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 schematic view of the whole process. This process includes 1) bone dissection, marrow retrieval, and mechanical isolation of marrow cells, 2) magnetic sorting of lineage negative (Lin-) cells, 3) seeding in liquid or methylcellulose hydrogel, and 4) resuspension of megakaryocytes grown in 3D gel for examination of proplatelet formation in liquid medium.
1. Bone collection from adult mice
NOTE: In this section, it is important to minimize microbial contamination.
<ol>
	<li>Prepare a 15 mL tube for bone collection with Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) containing 1% of the total volume of penicillin-streptomycin-glutamin (PSG) antibiotic mix (penicillin 10000 U/mL, streptomycin 100000 &#181;g/mL and L-glutamin 29.2 mg/mL). 
	NOTE:&#160;If all the mice used have the same genotype, pool all bones in the same tube containing 1 mL of DMEM - PSG 1% per number of mice. Antibiotics are important to prevent possible bacteria proliferation during the time of bone sampling.</li>
	<li>Fill a 50 mL tube with ethanol 70% for bone disinfection and another one for rinsing instruments during the procedure. Use sterilized dissection instruments.</li>
	<li>Anesthetize the mice using isoflurane inhalation (4%) and rapidly proceed to cervical dislocation to euthanize the mice. Rapidly immerse the body in 70% ethanol to disinfect and avoid microbial contamination.</li>
	<li>Rapidly dissect out the tibias and femurs.</li>
	<li>Using a scalpel, cut away the epiphyses of the ankle side end for the tibia and of the hip side end for the femur.</li>
	<li>Immerse the bones for one second in 70% ethanol before immersing them in DMEM medium containing 1% PSG.</li>
</ol>
2. Marrow dissociation and Lin- cells isolation
NOTE: This part of the protocol is performed under a laminar flow hood. For one culture, all the wells are part of the same experiment and cannot be considered as independent biological replicates. The cells from all mice are pooled together to ensure the homogeneity of all the wells and to be able to compare them to each other while eliminating possible inter-individual variability. For independent biological replicates, the culture must be repeated.
<ol>
	<li>Place the bones in a Petri dish and rise them twice in sterile Dulbecco&#39;s phosphate-buffered saline (DPBS) to remove potential contaminants.</li>
	<li>Prepare DMEM - 1% PSG in a 50 mL tube. 
	NOTE: Provide 2 mL of DMEM - 1% PSG per mice used for the experiment.</li>
	<li>Fill a 5 mL syringe equipped with a 21-gauge needle with DMEM - 1% PSG.</li>
	<li>Holding the bone with forceps, introduce only the bevel of the needle at the knee side end. 
	NOTE: The knee side epiphysis should remain intact from the dissection, leaving a small cavity in its center through which to insert the needle. The remaining epiphysis will maintain the bone attached to the needle during flushing. Be careful not to introduce more than the bevel in the bone as it might squash and damage the marrow.</li>
	<li>Quickly press the syringe plunger to flush the marrow out into a 50 mL tube. 
	NOTE:&#160;To avoid splashes and facilitate&#160;the marrow flush and liberation place the free end of the bone on the tube wall, immersed in DMEM - 1% PSG. In practice, a volume between 500 &#181;L and 1 mL is generally sufficient to expel the marrow from the bone. When the marrow has been totally expelled, the bone has become white. In case the marrow has not been totally expelled from the diaphysis as judged by some remaining red color, it is possible to repeat the flush with fresh medium.</li>
	<li>Repeat steps 2.4. and 2.5. for all bones, refilling the 5 mL syringe with DMEM - 1% PSG if necessary.</li>
	<li>Use the same 5 mL syringe with the 21-gauge needle to transfer the total volume of medium containing flushed marrow into round bottomed 10 mL tubes. 
	NOTE: It is not absolutely necessary to switch to a round bottomed 10 mL tube but it makes it easier to proceed to the following dissociation steps. Do not hesitate to change syringe and/or needle if risk of contamination is suspected.</li>
	<li>Proceed to cell dissociation by aspirating and expelling the medium and marrow cells successively two times through a 21-gauge needle, three times through a 23-gauge and once through a 25-gauge needle. 
	NOTE: Avoid air bubbles as it may be detrimental for the cells.</li>
	<li>Transfer the suspension into 15 mL tubes.</li>
	<li>Measure cell number and check the viability using an automated cell counter or a cell chamber for manual counting in the presence of trypan blue to exclude dead cells.</li>
