Biopolym. Cell. 2020; 36(3):182-196.
http://dx.doi.org/10.7124/bc.000A29
Reviews
Three-dimensional cell cultivation systems
1, 2Sukach O. M., 1Shevchenko M. V.
  1. Institute for Problems of Cryobiology and Cryomedicine, NAS of Ukraine
    23, Pereyaslavskaya Str., Kharkiv, Ukraine, 61015
  2. G. S. Skovoroda Kharkiv National Pedagogical University
    29, Alchevskyh Str., Kharkiv, Ukraine, 61002

Abstract

This review discusses the characteristics of three-dimensional cell culture systems on and without carriers (scaffolds). Scaffolds are used to simulate the extracellular matrix, as well as to reproduce the natural physical and structural microenvironment of cells, similar to living tissue. The review examines the types of scaffolds (hard and gel-like, natural and artificial, degradable and non-degradable), their characteristics, advantages and disadvantages, features of cell distribution in them. The use of decellularized and devitalized organs and tissues as scaffolds is discussed. The review also considers matrix-free cultivation of cells in the composition of three-dimensional multicellular structures – spheroids. The structure and biology of spheroids is discussed. The features of spheroid formation under static (self-assembly) and dynamic (under the influence of external forces) cultivation conditions are considered. The role of spheroid size for cell survival is discussed.
Keywords: cells, 3D cell culture, scaffolds, spheroids
Full text: (PDF, in English)

References

[1] Mahmud G, Campbell C J, Bishop K J M, Komarova Y A, Chaga O, Soh S, Huda S, Kandere-Grzybowska K, Grzybowski B A. Directing cell motions on micropatterned ratchets. Nat Phys. 2009; 5:606-12.
[2] Kilian K, Bugarija B, Lahn BT, Mrksich M. Geometric cues for directing the differentiation of mesenchymal stem cells. Proc Natl Acad Sci USA. 2010; 107(11):4872-7.
[3] Mseka T, Bamburg JR, Cramer LP. ADF/cofilin family proteins control formation of oriented actin-filament bundles in the cell body to trigger fibroblast polarization. J Cell Sci. 2007; 120(Pt 24):4332-44.
[4] Lane SW, Williams DA, Watt FM. Modulating the stem cell niche for tissue regeneration. Nat Biotechnol. 2014; 32(8):795-803.
[5] Donnelly H, Salmeron-Sanchez M, Dalby MJ. Designing stem cell niches for differentiation and self-renewal. J R Soc Interface. 2018; 15(145):20180388.
[6] Kleinman HK, Philp D, Hoffman MP. Role of the extracellular matrix in morphogenesis. Cur Opin Biotechnol. 2003; 14(5):526-32.
[7] Hynes RO. The extracellular matrix: not just pretty fibrils. Science. 2009; 326(5957):1216-19.
[8] Daley WP, Peters SB, Larsen M. Extracellular matrix dynamics in development and regenerative medicine. Journal of cell science. 2008; 121(Pt 3):255-64.
[9] Edelman G. Cell adhesion molecules. Science. 1983; 219(4584):450-57.
[10] Homrich M, Gotthard I, Wobst H, Diestel S. Cell adhesion molecules and ubiquitination functions and signific-ance. Biology (Basel). 2016; 5(1):1.
[11] Hynes RO. Cell adhesion: old and new questions. Trends Cell Biol. 1999; 9(12): M33-M37.
[12] Halbleib JM, Nelson WJ. Cadherins in development: cell adhesion, sorting, and tissue morphogenesis. Genes Dev. 2006; 20(23):3199-214.
[13] Nelson CM, Bissell MJ. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates develop-ment, homeostasis, and cancer. Annu Rev Cell Dev Biol. 2006; 22:287-309.
[14] Meyers J, Craig J, Odde DJ. Potential for control of signaling pathways via cell size and shape. Curr Biol. 2003; 16(17):1685-93.
