Patent ID: 7727764

Claim:
A two step method of non-isopycnic cell isolation and purification comprises the steps of: initiating a first step of cell isolation by adding a sample of blood of a defined volume to an initial preparation tube having a corresponding ratio volume of ethylenediaminetetraacetic acid, EDTA solution to produce a volume of anti-coagulated blood; mixing gently by sealing the end of the preparation tube, inverting the preparation tube once so as not to shake or stir violently to mix the combination of blood and EDTA solution while avoiding changing the cell density; taking a predetermined volume of the anti-coagulated blood from the initial preparation tube and gently placing in a first tube containing a selected defined volume of percoll stock solution, PSS, wherein the selected defined volume of PSS is taken from a group of defined volumes of PSS, each defined volume of PSS establishes a specific cell type to be isolated and purified; mix gently by sealing the end and inverting the first tube once; centrifuging the tube for a first predetermined time and predetermined speed in the absence of isopycnic cell layering to form a mixed volume of supernatant of plasma/PSS and a lower bottom sedimented volume of red blood cells; extracting the supernatant of plasma/PSS containing the cell type to be isolated to within 5 mm to 10 mm of an interface between the sedimented red blood cells and the supernatant for transfer into a second tube completing the first step of cell isolation; initiating a second step of cell isolation by transferring an appropriate pre-selected volumetric amount of the supernatant into the second tube holding a defined volume of physiological media to form a solution of the supernatant of plasma/PSS and the physiological media; mixing the solutions gently by sealing the end of the second tube and inverting the second tube once; centrifuging for a second predetermined speed and time in the absence of isopycnic cell layering; and pouring off the supernatant and at the bottom of the tube will be a cell button containing a quantity of the selected purified cells in a high percentage and a low percentage of some contaminating non selected cells, the selected purified cells being purified in the absence of chemical washing and normal cell layering thereby completing the non-isopycnic cell isolation in two steps.