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 comprising:
providing adipose tissue in a centrifuge;
directing a flow of a solution of collagenase to the centrifuge, said flow passes through a first valve and a second valve, wherein a flow path from the second valve to a first filter located between the first valve and the second valve is closed;
incubating the adipose tissue and the collagenase for a predetermined time period to allow for the digestion of the adipose tissue by the collagenase to form a digested material;
filtering the digested material by removing a plurality of fibrous tissue via the first filter to form a filtered digested material, wherein the digested material is directed to flow through the first filter and the second valve;
disposing the filtered digested material in the centrifuge, wherein the filtered digested material is directed to the centrifuge through the first valve;
separating a plurality of cells from the filtered digested material in the centrifuge; and
collecting the plurality of cells.
2. The method of claim 1 , wherein the plurality of cells collected comprises one or more of fibroblasts, smooth muscle cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, tissue-specific parenchymal cells, endothelial cells, urothelial cells, or adipose derived stem cells.
3. The method of claim 1 , further comprising: disposing a first mixture into the centrifuge to create a suspension comprising the plurality of cells collected and filtering the suspension via a second filter.
4. The method of claim 3 , further comprising forming a graft from the plurality of collected cells, wherein the graft is formed by a method comprising:
pressure sodding a graft substrate by applying a sustained low magnitude pressure to the suspension as the suspension flows across the graft substrate to form a graft;
circulating a second mixture over the pressure sodded graft substrate; and
harvesting the graft, wherein the second mixture is circulated over the pressure sodded graft substrate until the graft is harvested.
5. The method of claim 4 , wherein the graft substrate is pretreated with at least one of Medium 199 (M199), Medium 199 with Earle's salts (M199E), phosphate buffered saline (PBS), Saline, or Divalent-Cation Free Dulbecco's phosphate buffered saline (DPBS) prior to pressure sodding the graft substrate.
6. The method of claim 4 , wherein the graft substrate comprises skin, cartilage, bone, bone marrow, tendon, ligament, gastrointestinal tract, genitourinary tracts, liver, pancreas, kidney, adrenal gland, mucosal epithelium, or nerve grafts.
7. The method of claim 4 , wherein the graft substrate is pressure sodded with at least 200,000 cells.
8. The method of claim 1 , wherein a portion of the digested material is disposed in a pristine waste bag subsequent to filtering through the first filter.
9. A method of preparing a graft, comprising:
(a) providing adipose tissue in a centrifuge;
(b) directing a flow of a solution of collagenase to the centrifuge, said flow passes through a first valve and a second valve, wherein a flow path from the second valve to a first filter located between the first valve and the second valve is closed;
(c) incubating the adipose tissue and the collagenase for a predetermined time period to allow for the digestion of the adipose tissue by the collagenase to form a digested material;
(d) filtering the digested material by removing a plurality of fibrous tissue via the first filter to form a filtered digested material, wherein the digested material is directed to flow through the first filter and the second valve;
(e) disposing the filtered digested material in the centrifuge, wherein the filtered digested material is directed to the centrifuge through the first valve;
(f) separating a plurality of endothelial cells from the filtered digested material in the centrifuge;
(g) disposing a first mixture into the centrifuge to create a suspension comprising the plurality of endothelial cells;
(h) filtering the suspension via a second filter;
(i) pressure sodding a graft scaffold by applying a sustained low magnitude pressure to a graft scaffold in a graft chamber to form a graft;
(j) circulating a second mixture over the pressure sodded graft scaffold; and
(k) harvesting the graft, wherein the second mixture is circulated over the pressure sodded graft scaffold until the graft is harvested.
10. The method of claim 8 , wherein a portion of the digested material in the pristine waste bag is recycled into the centrifuge.
11. The method of claim 9 , wherein the first mixture comprises M199E and serum.
12. The method of claim 9 , wherein the first mixture comprises M199E and serum in a ratio of M199E:serum of 6:1.
13. The method of claim 9 , wherein the first mixture comprises serum and at least one of M199, M199E, PBS, Saline, or Divalent-Cation Free DPBS.
14. The method of claim 9 , wherein after step (f), a plurality of endothelial cells is disposed in a tube to determine, via an optical sensor, a location and a volume of the plurality of endothelial cells.
15. The method of claim 14 , wherein the plurality of endothelial cells is disposed into the centrifuge subsequent to determining the location and the volume of said endothelial cells.
16. The method of claim 9 , wherein the first filter is used to remove material greater than 100 μm.
17. The method of claim 9 , wherein the second filter is used to remove material greater than 30 μm.
18. The method of claim 9 , wherein after step (j), a plurality of endothelial cells is purged from the graft scaffold.
19. The method of claim 9 , wherein the second mixture comprises M199 and serum.