[15] Birgersdotter A, Sandberg R, Ernberg I. Gene expression perturbation in vitro - a growing case for three-dimensional (3D) culture systems. Semin Cancer Biol. 2005; 15(5): 405-12.
[16] Gomez-Lechon M, Jover R, Donato T, Ponsoda X, Rodriquez C, Stenzel KG, Klocke R, Paul D, Guillen L, Bort R, Castell JV. Long-term expression of differentiated functions in hepatocytes cultured in three-dimensional colla-gen matrix. J Cell Physiol. 1998; 177(4):553-62.
[17] von der Mark K, Gauss V, von der Mark H, Muller P. Relationship between cell shape and type of collagen syn-thesized as chondrocytes lose their cartilage phenotype in culture. Nature. 1977; 267:531-2.
[18] Petersen OW, Ronnov-Jessen L, Howlett AR, Bissel MJ. Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci USA. 1992; 89(19):9064-8.
[19] Tan W, Krishnaraj R, Desai TA. Evaluation of nanostructured composite collagen-chitosan matrices for tissue engineering. Tissue Eng. 2001; 7(2):203-10.
[20] Shruti S, Salinas AJ, Lusvardi G, Malavasi G, Menabue L, Vallet-Regi M. Mesoporous bioactive scaffolds pre-pared with cerium-, gallium- and zinc-containing glasses. Acta Biomater. 2013; 9(1):4836-44.
[21] Wei G, Ma PX. Structure and properties of nano-hydroxyapatite/polymer composite scaffolds for bone tissue engineering. Biomaterials. 2004; 25(19):4749-57.
[22] Boyan BD, Hummert TW, Dean DD, Schwartz Z. Role of material surfaces in regulating bone and cartilage cell response. Biomaterials. 1996; 17(2):137-46.
[23] Lee M, Wu BM, Dunn J.C. Effect of scaffold architecture and pore size on smooth muscle cell growth. J Biomed Mater Res A. 2008; 87(4):1010-16.
[24] Marrella A, Lee TY, Lee DH, Karuthedom S, Syla D, Chawla A, Khademhosseini A, Jang HL. Engineering vascu-larized and innervated bone biomaterials for improved skeletal tissue regeneration. Mater. Today. 2018; 21(4):362-76.
[25] Dhandayuthapani B, Yoshida Y, Maekawa T, Kumar DS. Polymeric scaffolds in tissue engineering application: A review. Int. J. Polym. Sci. 2011; 2011:290602.
[26] Lee P, Tran K, Chang W, Fang Y-L, Zhou G., Junka R, Shelke NB, Yu X, Kumbar SG. Bioactive polymeric scaf-folds for osteochondral tissue engineering: in vitro evaluation of the effect of culture media on bone marrow stromal cells. Polym. Adv. Technol. 2015; 26(12):1476-85.
[27] Lee P, Tran K, Zhou G, Bedi A, Shelke NB, Yu X, Kumbar SG. Guided differentiation of bone marrow stromal cells on co-cultured cartilage and bone scaffolds. Soft Matter. 2015; 11:7648-55.
[28] Caicedo-Carvajal CE, Liu Q, Remache Y, Goy A, Suh KS. Cancer tissue engineering: A novel 3D polystyrene scaffold for in vitro isolation and amplification of lymphoma cancer cells from heterogeneous cell mixtures. J Tissue Eng. 2011; 2011:362326.
[29] Surmeneva MA, Surmenev RA,. Chudinova EA, Koptioug A, Tkachev MS, Gorodzha SN, Rännar L-E. Fabrication of multiple-layered gradient cellular metal scaffold via electron beam melting for segmental bone reconstruction. Mater Des. 2017; 133:195-204.
[30] Hench LL. Bioceramics: from concept to clinic. J Am Ceram Soc. 1991; 74(7):1487-510.
