Apparatus and method for separating and concentrating fluids containing multiple components

Abstract

An apparatus that allows for separating and collecting a fraction of a sample. The apparatus, when used with a centrifuge, allows for the creation of at least three fractions in the apparatus. It also provides for a new method of extracting the buffy coat phase from a whole blood sample. A buoy system that may include a first buoy portion and a second buoy member operably interconnected may be used to form at least three fractions from a sample during a substantially single centrifugation process. Therefore, the separation of various fractions may be substantially quick and efficient.

1 - 32 . (canceled) 
     
     
         33 . A method for concentrating bone marrow aspirate, the method comprising:
 obtaining a volume of bone marrow aspirate;   loading the volume of bone marrow aspirate into a separator comprising two buoys and an extraction port, the buoys operable to substantially physically separate the aspirate into three or more fractions and the extraction port operable to allow extraction of a fraction between the buoys without substantially commingling the fraction with the other fractions in the separator;   centrifuging the separator; wherein the buoys separate the bone marrow aspirate creating the fraction between the buoys comprising a bone marrow aspirate concentrate, wherein the bone marrow aspirate concentrate includes red blood cells and white blood cells; and   extracting the fraction between the buoys of the bone marrow aspirate concentrate through the extraction port.   
     
     
         34 . The method according to  claim 33  wherein the at least one fraction comprises at least one of the group consisting essentially of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells. 
     
     
         35 . The method according to  claim 33  wherein an amount of nucleated cells in the bone marrow aspirate concentrate is greater than or equal to 4 times an amount of nucleated cells found in the volume of bone marrow aspirate. 
     
     
         36 . The method according to  claim 33  further comprising applying the bone marrow aspirate concentrate into a patient. 
     
     
         37 . The method according to  claim 64  wherein the combined volume comprises at least one of the group consisting essentially of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells. 
     
     
         38 . The method according to  claim 64  wherein an amount of nucleated cells in the combined volume is greater than or equal to 4 times an amount of nucleated cells in the volume of bone marrow aspirate and the volume of blood. 
     
     
         39 . A method for treating a defect in a mammal using a concentrated bone marrow aspirate, the method comprising:
 drawing a volume of bone marrow aspirate from the mammal;   loading the volume of bone marrow aspirate into a separator comprising two buoys, the separator being operable to separate the aspirate into three or more fractions;   centrifuging the separator;   separating the volume of bone marrow aspirate into a plurality of fractions including a fraction comprising a concentrated bone marrow aspirate;   extracting the fraction comprising a concentrated bone marrow aspirate; and   applying the fraction comprising a concentrated bone marrow aspirate to site of the defect.   
     
     
         40 . The method according to  claim 39  wherein the fraction comprising a concentrated bone marrow aspirate is essentially buffy coat including a plurality of undifferentiated cells. 
     
     
         41 . The method according to  claim 39  wherein the fraction comprises at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells. 
     
     
         42 . The method according to  claim 39  further comprising combining the fraction with a carrier. 
     
     
         43 . The method according to  claim 42  wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof. 
     
     
         44 . The method according to  claim 39  further comprising combining an activating agent with the fraction. 
     
     
         45 . The method according to  claim 39  further comprising combining a pharmaceutical agent with the fraction. 
     
     
         46 . A method for treating a defect in an animal, the method comprising:
 obtaining a volume of bone marrow aspirate and a volume of whole blood from the animal;
 loading the volume of bone marrow aspirate into a first separator comprising two buoys, the separator being operable to separate the aspirate into three or more fractions; 
 loading the volume of whole blood into a second separator comprising two buoys, the separator being operable to separate the aspirate into three or more fractions; 
 centrifuging the first separator and the second separators, thereby separating the volume of bone marrow aspirate into a plurality of fractions and separating the volume of whole blood into a plurality of fractions; 
 collecting a fraction of the bone marrow aspirate; 
 collecting a fraction of the whole blood; and 
 applying at least one of the fraction of bone marrow aspirate and the fraction of whole blood to site of the defect. 
   
     
     
         47 . The method according to  claim 46  wherein the at least one of the fraction of bone marrow aspirate and the fraction of whole blood comprises at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells. 
     
     
         48 . The method according to  claim 46  further comprising combining the at least one of the fraction of bone marrow aspirate and the fraction of whole blood with a carrier. 
     
     
         49 . The method according to  claim 48  wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof. 
     
