Microfluidic Device And Method For Generating A Skin Construct

Abstract

A device and method are provided for generating a skin construct. The device includes a body having a chamber formed therein. The second portion of the chamber is filled with a polymerizable material and the material is allowed to polymerize within the second portion of the chamber. A cell media is deposited in the first portion of the chamber and a first portion of the cell media forms a skin construct on the polymerized material. The third portion of the chamber is filled with a media which contacts the polymerized material. A second portion of the cell media is removed from the first portion of the chamber such that ambient air in the first portion of the chamber forms an interface with the skin construct. The body may be rotated so as to allow the skin construct to be formed on the polymerized material and to be imaged with an imaging device.

Description

FIELD OF THE INVENTION

This invention relates generally to microfluidic devices, and in particular, to a microfluidic device and method for generating a skin construct.

BACKGROUND AND SUMMARY OF THE INVENTION

Research into new therapies for skin diseases is challenging for a number of reasons. As is known, skin cells are exposed to both atmospheric air and liquids within the body. However, conventional cell culture models do not accurately mimic the in vivo conditions necessary for insuring the reliability, validity, and transferability of the research. As such, dermatology and pulmonary researchers have turned to air-liquid interface systems to allow the researchers to more accurately mimic in vivo conditions.

Typically, an air-liquid interface system utilizes a transwell plate having inserts positioned in each well. The insert divides each well in upper and lower compartments, which are separated by a porous filter support. The lower compartment is filled with a liquid media and cells are cultured on porous filter support. It can be appreciated that one side of the cell culture is continuously exposed to the liquid media in the second compartment, while the opposite side of the cell culture is surrounded by air. This arrangement, in turn, allows the researcher to produce a skin layer in an environment that more closely resembles in vivo conditions.

While functional for their intended purpose, these prior air-liquid interface systems have certain limitations. For example, air-liquid interface system utilizing transwell plates have a limited capacity for multiple assays due in part to the difficulty of extracting cells from them. Further, transwell plates are expensive to manufacture, the manufacturing process is slow, and the transwell plates do not allow for easy imaging in 3-dimensions. Consequently, there exists a need to provide an air-liquid interface system for generating optically transparent skin that is simple and inexpensive to manufacture and allows for easy 3-dimensional imaging.

Therefore, it is primary object and feature of the present invention to provide a microfluidic device and method for generating a skin construct.

It is a further object and feature of the present invention to provide a microfluidic device and method for generating a skin construct in an environment that more closely resembles in vivo conditions.

It is a still further object and feature of the present invention to provide a microfluidic device and method for generating a skin construct that is simple and inexpensive to manufacture and allows for easy 3-dimensional imaging.

In accordance with the present invention, a device is provided for generating a skin construct. The device includes a body having a chamber formed therein. The chamber is defined by opposing first and second surfaces interconnected and spaced by an inner surface. The chamber includes a first portion adapted for receiving a cell culture therein, a second portion adapted for receiving a polymerizable material therein and a third portion adapted for receiving a media therein. A first flow restriction projects from the inner surface between the first and second portions of the chamber. The first flow restriction is configured to discourage flow of polymerizable material from the second portion of the chamber to the first portion of the chamber. A second flow restriction projects from the inner surface between the second and third portions of the chamber. The second flow restriction is configured to discourage flow of polymerizable material from the second portion of the chamber to the third portion of the chamber. A cell port extends through the first surface and communicates with the first portion of the chamber. A material port extends through the first surface and communicates with the second portion of the chamber. A media port extends through the first surface and communicates with the third portion of the chamber. The first, second and third portions of the chamber have corresponding lengths. The length of the second portion of the chamber is less than the length of the first portion of the chamber. Similarly, the length of the second portion of the chamber is less than the length of the third portion of the chamber.

The first flow restriction is defined by first and second projections extending from the inner surface towards each other and being spaced by a length. The length between the first and second projections is less than the length of the second chamber. Similarly, the second flow restriction is defined by first and second projections extending from the inner surface towards each other and being spaced by a length. The length between the first and second projections of the second flow restriction is less than the length of the second chamber.

The first, second and third portions of the chamber also have corresponding widths. The width of the second portion of the chamber is greater than the width of the first portion of the chamber. In addition, the width of the second portion of the chamber is greater than the width of the third portion of the chamber.