[31] Fielding GA, Bandyopadhyay A, Bose S. Effects of silica and zinc oxide doping on mechanical and biological properties of 3D printed tricalcium phosphate tissue engineering scaffolds. Dent Mater. 2012; 28(2):113-22.
[32] Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: state of the art. Tissue engineering. Part B, Reviews. 2008; 14(1):61-86.
[33] Sin DC, Miao X, Liu G, Wei F, Chadwick G, Yan C, Friis T. Polyurethane (PU) scaffolds prepared by solvent casting/particulate leaching (SCPL) combined with centrifugation . Mat Sci Eng C. 2010; 30(1):78-85.
[34] Chen J, Ye J, Liao X, Li S, Xiao W, Yang Q, Li G. Organic solvent free preparation of porous scaffolds based on the phase morphology control using supercritical CO2 . J Supercrit Fluid. 2019; 149:88-96.
[35] Um C, Fang D, Hsiao B. S, Okamoto A, Chu B. Electro-spinning and electro-blowing of hyaluronic acid. Bioma-cromolecules 2004; 5(4):1428-36.
[36] Ge Z, Tian X, Heng BC, Fan V, Yeo JF, Cao T. Histological evaluation of osteogenesis of 3D-printed polylac-tic-co-glycolic acid (PLGA) scaffolds in a rabbit model. Biomed Mater. 2009; 4(2):021001.
[37] Zagris N. Extracellular matrix in development of the early embryo. Micron. 2001; 32(4):427-38.
[38] Aumailley M, Gayraud B. Structure and biological activity of the extracellular matrix. J Mol Med.1998; 76(3-4):253-65.
[39] Stevens MM, George JH. Exploring and engineering the cell surface interface. Science.2005; 310(5751):1135-8.
[40] Curtis A, Wilkinson C. New depths in cell behaviour: reactions of cells to nanotopography. Biochem Soc Symp. 1999; 65:15-26.
[41] Haycock JW. 3D cell culture: a review of current approaches and techniques. Methods Mol Biol. 2011; 695:1-15.
[42] Lan PX, Lee JW, Seol Y-J, Cho D-W. Development of 3D PPF/DEF scaffolds using micro-stereolithography and surface modification. J Mater Sci Mater Med. 2009; 20(1):271-9.
[43] Liu X, Won Y, Ma PX. Porogen-induced surface modification of nano-fibrous poly(L-lactic acid) scaffolds for tissue engineering. Biomaterials. 2006; 27(21):3980-7.
[44] Siow K, Britcher L, Kumar S, Griesser H. Plasma methods for the generation of chemically reactive surfaces for biomolecule immobilisation and cell colonisation - a review. Plasma Process Polym. 2006; 3(6-7):392-418.
[45] Zhao F, Ma T. Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic seed-ing and construct development. Biotechnol Bioeng. 2005; 91 (4):482-93.
[46] van den Dolder J, Spauwen PH, Jansen JA. Evaluation of various seeding techniques for culturing osteogenic cells on titanium fiber mesh. Tissue Eng. 2003; 9(2):315-25.
[47] Choong CS, Hutmacher DW, Triffitt JT. Co-culture of bone marrow fibroblasts and endothelial cells on modified polycaprolactone substrates for enhanced potentials in bone tissue engineering. Tissue Eng. 2006; 12(9):2521-31.
[48] Wan Y, Wang Y, Liu Z, Qu X, Han B, Bei J, Wang S. Adhesion and proliferation of OCT-1 osteoblast-like cells on micro- and nano-scale topography structure poly(L-lactide). Biomaterials. 2005; 26(21):4453-9.
[49] Honda MJ, Yada T, Ueda M, Kimata K. Cartilage formation by serial passaged cultured chondrocytes in a new scaffold: hybrid 75:25 poly(L-lactide-epsilon-caprolactone) sponge. J Oral Maxillofac Surg. 2004; 62(12):1510-6.
