Abstract:
The present invention relates to a device and method for facilitating high throughput transcellular flux testing of compounds, such as pharmaceuticals or drugs, other compounds, or compound combinations. In one embodiment, the system and methods of the present invention may be used to identify the optimal components (e.g., solvents, carriers, transport enhancers, adhesives, additives, inhibitors, or other excipients) for pharmaceutical compositions or formulations that are delivered to a patient via tissue transport, including without limitation, pharmaceutical compositions or formulations administered or delivered transcellularly, topically, and ocularly.

Description:
RELATED APPLICATIONS  
       [0001]     This application claims the benefit of U.S. Provisional Application No. 60/441,358 filed Jan. 21, 2003. 
     
    
     TECHNICAL FIELD OF THE INVENTION  
       [0002]     Generally this invention relates to a device and method for in vitro testing. More specifically, this invention relates to a device and method for high throughput transcellular testing.  
       DESCRIPTION OF RELATED ART  
       [0003]     Currently, there are numerous incurable diseases and new diseases and new forms of diseases are being discovered often. Accordingly, research and development of new and more effective drugs and pharmaceuticals is highly important. One important aspect of drug and pharmaceutical research and development relates to methods for delivering or administering drugs into a patient.  
         [0004]     Traditional routes of drug administration include inhalation, intranasal, oral, rectal, vaginal, topical, infusion, and injection. A relatively recent advancement in drug administration is the administration of a drug directly across the skin of a patient, otherwise known as transdermal drug delivery. Typically, in transdermal delivery a drug is positioned on the outermost layer of a patient&#39;s skin (epidermis) and thereafter transfers through the skin and into the patient&#39;s body, typically via simple diffusion.  
         [0005]     Transdermal delivery is a desirable delivery technique as it offers many advantages over other methods of drug delivery. For example, an advantage over injection delivery is the reduction of contamination and the ease of disposal, as compared to traditional syringe needles. In addition, the unpleasantness of receiving injections is avoided, leading to improved patient compliance of drug regimens. Furthermore, transdermal delivery is particularly useful for drugs that require repeated administration, such as insulin for diabetes, or the like.  
         [0006]     Another advantage of transdermal drug delivery is the ability to maintain a constant drug concentration within the body over a long period of time, such as several days or weeks. Other delivery methods, such as oral or pulmonary delivery, typically require the drug to be administered repeatedly to sustain the proper drug concentration within the body. With traditional drug delivery methods concentration of the drug in the body spikes to a high level shortly after administration. These “spikes” can cause toxicity problems, thereby, making some otherwise viable drugs a less preferred treatment option. Unlike traditional drug delivery, transcellular drug delivery delivers a substantially constant flow of drug to the body over an extended period of time from days to weeks, thereby reducing the toxicity problems.  
         [0007]     Another advantage of drugs administered using transdermal delivery is that they bypass the first-pass metabolism in the liver and avoid other degradation pathways such as the low pH&#39;s and enzymes present in the gastrointestinal tract. These biological barriers are avoided because transdermally administered drugs diffuse through the skin and directly into the blood stream without passing through the gastrointestinal tract.  
         [0008]     An example of a transdermal drug delivery system currently in use is the D-Trans® system made by ALZA Corp. Mountain View, Calif. The D-Trans® system typically incorporates a series of thin, flexible films which include a backing layer, a drug reservoir, a rate-controlling film, and an adhesive. One drug this system is effective in delivering is nicotine. Two ALZA Corp. products, NicoDerm® CQ® and Clear NicoDerm® CQ® deliver nicotine to patients through the D-Trans® system to control nicotine withdrawal incurred by individuals attempting to quit smoking. In use, the nicotine stored in the drug reservoir transfers through the rate-controlling film and is absorbed or permeates through the skin of the user and into the blood stream.  
         [0009]     However, transdermal drug delivery has its drawbacks. For example, it is difficult to transfer the drug across the epidermis of the skin of a patient. The skin is the largest organ of the body and is naturally highly impermeable to prevent loss of water and electrolytes and to prevent the body from being invaded by foreign substances such as bacteria, viruses, liquids, and other compounds and materials. Accordingly, the natural barrier to permeability, the skin, also severely restricts the potential for transdermal delivery to a wide array of drugs.  
         [0010]     The skin is generally subdivided into two main layers: the outer layer being the epidermis and the inner layer being the dermis. The epidermis is about 50 to 100 micrometers thick. The dermis varies from 1 to 3 millimeters in thickness. The blood capillaries are housed in the dermis and, therefore, it is the goal of transdermal drug delivery to get the drug to cross the epidermis and enter the dermis such that the drug can enter the blood stream for systemic delivery.  
