Abstract:
In a general aspect, an apparatus for washing biological material is provided which includes an outer sleeve, and an inner sleeve disposed within the outer sleeve. The outer sleeve has an open end for receiving the biological material, and an opposed closed end. The inner sleeve is detachably and slidably positioned within the outer sleeve, and includes a first port for receiving and dispensing washing liquid, a second port defining a liquid flow path between an interior of the inner sleeve and an interior of the outer sleeve, and a filter disposed in the second port. The filter allows passage of washing liquid but not passage of the biological material therethrough. A chamber, formed between the filter and the closed end of the outer sleeve, is configured to permit flow of washing liquid via the filter while retaining the biological material therein.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of Taiwanese Application No. 96123957 filed Jul. 2, 2007. The subject matter of this priority document is incorporated by reference herein. 
     BACKGROUND 
     Advances in medical technologies have offered new promises in the field of tissue regeneration. Recently, for example, bioengineered cartilage tissue has been surgically implanted in patients to repair cartilage in injured knees. In some situations, to engineer such an implant, primary cells are harvested from a donor tissue and cultured on scaffolds to form new tissues. During this process, harvested and cultured cells/tissues are frequently washed and filtered to remove chemical reagents and prevent contamination. Conventional washing apparatus include laboratory centrifuges that use centrifugal forces to separate tissue/cell from mixtures. However, laboratory centrifuges are not suitable for use in operating rooms. 
     SUMMARY 
     In one aspect, in general, an apparatus for washing biological material is provided which includes an outer sleeve, and an inner sleeve disposed within the outer sleeve. The outer sleeve has an open end for receiving the biological material, and an opposed closed end. The inner sleeve is detachably and slidably positioned within the outer sleeve, and includes a first port for receiving and dispensing washing liquid, a second port defining a liquid flow path between an interior of the inner sleeve and an interior of the outer sleeve, and a filter disposed in the second port. The filter allows passage of washing liquid but not passage of the biological material therethrough. A chamber, formed between the filter and the closed end of the outer sleeve, is configured to permit flow of washing liquid via the filter while retaining the biological material therein. 
     Embodiments may include one or more of the following features. 
     The outer sleeve includes a first sleeve piece and a second sleeve piece detachably connected to the first sleeve piece. The inner sleeve includes a resilient member that sealingly engages an inner peripheral surface of the outer sleeve. The resilient member includes an annular rubber member. 
     The outer sleeve comprises a base member and an extension member. The base member has an open end and an opposed closed end, and the extension member has open, first and second ends which are respectively opposed. The first end of the extension member is configured to detachably connect to the open end of the base member. The second end of the extension member is configured to detachably connect to another extension member. 
     The first port of the inner sleeve includes a stop portion, the stop portion configured to limit relative axial motion of the inner sleeve with respect to the outer sleeve. 
     The filter may be removably disposed in the second port. The filter may be a mesh filter having a plurality of mesh openings. The size of the plurality of mesh openings ranges from 100 μm to 3000 μm in diameter. Alternatively, the filter may be a membrane filter having a plurality of membrane openings. The size of the plurality of membrane openings ranges from 5 μm to 20 μm in diameter. 
     In another aspect, in general, a method of washing biological material is provided. The method includes the following steps: Providing biological material; placing the biological material into a chamber; and driving washing liquid in and out of the chamber through a filter by expanding and contracting the chamber, while retaining the biological material within the chamber. 
     Embodiments may include one or more of the following additional steps: Providing said washing liquid inside the chamber; discharging said washing liquid from the chamber through the filter; providing fresh washing liquid to the chamber after discharging said washing liquid; agitating the washing liquid within the chamber; and collecting said biological material after discharging said washing liquid. 
     In some embodiments, the method further includes one or more of the following steps: Providing an outer sleeve having an open end for receiving the biological material and an opposed closed end; and providing an inner sleeve detachably and slidably positioned within the outer sleeve. In some embodiments, the inner sleeve includes a first port for receiving and dispensing said washing liquid, and a second port defining a liquid flow path between an interior of the inner sleeve and an interior of the outer sleeve. In addition, the filter is disposed in the second port, and the chamber is formed between the filter and the closed end of the outer sleeve. 
     Embodiments may further include the following feature. The outer sleeve includes a first sleeve piece and a second sleeve piece detachably connected to the first sleeve piece, and the method includes the further method step of detaching the first sleeve piece from the second sleeve piece to collect said biological material. 
     Other features and advantages of the invention are apparent from the following description, and from the claims. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a sectional view of an apparatus for washing biological material. 