[50] Hofmann A, Konrad L, Gotzen L, Printz H, Ramaswamy A, Hofmann C. Bioengineering human bone tissue using autogenous osteoblasts cultured on different biomatrices. J Biomed Mater Res A. 2003; 67(1):191-9.
[51] Shen H, Hu X, Yang F, Bei J, Wang S. Combining oxygen plasma treatment with anchorage of cationized gelatin for enhancing cell affinity of poly(lactide-co-glycolide). Biomaterials. 2007; 28(29):4219-30.
[52] Spalazzi JP, Doty SB, Moffat KL, Levine WN, Lu HH. Development of controlled matrix heterogeneity on a tri-phasic scaffold for orthopedic interface tissue engineering. Tissue Eng. 2006; 12(12):3497-508.
[53] Zhu J, Marchant RE. Design properties of hydrogel tissue-engineering scaffolds. Expert Rev Med Devices. 2011; 8(5):607-26.
[54] Slaughter BV, Khurshid SS, Fisher OZ, Khademhosseini A, Peppas NA. Hydrogels in regenerative medicine. Adv Mater. 2009; 21(32-33):3307-29.
[55] Cushing MC, Anseth KS. Hydrogel cell culture. Science. 2007; 316(5828):1133-4.
[56] Nuttelman CR, Rice MA, Rydholm AE, Salinas CN, Shah DN, Anseth KS. Macromolecular monomers for the synthesis of hydrogel niches and their application in cell encapsulation and tissue engineering. Prog Polym Sci. 2008; 33(2):167-70.
[57] Hoffman AS. Hydrogels for biomedical applications. Adv Drug Deliver Rev. 2002; 43(1):3-12.
[58] Liu SQ, Tay R, Khan M, Ee PLR, Hedrick JL, Yang YY. Synthetic hydrogels for controlled stem cell differentiation. Soft Matter. 2010; 6(1):67-81.
[59] Nguyen TK, West JL. Photopolymerizable hydrogels for tissue engineering applications. Biomaterials. 2002; 23(22):4307-14.
[60] Dawson E, Mapili G, Erickson K, Taqvi S, Roy K. Biomaterials for stem cell differentiation. Adv Drug Deliv Rev. 2008; 60(2):215-28.
[61] Tibbitt MW, Anseth KS. Hydrogels as extracellular matrix mimics for 3D cell culture. Biotechnol Bioeng. 2009; 103(4):655-63.
[62] Sengupta D, Heilshorn S. Protein-engineered biomaterials: Highly tunable tissue engineering scaffolds. Tissue Eng Part B Rev. 2010; 16(3):285-93.
[63] MacEwan SR, Chilkoti A. Elastin-like polypeptides: biomedical applications of tunable biopolymers. Pept Sci. 2010; 94(1):60-77.
[64] Romano NH, Sengupta D, Chung C, Heilshorn SC. Protein-engineering biomaterials: nanoscale mimcs of the extracellular matrix. Bioichim Biophy Acta. 2011; 1810(3):339-49.
[65] Greish K, Araki K, Li D, O'Malley Jr. BW., R. Dandu, J. Frandsen, J. Cappello, Ghandeharia H.Silk-elastinlike protein polymer hydrogels for localized adenoviral gene therapy of head and neck tumors. Biomacromolecules. 2009; 10(8):2183.
[66] Banta S, Wheeldon IR, Blenner M. Protein engineering in the development of functional hydrogels. Annu Rev Biomed Eng. 2010; 12:167-186.
[67] Kaufmann D, Fiedler A, Junger A, Auernheimer J, Kessler H, Weberskirch R. Chemical conjugation of linear and cyclic RGD moieties to a recombinant elastin-mimetic polypeptide - a versatile approach towards bioactive pro-tein hydrogels. Macromol Biosci. 2008; 8(6):577-88.
[68] Leach JB, Bivens KA, Patrick CW, Schmidt CE. Photocrosslinked hyaluronic acid hydrogels: Natural, biodegrad-able tissue engineering scaffolds. Biotechnol Bioeng. 2003; 82(5):578-89.
[69] Ramamurthi A, Vesely I. Ultraviolet light-induced modification of crosslinked hyaluronan gels. J Biomed Mater Res A. 2003; 66(2):317-29.
[70] Denizli BK, Can HK, Rzaev ZMO, Guner A. Preparation conditions and swelling equilibria of dextran hydrogels prepared by some crosslinked agents. Polymer. 2004; 45(19):6431-35.
[71] Kuo CK, Ma PX. Ionically crosslinked alginate hydrogels as scaffolds for tissue engineering: part I. Structure, gelation rate and mechanical properties. Biomaterials. 2001; 22(6):511-21.
[72] Kim IY, Seo SJ, Moon HS, Yoo MK, Park IY, Kim BC, Cho CS. Chitosan and its derivatives for tissue engineering applications. Biotechnol Adv. 2008; 26(1):1-21.
[73] Liang Y, Liu W, Han B, Yang C, Ma Q, Song F, Bi Q. An in situ formed biodegradable hydrogel for reconstruction of the corneal endothelium. Colloids Surf B. 2011; 82(1):1-7.
[74] Um SH, Lee JB, Park N, Kwon SY, Umbach CC, Luo D. Enzyme-catalysed assembly of DNA hydrogel. Nat Mater. 2006; 5(10):797-801.
[75] Xing Y, Cheng E, Yang Y, Chen P, Zhang T, Sun Y, Yang Z, Liu D. Self-assembled DNA hydrogels with designable thermal and enzymatic responsiveness. Adv Mater. 2011; 23(9):1117-21.
[76] Kleinman HK, Martin GR. Matrigel: basement membrane matrix with biological activity. Semin Cancer Biol. 2005; 15(5):378-86.
[77] Bavaresco B, Comín R, Salvatierra NA, Cid MP. Three-dimensional printing of collagen and hyaluronic acid scaffolds with dehydrothermal treatment crosslinking. Compos. Commun. 2020; 19:1-5.
[78] Dhandayuthapani B, Krishnan UM, Sethuraman S. Fabrication and characterization of chitosan-gelatin blend nanofiber for skin tissue engineering. J Biomed Mater Res B Appl Biomater. 2010; 94(1):264-72.
[79] Lee CK, Shin SR, Lee SH, Jeon JH, So I, Kang TM, Kim SI, Mun JY, Han SS, Spinks GM, Wallace GG, Kim SJ. DNA hydrogel fiber with self-entanglement prepared by using an ionic liquid. Angew Chem Int Ed. 2008; 47(13):2470-4.
[80] Ifkovits JL, Burdick JA. Review: photopolymerizable and degradable biomaterials for tissue engineering applica-tions. Tissue Eng. 2007; 13(10):2369-85.
[81] Sawhney AS, Pathak CP, Hubbell JA. Bioerodible hydrogels based on photopolymerized poly(ethylene gly-col)-co-poly(.alpha.-hydroxy acid) diacrylate macromers. Macromolecules. 1993; 26:581-7.
[82] Martens P, Anseth KS. Characterization of hydrogels formed from acrylate modified poly(vinyl alcohol) macro-mers. Polymer. 2000; 41:7715-22.
[83] Atzet S, Curtin S, Trinh P, Bryant S, Ratner B. Degradable poly(2-hydroxyethyl methacry-late)-co-polycaprolactone hydrogels for tissue engineering scaffolds. Biomacromolecules. 2008; 9(12):3370-7.
[84] HyukIm S, JeongPark Su,,Chung JJ, Jung Y, HyunKim S. Creation of polylactide vascular scaffolds with high compressive strength using a novel melt-tube drawing method. Polymer. 2019; 166:130-7.
[85] Yang F, Williams CG, Wang D, Lee H, Manson PN, Elisseeff J. The effect of incorporating RGD adhesive peptide in polyethylene glycol diacrylate hydrogel on osteogenesis of bone marrow stromal cells. Biomaterials. 2005; 26(30):5991-8.