	<li>Centrifuge the 15 mL tubes for 7 min at 300 x g. Using a 1 mL transfer pipette, carefully pipette out and discard the supernatant.</li>
	<li>Isolate stem and progenitor cells by negative immunomagnetic sorting using a mouse hematopoietic cell isolation kit. 
	NOTE: The aim of this cell sorting is to retrieve the cells that are negative for all the selection antibodies (CD5, CD11b, CD19, CD45R/B220, Ly6G/C(Gr-1), TER119, 7-4) and therefore to eliminate the cells that are already engaged in a differentiation lineage other than the megakaryocytic one.</li>
	<li>Following the kit instructions, resuspend the cellular pellet in freshly prepared M medium (PBS with 2% of the final volume of fetal bovine serum (FBS), EDTA 1 mM) to a concentration of 1 &#215; 108 cells/mL and distribute the suspension in round bottomed 5 mL polystyrene tubes to a maximum volume of 2 mL.</li>
	<li>Add to the polystyrene tubes: normal rat serum at a concentration of 50 &#181;L/mL as well as the biotinylated antibody mix (CD5, CD11b, CD19, CD45R/B220, Ly6G/C(Gr-1), TER119, 7-4) at a concentration of 50 &#181;L of mix per mL and homogenize by gently flicking the tubes. 
	NOTE: These antibodies will bind to cells already engaged into a differentiation pathway except the megakaryocytic pathway.</li>
	<li>Incubate the tubes on ice for 15 min.</li>
	<li>Add streptavidin-coated magnetic beads at a concentration of 75 &#181;L/mL and homogenize by gently flicking the tubes.</li>
	<li>Incubate again on ice for 10 min.</li>
	<li>If necessary, adjust to a final volume of 2.5 mL per tube with M medium.</li>
	<li>Homogenize the suspension by gently flicking the tube just before placing them, without their caps, inside a magnet and wait for three minutes. 
	NOTE: The cells already engaged into a differentiation pathway and coated with magnetic beads will be retained on the wall of the tube inside the magnet.</li>
	<li>Invert magnet and tube to transfer the tube content into a new round-bottomed 5 mL polystyrene tube. 
	NOTE:&#160;Do not take the tube out of the magnet for the transfer; it is done by inverting the magnet with the tube still in. Use a steady movement and do not shake the tube.</li>
	<li>Discard the initial tube containing undesired magnetic-labeled cells and place the new one, without its cap, in the magnet for three more minutes.</li>
	<li>Proceeding as in step 2.20, transfer the isolated Lin&#8722; cells into a new 15 mL tube. 
	NOTE:&#160;If several 5 mL polystyrene tubes have been used for the previous steps, pool all the cells in the same 15 mL tube.&#160;The cells recovered after the cell sorting are hematopoietic stem cells and progenitors. The presence of thrombopoietin (TPO), the major physiological regulator of megakaryopoiesis 10, will direct the cell differentiation toward the megakaryocytic cell line.</li>
	<li>Measure the Lin&#8722; cell number and viability as in step 2.10.</li>
	<li>Calculate the required volume of cell suspension to centrifuge in order to have 1 x 106 viable cells x Well Number, Well Number being the number of wells to seed per condition.</li>
	<li>Prepare one tube per condition with the appropriate volume of cell suspension and centrifuge at 300 x g for 7 min.</li>
	<li>For liquid cultures, discard the supernatant and resuspend the cell pellet in complete culture medium (DMEM, PSG 1% of the final volume, FBS 10% of the final volume, hirudin 100 U/mL, TPO 50 ng/mL) to achieve the final concentration of 2 &#215; 106 viable cells/mL (equal to 1 &#215; 106 cells per 500 &#181;L well). Incubate the cells at 37 &#176;C under 5% CO2. (= day 0 of culture) 
	&#8203;NOTE: See the next paragraph for methylcellulose cultures As an example, to prepare complete culture medium for one well, use 435 &#181;L of DMEM, 50 &#181;L of 100% FBS for 10% final, 5 &#181;L of 100% PSG for 1% final, 5 &#181;L of 10 000 U/mL for 100 U/mL final and 5 &#181;L of 5 &#181;g/mL TPO for 50 ng/mL final. 4-well or 24-well culture plates are typically used as their well diameter is a good fit for the 500 &#181;L needed per well.</li>
</ol>
3. Cell embedding in methylcellulose hydrogel
NOTE: Please note that the following protocol describes the method to obtain a single well of hydrogel cell culture, adapt to the number of wells needed.
<ol>
	<li>Thaw 1 mL aliquots of 3% methylcellulose stock solution at room temperature.&#160;Prepare one separate extra aliquot of methylcellulose for syringe coating. 
	NOTE: At a concentration of 3%, methylcellulose remains liquid at room temperature (20-25 &#176;C).</li>
