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 D&#233;veloppement en M&#233;decine et Sant&#233; Publique) and by an ARN grant (ANR-18-CE14-0037 PlatForMechanics). Julie Boscher is a recipient from the Fondation pour la Recherche M&#233;dicale (FRM grant number FDT202012010422).

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 Fran&#231;ais 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...

<ol>
	<li>Doolin, M. T., Moriarty, R. A., Stroka, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanosensing+of+Mechanical+Confinement+by+Mesenchymal-Like+Cells.">Mechanosensing of Mechanical Confinement by Mesenchymal-Like Cells.</a> Frontiers in Physiology. 11, (2020).</li><li>Wang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Stiffness+Modulates+Patient-Derived+Glioblastoma+Cell+Fates+in+Three-Dimensional+Hydrogels.">Matrix Stiffness Modulates Patient-Derived Glioblastoma Cell Fates in Three-Dimensional Hydrogels.</a> Tissue Engineering Part A. , (2020).</li><li>Doyle, A. D., Yamada, K. 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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+combined+influence+of+substrate+elasticity+and+ligand+density+on+the+viability+and+biophysical+properties+of+hematopoietic+stem+and+progenitor+cells.">The combined influence of substrate elasticity and ligand density on the viability and biophysical properties of hematopoietic stem and progenitor cells.</a> Biomaterials. 33 (18), 4460-4468 (2012).</li><li>Shin, J. -. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contractile+forces+sustain+and+polarize+hematopoiesis+from+stem+and+progenitor+cells.">Contractile forces sustain and polarize hematopoiesis from stem and progenitor cells.</a> Cell stem cell. 14 (1), 81-93 (2014).</li><li>Boscher, J., Guinard, I., Eckly, A., Lanza, F., L&#233;on, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+platelet+formation+at+a+glance.">Blood platelet formation at a glance.</a> Journal of cell science. 133 (20), (2020).</li><li>Aguilar, A., Boscher, J., Pertuy, F., Gachet, C., L&#233;on, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+culture+in+a+methylcellulose-based+hydrogel+to+study+the+impact+of+stiffness+on+megakaryocyte+differentiation.">Three-dimensional culture in a methylcellulose-based hydrogel to study the impact of stiffness on megakaryocyte differentiation.</a> Methods in Molecular Biology. 1812, 139-153 (2018).</li><li>Aguilar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+environmental+stiffness+for+megakaryocyte+differentiation+and+proplatelet+formation.">Importance of environmental stiffness for megakaryocyte differentiation and proplatelet formation.</a> Blood. 128, 2022-2032 (2016).</li><li>Hitchcock, I. S., Kaushansky, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thrombopoietin+from+beginning+to+end.">Thrombopoietin from beginning to end.</a> British Journal of Haematology. 165 (2), 259-268 (2014).</li><li>Leiva, O., Leon, C., Kah Ng, S., Mangin, P., Gachet, C., Ravid, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+extracellular+matrix+stiffness+in+megakaryocyte+and+platelet+development+and+function.">The role of extracellular matrix stiffness in megakaryocyte and platelet development and function.</a> American Journal of Hematology. 93 (3), 430-441 (2018).</li><li>Jansen, L. E., Birch, N. P., Schiffman, J. D., Crosby, A. J., Peyton, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanics+of+intact+bone+marrow.">Mechanics of intact bone marrow.</a> Journal of the Mechanical Behavior of Biomedical Materials. 50, 299-307 (2015).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+megakaryocyte+morphology+and+proplatelet+formation+in+mice+with+megakaryocyte-restricted+MYH9+inactivation.">Abnormal megakaryocyte morphology and proplatelet formation in mice with megakaryocyte-restricted MYH9 inactivation.</a> Blood. 113 (14), 3182-3189 (2009).</li><li>Eckly, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proplatelet+formation+deficit+and+megakaryocyte+death+contribute+to+thrombocytopenia+in+Myh9+knockout+mice.">Proplatelet formation deficit and megakaryocyte death contribute to thrombocytopenia in Myh9 knockout mice.</a> Journal of Thrombosis and Haemostasis. 8 (10), 2243-2251 (2010).</li></ol>

The authors have nothing to disclose.

