Abstract:
An apparatus and method is described for seeding cells on a sample or specimen. The cells may be selectively and locally seeded on an upper and lower surface of a planar sample or specimen or on either or both of an interior luminal surface and exterior surface of a hollow sample or specimen. The apparatus includes a chamber suitable for cell seeding, cell growth, and cell conditioning.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the filing benefit and priority of U.S. Non-Provisional application Ser. No. 12/402,427 filed Mar. 11, 2009, the contents of which are incorporated herein by reference in its entirety. 
    
    
     FEDERAL SPONSORSHIP 
     Not Applicable 
     JOINT RESEARCH AGREEMENT 
     Not Applicable 
     TECHNICAL FIELD 
     This invention pertains generally to a method and apparatus to deposit cells within a chamber. This invention also pertains to a portable chamber for cell growth capable of maintaining a sterile system dosed to an external environment. 
     BACKGROUND 
     Generally, growth of cells on tissue, vascular grafts, biomedical prosthesis, substrate, and other medical devices (hereinafter referred to simply as a sample or specimen) has previously been described. Prior devices capable of depositing and growing cells typically submerge the sample with a cell suspended media, attempting to deposit cells uniformly on the entire sample. Often, pressure or other forces are used to influence adhesion of the cells on the sample. Many of these devices are designed for depositing cells onto a particularly shaped sample. By way of example, prior devices describe seeding cells on one surface of a biological vascular graft. 
     Other prior devices describe techniques for depositing a complex arrangement of an array of cells onto relatively planar substrates. For example, a multi step approach to deposit cells has been utilized to thereby build layers of proteins and cells utilizing masks to control the location of deposition and exposure of the cells on an exterior surface of the substrate. Other printing methods have been contemplated to, in essence, print cells onto an exterior surface of a Petri dish, glass, paper, plastic or other relatively planar substrate. 
     SUMMARY 
     Embodiments of the invention include an apparatus and method for a localized deposition of cells in a predefined pattern onto either a planar or three dimensional specimen or sample, including a tissue construct, vascular graft, biomedical prosthesis, or other medical device. The sample may be rotated while the cells are deposited onto the sample. Alternatively, the sample may be actuated linearly to deposit a row of cells in a straight line on the sample. In an embodiment of the invention, a tubular specimen or sample having a lumen may have cells deposited on one or both of an inner luminal surface and an exterior surface of the sample. Further, the tubular sample may be rotated or linearly actuated to thereby deposit cells locally on the sample to create a ring or linear pattern on either or both of the interior and exterior of the tubular sample. Alternatively, the sample may be both rotated and linearly actuated while depositing localized cells on the sample to create multiple variations of curvilinear patterns on the three dimensional sample. 
     Also described herein is an interchangeable, portable, chamber system that is capable of holding and rotating the sample within the chamber. The cells may be deposited on the sample contained within a sealed or pressurized chamber. The chamber provides for multiple ports to facilitate the delivery of nutrients, fluids, or gases within the chamber. Further, the chamber is suitable for use with other instrumented and servo controlled devices to allow for conditioning the sample and is particularly well suited for use with the bioreactors described in U.S. Pat. Nos. 7,410,792 and 7,348,175. 
     The accompanying drawings, which are incorporated in and constitute a portion of this specification, illustrate embodiments of the invention and, together with the detailed description, serve to further explain the invention. The embodiments illustrated herein are presently preferred; however, it should be understood, that the invention is not limited to the precise arrangements and instrumentalities shown. For a fuller understanding of the nature and advantages of the invention, reference should be made to the detailed description in conjunction with the accompanying drawings. 
    
    
     
       DESCRIPTION OF THE DRAWINGS 
       In the various figures, which are not necessarily drawn to scale, like numerals throughout the figures identify substantially similar components. 
