Abstract:
A method for preserving blood platelets is described. The method uses 1%-3% gelatin in the platelet preservation medium, and storing the platelets in the preservation medium at temperatures below 0° C. and at least 70 atmospheres pressure for at least one day.

Description:
BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention relates to method and apparatus for preserving biological materials. More particularly, the invention relates to method and apparatus for preserving biological materials which employs a combination of a preservation solution, high pressure, and low temperature. 
     2. Description of Related Art 
     Whole blood and blood components including leukocytes, erythrocytes, thrombocytes, and plasma, need to be preserved and stored until needed for use. Skin and other tissue, kidneys, hearts, livers, and other organs need to preserved and stored until needed for use. These and other biological materials are preserved and stored using both freezing and non-freezing temperatures. 
     Freezing temperatures require the use of cryoprotectors such as DMSO (dimethyl sulfoxide) and Thrombosol™ to prevent damage to these biological materials. However, these cryoprotectors are cytotoxic, and typically leave a significant portion of the materials with either reduced or no functional ability. Moreover, cryoprotectors usually require time-consuming preparation, such as rinsing processes, before the materials can be used, and cryoprotector residues often still remain afterwards. Freezing processes can store erythrocytes for more than 30 days, and leukocytes up to 12 hours only. 
     Non-freezing temperatures limit the amount of time biological materials may be stored and preserved. In addition, non-freezing temperatures may require additional protocols. For example, platelets require mechanical agitation to prevent clumping, and can be stored this way for up to 5 days only. 
     What is needed are a method and apparatus for preserving biological materials for greater periods of time than with currently available methods and apparatus. 
     SUMMARY OF THE INVENTION 
     The present invention describes a method for preserving biological materials. The method includes applying a preservation solution to a biological material, and then storing the biological material at a pressure greater than 70 atm and a temperature less than 10° C. 
     The present invention also describes an apparatus for preserving biological materials. The apparatus includes a chamber having a mouth and a lip, the lip having an inside surface and a top surface, the inside surface and the top surface of the lip meeting at a first radius, the top surface of the lip having a channel. The apparatus also includes a cover configured to mate with and seal the chamber, the cover having a bottom surface, the bottom surface having a protrusion and a sealing structure, the bottom surface of the cover and the protrusion meeting at a second radius, the protrusion being inserted into the mouth of the chamber when the cover is mated with the chamber, the protrusion having a side surface, the side surface of the protrusion and the inside surface of the lip defining a first gap and being substantially parallel when the cover is mated with the chamber, the bottom surface of the cover and the top surface of the lip defining a second gap and being substantially parallel when the cover is mated with the chamber, the second gap having a length greater than a width of the first gap, the sealing structure being inserted into the channel of the lip when the cover is mated with the chamber. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     FIG. 1 shows a graph of In K versus temperature for rate of biochemical reaction. 
     FIG. 2 the phase transition lines for plasma and a 2.5% NaCl solution. 
     FIG. 3 shows another set of phase transition lines. 
     FIG. 4 shows a cross-sectional view of a biological material preservation apparatus of the present invention. 
     FIGS. 5A-5B shows a side cutaway and top views, respectively, of the chamber of the preservation apparatus. 
     FIG. 6 shows a side view of the cover of the preservation apparatus. 
     FIGS. 7A-7C show a cover retaining device of the preservation apparatus. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The method of the present invention is capable of preserving blood, tissue, organs, and other biological materials for greater periods of time than with currently available methods. This is achieved by using a combination of: (1) a preservation solution having a gel state at the low storage temperatures employed; (2) high storage pressures greater than 70 atm; and (3) low storage temperatures less than 10° C. 
     I. Introduction 
     Temperature is one of the most important parameters to be considered when storing living biological materials. When the temperature inside a cell drops too low, irreversible biochemical and structural changes occur. Several hundred biochemical reactions take place concurrently in the living cell. The rate of these biochemical reactions depends on several factors, including pressure, temperature, viscosity of the environment, pH, and concentrations of reactive molecules. 
     A metabolic process typically includes a series of intermediate processes, in which a substrate S is converted into a series of intermediate products X 1 , X 2 , X 3  . . . before being converted into a final product P. For each of these intermediate processes, the reactions may be catalyzed with different enzymes E o , E 1 , E 2  . . . : 
     
       
         
           E 
           o 
           E 
           1 
           E 
           2 
         
       
     
     
       
           S→X   1   →X   2   →X   3    . . . →P   Equation (1) 
       