In accordance with a further aspect of the present invention, a method is provided for generating a skin construct. The method includes the step of providing a chamber in a body. The chamber has first, second and third portions. The second portion of the chamber is filed with a polymerizable material and the material is allowed to polymerize within the second portion of the chamber. A cell media is deposited in the first portion of the chamber. A first portion of the cell media forms a skin construct on the polymerized material. The third portion of the chamber is filed with a media. The media contacts the polymerized material.

A second portion of the cell media is removed from the first portion of the chamber such that ambient air in the first portion of the chamber forms an interface with the skin construct. A flow of polymerizable material into the first portion of the chamber as the second chamber is filled with the polymerizable material is discouraged and a flow of polymerizable material into the third portion of the chamber as the second chamber is filled with the polymerizable material is discouraged.

In accordance with a still further aspect of the present invention, a method is provided for generating and imaging a skin construct. The method includes the step of providing a chamber in a body. The chamber is defined between a first wall and a second wall, and has first, second and third portions. The body is orientated such that the second wall lies in a generally horizontal plane. The second portion of the chamber is filled with a polymerizable material and the material is allowed to polymerize within the second portion of the chamber. A cell media is deposited in the first portion of the chamber and the body is rotated such that the first and second wall lie in generally vertical planes. A first portion of the cell media forms a skin construct on the polymerized material. The third portion of the chamber is filled with a media. The media contacts the polymerized material. A second portion of the cell media is removed from the first portion of the chamber such that ambient air in the first portion of the chamber forms an interface with the skin construct. The skin construct is imaged with an imaging device.

The body includes a cell port extending through the first wall and communicating with the first portion of the chamber. A material port extends through the first surface and communicates with the second portion of the chamber. A media port extends through the first surface and communicates with the third portion of the chamber. It is contemplated to discourage a flow of polymerizable material in the first portion of the chamber as the second chamber is filled with the polymerizable material and to discourage a flow of polymerizable material in the third portion of the chamber as the second chamber is filled with the polymerizable material. More specifically, the body may include at least one projection extending into chamber. The at least one projection discourages the flow of polymerizable material into the first portion of the chamber as the second chamber is filled with the polymerizable material. In addition, the at least one projection discourages the flow of polymerizable material into the third portion of the chamber as the second chamber is filed with the polymerizable material.

The first, second and third portions of the chamber have corresponding lengths. The length of the second portion of the chamber is less than the length of the first portion of the chamber. Similarly, the length of the second portion of the chamber is less than the length of the third portion of the chamber. In addition, the first, second and third portions of the chamber have corresponding widths. The width of the second portion of the chamber is greater than the width of the first portion of the chamber and the width of the second portion of the chamber is greater than the width of the third portion of the chamber.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings furnished herewith illustrate a preferred construction of the present invention in which the above advantages and features are clearly disclosed as well as others which will be readily understood from the following description of the illustrated embodiment.

In the drawings:

FIG. 1 is a top plan view of well plate including a microfluidic device in accordance with the present invention;

FIG. 2 is an isometric view of a microfluidic device in accordance with the present invention;

FIG. 3 is a cross-sectional view of the microfluidic device of the present invention taken along line 3-3 of FIG. 2;

FIG. 4 is a cross-sectional view of the microfluidic device of the present invention taken along line 4-4 of FIG. 3;

FIG. 5A is a cross-sectional view, similar to FIG. 4, showing an initial step for effectuating the methodology of the present invention;

FIG. 5B is a cross-sectional view of the microfluidic device, similar to FIG. 5A, showing a subsequent step for effectuating the methodology of the present invention;

FIG. 5C is a cross-sectional view of the microfluidic device, similar to FIG. 5B, showing a further step for effectuating the methodology of the present invention;

FIG. 5D is a cross-sectional view of the microfluidic device, similar to FIG. 5C, showing a still further step for effectuating the methodology of the present invention;

FIG. 5E is a cross-sectional view of the microfluidic device, similar to FIG. 5D, showing a still further step for effectuating the methodology of the present invention;

FIG. 5F is a cross-sectional view of the microfluidic device, similar to FIG. 5E, showing a still further step for effectuating the methodology of the present invention; and

FIG. 6 is a cross-sectional view of the microfluidic device of the present invention, similar to FIG. 3, showing a skin construct fabricated in the microfluidic device.

