Abstract:
A cell culture device for inducing shear stress and on substrate strain on cells. The device includes a cell culture membrane and a flow pathway for moving fluid across cells growing on the membrane to apply shear stress on the cells.

Description:
RELATED APPLICATION 
     This application claims the benefit of U.S. Provisional Patent Application Serial No. 60/111,023, filed Dec. 4, 1998, entitled “Apparatus for Growing Cells in Culture Under Shear Stress and/or Strain.” 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The present invention is directed toward a cell culture assembly used in the biomedical science field of tissue engineering and, more specifically, to a cell culture assembly through which fluid may flow for applying deformations to cells that include fluid-induced stress or substrate-induced strain to cultured cells. 
     2. Prior Art 
     In the human body, many cells are constantly subjected to stress from fluid flow. Fluid flow in the body includes blood flow through the vasculature, lymph in the lymphatics, cerebrospinal fluid flow, any secretion in ducts, and also the movement of interstitial fluid in the matrix between and among cells in any tissue. Stressing cells in culture simulates the in vivo environment, causing dramatic morphologic changes and biomechanical responses in the cells. There are both long and short-term changes that occur when cells are stressed in culture, such as alterations in the rate and amount of protein expression and secretion, the rate of cell division and alignment, changes in energy metabolism, changes in rates of macromolecular synthesis or degradation, and other changes in biochemistry and bioenergetics. Prior devices have been developed for applying substrate deformation on cells and applying fluid-induced shear stress by subjecting the cells to fluid flow. However, none of these devices have allowed for alternating or simultaneous application of both types of mechanical loading of cells in vitro and for quantitation of the applied stresses and strains. 
     A need remains for a cell culture assembly in which cells may be cultured and subjected to fluid-induced shear stress which is precisely controlled. 
     SUMMARY OF THE INVENTION 
     Accordingly, I have developed a cell culture assembly including a body having a flow surface extending across an upper surface of the body. The top surface of the body may also be used as a flow surface on which cells may be cultured. Moreover, a flexible membrane may be clamped by the body and also be used as a flow surface on which cells may be cultured. This rubber membrane may also be deformed by vacuum so this cell receives substrate tension in unconstrained distension may be deformed by stretching across a planar faced post so that the flexible substrate is deformed equibiaxially. Positive pressure may also be applied to deform the flexible membrane upward to apply a compressive deformation to overlying cells cultured on the top member. Both fluid stress and substrate strain may also be delivered simultaneously as often occurs in blood vessels or in other tissues. 
     The body further defines a passageway in fluid communication with the flow surface and a cover member covering the flow surface. The flow surface of the body and the cover member thereby define a flow chamber through which fluid may flow. A cell culture surface is positioned on the flow surface or on the cover or both. Cells cultured on the cell culture surface are subject to shear stress when fluid flows through the passageway and the flow chamber. 
     In one embodiment of the invention, the body has an upper surface defining a first opening therethrough. The assembly further includes a base attached to the body and a cell culture membrane fixed between the base and the body whereby the membrane covers the first opening, such that when fluid flows through the body passageway, the fluid passes across the membrane thereby inducing shear stress on cells growing on the membrane. The body passageway includes a pair of bores defined in the body on opposing sides of the first opening, wherein each bore extends between a side of the body and the upper surface. The upper surface defines a pair of second openings, preferably in the form of slits, on opposing sides of the first opening and each second opening is in fluid communication with one of the bores. A gasket is positioned on the body upper surface and surrounds the first opening and the second openings. The gasket is configured to retain fluid flowing out of one of the second openings and into the other second opening. A port is defined in the body for connection to a pressure supply. The body upper surface further defines an annular channel in fluid communication with the port. The gasket overlies the channel and the cover overlies the gasket. The gasket defines a plurality of holes which overlie the annular channel such that the cover seats on the gasket when negative pressure is applied to the port. Alternatively, the upper surface may be clamped by overlying pressure to the gasket and body by conventional assemblies such as a plate and fasteners. 
