Abstract:
A orbital shaker for cell extraction includes a digestion chamber and structure for translating the digestion chamber. Structure is also provided for rotating the digestion chamber. A method of performing cell extraction is also disclosed.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS  
       [0001]    This application claims priority from U.S. Provisional Application No. 60/359,298, filed Feb. 22, 2002. The foregoing is incorporated herein by reference. 
     
    
     
       STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT  
         [0002]    Not applicable.  
         BACKGROUND OF THE INVENTION  
         [0003]    Scientists are currently researching possible applications for isolated cells from parent organs, such as the liver, spleen, kidney, adrenal, and pancreas. Some research that has been conducted on the clinical application of isolated cells has involved groups of cells called the Islets of Langerhans that have been isolated from the pancreas. An application for the Islet of Langerhans cells is as a treatment for diabetic patients. Patients with diabetes have Islets of Langerhans that do not function properly, and therefore, do not produce enough insulin. Some clinical research is aimed at developing a procedure for transplanting functioning Islets of Langerhans into diabetic patients to restore the insulin producing ability of the pancreas. Clinical research of such requires isolated Islet of Langerhans cells, but these cells must be isolated while still viable. Viable isolated cells are mostly obtained from organs of the very recently deceased. The apparatus and method for isolating the cells should be able to extract isolated cells with as little damage to the cells as possible.  
           [0004]    Many different methods and approaches have been attempted to isolate individual cells from their respective parent organs. Prior methods have produced isolated cells with some cell destruction. This cell destruction can result from the relatively severe mechanical stimulation that is used to isolate cells from an organ.  
           [0005]    One method that attempts to overcome the loss of damaged cells due to relatively severe mechanical stress is described in U.S. Pat. No. 5,079,160, to Lacy, et al. The method disclosed by Lacy, et al. comprises the steps of: placing an organ or a piece of an organ in a digestion chamber along with marble agitators; distending the organ or a piece of the organ with physiologically compatible medium containing a protease; continuously recirculating that medium; and separating the isolated cells. The marble agitators greatly increase the amount of undamaged cells obtained through isolation without reducing the quality of the isolated cells obtained by gently agitating the organ. Moreover, the marbles are an appropriate size, weight, and density for obtaining beneficial results as compared to other agitators of varying size, weight, and density which can cause severe mechanical disruption of the organ tissue resulting in some cells being destroyed.  
         SUMMARY OF THE INVENTION  
         [0006]    An orbital shaker for cell extraction, includes an engagement structure for engaging a digestion chamber; a translational drive structure for translating the digestion chamber; and rotational drive structure for rotating the digestion chamber.  
           [0007]    A method for cell extraction includes the steps of providing tissue in a digestion chamber; supplying a digestive medium to the digestion chamber; and using an orbital shaker apparatus to translate the digestion chamber while rotating the digestion chamber.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0008]    There is shown in the drawings embodiments which are presently preferred, it being understood, however, that the invention can be embodied in other forms without departing from the spirit or essential attributes thereof.  
         [0009]    [0009]FIG. 1 is a perspective view of a orbital shaker for cell extraction.  
         [0010]    [0010]FIG. 2 is a perspective view, with an external housing removed to show internal features.  
         [0011]    [0011]FIG. 3 is a front elevation view.  
         [0012]    [0012]FIG. 4 is a rear elevation view.  
         [0013]    [0013]FIG. 5 is a top plan view.  
         [0014]    [0014]FIG. 6 is a left side elevation view.  
         [0015]    [0015]FIG. 7 is a right side elevation view.  
         [0016]    [0016]FIG. 8 is a perspective view of a rotational stroke adjustment mechanism.  
     
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0017]    Organ and tissue dissociation procedures require a combination of enzymatic or chemical reagents in a solution (at a certain temperature that is optimal for the effect of the selected reagent) and mechanical forces, to enhance the tissue disaggregation and dispersion process into progressively smaller tissue fragments, clusters of cells and even single cell products.  