DETAILED DESCRIPTION OF THE DRAWINGS

Referring to FIG. 1, a well plate adapted for receiving a plurality of microfluidic devices for generating a skin construct is generally designated by the reference numeral 10. It is contemplated for well-plate 10 to include a predetermined number of microfluidic devices 12 formed in outer surface 14 thereof. In the depicted embodiment, well plate 10 includes twenty (20) microfluidic devices arranged in rows and columns, however, microfluidic devices 12 in well plate 10 may be arrange in a different configurations and the well plate 10 may include any number of microfluidic devices 12 therein without deviating from the scope of the present invention. As hereinafter described, microfluidic devices 12 are identical in structure, and as such, the description of microfluidic device 12 hereinafter provided is intended to fully describe each of the microfluidic devices 12 formed in well plate 10, as if fully described herein.

It is contemplated for microfluidic devices 12 to be fabricated within well plate 10 or may be fabricated individually and deposited within a corresponding well 18 in outer surface 14 of well plate 10. Referring to FIGS. 2-4, each microfluidic device 12 is defined by outer wall 20 having upper and lower ends 22 and 24, respectively. Outer wall 20 may be of uniform thickness or have varying thicknesses without deviating from the scope of the present invention. Lower end 24 of outer wall 20 of microfluidic device 12 is closed by end wall 26 having inner and outer surfaces 28 and 30, respectively. It is contemplated for end wall 26 to be removably connected to lower end 24 of outer wall 20 of microfluidic device 12 to facilitate access to the interior of microfluidic device 12.

Access wall 32 extends radially inward from inner surface 34 of outer wall 20 adjacent upper end 22 thereof and lies in a place generally parallel to end wall 26. Inner surface 36 of access wall 32, inner surface 28 of end wall 26, and inner surface 34 of outer wall 20 define chamber 40 therebetween. As hereinafter described, chamber 40 is defined by first portion 42 adapted for receiving a cell culture therein, second portion 44 adapted for receiving a polymerizable material, e.g. a hydrogel, therein and third portion 46 adapted for receiving a media, e.g. a growth media or nutrients, therein. As best seen in FIG. 4, first portion 42 of chamber 40 is defined by corresponding first portion 48 of inner surface 34 of outer wall 20, second portion 44 of chamber 40 is defined by corresponding second portion 50 of inner surface 34 of outer wall 20, and third portion 46 of chamber 40 is defined by corresponding third portion 52 of inner surface 34 of outer wall 20.

Referring to FIGS. 3-4, chamber 40 has a depth DC in the range of 0.1 millimeters (mm) to 1 mm; an overall length LC, preferably, in the range of 5 mm to 20 mm; and an overall width WC, preferably, in the range 1.2 mm to 5 mm. Depth DC of chamber 40 ensures the skin construct to be formed, FIGS. 5F and 6, has adequate surface area to provide an air-liquid interface, as hereinafter described. First, second and third portions 42, 44 and 46, respectively, of chamber 40 have corresponding lengths L1, L2 and L3, respectively, and corresponding widths W1, W2 and W3, respectively. Preferably, length L2 of second portion 44 of chamber 40 is less than length L1 of first portion 42 of chamber 40 and length L2 of second portion 44 of chamber 40 is less than length L3 of third portion 46 of chamber 40. By way of example, length L1 of first portion 42 of chamber 40 is in the range of 2 mm to 15 mm; length L2 of second portion 44 of chamber 40 is in the range of 2 mm to 10 mm; and length L3 of third portion 46 of chamber 40 is in the range of 5 mm to 20 mm. Lengths L1, L2 and L3 of first, second and third portions 42, 44 and 46, respectively, of chamber 40 are broadly selected to recapitulate physiology of human skin, as hereinafter described.

It is contemplated for width W2 of second portion 44 of chamber 40 is greater than width W1 of first portion 42 of chamber 40 and width W2 of second portion 44 of chamber 40 is less than width W3 of third portion 46 of chamber 40. By way of example, width W1 of first portion 42 of chamber 40 is in the range of 0.25 mm to 2 mm; width W2 of second portion 44 of chamber 40 is in the range of 0.6 mm to 3 mm; and width W3 of third portion 46 of chamber 40 is in the range of 0.25 mm to 2 mm. Widths W1, W2 and W3 of first, second and third portions 42, 44 and 46, respectively, of chamber 40 are selected to maximize surface area for cell adherence and nutrient diffusion, as hereinafter described.