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...

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 in the orientation of stem cells towards various differentiation lineages. Since then, the impact of mechanical cues on cell fate and behavior has become increasingly recognized and studied. Despite it being one of the softest tissues of the organism, the bone marrow has a 3D structural organization that is confined inside the bone. Marrow stiffness, although technically difficult to measure precisely, is estimated to lie between 15 and 300 Pa 5, 6. Within the stroma, cells are tightly confined to one another. In addition, most of them are migrating toward the sinusoid vessels to enter the blood circulation. These conditions create additional mechanical constraints on adjacent cells, which have to adapt to these forces. Mechanical cues represent an important parameter whose consequences on megakaryocyte differentiation and proplatelet formation have just recently been explored. Although megakaryocytes can differentiate in vitro in traditional liquid culture, they do not reach the degree of maturation observed in vivo, in part due to the absence of the mechanical cues from the 3D environment 7. Growing progenitors embedded in hydrogel brings 3D mechanical cues that are lacking in liquid milieu.
Hydrogels have been widely used for several decades in the hematological field, notably to grow cells in colony forming assays to quantify hematopoietic progenitors. However, such hydrogels have seldom been used to explore the biological impact of the 3D mechanical environment on maturation and differentiation of hematopoietic cells. Over the past few years our laboratory has developed a 3D culture model using a methylcellulose-based hydrogel 8. This nonreactive physical gel is a useful tool to mimic the physical constraints of the native megakaryocyte environment. It is derived from cellulose by replacement of hydroxyl residues (-OH) by methoxide groups (-OCH3). Both the degree of methyl substitution and the methylcellulose concentration determine the hydrogel stiffness once it has jellified. During the development stage of this technique, it was demonstrated that a Young&#39;s modulus in the range of 30 to 60 Pa is the optimal gel stiffness for megakaryocyte growth 9.
The following protocol describes a method to grow mouse megakaryocytic progenitors in a 3D methylcellulose hydrogel. It has been previously shown that compared with standard liquid culture, this hydrogel culture increases the degree of megakaryocyte polyploidization, improves the maturation and intracellular organization, and increases the capacity of megakaryocytes to extend proplatelets once resuspended in a liquid medium 9. This manuscript describes in detail the protocol for the isolation of mouse bone marrow Lin&#8722; cells and their embedding in a methylcellulose hydrogel for 3D culture as well as the quantification of their capacity to produce proplatelets and the recovery of the cells for further analyses.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18-gauge needles</td>