	<li>Coat a 1 mL Luer lock syringe equipped with an 18-gauge needle with methylcellulose by drawing 1 mL of methylcellulose from the extra aliquot. Totally expel the methylcellulose. 
	NOTE: This coating step ensures that the volume of methylcellulose collected in step 3.3 is exact.</li>
	<li>With the same syringe and needle but using a new methylcellulose aliquot, draw the appropriate volume of methylcellulose (Figure 2A). 
	NOTE: To achieve a final concentration of 2% methylcellulose in a final volume of 500 &#181;L per well, 333 &#181;L of 3% methylcellulose is required.</li>
	<li>Cautiously remove the needle. Using sterilized forceps, screw a Luer lock connector onto the end of the syringe (Figure 2B-C).</li>
	<li>Attach a second, non-coated, 1 mL Luer lock syringe to the Luer lock connector in order to connect the two syringes together (Figure 2D). 
	NOTE: There is no need to coat this second syringe.</li>
	<li>Equally distribute the methylcellulose volume between the two syringes (Figure 2E) and put them aside until step 3.11.</li>
	<li>Prepare the concentrated DMEM culture medium so as to obtain in the final methylcellulose volume (step 3.11) a concentration identical to the one of the liquid culture medium for each compound (PSG 1% of the final volume, FBS 10% of the final volume, hirudin 100 U/mL, TPO 50 ng/mL).
	<ol>
		<li>Prepare 167 &#181;L of concentrated culture medium per final well of 1 &#215; 106 cells. This volume of medium is calculated so as to obtain a final methylcellulose concentration of 2%. The total volume in the well will be 500 &#181;L (167 &#181;L of cell suspension in concentrated culture medium + 333 &#181;L of methylcellulose) and all the components will have a concentration identical to that in liquid wells.</li>
		<li>As an example, to prepare complete culture medium for one well, use 102 &#181;L of DMEM, 50 &#181;L of 100% FBS for 10% final, 5 &#181;L of 100% PSG for 1% final, 5 &#181;L of 10 000 U/mL for 100 U/mL final and 5 &#181;L of 5 &#181;g/mL TPO for 50 ng/mL final. It gives a volume of 167 &#181;L used to resuspend the cells and with the addition of 333 &#181;L of methylcellulose the final volume will be 500 &#181;L.</li>
	</ol>
	</li>
	<li>After completing the centrifugation step 2.26, discard the supernatant and resuspend the cell pellet in the concentrated culture medium at a ratio of 1 &#215; 106 cells per 167 &#181;L.</li>
	<li>Take back the syringes and disconnect one of them from the connector.</li>
	<li>Pipette 167 &#181;L of the cell suspension.</li>
	<li>Add the cell suspension directly into the syringe connector (Figure 2F), making sure not to introduce air bubbles. 
	NOTE:&#160;While adding the cell suspension, slowly draw the syringe plunger simultaneously to free some space for the cell suspension.</li>
	<li>Carefully reconnect the two syringes (Figure 2G) without losing any suspension in the screw thread. 
	NOTE: Prior to the reconnection, draw the plunger in order to leave the connector half empty and let enough space for the second syringe to connect without the suspension overflowing.</li>
	<li>Slowly homogenize the methylcellulose medium with the cell suspension with ten back-and-forth plunger movements between the two syringes (Figure 2H).</li>
	<li>Draw the total volume into one syringe and disconnect the two syringes, leaving the connector on the empty one.</li>
	<li>Empty the content of the syringe into a well of a 4-well plate (Figure 2I).</li>
	<li>Incubate the cells at 37 &#176;C under 5% CO2 (= day 0 of culture). 
	NOTE:&#160;It is possible to prepare two methylcellulose wells with one pair of syringes. Increase by two the volume of methylcellulose and the volume of cell suspension to have 2 x106 cells. After completing step 3.13. distribute the volume equally between the two syringes to have 500 &#181;L in each of them. Disconnect them and empty the one without the connector in a culture well. Reconnect the syringes to transfer the volume from the one that kept the connector to the other. Disconnect the syringes, the one with the 500 &#181;L should not have the connector attached, and seed the cells in a second culture well.&#160;The 3% methylcellulose is purchased as a stock solution in Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) while concentrated cells are suspended in DMEM. Comparative tests have been initially done to make sure that this mixed medium had no impact on the outcome of the experiment, especially compared to the liquid culture in 100% DMEM.</li>
</ol>
4. Cell Resuspension for Proplatelet Analysis
