title: Fabrication of multi-well chips for spheroid cultures and implantable constructs through rapid prototyping techniques.
abstract: Three-dimensional (3D) culture models are widely used in basic and translational research. In this study, to generate and culture multiple 3D cell spheroids, we exploited laser ablation and replica molding for the fabrication of polydimethylsiloxane (PDMS) multi-well chips, which were validated using articular chondrocytes (ACs). Multi-well ACs spheroids were comparable or superior to standard spheroids, as revealed by glycosaminoglycan and type-II collagen deposition. Moreover, the use of our multi-well chips significantly reduced the operation time for cell seeding and medium refresh. Exploiting a similar approach, we used clinical-grade fibrin to generate implantable multi-well constructs allowing for the precise distribution of multiple cell types. Multi-well fibrin constructs were seeded with ACs generating high cell density regions, as shown by histology and cell fluorescent staining. Multi-well constructs were compared to standard constructs with homogeneously distributed ACs. After 7 days in vitro, expression of SOX9, ACAN, COL2A1, and COMP was increased in both constructs, with multi-well constructs expressing significantly higher levels of chondrogenic genes than standard constructs. After 5 weeks in vivo, we found that despite a dramatic size reduction, the cell distribution pattern was maintained and glycosaminoglycan content per wet weight was significantly increased respect to pre-implantation samples. In conclusion, multi-well chips for the generation and culture of multiple cell spheroids can be fabricated by low-cost rapid prototyping techniques. Furthermore, these techniques can be used to generate implantable constructs with defined architecture and controlled cell distribution, allowing for in vitro and in vivo investigation of cell interactions in a 3D environment.
authors: Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Via R. Galeazzi 4, 20161, Milan, Italy.
authors: Silvia
authors: S
authors: Lopa
authors: Department of Electronics, Information, and Bioengineering, Politecnico di Milano, Milan, 20133, Italy.
authors: Francesco
authors: F
authors: Piraino
authors: Tissue Regeneration Department, University of Twente, 7522 NB, Enschede, The Netherlands.
authors: Raymond J
authors: RJ
authors: Kemp
authors: Department of Electronics, Information, and Bioengineering, Politecnico di Milano, Milan, 20133, Italy.
authors: Clelia
authors: C
authors: Di Caro
authors: Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Via R. Galeazzi 4, 20161, Milan, Italy.
authors: Arianna B
authors: AB
authors: Lovati
authors: IRCCS Galeazzi Orthopaedic Institute, Milan, 20161, Italy.
authors: Alessia
authors: A
authors: Di Giancamillo
authors: Tissue Regeneration Department, University of Twente, 7522 NB, Enschede, The Netherlands.|Department of Complex Tissue Regeneration, Maastricht University, 6200 MD, Maastricht, The Netherlands.
authors: Lorenzo
authors: L
authors: Moroni
authors: IRCCS Galeazzi Orthopaedic Institute, Milan, 20161, Italy.|Department of Biomedical Sciences for Health, Università degli Studi di Milano, Milan, 20161, Italy.
authors: Giuseppe M
authors: GM
authors: Peretti
authors: Department of Electronics, Information, and Bioengineering, Politecnico di Milano, Milan, 20133, Italy.
authors: Marco
authors: M
authors: Rasponi
authors: Cell and Tissue Engineering Laboratory, IRCCS Galeazzi Orthopaedic Institute, Via R. Galeazzi 4, 20161, Milan, Italy. matteo.moretti@grupposandonato.it.
authors: Matteo
authors: M
authors: Moretti
mesh_terms: D002452:Cell Count; D018929:Cell Culture Techniques / Q000295:instrumentation* / Q000379:methods*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000502:physiology*; D020869:Gene Expression Profiling; D006801:Humans; D013997:Time Factors
keywords: 3D model; cell spheroid; implantable scaffold; rapid prototyping; tissue engineering