         FIG. 2A  is a perspective view of the outer sleeve of the apparatus shown in  FIG. 1 . 
         FIG. 2B  is a sectional view of the outer sleeve of  FIG. 2A . 
         FIG. 3A  is a perspective view of the inner sleeve of the apparatus shown in  FIG. 1 . 
         FIG. 3B  is a sectional view of the inner sleeve of  FIG. 3A . 
         FIG. 4  is a flow chart illustrating a method of washing biological material using the apparatus shown in  FIG. 1 . 
         FIGS. 5A and 5B  depict exemplary operation modes of the washing apparatus. 
     
    
    
     DETAILED DESCRIPTION 
     Referring to  FIG. 1 , in a preferred embodiment, a washing apparatus  100  includes an inner sleeve  20  detachably and slidably positioned in an outer sleeve  10 . The inner sleeve  20  and the outer sleeve  10  are elongate tubular members, and in some embodiments are circular in cross section. 
     Referring to  FIGS. 2A and 2B , the outer sleeve  10  includes a first sleeve piece  11  and a second sleeve piece  12 . The second sleeve piece  12  serves as a base member, and includes an open upper end  18 , and a closed lower end  19 . The terms “upper” and “lower” are used here and throughout this document for descriptive purposes rather than to imply any absolute relative orientation. The first sleeve piece  11  serves as an extension member, and includes opposed upper  14  and lower  15  ends. Both the upper and lower ends  14 ,  15  are open. The opening in the upper end  14  is identified as an opening  13 . The lower end  15  of the first sleeve piece  11  is detachably connected to the open upper end  18  of the second sleeve piece  12 . 
     In some embodiments, the first and second sleeve pieces are connected, e.g., by threads, although it is within the scope of the invention to detachably connect the first and second sleeves  11 ,  12  by other conventional means, such as press-fit engagement. In some embodiments, the outer periphery of the upper end  18  of the second sleeve piece  12  is provided with threads  28  configured to cooperatively engage complementary threads  27  formed on the inner periphery of the lower end  15  of the first sleeve piece  11 . In some embodiments, the outer periphery of the upper end  14  of the first sleeve piece is provided with threads  26  configured to cooperatively engage complementary threads of an additional extension member (not shown). 
     The outer sleeve  10  is configured to receive, through the opening  13 , biological material that needs to be washed. When washing is completed, the first sleeve piece  11  can be detached from the second sleeve piece  12  in which the biological material is collected. 
     Referring to  FIGS. 3A and 3B , the inner sleeve  20  has a first port  22  formed in an upper end  44  thereof, and a second port  23  formed in a lower end  42  thereof. The first port  22  is configured to receive washing liquid (e.g., saline), while the second port  23  defines a flow path for liquid exchange between the interiors of the inner and outer sleeves  20  and  10 . The upper end  44  of the inner sleeve  20  includes a stop member  25 . The stop member  25  also serves as a handle. 
     The stop member  25  is an outwardly extending protrusion having an outer dimension that is greater than the dimension of the opening  13  in the upper end of the outer sleeve  10 . The stop member  25  determines a maximum distance that the inner sleeve  20  is allowed to travel within in the outer sleeve  10 . The inner sleeve  20  further includes a tubular body  21  extending from the stop member  25 . In some embodiments, the tubular body  21  has an axial dimension that is less than that of the outer sleeve  10 , so that when the inner sleeve  20  is inserted into the outer sleeve  20  to the extent permitted by the stop member  25 , the lower end  42  of the inner sleeve  20  is spaced apart from the closed lower end  19  of the outer sleeve. That is, a chamber  50  is formed within the outer sleeve between the lower end  42  of the inner sleeve  20  and the closed lower end  19  of the outer sleeve, in which the biological material may be collected. Thus, the stop member  25  is provided so as to limit the extent to which the inner sleeve moves relative to the outer sleeve  10 , whereby mishandling of the apparatus  100  that may damage the biological material is avoided. 
     The chamber  50  varies in size based on the relative positions of the inner and outer sleeves  10 ,  20 . 
     The inner sleeve  20  further includes a filter structure  32  disposed in the second port  23 . Thus, an upper boundary of the chamber  50  is provided by the filter structure  32 . Biological material  40  can be placed in the outer sleeve  10  and confined in the chamber  50  by the filter  30  during washing process (see  FIG. 5A ). For purposes of this disclosure, biological material  40  may include, but is not limited to, one or more of cells or lysates thereof, and tissues. 