			<td>Sigma-Aldrich</td>
			<td>1001735825</td>
			<td></td>
		</tr>
		<tr>
			<td>21-gauge needles</td>
			<td>BD Microlance</td>
			<td>301155</td>
			<td></td>
		</tr>
		<tr>
			<td>23-gauge needles</td>
			<td>Terumo</td>
			<td>AN*2332R1</td>
			<td></td>
		</tr>
		<tr>
			<td>25-gauge neeldes</td>
			<td>BD Microlance</td>
			<td>300400</td>
			<td></td>
		</tr>
		<tr>
			<td>4-well culture dishes</td>
			<td>Thermo Scientific</td>
			<td>144444</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL syringes</td>
			<td>Terumo</td>
			<td>SS+05S1</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoclips</td>
			<td>Microm Microtech</td>
			<td>F/CLIPSH</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytofunnels equiped with filter cards</td>
			<td>Microm Microtech</td>
			<td>F/JC304</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytospin centrifuge</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Cytospin 4</td>
		</tr>
		<tr>
			<td>Dakopen</td>
			<td>Dako</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM 1x</td>
			<td>Gibco, Life Technologies</td>
			<td>41 966-029</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td>Sterile Dulbecco&#8217;s phosphate-buffered saline</td>
		</tr>
		<tr>
			<td>EasySep magnets</td>
			<td>Stem Cell Technologies</td>
			<td>18000</td>
			<td></td>
		</tr>
		<tr>
			<td>EasySep Mouse Hematopoietic Progenitor Cell isolation Kit</td>
			<td>Stem Cell Technologies</td>
			<td>19856A</td>
			<td>biotinylated antibodies (CD5,CD11b, CD19, CD45R/B220, Ly6G/C(Gr-1), TER119,7&#8211;4) and streptavidin-coated magnetic beads</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Invitrogen</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Healthcare Life Science</td>
			<td>SH30071.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer lock 1 mL syringes</td>
			<td>Sigma-Aldrich</td>
			<td>Z551546-100EA</td>
			<td>or 309628 syringes from BD MEDICAL</td>
		</tr>
		<tr>
			<td>Luer lock syringes connectors</td>
			<td>Fisher Scientific</td>
			<td>11891120</td>
			<td></td>
		</tr>
		<tr>
			<td>MC 3%</td>
			<td>R&#38;D systems</td>
			<td>HSC001</td>
			<td></td>
		</tr>
		<tr>
			<td>Polylysin coated slides</td>
			<td>Thermo Scientific</td>
			<td>J2800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>PSG 100x</td>
			<td>Gibco, Life Technologies</td>
			<td>1037-016</td>
			<td>10,000 units/mL penicillin, 10,000 &#956;g/mL streptomycin and 29.2 mg/mL glutamine</td>
		</tr>
		<tr>
			<td>Rat serum</td>
			<td>Stem Cell Technologies</td>
			<td>13551</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant hirudin</td>
			<td>Transg&#232;ne</td>
			<td>rHV2-Lys47</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human trombopoietin (rhTPO)</td>
			<td>Stem Cell Technologies</td>
			<td>2822</td>
			<td>10,000 units/mL</td>
		</tr>
		<tr>
			<td>Round bottomed 10 mL plastique tubes</td>
			<td>Falcon</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Round bottomed 5 mL polystyrene tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