         FIG. 1  is a perspective view of a cell seeding module in accordance with an embodiment of the invention; 
         FIG. 2  is a partial sectional view of the cell seeding module of the type shown in  FIG. 1 ; 
         FIG. 3  is a top plan view of the cell seeding module of the type shown in  FIG. 1 ; 
         FIG. 4  is a perspective view of a linear actuator system suitable for use in the cell seeding module of the type shown in  FIG. 1 ; 
         FIG. 5  is an exploded perspective view of a linear actuator system of the type shown in  FIG. 4 ; 
         FIG. 6  is a perspective view of a chamber grip actuator suitable for use in the cell seeding module of the type shown in  FIG. 1 ; 
         FIG. 7  is an exploded perspective view of a chamber grip actuator of the type shown in  FIG. 6 ; 
         FIG. 8  is a perspective view of an interchangeable, portable, bioreactor chamber suitable for use with a chamber grip actuator of the type shown in  FIG. 6 ; 
         FIG. 9  is an exploded perspective view of a bioreactor chamber of the type shown in  FIG. 8 ; 
         FIG. 10  is a perspective view of a delivery system suitable for use in the cell seeding module of the type shown in  FIG. 1 ; and 
         FIG. 11  is an exploded perspective view of a delivery system of the type shown in  FIG. 10 , 
     
    
    
     DETAILED DESCRIPTION 
     The following description provides detail of various embodiments of the invention, one or more examples of which are set forth below. Each of these embodiments are provided by way of explanation of the invention, and not intended to be a limitation of the invention. Further, those skilled in the art will appreciate that various modifications and variations may be made in the present invention without departing from the scope or spirit of the invention. By way of example, those skilled in the art will recognize that features illustrated or described as part of one embodiment, may be used in another embodiment to yield a still further embodiment. Thus, it is intended that the present invention also cover such modifications and variations that come within the scope of the appended claims and their equivalents. 
     The cell seeding and growing apparatus of the present invention include a chamber having one or more grips rotatable along a longitudinal axis of the chamber and grips. The grips may be contained within the chamber and rotated independently or in unison within the chamber without rotating the chamber itself. Also included are one or more delivery systems, each delivery system including a delivery conduit having a portion aligned within the chamber. Further included are one or more linear actuators that linearly displace the grips relative to the delivery conduits and one or more rotary actuators that rotate the grips within said chamber relative to the delivery conduits. The linear actuator system may interlock with the chamber and, additionally, may interlock with the rotary actuator. 
     Alternatively, the cell seeding and growing apparatus may include one or more grips (independent and without being enclosed in a chamber system). The grips may be rotated independently or in unison about an approximately longitudinal axis. Also included is a delivery systems that has an approximately longitudinal axis and includes one or more delivery conduits aligned in linear relation with the longitudinal axis of the grips. The delivery conduits may further be aligned between grips. An actuator system may be included that interlinks the grips and rotates the grips either independently or in unison along a longitudinal axis of the grips. Another actuator system may be included to increase or decrease a distance between the grips and the delivery system. As the distance is increased or decreased, at least one of the grips and delivery system are displaced along a longitudinal axis of the same. Further the actuator systems may interlock and may be interchanged with other systems. 
     Alternatively, the system for seeding cells on a sample may include a chamber including at least two opposing grips for holding a sample, a rotary system coupled to the opposing grips (to rotate the grips about a longitudinal axis of the grips), one or more delivery systems (to deliver a media between the grips), and a linear actuator that linearly displaces at least one of the opposing grips and the delivery systems to thereby increase or decrease a distance between the grips and the delivery systems. The delivery systems may include a delivery conduit having at least a portion of the delivery conduit positioned within the chamber between the grips. The chamber may include one or more ports suitable for receiving at least one of a fluid delivery, a gas delivery, a temperature transducer, a flow transducer, a pressure transducer, or a linear displacement transducer. Further, the rotary system may interlock with the chamber and may also interlock with the linear actuator. 
     A user of the apparatus may use the apparatus to deliver or otherwise deposit media to a localized site on the sample. The media may include cells suspended in a solution, nutrients, fluids, gases or other substance. The sample may be secured to the grips in a manner suitable to rotate the sample around an axis of the sample, where the axis of the sample may be aligned approximately parallel with a surface of the sample to which the media is to be delivered. After selecting a sample and securing the sample to grips, the user may selectively rotate or linearly displace the sample along the sample axis. One or more delivery conduits may be used to deliver selected media to the sample. An embodiment of the invention includes aligning a first delivery end of a first delivery conduit of a delivery system with the sample. Additionally, a second delivery conduit, coupled to the same or separate delivery system, may be aligned with the sample. A site of the sample may be displaced relative to an end of the delivery conduit by rotating the sample, linearly displacing the sample along the axis of the sample or linearly displacing the conduit, while simultaneously or intermittently delivering media to the sample. Those skilled in the art will appreciate that it may be desired to deliver or deposit media on a tubular sample having a lumen extending through at least a portion of the sample. The user may align a second delivery conduit to either an exterior surface of the sample or to a surface within a lumen of the sample. 