         [0011]     The epidermis is categorized into several layers. The outermost layer of epidermis is called the stratum corneum. The stratum corneum is comprised of dead cells called corneocytes or keratinocytes. The stratum corneum is commonly modeled or described as a brick wall. The “bricks” are the flattened, dead corneocytes. Typically, there are about 10 to 15 corneocytes stacked vertically across the stratum corneum. The corneocytes are encased in sheets of lipid bilayers (the “mortar”). The lipid bilayer sheets are separated by approximately 50 nm. Typically, there are about 4 to 8 lipid bilayers between each pair of corneocytes. The lipid matrix is primarily composed of ceramides, sphingolipids, cholesterol, fatty acids, and sterols, with very little water present.  
         [0012]     Although it is the thinnest layer of the skin, the stratum corneum is the primary barrier to entry of molecules or microorganisms into the body. Once the molecules have crossed the stratum corneum, diffusion across the remaining layers of the epidermis and dermis to the blood vessels occurs rapidly. Thus, most of the attention in transdermal drug delivery research has been focused on transporting molecules and drugs across the stratum corneum.  
         [0013]     Another drawback of transdermal drug delivery is that currently, the technique is effective only with small, lipophilic molecules which readily permeate the skin. However, many substances have been developed to enhance the molecular transport rates of less permeable drugs. These substances are known as chemical enhancers or penetration enhancers. Chemical enhancers attempt to increase the flux of a drug through the skin by increasing the solubility of a drug in the stratum corneum or by increasing the permeability of the drug in the stratum corneum.  
         [0014]     Selecting a proper enhancer is both difficult and complicated, as there is a myriad of possible enhancer/drug combinations. Not all enhancers are suitable for use with all drug molecules as some might interact with the drug molecule and cause an undesirable effect within the body. Further, some combinations of enhancers may improve drug flux beyond the expected flux rate and therefore result in too high of a drug concentration being delivered over too short of an interval for effective or safe treatment.  
         [0015]     A further drawback is the adhesive used with transdermal delivery patches. The adhesive is required to keep the patch in place on a patient, however, there are many different forms of adhesives that can be used. Typically, it is difficult to select which adhesive to use with any particular drug, and/or drug and enhancer combination, because there may be a chemical interaction between the various chemical compounds.  
         [0016]     Currently, the choice of appropriate enhancers, adhesives, and their relative proportion with respect to the drug is determined by general guidelines from what is known to be safe and what may have been effective with other drugs. The vast majority of the formulation development is made through trial and error experimentation, as the current transdermal testing devices are inadequate.  
         [0017]     To date, the devices for transdermal testing are relatively large, inefficient, ineffective, costly, and prone to error. In particular, two types of transcellular testing devices are currently in use, the Ussing chamber and the “filter insert” device. These devices will be briefly explained along with their associated drawbacks.  
         [0018]     The Ussing chamber, invented by Dr. Hans Ussing is configured for transcellular testing and consists of two hemi-chambers or reservoirs with open ends that are separated by a tissue or cellular layer located on a permeable membrane. In use, the two open ended reservoirs are clamped together with the tissue layer on the permeable membrane pinched between the reservoirs. One reservoir, the testing reservoir, is filled with a particular solution containing some drug or pharmaceutical composition (with or without enhancers or other additives) and the other reservoir, the sampling reservoir, is filled with a neutral solution, such as a saline type solution. Over a given time interval, samples are withdrawn from the sampling reservoir to determine what compounds, if any have diffused from the testing reservoir, across the tissue layer, and into the sampling reservoir.  
         [0019]     The Ussing chamber system, however, has several drawbacks. The Ussing chamber is not compatible with high throughput testing regimes and, therefore, amenable to testing only a handful of compounds or substances. This is unacceptable given the myriad of drug, enhancer, adhesive, or the like components and combinations of these components that require testing. As a result, testing only a few samples at any particular time is both inefficient and ineffective. Furthermore, the relatively large dimensions of the Ussing chamber device require a large amount of laboratory space, many technicians, and a large quantity of resources. This substantially increases the cost and time required to conduct the necessary testing of new drug delivery compositions.  
         [0020]     Another drawback of the Ussing chamber device comes about while clamping the membrane, with the cellular layer thereon, between the reservoirs. When the two reservoirs are clamped together damage frequently occurs to the tissue or cellular layer. The damage most often occurs near the edges of the reservoir, where the reservoirs pinch the cellular layer together to form a tight seal. This clamping damage typically produces a “gap” in the cellular layer between the abutting reservoirs. This “gap” functions as an open passage through which the compounds in each reservoir may freely transfer, thereby bypassing transfer through the cellular layer and compromising the experiment results. Therefore, a high throughput transcellular testing device would be highly desirable.  
