1 Scientific RepoRts | 5:10288 | DOi: 10.1038/srep10288 www.nature.com/scientificreports High-Throughput Microluidic platform for 3 D Cultures of Mesenchymal Stem Cells, Towards engineering Developmental processes paola occhetta 1,2,* , Matteo Centola 2,*,† , Beatrice tonnarelli 2 , Alberto Redaelli 1 , Ivan Martin 2 & Marco Rasponi 1 The development of in vitro models to screen the efect of diferent concentrations, combinations and temporal sequences of morpho-regulatory factors on stem/progenitor cells is crucial to investigate and possibly recapitulate developmental processes with adult cells. Here, we designed and validated a microluidic platform to (i) allow cellular condensation, (ii) culture 3D micromasses of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) under continuous low perfusion, and (ii) deliver deined concentrations of morphogens to speciic culture units. Condensation of hBM-MSCs was obtained within 3 hours, generating micromasses in uniform sizes (56.2 ± 3.9 μm). As compared to traditional macromass pellet cultures, exposure to morphogens involved in the irst phases of embryonic limb development (i.e. Wnt and FGF pathways) yielded more uniform cell response throughout the 3D structures of perfused micromasses (PMMs), and a 34-fold higher percentage of proliferating cells at day 7. The use of a logarithmic serial dilution generator allowed to identify an unexpected concentration of TGFβ3 (0.1 ng/ml) permissive to hBM- MSCs proliferation and inductive to chondrogenesis. This proof-of-principle study supports the described microluidic system as a tool to investigate processes involved in mesenchymal progenitor cells diferentiation, towards a ‘developmental engineering’ approach for skeletal tissue regeneration. he in vitro recapitulation of key mechanisms and temporal sequence of events involved in embryonic organogenesis is increasingly being recognized of great importance in the ield of Tissue Engineering 1 and more in general of regenerative medicine 2,3 . Several techniques for regenerating functional tissues have indeed found inspiration from developmental biology paradigms 4 , giving rise to the “so-called” ield of developmental engineering 5 . In the context of skeletal tissues, this approach inspired the use of embryonic stem cells 6 or human adult bone marrow-derived mesenchymal stem/stromal cells (hBM-MSCs) 7–10 to recapitulate endochondral ossiication processes through the early stages of limb development - namely cell condensation, undiferentiated proliferation of a mesenchymal cell popu- lation and pre-chondrogenesis. During development, these steps are tightly regulated by the interplay of speciic signaling pathways – namely, Wnt/β-catenin, FGF and TGFβ/BMP – deining complex and spatio-temporal gradients 11 . In detail, the proper activation of Wnt-canonical and FGF pathways initially 1 Department of electronics, information and Bioengineering, Politecnico di Milano, Milano, italy. 2 Departments of Surgery and of Biomedicine, University Hospital Basel, University of Basel, Basel, Switzerland. * these authors contributed equally to this work. † current address: Musculoskeletal Disease Area, novartis institutes for BioMedical Research, 4002 Basel, Switzerland. correspondence and requests for materials should be addressed to M.R. (email: marco.rasponi@polimi.it) Received: 16 September 2014 Accepted: 09 April 2015 Published: 18 May 2015 opeN