title: The microwell-mesh: A novel device and protocol for the high throughput manufacturing of cartilage microtissues.
abstract: Microwell platforms are frequently described for the efficient and uniform manufacture of 3-dimensional (3D) multicellular microtissues. Multiple partial or complete medium exchanges can displace microtissues from discrete microwells, and this can result in either the loss of microtissues from culture, or microtissue amalgamation when displaced microtissues fall into common microwells. Herein we describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells; the microwell-mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells. To demonstrate the utility of this platform, we used the microwell-mesh to manufacture hundreds of cartilage microtissues, each formed from 5 × 10(3) bone marrow-derived mesenchymal stem/stromal cells (MSC). The microwell-mesh enabled reliable microtissue retention over 21-day cultures that included multiple full medium exchanges. Cartilage-like matrix formation was more rapid and homogeneous in microtissues than in conventional large diameter control cartilage pellets formed from 2 × 10(5) MSC each. The microwell-mesh platform offers an elegant mechanism to retain microtissues in microwells, and we believe that this improvement will make this platform useful in 3D culture protocols that require multiple medium exchanges, such as those that mimic specific developmental processes or complex sequential drug exposures.
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology at The Translational Research Institute, Brisbane, QLD, Australia. Electronic address: k.futrega@qut.edu.au.
authors.forename: Kathryn
authors.initials: K
authors.lastname: Futrega
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology at The Translational Research Institute, Brisbane, QLD, Australia. Electronic address: james.palmer@qut.edu.au.
authors.forename: James S
authors.initials: JS
authors.lastname: Palmer
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology at The Translational Research Institute, Brisbane, QLD, Australia. Electronic address: mackenziekinney@gmail.com.
authors.forename: Mackenzie
authors.initials: M
authors.lastname: Kinney
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology at The Translational Research Institute, Brisbane, QLD, Australia; School of Chemistry, Physics and Mechanical Engineering, Science and Engineering Faculty, Queensland University of Technology Brisbane, QLD, Australia. Electronic address: b.lott@qut.edu.au.
authors.forename: William B
authors.initials: WB
authors.lastname: Lott
authors.affiliation: Department of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine, University of Calgary, Alberta, Canada. Electronic address: mdungrin@ucalgary.ca.
authors.forename: Mark D
authors.initials: MD
authors.lastname: Ungrin
authors.affiliation: Institute of Biomaterials and Biomedical Engineering, University of Toronto, Ontario, Canada. Electronic address: peter.zandstra@utoronto.ca.
authors.forename: Peter W
authors.initials: PW
authors.lastname: Zandstra
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology at The Translational Research Institute, Brisbane, QLD, Australia; Mater Medical Research - University of Queensland, Brisbane, QLD, Australia. Electronic address: michael.doran@qut.edu.au.
authors.forename: Michael R
authors.initials: MR
authors.lastname: Doran
mesh_terms: D061249:Batch Cell Culture Techniques / Q000295:instrumentation*; D002356:Cartilage / Q000166:cytology* / Q000254:growth & development*; D002454:Cell Differentiation / Q000502:physiology; D002469:Cell Separation / Q000295:instrumentation; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000502:physiology; D020219:Chondrogenesis / Q000502:physiology; D004867:Equipment Design; D019544:Equipment Failure Analysis; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology; D008904:Miniaturization; D023822:Tissue Engineering / Q000295:instrumentation; D054457:Tissue Scaffolds; D014462:Ultrafiltration / Q000295:instrumentation
keywords: 3D culture; Cartilage; Chondrocyte; Differentiation; Mesenchymal stromal cell; Microtissue; Microwell; Stem cell