         [0021]     The “filter insert” device currently in use consists of a sleeve or cylindrical tube where one end of the sleeve is closed off by a permeable membrane with a cellular layer grown across it. The other end of the sleeve is left open for receiving, sampling, or depositing substances. In use, the sleeve is placed, cellular end first, into a reservoir containing a sample solution. The sample in the reservoir then diffuses through the cellular layer comprising the end of the sleeve, and into the sleeve. Sampling and subsequent analysis of the resulting composition in the sleeve is used to determine the diffusion and flux rate through the cellular layer.  
         [0022]     However, the “filter insert” device has many of the same disadvantages of the Ussing chamber. First, the “filter insert” device is not compatible with high throughput and requires large quantities of testing materials. Second, the cellular layer used in the device is grown over a permeable membrane and a junction where the permeable membrane attaches to the sleeve. At the permeable membrane/sleeve junction there is often incomplete cellular growth creating “gaps” between the reservoir and the sleeve where the substance can freely pass into the sleeve without diffusing through the cellular layer.  
         [0023]     Furthermore, as a natural characteristic of cell cultures, the cellular layer does not grow uniformly across different base materials. Therefore, because the cellular layer is not uniform, the rate of transport or diffusion is not a constant throughout the cellular layer and any flux rate calculation not accounting for this will be flawed.  
         [0024]     Like the Ussing device, the “filter insert” configuration requires costly equipment and space, multiple operators to perform the desired experiments, and is also prone to error. As a result, innovation related to transdermal drug delivery compositions has been delayed. In light of the above, a device and method that addresses the above described drawbacks would be highly desirable. Specifically a device that can facilitate accurate high-throughput testing of transcellular drug flux would be highly desirable.  
       SUMMARY OF THE INVENTION  
       [0025]     According to the invention there is provided a device for accurate high throughput testing of transcellular drug flux.  
         [0026]     The present invention relates to a device and method for facilitating high throughput transcellular flux testing of compounds, such as pharmaceuticals or drugs, other compounds, or compound combinations. In one embodiment, the system and methods of the present invention may be used to identify the optimal components (e.g., solvents, carriers, transport enhancers, adhesives, additives, inhibitors, or other excipients) for pharmaceutical compositions or formulations that are delivered to a patient via tissue transport, including without limitation, pharmaceutical compositions or formulations administered or delivered transcellularly (e.g., in the form of a transcellular delivery device), topically (e.g., in the form of ointments, lotions, gels, and solutions), and ocularly (e.g., in the form of a solution). As used herein, “high throughput” refers to the number of samples generated or screened as described herein, typically at least 10, more typically at least 50 to 100, and preferably more than 1000 samples tested simultaneously in the same device.  
         [0027]     The transcellular testing device of the present invention addresses the drawbacks of current devices and methods, because it does not damage the cellular layer, contains a uniform cellular layer that produces uniform diffusion rates, and can be used for high throughput testing. The transcellular testing device of the present invention does not damage the cellular layer because there is no clamping or crushing of the cellular layer between components of the device. Therefore there are no gaps in the cellular layer, through which the testing substances can freely flow without being diffused through the cellular layer. This results in more accurate results than the traditional methods. Furthermore, there is no variation in the cellular layer because the surface on which the cellular layer is grown is uniform. This produces uniform transportation or diffusion across the entire cellular layer and yields more accurate results than traditional methods. Finally, the transcellular testing device is capable of use in high throughput testing. This results in the ability to test multiple variations of compounds and substances in a rapid manner, thereby resulting in an increased number of suitable transcellularly administered drugs.  
         [0028]     In one aspect, the present invention incorporates a transcellular testing device, comprising: 
        (a) a membrane having a first membrane surface opposing a second membrane surface, wherein said first membrane surface is configured for cellular adhesion; and     (b) a hydrophobic layer having a first hydrophobic layer surface opposing a second hydrophobic layer surface wherein said second hydrophobic layer surface is coupled to said second membrane surface, and wherein said hydrophobic layer comprises at least one discrete independent testing unit having; 
            (i) a first opening in said first hydrophobic layer surface;     (ii) a second opening in said second hydrophobic layer surface; and     (iii) a passageway defined by said hydrophobic layer between said first opening and said second opening.   
               
 
     
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0034]     The features and advantages of the present invention will be better understood by reference to the following detailed description, which should be read in conjunction with the accompanying drawings in which:  
         [0035]      FIG. 1  is an oblique view of a modified membrane according to the present invention;  
         [0036]      FIG. 2A  is a cross sectional view of a transcellular testing device, according to an embodiment of the invention, as taken along the line X-X′ of  FIG. 1 ;  
         [0037]      FIG. 2B  is a cross sectional view of another transcellular testing device, according to another embodiment of the invention, as taken along the line X-X′ of  FIG. 1 ;  
         [0038]      FIG. 2C  is a cross sectional view of yet another transcellular testing device, according to yet another embodiment of the invention, as taken along the line X-X′ of  FIG. 1 ;  
         [0039]      FIG. 2D  is a cross sectional view of even another transcellular testing device, according to even another embodiment of the invention, as taken along the line X-X′ of  FIG. 1 ;  
         [0040]      FIG. 2E  is a cross sectional view of a further transcellular testing device, according to a further embodiment of the invention, as taken along the line X-X′ of  FIG. 1 ;  
         [0041]      FIG. 2F  is a cross sectional view of still another transcellular testing device, according to still another embodiment of the invention, as taken along the line X-X′ of  FIG. 1 ; and  
         [0042]      FIG. 3  is a flow chart of a method for making and using a transcellular testing device, according to the present invention. 
