Abstract:
A container holding, and a method of storing, freeze-dried biological samples. The container includes a chamber having an upper portion and a lower portion. The chamber includes a wall, and the lower portion of the chamber is fluidly connected to the upper portion of the chamber such that, when liquid is received at the upper portion, the received liquid can pass to and accumulate in the lower portion. Further, the freeze-dried material is located in the lower portion, and the container includes a physical structure in the form of a stop protruding inwards from the wall, the physical structure being for inhibiting the freeze-dried material from moving from the lower portion of the chamber to the upper portion of the chamber.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a filing under 35 U.S.C. 371 of international application number PCT/EP2012/051098, filed Jan. 25, 2012, published on Aug. 2, 2012 as WO 2012/101150, which claims priority to 1101488.3 filed in Great Britain on Jan. 28, 2011. 
     FIELD OF THE INVENTION 
     The present invention relates to containers for holding and methods for storing freeze-dried biological samples. 
     BACKGROUND OF THE INVENTION 
     Biological samples, such as tissue or cellular extracts or lysates, enzymes, proteins, peptides, nucleic acids, fatty acids, glycerides, carbohydrates, oligosaccharides and saccharides, are commonly prepared at a first site, stored in a container, and transported to a second site for processing. One example of this is in diagnostic devices and/or in DNA/RNA amplification processes. This may require that the biological sample, which may be an enzyme such as a polymerase or reverse transcriptase, is stored for significant periods of time in the container and/or subject to vibrations and/or other external forces during transportation. 
     A convenient way of supplying such biological samples is to freeze-dry the biological sample in the container prior to transportation. The container is typically a tube of cylindrical cross-section having a single internal chamber. The freeze-drying process typically involves freezing a liquid sample, reducing the ambient pressure, and then gradually adding enough heat to allow sublimation of the water contained within the frozen liquid. The result is a coagulated, dehydrated sample formed at the bottom of the container; the container is then sealed and the biological sample (which may contain an enzyme) is stored, and transported, in this freeze-dried state. 
     When the biological sample is subsequently required for processing, it can be reconstituted by adding water to the freeze-dried material. The amount of biological sample dissolved for a given volume of water added can be critical; for example, if the amount of biological sample dissolved is too small, the strength of the solution may be insufficient. 
     However, it has been observed that when the freeze-dried material is supplied in existing tubes, some of the freeze-dried material remains un-dissolved when water is subsequently added. This can result in a solution of insufficient strength, as mentioned above. 
     Furthermore, biological samples freeze-dried, stored and transported in this way are often of high value; accordingly, un-dissolved biological sample is wasteful and can increase costs. 
     It is an object of the present invention to at least mitigate some of the problems of the prior art. 
     SUMMARY OF THE INVENTION 
     In accordance with a first aspect of the present invention, there is provided a container holding a freeze-dried material comprising a biological sample, the container comprising a chamber having an upper portion and a lower portion, the chamber comprising a wall and the lower portion being fluidly connected to the upper portion such that, when liquid is received at the upper portion, the received liquid can pass to and accumulate in the lower portion, wherein the freeze-dried material is located in said lower portion, and the container comprises a physical structure protruding inwards from the wall, the physical structure being for inhibiting the freeze-dried material from moving from the lower portion of the chamber to the upper portion of the chamber, and wherein the freeze-dried material defines a first cross-section and the physical structure comprises a stop extending inwards from an internal wall of said chamber, thereby defining a second cross-section, the second cross-section having a dimension smaller than a corresponding dimension of the first cross-section. 
     Since the freeze-dried material is inhibited from moving out of the lower portion of the chamber, it can be contained within a given volume, ensuring that when the material is subsequently reconstituted by the insertion of a liquid into the container, all of the freeze-dried material can be dissolved in the liquid, ensuring that the reconstituted solution is of the required strength. Further, since a physical structure is used to prevent the movement of the freeze-dried material, the use of a chemical, such a bonding agent, which may interfere with the composition of the reconstituted material can be avoided. 
