Abstract: Three-dimensional (3D) cell culture methods have been validated that can replicate the tumor environment in vivo to a large extent, providing an effective tool for studying tumors. In this study, we demonstrated the use of standard laboratory pipette tips as micro vessels for generating 3D cell spheroids. No microfabrication or wet-chemistry surface modifications were involved in the procedure. Spheroids consisting of single or multiple cell types were generated within 24 h just by pipetting and incubating a cell suspension in pipette tips. Scanning electron microscope and optical microscope proved that the cells grew together tightly, and suggested that while gravity force might have initiated the sedimentation of cells at the bottom of the tip, the active aggregation of cells to form tight cell-cell interactions drove the formation of spheroids. Using common laboratory micropipettes and pipette tips, the rate of spheroid generation and the generation reproducibility was characterized from five boxes each with 80 tips. The ease of transferring reagents allowed modeling of the growth of microvascular endothelial cells in tumor spheroids. Moreover, the pairing and fusion of tumor spheroids could be manipulated in the pipette tips, suggesting the potential for building and assembling heterogeneous micro-tumor tissues in vitro to mimic solid tumors in vivo. This study demonstrated that spheroids can be readily and cost-effectively generated in standard biological laboratories in a timely manner using pipette tips.

使用移液器吸头培养三维肿瘤球

[1]AljadiZ, AvalNA, KumarT, et al., 2022. Layer-by-layer cellulose nanofibrils: a new coating strategy for development and characterization of tumor spheroids as a model for in vitro anticancer drug screening. Macromolecular Bioscience, 22(10):2200137.

[2]AltschulerSJ, WuLF, 2010. Cellular heterogeneity: do differences make a difference? Cell, 141(4):559-563.

[3]BiałkowskaK, KomorowskiP, BryszewskaM, et al., 2020. Spheroids as a type of three-dimensional cell cultures—examples of methods of preparation and the most important application. International Journal of Molecular Sciences, 21(17):6225.

[4]BrayF, LaversanneM, WeiderpassE, et al., 2021. The ever-increasing importance of cancer as a leading cause of premature death worldwide. Cancer, 127(16):3029-3030.

[5]BreslinS, O’driscollL, 2013. Three-dimensional cell culture: the missing link in drug discovery. Drug Discovery Today, 18(5-6):240-249.

[6]BustamanteDJ, BasileEJ, HildrethBM, et al., 2021. Biofabrication of spheroids fusion-based tumor models: computational simulation of glucose effects. Biofabrication, 13(3):035010.

[7]CarvalhoBG, VitFF, CarvalhoHF, et al., 2022. Layer-by-layer biomimetic microgels for 3D cell culture and nonviral gene delivery. Biomacromolecules, 23(4):1545-1556.

[8]CastiauxAD, SpenceDM, MartinRS, 2019. Review of 3D cell culture with analysis in microfluidic systems. Analytical Methods, 11(33):4220-4232.

[9]CavoM, Delle CaveD, D’AmoneE, et al., 2020. A synergic approach to enhance long-term culture and manipulation of MiaPaCa-2 pancreatic cancer spheroids. Scientific Reports, 10(1):10192.

[10]CostaEC, GasparVM, CoutinhoP, et al., 2014. Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models. Biotechnology and Bioengineering, 111(8):1672-1685.

[11]CostaEC, MoreiraAF, de Melo-DiogoD, et al., 2016. 3D tumor spheroids: an overview on the tools and techniques used for their analysis. Biotechnology Advances, 34(8):1427-1441.

[12]CuiHJ, WangXX, WesslowskiJ, et al., 2021. Assembly of multi-spheroid cellular architectures by programmable droplet merging. Advanced Materrials, 33(4):2006434.

[13]DadgarN, Gonzalez-SuarezAM, FattahiP, et al., 2020. A microfluidic platform for cultivating ovarian cancer spheroids and testing their responses to chemotherapies. Microsystems & Nanoengineering, 6:93.

[14]do AmaralJB, Rezende-TeixeiraP, FreitasVM, et al., 2011. MCF-7 cells as a three-dimensional model for the study of human breast cancer. Tissue Engineering Part C: Methods, 17(11):1097-1107.

[15]FolkmanJ, 1995. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nature Medicine, 1(1):27-30.

[16]FuJJ, LvXH, WangLX, et al., 2021. Cutting and bonding Parafilm^® to fast prototyping flexible hanging drop chips for 3D spheroid cultures. Cellular and Molecular Bioengineering, 14(2):187-199.

[17]FukudaY, AkagiT, AsaokaT, et al., 2018. Layer-by-layer cell coating technique using extracellular matrix facilitates rapid fabrication and function of pancreatic β-cell spheroids. Biomaterials, 160:82-91.

[18]HanY, LiuXM, LiuH, et al., 2006. Cultivation of recombinant Chinese hamster ovary cells grown as suspended aggregates in stirred vessels. Journal of Bioscience and Bioengineering, 102(5):430-435.

[19]HicklinDJ, EllisLM, 2005. Role of the vascular endothelial growth factor pathway in tumor growth and angiogenesis. Journal of Clinical Oncology, 23(5):1011-1027.

[20]HoriguchiI, SakaiY, 2015. Alginate encapsulation of pluripotent stem cells using a co-axial nozzle. Journal of Visualized Experiments, (101):e52835.