     
    
       [0043]     Like reference numerals refer to corresponding parts throughout the views of the drawings.  
       DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT  
       [0044]     Transcellular testing devices  200 A- 200 F ( FIG. 2A-2F ) of the present invention preferably comprise a permeable membrane (hereinafter “membrane”), a cellular layer, a hydrophobic layer with passageways through it, donor substances, and receivers. In use, chemical substances including drug molecules, pharmaceuticals, enhancers, adhesives, and other additives positioned in the donor substance, diffuse out of the donor substance, through the cellular layer, through the membrane, and into the receivers. Samples are taken from the receivers and analyzed to determine the composition of the chemical substances that have diffused across the cellular layer. Also, the flux rate for the tested composition is determined. Further details describing the making and using of the transcellular testing devices  200 A- 200 F can be found below in relation to  FIG. 3 .  
         [0045]      FIG. 1  shows a modified membrane  100  of the transcellular testing devices  200 A- 200 F ( FIG. 2A-2F ) according to an embodiment of the present invention. In this configuration, a membrane  102  is bound to a hydrophobic layer  110 . The hydrophobic layer  110  forms an array of passageways  130  for transcellular testing. Further details of the structure of the transcellular testing devices  200 A- 200 F can be found below. It will be appreciated by those skilled in the art that the array of passageways  130  can be manufactured to inter-operate with standard testing or dispensing machinery, such as machinery used in conjunction with standard microtiter plates.  
         [0046]      FIG. 2A-2F  are cross sections of different embodiments of the transcellular testing devices  200 A- 200 F, as taken along line X-X′ of  FIG. 1 . The transcellular testing devices  200 A- 200 F generally include the membrane  102  and the hydrophobic layer  110  defining passageways  130  therethrough, as described below. Initially, the transcellular testing devices  200 A- 200 F will be described in detail with respect to  FIG. 2A , thereafter, alternative embodiments will be described in detail.  
         [0047]      FIG. 2A  shows a transcellular testing device  200 A according to an embodiment of the invention. The transcellular testing device  200 A includes a membrane  102  having a first membrane surface  104  opposing a second membrane surface  106 . It is preferable that the first and second membrane surfaces are substantially planar. The membrane  102  is any hydrophilic porous membrane typically used in medical or pharmaceutical laboratories. Suitable examples of the membrane  102  are the HAWP membrane filter made by Millipore Corp. of Massachusetts; the Nuclepore® polycarbonate membrane filters made by Whatman of Massachusetts; or the Cyclopore® membrane made by Becton Dickinson of New Jersey.  
         [0048]     The membrane  102  is preferably between about 40 and about 200 mm wide (as taken along the x axis) and about 50 and about 300 mm long (as taken along the y axis, shown in  FIG. 1 ). However, in a preferred embodiment the membrane  102  has substantially the same width and length as a standard microtiter plate, and is preferably between about 25 micrometers and about 2 mm thick (along the z axis), and more preferably about 0.15 mm thick. For example, a width of about 85.5 mm and a length of about 127.8 mm are preferred membrane dimensions.  
         [0049]     The membrane  102  is also porous, thereby allowing fluids and particles smaller than the membrane&#39;s pore size to pass therethrough. In a preferred embodiment, the pore size of the membrane  102  is dependent upon the size of pores required for cellular growth and adherence to the membrane  102 . An example of pore size that effectuates cell growth and adherence is pores between about 0.1 micrometers and about 10 micrometers in diameter, and more preferably about 0.1 to about 5 micrometers.  
         [0050]     In another embodiment, the membrane  102  can be any material that is permeable to fluids, whether or not it is able to sustain cellular growth and attachment. However, in this embodiment, the first membrane surface  104  is treated with a substance that promotes cellular growth and adhesion. For example, the first membrane surface  104  may be treated with a substance that has a rough or porous structure and has properties that are compatible with cellular growth and adhesion. For example, the first membrane surface  104  may be treated with a variety of biologically compatible materials, such as collagen type I, or the like.  
         [0051]     The second membrane surface  106  is treated with a hydrophobic layer  110 . The hydrophobic layer  110  is any hydrophobic, biologically and chemically inert material. A suitable hydrophobic layer  110  is TEFLON® made by Dupont. Other suitable materials include wax, polypropylene, polyethylene, polyvinyl chloride (PVC), or the like.  