First portion 48 of inner surface 34 of outer wall 20 includes opposing, first and second end faces 60 and 62, respectively, which are generally parallel. First end face 60 extends between first and second terminal edges 60a and 60b, respectively. Similarly, second end face 62 extends between first and second terminal edges 62a and 62b, respectively. Side face 64 is generally perpendicular to first and second end faces 60 and 62, respectively, and terminates at first and second terminal edges 64a and 64b, respectively. First arcuate face 66 extends between first terminal edge 60a of first end face 60 and first terminal edge 64a of side face 64 so as to define cell port 70 of first portion 42 of chamber 40. Second arcuate face 68 extends between first terminal edge 62a of second end face 62 and second terminal edge 64b of side face 64 so as to define cell port 72 of first portion 42 of chamber 40.

First portion 48 of inner surface 34 of outer wall 20 further includes first and second restriction faces 74 and 76, respectively, which lie in a common plane generally parallel to and spaced from side face 64. More specifically, first restriction face 74 projects from second terminal edge 60b of first end face 60 and second restriction face 76 projects from second terminal edge 62b of second end face 62. Terminal edges 74a and 76a of first and second restriction faces 74 and 76, respectively, define an opening 78 between first and second portions 42 and 44, respectively, of chamber 40 so as to allow communication therebetween. Opening 78 has a length L4 less than length L2 of second portion 44 of chamber 40, and preferably, in the range of 2 mm to 15 mm, such that first and second restriction faces 74 and 76, respectively, act as pinning features to ensure polymerizable material received in second portion 44 of chamber 40 is constrained therein and does not invade first portion 42 of chamber 40, as hereinafter described.

Second portion 50 of inner surface 34 of outer wall 20 includes opposing, first and second end faces 80 and 82, respectively, which are generally parallel to each other. First end face 80 extends between first and second terminal edges 80a and 80b, respectively. Similarly, second end face 82 extends between first and second terminal edges 82a and 82b, respectively. Angular faces 84 and 86, respectively, project from corresponding first and second terminal edges 80a and 80b, respectively, of first end face 80 and diverge from each other. Angular face 84 terminates at and intersects terminal edge 74a of first restriction face 74. It can be understood that angular face 84 and first restriction face 74 define an acute angle therebetween. Angular face 86 terminates at terminal edge 86a.

Angular faces 88 and 90, respectively, project from corresponding first and second terminal edges 82a and 82b, respectively, of second end face 82 and diverge from each other. Angular face 88 terminates at terminal edge 76a of second restriction face 76. It can be understood that angular face 88 and second restriction face 76 define an acute angle therebetween. Angular face 90 terminates at terminal edge 90a. Terminal edges 88a and 90a of angular faces 88 and 90, respectively, define an opening 92 between second and third portions 44 and 46, respectively, of chamber 40 so as to allow communication therebetween.

Third portion 52 of inner surface 34 of outer wall 20 includes side face 94 which is generally parallel to side face 64 and terminates at first and second terminal edges 94a and 94b, respectively. Third portion 52 of inner surface 34 of outer wall 20 further includes first and second restriction faces 96 and 98, respectively, which lie in a common plane and generally parallel to and spaced from side face 94. More specifically, first restriction face 96 projects from terminal edge 88a of angular face 88 and terminates at terminal edge 96a. Second restriction face 98 projects from terminal edge 90a of angular face 90 and terminal edge 98a. Opening 92 has a length L5 less than length L3 of third portion 46 of chamber 40, and preferably, in the range of 2 mm to 15 mm, such that first and second restriction faces 96 and 98, respectively, act as pinning features to ensure polymerizable material received in second portion 44 of chamber 40 is constrained therein and does not invade third portion 46 of chamber 40.

First arcuate face 100 extends between first terminal edge 94a of side face 94 and terminal edge 96a of first restriction face 96 so as to define media port 102 of third portion 46 of chamber 40. Second arcuate face 104 extends between second terminal edge 94b of side face 94 and terminal edge 98a of second restriction face 98 so as to define media port 106 of third portion 46 of chamber 40.

Referring to FIG. 2, access wall 32 includes apertures 110 and 112, respectively, which are axially aligned with and communicate with corresponding first and second cell ports 70 and 72, respectively, of first portion 42 of chamber 40. Media port or aperture 114 extends through access wall 32 and communicates with second portion 44 of chamber 40. Apertures 116 and 118, respectively, which are axially aligned with and communicate with corresponding media ports 102 and 106, respectively, of third portion 46 of chamber 40. It is contemplated for apertures 110, 112, 114, 116, and 118 to have diameters in the range of 0.1 mm to 2 mm to ensure ease of loading of first, second, and third portions 42, 44 and 46, respectively, of chamber 40, as hereinafter described.