NOTE: Analysis of the capacity to form proplatelets has to be performed under comparable conditions between liquid and methylcellulose grown megakaryocytes. The physical constraints exerted by the methylcellulose hydrogel inhibit proplatelet extension. Therefore, methylcellulose-grown cells are resuspended in fresh liquid medium on day 3 of culture to allow them to extend proplatelets. Methylcellulose hydrogel is a physical hydrogel that is easily diluted upon liquid medium addition. Importantly, to avoid artifacts from resuspension and centrifugation, cells in the control liquid medium condition have to be treated simultaneously in the same way as methylcellulose-grown cells. Refer to the schematic representation of the experiment (Figure 1).
<ol>
	<li>Prepare 10 mL of DMEM - 1% PSG preheated at 37 &#176;C in a 15 mL tube for each well to resuspend.</li>
	<li>Cautiously resuspend the cells from each well in the 10 mL of DMEM - 1% PSG. 
	NOTE: Gently do several up-and-down movements to dilute the methylcellulose completely. For the liquid wells make sure to collect all the cells deposited at the bottom of the well.</li>
	<li>Centrifuge the tubes 5 min at 300 &#215; g.</li>
	<li>Meanwhile prepare complete culture medium (DMEM, PSG 1% of the final volume, FBS 10% of the final volume, hirudin 100 U/mL, TPO 50 ng/mL). 
	NOTE: At this step each well is retrieved to be diluted by half, therefore prepare 1 mL of complete culture medium per well.</li>
	<li>Discard the supernatant and resuspend the cell pellet in 1 mL of culture medium for each tube.</li>
	<li>Reseed 500 &#181;L of cell suspension per well in a 4 or 24-well plate and incubate at 37 &#176;C under 5% CO2. 
	NOTE: From one initial well, obtain 2 wells for proplatelet visualization in duplicate. Please note that as these duplicates originate from the same sample they cannot be considered as independent replicates.</li>
	<li>24 hours after reseeding, on day 4 of culture, randomly acquire 10 images per well using bright field microscopy and the 20&#215; objective. 
	NOTE: The cells tend to group at the center of the well, make sure not to have too many cells on the field as it may render proplatelet visualization and quantification difficult. Make sure to capture at least 5 megakaryocytes per field.</li>
	<li>Count the total number of megakaryocytes and of megakaryocytes extending proplatelets in each image and calculate the proportion of megakaryocytes extending proplatelets. 
	NOTE:&#160;The quantification is not automated; perform cell counting manually. Counting can be facilitated by the use of the cell counter plugin of ImageJ to click on the cells in order to mark them as they are counted. 10 fields acquired per well and wells in duplicate represent approximately 150-300 megakaryocytes per condition.</li>
</ol>
5. Cell fixation and retrieval for future analyses
CAUTION: This protocol uses fixatives which must be handled under a fume hood, wearing protective equipment.
NOTE: The aim is to maintain intact the gel constraints applied on the cells until they are fully fixed. Therefore, and regardless of the fixative used, it must be added in the well on top of the methylcellulose, without disturbing the gel. The same protocol is applied to liquid cultures.
<ol>
	<li>Add a volume of fixative solution equal to the seeded volume (500 &#181;L in this protocol), on top of the methylcellulose without disrupting the gel. Wait for the appropriate time according to the fixative used (at least 10 min). 
	NOTE: The fixative diffusion throughout the gel should be very rapid as revealed by a rapid change in the gel color (from pink to a yellow-orange shade). Paraformaldehyde (8% in DPBS, 500 &#181;L per well) is usually used for immunolabeling, while glutaraldhehyde (5% in cacodylate buffer, 500 &#181;L per well) is used for electron microscopy analysis.</li>
	<li>Using a P1000 pipette gently do several up-and-down pipettings with the fixative and the gel so as to homogeneously dilute the methylcellulose.</li>
	<li>Using the same pipette and tip, transfer all the volume from the well into a 15 mL tube containing 10 mL of DPBS and homogenize.</li>
	<li>Centrifuge the mixture at 300 &#215; g for 7 min. 
	NOTE: A second wash step might be needed to eliminate all the methylcellulose</li>
	<li>Discard the supernatant and resuspend the megakaryocyte pellet in the appropriate medium according to the desired analysis (immunolabeling, flow cytometry9, electron microscopy &#8230;) (for electron microscopy see also the paper method &#34;In situ exploration of the major steps of megakaryopoiesis using transmission electron microscopy&#34; in this JoVE issue).</li>
</ol>

