Mixed cell populations for tissue repair and separation technique for cell processing
View Patent ↗The present invention provides a fluid exchange cell culture technique and tissue repair cells (TRCs) made by these methods, as well as methods using these cells. The method includes a new wash step which increases the tissue repair properties of the TRCs of the invention. This wash step allows for the production of TRC populations with greater tissue repair and anti-inflammatory capabilities. Embodiments of the present invention include a post-culture process for cultured cells that preferably includes the steps of: a wash process for removing unwanted residual culture components, a volume reduction process, and a harvesting process to remove cultured cells. Preferably, all these steps are performed within a aseptically closed cell culture chamber by implementing a separation method that minimizes mechanical disruption of the cells and is simple to automate. The harvested cells may then be concentrated to a final volume for the intended use. In such embodiments, the final composition is a substantially purified and concentrated cell mixture suspended in a physiologic solution suitable for immediate use in humans without further washing, volume reduction, or processing. Embodiments are also applicable to harvesting (and/or washing) particles within a liquid or solution within a chamber.
1. A method for processing cells in a biochamber, the method comprising:
a) culturing said cells with culture media for a predetermined time period;
b) displacing the culture media from the biochamber into a waste container by introducing a biocompatible first rinse solution;
c) replacing the first rinse solution with a cell harvest solution comprising an enzyme that allows for the detachment of cells from each other and/or from the biochamber surface;
d) incubating the contents of the biochamber for a predetermined period of time to allow the detachment of cells adhered to each other and/or the biochamber surface, wherein said cells remain settled on the surface;
e) displacing the cell harvest solution into a waste container by introducing a second rinse solution into the biochamber, wherein said cells remain settled on the surface; and
f) agitating the biochamber to bring settled cells into suspension,
wherein said method produces a mixed cell population that is at least 80% viable and at least 5% of the cells express CD90.
2. The method according to claim 1 , wherein the second rinse solution comprises a solution capable of being injectable into a human.
3. The method for processing cultured cells according to claim 1 , further comprising:
g) draining the solution with suspended cells into a cell collection container.
4. The method for processing cultured cells according to claim 3 , further comprising, subsequent to g) draining the solution with suspended cells into a cell collection container:
(h) adding an additional amount of the second rinse solution to the biochamber, and
(i) agitating the biochamber to bring residual settled cells into suspension.
5. The method for processing cultured cells according to claim 4 , further comprising:
j) draining the solution with suspended cells into a cell collection container.
6. The method for processing cultured cells according to claim 1 , further comprising: displacing a portion of the second rinse solution with a gas to obtain a predetermined reduced liquid volume in the biochamber.
7. The method according to claim 6 wherein the flow rate for the introduction of solutions and/or gases added to the biochamber is between about 0.03 to about 1.0 volume exchanges/min.
8. The method according to claim 6 , wherein the flow rate for the introduction of solutions and/or gases added to the biochamber is between about 0.50 to about 0.75 volume exchanges/min.
9. The method according to claim 1 , wherein liquids/solutions or gases are introduced into the biochamber according to a radial plug flow.
10. The method according to claim 1 , wherein said cells are derived from mononuclear cells.
11. The method according to claim 10 , wherein said mononuclear cells are bone marrow, peripheral blood, umbilical cord blood or fetal liver.