Referring to FIGS. 5A-5F, in operation, microfluidic device 12 (or well-plate 10 housing microfluidic device 12) is positioned such that end wall 26 is lies in a generally horizontal plane, FIG. 5A. Polymerizable material 120 is loaded into second portion 44 of chamber 40 through aperture 114 in access wall 32 in any conventional matter, for example, using pipette 122, FIG. 5B. As noted above, first and second restriction faces 74 and 76, respectively, act as pinning features to ensure polymerizable material 120 received in second portion 44 of chamber 40 is constrained therein and does not invade first portion 42 of chamber 40. Similarly, first and second restriction faces 96 and 98, respectively, act as pinning features to ensure polymerizable material 20 received in second portion 44 of chamber 40 is constrained therein and does not invade third portion 46 of chamber 40.

After a time period, e.g. fifteen (15) minutes, polymerizable material 120 received in second portion 44 of chamber 40 polymerizes. Once polymerizable material 120 received in second portion 44 of chamber 40 is polymerized, cell culture 124 is loaded into first portion 42 of chamber 40 at least one of apertures 110 and 112, respectively, (e.g., aperture 112) through access wall 32, in any conventional matter, for example, using pipette 126, FIG. 5C. Once cell culture 124 is loaded into first portion 42 of chamber 40, microfluidic device 12 is rotated 90 degrees such that end wall 26 of microfluidic device 12 is lies in a generally vertical plane, FIG. 5D. It can be appreciated that cell culture 124 contacts polymerized material 120 through opening 78 between first and second portions 42 and 44, respectively, of chamber 40. Cells 128 in cell culture 124 are allowed to settle and adhere to polymerizable material 120.

As cells 128 in cell culture 124 settle and adhere to polymerizable material 120, media 130 is loaded into third portion 46 of chamber 40 through at least one of apertures 116 and 118, respectively, in access wall 32, in any conventional matter, for example, using pipette 132, FIG. 5E. With media 130 in third portion 46 of chamber 40, It can be appreciated understood that media 130 in third portion 46 of chamber 40 contacts polymerized material 120 in second portion 44 of chamber 40 through opening 92 between second and third portions 44 and 46, respectively, of chamber 40. Media 130 then permeates polymerized material 120, for reasons hereinafter described.

Once cells 128 in cell culture 124 settle and adhere to polymerized material 120, a layer or skin construct 134 of cells 128 is formed along the intersection of cell culture 124 and polymerizable material 120. Thereafter, cell culture 124 in first portion 42 of chamber 40 is removed therefrom, e.g. using a vacuum or the like, thereby establishing an air interface between skin construct 134 and the environment within first portion 42 of chamber 40. Similarly, media 130 permeating polymerized material 120 communicates with skin construct 134 through opening 78 between first and second portions 42 and 44, respectively, of chamber 40, thereby forming a liquid interface between skin construct 134 and media 130 permeating polymerized material 120 in second portion 44 of chamber 40.

As described, it can be appreciated that one side of skin construct 134 is continuously exposed to media in the second portion of 44 of chamber 40, while the opposite side of skin construct 134 is surrounded by air. This arrangement, in turn, provides a researcher with an environment for skin construct 134 that more closely resembles in vivo conditions. Thereafter, image device 140 may be used to image skin construct 134 from various locations to allow a user to consider the effects of the media on skin construct 134.

Various modes of carrying out the invention are contemplated as being within the scope of the following claims particularly pointing out and distinctly claiming the subject matter that is regarded as the invention.

Claims

1. A device for generating a skin construct, comprising a body including: a chamber formed therein and being defined by opposing first and second surfaces interconnected and spaced by an inner surface, the chamber including a first portion adapted for receiving a cell culture therein, a second portion adapted for receiving a polymerizable material therein and a third portion adapted for receiving a media therein;a first flow restriction projecting from the inner surface between the first and second portions of the chamber, the first flow restriction configured to discourage flow of polymerizable material from the second portion of the chamber to the first portion of the chamber; anda second flow restriction projecting from the inner surface between the second and third portions of the chamber, the second flow restriction configured to discourage flow of polymerizable material from the second portion of the chamber to the third portion of the chamber.

2. The device of claim 1 further comprising a cell port extending through the first surface and communicating with the first portion of the chamber.