<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. 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+via+cell-matrix+adhesions+in+3D+microenvironments.">Mechanosensing via cell-matrix adhesions in 3D microenvironments.</a> Experimental Cell Research. 343 (1), 60-66 (2016).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Elasticity+Directs+Stem+Cell+Lineage+Specification.">Matrix Elasticity Directs Stem Cell Lineage Specification.</a> Cell. 126 (4), 677-689 (2006).</li><li>Choi, J. S., Harley, B. A. 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 ...

megakaryocyte-culture-3d-methylcellulose-based-hydrogel-to-improve

Research

JoVE Journal

Biology

2.9K Views.  Université 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.

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

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Endothelial Cell Co-culture Mediates Maturation of Human Embryonic Stem Cell to Pancreatic Insulin Producing Cells in a Directed Differentiation Approach

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are pluripotent, thus having the potential to differentiate into multiple lineages. Such properties make ESCs extremely attractive for cell based therapy and regenerative treatment applications 1. However for its full potential to be realized the cells have to be differentiated into mature and functional phenotypes, which is a daunting task. A promising approach in inducing cellular differentiation is to closely mimic the path of organogenesis in the in vitro setting. Pancreatic development is known to occur in specific stages 2, starting with endoderm, which can develop into several organs, including liver and pancreas. Endoderm induction can be achieved by modulation of the nodal pathway through addition of Activin A 3 in combination with several growth factors 4-7. Definitive endoderm cells then undergo pancreatic commitment by inhibition of sonic hedgehog inhibition, which can be achieved in vitro by addition of cyclopamine 8. Pancreatic maturation is mediated by several parallel events including inhibition of notch signaling; aggregation of pancreatic progenitors into 3-dimentional clusters; induction of vascularization; to name a few. By far the most successful in vitro maturation of ESC derived pancreatic progenitor cells have been achieved through inhibition of notch signaling by DAPT supplementation 9. Although successful, this results in low yield of the mature phenotype with reduced functionality. A less studied area is the effect of endothelial cell signaling in pancreatic maturation, which is increasingly being appreciated as an important contributing factor in in-vivo pancreatic islet maturation 10,11.
The current study explores such effect of endothelial cell signaling in maturation of human ESC derived pancreatic progenitor cells into insulin producing islet-like cells. We report a multi-stage directed differentiation protocol where the human ESCs are first induced towards endoderm by Activin A along with inhibition of PI3K pathway. Pancreatic specification of endoderm cells is achieved by inhibition of sonic hedgehog signaling by Cyclopamine along with retinoid induction by addition of Retinoic Acid. The final stage of maturation is induced by endothelial cell signaling achieved by a co-culture configuration. While several endothelial cells have been tested in the co-culture, herein we present our data with rat heart microvascular endothelial Cells (RHMVEC), primarily for the ease of analysis.

The current study describes a directed differentiation approach in inducing pancreatic differentiation of human embryonic stem cells. Of great significance is the finding that endothelial cell co-culture mediates maturation of human embryonic stem cell derived pancreatic progenitors into insulin expressing cells.

