sciences, 2007, 3(5): 303-317
[6] ZHAO J, ZHOU H, HUANG W, et al. Cell morphological analysis of SARS-CoV-2infection by transmission electron microscopy[J]. Journal of Thoracic Disease, 2020,12(8): 4368.
[7] MOFRAD M R K, KARCHER H, KAMM R D. Continuum elastic or viscoelasticmodels for the cell[J]. Cytoskeletal Mechanics, 2009: 71–83.
[8] GENNISSON J L, GRENIER N, COMBE C, et al. Supersonic shear wave elastographyof in vivo pig kidney: influence of blood pressure, urinary pressure and tissueanisotropy[J]. Ultrasound in Medicine and Biology, 2012, 38(9): 1559-1567.
[9] W.L. NYBORG, Acoustic streaming, in: W.P. Mason [Ed]. Physical Acoustics,Academic Press, 1965, 265–383.
[10] W.L. NYBORG，Mechanisms for nonthermal effects of sound[J]. The Journal of theAcoustical Society of America, 1968, 44(5): 1302–1309.
[11] HILL C R. Calibration of ultrasonic beams for bio-medical applications[J]. Physics inMedicine and Biology, 1970, 15(2): 241.
[12] NIGHTINGALE KATHRYN, SOO MARYSCOTT, NIGHTINGALE ROGER, et al.Acoustic radiation force impulse imaging: in vivo demonstration of clinicalfeasibility[J]. Ultrasound in Medicine and Biology,2002,28(2):227-235.
[13] A.P. SARVAZYAN, O.V. RUDENKO, S.D. SWANSON, et al. Shear wave elasticityimaging: a new ultrasonic technology of medical diagnostics[J]. Ultrasound inMedicine. Biology, 1998, 24(9): 1419–1435.
[14] J. BERCOFF, M. TANTER, M. FINK, Supersonic shear imaging: a new technique forsoft tissue elasticity mapping[J]. IEEE Transactions on Ultrasonics, Ferroelectrics andFrequency Control, 2004, 51(4): 396–409.
[15] J. BERCOFF, M. PERNOT, M. TANTER, et al. Monitoring thermally-induced lesionswith supersonic shear imaging[J]. Ultrasonic Imaging, 2004, 26(2): 71–84.
[16] Maleke C, Konofagou E E., Harmonic motion imaging for focused ultrasound(HMIFU): a fully integrated technique for sonication and monitoring of thermalablation in tissues[J]. Physics in Medicine and Biology, 2008, 53(6): 1773-1793
[17] DAYTON P, KLIBANOV A, BRANDENBURGER G, et al. Acoustic radiation forcein vivo: a mechanism to assist targeting of microbubbles.[J]. Ultrasound in Medicineand Biology, 1999, 25(8): 1195-1201.
[18] J. HU, A.K. SANTOSO, A pi-shaped ultrasonic tweezers concept for manipulation ofsmall particles, IEEE Trans. IEEE Transactions on Ultrasonics, Ferroelectrics andFrequency Control, 2004, 51 (11), pp. 499-507.
[19] LEE J, HA K, SHUNG KK. A theoretical study of the feasibility of acoustical tweez ers:Ray acoustics approach[J]. The Journal of the Acoustical Society of America, 2005,117(5): 3273-3280.
[20] ZHAO, ZHENYU, LI, GEN, RUAN, HUITONG, et al. Capturing Magnesium Ions viaMicrofluidic Hydrogel Microspheres for Promoting Cancellous Bone Regeneration[J].ACS Nano, 2021, 15(8): 13041-13054.
[21] YIN, JUN, YAN, MENGLING, WANG, YANCHENG, et al. 3D Bioprinting of LowConcentration Cell-Laden Gelatin Methacrylate (GelMA) Bioinks with a Two-StepCross-linking Strategy[J]. Applied Materials and Interfaces, 2018, 10(8): 6849-6857.
[22] 孙伟皓.基于压痕实验的细胞弹性模量拟合公式修正及应用[D]. 辽宁:大连理工大学,2021.
[23] GUO Y, BAE J, FANG Z, et al. Hydrogels and hydrogel-derived materials for energyand water sustainability[J]. Chemical Reviews, 2020, 120(15): 7642-7707.
[24] 李一凡,刘捷,李政雄. PVA 水凝胶制备,改性及在生物医学工程中的研究进展[J].硅谷,2012.
[25] OLIVERA S, MURALIDHARA H B, VENKATESH K, et al. Potential applications ofcellulose and chitosan nanoparticles/composites in wastewater treatment: A review[J].Carbohydrate Polymers, 2016, 153: 600-618.