     In addition, because the chamber has upper and lower portions, with the freeze-dried material being kept in the lower chamber, the freeze-dried material can be kept away from any seal applied to the upper portion; this avoids the contamination which may result from such contact. 
     Alternatively or additionally, in some embodiments the freeze-dried material defines a first cross-section and the physical structure comprises a stop extending inwards from an internal wall of said chamber, the stop defining a second cross-section, the second cross-section having a dimension smaller than a corresponding dimension of the first cross-section. The stop may define a boundary between the upper portion of the chamber and the lower portion of the chamber. The stop may thus define a volume of the container within which the freeze-dried material is maintained. In some embodiments, the stop comprises a collar that is integrally formed with the chamber. Alternatively, the stop may comprise a movable insert, the movable insert being movable within said chamber, thereby varying the extent or position of said boundary. Using a movable collar enables the volume of the chamber within which the freeze-dried material is maintained to be varied. 
     Preferably, the container comprises a physical structure comprising a textured surface, which further increases the bonding surface area between the freeze-dried biological sample and the chamber. 
     The container may be of a plastics material. 
     The chamber may have a substantially circular cross-section. 
     Preferably, the upper portion extends from a first end of the container, and the container comprises a seal at the first end. 
     In accordance with a second aspect of the present invention, there is provided an apparatus storing a freeze-dried material comprising a biological sample, the apparatus comprising; a plurality of containers according to the first aspect of the invention; and a base plate supporting the plurality of containers. 
     The apparatus may comprise a base plate of a plastics material. 
     In accordance with a third aspect of the present invention, there is provided a method of storing a biological sample, comprising: providing a container, the container comprising a chamber having an upper portion and a lower portion, the chamber comprising a wall and the lower portion being fluidly connected to the upper portion such that, when liquid is received at the upper portion, the received liquid can pass to and accumulate in the lower portion; inserting a material containing a biological sample into the chamber and accumulating the liquid biological sample in the lower portion of the chamber; and performing a freeze-drying process on the material, whereby a freeze-dried material comprising said biological sample is formed in the lower portion, wherein the container comprises a physical structure protruding inwards from the wall, the physical structure being for inhibiting the freeze-dried material from moving out of the lower portion, and wherein the freeze-dried material defines a first cross-section and the physical structure comprises a stop extending inwards from an internal wall of said chamber, thereby defining a second cross-section, the second cross-section having a dimension smaller than a corresponding dimension of the first cross-section. 
     In accordance with a fourth aspect of the present invention, there is provided a container holding a freeze-dried biological sample, the container comprising a chamber having an upper portion and a lower portion, the lower portion comprising a wall and being fluidly connected to the upper portion such that, when liquid is received at the upper portion, the received liquid can pass to and accumulate in the lower portion, wherein the freeze-dried biological sample is located in said lower portion, and the wall comprises a rough surface to which the freeze-dried biological sample is bonded, whereby the freeze-dried biological sample is prevented from moving from the lower portion to the upper portion. 
     A further aspect of the invention provides the use of the container of the first aspect, and/or the array of the second aspect, and/or the container of the fourth aspect for processing a biological sample. 