         [0052]     The hydrophobic layer  110  has a first hydrophobic layer surface  111  and an opposing second hydrophobic layer surface  112 . The hydrophobic layer  110  preferably has at least the same width and length as the membrane  102 . The hydrophobic layer  110  is also preferably substantially planar.  
         [0053]     The second hydrophobic layer surface  112 , is coupled to the second membrane surface  106  by any of several processes including, but not limited to, heat bonding, adhesive attachment, vapor deposition, or the like. A preferred process of coupling the hydrophobic layer  110  to the membrane  102  is by heat sealing parafilm to the second membrane surface  106 .  
         [0054]     The first hydrophobic layer surface  111  has at least one first opening  135  ( FIG. 2A ). The second hydrophobic layer surface  112  has at least one second opening  136  ( FIG. 2A ). The first opening  135  and the second opening  136  are connected to each other, forming a passageway  130  through the hydrophobic layer  110 , such that the passageway runs parallel to the z-axis. Accordingly, once the hydrophobic layer  110  is coupled to the second membrane surface  106 , permeability through the transcellular testing device  200 A is restricted to the passageways  130  through the hydrophobic layer  110 .  
         [0055]     In a preferred embodiment, the hydrophobic layer  110  defines an array of passageways  130 . The number of passageways  130  varies depending of the surface area of the membrane  102 , the diameter of the passageways  130 , and the distance between the passageways  130 . In a preferred embodiment the array of passageways  130  are configured to mate with a standard array pin replicator or multi-channel pipettor such as those used to dispense fluids into a microtiter plate. In a preferred embodiment of the present invention, there are 24, 96, 384, or 1536 passageways  130  through the hydrophobic layer  110 . In use, the passageways  130  are positioned such that they mate or align with a standard 24, 96, 384, or 1536 pin replicator or multi-channel pipettor.  
         [0056]      FIG. 2A  also shows a confluent cellular layer of cells  220  grown onto the first membrane surface  104 . By “confluent layer” it is meant that the cells are organized such that there exists a continuous layer of cells across the first membrane surface  104 , where each cell abuts another cell, and there are no gaps or spaces between any two cells. This is otherwise known as “lawn growth”. Therefore, the confluent cellular layer of cells  220  forms a substantially planar cellular layer on the first membrane surface  104 . Thus, for anything to permeate or diffuse through the transcellular testing device  200 A, it must transport or diffuse through the confluent cellular layer of cells  220 .  
         [0057]     Because different skin locations provide different levels of permeability to foreign substances, such as ocular tissue compared to the epidermis on one&#39;s arm, the rate of transcellular diffusion varies accordingly. Therefore, transcellular testing of a drug compound must simulate the location in which the drug will be delivered. To do this, the thickness of the confluent cellular layer of cells  220  and the type of cells used to generate the confluent cellular layer of cells  220  are varied with respect to the test being performed. A suitable example of the cell type used in the confluent cellular layer of cells  220  is Caco 2 cells from the epithelial cell line. Other examples of cell types used in the confluent cellular layer of cells  220  include epithelial Madin-Darby canine kidney (MDCK) cells and human epidermal keratinocytes (HEK).  
         [0058]     The confluent cellular layer of cells  220  is grown directly onto the first membrane surface  104  (as described in detail with respect to  FIG. 3 ) and the cells of the confluent cellular layer of cells  220  attach to the pores of the membrane  102 , as described above, thereby anchoring the confluent cellular layer of cells  220  to the membrane  102 . It is preferable that the confluent cellular layer of cells  220  is a monolayer of cells.  
         [0059]     The transcellular testing device  200 A also preferably includes at least one donor substance  240  positioned directly onto the confluent cellular layer of cells  220 . The donor substances  240  contain chemical substances such as drugs, inhibitors, activators, and excipients dissolved in a salt solution. The donor substance  240  is typically comprised of a cell compatible matrix, of the appropriate texture, and contains a water content equal to or greater than 70 percent. Suitable compounds include hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methacrylate, or the like. A suitable example of the composition of the donor substance  240  is a polymerized Mowiol® 28-99 made by Clariant International Ltd. of Switzerland, to which the test substances are added. It is also preferable that the salt concentration of the donor substance  240  is isotonic.  
         [0060]     In a preferred embodiment of the invention there are multiple, or an array of donor substances  240 . Each donor substance  240  preferably substantially aligns with a passageway  130  on the second membrane surface  106 . In this configuration, each donor substance  240  has roughly the same diameter as the first opening  135 , the second opening  136 , and the passageway  130 .  
