Abstract:
An apparatus and method is described for culturing and conditioning cells on a sample. The apparatus includes a fluid delivery system that transmits gas to a first surface of a sample and transmits liquid to an opposite surface of the sample without transmitting liquid onto the first surface. Further, while culturing cells on the sample, the apparatus enables a constant or variable tension and shear stress applied to the sample. The apparatus may thus, for example, be used to culture cells on an external surface of a tissue construct with air and culture cells underneath the tissue construct with a cell growth media. The fluid delivery system may alternatively transmit liquid cell growth media underneath and on the sides of the tissue construct without flowing onto a designated external surface of the tissue construct. In this manner, cells may be cultured on the tissue construct while stretching and relaxing the tissue and further simulating a dissimilar environment on opposite surfaces of the tissue construct.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    Not Applicable 
       FEDERAL SPONSORSHIP 
       [0002]    Not Applicable 
       JOINT RESEARCH AGREEMENT 
       [0003]    Not Applicable 
       TECHNICAL FIELD 
       [0004]    This invention pertains generally to a system and method of culturing and conditioning cells on a sample. This invention further pertains to a culturing of cells on a sample while simulating multiple environmental conditions on select surfaces of the sample, and subjecting the sample to variable tensions, stresses, and strains. 
       BACKGROUND 
       [0005]    Generally, growing or culturing cells on a tissue sample has previously been described. In the past, cells have been cultured on a selected matrix, tissue sample, vascular graft, biomedical prosthesis, substrate, medical device or other specimen. Preconditioning biological implants has been found to be beneficial in restoring function for tissue grafts, implantable biomaterials, and tissue engineered constructs. It has also been found that tissue cells cultured in dynamic environments are more likely to tolerate physiological conditions of the human body. Further, it is now recognized that it is advantageous to provide a dynamic environment that allows a constant or varying strain or other forces applied to the specimen while culturing cells on the specimen. Whether to apply a constant or varying strain is dependent upon the implant location and type of construct to develop. For example, prior to implantation, skin tissue constructs often lack the proper mechanical properties and cellular function to become fully integrated and functional. Once implanted, the skin tissue construct may routinely undergo stretching and relaxing. Restoration of the skin tissue function is more likely if the implant tissue possesses mechanical properties similar to the surrounding host tissue and if the various layers of the cells include preferred characteristics to properly integrate into host tissue. 
         [0006]    Prior systems have seeded cells on a specimen and then subsequently supplied nutrients to the cells for growth. These systems typically either submerge the specimen in growth media or isolate opposing surfaces of the specimen by clamping around an entire perimeter of the specimen or sandwiching the specimen around the perimeter between a frame or mold. It is now recognized that it may be advantageous to restrain or grip the specimen and deliver growth media in a way that a surface and sides of the specimen may have media directed thereto, while simultaneously directing gas to an opposing surface, and further stretching or relaxing the specimen. 
       SUMMARY 
       [0007]    Embodiments according to aspects of the invention include an apparatus and method for selectively delivering gases to a first selected surface of a sample, delivering liquids to an opposing surface, and delivering either gas or liquid to edges of the sample. Further, while fluids are delivered to the sample, a static, variable, or intermittent force is applied to the sample. The sample may consist of, without limitation, a harvested tissue, a tissue engineered construct (including, but not limited to skin, cornea, and lung tissue), scaffold, or other specimen (hereinafter referred to generally as a sample). 
         [0008]    In an embodiment of the invention, liquid is delivered to a receptacle or reservoir within a chamber. A sample is suspended above the receptacle in a manner such that a lower surface of the sample contacts the liquid. The amount of liquid in the receptacle is controlled by overflows or spillways so that liquid does not overflow onto the upper surface of the sample. Inserts are placed in the receptacle to vary the liquid capacity of the receptacle. Modifying the liquid capacity facilitates applying various fluid shear stresses to the underside of the sample while using identical volumetric flow rates. Liquid that overflows from the receptacle flows into a second receptacle and, from there, drains from the chamber. Fluid ports may be used for perfusion or creating a pressure differential between the interior and exterior of the chamber. Grips retain the sample and include an adjustable separation distance between the grips to accommodate varied lengths of samples. 