Embryonic stem cells (ESC) have two main characteristics: they can be indefinitely propagated in vitro in an undifferentiated state and they are ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

An Ex vivo Culture System to Study Thyroid Development

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are produced by differentiated thyrocytes, organized in closed spheres called follicles, while calcitonin is synthesized by C-cells, interspersed in between the follicles and a dense network of blood capillaries. Although adult thyroid architecture and functions have been extensively described and studied, the formation of the &#8220;angio-follicular&#8221; units, the distribution of C-cells in the parenchyma and the paracrine communications between epithelial and endothelial cells is far from being understood.
This method describes the sequential steps of mouse embryonic thyroid anlagen dissection and its culture on semiporous filters or on microscopy plastic slides. Within a period of four days, this culture system faithfully recapitulates in vivo thyroid development. Indeed, (i) bilobation of the organ occurs (for e12.5 explants), (ii) thyrocytes precursors organize into follicles and polarize, (iii) thyrocytes and C-cells differentiate, and (iv) endothelial cells present in the microdissected tissue proliferate, migrate into the thyroid lobes, and closely associate with the epithelial cells, as they do in vivo.
Thyroid tissues can be obtained from wild type, knockout or fluorescent transgenic embryos. Moreover, explants culture can be manipulated by addition of inhibitors, blocking antibodies, growth factors, or even cells or conditioned medium. Ex vivo development can be analyzed in real-time, or at any time of the culture by immunostaining and RT-qPCR.
In conclusion, thyroid explant culture combined with downstream whole-mount or on sections imaging and gene expression profiling provides a powerful system for manipulating and studying morphogenetic and differentiation events of thyroid organogenesis.

An Ex vivo Culture System to Study Thyroid Development

This protocol describes dissection of mouse embryonic thyroid anlagen and the culture of explants on semiporous filters or on microscopy plastic slides. This system is ideal to study morphogenetic or differentiation events occurring during thyroid development of wild type or knockout embryos, and is amenable to gain- and loss-of-function experiments.

The thyroid is a bilobated endocrine gland localized at the base of the neck, producing the thyroid hormones T3, T4, and calcitonin. T3 and T4 are ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a versatile analytical tool for characterizing viscoelastic mechanical properties for a variety of materials ranging from hard (plastic, glass, metal, etc.) surfaces to cells on any substrate. It has been widely used to measure the stiffness of cells, but less frequently used to measure the stiffness of aortas. In this paper, we will describe the procedures for using AFM in contact mode to measure the ex vivo elastic modulus of unloaded mouse arteries. We describe our procedure for isolation of mouse aortas, and then provide detailed information for the AFM analysis. This includes step-by-step instructions for alignment of the laser beam, calibration of the spring constant and deflection sensitivity of the AFM probe, and acquisition of force curves. We also provide a detailed protocol for data analysis of the force curves.

Measuring the Stiffness of Ex Vivo Mouse Aortas Using Atomic Force Microscopy

We present detailed protocols for isolation of aortas from mouse and measurement of their elastic modulus using atomic force microscopy.

Arterial stiffening is a significant risk factor and biomarker for cardiovascular disease and a hallmark of aging. Atomic force microscopy (AFM) is a ...

Isolation and 3D Collagen Sandwich Culture of Primary Mouse Hepatocytes to Study the Role of Cytoskeleton in Bile Canalicular Formation In Vitro

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or more hepatocytes contribute apical membranes to form a bile canalicular network through which bile is secreted. Hepatocyte polarization is essential for correct canalicular formation and depends on interactions between the hepatocyte cytoskeleton, cell-cell contacts, and the extracellular matrix. In vitro studies of hepatocyte cytoskeleton involvement in canaliculi formation and its response to pathological situations are handicapped by the lack of cell culture, which would closely resemble the canaliculi network structure in vivo. Described here is a protocol for the isolation of mouse hepatocytes from the adult mouse liver using a modified collagenase perfusion technique. Also described is the production of culture in a 3D collagen sandwich setting, which is used for immunolabeling of cytoskeletal components to study bile canalicular formation and its response to treatments in vitro. It is shown that hepatocyte 3D collagen sandwich cultures respond to treatments with toxins (ethanol) or actin cytoskeleton altering drugs (e.g., blebbistatin) and serve as a valuable tool for in vitro studies of bile canaliculi formation and function.

Presented here is a protocol for the isolation of mouse hepatocytes from adult mouse livers using a modified collagenase perfusion technique. Also described is the long-term culture of hepatocytes in a 3D collagen sandwich setting as well as immunolabeling of the cytoskeletal components to study bile canalicular formation and its response to treatment.