     
         50 . The method according to  claim 48  further comprising adding an activating agent to the at least one of the fraction of bone marrow aspirate and the fraction of whole blood. 
     
     
         51 . The method according to  claim 48  further comprising adding a pharmaceutical agent to the at least one of the fraction of bone marrow aspirate and the fraction of whole blood. 
     
     
         52 . A method for treating a defect in a patient, the method comprising:
 drawing a volume of bone marrow aspirate and a volume of whole blood from the patient;   adding a first anticoagulant to the volume of bone marrow aspirate;   adding a second anticoagulant to the volume of whole blood;   loading the volume of bone marrow aspirate and the volume of whole blood into a separator comprising two buoys, the separator being operable to separate the volume of bone marrow aspirate and the volume of whole blood into three or more fractions;   centrifuging the separator, separating the volume of bone marrow aspirate and the volume of whole blood into a plurality of fractions;   withdrawing a fraction comprising at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells; and   applying the fraction to site of the defect.   
     
     
         53 . The method according to  claim 52  wherein the fraction is essentially buffy coat including a plurality of undifferentiated cells. 
     
     
         54 . The method according to  claim 52  further comprising combining the fraction with a carrier. 
     
     
         55 . The method according to  claim 63  wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof. 
     
     
         56 . The method according to  claim 52  further comprising combining an activating agent with the fraction. 
     
     
         57 . The method according to  claim 52  further comprising combining a pharmaceutical agent with the fraction. 
     
     
         58 . A method of treating a patient with combination of a concentrated bone marrow aspirate and buffy coat, the method comprising:
 obtaining a volume of a whole blood from the patient;   obtaining a volume of a bone marrow aspirate from the patient;   forming a buffy coat fraction of the whole blood;   forming a concentrated bone marrow aspirate fraction of the bone marrow aspirate; and   
       applying at least one of the buffy coat fraction or the concentrated bone marrow aspirate fraction to the patient. 
     
     
         59 . The method according to  claim 58 , wherein the forming a first fraction of the first whole material and the forming a second fraction of the second whole material includes:
 loading the volume of whole blood and the volume of bone marrow aspirate in a container; and   applying a force to the container to form at least three fractions.   
     
     
         60 . The method according to  claim 58 , wherein the forming a first fraction of the first whole material and the forming a second fraction of the second whole material includes:
 loading the volume of whole blood and the volume of bone marrow aspirate in the container having a separating member, the separating member having a specific gravity substantially dependent upon at least one of the at least three fractions; and   centrifuging the container to move the separating member to a selected position relative to the at least one of the volume of whole blood and the volume of bone marrow aspirate to substantially physically separate the at least three fractions.   
     
     
         61 . The method according to  claim 58  wherein the at least one of the first fraction or the second fraction comprises at least one of the group consisting of hematopoietic stem cells, stromal stem cells, mesenchymal stem cells, endothelial progenitor cells, red blood cells, white blood cells, fibroblasts, reticulacytes, adipose cells, and endothelial cells. 
     
     
         62 . The method according to  claim 58  further comprising combining the at least one of the first fraction or the second fraction with a carrier. 
     
     
         63 . The method according to  claim 62  wherein the carrier is selected from the group consisting essentially of collagen, hydrogel, a bioabsorbable polymer, a biopolymer, water, buffered solution, fibrin, concentrated fibrin, demineralized bone matrix, gelatin, porous calcium based ceramics, porous metal, synthetic fiber matrices, resorbable matrices, autogeneic tissue, allogenic tissue, xenogeneic tissue, and combinations thereof. 
     
     
         64 . A method for preparing a bone marrow aspirate concentrate and a peripheral blood concentrate, the method comprising:
 collecting a volume of bone marrow aspirate and a volume of peripheral blood from a patient to create a combined volume;   loading the combined volume of bone marrow aspirate and blood into a separator comprising two buoys and an extraction port, the buoys being operable to substantially physically separate the combined volume of aspirate and blood into three or more fractions and the extraction port operable to allow extraction of a fraction between the buoys without substantially commingling the fraction with the other fractions in the separator;   centrifuging the separator, wherein the buoys separate the combined volume to create the fraction between the buoys comprising a bone marrow aspirate concentrate and a peripheral blood concentrate fraction comprising red blood cells and white blood cells; and   withdrawing the fraction between the buoys of bone marrow aspirate concentrate and peripheral blood concentrate through the extraction port.   
     
     
         65 . The method according to  claim 64  further comprising applying the combined volume into a patient.