     Further features and advantages of the invention will become apparent from the following description of preferred embodiments of the invention, given by way of example only, which is made with reference to the accompanying drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  shows a cross-section view of a container holding a freeze-dried material; 
         FIG. 2 a    shows a perspective view of another container for holding a freeze-dried material; 
         FIG. 2 b    shows orthographic projections of the container of  FIG. 2 a    holding a freeze-dried material; 
         FIG. 3 a    shows a perspective view of a container for holding a freeze-dried material comprising a biological sample according to a first embodiment of the present invention; 
         FIG. 3 b    shows orthographic projections of a container holding a freeze-dried material comprising a biological sample according the first embodiment; 
         FIG. 4  shows a perspective view of a container for holding a freeze-dried material comprising a biological sample according to a second embodiment of the present invention; 
         FIG. 5 a    shows a perspective view of an array of containers for holding a freeze-dried material comprising a biological sample according to embodiments of the present invention; 
         FIG. 5 b    shows a perspective view of an array of containers for holding a freeze-dried material comprising a biological sample according to embodiments of the present invention; 
         FIG. 5 c    shows a plan view of an array of containers for holding a freeze-dried material comprising a biological sample according to embodiments of the present invention; 
         FIG. 5 d    shows a side view of an array of containers for holding freeze-dried biological samples according to embodiments of the present invention; 
         FIG. 6 a    shows a perspective view of an alternative arrangement in which there is no physical structure holding a freeze-dried material comprising a biological sample; and 
         FIG. 6 b    shows an orthographic projection of an alternative arrangement in which there is no physical structure holding a freeze-dried material comprising a biological sample. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     In  FIG. 1 , a container in the form of a tube  100  is provided, the tube  100  comprising a chamber  102  having a wall  104 , an upper portion  106 , and a lower portion  108 , the lower portion  106  holding a freeze-dried material comprising a biological sample; the freeze-dried material is hereinafter referred to as a cake  110 . Typically, the cake  110  is in a disc-shaped form. The tube  100  comprises a physical structure  112  to inhibit movement of the freeze-dried biological sample from the lower portion  108  of the chamber  102  to the upper portion  106  of the chamber  102 . The tube  100  comprises a first end  114 , which may initially be open in order to enable insertion of the material prior to freeze-drying, and subsequently closed with a seal (not shown). 
     The external dimensions of the tube  100  may be fixed at an industry standard for ease of handling, for example, automated handling by existing laboratory equipment. Typically, the tube  100  is 8 mm in diameter and 18.3 mm long. Typically, the internal dimensions of the tube are large enough in both diameter and depth to accommodate standard dispensing tools, for example pipetting needles in automated dispensing systems; this has the advantage that there is no need for readjustment or calibration of existing tools. 
     The biological sample may comprise an enzyme, such as a polymerase, reverse transcriptase or any other enzyme, blood, tissue, serum or any other biological substance. 
     In  FIGS. 2 a  and 2 b   , a tube  100   a  is provided in which a physical structure  112  in the form of protrusions, herein referred to as fins  200 , extends inwards from the wall  104   a  of the chamber  102   a , and extends longitudinally along a direction parallel to the axis L of the tube  100   a.    
       FIG. 2 a    shows the tube  100   a  prior to insertion of the biological sample. The tube  100   a  is initially open at a first end  114   a , from which the upper portion  106   a  extends. During preparation, a material (typically in liquid form) containing the biological sample, is inserted into the chamber  102   a  via the first end  114   a  and allowed to accumulate in the lower portion  108   a . A freeze-drying process is then performed so that the material is dehydrated in the lower portion  108   a , and in and around the spaces between the fins  200 , thereby forming the cake  110  shown in  FIG. 2 b   . The freeze-drying process bonds the cake  110  to the fins  200 . The additional contact area between the cake  110  and the internal surface of the tube  100   a  provided by the fins  200  increases the strength of the bond between the cake  110  and the tube  100   a , thereby increasing the ability of the tube  100   a  to hold the freeze-dried cake  110  in place. Further, the shape of the freeze-dried cake  110  comprises recesses into which the fins  200  fit. This interlocking fit between the freeze-dried cake  110  and the fins  200  prevents rotation of the freeze-dried cake  110  about the central longitudinal axis L of the tube  100   a , further reducing the likelihood of detachment. 
     The first end  114   a  may be sealed, subsequent to insertion of the material that is to be freeze-dried with a seal (not shown). The seal, which may be an impermeable seal, may be made of flexible foil, polymer laminate and/or any other suitable material, and may be held in place with a permanent or semi-permanent adhesive. When the freeze-dried material is required for subsequent processing, the seal may be removed, or penetrated by a dispensing needle, for example. Since the cake  110  is kept in the lower portion  108   a  of the chamber  102   a , it is kept away from the seal. This is advantageous because contact with the seal can cause contamination of the freeze-dried biological sample. 