3. The device of claim 1 further comprising a material port extending through the first surface and communicating with the second portion of the chamber.

4. The device of claim 1 further comprising a media port extending through the first surface and communicating with the third portion of the chamber.

5. The device of claim 1 wherein the first, second and third portions of the chamber have corresponding lengths, the length of the second portion of the chamber being less than the length of the first portion of the chamber.

6. The device of claim 1 wherein the first, second and third portions of the chamber have corresponding lengths, the length of the second portion of the chamber being less than the length of the third portion of the chamber.

7. The device of claim 5 wherein the first flow restriction is defined by first and second projections extending from the inner surface towards each other and being spaced by a length, the length between the first and second projections being less than the length of the second chamber.

8. The device of claim 7 wherein the second flow restriction is defined by first and second projections extending from the inner surface towards each other and being spaced by a length, the length between the first and second projections of the second flow restriction being less than the length of the second chamber.

9. The device of claim 1 wherein the first, second and third portions of the chamber have corresponding widths, the width of the second portion of the chamber being greater than the width of the first portion of the chamber.

10. The device of claim 1 wherein the first, second and third portions of the chamber have corresponding widths, the width of the second portion of the chamber being greater than the width of the third portion of the chamber.

11. A method for generating a skin construct, comprising the steps of: providing a chamber in a body, the chamber having first, second and third portions;filing the second portion of the chamber with a polymerizable material and allowing the material to polymerize within the second portion of the chamber;depositing a cell media in the first portion of the chamber, a first portion of the cell media forming a skin construct on the polymerized material; andfilling the third portion of the chamber with a media, the media contacting the polymerized material.

11. The method of claim 10 comprising the additional step of removing a second portion of the cell media from the first portion of the chamber such that ambient air in the first portion of the chamber forms an interface with the skin construct.

12. The method of claim 10 comprising the additional step of discouraging a flow of polymerizable material into the first portion of the chamber as the second chamber is filled with the polymerizable material.

13. The method of claim 12 comprising the additional step of discouraging a flow of polymerizable material into the third portion of the chamber as the second chamber is filled with the polymerizable material.

14. A method for generating and imaging a skin construct, comprising the steps of: providing a chamber in a body, the chamber defined between a first wall and a second wall and having first, second and third portions;orientating the body such that the second wall lies in a generally horizontal plane;filing the second portion of the chamber with a polymerizable material and allowing the material to polymerize within the second portion of the chamber;depositing a cell media in the first portion of the chamber and rotating the body such that the first and second wall lie in generally vertical planes whereby a first portion of the cell media forming a skin construct on the polymerized material;filling the third portion of the chamber with a media, the media contacting the polymerized material;removing a second portion of the cell media from the first portion of the chamber such that ambient air in the first portion of the chamber forms an interface with the skin construct; andimaging the skin construct with an imaging device.

15. The method of claim 14 wherein the body includes: a cell port extending through the first wall and communicating with the first portion of the chamber;a material port extending through the first surface and communicating with the second portion of the chamber; anda media port extending through the first surface and communicating with the third portion of the chamber.

16. The method of claim 14 comprising the additional step of discouraging a flow of polymerizable material in the first portion of the chamber as the second chamber is filled with the polymerizable material.

17. The method of claim 16 comprising the additional step of discouraging a flow of polymerizable material in the third portion of the chamber as the second chamber is filled with the polymerizable material.

18. The method of claim 14 wherein the first, second and third portions of the chamber have corresponding lengths, the length of the second portion of the chamber being less than the length of the first portion of the chamber.

19. The method of claim 14 wherein the first, second and third portions of the chamber have corresponding lengths, the length of the second portion of the chamber being less than the length of the third portion of the chamber.

20. The method of claim 14 wherein the body includes at least one projection extending into chamber, the at least one projection discouraging a flow of polymerizable material into the first portion of the chamber as the second chamber is filled with the polymerizable material.

21. The method of claim 14 wherein the body includes at least one projection extending into chamber, the at least one projection discouraging a flow of polymerizable material into the third portion of the chamber as the second chamber is filed with the polymerizable material.

22. The method of claim 14 wherein the first, second and third portions of the chamber have corresponding widths, the width of the second portion of the chamber being greater than the width of the first portion of the chamber.

23. The method of claim 14 wherein the first, second and third portions of the chamber have corresponding widths, the width of the second portion of the chamber being greater than the width of the third portion of the chamber.