Cell separation apparatus and methods of use
The present invention provides automated devices for use in supporting various cell therapies and tissue engineering methods. The present invention provides an automated cell separation apparatus capable of separating cells from a tissue sample for use in cell therapies and/or tissue engineering. The cell separation apparatus can be used in combination with complementary devices such as cell collection device and/or a sodding apparatus to support various therapies. The automated apparatus includes media and tissue dissociating chemical reservoirs, filters, a cell separator and a perfusion flow loop through a graft chamber which supports a graft substrate or other endovascular device. The present invention further provides methods for using the tissue grafts and cell samples prepared by the devices described herein in a multitude of therapies including revascularization, regeneration and reconstruction of tissues and organs, as well as treatment and prevention of diseases.
1. A method for separating cells comprising:
a) adding an amount of tissue comprising fat cells to an inner bow of a cell separation apparatus, the cell separation apparatus comprising:
(i) a medium reservoir;
(ii) a cell processing device comprising (A) at least one inlet and (B) at least one outlet, (C)a first lobe and (D) a second lobe;
(iii) at least one pump; and
(iv) at least one valve adapted to divert or prevent fluid flow, wherein the at least one pump and at least one valve are in fluid communication with one another; and wherein the inner bowl is in fluid communication with an outer bowl;
b) heating the tissue to a temperature of about 37°C;
c) adding an effective amount of collagenase to the inner bowl;
d) digesting the tissue by oscillating the inner bowl for an effective amount of time;
e) removing fibrous tissue from the digested tissue;
f) spinning the inner bowl at about 3100 RPM for about 5 minutes;
g) displacing the fat cells to the center of the inner bowl by adding fluid to the inner bowl;
h) directing the fat cells through at least one aperture in the inner bowl into the outer bowl by centrifugal action;
i) rinsing the inner bowl with fluid;
j) directing at least one cell pellet from at least one lobe of the inner bowl to the bottom or the inner bowl;
k) optionally, adding fluid to the inner bowl and spinning the inner bowl at about 3100 RPM for about 5 minutes; and
l) collecting the at least one cell pellet.
2. The method of claim 1 , wherein the tissue is selected from the group consisting of adipose tissue, bone marrow, skin, and skeletal muscle.
3. The method of claim 1 , wherein the tissue is adipose tissue.
4. The method of claim 1 , wherein the fluid is a solution containing a physiological concentration of sodium chloride at a physiological pH.
5. The method of claim 1 , wherein the fluid is selected from the group consisting of M199, M199E serum, saline, Hartmann's solution, di-cation-free DPBS and effective mixtures thereof.
6. The method of claim 1 , wherein the cell pellet is dislodged from at least one lobe of the inner bowl using an adjustable spray nozzle.
7. The method of claim 1 , wherein fluid is added and removed from the bowl using at least one rotary coupling transport tube.