The application of microfluidic devices as next-generation cell and tissue culture systems has increased impressively in the last decades. With that, a plethora of materials as well as fabrication methods for these devices have emerged. Here, we describe the rapid prototyping of microfluidic devices, using micromilling and vapour-assisted thermal bonding of polymethyl methacrylate (PMMA), to create a spheroid-on-a-chip culture system. Surface roughness of the micromilled structures was assessed using scanning electron microscopy (SEM) and atomic force microscopy (AFM), showing that the fabrication procedure can impact the surface quality of micromilled substrates with milling tracks that can be readily observed in micromilled channels. A roughness of approximately 153 nm was created. Chloroform vapour-assisted bonding was used for simultaneous surface smoothing and bonding. A 30-s treatment with chloroform-vapour was able to reduce the surface roughness and smooth it to approximately 39 nm roughness. Subsequent bonding of multilayer PMMA-based microfluidic chips created a durable assembly, as shown by tensile testing. MDA-MB-231 breast cancer cells were cultured as multicellular tumour spheroids in the device and their characteristics evaluated using immunofluorescence staining. Spheroids could be successfully maintained for at least three weeks. They consisted of a characteristic hypoxic core, along with expression of the quiescence marker, p27kip1. This core was surrounded by a ring of Ki67-positive, proliferative cells. Overall, the method described represents a versatile approach to generate microfluidic devices compatible with biological applications.

Department of Surgery University Medical Center Groningen Groningen The Netherlands

Institute of Physics Czech Academy of Sciences Prague Czech Republic

Pharmaceutical Analysis Groningen Research Institute of Pharmacy University of Groningen Groningen The Netherlands

W J Kolff Institute for Biomedical Engineering and Materials Science University Medical Center Groningen Groningen The Netherlands

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Wu Q, et al. Organ-on-a-chip: Recent breakthroughs and future prospects. Biomed. Eng. Online. 2020;19:9. doi: 10.1186/s12938-020-0752-0. PubMed DOI PMC

Leung CM, et al. A guide to the organ-on-a-chip. Nat. Rev. Methods Prim. 2022;2:33. doi: 10.1038/s43586-022-00118-6. DOI

Xia Y, Whitesides GM. Soft lithography. Angew. Chem. Int. Ed. Engl. 1998;37:550–575. doi: 10.1002/(SICI)1521-3773(19980316)37:5<550::AID-ANIE550>3.0.CO;2-G. PubMed DOI

Masuda S, Washizu M, Nanba T. Novel method of cell fusion in field constriction area in fluid integration circuit. IEEE Trans. Industry Appl. 1989;25:732–737. doi: 10.1109/28.31255. DOI

Sia SK, Whitesides GM. Microfluidic devices fabricated in poly(dimethylsiloxane) for biological studies. Electrophoresis. 2003;24:3563–3576. doi: 10.1002/elps.200305584. PubMed DOI

Silverio, V. & Cardoso de Freitas, S. in Complex Fluid-Flows in Microfluidics (ed Francisco José Galindo-Rosales) 25-51, doi: 10.1007/978-3-319-59593-1_2 (Springer International Publishing, 2018)

Ren K, Zhou J, Wu H. Materials for microfluidic chip fabrication. Acc. Chem. Res. 2013;46:2396–2406. doi: 10.1021/ar300314s. PubMed DOI

Toepke MW, Beebe DJ. PDMS absorption of small molecules and consequences in microfluidic applications. Lab Chip. 2006;6:1484–1486. doi: 10.1039/b612140c. PubMed DOI

van Meer BJ, et al. Small molecule absorption by PDMS in the context of drug response bioassays. Biochem. Biophys. Res. Commun. 2017;482:323–328. doi: 10.1016/j.bbrc.2016.11.062. PubMed DOI PMC

Moore TA, Brodersen P, Young EWK. Multiple myeloma cell drug responses differ in thermoplastic vs PDMS microfluidic devices. Anal. Chem. 2017;89:11391–11398. doi: 10.1021/acs.analchem.7b02351. PubMed DOI