[86] Atzet S, Curtin S, Trinh P, Bryant S, Ratner B. Degradation poly(2-hydroxyl methacrylate)-co-polycaprolactone hydrogels for tissue engineering scaffolds. Biomacromolecules. 2008; 9(12):3370-7.
[87] Bryant SJ, Anseth KS. Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels. J Biomed Mater Res. 2002; 59(1):63-72.
[88] Tan H, Marra KG. Injectable, biodegradable hydrogels for tissue engineering applications. Materials (Basel). 2010; 3(3):1746-67.
[89] Zhu J. Bioactive modification of poly(ethylene glycol) hydrogels for tissue engineering. Biomaterials. 2010; 31(17):4639-56.
[90] Fisher KE, Pop A, Koh W, Anthis NJ, Saunders WB, Davis GE. Tumor cell invasion of collagen matrices requires coordinate lipid agonist-induced G-protein and membrane-type matrix metalloproteinase-1-dependent signaling. Mol Cancer. 2006; 5:69.
[91] Levis HJ, Massie I, Dziasko MA, Kaasi A, Daniels JT. Rapid tissue engineering of biomimetic human corneal limbal crypts with 3D niche architecture. Biomaterials. 2013; 34(35):8860-8.
[92] Rana D, Zreiqat H, Benkirane-Jessel N, Ramakrishna S, Ramalingam M. Development of decellularized scaffolds for stem cell-driven tissue engineering. J Tissue Eng Regenerat Med. 2015; 11(4): 942-65.
[93] Groebe K, Mueller-Klieser W. Distributions of oxygen, nutrient, and metabolic waste concentrations in multicel-lular spheroids and their dependence on spheroid parameters. Eur Biophys J. 1991; 19(4):169-81.
[94] Takahashi K, Mitsui M, Takeuchi K, Uwabe Y, Kobayashi K, Sawasaki Y, Matsuoka T. Preservation of the cha-racteristics of the cultured human type II alveolar epithelial cells. Lung. 2004;182(4):213-26.
[95] Brophy CM, Luebke-Wheeler JL, Amiot BP, Khan H, Remmel RP, Rinaldo P, Nyberg SL. Rat hepatocyte sphero-ids formed by rocked technique maintain differentiated hepatocyte gene expression and function. Hepatology. 2009; 49(2): 578-6.
[96] Ninomiya H, Winklbauer R. Epithelial coating controls mesenchymal shape change through tissue-positioning effects and reduction of surface-minimizing tension. Nat. Cell Biol. 2008; 10(1):61-71.
[97] Marga F, Neagu A, Kostzin I, Forgacs G. Developmental biology and tissue engineering. Birth Defects Res. Part C. 2007; 81(4):320-8.
[98] Lin R-Z, Chou L-F, Chien C-CM, Chang H-Y. Dynamic analysis of hepatoma spheroid formation: roles of E-cadherin and b1-integrin. Cell Tissue Res. 2006; 324(3):411-22.
[99] Sukach AN. Characteristics of human embryonic neuronal cells procured by nonenzyme method. Tsitologiia. 2005; 47(3):207-13.
[100] Ivascu A, Kubbies M. Rapid generation of single-tumor spheroids for high-throughput cell function and tox-icity analysis. J. Biomol. Screen. 2006; 11(8):922-32.
[101] Ma HL, Jiang Q, Han S, Wu Y, Cui Tomshine J, Wang D, Gan Y, Zou G, Liang X. Multicellular tumor sphe-roids as an in vivo-like tumor model for three-dimensional imaging of chemotherapeutic and nano material cellu-lar penetration. Mol. Imaging. 2012; 11 (6):487-98.
[102] Cui X, Dini S, Dai S, Bi J, Binder BJ, Green JEF, Zhang H. A mechanistic study on tumour spheroid forma-tion in thermosensitive hydrogels: experiments and mathematical modelling. RSC Adv. 2016; 6(77):73282-91.