[21]IvascuA, KubbiesM, 2006. Rapid generation of single-tumor spheroids for high-throughput cell function and toxicity analysis. Journal of Biomolecular Screening, 11(8):‍922-932.

[22]JakabK, NorotteC, MargaF, et al., 2010. Tissue engineering by self-assembly and bio-printing of living cells. Biofabrication, 2(2):022001.

[23]JeongSY, LeeJH, ShinY, et al., 2016. Co-culture of tumor spheroids and fibroblasts in a collagen matrix-incorporated microfluidic chip mimics reciprocal activation in solid tumor microenvironment. PLoS One, 11(7):e0159013.

[24]JinZ, LiX, LiuB, et al., 2022. Coaxial bioprinted microfibers with mesenchymal stem cells for glioma microenvironment simulation. Bio-Design and Manufacturing, 5:348-357.

[25]KadletzL, HeiduschkaG, DomayerJ, et al., 2015. Evaluation of spheroid head and neck squamous cell carcinoma cell models in comparison to monolayer cultures. Oncology Letters, 10(3):1281-1286.

[26]KelmJM, TimminsNE, BrownCJ, et al., 2003. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnology and Bioengineering, 83(2):173-180.

[27]KhotMI, LevensteinMA, de BoerGN, et al., 2020. Characterising a PDMS based 3D cell culturing microfluidic platform for screening chemotherapeutic drug cytotoxic activity. Scientific Reports, 10(1):15915.

[28]KoshelevaNV, EfremovYM, ShavkutaBS, et al., 2020. Cell spheroid fusion: beyond liquid drops model. Scientific Reports, 10(1):12614.

[29]LeeHJ, MunS, PhamDM, et al., 2021. Extracellular matrix-based hydrogels to tailoring tumor organoids. ACS Biomaterials Science & Engineering, 7(9):4128-4135.

[30]LeeJ, ShinD, RohJL, 2018. Development of an in vitro cell-sheet cancer model for chemotherapeutic screening. Theranostics, 8(14):3964-3973.

[31]LeeTJ, BhangSH, LaWG, et al., 2011. Spinner-flask culture induces redifferentiation of de-differentiated chondrocytes. Biotechnology Letters, 33(4):829-836.

[32]LeiKF, LinBY, TsangNM, 2017. Real-time and label-free impedimetric analysis of the formation and drug testing of tumor spheroids formed via the liquid overlay technique. RSC Advances, 7(23):13939-13946.

[33]LiM, SongX, JinS, et al., 2021. 3D tumor model biofabrication. Bio-Design and Manufacturing, 4:526-540.

[34]LiuXM, LiuH, WuBC, et al., 2006. Suspended aggregates as an immobilization mode for high-density perfusion culture of HEK 293 cells in a stirred tank bioreactor. Applied Microbiology and Biotechnology, 72(6):1144-1151.

[35]MetzgerW, SossongD, BächleA, et al., 2011. The liquid overlay technique is the key to formation of co-culture spheroids consisting of primary osteoblasts, fibroblasts and endothelial cells. Cytotherapy, 13(8):1000-1012.

[36]MuellerM, RasoulinejadS, GargS, et al., 2020. The importance of cell-cell interaction dynamics in bottom-up tissue engineering: concepts of colloidal self-assembly in the fabrication of multicellular architectures. Nano Letters, 20(4):2257-2263.

[37]NunesAS, BarrosAS, CostaEC, et al., 2019. 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs. Biotechnology and Bioengineering, 116(1):206-226.

[38]QuailDF, JoyceJA, 2013. Microenvironmental regulation of tumor progression and metastasis. Nature Medicine, 19(11):1423-1437.

[39]RourouS, van der ArkA, van der VeldenT, et al., 2007. A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions. Vaccine, 25(19):3879-3889.

[40]RyanHE, McNultyW, ElsonD, et al., 2000. Hypoxia-inducible factor-1α is a positive factor in solid tumor growth. Cancer Research, 60(15):4010-4015.

[41]SarkarS, HornG, MoultonK, et al., 2013. Cancer development, progression, and therapy: an epigenetic overview. International Journal of Molecular Sciences, 14(10):‍21087-21113.

[42]ShriM, AgrawalH, RaniP, et al., 2017. Hanging drop, a best three-dimensional (3D) culture method for primary buffalo and sheep hepatocytes. Scientific Reports, 7(1):1203.

[43]SodekKL, RinguetteMJ, BrownTJ, 2009. Compact spheroid formation by ovarian cancer cells is associated with contractile behavior and an invasive phenotype. International Journal of Cancer, 124(9):2060-2070.

[44]TangR, MurrayCW, LindeIL, et al., 2020. A versatile system to record cell-cell interactions. eLife, 9:e61080.

[45]TungYC, HsiaoAY, AllenSG, et al., 2011. High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array. Analyst, 136(3):473-478.

[46]WangJ, MiaoY, HuangY, et al., 2018. Bottom-up nanoencapsulation from single cells to tunable and scalable cellular spheroids for hair follicle regeneration. Advanced Healthcare Materials, 7(3):170047.

[47]YangYJ, WuHC, JiaJB, et al., 2019. Scaffold-based 3-D cell culture imaging using a miniature electrical impedance tomography sensor. IEEE Sensors Journal, 19(20):‍9071-9080.