Abstract:
A method of injecting cells into a biological tissue comprising thrusting injection needle ( 200 ) under microvibration into tissue ( 5 ) secured by suction to tissue suction means ( 210 ) and effecting injection of a cell suspension into the tissue through the needle. There is also provided an apparatus for cell injection into the biological tissue.

Description:
FILED OF THE INVENTION 
     This invention is in the field of regenerative medical technology in which the function of a particular organ or tissue of a patient is normalized by transplantation when the function is lost or otherwise in disorder. More particularly it relates to a method and system for injecting cells into biological tissues to be transplanted. 
     BACKGROUND ART 
     For regenerative treatments, native tissue segments of human or mammalian origin have been used clinically as such or after decellularizing or treating with cell-fixing agents such as glutraldehyde. It is advantageous in the regerative treatment to use a regeneratable hybrid tissue prepared by injecting autologous cells of the recipient into the tissue and allowing the cells to grow therein. This method may not only avoid recipient&#39;s immune response to the transplanted tissue but also promote autogenesis of the transplanted tissue. 
     In the preparation of the regeneratable hybrid tissues, a cell suspension is injected manually using a syringe. However, many transplatable tissues have elastically deformable surfaces and, therefore, it is not easy even for skilled operators to pierce the tissue with a syringe needle accurately and precisely in position. 
     A method and device adapted for injecting medical solutions and other liquid into such elastically deformable tissues is disclosed in JP-A-03322568 and JP-A-200146500. The method and device injects a liquid while vibrating the needle ultrasonically. 
     In the preparation of the regeneratable hybrid tissues, it is imperative to inject a cell suspension into the tissue in a volume in the order of microliters at many target positions space apart a distance in the order of micrometers with a distribution density which may often reach up to several thousands per square centimeter. It will be impossible in practice to prepare such a regeneratable hybrid tissue with the known method and device by simply vibrating the needle. 
     There is another problem associated with the known method and device. Regeneratable hybrid tissues are often prepared from pulsating mammalian heart walls or relatively thin blood vessel walls. Since these tissues may move back and forth in the direction perpendicular to the general plane of the tissue, it is difficult to locate the vibrating needle tip at a desired depth and the needle tip may often penetrate through the tissue thoroughly by the vibration. 
     DISCLOSURE OF THE INVENTION 
     In order to eliminate or ameliorate the above and other problems, the method and system of the present invention is characterized by holding at least a portion of elastically deformable tissue surface by suction with tissue suction means, piercing the tissue with a microvibrating needle, and injecting a cell suspension through the needle at a desired position within the tissue. 
     In order to inject the cell suspension in a metered volume, the method and system of the present invention utilizes means for precisely metering the cell suspension in combination with the microvibrating needle. 
     In order to inject the cell suspension into a desired position within the tissue precisely and automatically, the method and system of the present invention utilizes means for automatically positioning the needle tip at a particular point in the three dimensional coordinates 
     Using the method and system of the present invention, (1) it is possible to inject cell directly into a site of recipient that needs thereof during a surgical operation, (2) it is possible to treat either allogenic soft tissues from brain death or organ death donors or xenogenic soft tissues of porcine or bovine origin, and (3) it is possible to inject cells into artificial or synthetic tissues as well. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a perspective view of the entire system for automatic injection according to the present invention, and 
         FIG. 2  is a cross-sectional view of the system taken along the line X-X of  FIG. 1 . 
     
    
    
     DESCRIPTION OF PREFERRED EMBODIMENTS 
     Now a preferred embodiment will be described by making reference to the accompanying drawings.  FIG. 1  shows the entire system  1  for automatic injection of cells according to the present invention. The system  1  comprises an injection device  2  provided with a microbivrating needle  200 . The system  1  also comprises a device  3  for precisely positioning the injection device  2  in three-dimensional directions. 
     The microvibrating injection device  2  comprises, as described below in detail, a vibrating needle  200  provided with a microvibrator  202  and a suction housing  210  for holding tissue segment  5  by suctioning the tissue surface in place for piercing the tissue with the needle  200  centrally and slidably extending through the suction housing  210 . 
