Closed System Separation of Adherent Bone Marrow Stem Cells for Regenerative Medicine Applications

Abstract

A method for isolating and processing bone marrow derived stem cells, including the steps of: (a) collecting a biological sample containing adherent bone marrow stem cells in a receptacle with interior walls coated with a cell-adherent substrate; (b) incubating the bone marrow cells on the adherent substrate so that a layer of adherent bone marrow stem cells adheres to the substrate; (c) washing any non-adherent cells from the substrate; and (d) collecting the bone marrow stem cell layer. Isolation kits and use of bone marrow cells harvested for cell therapies are also described.

What is claimed is: 
     
         1 . A method for isolating and processing bone marrow derived stem cells comprising the steps of:
 (a) collecting a biological sample containing adherent bone marrow stem cells in a receptacle with interior walls coated with a cell-adherent substrate;   (b) incubating the bone marrow cells on the adherent substrate so that a layer of adherent bone marrow stem cells adheres to the substrate;   (c) washing any non-adherent cells from said substrate; and   (d) collecting said bone marrow stem cell layer.   
     
     
         2 . The method of  claim 1 , wherein said biological sample comprises red blood cells and the step of collecting a biological sample further includes the step of:
 receiving from a subject a biological sample containing red blood cells and bone marrow stem cells and separating the red blood cells from the biological sample before the bone marrow stem cells are incubated.   
     
     
         3 . The method of  claim 1 , wherein said sample is autologous. 
     
     
         4 . The method of  claim 1  wherein said biological sample comprises umbilical cord blood or bone marrow aspirates. 
     
     
         5 . The method of  claim 1 , wherein said cell-adherent substrate comprises a polymeric substrate coated with a cell-adherent biopolymer, polypeptide, protein or polysaccharide. 
     
     
         6 . The method of  claim 5 , wherein said polymer is corona discharge treated prior to coating with said cell-adherent biopolymer, polypeptide, protein or polysaccharide. 
     
     
         7 . The method of  claim 5 , wherein said cell-adherent surface coating comprises a coating of one or more basement membrane proteins. 
     
     
         8 . The method of  claim 7 , wherein said basement membrane proteins are selected from the group consisting of fibronectin, collagen, laminin, keratin, fibrin and fibrinogen. 
     
     
         9 . The method of  claim 5 , wherein said substrate is coated with gelatin. 
     
     
         10 . The method of  claim 5 , wherein said cell-adherent surface coating comprises a coating of one or more polysaccharides selected from the group consisting of hyaluronic acid, heparin sulfate, chondroitin sulfate and agarose. 
     
     
         11 . The method of  claim 5 , wherein said substrate is coated with poly-L-lysine or poly-D-lysine. 
     
     
         12 . The method of  claim 2 , wherein said red blood cells are separated by mixing said sample with a growth medium and diluting the mixture with an amount of plasma blood volume expander effective to separate said red blood cells therefrom. 
     
     
         13 . The method of  claim 12 , wherein said sample is mixed about 1:1 with ex vivo 10 growth medium. 
     
     
         14 . The method of  claim 12  wherein said sample mixed with growth medium is mixed with said plasma blood volume expander in a first cell collection bag separate from said receptacle. 
     
     
         15 . The method of  claim 1 , wherein said cell adherent substrate is selected from the group consisting of cell-adherent biopolymers, polypeptides, proteins and polysaccharides. 
     
     
         16 . The method of  claim 15 , wherein said cells are mixed with a growth medium in said receptacle. 
     
     
         17 . The method of  claim 1 , wherein said cells are incubated on said substrate between about two hours and about five days. 
     
     
         18 . The method of  claim 1 , wherein said non-adherent cells are washed from said substrate by replacing said growth medium. 
     
     
         19 . The method of  claim 18 , wherein said replacing step is performed more than once. 
     
     
         20 . The method of  claim 1 , wherein said bone marrow stem cells are collected by incubating said adherent cell layer with a cell detachment solution. 
     
     
         21 . The method of  claim 1 , further comprising the step of suspending said collected bone marrow stem cells in a pharmaceutically acceptable saline solution. 
     
     
         22 . The method of  claim 1 , further comprising the step of checking the collected bone marrow stem cells for at least one of cell phenotype, viability, and sterility. 
     
     
         23 . A method of regenerative therapy comprising administering bone marrow stem cells to tissues from which regeneration can be elicited by said stem cells, wherein the improvement comprises administering adherent bone marrow stem cells collected by the method of  claim 1 . 
     
     
         24 . A stem cell isolation kit, comprising:
 (a) a first cell collection bag containing a predetermined quantity of a plasma blood volume expander;   (b) a second cell collection bag comprising an interior surface coated with a cell-adherent surface layer and containing a predetermined quantity of growth medium; and   (c) sterile connection means for transferring the contents of said first bag to said second bag.   
     
     
         25 . The kit of  claim 24 , further comprising at least one container comprising a predetermined amount of growth medium. 
     
     
         26 . The kit of  claim 25 , wherein said growth medium in said container comprises RPMI-1640. 
     
     
         27 . The kit of  claim 24 , further comprising a container of cell detachment solution. 
     
     
         28 . The kit of  claim 24 , further comprising aspirating means for collecting bone marrow aspirates. 
     
     
         29 . The kit of  claim 24 , wherein the interior surface of said collection bag is corona discharge treated before being coated with said cell-adherent surface layer. 
     
     
         30 . The kit of  claim 24 , wherein said cell-adherent surface layer comprises a cell-adherent biopolymer, polypeptide, protein or polysaccharide. 
     
     
         31 . The kit of  claim 30 , wherein said cell-adherent surface layer comprises a coating of one or more basement membrane proteins. 
     
     
         32 . The kit of  claim 31 , wherein said basement membrane proteins are selected from the group consisting of fibronectin, collagen, laminin, keratin, fibrin and fibrinogen. 
     
     
         33 . The kit of  claim 30 , wherein said cell-adherent surface layer comprises a coating of one or more polysaccharides selected from the group consisting of hyaluronic acid, heparin sulfate, chondroitin sulfate and agarose. 
     
     
         34 . The kit of  claim 30 , wherein said cell-adherent surface layer comprises poly-L-lysine.