Hepatocytes are the central cells of the liver responsible for its metabolic function. As such, they form a uniquely polarized epithelium, in which two or ...

An Adipocyte Cell Culture Model to Study the Impact of Protein and Micro-RNA Modulation on Adipocyte Function

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights the need to better understand the molecular mechanism involved in adipocyte dysfunction to develop new therapies against obesity-related diseases. Modulating the expression of proteins and micro-RNAs in adipocytes remains highly challenging. This paper describes a protocol to differentiate murine fibroblasts into mature adipocytes and to modulate the expression of proteins and micro-RNAs in mature adipocytes through reverse-transfection using small-interfering RNA (siRNA) and micro-RNA mimicking (miR mimic) oligonucleotides. This reverse-transfection protocol involves the incubation of the transfection reagent and the oligonucleotides to form a complex in the cell culture plate to which the mature adipocytes are added. The adipocytes are then allowed to reattach to the adherent plate surface in the presence of the oligonucleotides/transfection reagent complex. Functional analyses such as the study of insulin signaling, glucose uptake, lipogenesis, and lipolysis can be performed on the transfected 3T3-L1 mature adipocytes to study the impact of protein or micro-RNA manipulation on adipocyte function.

Presented here is a protocol to deliver oligonucleotides such as small-interfering RNA (siRNA), micro-RNA mimics (miRs), or anti-micro-RNA (anti-miR) into mature adipocytes to modulate protein and micro-RNA expression.

Alteration of adipocyte function contributes to the pathogenesis of metabolic diseases including Type 2 diabetes and insulin resistance. This highlights ...

Large-Scale Preparation of Synovial Fluid Mesenchymal Stem Cell-Derived Exosomes by 3D Bioreactor Culture

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. Exosomes for clinical application require large-scale production. To this aim, human synovial fluid MSCs (hSF-MSCs) were grown on microcarrier beads, and then cultured in a dynamic three-dimension (3D) culture system. Through utilizing 3D dynamic culture, this protocol successfully obtained large-scale exosomes from SF-MSC culture supernatants. Exosomes were harvested by ultracentrifugation and verified by a transmission electron microscope, nanoparticle transmission assay, and western blotting. Also, the microbiological safety of exosomes was detected. Results of exosome detection suggest that this approach can produce a large number of good manufacturing practices (GMP)-grade exosomes. These exosomes could be utilized in exosome biology research and clinical osteoarthritis treatment.

Here, we present a protocol to produce a large number of GMP-grade exosomes from synovial fluid mesenchymal stem cells using a 3D bioreactor.

Exosomes secreted by mesenchymal stem cells (MSCs) have been suggested as promising candidates for cartilage injuries and osteoarthritis treatment. ...

Leukodepletion Filters-Derived CD34+ Cells As a Cell Source to Study Megakaryocyte Differentiation and Platelet Formation

The in vitro expansion and differentiation of human hematopoietic progenitors into megakaryocytes capable of elongating proplatelets and releasing platelets allows an in-depth study of the mechanisms underlying platelet biogenesis. Available culture protocols are mostly based on hematopoietic progenitors derived from bone marrow or cord blood raising a number of ethical, technical, and economic concerns. If there are already available protocols for obtaining CD34 cells from peripheral blood, this manuscript proposes a straightforward and optimized protocol for obtaining CD34+ cells from leukodepletion filters readily available in blood centers. These cells are isolated from leukodepletion filters used in the preparation of blood transfusion products, corresponding to eight blood donations. These filters are meant to be discarded. A detailed procedure to collect hematopoietic progenitors identified as CD34+ cells from these filters is described. The method to obtain mature megakaryocytes extending proplatelets while discussing their phenotypic evolution is also detailed. Finally, the protocol present a calibrated pipetting method, to efficiently release platelets that are morphologically and functionally similar to native ones. This protocol can serve as a basis for evaluating pharmacological compounds acting at various steps of the process to dissect the underlying mechanisms and approach the in vivo platelet yields.

This protocol describes in detail all the steps involved in obtaining leukofilter-derived CD34+ hematopoietic progenitors and their in vitro differentiation and maturation into proplatelet-bearing megakaryocytes that are able to release platelets in the culture medium. This procedure is useful for in-depth analysis of cellular and molecular mechanisms controlling megakaryopoiesis.