     
     Under normal conditions, the volume of substrate S transformed per unit of time equals the volume of product P obtained per unit of time:                -            [   S   ]            t         =     +            [   P   ]            t                 Equation                   (   2   )                                  
     where [S] and [P] are the concentrations of substrate S and product P . The concentration of the intermediate products [X 1 ], [X 2 ], [X 3 ] under such conditions should also be constant:                       [     X   1     ]            t       =              [     X   2     ]            t       =              [     X   3     ]            t       =   0               Equation                   (   3   )                                  
     Therefore, for each intermediate product, its rate of formation equals its rate of transformation. The concentrations of each intermediate product may be expressed in terms of the rates of formation and transformation:                       [     X   3     ]            t       =       -            [     X   2     ]            t         =       K   2     ·     [     X   2     ]                 Equation                   (   4   )                                  
     where K 2  is the constant of rate reaction constant of transformation of product X 2  and formation of product X 3 . For steady state:                -            [   S   ]            t         =       +            [     X   1     ]            t         =       +            [     X   2     ]            t         =         +            [     X   3     ]            t                       …     =     +            [   P   ]            t                       Equation                   (   5   )                                  
     From the above it follows that: 
     
       
           K   1   ·[X   1   ]=K   2   ·[X   2 ] 
       
     
     
       
         [ X   1   ]:[X   2   ]=K   2   :K   1   Equation (6) 
       
     
     Therefore, the concentration of each intermediate product is determined by its rate constants of formation and transformation. 
     Temperature dependence is defined by constant K of the rate of chemical reaction to Arrenius: 
     
       
           K=A·e   −EIRT   Equation (7) 
       