megakaryocyte culture in 3d methylcellulose-based hydrogel to improve cell maturation and study the impact of stiffness and confinement

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.

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 (<strong clas...

Watch this Scientific Journal Video about Megakaryocyte Culture in 3D Methylcellulose-Based Hydrogel to Improve Cell Maturation and Study the Impact of Stiffness and Confinement at JoVE.com

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

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 ...

This protocol allows for in vitro evaluation of the impact of confinement and medium stiffness to which native megakaryocytes are exposed to in the bone marrow on their behavior and maturation. The main advantage of this technique is a simplified model which eliminates cell-cell and cell matrix interactions and focuses on the mechanical aspects. Good laboratory practice and sterile working conditions are to be strictly observed.Indeed, even a slight contamination of the hydrogel can have a dramatic impact on megakaryocyte differentiation. To obtain a single well of hydrogel cell culture, begin by thawing two one milliliter aliquots of 3%methylcellulose at room temperature, then coat a one milliliter Luer lock syringe with the methylcellulose by first drawing one milliliter and then expelling all of it. Next, using the same syringe and needle, draw 333 microliters of methylcellulose from a fresh aliquot.After carefully removing the needle, use sterilized forceps to screw a Luer lock connector onto the end of the syringe, then attach a second non-coated one milliliter Luer lock syringe to the connector. Equally distribute the methylcellulose between the two syringes and set them aside. Next, resuspend the previously isolated mouse lineage negative hematopoietic stem and progenitor cells in the concentrated culture medium at a density of one times 10 to the six cells per 167 microliters.Disconnect one of the syringes from the connector and pipette 167 microliters of the cell suspension directly into the connector while simultaneously drawing the syringe plunger slowly to make room for the cell suspension. After adding all of the cell suspension into the connector, draw the plunger further to withdraw the suspension from the connector and carefully reconnect the second syringe, taking care not to lose any suspension in the screw thread. To homogenize the methylcellulose medium with the cell suspension, slowly move the plungers back and forth between the two syringes 10 times, then draw the total volume into one syringe and disconnect the two syringes, leaving the connector on the empty one.Empty the contents of the syringe into one well of a four-well plate and incubate the plate at 37 degrees under 5%carbon dioxide. To analyze proplatelets, on day three of culture, carefully transfer all cells from one well into a 15 milliliter tube containing 10 milliliters of DMEM with 1%PSG. Resuspend the cells by gently pipetting to completely dilute the methylcellulose, then centrifuge the tube for five minutes at 300 times G at room temperature.After discarding the supernatant, resuspend the cell pellet in one milliliter of complete culture medium and re-seed the cells at 500 microliters per well of a four-well plate. Incubate the plate at 37 degrees Celsius under 5%carbon dioxide. On the following day, using a Brightfield microscope with a 20X objective, randomly acquire 10 images per well, making sure not to have too many cells in the field of view and capturing at least five megakaryocytes per field.Using ImageJ, count the total number of megakaryocytes and those extending proplatelets in each image to calculate the proportion of megakaryocytes extending proplatelets. To fix the cells for future analyses, add the fixative solution on top of the methylcellulose without disrupting the gel. After waiting for an appropriate time depending on the fixative used, use a P1000 pipette to gently pipette the fixative and gel until the methylcellulose has been homogeneously diluted.And using the same pipette tip, transfer all the contents of the well into a 15 milliliter tube containing 10 milliliters of DPBS and homogenize by mixing. Centrifuge the mixture. Discard the supernatant and resuspend the megakaryocyte pellet in a medium appropriate for the desired analysis.By day three of culture, megakaryocytes in the liquid medium have sedimented at the bottom of the well and are in contact with the stiff plastic surface, as well as other cells. In contrast, cells embedded in the methylcellulose hydrogel are homogeneously distributed and isolated from neighboring cells. An analysis of the mean diameter of megakaryocytes under different culture conditions shows that 2%methylcellulose slightly increases the mean megakaryocyte diameter compared to the liquid culture.However, increasing the methylcellulose concentration by 0.5%impairs megakaryocyte differentiation as indicated by a smaller mean diameter. In representative transmission electron microscopy images, the intracytoplasmic membranes in megakaryocytes differentiated in vivo within the bone marrow appear to be closely opposed with delineated cytoplasmic territories. In liquid culture, the membranes mostly have a small round oval appearance or elongated vesicles without delimitation of cytoplasmic territories.In contrast, the 2%methylcellulose culture has closely opposed membranes with delimiting cytoplasmic territories resembling the in situ structure. By day four of culture, megakaryocytes previously cultured in hydrogel show increased proplatelet formation compared to those cultured in liquid medium. The mean proportion of megakaryocyte extending proplatelets is usually around 15 to 20%with a liquid pre-culture compared to 35 to 40%with a methylcellulose hydrogel pre-culture.When attempting this procedure, it is important to pipette the appropriate volumes very precisely as a minor change in the final methylcellulose concentration can significantly modify the media stiffness. Following cell maturation in the hydrogel, megakaryocytes can be recovered for ploidy and cell marker analysis by flow cytometry or fixed for electron microscopy or immunostaining of protein of interest.

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2.8K visualizações.  Université de Strasbourg, INSERM, EFS Grand Est, BPPS UMR-S 1255, FMTS. Este protocolo permite a avaliação in vitro do impacto do confinamento e da rigidez média a que megacariócitos nativos são expostos na medula óssea sobre seu comportamento e maturação. A principal vantagem dessa técnica é um modelo simplificado que elimina interações células-células e matrizes celulares e foca nos aspectos mecânicos. Boas práticas laboratoriais e condições de trabalho estéreis devem ser estritamente observadas.De fato, mesmo uma ligeira contaminaçã...