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.
