Abstract:
A class of designs is provided for a permeable support where the design principle includes a permeable support layer, such as a membrane, bonded midway between two arrays of wells or multiwell plates, attached at their bottom portions, forming a double-sided multiwell plate. Thus, opposite facing wells on either side of the permeable support layer are accessible by inversion of the double-sided multiwell plate. Well fluid is held in place in the novel multiwell plate by capillary forces in the case of aligned upper and lower well arrays or by surface tension on patterned well regions on a permeable membrane layer. No additional components are necessary to form compartments for fluid retention. These new plate designs allow the surface area of the permeable support layer to be maximized, eliminate potential wicking and cross contamination issues that may arise from multiple component sidewalls, and take advantage of the small well diameters to retain fluid by utilizing the effects of surface tension or capillary forces.

Description:
FIELD OF THE INVENTION 
       [0001]    This invention relates generally to cell culture labware, such as multiwell plates, and more particularly to a class of designs for Transwell® Permeable Supports or permeable supports within multiwell plates useful for high throughput screening. 
       BACKGROUND OF THE INVENTION 
       [0002]    Multiwell plate systems that include permeable supports (or membranes), such as those found in Corning® Transwell® Permeable Supports, facilitate assessment of new chemical entities through the use of high-throughput screening. These multiwell plate systems with permeable supports are used for many types of studies, including drug transport and cell migration. In drug transport studies, intestinal cells (e.g. CaCO2) are grown into a confluent monolayer on the permeable support until they differentiate to form tight junctions. A molecule of interest is then introduced on one side of the cell sheet to see if it can be actively transported across the sheet to the other side where it is detected. 
         [0003]    Permeable supports are also useful for cell migration studies, where a chemo-attractant is placed in a compartment adjoining a cell monolayer, and the migration of cells toward the chemo-attractant is detected. Chemical entities are typically tested to determine the ability to block migration. 
         [0004]    Alternatively, permeability can be assessed using techniques known in the art such as Parallel Artificial Membrane Assay (PAMPA) or Immobilized Artificial Membranes (IAM). No cell culture is necessary for these applications. Instead, the permeable support is typically coated with a solution of lipid in an inert organic solvent. The molecule of interest is placed on one side of the coated permeable support and its passage to the other side is monitored. 
         [0005]    High throughput screening permeable support systems are currently available with 96 well tests being carried out in parallel, and require multiple components including a Transwell plate and a receiver or reservoir plate in which the Transwell resides. In order to increase throughput, there is interest in the field to increase the well density of the multiwell plate, to higher densities such as 384 well and 1536 well formats, for example. In the current art, the permeable support is typically suspended at the bottom of a well. The well or multiwell plate with the permeable support is then placed inside another well or reservoir plate having a solid bottom, enabling fluid retention. The increase in the number of wells leads to a miniaturization of the wells in these higher density formats; accordingly, the total membrane area for cell growth within the well becomes limited and the close proximity of the sidewalls in the multiple components can lead to wicking or cross contamination issues. 
         [0006]    The design and manufacturing challenges seen with 96 wells become even more pronounced and difficult as one proceeds to 384 and 1536 well formats. Hence, it is desirable to overcome the problem of wicking or contamination in multiwell plates. Additionally, it is desirable to reduce the number of discrete parts in the permeable support. 
         [0007]    Prior art approaches for performing the above described desired capabilities that are known in the art include the following examples. 
         [0008]    Different types of filtration devices are found in U.S. Pat. No. 5,047,215, entitled, “Multi Well Test Plate”; U.S. Pat. No. 4,304,865, entitled “Harvesting Material from Micro-Culture Plates”; and U.S. