Mixed Cell Populations For Tissue Repair And Separation Technique For Cell Processing

Abstract

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.

We claim: 
     
         1 . An isolated cell composition for tissue repair comprising:
 (a) a mixed population of mononucleated cells, wherein;
 (i) the mononucleated cells are derived from bone marrow, 
 (ii) at least 70% of the mononucleated cells are viable, and 
 (iii) at least 5% of the viable mononucleated cells are CD90 + , with the remaining viable mononucleated cells being CD45 + , 
   (b) less than 5 μg/ml of bovine serum albumin; and   (c) less than 5 μg/ml of an enzymatically active harvest reagent.   
     
     
         2 . The isolated cell composition of  claim 1 , wherein at least 1% of the viable mononucleated cells are CD14 +  and CD45 + . 
     
     
         3 . The isolated cell composition of  claim 1 , wherein a total number of the viable mononucelated cells is at least 35 million. 
     
     
         4 . The isolated cell composition of  claim 3 , wherein the total number of the viable mononucelated cells is less than 300 million. 
     
     
         5 . The isolated cell composition of  claim 1 , further comprising cells expressing CD235a. 
     
     
         6 . The isolated cell composition of  claim 1 , wherein a concentration of the bovine serum albumin is less than 3 μg/ml. 
     
     
         7 . The isolated cell composition of  claim 1 , wherein the isolated cell composition is substantially free of  mycoplasma , endotoxin, and/or microbial contamination. 
     
     
         8 . The isolated cell composition of  claim 7 , wherein the isolated cell composition comprises less than 350 endotoxin unit (EU). 
     
     
         9 . The isolated cell composition of  claim 7 , wherein the isolated cell composition is determined to be substantially free of microbial contamination by
 (a) inoculating a medium with the isolated cell composition,   (b) incubating the medium at 37° C. for at least 1, 3, 7, or 14 days, and   (c) visually inspecting a turbidity of the medium.   
     
     
         10 . The isolated cell composition of  claim 7 , wherein the isolated cell composition is determined to be substantially free of  mycoplasma  by
 (a) inoculating a medium with the isolated cell composition,   (b) incubating the medium at 37° C. for at least 1, 3, 7, or 14 days,   (c) distributing the medium over agar plates at day 1, 3, 7, or 14 and   (d) microscopically inspecting the plates.   
     
     
         11 . The isolated cell composition of  claim 7 , wherein the isolated cell composition is determined to be substantially free of  mycoplasma  by
 (a) inoculating a medium with the isolated cell composition,   (b) incubating the medium at 37° C. for at least 1, 3, 7, or 14 days, and   (c) visualizing  mycoplasma  in the medium by using a DNA-binding fluorochrome assay.   
     
     
         12 . The isolated cell composition of  claim 1 , wherein a total volume of the isolated cell composition is from about 5 mL to about 15 mL. 
     
     
         13 . The isolated cell composition of  claim 1 , wherein a total volume of the isolated cell composition is about 7.5 mL. 
     
     
         14 . The isolated cell composition of  claim 1 , wherein the isolated cell composition is cultured in Iscove's modified Dulbecco's medium (IMDM). 
     
     
         15 . The isolated cell composition of  claim 14 , wherein the medium further comprises 10% fetal bovine serum (FBS) and 10% horse serum. 
     
     
         16 . The isolated cell composition of  claim 15 , wherein the medium further comprises hydrocortisone and L-glutamine. 
     
     
         17 . The isolated cell composition of  claim 16 , wherein hydrocortisone is present at a concentration of about 5 μM. 
     
     
         18 . The isolated cell composition of  claim 16 , wherein L-glutamine is present at a concentration of about 4 mM. 
     
     
         19 . The isolated cell composition of  claim 1 , wherein the percentage of viable mononucleated cells in the isolated cell composition is determined by staining the isolated cell composition with propidium iodide. 
     
     
         20 . The composition of  claim 1 , wherein the cell composition is useful in treating pathologies selected from the group consisting of ischemic conditions, conditions requiring organ or tissue regeneration, inflammatory diseases and auto-immune diseases. 
     
     
         21 . The composition of  claim 20 , wherein the ischemic conditions are selected from the group consisting of limb ischemia, congestive heart failure, cardiac ischemia, kidney ischemia and ESRD, stroke, and ischemia of the eye. 
     
     
         22 . The composition of  claim 20 , wherein the organ or tissue regeneration is selected from the group consisting of regeneration of liver, pancreas, lung, salivary gland, blood vessel, bone, skin, cartilage, tendon, ligament, brain, hair, kidney, muscle, cardiac muscle, nerve, and limb. 
     
     
         23 . The composition of  claim 20 , wherein the inflammatory disease is selected from the group consisting of heart disease, diabetes, spinal cord injury, rheumatoid arthritis, osteo-arthritis, inflammation due to hip replacement or revision, Crohn's disease, and graft versus host disease. 
     
     
         24 . The composition of  claim 20 , wherein the auto-immune disease is selected from the group consisting of type 1 diabetes, psoriasis, systemic lupus, and multiple sclerosis. 
     
     
         25 . The composition of  claim 1 , for tissue regeneration or repair. 
     
     
         26 . The composition of  claim 25 , wherein said tissue is selected from the group consisting of cardiac tissue, bone tissue, neuronal tissue, skin tissue, lung tissue, salivary gland tissue, liver tissue, and pancreatic tissue. 
     
     
         27 . The composition of  claim 1 , for treating ischemic disorders. 
     
     
         28 . The composition of  claim 1 , for inducing angiogenesis in a tissue.