         [0018]    The mechanical action is generally performed by digestion chambers that contain digestion enhancers such as marbles. The organ or tissue is placed into the selected chamber with the digestion solution (e.g., containing collagenase or specific enzyme blends). In order to mix evenly the solution and evenly increase the temperature to the desired target range, as well as to enhance the mechanical tissue dispersion within the digestion chamber, a double action (rotational and translational), variable speed and variable rate shaker is used to replace the manual movement currently utilized in several tissue processing facilities, for example to process human pancreata for the production of human islets for transplantation or research applications.  
         [0019]    The invention has been developed to provide the necessary shaking of the chamber in a consistent and automated vertical and/or rotational manner during the isolation of beta cells from the digestion of a pancreas or of other cells from other tissues or organs. The double action (rotational and translational movement), variable speed and variable rate shaker replaces the existing shaking of the chamber by human hands which cannot provide the consistency and speed required to obtain more desirable results during the digestion of the pancreas. This shaker can perform virtually an unlimited combination of motion movements at different amplitudes and frequencies, making it an excellent machine to apply the different movements and strengths of mechanical forces that are needed during an organ and tissue dispersion process. This is particularly important because of the great variability in organ characteristics with age, conditions of the tissue and several other variables including but not limited to organ procurement, body mass composition of the donor, preservation solution, cold ischemia time before processing and the like. It is therefore critically important for the operator to be able to change the movement and the intensity of the mechanical enhancing process during tissue and organ digestion processes.  
         [0020]    The vertical motion is controlled by a straight line Watts mechanism driven by an electric motor. The rotational movement is controlled by a separate motor connected to several linkages. Both the amplitude of the translational and rotational movement can be adjusted individually depending on the required movement of the chamber throughout the progress of the digestion of the pancreas in the chamber.  
         [0021]    This ability to modify the amplitude, rate and combined (translational and rotational) movement of the shaker is very important even for an individual organ or tissue processing, at different times during the digestion process. For example in the case of pancreas digestion the movement is more gentle at the beginning of the process and increases in the final stages of the tissue dispersion procedure. These requirements also change when organs of different species are processed. For example, porcine islet separation from porcine pancreata requires a more gentle dissociation process compared to human pancreas processing.  
         [0022]    The translational motion can be in any direction. In one aspect, the translational motion is a reciprocal motion in a substantially vertical plane. The reciprocal motion can be substantially vertical.  
         [0023]    The rotational motion is imparted to the digestion chamber preferably while the digestion chamber is translating so as to provide optimal cell extraction.  
         [0024]    Structure is preferably provided for varying the translational and rotational speed and distance. In one aspect, the translational motion is between about 1″ and about 5″, preferably 1.8″-4.2″. The vertical oscillation frequency can be between 0 and 250 cycles per minute. The rotational motion can be between 0° and 270°. The rotational frequency can be between 0 and 60 cycles per minute. Other translational and rotational distances and frequencies are possible.  
         [0025]    The structure for imparting translational and rotational motion to the digestion chamber can be any suitable structure. The drive mechanism can be electric, mechanical, electromechanical, pneumatic, hydraulic, or other suitable structures. Electric solenoids can be designed to impart rotational and translational motion to the digestion chamber.  
         [0026]    An orbital shaker for cell extraction  10  is shown in FIGS.  1 - 7 . A digestion chamber  14  is provided for retaining the organ or other tissue from which cells are to be extracted. As is known in the art, the digestion chamber  14  can be provided with inlet and outlet ports for a digestive medium and other structure for facilitating the cell extraction process. A heating surface  18  can be provided for heating the medium by wrapping a supply conduit (not shown) around the heating surface  18  upstream of the connection to the digestion chamber  14 . The digestion chamber  14  is mounted an engagement arm  22  which can extend through a suitable opening  26  in the housing  30 . A chamber clamp  34  can be provided on the engagement arm  22  for engaging the digestion chamber  14 . The engagement clamp  34  permits removal of the digestion chamber  14  for servicing during the extraction process and for cleaning and sterilization.  