     When the biological sample is subsequently reconstituted, by inserting, for example, water into the tube  100   a , provided that sufficient volume of water is inserted to fill the lower portion  108   b , it can be ensured that substantially all of the cake  110  is dissolved in the water. 
       FIGS. 3 a  and 3 b    show an embodiment of the present invention in which the tube  100   b  comprises a stop in the form of a collar  300  extending inwards from the wall  104   b  of the chamber  102   b .  FIG. 3 a    shows the tube  100   b  prior to insertion of the biological sample. As was described above in relation to  FIGS. 2 a  and 2 b   , the tube  100   b  is initially open at a first end  114   b , from which the upper portion  106   b  extends. 
     During preparation, a material (typically in liquid form) containing the biological sample, is inserted into the chamber  102   b  via the first end  114   b  and allowed to accumulate in the lower portion  108   b . A freeze-drying process is then performed so that the material coagulates in the lower portion  108   b , below the collar  300 , thereby forming the cake  110  shown in  FIG. 3 b   , the cake  110  being bonded to the wall  104   b  of the chamber  102   b.    
     The cake  110  is formed in the lower portion  108   b  below the collar  300  such that it has a larger external diameter than the internal diameter of the collar  300 . In this way, the cake  110  is inhibited from moving through the aperture formed by the collar  300  and is kept in the lower portion  108   b  of the tube  100   b . Thus, the position of the collar  300  defines the boundary between the upper portion  106   b  and the lower portion  108   b , and the volume of the lower portion  108   b  within which the cake  110  is held. When the biological sample is subsequently reconstituted, by inserting, for example, water into the tube  100   b , provided that sufficient volume of water is inserted to fill the tube  100   b  up to or higher than the level of the collar  300 , it can be ensured that substantially all of the cake  110  is dissolved in the water. 
     The first end  114   b  may be sealed, subsequent to insertion of the material that is to be freeze-dried with a seal as described above in relation to  FIGS. 2 a    and  2   b.    
     Typically, the collar  300  comprises a substantially semicircular rib with a radius of 0.24 mm running parallel to, and 4.2 mm above, the base of the chamber  102   b  of the tube  100   b.    
     The collar  300  described in this example is integrally formed with the wall  104   b  of the tube  100   b . However, in some embodiments, a collar is provided by a separate component which is inserted into the tube  100   b . This separate component may take the form of a ring, and may be held in place by an interference fit. The position separate component within the tube may be adjustable, allowing adjustment of the boundary between the upper portion  106   b  and the lower portion  108   b  of the chamber  102   b  and, therefore, the volume of the lower portion  108   b.    
     In one embodiment, shown in  FIG. 4 , rather than using a physical structure  112  to inhibit movement of the cake, the surface of the internal walls  104   c  of the lower portion  108   c  of a tube  100   c  comprises a textured portion  400  to improve the adhesion of the freeze-dried cake  110  onto the internal walls  104   c  of the tube  100   c . The textured portion  400  effectively increases the contact area between the freeze-dried cake  110  and the wall  104  of the tube  100   c . The textured portion  400  may comprise a regular pattern of surface features, such as a knurled surface, or may comprise a rough surface with randomly varying surface features. The additional adhesion provided by the increased surface area increases the force required to detach the cake  110  from the tube  100   c  and, therefore, reduces the likelihood of detachment. The cake  110  may be formed using a freeze-drying process as described above in relation to  FIGS. 2 a    to  3   b.    
     In this embodiment no other physical structures  112  are present in the lower portion  108   c  of the tube  100   c  and the freeze-dried cake  110  is held in place solely by adhesion to the interior walls of the tube  100   c . However, it will be understood that the textured portion  400  may be used in combination with any of the physical structures  200 ,  300  in the embodiments described above, or indeed with any other form of physical structure for inhibiting the freeze-dried material from moving from the lower portion  108  of the chamber  102  to the upper portion  106  of the chamber  102 . For example, the fins  200  described above in relation to  FIGS. 2 a  and 2 b    may themselves be textured in order to further increase the strength of the bond between the cake and the fins  200 ; in the embodiment described above in relation to  FIGS. 3 a  and 3 b   , the textured portion could be positioned in the lower portion  108   b  below the collar  300 . 