     Turning attention now to the Figures, embodiments of the cell seeding module or system  10  of the present invention will now be described in more detail and are generally shown in  FIGS. 1-3 . Cell seeding module  10  includes a linear actuator system  20 , a chamber grip actuator  30 , an interchangeable chamber  40 , cell delivery systems or modules  50 , and controller  60 . The cell delivery systems  50  are shown electrically coupled in parallel to controller  60  through electric leads  62 - 66 . Although no shown, the linear actuator system  20  and chamber grip actuator  30  may also be electrically coupled to controller  60  or alternatively may be coupled to independent controllers. 
       FIGS. 4 and 5  show generally an embodiment of the linear actuator system  20 . Base plate  202  includes foot pads  212  attached to a bottom of the base plate  202 . The foot pads  212  are slightly compressible so that when the cell seeding system  10  rests on an uneven surface, compression of the foot pads  212  compensate for the uneven surface and may be adjusted to keep the cell seeding system  10  level. Linear stage  204  is attached to base plate  202  and includes a worm  206  and worm gear base  208 . Stepper motor  210  is coupled to worm  206 . When stepper motor  210  is activated, the worm gear base  208  is actuated along worm  206  in either direction, depending upon the direction the worm  206  is turned by stepper motor  210 . A mover base plate  214  is attached to the worm gear base  208 , and includes guide pegs  216  attached to an upper surface of the mover base plate  214 . The guide pegs  216  interlock with a mover top plate  302  of the chamber grip actuator system  30  (see also  FIGS. 6 and 7 ). The interlocking of the linear actuator  20  and chamber grip actuator  30 , provides for a quick interconnect and removal of the chamber system. 
       FIGS. 6 and 7  show an embodiment of the chamber grip actuator  30 . Mover top plate  302  includes drive bar guide standoffs  306 , drive bar guide  314  and motor plate  316  attached thereto. Drive bar  304  is positioned through apertures of the drive bar guide standoffs  306 , drive bar guide  314  and motor plate  316  and an end of the drive bar  304  is coupled to rotary stepper motor  318 . Drive gears  310  may be press fit or otherwise affixed to the drive bar  304 . Bearings  308  are positioned within the apertures of drive bar guide standoffs  306  and drive bar guide  314 . Drive bar guide  304  extends through bearings  308  which provides for a stable rotation of drive bar  304 . Shims  312  are positioned between drive gear  310  and bearings  308  to ensure a space between drive gear  310 , drive bar guide standoffs  306 , and drive bar guide  314 , and to reduce wear or rubbing of the drive gear  310  against the same. Chamber holder  320  is attached to a top portion of drive bar guide standoffs  306 . Knurled nuts  430  are sized to fit within apertures extending through a top portion of chamber holder  320  and interlock or engage interchangeable chamber  40  with chamber grip actuator  30  (see also  FIGS. 8 and 9 ). The interlock of the chamber grip actuator  30  and interchangeable chamber  40  provides for a quick interconnect of the chamber system. Activating rotary stepper motor  318  rotates drive bar  304  which consequently rotates drive gear  310 . 
       FIGS. 8 and 9  show an embodiment of the interchangeable, portable, autoclavable, bioreactor chamber  40 . First sealing plate  402 , chamber body  404 , and second sealing plate  406  are held together by knurled nuts  430  and threaded rods  428  which extend through first sealing plate  402 , chamber body  404 , and second sealing plate  406 . Knurled nuts  430  are positioned on each end of threaded rod  428  and may be turned to tighten the first sealing plate  402  and second sealing plate  406  against the chamber body  404 . A seal may be positioned between the first sealing plate  402  and chamber body  404  and between the chamber body  404  and second sealing plate  406  to provide a seal between the respective same. The first and second sealing plates  402  and  406  may be of a suitable construction and one or both may be made of a transparent material. 