     The base comprises an annular member defining a chamber and having a wall with a top surface on which the membrane is seated. An insert is received within the chamber. The insert includes a support member with a support surface for supporting a portion of the membrane. The wall of the base defines an aperture and the insert defines an insert passageway extending between a side of the insert and the insert support surface where the insert passageway is in fluid communication with the aperture of the base wall. When negative pressure is applied to the chamber through the aperture, the membrane is urged against the insert support surface. Preferably, the insert includes a post spaced apart from the support member thereby defining an annular gap between the post and the support member. An opening defined in the support member is in fluid communication with the gap. Preferably, an upper surface of the post is lower than the support surface and an upper surface of the portion of the membrane supported by the support surface is in a plane of the upper surface of the body. 
     In another embodiment of the invention, the body includes a flow member and a pair of end members attached to opposing ends of the flow member, where the opposing ends of the flow member each define a recess, and where each flow surface extends between the recesses in the ends of the flow member. In this arrangement, the openings are defined in the end members and are in fluid communication with the recesses. An annular channel is defined in an upper surface of the body and surrounds the flow surface. The body defines a port in fluid communication with the channel whereby when negative pressure is applied to the port, the cover is urged toward the body. A gasket defining an opening aligned with the flow surface and defining a plurality of holes therethrough is positioned between the body and the cover. The gasket opening overlies the flow surface and the holes overlie the channel, such that when negative pressure is applied to the port, the cover sealingly seats on the gasket and the gasket sealingly seats on the body. The flow chamber is defined by the gasket, the cover and the recess. The body may include a plurality of flow surfaces with the channel surrounding each flow surface defined in the body, and the gasket defining a plurality of openings each overlying a flow surface. Each end of the body then defines a plurality of openings aligned with each of the flow surfaces. 
    
    
     DESCRIPTION OF THE DRAWINGS 
     A complete understanding of the invention will be obtained from the following description when taken in connection with the accompanying drawing figures wherein like reference characters identify like parts throughout. 
     FIG. 1 is an exploded perspective view of a cell culture assembly made in accordance with the present invention including a body, an insert and a base; 
     FIG. 2 illustrates a perspective view of the cell culture assembly depicted in FIG. 1, partially assembled; 
     FIG. 3 is a plan view of the body shown in FIG. 1; 
     FIG. 4 is a partial sectional view of the body taken along lines IV—IV in FIG. 3; 
     FIG. 5 is a partial sectional view of the body taken along lines V—V in FIG. 3; 
     FIG. 6 is a plan view of the insert shown in FIG. 1; 
     FIG. 7 is a side view of the insert shown in FIG. 6; 
     FIG. 8 is a cross-sectional view of the insert taken along lines VIII—VIII shown in FIG. 6; 
     FIG. 9 is a plan view of an alternative insert made in accordance with the present invention; 
     FIG. 10 is an end view of the insert shown in FIG. 9; 
     FIG. 11 is a cross-sectional view of the insert taken along lines XI—XI in FIG. 9; 
     FIG. 12 is an exploded perspective view of another embodiment of a cell culture assembly made in accordance with the present invention including an insert; 
     FIG. 13 is a plan view of the insert shown in FIG. 12; 
     FIG. 14 is a side view of the insert shown in FIG. 13; 
     FIG. 15 is cross-sectional view of the insert taken along lines XV—XV in FIG. 13; 
     FIG. 16 is an exploded perspective view of another embodiment of the cell culture assembly made in accordance with the present invention including a cover, a gasket and a body; 
     FIG. 17 is a perspective view of the body shown in FIG. 16; 
     FIG. 18 is an exploded perspective view of the body shown in FIG. 17 including a flow member and end pieces; 
     FIG. 19 is a plan view of the flow member shown in FIG. 18; and 
     FIG. 20 is a cross-sectional view of the flow member taken along line XX—XX in FIG.  19 . 
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     For purposes of the description hereinafter, the terms “upper”, “lower”, “right”, “left”, “vertical”, “horizontal”, “top”, “bottom” and derivatives thereof shall relate to the invention as it is oriented in the drawing figures. However, it is to be understood that the invention may assume various alternative variations and step sequences, except where expressly specified to the contrary. It is also to be understood that the specific devices and processes illustrated in the attached drawings, and described in the following specification, are simply exemplary embodiments of the invention. Hence, specific dimensions and other physical characteristics related to the embodiments disclosed herein are not to be considered as limiting. 
