An In Vitro Method for the Differentiation of Megakaryocytes and Platelet Formation

For megakaryocyte differentiation, seed 5 x 10⁵ CD34 positive cells per milliliter in 2 milliliters of serum-free medium, supplemented with recombinant human thrombopoietin per well, in a 12-well plate for their incubation at 37 degrees Celsius and 5% carbon dioxide in a humidified atmosphere.

At the appropriate differentiation monitoring time points, harvest the cells from two to three wells per time point without disturbing the cells in the other wells, and stain the collected cells with anti-CD41 and anti-CD42a antibodies to determine the percentage of mature megakaryocytes within the cultures.

For microscopic visualization of the cell surface markers, wash the harvested cells in 1 milliliter of separation buffer, and resuspend the pellet in 100 microliters of fresh buffer. Use a cytospin to seed the cells onto a glass slide, and fix the cells to the slide with a 30-second submersion in methanol. After air-drying, label the cells with 20 microliters of DAPI-supplemented mounting medium, and cover the cells with a coverslip for visualization by fluorescent microscopy.

To count the proplatelets, harvest the cells at day 8 or 9 of differentiation, and seed the collected cells at 1 x 10⁴ cells per 200 microliters of fresh serum-free medium supplemented with recombinant human thrombopoietin per well in a 48-well plate. After 5 days at 37 degrees Celsius and 5% carbon dioxide, count the number of proplatelet-bearing megakaryocytes per well on an inverted light microscope using the 10 or 20 times objective.

To analyze the platelet activation, on day 14 or 15 of culture, gently mix the cells with a Pasteur pipette, and collect 100 microliters of cells. Add 1 milliliter of Tyrode's buffer to the cells, and pellet them by centrifugation. Collect the supernatant for centrifugation to pellet the platelet-sized particles, and resuspend the particles in 100 microliters of fresh Tyrode's buffer. Then, label the particles with anti-PAC-1 antibody. Activate them with adenosine diphosphate for 20 minutes at room temperature, and analyze the percentage of PAC-1 positive events by flow cytometry.