         [0061]      FIG. 2A  also shows the receivers  250 . In a preferred embodiment the receivers  250  are fluid droplets containing a balanced salt solution. In use, the receivers  250  are deposited within the first opening  135  of each passageway  130 . Each passageway  130  holds each receiver  250 . In a preferred embodiment the salt solution of the receivers  250  is isotonic. Also, in a preferred embodiment there are 24, 96, 384, or 1536 receivers  250 .  
         [0062]     It is preferable that each combination of aligned donor substance  240  and passageway  130  forms a discrete independent testing unit  260  such that each discrete independent testing unit  260  can test a different drug, inhibitor, adhesive, and additive combination simultaneously. For example, in use, substances dissolved in the donor substances  240  diffuse out of the donor substances  240 , transfer across the confluent cellular layer of cells  220  in the direction depicted by the arrow  280 , permeate through the membrane  102 , and into the receivers  250  located within each passageway  130 . In this embodiment, each combination of donor substance  240 , passageway  130 , and receiver  250  forms a discrete independent testing unit  260 .  
         [0063]      FIG. 2B  is another embodiment of a transcellular testing device  200 B, according to another embodiment of the invention. In this embodiment the hydrophobic layer  110 B passes through the second membrane surface  106 B and is embedded into the membrane  102 B. (Second hydrophobic layer surface  112 B is embedded into membrane  102 B.) The hydrophobic layer  110 B penetrates between about 10 micrometers and about 1 mm, and more preferably about 0.1 mm. In use, this forms a pseudo well or channel that helps maintain each passageway  130  as a discrete independent testing unit  260 . Because the hydrophobic layer  110 B is embedded into the membrane  102 B, the testing substances of one discrete independent testing unit  260  is less likely to mix with the testing substances of a neighboring testing unit.  
         [0064]      FIG. 2C  is yet another embodiment of a transcellular testing device  200 C, according to yet another embodiment of the invention. In this embodiment the thickness of the hydrophobic layer  110 C is increased as compared to the embodiment corresponding to  FIG. 2A . It is preferable that this hydrophobic layer  110 C is between about 5 mm and about 20 mm thick, and more preferably about 10 mm thick.  
         [0065]     Due to the increased thickness the volume of the receiver  250 C can be greater. In use, due to the larger volume of the receivers  250 C, multiple samples can be withdrawn over time from each receiver  250 C for analysis. In this embodiment, the transcellular testing device  200 C is configured for transcellular testing over longer periods of time, such as days or weeks.  
         [0066]      FIG. 2D  is even another embodiment of a transcellular testing device  200 D, according to even another embodiment of the invention. In this embodiment the hydrophobic layer  110 D is relatively thick, as described with respect to  FIG. 2C . It is preferable that this hydrophobic layer  110 D is between about 5 mm and about 20 mm thick, and more preferably about 11 to about 13 mm thick. In use, the volume of the receivers  250 D is increased due to the increased thickness of the hydrophobic layer  110 D. Therefore, multiple samples can be withdrawn over time from the receivers  250 D for analysis. In this embodiment, the transcellular testing device  200 D is configured for transcellular testing over longer periods of time, such as days or weeks.  
         [0067]     Also, in this embodiment, the hydrophobic layer  110 D is embedded through the second membrane surface  106 D and into the membrane  102 D. (Second hydrophobic layer surface  112 D is embedded into membrane  102 D.) The hydrophobic layer  110 D penetrates between about 0.01 mm and about 1 mm, and more preferably about 0.1 mm into the membrane  102 D. In use, this forms a pseudo well or channel that helps maintain each passageway  130  as a discrete independent testing unit  260 . In use, each donor substance  240  preferably contains a different testing substance that diffuses to its associated receiver  250 D. Because the hydrophobic layer  110 D is embedded into the membrane  102 D, the substances being tested in one discrete independent testing unit  260  are less likely to mix with the testing substances of a neighboring testing unit.  
         [0068]      FIG. 2E  is a further embodiment of a transcellular testing device  200 E, according to a further embodiment of the invention. In this embodiment, the hydrophobic layer  110 E is coupled to the second membrane surface  106 , and an additional hydrophobic layer  217 E is coupled to the first membrane surface  104 . The hydrophobic layer  110 E defines an array of passageways  130  therethrough, as described above. Similarly, the additional hydrophobic layer  217 E defines an array of additional passageways  131 E,  132 E, and  133 E therethrough. The passageways  130  are substantially aligned with the additional passageways  131 E,  132 E, and  133 E, such that a donor substance  240  flowing in the direction of the arrow  280  (parallel to the z-axis) passes through the additional passageway  131 E,  132 E, or  133 E, then continues into the passageways  130 . Thereby, a plurality of permeable channel or conduit like discrete independent testing units  260  is formed. The first and second membrane surfaces at the openings of each passageway  130  and each additional passageway  131 E,  132 E, and  133 E remain untreated.  