     A gear holder  414  is engaged to each end of the chamber body  404 . Grip holder  418  is positioned through apertures of the gear holder  414  and chamber body  404 . A follower gear  412  may be press fit or otherwise affixed to the grip holder  418 . Bearings  408  are positioned within the apertures of gear holder  414 . Grip holder  418  extends through bearings  408  which provides for a stable rotation of grip holder  418 . Shims  410  are positioned between follower gear  412  and bearings  408  to ensure a space between follower gear  412 , bearings  408  and gear holder  414  to reduce wear or rubbing of the follower gear  412  against the same. Likewise, grip holder  420  is positioned through apertures of another gear holder  414  and an opposite end of chamber body  404 . A follower gear  412  may be press fit or otherwise affixed to the grip holder  420 . Bearings  408  are positioned within the apertures of gear holder  414 . Grip holder  420  extends through bearings  408  which provides for a stable rotation of grip holder  420 . Shims  410  are positioned between follower gear  412  and bearings  408  to ensure a space between follower gear  412 , bearings  408  and gear holder  414  to reduce wear or rubbing of the follower gear  412  against the same. Those skilled in the art will appreciate that the gear holders  414  may be attached instead to the chamber holder  320  and modified to interlock with chamber body  404 . 
     Hollow grip holder  418  and  420  includes a grip  416  coupled to a first end of the respective grip holders  418  and  420  (first and second grips) and includes a Luer fitting  422  attached to an opposite end of each respective grip holder  418  and  420 . Grip  416  may be a suitable construction adapted for holding a tissue, vascular grafts, biomedical prosthesis, medical devices or other desired specimen or sample. A tubular sample may slip over an end of the grip  416  and a relatively planar sample may, by way of example and without limitation, be sutured to an end of the grip  416 . Also, grip  416  may include an aperture extending though a center axis of the grip to provide a passage between an interior lumen of the sample and the opposite end of the hollow grip holder  418  or  420 . The grip ends of the grip holder  418  and  420  are positioned within an interior cavity of the chamber body  404  and may extend further or less within the cavity of chamber body  404  to accommodate samples of varying lengths. The grip holders  418  and  420  and grips  416  rotate within the cavity about a longitudinal axis of the chamber  30  or grip  416 , without rotating the chamber  30  or chamber body  404 . 
     Gear holder  414  further includes a gear lock pin  424  that may be actuated to engage within aperture  426  of follower gear  412 . By engaging the lock pin  424  within the aperture  426  of follower gear  412 , the grip holders may be restricted from rotating. Additional Luer fittings may be coupled to the chamber body and may be capped or may be utilized as ports suitable for receiving at least one of a fluid delivery, a gas delivery, a temperature transducer, a flow transducer, a pressure transducer, or linear displacement transducers. When first sealing plate  402  is engaged with chamber holder  320 , drive gear  310  and follower gear  412  align and engage. Thus, when rotary stepper motor  318  rotates drive gear  310 , follower gear  412  rotates which in turn rotates grip holders  418  and  420  and grips  416  in unison. Ultimately, the rotary or chamber actuation system rotates the sample coupled to grips  416  about a longitudinal axis of the chamber. 
       FIGS. 10 and 11  show an embodiment of the cell delivery module  50 . A standoff  502  of each cell delivery module  50  is attached to the base plate  202  of the linear actuator  20  (shown in  FIGS. 1 and 2 ). A base plate  504  is attached to the standoff  502  and stepper motor  506  or a manual linear stage (not shown) is attached thereto. A mover plate  522  is attached to stepper motor  506  via a worm drive (not shown) that linearly actuates the mover plate  522  back and forth in a longitudinal direction relative to the stepper motor  506 . A syringe holder front plate  508  is attached to an end of the stepper motor  506 . A syringe base plate  510  is fixed to the syringe holder front plate  508  and a syringe top plate  512  is attached to the base plate  510  with a hinge  516 . A top plate catch  514  is positioned between the top plate  512  and base plate  510  to engage the top plate to the base plate. A top plate catch  514  may, for example without limitation, be a magnetic catch of known suitable construction. A push block plate  520  and syringe push block  518  are attached to mover plate  522 . A syringe  524  of known suitable construction may be positioned between the top plate  512  and base plate  510 , wherein a hollow needle or delivery conduit  530  and coupling  528  are attached in fluid communication to an end of the syringe  524 . The hollow needle  530  may be made of a known suitable material, with a hollow stainless steel tube being preferred. The opposite end of syringe  524  includes a plunger  526  that draws fluid or gas into and pushes fluid or gas out of the opposite end of the syringe  524 . Syringe push block  518  is actuated by stepper motor  506  and engages with an end of the plunger  526 . 