         [0009]    In an embodiment of the invention, the apparatus includes a combination gas and liquid bioreactor. The bioreactor includes a chamber; grips to retain a construct within the chamber; a fluid delivery system that transmits gases to an upper portion of the construct and transmits liquids to a lower portion of the construct; and an actuator linked to a portion of the grips to selectively provide a variable and static force on the construct. The liquid may be transmitted to the lower portion of the construct without transmitting liquid to the upper portion of the construct. Further, the fluid delivery system includes overflow controls to control a volume of liquid transmitted within the chamber. Also, a liquid capacity within the chamber is adjustable. 
         [0010]    An aspect of the invention includes gripping a construct within a chamber to culture and condition a construct by simulating multiple environmental conditions on select surfaces of the sample, and subjecting the sample to variable tensions, stresses, and strains. A further aspect of the invention controls a volume of growth media within the chamber to selectively deliver the media to one or more surfaces of the construct while the construct is repeatedly actuated between strained and relaxed positions. A further aspect of the invention includes modulating or mitigating a condensation on a surface within the chamber. 
         [0011]    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 
         [0012]    In the various figures, which are not necessarily drawn to scale, like numerals throughout the figures identify substantially similar components. 
           [0013]      FIG. 1  is a perspective view of the bioreactor interface in accordance with an embodiment of the invention; 
           [0014]      FIG. 2  is a perspective view of the bioreactor chamber of the type shown in  FIG. 1 ; 
           [0015]      FIG. 3  is a top plan view of the bioreactor chamber of the type shown in  FIG. 2  with the cover removed; 
           [0016]      FIG. 4  is a partial sectional perspective view of the bioreactor chamber of the type shown in  FIG. 2 , with the cover removed; 
           [0017]      FIG. 5  is an enlarged detail of that portion of the chamber encircled and identified as “ 5 - 5 ” in  FIG. 4 ; 
           [0018]      FIG. 6  is a partial sectional side view of the bioreactor chamber of the type shown in  FIG. 4 , with the cover removed; 
           [0019]      FIG. 7  is a partial sectional perspective view of the bioreactor chamber of the type shown in  FIG. 4 , with the cover removed, and a grip in its aft position; 
           [0020]      FIG. 8  is a partial sectional perspective view of the bioreactor chamber of the type shown in  FIG. 4 , with a grip in its forward position; 
           [0021]      FIG. 9  is a partial sectional perspective view of the bioreactor chamber of the type shown in  FIG. 7 , with an insert positioned within a liquid receptacle; 
           [0022]      FIG. 10  is an enlarged detail of that portion of the chamber encircled and identified as “ 10 - 10 ” in  FIG. 9 ; 
           [0023]      FIG. 11  is an alternate embodiment of the enlarged detail of the chamber portion illustrated in  FIG. 10 ; 
           [0024]      FIG. 12  is a partial sectional end view of an alternate embodiment of the bioreactor chamber of the invention; 
           [0025]      FIG. 13  is perspective of the insert of the type illustrated in  FIG. 12 ; 
           [0026]      FIG. 14  is a partial sectional end view of an alternate embodiment of the bioreactor chamber of the invention; 
           [0027]      FIG.15  is perspective of the insert of the type illustrated in  FIG. 14 ; 
           [0028]      FIG. 16  is a perspective view of an alternate embodiment of the bioreactor chamber of the invention; 
           [0029]      FIG. 17  is a perspective view of an alternate embodiment of the bioreactor chamber of the invention; 
           [0030]      FIG. 18  is a perspective view of an alternate embodiment of the bioreactor chamber of the invention; and 
           [0031]      FIG. 19  is a perspective view of an alternate embodiment of the bioreactor chamber of the invention. 
       
    
    
     DETAILED DESCRIPTION 
       [0032]    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. Furthermore, 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. 
         [0033]    The bioreactor of the present invention includes a chamber capable of retaining fluids within the chamber, a fluid delivery system, spaced apart grips, and an actuation system capable of repeatedly actuating the grips between multiple positions or force levels. The overall size or “footprint” of the bioreactor is designed so that the bioreactor fits within commercially available incubators. A sample of varying size and shape may be retained in the chamber between the grips. A variety of liquids (including growth media) and gases may be delivered to the sample held within the chamber to simulate a variety of natural environments the sample may encounter after implantation or during intended use. Further, the fluid delivery system delivers fluid to a select surface of the construct and the internal chamber design inhibits liquid from overflowing onto surfaces other than on the preselected surfaces. The internal chamber design also inhibits overflow of liquids while the sample is actuated between a first stretched position and second relaxed position. In this manner cells, for example, may be seeded on select surfaces of the sample and then growth media and gases may be delivered to select surfaces while the cells are cultured and the sample is stimulated by mechanical actuation and alternatively shear stresses. 