     The present invention includes a cell culture assembly for applying shear stress to cells. One embodiment of the invention is the cell culture assembly  2  shown in FIGS. 1 and 2 which includes a body  10  through which fluid may flow. The body  10  shown in detail in FIGS. 2-5 includes an upper surface  12  which defines a central opening  14  surrounding a rim  15  centrally located in a central planar portion  16  of the upper surface  12 . The opening  14  and rim  15  are preferably rectangular in shape. The planar portion  16  is disposed between a pair of integrally formed housings  17 . Preferably, the body  10  is formed from aluminum, but stainless steel, lucite or other like materials may be used in the fabrication. 
     Referring to FIGS. 4 and 5, a bore  18  is defined in each of the housings  17  on opposing ends of the body  10 . Each bore  18  is in fluid communication with a slit  20  defined in the upper surface  12 . The bores  18  are each preferably internally threaded to accept a fitting  22 . The fittings  22  may constitute quick disconnect valves instead of the particular fitting shown in FIGS. 3 and 5. Also defined in the upper surface  12  is a continuous channel  24 , preferably having a rectangular configuration. The channel  24  surrounds the central opening  14  and the slits  20 . The shorter sides of the rectangular channel  24  are each in fluid communication with a vacuum opening  26  defined in each of the ends of the body  10 . The vacuum openings  26  in the ends of the body are preferably internally threaded to accept a vacuum fitting  28  at one end and a sealing nut  30  at the other end. Either end of the body  10  may accept either of the vacuum fitting  28  or the nut  30 . 
     Referring to FIGS. 1 and 2, overlying the body  10  is a gasket  40 , preferably having a configuration which coordinates with the configuration of the channel  24 . The gasket  40  shown in FIGS. 1 and 2 is rectangular in shape so that the gasket  40  covers the channel  24  while leaving exposed the slits  20  and the central opening  14 . The gasket  40  is preferably formed from a silicone rubber and includes a two-sided acrylic-silicone mastic (not shown) adhered to one side. The silicone side of the mastic is adhered to the gasket  40  and the acrylic side is adhered to the upper surface  12  of the body  10 . A plurality of apertures  42  is defined in the gasket  40  at locations whereby the apertures  42  are in overlying relation to the channel  24  defined in the upper surface  12  of the body  10 . The gasket  40  is shown in FIG. 2 as covering a portion of the upper surface  12 . However, the gasket may completely cover the entire upper surface  12  to enhance the seal formed thereby. A cover  50  such as a glass microscope slide is positioned over the gasket  40 . Upon application of a vacuum through one of the vacuum openings  26  in an end of the body  10  via the vacuum fitting  28 , air is drawn through the apertures  42  in the gasket  40 , through the channel  24  in the upper surface  12  and out through the vacuum opening  26  in the side of the body  10  and through the vacuum fitting  28 . This causes the cover  50  to be pulled down in sealing engagement with the gasket  40 . 
     Alternatively, the cover  50  may be clamped to the gasket  40  and body  10 . Conventional clamping assemblies may be used to clamp the cover  50  to the gasket  40  and the body  10 . 
     The cell culture assembly  2  further includes a base  60  as disclosed in co-pending U.S. application Ser. No. 09/201,570, now U.S. Pat. No. 5,048,723, entitled “Culture Plate for Applying Mechanical Load to Cell Cultures,” incorporated herein by reference. The body  60  has a wall  62 , preferably cylindrical in configuration, extending from a planar member  64  and defining a cylindrical well  66 . The planar member  64  defines a base opening  68  having a diameter which preferably is smaller than an inner diameter of the well  66 . The planar member  64  preferably is sized and configured for placement on the objective of a microscope. A groove  70  is defined in a top surface of the wall  62  and is sized to accept an O-ring  72 . The top surface of the wall  62  also defines a plurality of threaded holes  73  used in assembling the assembly  2  as described below. A spacer  74  preferably formed from silicone in a ring shape is positioned within the well  66  on the planar member  64 . A pressure differential supply fitting  76  extends outwardly from the wall  62  and communicates with the well  66  via an opening through the wall  62 . An insert  80  (described in detail below) is received within the well  66  and seats on the spacer  74 . 
