An easy-to-use and versatile method for building cell-laden microfibres

摘要

Introduction: Many in vivo tissues are composed of microscale alignements of structures composed of cells associated or not with extracellular matrix elements (i.e. microcapillaries, nerves network, muscle fibres...). Several microfabrication methods have been proposed in an effort to produce substrates with different sizes and shapes, to promote tissue-specific cell culture organization for building complex tissues. Fibre-shaped materials are useful in creating different functional three-dimensional structures that could mimick a 3D network and facilitate assemblies with other constructs to produce a functional tissue. Onoe et al. have first reported the current and future trends in the fabricating of fibre-shaped cellular structures. Different strategies, more or less complex, have been proposed for building hydrogel microfibres containing numerous cell types, i.e. by extrusion, by a laminar flow or by electrospinning. However, those methods require microfluidic chambers, specific pump and numerous adaptations of the protocols for fabricating fibres and electrospinnig techniques still have also limitations such as the inability to control the direction of the micofibre alignment. Here, we present a new ready-to-use method for producing microfibres on the core shell fibre - based approach, composed of calcium alginate and type Ⅰcollagen. Material and Methods: After the preparation of the calcium-alginate shell, the core of the fabricated microfibre consists of encapsulated cells within collagen in a capillary of 100μm diameter that is introduced within the calcium-alginate shell. The removal of the calcium alginate shell after 24 hours leads to a stable and reproducible fibre shaped cellular construct. We investigated the characterization, the morphology and the functionalities of the cell fibres using a multipotent mouse bone marrow stromal precursor cells, the D1 cell line as cell culture model. Results: The fabricated microfibres are homogenous in size and maintain their shape even after the calcium-alginate shell was removed. Microfibres with controllable diameter and length have been generated. At day 21, almost of the cells remained viable. Representative 3D reconstruction confocal images revealed that D1 cells are mainly distributed around the collagen and located in the periphery of the fibres (Fig. 1). Staining of F-actin with fluorescent phalloidin combined with DAPI staining revealed well oriented actin filaments with time of culture from D1 as well a uniform cellular orientation, observed without any fluid flow (Fig. 2). Von Kossa (VK) staining observations show mineralization of the D1 cell-fibres cultured without osteoinductive factors up to 15 days of culture whereas mineralization is observed after 7 days of fibre culture in osteoinduction conditions. Conclusion: We demonstrated the versatility of this method and the functionality of our cell fibres. Our method can allow creation of fibres using various cell types of primary cells including human mesenchymal stem cells, endothelial cells, or different cell lines.