         [0034]    The grips contained within the chamber are mechanically coupled to actuators, for example magnetic forcers, stepper motors or other motor of known suitable construction, to selectively deliver axial and linear displacement to the sample. A controller and feedback device may be utilized to control the actuators and selectively apply forces and displacements to the sample. The controller allows the user to selectively define a displacement distance or applied load on the sample. Also, the user of the apparatus may use the bioreactor to culture cells of a skin, lung, or cornea tissue while simulating the stretching and relaxing of skin, lung or cornea tissue, for example. Those skilled in the art will appreciate that it may be desired to expose one side of the skin, for example, to oxygen and carbon dioxide, while exposing the other side to cell growth media, to thereby simulate a natural environment for the skin tissue. 
         [0035]    Turning attention now to the Figures, embodiments of the bioreactor or system  10  of the present invention will now be described in more detail.  FIG. 1  generally shows the bioreactor  10  including a chamber  20 , linear actuator  30 , linear variable differential transformer (LVDT)  40 , linear displacement transducer (LDT)  50 , linkage  60 , and load cell  70 . Although the chamber  20  is shown in Figures as opaque, those skilled in the art will appreciate that the cover  202  and other portions of the chamber  20  may be constructed from a translucent material. The linear or axial actuator  30  is mechanically coupled to one end of a bar linkage  80  and the second end of linkage  80  is coupled to a first end of alignment linkage  60 . A second end of alignment linkage  60  is coupled to a load cell  70 . The load cell  70  is further coupled to a drive bar  248  which is coupled to an elbow  254  and grips  240 . The first end of the alignment linkage  60  or second end of the linkage  80  may be coupled to an extender  90 . The extender  90  includes rollers  92  and a magnetic strip, sensing media or other indicia associated therewith. Those skilled in the art will appreciate that an optical sensor could replace the LDT  50 , however, when the bioreactor is used in humid environments sensing magnetic indicia is preferred over sensing physical markings or indicia. When the extender  90  slides under LDT  50 , the amount the extender  90  (and ultimately grips  240 ) is displaced may be precisely determined from the LDT  50  output. The actuator  30  may be of a servo pneumatic, electromechanical flexure bearing or electromechanical linear screw motors of known suitable construction. The mechanical coupling between the various components may be aligned so that a grip  240  may be actuated fore and aft in a controlled finite manner. 
         [0036]    A controller (not shown, but of suitable construction) may be electrically coupled to the linear actuator  30 , LVDT  40 , LDT  50 , and load cell  70  so that feedback and analysis loops may be incorporated into the controller to selectively provide repetitive, continuous, or intermittent stimulus to a sample or construct  24  held in place between grips  240 . The displacement feedback may be controlled from either or both the LVDT  40  and LDT  50  output to offset any step function observed from actuator  30  displacement. Those skilled in the art will appreciate that the redundancy with the LVDT  40  and LDT  50  may not be required for adequate function. A controller may also be utilized to control actuator  30  to thereby alter a separation distance between the grips  240  or to apply a selected load to the sample  24 . A controller may further enable a user to maintain the position of the grips  240  in a fixed position to thereby translate a fixed tension on the sample  24 . In this manner a variety of stimulus sequences may be applied to a selected sample  24 . 
         [0037]    It will be appreciated by those skilled in the art that setting, monitoring and controlling the separation distance between the grips enables finite control of the tension applied to the sample. Alternatively, setting, monitoring, and controlling a load on the sample allows the user to apply a consistent load on the sample independent of a particular tensile strength of the sample. Further, additional sensors may be electrically coupled to the controller to detect the position of the grips  240 . The grip displacement could be measured with, by way of illustration and without limitation, an LVDT, laser PSD, incremental encoder, or other measurement feedback device of known suitable construction. Under load control, the controller adjusts the separation distance and positions of the grips  240  so that a known force (common preload) may be applied to all samples  24 . The load cell  70  may also be utilized to control the force applied to the sample. 