Carter S-SD, et al. PDMS leaching and its implications for on-chip studies focusing on bone regeneration applications. Organs-on-a-Chip. 2020;2:100004. doi: 10.1016/j.ooc.2020.100004. DOI

Halldorsson S, Lucumi E, Gomez-Sjoberg R, Fleming RMT. Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices. Biosens. Bioelectron. 2015;63:218–231. doi: 10.1016/j.bios.2014.07.029. PubMed DOI

Gencturk E, Mutlu S, Ulgen KO. Advances in microfluidic devices made from thermoplastics used in cell biology and analyses. Biomicrofluidics. 2017;11:051502. doi: 10.1063/1.4998604. PubMed DOI PMC

Tsao CW. Polymer microfluidics: Simple, low-cost fabrication process bridging academic lab research to commercialized production. Micromachines (Basel) 2016 doi: 10.3390/mi7120225. PubMed DOI PMC

Li Y, et al. Effects of direct current electric fields on lung cancer cell electrotaxis in a PMMA-based microfluidic device. Anal. Bioanal. Chem. 2017;409:2163–2178. doi: 10.1007/s00216-016-0162-0. PubMed DOI

Humayun M, Chow CW, Young EWK. Microfluidic lung airway-on-a-chip with arrayable suspended gels for studying epithelial and smooth muscle cell interactions. Lab Chip. 2018;18:1298–1309. doi: 10.1039/c7lc01357d. PubMed DOI

Wong JF, Mohan MD, Young EWK, Simmons CA. Integrated electrochemical measurement of endothelial permeability in a 3D hydrogel-based microfluidic vascular model. Biosens. Bioelectron. 2020;147:111757. doi: 10.1016/j.bios.2019.111757. PubMed DOI

Shah P, et al. A microfluidics-based in vitro model of the gastrointestinal human-microbe interface. Nat. Commun. 2016;7:11535. doi: 10.1038/ncomms11535. PubMed DOI PMC

Bruijns B, Veciana A, Tiggelaar R, Gardeniers H. Cyclic olefin copolymer microfluidic devices for forensic applications. Biosensors (Basel) 2019 doi: 10.3390/bios9030085. PubMed DOI PMC

Guckenberger DJ, de Groot TE, Wan AM, Beebe DJ, Young EW. Micromilling: A method for ultra-rapid prototyping of plastic microfluidic devices. Lab Chip. 2015;15:2364–2378. doi: 10.1039/c5lc00234f. PubMed DOI PMC

Lashkaripour A, Silva R, Densmore D. Desktop micromilled microfluidics. Microfluid. Nanofluid. 2018;22:31. doi: 10.1007/s10404-018-2048-2. DOI

Chai M, Cui R, Liu J, Zhang Y, Fan Y. Polyformaldehyde-based microfluidics and application in enhanced oil recovery. Microsyst. Technol. 2022;28:947–954. doi: 10.1007/s00542-021-05243-y. DOI

Shallan AI, Smejkal P, Corban M, Guijt RM, Breadmore MC. Cost-effective three-dimensional printing of visibly transparent microchips within minutes. Anal. Chem. 2014;86:3124–3130. doi: 10.1021/ac4041857. PubMed DOI

Prentner, S. et al. Effects of channel surface finish on blood flow in microfluidic devices. In 2009 Symposium on Design, Test, Integration & Packaging of MEMS/MOEMS. 51–54 (2009).

De Marco C, et al. Solvent vapor treatment controls surface wettability in PMMA femtosecond-laser-ablated microchannels. Microfluid . Nanofluid. 2013;14:171–176. doi: 10.1007/s10404-012-1035-2. DOI

Ogilvie IRG, et al. Reduction of surface roughness for optical quality microfluidic devices in PMMA and COC. J. Micromech. Microeng. 2010;20:065016. doi: 10.1088/0960-1317/20/6/065016. DOI

Matellan C, del Río Hernández AE. Cost-effective rapid prototyping and assembly of poly(methyl methacrylate) microfluidic devices. Sci. Rep. 2018;8:6971. doi: 10.1038/s41598-018-25202-4. PubMed DOI PMC