     
     where 
     A is the constant coefficient in some temperature interval; 
     E is the activating energy of chemical reaction per 1 mol of the substance; 
     R is the universal gas constant; and 
     T is the absolute temperature. 
     For most biochemical reactions, E&gt;&gt;RT . Taking the natural logarithm of both sides of Equation (7) gives:                ln                 K     =       ln                 A     -     E   RT               Equation                   (   8   )                                  
     FIG. 1 shows a graph of In K versus temperature. From 30° C. to 37° C., A is constant. For different chemical reactions E and A are different. As temperature decreases, there is a misbalance of reactions rates and Equation (5) no longer holds. This means the intermediate product concentrations corresponding to each of the biochemical reactions begin to change. This begins breakdown of cell structures, including the cell membrane, and can end in cell death. 
     Chemical reactions are either exothermic or endothermic, i.e. they either give off or absorb energy. Reactions taking place during hydrolysis can release large amounts of energy. The oxidation of  1  mol of glucose releases 2883 kJ of energy. Should the biochemical reaction rates slow down too much, irreversible process begin to take place finally leading to total destruction of the cell. Therefore, coefficient A becomes a function of temperature T. 
     As the temperature drops below 20° C., the lipid bi-lay of the cell membrane undergoes a phase transmission from a colloid to a gel. The viscosity of a gel is much higher then that of its colloid. Consequently, rates of diffusion and active transportation of molecules through the cell membrane decrease sharply, resulting in a slowing down of the rate of biochemical reactions in a cell. As a result of the phase transformation of the cell membrane, the surface area of the lipid bi-lay surface and cell size reduce considerably due to the loss of water from the cell. 
     As the density of osmo-active substances increases, water molecules return to the cell thereby increasing osmotic pressure. Membrane tension reaches a critical point and may lose its barrier function. Membrane damage develops, resulting in morphological and structural changes, as well as loss of the ability for active adaptation. 
     As the temperature drops below 8° C., the cell cytoplasma undergoes a phase transformation into a gel. At this temperature, there is a sharp decrease in diffusion rate and active transportation of molecules, as well as in biochemical reaction rates. 
     As the temperature falls below −3° C., water crystallization begins to occur both inside and outside the cell. In the absence of cryoprotectors, water crystallization outside the cell leads to cell dehydration, decreased cell size, and increased concentrations of salt and other substances inside the cell. Water crystallization inside the cell results in structural cell membrane destruction. 
     In light of the above, the method and apparatus of the present invention seek to achieve the following: 
     1. Using a preservation solution which forms a viscous gel to mechanically suspend the biological materials. 
     2. Storing the biological materials at the lowest possible temperature while maintaining them in a liquid state. Under these conditions, the rate of biochemical reactions are relatively slow and therefore, the rates of change in the concentrations of intermediate products is small. 
     3. Slowly cooling solutions with platelets to allow free and safe water flow from the cell to prevent membrane tension from reaching a bursting point during the phase transmission from a colloid to a gel. On the other hand, the cooling rate should be high enough to prevent intermediate biochemical reactions from causing irreversible changes in cell structure. 
     II. Method 
     One embodiment of a method for preserving a biological material comprises: (1) applying a preservation solution to the biological material; (2) storing the biological material at a low temperature and a high pressure. 
     The preservation solution includes 1 to 3% gelatin. As it is cooled, the gelatin undergoes a phase transformation between 8° C. and 15° C. from a colloid to a gel. This gel suspends cells in the preservation solution. The gel reduces sedimentation and clumping of platelets. The gel also mechanically supports the cell membrane and reduces deformation of the membrane when the interior volume of the cell changes during the cooling process. The gel also lowers cell metabolism by decreasing exchange between the cell and its environment. 
     The preservation solution may also include sucrose, glucose, and/or sodium chloride. The sucrose repairs damage in the cell membrane caused by the cooling process. The glucose provides nutrients to sustain cell metabolism in the oxygen-poor conditions caused by the cooling process. Glycolysis produces 208 J/mol. The sucrose and glucose also bind water, thus promoting gel formation and inhibiting osmotic pressure build-up within the cell. 
     The sodium chloride prevents hemolysis by inhibiting the flow of water to the platelets during cooling. As the platelets are cooled below 20° C., the cytoplasma changes from a colloid to a gel, and free water leaves the cell. As the platelets are cooled even further, the hypertonic concentration of NaCl prevents water from reentering the platelets. The sodium chloride also lowers the freezing point of blood plasma by 2.5° C. 
     In one embodiment, the preservation solution includes 1.0 to 3.0% gelatin, 1.0 to 2.0% glucose, 1.0 to 3.0% sucrose, and 0.2 to 0.6% NaCl. In another embodiment, the preservation solution includes 2.9% gelatin, 0.44% sucrose, 1.17% glucose, and 0.49% NaCl. 
     The biological material is stored at a pressure greater than 70 atm and a temperature less than 10° C. In one embodiment, the biological material is stored at a pressure in the range of 70 to 1000 atm and a temperature in the range of −12° C. to 0° C. In another embodiment, the biological material is stored at a pressure in the range of 400 to 500 atm and a temperature in the range of −8° C. to −7° C. 
     FIG. 2 shows the phase transition lines for plasma and a 2.5% NaCl solution. At normal pressures, plasma freezes at −2.5° C. Plasma contains various chemical which lower the freezing point by interfering with the formation of the crystal lattice structure of ice. Cell structures can be cooled to −4° C. to −3° C. without water crystallization into cytoplasma. At normal pressures, the 2.5% NaCl solution freezes at −1.7° C. 
     FIG. 3 shows the phase transition lines for water and a 2.5% NaCl solution. The addition of NaCl to water as lowers the freezing point, and thus allows lower temperatures to be achieved for a given pressure. The method line shows one example of how a biological material may be subjected to a combination of high pressure and low temperature to prevent freezing. 
     III. Apparatus 
     FIG. 4 shows an assembled view of one embodiment of a biological material preservation apparatus  100  of the present invention. Preservation apparatus  100  includes a chamber  110  and a cover  130 . 