         [0069]     The confluent cellular layer of cells  221 E,  222 E, and  223 E are grown on the first membrane surface  104  within each additional passageway  131 E,  132 E, and  133 E. The confluent cellular layer of cells  221 E,  222 E, and  223 E attaches to the membrane  102  at the opening in the additional hydrophobic layer  217 E formed by the additional passageways  131 E,  132 E, and  133 E. Therefore, in use, each of the additional passageways  131 E,  132 E and  133 E, on the first membrane surface  104  has its own discrete confluent cellular layer of cells  221 E,  222 E, and  223 E that fully covers each additional passageway  131 E,  132 E, and  133 E.  
         [0070]     It should be stressed that the second hydrophobic layer surface  112  remain free from any cell culture medium used to grow the confluent cellular layer of cells  220 , as described with respect to  FIG. 3 . This is because cellular proteins will stick to the second hydrophobic layer surface  112  and alter the surface characteristics, making the layer semi hydrophilic, thereby, causing the samples in the receivers  250  to deviate from discrete fluid droplets within each passageway  130 .  
         [0071]      FIG. 2F  is still another embodiment of a transcellular testing device  200 F, according to still another embodiment of the invention. This embodiment is similar to that of  FIG. 2E  except the hydrophobic layer  110 F and the additional hydrophobic layer  217 F are both embedded into the membrane  102 F, as described with respect to  FIG. 2B . In this configuration there is a channel formed by the hydrophobic layer  110 F and the additional hydrophobic layer  217 F, such that substances are less likely to migrate from one discrete independent testing unit  260  to a neighboring discrete independent testing unit. Furthermore, in this embodiment, the hydrophobic layer  110 F is between about 5 mm and about 20 mm thick, and more preferably about 11 to about 13 mm thick. In this configuration, the receivers  250  preferably contain a balanced salt solution. Also, in this configuration, the receivers  250  contain sufficient solution to sustain prolonged testing and the extraction of multiple samples for analysis as described with respect to  FIG. 2C .  
         [0072]      FIG. 3  is a flow chart of the method  300  for making and using a transcellular testing device of the present invention. Once a membrane has been provided, as described above, a hydrophobic layer  110  ( FIG. 2A-2F ) is coupled to the second membrane surface  106  ( FIG. 2A-2F ), at step  310 . The hydrophobic layer  110  defines passageways  130  ( FIG. 2A-2F ) therethrough, such that as the hydrophobic layer  110  ( FIG. 2A-2F ) is coupled to the second membrane surface  106  ( FIG. 2A-2F ), areas of the membrane  102  ( FIG. 2A-2F ) do not couple with the hydrophobic layer  110  ( FIG. 2A-2F ).  
         [0073]     In an alternative embodiment, separate hydrophobic layers are coupled to opposing surfaces of the membrane  102 . In this configuration, each hydrophobic layer defines passageways therethrough. Once the hydrophobic layers are coupled to their respective surface of the membrane, the passageways on one surface of the membrane  102  are in substantial alignment with the passageways on the opposing surface of the membrane.  
         [0074]     In yet another embodiment, also at step  310 , coupling of the hydrophobic layer includes embedding the hydrophobic layer into the membrane. In this configuration, the hydrophobic layer  110  penetrates at least partially through the membrane surface and is embedded into the membrane. The hydrophobic layer  110  penetrates between about 0.01 and about 1 mm, and more preferably about 0.1 mm. In use, this forms a pseudo well or channel that helps maintain each passageway  130  in a discrete independent testing unit  260  ( FIG. 2A-2F ).  
         [0075]     In another embodiment, also at step  310 , the separate hydrophobic layers coupled to opposing surfaces of the membrane both penetrate through the membrane surfaces, respectively, and embed into the membrane. For example, the hydrophobic layer  110  ( FIG. 2F ) is embedded through the second membrane surface  106  and embedded into the membrane  102 . The additional hydrophobic layer  217  ( FIG. 2F ) penetrates the first membrane surface  104  and is embedded into the membrane  102 . Both hydrophobic layers are preferably embedded between about 0.01 mm and about 1 mm, and more preferably about 0.1 mm into the membrane  102 .  
         [0076]     At step  320 , a confluent cellular layer of cells  220  ( FIG. 2A-2F ) is grown on at least one surface of a membrane  102 . The confluent cellular layer of cells is a continuous layer covering substantially the entire membrane surface. The cells of the confluent cellular layer of cells abut one another such that there are no gaps between the cells. Therefore, for donor substances to pass through the confluent cellular layer of cells, the donor substance must pass through at least one cell.  
