An Innovative 3D-Printed Insert Designed to Enable Straightforward 2D and 3D Cell Cultures.

The classical analyses of indirect communication between different cell types necessitate the use of conditioned media. Moreover, the production of conditioned media remains time-consuming and far from physiological and pathological conditions. Although a few models of co-culture are commercially available, they remain restricted to specific assays and are mostly for two types of cells. Here, 3D-printed inserts are used that are compatible with numerous functional assays. The insert allows the separation of one well of a 6-well plate into four compartments. A wide range of combinations can be set. Moreover, windows are designed in each wall of the compartments so that potential intercellular communication between every compartment is possible in the culture medium in a volume-dependent manner. For example, paracrine intercellular communication can be studied between four cell types in monolayer, in 3D (spheroids), or by combining both. In addition, a mix of different cell types can be seeded in the same compartment in 2D or 3D (organoids) format. The absence of a bottom in the 3D-printed inserts allows the usual culture conditions on the plate, possible coating on the plate containing the insert, and direct visualization by optical microscopy. The multiple compartments provide the possibility to collect different cell types independently or to use, in each compartment, different reagents for RNA or protein extraction. In this study, a detailed methodology is provided to use the new 3D-printed insert as a co-culture system. To demonstrate several capacities of this flexible and simple model, previously published functional assays of cell communication were performed in the new 3D-printed inserts and were demonstrated to be reproducible. The 3D-printed inserts and the conventional cell culture using conditioned media led to similar results. In conclusion, the 3D-printed insert is a simple device that can be adapted to numerous models of co-cultures with adherent cell types.

Regulation of stem cell fate by HSPGs: implication in hair follicle cycling.

Heparan sulfate proteoglycans (HSPGs) are part of proteoglycan family. They are composed of heparan sulfate (HS)-type glycosaminoglycan (GAG) chains covalently linked to a core protein. By interacting with growth factors and/or receptors, they regulate numerous pathways including Wnt, hedgehog (Hh), bone morphogenic protein (BMP) and fibroblast growth factor (FGF) pathways. They act as inhibitor or activator of these pathways to modulate embryonic and adult stem cell fate during organ morphogenesis, regeneration and homeostasis. This review summarizes the knowledge on HSPG structure and classification and explores several signaling pathways regulated by HSPGs in stem cell fate. A specific focus on hair follicle stem cell fate and the possibility to target HSPGs in order to tackle hair loss are discussed in more dermatological and cosmeceutical perspectives.