     Any of the tubes  100  described above may be used as individual containers or may be arranged, as in  FIG. 5 a   , in an array  500  for subsequent parallel or batch processing. In the embodiment shown, the tubes  100  are arranged in an 8×12 rectangular array on a base plate  502  with external dimensions corresponding to the SBS industrial standard footprint. Typically, the base plate  502  is 127.76 mm long and 85.48 mm wide. 
     The array  500  may comprise an impermeable seal  504 , which covers the open first ends  114  of each of the tubes  100  as shown in  FIG. 5 b   . In this example, the seal  504  may comprise a continuous sheet in contact with each of the tubes  100 ; alternatively, the tubes could be individually sealed, as described above. The seal  504  may be held in place with a permanent or semi-permanent adhesive. The seal  504  may be made from a flexible foil/polymer laminate or any other suitable material. The seal  504  may be penetrated by a dispensing needle or removed from the tubes  100  prior to use by breaking the semi-permanent adhesive bond. 
     The base plate  502  of the array  500  may comprise identifying grid-coordinates  506 , as shown in  FIG. 5 c   , correlating with the positions of each of the tubes  100  to enable identification and addressing of individual samples. 
     The base plate  502  of the array  500  may comprise an identification tag  508  comprising coded computer readable identification information, as shown in  FIG. 5 d   . The tag  508  may comprise a barcode  508   a  that can be scanned and compared with a database of sample identification codes; other types of tag  508  may be used, for example an RFID tag. 
     Crush ribs may be added to the external walls of the tube  100  to increase the structural strength of the tube  100  without increasing the overall external dimensions of the tube  100 . 
     The tube  100  and array  500  may comprise a chemically and biologically inert material that can withstand temperatures in the range −40° C. to 50° C. or more. Suitable materials include polycarbonate, polystyrene, or polypropylene. The tube  100  may be manufactured using an injection moulding process or by any other appropriate method. The array  500  may be manufactured as a single component using an injection moulding process or the tubes  100  and the base plate  502  may be manufactured as discrete parts and assembled and fixed together by, for example, an ultrasonic weld or by any other appropriate method. 
       FIGS. 6 a  and 6 b    show an alternative tube  100   d  in which no physical structure  112  inhibits movement of the cake  110 . In this arrangement, the tube has a chamber  102   d  having a volume arranged to be significantly lower than the external volume of a tube  100   d  but not significantly larger than the volume of liquid required to reconstitute the cake  110 . This allows the external dimensions of the tube  100   d  to be arranged to, for example, satisfy an industry standard, but the internal volume  102   d  to be reduced, thereby reducing the volume within which the cake  110  can move. Typically, while the external length of the tube  100   d  remains at 18.3 mm, the internal depth of the tube  100   d  is reduced to 6.9 mm. The effect of this is to reduce the risk of the freeze-dried cake  110  occupying a volume that is not subsequently filled with a liquid during subsequent reconstitution of the biological material (for example, an enzyme). It will be understood that this arrangement may be used as an individual container or in an array  500 , as described above. 
     Embodiments of the present invention are typically to be used for DNA or RNA amplification procedures such as polymerase chain reaction (PCR) that have common application in a variety of fields including molecular biology, medicine and forensic science. 
     The above embodiments are to be understood as illustrative examples of the invention. Further embodiments of the invention are envisaged. For example, although in the embodiment described above with reference to  FIGS. 2 a  and 2 b   , there is no physical indication of a boundary between the lower portion  108   b  and the upper portion  106   b , in some embodiments an indication of the boundary may be provided, for example in the form of a mark or protrusion on the wall  104   b  of the chamber  114   b.    
     It is to be understood that any feature described in relation to any one embodiment may be used alone, or in combination with other features described, and may also be used in combination with one or more features of any other of the embodiments, or any combination of any other of the embodiments. Furthermore, equivalents and modifications not described above may also be employed without departing from the scope of the invention, which is defined in the accompanying claims.