Zhu X, Liu G, Guo Y, Tian Y. Study of PMMA thermal bonding. Microsyst. Technol. 2007;13:403–407. doi: 10.1007/s00542-006-0224-x. DOI

Chiang C-C, Immanuel PN, Chiu Y-H, Huang S-J. Heterogeneous bonding of PMMA and double-sided polished silicon wafers through H2O plasma treatment for microfluidic devices. Coatings. 2021 doi: 10.3390/coatings11050580. DOI

Zhang Y, Gao K, Fan Y. Application of a new UV curable adhesive for rapid bonding in thermoplastic-based microfluidics. Micro Nano Lett. 2019;14:211–214. doi: 10.1049/mnl.2018.5479. DOI

Bamshad A, Nikfarjam A, Khaleghi H. A new simple and fast thermally-solvent assisted method to bond PMMA–PMMA in micro-fluidics devices. J. Micromech. Microeng. 2016;26:065017. doi: 10.1088/0960-1317/26/6/065017. DOI

Mahmoodi SR, Sun PK, Mayer M, Besser RS. Gas-assisted thermal bonding of thermoplastics for the fabrication of microfluidic devices. Microsyst. Technol. 2019;25:3923–3932. doi: 10.1007/s00542-019-04380-9. DOI

Park T, Song IH, Park DS, You BH, Murphy MC. Thermoplastic fusion bonding using a pressure-assisted boiling point control system. Lab Chip. 2012;12:2799–2802. doi: 10.1039/c2lc40252a. PubMed DOI

Gong Y, Park JM, Lim J. An interference-assisted thermal bonding method for the fabrication of thermoplastic microfluidic devices. Micromachines (Basel) 2016 doi: 10.3390/mi7110211. PubMed DOI PMC

Tsao CW, Hromada L, Liu J, Kumar P, DeVoe DL. Low temperature bonding of PMMA and COC microfluidic substrates using UV/ozone surface treatment. Lab Chip. 2007;7:499–505. doi: 10.1039/b618901f. PubMed DOI

Wen X, Takahashi S, Hatakeyama K, Kamei KI. Evaluation of the effects of solvents used in the fabrication of microfluidic devices on cell cultures. Micromachines (Basel) 2021 doi: 10.3390/mi12050550. PubMed DOI PMC

Grimes DR, Kelly C, Bloch K, Partridge M. A method for estimating the oxygen consumption rate in multicellular tumour spheroids. J. R. Soc. Interface. 2014;11:20131124. doi: 10.1098/rsif.2013.1124. PubMed DOI PMC

Uxa S, et al. Ki-67 gene expression. Cell Death Differ. 2021;28:3357–3370. doi: 10.1039/b618901f. PubMed DOI PMC

Lloyd RV, et al. p27kip1: a multifunctional cyclin-dependent kinase inhibitor with prognostic significance in human cancers. Am. J. Pathol. 1999;154:313–323. doi: 10.1016/S0002-9440(10)65277-7. PubMed DOI PMC

Nagelkerke A, Bussink J, Sweep FCGJ, Span PN. Generation of multicellular tumor spheroids of breast cancer cells: How to go three-dimensional. Anal. Biochem. 2013;437:17–19. doi: 10.1016/j.ab.2013.02.004. PubMed DOI

Reichenbach IG, Bohley M, Sousa FJP, Aurich JC. Micromachining of PMMA—manufacturing of burr-free structures with single-edge ultra-small micro end mills. Int. J. Adv. Manuf. Technol. 2018;96:3665–3677. doi: 10.1007/s00170-018-1821-4. DOI

Chen P-C, Pan C-W, Lee W-C, Li K-M. Optimization of micromilling microchannels on a polycarbonate substrate. Int. J. Precis. Eng. Manuf. 2014;15:149–154. doi: 10.1007/s12541-013-0318-1. DOI

Yen DP, Ando Y, Shen K. A cost-effective micromilling platform for rapid prototyping of microdevices. Technology (Singap World Sci) 2016;4:234–239. doi: 10.1142/S2339547816200041. PubMed DOI PMC