     A flexible cell culture membrane  100  is disposed between the body  10  and the base  60 . The body  10  clamps the membrane  100  and the O-ring  72  against the top surface of the wall  62  using conventional means such as a plurality of screws  102  threaded through a perimeter of the body planar portion  16  and into the threaded holes  73  in the base  60 . A portion of the membrane  100  is exposed through the central opening  14 . Cells are culturable on the exposed portion of the membrane  100 . Alternatively, cells may be cultured on the cover  50 . The integrity of a seal formed between the body  10 , the membrane  100  and the base  60 , thereby forming a flow chamber, is in part achieved by use of the insert  80  as described below. 
     The insert  80  shown in FIG. 1 is shown in detail in FIGS. 6-8. The insert  80  includes a planar member  82  and a support member  84  bearing a support surface  86  extending therefrom. The support member  84  includes a ledge  87 . The ledge  87  is sized to accept and mate with the rim  15  of the planar portion  16  surrounding the central opening  14 . In this manner, the membrane  100  is clamped between the rim  15  of the body  10  and the ledge  87  of the insert  80 . The support member  84  defines an annular opening  88  surrounding a post  90 . As may be seen in FIG. 8, an upper surface of the post  90  is lower than the support surface  86 . A layer of lubricating material (not shown), such as silicone, may be positioned on the top of the post  90  such that the support surface  86  and surface of the lubricating material are in a common plane. A passageway  92  is defined in each of two opposing sides of the support member  84  with each passageway  92  being in fluid communication with the annular opening  88 . The post  90  may be one of a variety of geometrical shapes such as conical or frustoconical or have a constant rectangular or square cross-sectional configuration. 
     In operation, when a pressure differential, e.g., negative pressure, is applied to the pressure differential supply fitting  76 , air is drawn from the annular opening  88  through the passageways  92  and out the pressure differential supply fitting  76  so that the membrane  100  is urged downwardly over or upwardly above the support surface  86  and over the lubricating material covering the post  90 . Negative pressure is also applied to the vacuum fitting  28  on the body  10  to pull the cover  50  against the gasket  40  seated on the body  10 . Upon application of negative pressure to the base  60  and the body  10 , fluid at a selected flow rate is directed into one of the fittings  22 , through the corresponding bore  18 , up through the corresponding slit  20 , across the membrane  100  covering the central opening  14  and out through the opposing slit  20 , the other bore  18  and the other fitting  22 . The membrane  100  preferably extends through the central opening  14  in the body  10  so that the fluid flows smoothly across one portion of the upper surface  12  on one side of the central opening  14 , over cells growing on the membrane  100  and across the other portion of the upper surface  12 . In this manner, the amount of flow to induce stress on cells growing on the membrane  100  from fluid flow thereover may be altered and studied. The flow rate of fluid passing through the body  10  can be controlled and the shear stress on cells growing on the membrane  100  may be calculated. By varying the flow rate to the body  10 , varying degrees of shear stress may be applied to cells. Hence, the impact of shear stress on the cells can be determined quantitatively. The membrane  100  preferably is formed from a transparent material so that the entire assembly may be placed on a microscope. The effect of fluid flow and stress therefrom on cells growing on the membrane  100  may be actively studied. 
     Although not shown in the drawings, the assembly  2  preferably includes a fluid pump, preferably a peristaltic pump, one or more fluid pulse dampeners, a digital flow meter, valves to regulate fluid sampling of flow to the assembly  2  and a fluid reservoir. All components are connected with flexible tubing in a continuous or discontinuous flow loop. The fluid flowing through the assembly  2  may be recycled therethrough in a continuous loop. In this manner, substances secreted by the cultured cells interact with the cells. Alternatively, the fluid may not be returned to the assembly  2  so that secreted substances do not effect the cells. The assembly  2  may further include a sampling port into the fluid effluent leaving the body  10 . Fluid withdrawn can be biochemically analyzed. 
