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Biology

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

Published: August 26th, 2021

DOI:

10.3791/62511

1Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS

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.

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.

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 ....

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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 sc.......

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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 (

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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.......

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The authors would like to thank Fabien Pertuy and Alicia Aguilar who initially developed this technique in the lab, as well as Dominique Collin (Institut Charles Sadron - Strasbourg) who characterized the viscoelastic properties of the methylcellulose hydrogel. This work was supported by ARMESA (Association de Recherche et Développement en Médecine et Santé Publique) and by an ARN grant (ANR-18-CE14-0037 PlatForMechanics). Julie Boscher is a recipient from the Fondation pour la Recherche Médicale (FRM grant number FDT202012010422).

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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

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