     In some embodiments, a resilient member  24  is provided at the lower end  42  of the inner sleeve  20  that sealingly engages an inner peripheral surface of the outer sleeve  10 . The resilient member  24  is affixed to the outer peripheral surface of the inner sleeve  20 , and may consist of, for example, an annular rubber member (e.g., an o-ring). The resilient member  20  serves to seal the space between the inner peripheral surface of the outer sleeve  10  and the outer peripheral surface of the inner sleeve  20 , whereby washing fluid and biological material is prevented from exiting the chamber  50  other than through the filter  30 . 
     The filter structure  32  disposed in the second port  23  defines a liquid flow path between the interiors of the inner and outer sleeves  20 ,  10 . In some embodiments, the filter structure  32  includes a filter  30  affixed to and sandwiched between a wire mesh member  34  and support members  29  formed in the second port  23  ( FIGS. 3B ,  5 A, and  5 B). In other embodiments, the filter structure  32  includes a filter  30  affixed to and sandwiched between two wire mesh members (not shown). This arrangement provides additional support to the filter  30  to prevent filter breakage or deformation during the washing process. In still other embodiments, the filter  30  is mounted on a support structure (e.g., a wire mesh member) that is detachable from the second port  23  to allow reuse of the washing apparatus with replacement filters (not shown). 
     Depending on the implementation, various filters may be employed in the filter structure  32  of washing apparatus  100 . For example, when washing tissues obtained from cartilage, a mesh filter having a pore size of 100 μm may be suitable, while a mesh filter having a pore size in the range from 150 μm to 3000 μm may be suitable for tissues obtained from an umbilical cord. When the biological material to be washed consists of cells rather than tissues, it is preferable to choose membrane filters having a smaller pore size. For example, when washing cells, a membrane filter having a pore size of 10 μm may be suitable. In some embodiments, the membrane filters may include pore sizes in the range of 5 μm to 20 μm. 
     Referring to  FIG. 4 , a flow chart  400  illustrates a method of washing biological material using the apparatus  100  described above. 
     Initially, in step  410 , biological material  40  (e.g., biological tissue) is placed in the outer sleeve  10  through the opening  13 . In step  420 , the inner sleeve  20  is inserted in the outer sleeve  10  to form the chamber  50 , in which the biological material  40  is retained during the washing process. In step  430 , the chamber  50  is filled with washing liquid  60  suitable for this biological material  40 . The washing liquid  60  is provided through the first port  22  of the inner sleeve  20 . One example of a commonly used washing liquid  60  includes phosphate buffer saline (PBS), but the invention is not limited thereto. 
     Next, the washing process  440  is performed by driving washing liquid  60  in and out of the chamber  50  through the filter  30 . This process  440  includes e.g., moving the inner sleeve  20  in a first axial direction D 1  to compress the chamber  50  (step  442 ), and moving the inner sleeve  20  in a second axial direction D 2  to expand the chamber (step  444 ), as depicted in  FIGS. 5A and 5B , respectively. These two steps can be performed multiple times in an alternating manner to remove, for example, undesired chemical reagents contained in the biological material. In some applications, gentle agitation of the mixture (step  446 ) also helps improve washing efficiency. Gentle agitation may be provided after one or both of the compression step (step  442 ) or the expansion step (step  444 ). 
     During washing, the biological material  40  is substantially retained in the chamber  50 , while washing liquid  60  enters and exits the chamber  50  through the filter  30 . The rubber resilient member  24 , affixed at the exterior of the inner sleeve  20 , prevents fluid exchange between inner and outer sleeves  20 ,  10  other than through the liquid flow path provided by the filter  30 . 
     After the washing process  440 , washing liquid  60  in the chamber  50  is discharged through the filter  30  and subsequently the first port  22  to a waste container, as shown in step  450 . Discharge of the washing liquid  60  may be achieved, for example, by inverting the apparatus  100 . 
     In some applications, it is desirable to wash the biological material  40  multiple times to ensure successful removal of chemical reagents and other undesired substances. In these cases, after discharging the washing liquid  60  (step  450 ), fresh washing liquid  60 ′ is supplied to the chamber  50  through the first port  22  (step  430 ) to repeat the washing process  440  and the discharging step  450 . 
     When biological material  40  has been sufficiently washed (step  460 ), the inner sleeve  20  is detached from the outer sleeve  10  where the biological material  40  is collected (step  470 ). In some applications, the first and second sleeve pieces  11  and  12  are further disassembled to provide easy access to the biological material  40 . 
     OTHER EMBODIMENTS 
     All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features. 
     From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.