     The three-dimensional positioning device  3  is configured to displace the injection device  2  mounted thereon horizontally in a plane at a particular point in both X and Y coordinate directions accurately. The device  3  also move the injection device  2  vertically so that the needle  200  can pierce the tissue in a predetermined depth precisely. Such positioning devices are well known in the art and commercially available. 
     The positioning device  3  shown in  FIG. 1  comprises a frame member  31  on which a position controller  30  for positioning the injection device  2  in the Z coordinate direction is movably mounted. The positioning device  3  also comprises a bed  32  for mounting a tissue container  4  containing a tissue segment  5 . The frame member  31  comprises a pair of legs and a cross-bar secured between legs as shown. The controller  30  is slidable on the cross-bar in the X coordinate direction horizontally and moves the injection device  2  in the Z coordinate direction vertically The frame member  31  carrying the controller  30  is movable along the side edges of the bed  32  in the Y coordinate direction. 
     Now, the method of injecting cells into a tissue segment  5  will be described by making reference to the drawings. 
     The tissue segment  5  is placed in the container  4  while keeping wet or intact state. Then the container  4  is secured on the fixing bed  3  using a fixture (not shown). The positioning device  3  includes self-contained control means (not shown) for controlling the movement of both of the frame member  31  and the position controller  30  in the X, Y and Z coordinate directions. The data concerning desired positions and depth of the tissue to be pierced may be inputed in the control means. 
     Specifically, the vibrating needle  200  is located just above the tissue segment  5  by manually moving the frame member  31  and the injection device  2  horizontally and data concerning the position in X and Y directions at this moment are inputed in the control means as the reference position. Then the injection device  2  is gradually lowered manually until the needle  200  just contact with the surface of the tissue segment  5 . The height of needle  200  at this moment is also inputed in the control means as the reference position in the Z direction. 
     Then the suction housing  211  (see,  FIG. 2 ) is lowered to contact with the tissue  5  with the needle  200  being retracted to a position flush with or slightly above the bottom surface of the housing. The interior of the housing  211  is kept under negative pressure before lowering to hold the tissue  5  against the bottom of the suction housing  211  by suction force. 
     The vibrating needle  200  is movable in vertical direction independently from the suction housing  210 . The control means determines a distance in which the needle tip extends beyond the bottom of the suction chamber. This distance corresponds to the depth that the needle tip penetrate into the tissue. When the needle tip extends 1 mm, for instance, beyond the bottom of suction housing, then the needle tip penetrates into the tissue to a depth of 1 mm. 
     It is important at this moment that the tissue segment  5  is securely held on the bottom surface  212  by a suction force exerted through a plurality of holes  213  and that the needle tip penetrates the tissue while vibrating. This enables the position of upper surface of the tissue  5  which is flush with the bottom surface of suction housing  210  to be detected and also enables the penetration depth of the needle into the tissue to be accurately and precisely determined. Suctioning the tissue against the bottom surface  210  of suction housing assists to hold the tissue under-tension and, therefore, the needle can penetrate into otherwise deformable tissue segment to a desired depth accurately and precisely. 
     The injection needle is in fluid communication with means for precisely metering a cell suspension (not shown) and injects the metered volume of the cell suspension. Thereafter the needle is retracted to the original position and the negative pressure within the suction chamber  211  is released to detach the tissue  5  from the suction housing  210 . Then the injection device  2  is elevated away from the tissue. The three dimensional positioning device  3  and its control means repeat the above procedure as desired at various positions of the tissue  5  according a program for scanning over the tissue and these positions in the X and Y coordinates are determined relative to the reference position originally inputed in the control means. 