         [0038]    Referring now to  FIGS. 2-6 , the chamber  20  will be described in greater detail. The chamber  20  generally includes an enclosure  200 , cover  202 , latches  204 , vent or cover port  208 , fluid in port or conduit  210 , and fluid out port or conduit  212  (see  FIG. 2 ). The cover  202  is attached to the enclosure  200  with hinges  218  and latch extension  206  distributes the force of the latches to uniformly press the cover  202  against the enclosure  200 . A perimeter seal  216  is positioned around the perimeter opening of the enclosure  200  and is sandwiched between the cover  202  and enclosure  200  to allow the user to maintain a sterile environment within the chamber  20 . The lower portion of the enclosure  200  may include alignment members (not shown) to engage with the frame of the bioreactor  10 , thereby fixing the chamber in place with respect to the alignment of the actuator  30 , LVDT  40  and LDT  50 . Ports  208  and  210  may be used to deliver fluids (either liquid or gas) into the interior of the chamber  20 . Port  208  may include a valve to create a pressure differential between the exterior of the chamber and interior of the chamber  20 . Further, the port  208  may include a filter therein and may be utilized to evacuate vapors from the interior of the chamber  20 . 
         [0039]    The interior of the chamber  20  and enclosure  200  includes an inner liquid receptacle or trough  220  and outer receptacle or trough  230  (see  FIG. 3 ). The inner receptacle  220  includes sidewalls  224  extending from a floor  232  of the receptacle to a defined height  228  (see  FIG. 6 ). The sidewalls  224  include one or more slots or spillways  226  that may be used to control the height of the liquid contained within the inner receptacle  220 . The depth  244  of the spillway  226  defines the height of the liquid (see  FIG. 5 ). The location of the spillway and height of the liquid affects the ability to apply fluid shear stresses to the underside of the sample. The edges of the inner receptacle  220  sidewalls  224  are rounded to further aid a uniform flow of liquid over the sidewalls and spillway  226 . As the liquid reaches a height equal to the bottom of the spillway  226 , the liquid overflows into the outer receptacle  230 . If the flow of liquid is too great, the liquid flows through the spillways  226  and overflows over the top of sidewalls  224 . The outer sidewalls  222  and slanted floor  234  of the outer receptacle  230  directs the overflow liquid to the opening of the fluid out port  212  (see  FIG. 4 ). 
         [0040]    Grip  240  includes a compression knob  242  that may be turned to tighten a grip on a sample  24  sandwiched in the grip  240  (see  FIG. 6 ). The grip  240  may include an elbow  254  to provide an offset between the center axis of the drive bar  248  and the center axis of the sample  24  positioned between grips  240 . Thus, the bottom of the drive bar  248  or linear actuator is elevated above a maximum liquid level within the inner liquid receptacle. Pins  256  extend from the grip  240  and engage with apertures formed in the bottom of the spillway or in the drive bar  248 . The pins  256  allow for an efficient removal and placement of the grips within the chamber  20 . Drive bar  248  extends through a side of the enclosure  200  and is aligned within the chamber by drive bar guide  250 . Alternatively, the guide may be oversized so that the drive bar may be angled within the guide  250 . Drive bar lock  252  allows the user to adjust the separation distance between grips  240  to accommodate various lengths of sample  24 . Bellow  246  surrounds drive bar  248  and a first end of the bellow  246  seals around the drive bar  248  and a second end of the bellow seals to the enclosure  200 . Since the drive bar and grips include an offset, the bellows are elevated above the maximum level of liquid within the liquid receptacle and liquid is not disturbed by the bellows movement. A length of drive bar  248  that extends into chamber  20  may be controlled by controller, thus defining the maximum extension of the drive bar  248  into the chamber  20 . In this manner the position of the grips  240  may be controlled when the sample  24  is in a relaxed state. When the sample  24  is stretched, the bellow and grip  240  are in the aft position  264  (see  FIG. 7 ), and when the sample  24  is relaxed the bellow and grip  240  are in the forward position  262  (see  FIG. 8 ).  FIGS. 7 and 8  further illustrates varying lengths of sample positioned between grips  240 . The sample  24  illustrated in  FIG. 7  is longer than the sample illustrated in  FIG. 8 , and thus the position of the drive bar lock  252  varies depending upon the length of sample  24 . 
         [0041]    Referring now to  FIGS. 9-11 , an insert  280  is shown positioned within the inner receptacle  220 . When positioned within the receptacle  220 , the volume of fluid contained within the receptacle  220 , the effective capacity of the inner receptacle, and the depth of fluid within the receptacle  220  are all reduced. An end of the insert  280  may have a varying thickness  284  (compare  FIG. 10  and  FIG. 11 ) which further affects the depth of fluid, capacity of the receptacle, and volume of liquid within the receptacle  220 . By varying the depth of fluid within receptacle  220  the shear stress on the bottom surface may be modified. As the depth of fluid decreases the shear stress increases and as the depth of fluid increases the shear stress decreases. Ports  282  extend from a lower portion to an upper surface of the insert  280 . The end of the insert having ports  282  is positioned near the fluid in port  210  and directs the liquid through the insert to an upper surface of the insert  280 . 