     Having described the constructional features of embodiments of the invention, the mode of use will next be described. A user selects a desired sample or specimen and affixes the sample or specimen to the grip  416 . For purposes of illustration and without limitation, a tubular sample will be described having each end attached to grips  416 . A first delivery conduit  530  extends through Luer fitting  422 , grip holder  420  and grip  416 . An end of the delivery conduit  530  aligns within the chamber  30  and extends into the lumen of the sample. Those skilled in the art will appreciate that a nozzle may be attached to the end of the delivery conduit to control the flow and shape of the stream of fluid emitted from the end of the delivery conduit  530 . A second delivery conduit may be aligned or positioned above and adjacent an exterior surface of the sample. Once the first and second delivery conduits are aligned in the desired position stepper motor  506  may be activated to push the syringe plunger  526 , causing the contents of the syringe  524  to pass through the delivery conduit  530  and exit the open end of the delivery conduit  530 . In this manner, cells suspended in drops of fluid, contained with the syringe  524 , may be delivered to a selected and localized point on both the inner luminal surface of the sample and on an exterior of the sample. 
     Stepper motor  210  may be selectively activated to linearly displace grips  416  relative to delivery conduit  530  of delivery systems  50 . Thus, this linear actuator or actuator system linearly displaces the grips relative to the delivery system along a longitudinal axis of the grip, chamber, and delivery system, thereby increasing or decreasing a distance between the grip and delivery system. Chamber grip actuator  30  and interchangeable chamber  40  together provide a rotary system or actuator system that rotates the grip  416  within the chamber around a longitudinal axis of the grip or chamber. 
     Stepper motor  210  may be activated in conjunction with activating stepper motor  506 . In this manner drops of fluid are deposited on the exterior and interior of the sample in a straight line or row. Alternatively, rotary motor  318  may be activated in conjunction with activating stepper motor  506 . In this manner drops of fluid are deposited on the exterior and interior of the sample to forms rings around the exterior and interior of the sample. In yet another embodiment both stepper motor  210  and rotary motor  318  may be activated in conjunction with activating stepper motor  506 . In this manner, drops of fluid are deposited on the exterior and interior of the sample to create multiple variations of curvilinear patterns on the interior and exterior of the sample. Those skilled in the art will appreciate that altering the speeds of motors  210 ,  318 , and  506 , viscosity of the fluid, size of the cells, and size of the opening at the end of the delivery conduit  530  will all affect the amount of cells deposited at any given point on the sample. Further, motor  506  may be switched on an off while one or both of the other motors  210  and  318  remain activated to create row segments or curvilinear segments of cells on both the interior and exterior of the sample. Still further, the motors  506  on the first and second delivery systems may be switch on and off to vary the deposition of cells on the exterior of the sample in relation to the deposition of cells on the interior of the sample. 
     Those skilled in the art will further appreciate that different fluid suspended cells, fluid, pressurized air, gases or other media may be included in each delivery system syringe  524 . For example, each delivery system may include a different cell type. Further, the size of the delivery conduit  530  may be selected to accommodate the size of the chosen specimen and cells to be delivered. 
     Although not required, cells can typically be contained within a cell composition or liquid carrier for the cells. The cell composition can be in the form of a suspension, solution, or any suitable form. Examples of suitable liquid carriers include, but are not limited to, water, ionic buffer solutions and so forth. The use of a liquid carrier in the cell composition can ensure adequate hydration after depositing. 
     These and various other aspects and features of the invention are described with the intent to be illustrative, and not restrictive. This invention has been described herein with detail in order to comply with the patent statutes and to provide those skilled in the art with information needed to apply the novel principles and to construct and use such specialized components as are required. It is to be understood, however, that the invention can be carried out by specifically different constructions, and that various modifications, both as to the construction and operating procedures, can be accomplished without departing from the scope of the invention. Further, in the appended claims, the transitional terms comprising and including are used in the open ended sense in that elements in addition to those enumerated may also be present. Other examples will be apparent to those of skill in the art upon reviewing this document.