[103] Yuhas JM, Li AP, Martinez AO, Ladman AJ. A simplified method for production and growth of multicellular tumor spheroids. Cancer Res. 1977; 37(10):3639-43.
[104] Foty R. A simple hanging drop cell culture protocol for generation of 3D spheroids. Journal of Visualized Experiments. 2011; 51:2720.
[105] Oliveira MB, Neto AI, Correia CR, Rial-Hermida MI, Alvarez-Lorenzo C, Mano JF. Superhydrophobic chips for cell spheroids high-throughput generation and drug screening. ACS Appl. Mater. Interfaces. 2014; 6(12): 9488-95.
[106] McMillan KS, McCluskey AG, Sorensen A, Boyd M, Zagnoni M. Emulsion technologies for multicellular tumour spheroid radiation assay. Analyst. 2016; 141(1):100-10.
[107] Kim JB. Three-dimensional tissue culture models in cancer biology. Semin. Cancer Biol. 2005; 15:365-77.
[108] Ingram M, Techy GB, Saroufeem R, Yazan O, Narayan KS, Goodwin TJ, Spaulding GF. Three-dimensional growth patterns of various human tumor cell lines in simulated microgravity of a NASA bioreactor. In Vitro Cell. Dev. Biol. Anim. 1997; 33(6):459-66.
[109] Haisler WL, Timm DM, Gage JA, Tseng H, Killian TC., Souza GR. Three-dimensional cell culturing by magnetic levitation. NatProtoc. 2013; 8(10):1940-9.
[110] Brophy CM, Luebke-Wheeler JL, Amiot BP, Khan H, Remmel RP, Rinaldo P, Nyberg SL. Rat hepatocyte spheroids formed by rocked technique maintain differentiated hepatocyte gene expression and function. Hepatology. 2009; 49(2):578-86.
[111] Chen K, Wu M, Guo F, Li P, Chan CY, Mao Z, Li S, Ren L, Zhang R, Huang TJ. Rapid formation of sizecon-trollable multicellular spheroids via 3D acoustic tweezers. Lab. Chip. 2016; 16(14):2636-43.
[112] Alhasan L QiA, Al-Abboodi A, Rezk A, Chan PPY, Iliescu C, Yeo LY. Rapid enhancement of cellular spheroid assembly by acoustically driven microcentrifugation. ACS Biomater. Sci. Eng. 2016; 2:1013-22.
[113] Sebastian A, Buckle AM, Markx GH. Tissue engineering with electric fields: Immobilization of mammalian cells in multilayer aggregates using dielectrophoresis. Biotechnol. Bioeng. 2007; 98(3):694-700.
[114] Mironov V, Visconti RP, Kasyanov V, Forgacs G, Drake CJ, Markwald RR. Organ printing: tissue spheroids as building blocks. Biomaterials. 2009; 30(12):2164-74.
[115] Curcio E, Salern S o, Barbieri G, De Bartolo L, Drioli E, Bader A. Mass transfer and metabolic reactions in hepatocyte spheroids cultured in rotating wall gas-permeable membrane system. Biomaterials. 2007; 28(36): 5487-97.
[116] Griffith LG, Swartz MA. Capturing complex 3D-tissue physiology in vitro. Mol Cell Biol. 2006; 7(3):211-24.
[117] 121. Lin RZ, Chang HY. Recent advances in 3-D-multicellular spheroid culture for biomedical research. Biotechnology. 2008; 3(9-10):1172-84.
[118] Glicklis R, Merchuk JC, Cohen S. Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions. Biotechnol. Bioeng. 2004; 86(6):672-80.
[119] Anada T, Fukuda J, Sai Y, Suzuki O. An oxygen-permeable spheroid culture system for the prevention of central hypoxia and necrosis of spheroids. Biomaterials. 2012; 33(33):8430-41.