Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement

Summary

It is now acknowledged that the three-dimensional environment of cells can play an important role in their behavior, maturation and/or differentiation. This protocol describes a three-dimensional cell culture model designed to study the impact of physical containment and mechanical constraints on megakaryocytes.

Abstract

The 3D environment leading to both confinement and mechanical constraints is increasingly recognized as an important determinant of cell behavior. 3D culture has thus been developed to better approach the in vivo situation. Megakaryocytes differentiate from hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). The BM is one of the softest tissues of the body, confined inside the bone. The bone being poorly extensible at the cell scale, megakaryocytes are concomitantly subjected to a weak stiffness and high confinement. This protocol presents a method for the recovery of mouse lineage negative (Lin-) HSPCs by immuno-magnetic sorting and their differentiation into mature megakaryocytes in a 3D medium composed of methylcellulose. Methylcellulose is non-reactive towards megakaryocytes and its stiffness may be adjusted to that of normal bone marrow or increased to mimic a pathological fibrotic marrow. The process to recover the megakaryocytes for further cell analyses is also detailed in the protocol. Although proplatelet extension is prevented within the 3D milieu, it is described below how to resuspend the megakaryocytes in liquid medium and to quantify their capacity to extend proplatelets. Megakaryocytes grown in 3D hydrogel have a higher capacity to form proplatelets compared to those grown in a liquid milieu. This 3D culture allows i) to differentiate progenitors towards megakaryocytes reaching a higher maturation state, ii) to recapitulate phenotypes that may be observed in vivo but go unnoticed in classical liquid cultures, and iii) to study transduction pathways induced by the mechanical cues provided by a 3D environment.

Introduction

Cells in the body experience a complex 3D microenvironment and are subjected to the interplay between chemical and mechanophysical cues including stiffness from the tissue and confinement due to neighboring cells and surrounding matrix ^1,2,3. The importance of stiffness and confinement for cell behavior has only been recognized in the last decades. In 2006, the seminal work from Engler et al. ⁴ highlighted the importance of the mechanical environment for cell differentiation. The authors demonstrated that variation in cell substrate stiffness resulted ....

Protocol

All experiments should be performed in compliance with institutional guidelines for the care and use of laboratory animals. All protocols displayed in the video were carried out in strict accordance with the European law and the recommendations of the Review Board of the Etablissement Français du Sang (EFS). A first version of this protocol was originally published in 2018 in Methods in Molecular Biology ⁸.

NOTE: Figure 1 presents a schematic view of the whole process. This process includes 1) bone dissection, marrow retrieval, and mechanical isolation of marrow cells, 2) magnetic s....

Results

Data obtained using this protocol were originally published in Blood in 2016⁹.

According to the protocol, the cells were seeded in either liquid or methylcellulose hydrogel medium. Cells in liquid medium have all sedimented at the bottom of the well, in contact with the stiff plastic surface and sometime with other cells. In contrast, cells embedded in methylcellulose hydrogel are distributed homogeneously in the gel and are isolated from neighboring cells (

Discussion

In the previous decade, mechanobiology has raised more and more interest in many areas of biology. It is now commonly acknowledged that the mechanical environment surrounding the cells does play a role in their behavior, emphasizing the importance to study how megakaryocytes sense and respond to extracellular mechanical cues. It is challenging to accurately measure the stiffness of the bone marrow tissue in situ¹¹, especially if we consider the hematopoietic red marrow as it is located in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
18-gauge needles	Sigma-Aldrich	1001735825
21-gauge needles	BD Microlance	301155
23-gauge needles	Terumo	AN*2332R1
25-gauge neeldes	BD Microlance	300400
4-well culture dishes	Thermo Scientific	144444
5 mL syringes	Terumo	SS+05S1
Cytoclips	Microm Microtech	F/CLIPSH
Cytofunnels equiped with filter cards	Microm Microtech	F/JC304
Cytospin centrifuge	Thermo Scientific		Cytospin 4
Dakopen	Dako
DMEM 1x	Gibco, Life Technologies	41 966-029
DPBS	Life Technologies	14190-094	Sterile Dulbecco’s phosphate-buffered saline
EasySep magnets	Stem Cell Technologies	18000
EasySep Mouse Hematopoietic Progenitor Cell isolation Kit	Stem Cell Technologies	19856A	biotinylated antibodies (CD5,CD11b, CD19, CD45R/B220, Ly6G/C(Gr-1), TER119,7–4) and streptavidin-coated magnetic beads
EDTA	Invitrogen	15575-020
Fetal Bovine Serum	Healthcare Life Science	SH30071.01
Luer lock 1 mL syringes	Sigma-Aldrich	Z551546-100EA	or 309628 syringes from BD MEDICAL
Luer lock syringes connectors	Fisher Scientific	11891120
MC 3%	R&D systems	HSC001
Polylysin coated slides	Thermo Scientific	J2800AMNZ
PSG 100x	Gibco, Life Technologies	1037-016	10,000 units/mL penicillin, 10,000 μg/mL streptomycin and 29.2 mg/mL glutamine
Rat serum	Stem Cell Technologies	13551
Recombinant hirudin	Transgène	rHV2-Lys47
Recombinant human trombopoietin (rhTPO)	Stem Cell Technologies	2822	10,000 units/mL
Round bottomed 10 mL plastique tubes	Falcon	352054
Round bottomed 5 mL polystyrene tubes