Yuan X, Tao Z, Li H, Tian Y. Experimental investigation of surface roughness effects on flow behavior and heat transfer characteristics for circular microchannels. Chin. J. Aeronaut. 2016;29:1575–1581. doi: 10.1016/j.cja.2016.10.006. DOI

Tyagi P, Goulet T, Riso C, Garcia-Moreno F. Reducing surface roughness by chemical polishing of additively manufactured 3D printed 316 stainless steel components. Int. J. Adv. Manuf. Technol. 2019;100:2895–2900. doi: 10.1007/s00170-018-2890-0. DOI

Tsao CW, Wu ZK. Polymer microchannel and micromold surface polishing for rapid, low-quantity polydimethylsiloxane and thermoplastic microfluidic device fabrication. Polymers (Basel) 2020 doi: 10.3390/polym12112574. PubMed DOI PMC

Majhy B, Priyadarshini P, Sen AK. Effect of surface energy and roughness on cell adhesion and growth - facile surface modification for enhanced cell culture. RSC Adv. 2021;11:15467–15476. doi: 10.1039/d1ra02402g. PubMed DOI PMC

Akhil AV, et al. Vaporized solvent bonding of polymethyl methacrylate. J. Adhesion Sci. Technol. 2016;30:826–841. doi: 10.1080/01694243.2015.1125721. DOI

Nath S, Devi GR. Three-dimensional culture systems in cancer research: Focus on tumor spheroid model. Pharmacol. Ther. 2016;163:94–108. doi: 10.1016/j.pharmthera.2016.03.013. PubMed DOI PMC

Collins T, et al. Spheroid-on-chip microfluidic technology for the evaluation of the impact of continuous flow on metastatic potential in cancer models in vitro. Biomicrofluidics. 2021;15:044103. doi: 10.1063/5.0061373. PubMed DOI PMC

Khot MI, et al. Characterising a PDMS based 3D cell culturing microfluidic platform for screening chemotherapeutic drug cytotoxic activity. Sci. Rep. 2020;10:15915. doi: 10.1038/s41598-020-72952-1. PubMed DOI PMC

Aoun L, et al. Microdevice arrays of high aspect ratio poly(dimethylsiloxane) pillars for the investigation of multicellular tumour spheroid mechanical properties. Lab Chip. 2014;14:2344–2353. doi: 10.1039/c4lc00197d. PubMed DOI

Ruppen J, et al. A microfluidic platform for chemoresistive testing of multicellular pleural cancer spheroids. Lab Chip. 2014;14:1198–1205. doi: 10.1039/c3lc51093j. PubMed DOI

Liu W, Wang JC, Wang J. Controllable organization and high throughput production of recoverable 3D tumors using pneumatic microfluidics. Lab Chip. 2015;15:1195–1204. doi: 10.1039/c4lc01242a. PubMed DOI

Zhuang J, Zhang J, Minhao W, Zhang Y. A dynamic 3D tumor spheroid chip enables more accurate nanomedicine uptake evaluation. Adv. Sci. 2019 doi: 10.1002/advs.201901462. PubMed DOI PMC

Hirschhaeuser F, et al. Multicellular tumor spheroids: An underestimated tool is catching up again. J. Biotechnol. 2010;148:3–15. doi: 10.1016/j.jbiotec.2010.01.012. PubMed DOI

Mehta G, Hsiao AY, Ingram M, Luker GD, Takayama S. Opportunities and challenges for use of tumor spheroids as models to test drug delivery and efficacy. J. Controll. Release. 2012;164:192–204. doi: 10.1016/j.jconrel.2012.04.045. PubMed DOI PMC

Nagelkerke A, et al. Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response. Breast Cancer Res. 2013;15:R2. doi: 10.1186/bcr3373. PubMed DOI PMC

Moshksayan K, et al. Spheroids-on-a-chip: Recent advances and design considerations in microfluidic platforms for spheroid formation and culture. Sens. Actuators B: Chem. 2018;263:151–176. doi: 10.1016/j.snb.2018.01.223. DOI