     FIGS. 5A-5B show side cutaway and top views, respectively, of chamber  110 . Chamber  110  includes a mouth  111  and a lip  112 . Lip  112  includes an inside surface  113  and a top surface  114 . Inside surface  113  and top surface  114  meet at a first radius r 1 . Top surface  114  includes a channel  115 . Channel  115  may have a sealing device  116  seated at a bottom of channel  115 , such as an O-ring or rubber gasket. Chamber  110  may be manufactured in different sizes to accommodate a platelet bag, blood donation bag, heart, liver, kidney, or other bags and biological materials. 
     FIG. 6 shows a cutaway view of cover  130 . Cover  130  is configured to mate with and seal chamber  110 . Cover  130  includes a bottom surface  131 . Bottom surface  131  includes a protrusion  132  and a sealing structure  133 . Bottom surface  131  and protrusion  132  meet at a second radius r 2 . Protrusion  132  is inserted into mouth  111  of chamber  110  when cover  130  is mated with chamber  110 . Protrusion  132  includes a side surface  134 . Side surface  134  of protrusion  132  and inside surface  113  of lip  112  define a first gap  140  and are substantially parallel when cover  130  is mated with chamber  110 . Bottom surface  132  of cover  130  and top surface  118  of lip  114  define a second gap  141  and are substantially parallel when cover  130  is mated with chamber  110 . Second gap  141  has a length greater than a width of first gap  140 . Sealing structure  133  is inserted into channel  115  of lip  112  when cover  130  is mated with chamber  110 . 
     Cover  130  may be made to be a spherical section, which allows cover  130  to be made lighter and with less material than a flat cover  130  without sacrificing strength. When preservation apparatus  100  is filled with, for example, saline solution and then cooled below the freezing point, ice will begin to form along the walls of chamber  110  and cover  130 . Ice will form in first gap  140  and second gap  141  and help to seal chamber  110 . Thus, the high pressures within chamber  110  are largely borne by this ice seal, thus minimizing the need to make channel  115 , sealing device  116 , and sealing structure  133  extremely robust and capable of withstanding such high pressures. Channel  115 , sealing device  116 , and sealing structure  133  only need to withstand pressures of up to  10  atm before the ice seal takes over. The sizes of first gap  140  and second  141  are not critical, but may be minimized so that ice fills them before the seal is subjected to pressures above 10 atm. In one embodiment, first gap  140  and second gap  141  may be less than 2.0 mm in width. Chamber  110  includes a suspension device  117  which prevents biological material or bag placed within pressure chamber from coming into contact with the walls of chamber  110 . Suspension device  117  may be a net, a platform, a spacer, or any other suitable device. 
     Cover  130  may be designed to be sealed to chamber  110  directly, or with the aid of a cover retaining device  135 . Cover retaining device  135  may be designed to allow cover  130  to be installed and removed quickly and easily. Cover retaining device  135  may be coupled to chamber  110  via a bayonet-style connection, threads, or any other suitable coupling method. Cover retaining device  135  may include a centering pin  136  to keep cover retaining device  135  centered or attached to cover  130 . Cover retaining device  135  may also include holes  137  to allow a wrench or other tool to be used with cover retaining device  135 . Cover retaining device  135  may be produced in two separated pieces to simplify manufacturing. FIGS. 7A-7C show cutaway and top views of a two-piece cover retaining device  135 . 
     Preservation apparatus  100  may include a pressure gauge  150  with an elastic membrane  151  placed within chamber  110 . Pressure gauge  150  may include a relief valve  152  which prevents pressure within preservation apparatus  100  from exceeding a predetermined maximum. 
     EXAMPLE 
     The following is one example of the method of the present invention as used to preserve blood platelets. Heparin may be used as an anticoagulant before this process is begun. 
     1. Mix the platelets with a preservation solution of 2.9% gelatin, 0.44% sucrose, 1.17% glucose, and 0.49% NaCl. 
     2. Seal the platelets and preservation solution into a storage bag, making sure that any air has been pumped out The storage bag may be any standard platelet storage bag such as a flexible silicone rubber bag. 
     3. Cool the platelets and preservation solution to 15° C. within 1 hour. 
     Continuous agitation is required until the preservation solution becomes a gel. 
     4. Cool the storage bag to 6° C. to 8° C. within 1 to 1.5 hours. 
     5. Cool the preservation apparatus to 6° C. to 8° C. 
     6. Insert the storage bag into the preservation apparatus using the suspension device. 
     7. Fill the preservation apparatus with a pressure transfer fluid of 2.5% NaCl solution. 
     8. Seal the preservation apparatus, making sure it is completely full and no air is trapped inside. 
     9. Cool the preservation apparatus to −7.5±0.2° C. within 1.5 to 2 hours. The pressure transfer fluid is a fluid which expands when cooled or frozen, and thus will be able to exert a pressure upon the bag within the substantially fixed volume of the preservation apparatus. With the 2.5% NaCl solution, the water will begin to freeze at the walls of the pressure chamber. As the ice is formed at the walls of the pressure chamber, the expansion will create the high pressures required within the preservation apparatus, which will be transferred by the unfrozen fluid immediately surrounding the storage bag to the storage bag. The NaCl lowers the freezing point of the pressure transfer fluid, thus allowing the low temperatures required to be achieved before the entire volume of the pressure transfer fluid becomes frozen. The preservation solution has a lower freezing point than the pressure transfer fluid. The pressure inside the preservation apparatus will rise to 500 atm. As ice begins to form, pressure within the preservation apparatus will increase because ice and water are essentially non-compressible. The relationship between temperature and pressure here is consistent and predictable. The combination of the preservation solution, the high storage pressure, and the low storage temperature allows the platelets to be stored for up to 15 days. Erythrocytes may be stored up to 30 days and leukocytes up to 22 days using this method. 
     10. When the platelets are needed for use, allow the preservation apparatus to thaw completely at room temperature, approximately 20° C., before opening the preservation apparatus. Because the components in the preservation solution are all nontoxic, the platelets may be used immediately without further preparation. 
     The foregoing description of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations will be apparent. It is intended that the scope of the invention be defined by the following claims and their equivalents.