     Control of fluid flow is achieved by regulation of the pump flow rate, the bore size of the tubing and the opening and closing of a valve or valves positioned upstream of the flow chamber, downstream of the flow chamber or in both locations. Flow control may be achieved by regulation of the pump flow rate alone. The flow rate may range from picoliters to milliliters of total flow to continuous flow of fluid. Alternatively, the pump flow rate may be maintained at a constant rate and the valves may be opened and closed to direct fluid flow away from the flow chamber to provide rapid regulation of flow rate, particularly for rapid oscillations in the flow stream. Alternatively, the valves may be used to provide flow reversals to the flow chamber so that fluid enters the chamber from one direction at one instant then reverses direction and enters from the opposite side of the chamber at the next instant. These levels of flow control permits both continuous fluid flow, and discontinuous fluid flow, the latter as a pulsating flow or a flow reversal, as occurs in the vasculature, lymphatics and in interstitial fluid flow in tissues. The precise nature of the rate of fluid flow, and type of fluid flow may have unique consequences for the response(s) of the cells or tissue experiencing the deformation. This is particularly true when fluid flow is combined with substrate strain. This is an imperative point for cells that may be “conditioned” by this mechanical environment and transferred to a location in the body in a tissue engineering application where they must withstand the rigors of the mechanically active environment of the body. 
     FIGS. 9-11 show an alternative insert  180 . The insert  180  includes a support member  184  extending from a planar member  82  and including a substantially planar support surface  186  and a ledge  187 . The support surface  186  is preferably rectangular in configuration. A plurality of, preferably two, bores  188  extends longitudinally through the support member  184 . A plurality of, preferably four, holes  192  is defined in the support surface  186  and communicates with the bores  188 . When insert  180  is used instead of the insert  80  in the assembly  2 , the membrane  100  is clamped between the rim  15  of the body  10  and the ledge  187  of the insert  180  thereby maintaining the membrane  100  flat across the support surface  186 . Negative pressure applied to the fitting  76  pulls air through the holes  192  and out through the bores  188 , the well  66  and the fitting  76 . The insert  180  serves to ensure uniformity in the flatness of the membrane  100 . 
     Another embodiment of the invention is the cell culture assembly  202  shown in FIG.  12 . The cell culture assembly  202  includes a cover  50 , a gasket  40 , a membrane  100 , an O-ring  72  and a base  60  similar to those components described above in connection with assembly  2 . However, assembly  202  includes a body  210  having an upper surface  212  which defines a central opening  214  surrounding a rim  215  which is preferably circular in configuration and centrally located in a central portion  216 . All other components of the body  210  are similar to those of the body  10 . 
     An insert  280  is received within the well  66 . As shown in more detail in FIGS. 13-15, the insert  280  has a support member  284  with a preferably overall cylindrical shape. A ring-shaped support surface  286  stepped up from a ledge  287  supports the membrane  100  when the body  210  and the base  60  are clamped together with the membrane  100  therebetween. The ledge  287  is sized to accept and mate with the rim  215  of the body  210  surrounding the central opening  214 . In this manner, the membrane  100  is clamped between the central portion  216  and the ledge  287 . Cells may be cultured on the portion of the membrane  100  which is exposed through the central opening  214 . The support member  284  defines an annular opening  288  surrounding a post  290 . As can be best seen in FIG. 15, an upper surface of the post  290  is lower than the support surface  286 . As is true for insert  80 , a layer of lubricating material (not shown) may be placed on the top of the post  290  such that the support surface  286  and surface of the lubricating material are in a common plane. A plurality of, preferably four, holes  292  are defined in the support member  284  and communicate with the annular opening  288  and the well  66 . When negative pressure is applied to the fitting  76 , air is drawn from the annular opening  288  out through the holes  292 , the well  66  and the fitting  76  to pull the membrane  100  against the support surface  186 . 
     Yet another embodiment of the invention is shown in FIGS. 16-20. The cell culture assembly  302  shown in FIGS. 16 and 17 includes a preferably rectangular shaped body  310 , a gasket  340  and a cover  350 . 
     As shown in FIG. 18, the body  310  preferably includes a multi-sectional flow member  312  and a pair of end pieces  314 . The end pieces  314  are fixed to the flow member  312  using screws  316  (FIGS. 16 and 17) or other conventional securing mechanisms. The flow member  312  includes a top surface  318  and a pair of opposing end surfaces  320  (only one being shown). The top surface  318  includes a plurality of flow surfaces  321 . A plurality of recesses  322  corresponding in number to the number of flow surfaces  321  is defined in each of the end surfaces  320  and are in fluid communication with the flow surfaces  321 . The end pieces  314  each define a plurality of openings  324  and have a top surface  325 . Each opening  324  is aligned with one of the recesses  322  and is internally threaded to accept a fitting  326  (FIGS.  16  and  17 ). When the flow member  312  and the end pieces  314  are assembled as shown in FIG. 17, a plurality of pathways are formed for fluid to flow in the direction shown by arrow A via each set of fittings  326 , openings  324 , recesses  322  and flow surfaces  321  and out through the corresponding recesses  322 , openings  324  and fittings (not shown) in the opposite end of the body  310 . 
