Three-Dimensional Culture in a Methylcellulose-Based Hydrogel to Study the Impact of Stiffness on Megakaryocyte Differentiation.

Fiche publication


Date publication

janvier 2018

Journal

Methods in molecular biology (Clifton, N.J.)

Auteurs

Membres identifiés du Cancéropôle Est :
Dr GACHET Christian


Tous les auteurs :
Aguilar A, Boscher J, Pertuy F, Gachet C, Léon C

Résumé

The differentiation and maturation of megakaryocytes (MKs) occurs in a 3D environment where the cells must constantly adapt to the external physical and mechanical constraints during their development and migration to sinusoid vessels. In this chapter, we present a method for culture of mouse MKs from bone marrow hematopoietic progenitor cells in a methylcellulose 3D medium with a stiffness mimicking that of bone marrow. In addition, we describe how the MKs can be recovered to allow for analysis of their differentiation and maturation state by transmission electron microscopy, immunofluorescence or flow cytometry techniques and to evaluate their ability to form proplatelets. This approach allows (1) generation of MKs with a morphology that more closely resembles the MKs that differentiate in vivo, (2) recovery of megakaryocyte phenotypes sometimes observed in vivo but not found in classical liquid cultures, and (3) study of mechanotransduction pathways induced by the stiffness of the medium.

Mots clés

3D hydrogel culture, Lin− bone marrow progenitors, Megakaryocyte, Methylcellulose, Proplatelets, Stiffness

Référence

Methods Mol. Biol.. 2018 ;1812:139-153