         [0077]     A suitable example of growing a confluent cellular layer of cells  220  on a surface of the membrane  102  is described in relation to steps  322 ,  324 ,  326 , and  328 . At step  322 , the membrane  102  ( FIG. 2A-2F ) is placed second membrane surface  106  down on a bottom plate (not shown) and clamped around the perimeter with an open top frame (not shown). Because the open top frame (not shown) is simply a perimeter frame, the membrane clamped between the open top frame (not shown) and the bottom plate (not shown) is accessible. Therefore, the first membrane surface  104  is left exposed. The exterior junction between the open top frame and the bottom plate is then sealed, such that no substances deposited within the open top frame (not shown) can leak out between the open top frame and the bottom plate (not shown). For example, suitable sealing of the juncture between the open top frame and the bottom plate is by parafilm with or without a tape overlay.  
         [0078]     Next, a cell suspension in standard growth medium containing serum is seeded onto the exposed first membrane surface  104  ( FIG. 2A-2F ), at step  324 . In a preferred embodiment, the top frame (not shown) acts as a reservoir for the cell suspension and growth medium. The combination top frame, bottom plate, clamped membrane  102 , and cell suspension is then placed within a sterile container and incubated, at step  326 . In a preferred embodiment the cells of the cell suspension are incubated at about 37 degrees Celsius in approximately a 5 to 10 percent carbon dioxide atmosphere. The length of time for incubating varies depending on the time for attachment of the specific cell type to the membrane.  
         [0079]     Following incubation, at step  328 , the membrane  102  is removed from between the top frame (not shown) and the bottom plate (not shown) and floated, cell-side down (hydrophobic layer  110  side up) in a dish containing complete cellular growth medium. The cell cultures are maintained in this configuration with periodic replenishment of cell growth medium until the cellular layer is post-confluent, i.e., a confluent cellular layer of cells is grown, at step  328 . The rate of cell growth varies considerably with cell type and can take, for example, from 1 day to 30 days to obtain a post-confluent cellular layer.  
         [0080]     It is stressed that during steps  324 ,  326 , and  328  the second hydrophobic layer surface  112  ( FIG. 2A-2F ) remains free from any cell culture medium used to grow the confluent cellular layer of cells  220 . This is because cell culture medium contains serum and other proteins that adhere to the second hydrophobic layer surface  112  ( FIG. 2A-2F ) and alter its characteristics, rendering the hydrophobic nature of the material hydrophilic and therefore, unsuitable to maintain discrete receivers  250  ( FIG. 2A-2F ), as described above.  
         [0081]     The membrane  102  ( FIG. 2A-2F ), with the attached hydrophobic layer  110  ( FIG. 2A-2F ) and confluent cellular layer of cells  220  ( FIG. 2A-2F ) is then removed from the cell growth medium and positioned to receive at least one donor substance  240  ( FIG. 2A-2F ), at step  330 . Drug compounds to be tested or combinations of drugs, adhesives, enhancers, inhibitors, or the like are selected and incorporated into the donor substances  240  ( FIG. 2A-2F ).  
         [0082]     Donor substances  240  ( FIG. 2A-2F ) are then deposited onto the confluent cellular layer of cells  220  in substantial alignment with each passageway  130 . Receivers  250  ( FIG. 2A-2F ), as described above, are then positioned within the passageways  130  ( FIG. 2A-2F ), at step  340 . Transcellular testing is then conducted with the transcellular testing device  200 A- 200 F, at step  350 .  
         [0083]     At step  352 , the substances dissolved in the donor substances diffuse out of the donor substances  240  ( FIG. 2A-2F ), through the confluent cellular layer of cells  220  ( FIG. 2A-2F ), the membrane  102  ( FIG. 2A-2F ), and into the receivers  250  ( FIG. 2A-2F ). Samples are then retrieved from the receivers  250  ( FIG. 2A-2F ), at step  354 . In a preferred embodiment, at step  354 , aliquots from the receivers  250  ( FIG. 2A-2F ) are retrieved for analysis. In an alternative embodiment, at step  354 , the entire quantity of each of the receivers  250  ( FIG. 2A-2F ) is retrieved for analysis.  
         [0084]     Next, the samples from the receivers  250  ( FIG. 2A-2F ) are analyzed, at step  356 , to determine the substance concentration in the receivers  250  ( FIG. 2A-2F ) at definite time intervals to determine the concentration of donor substances retrieved from the receivers  250  and the flux rate for the particular substance tested through the particular confluent cellular layer of cells  220  ( FIG. 2A-2F ) used in the particular experiment protocol. The concentration can be determined using one or more of many techniques known to those skilled in the art including, for example, UV spectroscopy and HPLC.  
         [0085]     Although the present invention has been described in considerable detail with reference to certain preferred embodiments, other embodiments are possible. Therefore, the spirit and scope of the appended claims should not be limited to the description of the preferred embodiments contained herein. Modifications and variations of the invention described herein will be obvious to those skilled in the art from the foregoing detailed description and such modifications and variations are intended to come within the scope of the appended claims.