         [0042]    Having described the constructional features of embodiments of the invention, the mode of use will next be described. A user positions a selected sample  24  between grips  240 . The hold of the grip  240  on the sample  24  is tightened with the compression knob  242 . Liquids may be delivered through port  210  to an interior of the chamber. The fluid flows within the inner receptacle  220  until the amount of liquid exceeds the capacity of the receptacle  220  and overflows into outer receptacle  230 . The relative position of the bottom surface of the sample and the height of the liquid within the receptacle  220  may be adjusted so that only the bottom surface of the sample comes into fluid communication or contact with the liquid, both the bottom and sides of the sample contact the liquid, or the bottom, top, and sides of the sample contact the liquid. While the sample is in fluid communication with the liquid, the grips may be actuated between a fore and aft position. In this manner, the sample may be subjected to a controlled stimulus including variable and static forces, fluid shear forces, as well as tension or stretching force on the sample. The actuation and/or load on the sample may be repeated as desired. Additionally, while the sample is being subjected to forces and liquids, oxygen, carbon dioxide or other selected gas may be delivered through port  208 . The user may choose to deliver a variety of liquids including a growth media to assist in the culturing of the cells on the sample. 
         [0043]    In use, the gases within the chamber  20  may have a relatively high humidity. If an external temperature is less than a temperature of the gases within the chamber, a condensation may form on an interior surface of the chamber. Further, condensation on an underside of the cover of the chamber may accumulate and form droplets. During certain uses of the chamber  20 , it may be undesirable for droplets to release from the cover and fall onto a dry side of the construct  24 . Without limitation,  FIGS. 12-19  illustrate several alternative embodiments to mitigate or modulate a condensation within the chamber  20 . 
         [0044]    With reference to  FIGS. 12-13 , the underside of the cover  202  of enclosure  200  may be modified to include a contoured insert  320  attached to an underside of the cover  202 . The insert  320  includes a slope  322 . Droplets formed on the slope tend to migrate towards the lowest point of the slope before releasing from the slope. When the droplets release, they are aligned with the outer receptacle  230  and fall into the sloped floor  234  of the outer receptacle. Those skilled in the art will appreciate that the contour and slope  322  may be formed integral to the cover rather than requiring a separate insert. 
         [0045]    Alternatively, an independent contoured insert  330  may be positioned within the chamber. The insert  330  includes flanges  332  and  334  that extend downward, the bottoms of which rest on the slanting floor  234  of the outer receptacle  230 . The underside of the insert  330  includes a slope  336 . Droplets formed on the slope  336  tend to migrate down the slope  336  and down pillar  334  before releasing from the insert  330 . When the droplets release, they are aligned with the outer receptacle  230  and fall onto the sloped floor  234  of the outer receptacle. 
         [0046]    Shown in  FIG. 16  is a modified cover that includes a filter sheet  340  formed integral with the cover. The filter  340  is of the type to allow gas vapor to pass through the filter  340  to the exterior of the chamber  20 . Alternatively, as shown in  FIG. 17 , the cover may be modified to include a second filter or port  350 . With the cover engaged to the enclosure  200  and sealed with seal  216 , gases may be delivered under pressure through filter or port  208 . Port  350  may function as a relief valve allowing gases to escape from the interior of the chamber once an increased pressure exceeds a desired maximum. The likelihood of condensation will decrease as the pressure within the chamber is increased. Further, a humidity of gases delivered into the chamber may be selectively controlled. Also, the temperature of the gases may be controlled by continuously porting gas at a fixed temperature into the interior of the chamber. 
         [0047]    Alternatively, the temperature of chamber may be controlled so that condensation forming on an interior of the chamber is unlikely. Without limitation  FIGS. 18 and 19  illustrate embodiments for controlling the temperature of the chamber. The chamber shown in  FIG. 18  includes a plate  360  coupled to the top of the cover. The plate may be preheated or cooled, as desired, to control the temperature or compensate for the external air temperature surrounding the chamber.  FIG. 19  shows a thermal pad  370  resting on an upper portion of the chamber. The thermal pad or tape  370  includes leads  372  that deliver a power supply to thermal pad. Those skilled in the art will appreciate that the pad or tape  370  may be made integral with the cover. The other components of the chamber, including port or filter  208  remain functional and operate as shown and described herein. 
         [0048]    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.