This protocol proposes a simple method for obtaining human hematopoietic progenitors, and for inducing their differentiation into megakaryocytes. It can serve as a basis for better understanding of megakaryopoiesis This technique uses a source, but offers the advantage of being affordable and abundant for laboratory research in hematopoiesis. This method is used to better understand megakaryopoiesis for the cell to be used in therapy they will need to be prepared in the GMP conditions, which is currently not the case.
Understanding the steps including PBMC collection can only be fully complete with a visual demonstration. Before beginning an experiment, connect a leukoreduction filter to an empty 600 milliliter transfer bag and to a tubing set. In a bio safety cabinet inject 25 milliliters of filtered elution buffer per leukoreduction filter into the empty bag, and use a 30 milliliter syringe to gently aspirate the bag contents through the filter.
Transfer the collected red blood cells into a new 50 milliliter tube, and dilute the cell suspension by half with 2%dextran. Mix well to aggregate the blood cells and allow the cells to sediment for 30 minutes at room temperature. When the red blood cells have settled transfer the supernatant to a 50 milliliter tube and fill the tube with two millimolar PBS EDTA solution.
Gently overlay half of the supernatant onto 25 milliliters of density gradient medium in each of two 50 milliliter tubes without disturbing the gradient surface and separate the cells by density gradient centrifugation. Use a disposable pipette to transfer the cells from each interface to a new 50 milliliter tube. Wash the cells two times in 50 milliliters of two millimolar PBS EDTA per wash.
After the second wash, resuspend the pellets in a single 50 milliliter volume of fresh PBS EDTA for counting. Here is possible to stop the procedure by maintaining the collected cells under agitation at four degrees Celsius during the night. For CD34+cell selection, determine the cell number and collect the cells by centrifugation and resuspend the cell pellet in 300 microliters of two millimolar PBS EDTA per 10 to the eighth cells.
Add the appropriate volume of FC receptor blocking reagent and 50 microliters of CD34 micro-beads per 10 to the eighth cells. After 30 minutes at four degrees Celsius, wash the cells with two millimolar PBS EDTA, and resuspend the pellet in 500 microliters of fresh two millimolar PBS EDTA, per 10 to the eight cells. Humidify and appropriately size magnetic column with PBS EDTA, and then add the sample to the column.
Wash the column two times with three milliliters of two millimolar PBS EDTA per wash. Before eluding the CD34+cells, with two five milliliter volumes of two millimolar PBS EDTA per elution. To assess the purity of the magnetic bead selected cells, add two microliters of an appropriate human anti CD34 antibody to a 100 microliter aliquot of the isolated cells and mix well before incubating for 15 minutes at four degrees celsius.
Following incubation, wash the cells in PBS and resuspend the pellet in 200 microliters of PBS for analysis of the purity of the cell sample by flow cytometry. After counting, collect the CD34+cells by centrifugation and immediately resuspend the pellet in cold solution one to a density of 10 to the six cells per milliliter before quickly adding the cell suspension to cold solution two. Then immediately place the cryo tube in a minus 80 degree Celsius freezer for 24 hours before transferring the cryo tube to liquid nitrogen storage.
In this representative study, the cells were recovered by leukoreduction filtration as demonstrated with an approximate 95%viability. After magnetic bead selection, a greater than 90%pure CD34+cell population was obtained. Cell proliferation typically decreases after a week of culture, but with no significant changes in cell viability.
By day seven, the CD34+cells should begin expressing CD41, a specific and early marker or for megakaryocyte and platelet development. By day 10, the majority of the cells in the culture typically develop into mature CD41 expressing megakaryocytes. By day 13, megakaryocyte pro platelet extension and platelet release can be observed by light microscopy.
The total number of CD41, CD42, 8 positive platelets released into the culture can then be quantified using a calibrated number of fluorescent beads. This method paves the way for enhancing the underlying mechanisms of megakaryopoiesis and for increasing platelets yields in visual. Even if this method seems tedious, it is very simple you just need to be patient and prepare all the regions in advance.
Although our critical concerns megakaryopoiesis we obtain CD42 cells can be used in over hematopoietic pathways.
