System and method for extraction and cryopreservation of bone marrow

Abstract

Methods are provided for extracting bone marrow cells from bone obtained from deceased donors, for preparing the bone marrow for cryopreservation and for obtaining desired cells from cryopreserved and fresh bone marrow.

What is claimed is: 
     
         1 . A method for recovering cells from deceased donor bone marrow, comprising:
 obtaining bone from a deceased donor;   processing the bone to extract bone marrow cells from the bone;   obtaining a reduced density Ficoll solution having a density of 1.063-1.052 gm/mL;   introducing the reduced density Ficoll solution into a centrifuge tube to form a Ficoll gradient;   layering the extracted bone marrow cells over the Ficoll gradient in the centrifuge tube;   centrifuging the tubes containing the Ficoll gradient and bone marrow cells;   harvesting the buffy coat cells from within the centrifuge tubes; and   washing the harvested cells for subsequent use or processing.   
     
     
         2 . The method of  claim 1 , wherein the bone is a vertebral body. 
     
     
         3 . The method of  claim 1 , wherein the harvested cells are CD34+ cells. 
     
     
         4 . The method of  claim 1 , wherein the step of processing the bone comprises:
 cleaning the bone of soft tissue;   cutting the bone into pieces and then grinding the pieces;   filtering and rinsing the ground pieces of bone; and   centrifuging a suspension of the filtered and rinsed pieces of bone to concentrate bone marrow cells.   
     
     
         5 . The method of  claim 1 , wherein the step of obtaining a reduced density Ficoll comprises mixing Ficoll-Paque at a density of 1.077 g/mL with PLASMA-LYTE™ in a proportion to obtain a density of 1.063-1.052 g/mL. 
     
     
         6 . The method of  claim 1 , wherein the step of centrifuging the tubes includes centrifuging the tubes for 30 minutes at 400 g. 
     
     
         7 . The method of  claim 1 , wherein the step of washing the harvested cells includes washing the cells in phosphate-buffered saline (PBS) containing 0.5% human serum albumin (HSA) and 2 mM Ethylenediaminetetraacetic acid (EDTA). 
     
     
         8 . A method for obtaining bone marrow cells from deceased donor bone comprising:
 obtaining a bone from a deceased donor;   cleaning the bone of soft tissue;   grinding the bone into bone pieces;   filtering and rinsing the ground bone to produce a liquid composition;   centrifuging the liquid composition of the filtered and rinsed ground bone to concentrate bone marrow cells into a bone marrow cell composition; and   extracting the bone marrow cell composition into a sterile container.   
     
     
         9 . The method of  claim 8 , wherein the donor bone is one or more vertebral bodies and/or the ilium of the deceased donor. 
     
     
         10 . The method of  claim 8 , wherein the donor bone is freshly obtained and not frozen. 
     
     
         11 . The method of  claim 8 , wherein the donor bone is thawed after being frozen for transfer to a processing facility. 
     
     
         12 . The method of  claim 8 , wherein the step of cleaning the bone of soft tissue comprises:
 removing soft tissue from the bone using a tool; and then   submerging the bone in one or more solutions adapted to remove soft tissue and soft tissue cells from the bone.   
     
     
         13 . The method of  claim 12 , wherein the step of submerging the bone in one or more solutions comprising:
 submerging the bone in a bleach solution; and then   submerging the bone in a hydrogen peroxide solution.   
     
     
         14 . The method of  claim 12 , wherein the step of submerging the bone in one or more solutions includes;
 submerging the bone in a container;   detecting a level of foam within the container; and   repeating at least the step of submerging the bone in a hydrogen peroxide solution until no foam is detected.   
     
     
         15 . The method of  claim 14 , wherein an inert contrast dye is added to the hydrogen peroxide solution to enhance the visibility of any foam in the container. 
     
     
         16 . The method of  claim 8 , wherein the step of grinding the bone comprises:
 cutting the bone into fragments; and then   grinding the bone fragments in a bone grinder with a grind media.   
     
     
         17 . The method of  claim 16 , wherein the grind media comprises PLASMA-LYTE™ as a base with 10 U/mL heparin, 2.5% human serum albumin (HSA), and 3 U/mL Benzonase® reagent. 
     
     
         18 . The method of  claim 8 , wherein the step of filtering and rinsing the ground bone comprises:
 submerging the ground bone in grind media;   passing the ground bone and grind media through a series of sieves;   thereafter rinsing the sieves with grind media;   receiving the liquid composition passing through the sieves in a container; and then   transferring the liquid composition to a sterile container.   
     
     
         19 . The method of  claim 18 , wherein the series of sieves includes a first No. 40 sieve (425 μm) followed by a second No. 80 sieve (177 μm). 
     