         [0027]    Translational motion can be imparted by suitable structure such as motor  40 . The translational motor  40  rotates plate  44  which causes arms  48  to pivot. Arms  48  are joined to auxiliary link  52  which is pivotally mounted on shaft  56 . Pivoting of the arms  48  causes the auxiliary link  52  to pivot. Auxiliary link  52  is pivotally joined to extension link  60  at a pivot  64 . Extension link  60  is joined to drive link  68  at a pivot  72 . Drive link  68  is pivotally connected to bracket  74  at a pivot  80 . Engagement arm  22  passes through an aperture in bracket  74 . Drive link  68  is pivotally mounted on support bar  84 . A long link  88  can be pivotally mounted to support bar  84  and joined to drive link  68  by connection member  92 . Long link  88  is connected to rear bracket  76 . Engagement arm  22  passes through an aperture in rear bracket  76 .  
         [0028]    Pivoting of the auxiliary link  52  causes reciprocating movement of the extension link  60  and drive link  68 , which in turn causes reciprocating movement of front bracket  74  and rear bracket  76 . Engagement arm  22  is thereby reciprocated in a translational movement.  
         [0029]    The translational frequency is controlled by electronically adjusting the speed of the motor  40 . The amplitude is varied by adjusting the effective length of the arm  48 . The oscillating block  205  is attached to fly wheel  209  of motor  40 . The effective length of arm  48  is changed by the rotation of screw  204  that in turn moves the motor  40  and oscillating block  205  towards or away from the pivoting point  207  of arm  48 . This will adjust the throw of auxiliary link  52 . The screw  204  is rotated by motor  206  that is electronically controlled from the control panel.  
         [0030]    It is sometimes preferable that the digestion chamber be subjected to rotational motion as well as translational motion. The engagement arm  22  and clamp  34  can also be rotated. This can be accomplished by suitable structure. A rotational motor  100  rotates disc  104 . A connector  108  is pivotally connected to a link  112  which is pivotally connected to a gear drive link  116 . Gear drive link  116  is connected to gear  120 . Gear  120  engages drive gear  124 . Rotation of drive gear  124  rotates pulley  128  causing belt  132  to rotate pulley  134 . Pulley  134  is joined to a shaft  138 , and rotation of the shaft  138  causes rotation of pulley  142  which is also mounted to shaft  138 . Support links  144 ,  146  can be pivotally mounted between shaft  138  and support shaft  150 . A member  155  can secure support link  144  and  146  together.  
         [0031]    Rotation of the pulley  134  under the influence of belt  132  causes rotation of pulley  142  and corresponding movement of belt  158 . Belt  158  extends to a pulley  162  that is connected to engagement arm  22 . Movement of the pulley  162  thereby causes rotation of the engagement arm  22  and engagement clamp  34 .  
         [0032]    The rotational frequency is controlled by electronically adjusting the speed of the electric motor  100 . The rotational amplitude is changed by the rotation of arm  208 , as shown in FIG. 8. Movement of rotating arm  208  downward from the position shown in FIG. 8 will cause link  210  to also move downward, which will reduce the stroke length. By rotating arm  208 , the pivot point of arm  210  is changed thereby changing the oscillating stroke. The rotation of arm  208  is accomplished by electric motor  212  that is controlled by the rocker switch  214  situated on the control panel  170 .  
         [0033]    Suitable controls can be provided through a control panel  170 . The control panel  170  can have controls  174  for translational stroke length and controls  176  for rotational stroke length. Control  180  can be provided to vary translational stroke frequency and control  184  can be provided to vary the rotational frequency. Controls  188  can be provided for adjusting the temperature of the heater  18 . A display  192  can be provided to monitor temperature within the chamber  14 . It will be apparent that the translational and rotational motion can be imparted to the digestion chamber  14  through a number of different mechanisms. In another embodiment, suitable solenoids can replace the mechanical linkages and pulleys to provide translational and rotational movement of the digestion chamber.  
         [0034]    This invention can be embodied in other forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be had to the following claims rather than the foregoing specification as indicating the scope of the invention.