[26] DONG, SHUOXUN, WANG, YILI. Characterization and adsorption properties of alanthanum-loaded magnetic cationic hydrogel composite for fluoride removal[J].Water Research, 2016, 88: 852-860.
[27] 赵亚楠. 羟丙基壳聚糖/大豆蛋白质复合支架的构建及其组织工程应用研究[D].湖北:武汉大学,2019.
[28] J. BERCOFF, M. PERNOT, M. TANTER, et al. Monitoring thermally-induced lesionswith supersonic shear imaging, Ultrasonic Imaging[J]. 2004, 26(2): 71-84
[29] C. MALEKE, E.E. KONOFAGOU, Harmonic motion imaging for focused ultrasound(HMIFU): a fully integrated technique for sonication and monitoring of thermalablation in tissues, Physics Medicine Biology[J]. 2008, 53 (6): 1773-1793
[30] BIAN, JIANG, CAI, FENG, CHEN, HAO, et al. Modulation of Local OveractiveInflammation via Injectable Hydrogel Microspheres[J]. Nano Letters, 2021, 21(6):2690-2698.
[31] W. T. COAKLEY, J. J. HAWKES, M. A. SOBANSKI, et al. Analytical scale ultrasonicstanding wave manipulation of cells and microparticles[J]. Ultrasonics, 2000, 38(1/8):638-641.
[32] JEREMY J.HAWKES, ROBERT W.BARBER, DAVID R.EMERSON, et al.Continuous cell washing and mixing driven by an ultrasound standing wave within amicrofluidic channel[J]. Lab on a chip, 2004, 4(5): 446-452.
[33] 肖新才,褚良银,陈文梅,等. 聚(N-异丙基丙烯酰胺)温敏微球的粒径及单分散性[J].化工学报,2004..
[34] SAMANDARI M, ALIPANAH F, JAVANMARD S H, et al. One-step wettabilitypatterning of PDMS microchannels for generation of monodisperse alginatemicrobeads by in Situ external gelation in double emulsion microdroplets[J]. Sensorsand Actuators B: Chemical, 2019, 291: 418-425.
[35] DESAI, K.G.H. and PARK, H.J, Preparation and characterization of drug-loadedchitosan–tripolyphosphate microspheres by spray drying. Drug DevelopmentResearch., 2005, 64(2): 114-128.
[36] WALLS S C, BARICHIVICH W J, BROWN M E. Drought, deluge and declines: theimpact of precipitation extremes on amphibians in a changing climate[J/OL]. Biology,2013, 2(1): 399-418.
[37] 陈登飞,李娜,杨建. 利用 SPG 膜制备 O/W 型乳液实验研究[J]. 膜科学与技术,2007.
[38] DANOV K D, DANOVA D K, KRALCHEVSKY P A. Hydrodynamic forces acting ona microscopic emulsion drop growing at a capillary tip in relation to the process ofmembrane emulsification[J]. Journal of Colloid and Interface Science, 2007, 316(2):844-857.
[39] KATO R, TACHIBE M, SUGANO S, et al. High‐Hydroxypropylated Tapioca StarchImproves Insulin Resistance in Genetically Diabetic KKAy Mice[J]. Journal of FoodScience, 2009, 74(3): H89-H96.
[40] ESSIAMBRE R J. Arthur Ashkin: Father of the optical tweezers[J]. Proceedings ofthe National Academy of Sciences, 2021, 118(7): e2026827118.
[41] RAYLEIGH L. The Polishing of Glass Surfaces[J]. Journal of the Royal MicroscopicalSociety, 1905, 25(5): 567-568.
[42] WU J. Acoustical tweezers[J]. The Journal of the Acoustical Society of America, 1991,89(5): 2140-2143.
[43] CHAE S K, LEE C H, LEE S H, et al. Oil droplet generation in PDMS microchannelusing an amphiphilic continuous phase[J]. Lab on a Chip, 2009, 9(13): 1957-1961.
[44] QIAO Y, GONG M, WANG H, et al. Acoustic radiation force on a free elastic spherein a viscous fluid: Theory and experiments[J]. Physics of Fluids, 2021, 33(4): 047107.
[45] . LI M, LIU L, XI N, et al. Nanoscale imaging and mechanical analysis of Fc receptormediated macrophage phagocytosis against cancer cells[J]. Langmuir, 2014, 30(6):1609-1621.
[46] CHAFFER C L, WEINBERG R A. A perspective on cancer cell metastasis[J]. Science,2011, 331(6024): 1559-1564.