     A network of channels  328  having flow slits  329  is defined in the top surface of the body  310 . The network of channels  328  is continuous when the body  310  is fully assembled as shown in FIGS. 16 and 17. The flow member  312  has channels  328  which extend parallel to the flow surfaces  321  along the length of the flow member  312 . The end pieces  314  each have parallel channels  328  which mate with the channels  328  in the flow member  312  and a channel  328  perpendicular thereto which connects together each of the parallel channels  328 . As best shown in FIGS. 18 and 20, an opening  332  is defined in the flow member  312  and extends between opposing sides of the flow member  312 . The opening  332  is in fluid communication with the channels  328 . A vacuum fitting  334  (FIGS. 16 and 17) is received in one end of the opening  332  and a sealing nut (not shown) is received in the other end of the opening  332 . 
     The gasket  340 , similar in material construction to gasket  40  such as molded silicone rubber, is placed over the top surfaces  318  and  325  to cover the network of channels  328 . The gasket  340  defines a plurality of apertures  342  at spaced apart locations. When the gasket  340  is seated on the body  310 , the apertures  342  are in overlying relation to the flow slits  329  in the channels  328 . The gasket  340  also defines a plurality of flow openings  344  corresponding in number to the number of flow surfaces  321  of the flow member  312 . Each flow opening  344  overlies one of the flow surfaces  321  of the flow member  312 . The cover  350 , preferably made of glass or Mylar, is positioned over the gasket  340 . When assembled together, each set of a flow surface  321 , gasket flow opening  344  and the cover  350  forms a flow chamber in fluid communication at the ends thereof with recesses  322 . The thickness of the gasket  340  may be selected to provide a desired volume of fluid flowable through each flow opening  344 . The cover  350  may also be clamped to the body  310  using conventional external clamps. The cover  350  may be sized to completely cover the body  310  (as shown in FIG. 16) or be sized to cover only the gasket  340 . 
     In operation, fluid is supplied to the fittings  326  and flows through openings  324  in one end piece  314  and into the recesses  322  on one end of the flow member  312 , over the flow surfaces  321  thereby filling the flow openings  344 , into the other recesses  322  on the opposite end of the flow member  312  and out through the openings  324  in the other end piece  314 . Negative or positive pressure is applied to the vacuum fitting  334  which draws air through the apertures  342  and out through the opening  332  and vacuum fitting  334 . The cover  350  is pulled onto the gasket  340  thereby sealing the flow chambers formed at each flow opening  344 . Cells may be cultured directly on the flow surfaces  321  or on an underside of the cover  350 . Alternatively, a cell culture membrane (not shown), preferably formed from silicone, may be adhered to the underside of the cover  350 . The flow rate of fluid applied to each flow chamber may be varied. One or more of the flow chambers may be used at one time. The flow may be continuous in one direction, the flow may be pulsed or the flow may be occasionally or periodically reversed as described above with respect to the assembly  2 . In this manner, a variety of stresses may be applied to cultures of cells grown side-by-side in the assembly  302 . 
     An example of the relative dimensions of the components of the assembly  302  are as follows. When the flow member  312  is 6 inches wide and 3.15 inches long, the flow surfaces  321  are 0.65 inch wide. The channels  328  are 0.25 inch wide and the slits  329  are 0.05 inch wide. The recesses  322  extend 1.125 inch downwardly and have a depth of 0.06 inch. The opening  332  is centered 0.15 inch down from the top surface of the flow member  312 . 
     It will be readily appreciated by those skilled in the art that modifications may be made to the invention without departing from the concepts disclosed in the foregoing description. Such modifications are to be considered as included within the following claims unless the claims, by their language, expressly state otherwise. Accordingly, the particular embodiments described in detail herein are illustrative only and are not limiting to the scope of the invention which is to be given the full breadth of the appended claims and any and all equivalents thereof.