     
         20 . The method of  claim 8 , wherein the step of filtering and rinsing the ground bone comprises:
 submerging the ground bone in grind media within a first collection bag;   suspending the first collection bag and connecting the bottom of the first collection bag to a series of in-line filters and a second collection bag; and   passing the contents of the first collection bag through the in-line filters into the second collection bag.   
     
     
         21 . The method of  claim 20 , wherein the series of in-line filters includes two filters having either a 200 μm filter or a 500 μm filter. 
     
     
         22 . The method of  claim 21 , wherein in a first pass of the contents of the first collection bag the two filters are 200 μm filters and in a second pass of the contents of the second collection bag into a third collection bag the two filters are 500 μm filters. 
     
     
         23 . The method of  claim 8 , wherein the step of filtering and rinsing the ground bone includes removing the fat content of the ground bone. 
     
     
         24 . The method of  claim 23 , wherein the step of removing the fat content of the ground bone includes:
 placing a suspension of the ground bone in a collection bag;   centrifuging the collection bag so that a fat layer is formed at the top of the collection bag when suspended; and   removing a pellet of the bone marrow from the bottom of the suspended collection bag.   
     
     
         25 . The method of  claim 24 , wherein prior to removing the pellet a clip is placed on the bag below the fat layer to pinch the bag and prevent passage of the fat layer as the pellet is removed. 
     
     
         26 . The method of  claim 8 , further comprising the step of cryopreserving the bone marrow cell composition. 
     
     
         27 . The method of  claim 26 , wherein the step of cryopreserving the bone marrow cell composition comprises:
 preparing a predetermined volume of freeze media as a solution of a rinse media and a cryopreservation composition; and   introducing the freeze media into the sterile container containing the bone marrow cell composition at a predetermined rate.   
     
     
         28 . The method of  claim 27 , wherein the sterile container contains 60-70 mL of bone marrow cell composition and the predetermined volume of freeze media is calibrated for the volume of bone marrow cell composition. 
     
     
         29 . The method of  claim 27 , wherein the predetermined rate is ten percent (10%) of the predetermined volume of the freeze media per minute. 
     
     
         30 . The method of  claim 27 , wherein the cryopreservation composition can be one or more compositions selected from group including: dimethyl sulfoxide (DMSO); 1, 2 propane diol; ethylene glycol; glycerol; foramamide; ethanediol or butane 2, 3 diol; hydroxyethyl starch (HES), Dextran, sucrose, trehalose, lactose, raffinose, Ribotol, Mannitol and polyvinylpyrrolidone (PVP). 
     
     
         31 . The method of  claim 27 , wherein the rinse media can be one or more compositions selected from the group including: PlasmaLyte; Isolyte; and IMDM. 
     
     
         32 . The method of  claim 27 , wherein the freeze media further includes oxyrase. 
     
     
         33 . A bone cutting tool comprising:
 a fixed handle having an end configured to be gripped by a user and an opposite jaw end, said fixed handle defining a bone engaging recess at said jaw end;   a lever handle pivotably connected at a first pivot to the fixed handle to pivot toward and away from the fixed handle and configured to be gripped by the user while gripping the fixed handle to successively pivot the lever handle toward the fixed handle;   a knife element pivotably connected at a second pivot to the fixed handle and including a knife edge facing said bone engaging recess at said jaw end of said fixed handle, the knife element including a ratchet component disposed between said fixed handle and said lever handle, said ratchet component including a plurality of teeth;   a pawl component pivotably connected at a third pivot to said lever handle and arranged to engage each of said plurality of teeth at each successive pivot of said lever handle to said fixed handle by the user,   wherein each of said first pivot includes an elongated pin passing through openings in said fixed handle and said lever handle, said second pivot includes an elongated pin passing through openings in the fixed handle and the knife element, and said third pivot includes an elongated pin passing through openings in the pivot handle and the pawl component, and   wherein each pin is removably retained within the respective first, second and third pivot by at least one removable retaining ring such that the fixed handle, pivot handle, knife element and pawl component can be readily disassembled for cleaning and re-assembled after cleaning.   
     
     
         34 . The bone cutting tool of  claim 33 , wherein:
 the knife element includes an integral link; and   the tool further includes a free link pivotably connected to the lever handle at a fourth pivot and pivotably connected to said integral link, said fourth pivot including an elongated pin passing through openings in the pivot handle and the free link for ready disassembly.   
     
     
         35 . The bone cutting tool of  claim 33 , wherein the fixed handle, knife element, pivot handle, pawl component and free link are formed of stainless steel and the surfaces thereof are passivated. 
     
     
         36 . The bone cutting tool of  claim 35 , wherein the stainless steel is a hardened stainless steel.