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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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'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− 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.

Protokół

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 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.

1. Prepare a 15 mL tube for bone collection with Dulbecco's Modified Eagle's Medium (DMEM) containing 1% of the total volume of penicillin-streptomycin-glutamin (PSG) antibiotic mix (penicillin 10000 U/mL, streptomycin 100000 µg/mL and L-glutamin 29.2 mg/mL).
   NOTE: 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.
2. Fill a 50 mL tube with ethanol 70% for bone disinfection and another one for rinsing instruments during the procedure. Use sterilized dissection instruments.
3. 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.
4. Rapidly dissect out the tibias and femurs.
5. Using a scalpel, cut away the epiphyses of the ankle side end for the tibia and of the hip side end for the femur.
6. Immerse the bones for one second in 70% ethanol before immersing them in DMEM medium containing 1% PSG.

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.

1. Place the bones in a Petri dish and rise them twice in sterile Dulbecco's phosphate-buffered saline (DPBS) to remove potential contaminants.
2. Prepare DMEM - 1% PSG in a 50 mL tube.
   NOTE: Provide 2 mL of DMEM - 1% PSG per mice used for the experiment.
3. Fill a 5 mL syringe equipped with a 21-gauge needle with DMEM - 1% PSG.
4. 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.
5. Quickly press the syringe plunger to flush the marrow out into a 50 mL tube.
   NOTE: To avoid splashes and facilitate 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 µL 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.
6. Repeat steps 2.4. and 2.5. for all bones, refilling the 5 mL syringe with DMEM - 1% PSG if necessary.
7. 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.
8. 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.
9. Transfer the suspension into 15 mL tubes.
10. 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.
11. 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.
12. 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.
13. 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 × 10⁸ cells/mL and distribute the suspension in round bottomed 5 mL polystyrene tubes to a maximum volume of 2 mL.
14. Add to the polystyrene tubes: normal rat serum at a concentration of 50 µL/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 µL 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.
15. Incubate the tubes on ice for 15 min.
16. Add streptavidin-coated magnetic beads at a concentration of 75 µL/mL and homogenize by gently flicking the tubes.
17. Incubate again on ice for 10 min.
18. If necessary, adjust to a final volume of 2.5 mL per tube with M medium.
19. 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.
20. Invert magnet and tube to transfer the tube content into a new round-bottomed 5 mL polystyrene tube.
    NOTE: 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.
21. Discard the initial tube containing undesired magnetic-labeled cells and place the new one, without its cap, in the magnet for three more minutes.
22. Proceeding as in step 2.20, transfer the isolated Lin− cells into a new 15 mL tube.
    NOTE: If several 5 mL polystyrene tubes have been used for the previous steps, pool all the cells in the same 15 mL tube. 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.
23. Measure the Lin− cell number and viability as in step 2.10.
24. 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.
25. Prepare one tube per condition with the appropriate volume of cell suspension and centrifuge at 300 x g for 7 min.
26. 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 × 10⁶ viable cells/mL (equal to 1 × 10⁶ cells per 500 µL well). Incubate the cells at 37 °C under 5% CO2. (= day 0 of culture)
    ​NOTE: See the next paragraph for methylcellulose cultures As an example, to prepare complete culture medium for one well, use 435 µL of DMEM, 50 µL of 100% FBS for 10% final, 5 µL of 100% PSG for 1% final, 5 µL of 10 000 U/mL for 100 U/mL final and 5 µL of 5 µg/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 µL needed per well.

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.

1. Thaw 1 mL aliquots of 3% methylcellulose stock solution at room temperature. Prepare one separate extra aliquot of methylcellulose for syringe coating.
   NOTE: At a concentration of 3%, methylcellulose remains liquid at room temperature (20-25 °C).
2. 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.
3. 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 µL per well, 333 µL of 3% methylcellulose is required.
4. Cautiously remove the needle. Using sterilized forceps, screw a Luer lock connector onto the end of the syringe (Figure 2B-C).
5. 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.
6. Equally distribute the methylcellulose volume between the two syringes (Figure 2E) and put them aside until step 3.11.
7. 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).
   1. Prepare 167 µL of concentrated culture medium per final well of 1 × 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 µL (167 µL of cell suspension in concentrated culture medium + 333 µL of methylcellulose) and all the components will have a concentration identical to that in liquid wells.
   2. As an example, to prepare complete culture medium for one well, use 102 µL of DMEM, 50 µL of 100% FBS for 10% final, 5 µL of 100% PSG for 1% final, 5 µL of 10 000 U/mL for 100 U/mL final and 5 µL of 5 µg/mL TPO for 50 ng/mL final. It gives a volume of 167 µL used to resuspend the cells and with the addition of 333 µL of methylcellulose the final volume will be 500 µL.
8. 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 × 10⁶ cells per 167 µL.
9. Take back the syringes and disconnect one of them from the connector.
10. Pipette 167 µL of the cell suspension.
11. Add the cell suspension directly into the syringe connector (Figure 2F), making sure not to introduce air bubbles.
    NOTE: While adding the cell suspension, slowly draw the syringe plunger simultaneously to free some space for the cell suspension.
12. 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.
13. Slowly homogenize the methylcellulose medium with the cell suspension with ten back-and-forth plunger movements between the two syringes (Figure 2H).
14. Draw the total volume into one syringe and disconnect the two syringes, leaving the connector on the empty one.
15. Empty the content of the syringe into a well of a 4-well plate (Figure 2I).
16. Incubate the cells at 37 °C under 5% CO₂ (= day 0 of culture).
    NOTE: 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 µL 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 µL should not have the connector attached, and seed the cells in a second culture well. The 3% methylcellulose is purchased as a stock solution in Iscove's Modified Dulbecco'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.

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).

1. Prepare 10 mL of DMEM - 1% PSG preheated at 37 °C in a 15 mL tube for each well to resuspend.
2. 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.
3. Centrifuge the tubes 5 min at 300 × g.
4. 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.
5. Discard the supernatant and resuspend the cell pellet in 1 mL of culture medium for each tube.
6. Reseed 500 µL of cell suspension per well in a 4 or 24-well plate and incubate at 37 °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.
7. 24 hours after reseeding, on day 4 of culture, randomly acquire 10 images per well using bright field microscopy and the 20× 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.
8. Count the total number of megakaryocytes and of megakaryocytes extending proplatelets in each image and calculate the proportion of megakaryocytes extending proplatelets.
   NOTE: 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.

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.

1. Add a volume of fixative solution equal to the seeded volume (500 µL 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 µL per well) is usually used for immunolabeling, while glutaraldhehyde (5% in cacodylate buffer, 500 µL per well) is used for electron microscopy analysis.
2. Using a P1000 pipette gently do several up-and-down pipettings with the fixative and the gel so as to homogeneously dilute the methylcellulose.
3. 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.
4. Centrifuge the mixture at 300 × g for 7 min.
   NOTE: A second wash step might be needed to eliminate all the methylcellulose
5. Discard the supernatant and resuspend the megakaryocyte pellet in the appropriate medium according to the desired analysis (immunolabeling, flow cytometry⁹, electron microscopy …) (for electron microscopy see also the paper method "In situ exploration of the major steps of megakaryopoiesis using transmission electron microscopy" in this JoVE issue).

Wyniki

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 (

Dyskusje

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...

Materiały

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