     Turning to  FIG. 2 , the suction housing  210  is shown in cross-section taken along line X-X of  FIG. 1 . The injection device  2  comprises an injection needle provided with a vibrator  202  and a suction housing  210  enclosing the needle for holding the tissue segment  5 . The needle  200  is secured through a flange or piston  203  that allows the needle to slide in the housing  210  vertically independently from the housing. Specifically the housing  210  is directly carried by the positioning device  30  that controls the position of the housing  2  in Z coordinate direction while the needle  200  is friction fitted in the mating bore of the needle guide rod  201  which, in turn, is coupled to the means for elevating and descending the guide rod  201  (not shown) contained in the positioning device  30 . The needle  200  is connected in fluid communication with the means for precisely metering the cell suspension (not shown). 
     The metering means may be of conventional type and is commercially available. The metering means may be disposed internally or externally of the injection device. In the embodiment shown in  FIG. 1 , the metering means is connected externally to the injection device  2  via flexible tubing  205 . The external connection of the metering means simplifies the construction of the injection device and facilitates the maintenance or operation of both of the injection device and the metering means, particularly when filling and refilling the metering means with fresh cell suspension. This arrangement not only contributes to the reliability of the entire system and also eliminates need for those accessories which are otherwise susceptible to failure or malfunction. 
     The suction housing  210  defines the suction chamber  211  therein and an opening  214  communicating with an external pressure source (not shown) to create negative or positive pressure within the suction chamber  211 . The pressure source may be a pump or a pair of negative and positive tanks. In the embodiment shown in  FIG. 2 , the pressure source is connected to the opening  214  in fluid communication via a plug  215  and a tubing or hose  216  to create negative pressure and positive pressure or the atmospheric pressure within the suction chamber  211  alternately for attracting and detaching the tissue segment to and from the suction housing  210 . 
     The suction housing  210  defines a plurality of throughholes  213  in the bottom wall for applying negative or positive pressure to the tissue segment  5 . The outer face of the bottom wall is flat in the X and Y coordinate directions to attract the tissue outer surface in alignment with the bottom face  212  of the suction housing  210 . 
     The top and bottom walls of the suction housing  210  are provided with sliding sleeve rings  217  and  218 , respectively to maintain the chamber  211  air tight. The needle  200  is slidably received in the bottom sleeve ring  217  while maintaining air tight contact between them. Similarly, the top sleeve ring  218  slidably receive the needle guide rod  201  while maintaining air tight contact between them. 
     As described above, the needle  200  is secured to the flange or piston  203  that reciprocates vertically in the suction chamber  211 . The reciprocating flange  202  is connected, in turn, to the needle guide rod  201  carrying the needle  200 . Thus the needle and guide rod assembly may be elevated and descended within the suction chamber  211  by its own drive means self-contained in the positioning device  30  independently from the movement of the suction housing  210 . However, how long the needle tip should be extended or retracted is determined based on the bottom surface  212  of the housing  210 . Preferably, the flange or piston  203  has one or more vent holes to equalize the pressure in the suction chamber  211  above and below the flange  203 . This facilitates the reciprocation of the flange  203  in the suction chamber. 
     The needle vibrator  202  is disposed around the needle  200  below the flange  203 , preferably adjacent to the flange. Any of known microvibrators may be employed including, mechanical vibrators, electrical oscillators, ultrasonic vibrators and the like. 
     The invention is not limited to the embodiments specifically shown and described herein. For example, tissue segments could be held in position by using adhesives having no adverse affects to the tissue or magnetically attracting a container for the tissue. 
     EXAMPLES 
     Example 1 
     Using the method and system of the present invention, a cell suspension (105 cell/ml) was automatically and precisely injected into porcine heart muscle at 2,500 locations each spaced apart a distance of 200 μm in the area of 1×1 cm square at a dose of 200 picoliters/location by inserting the injection needle a depth of 2 mm. 
     Example 2 
     Using the method and system of the present invention, a cell suspension (105 cells/ml) was automatically and precisely injected into pulsating porcine heart wall at 50 locations arranged in row each spaced apart a distance of 200 μm at a dose of 100 picoliters/location by inserting the injection needle a depth of 5 mm.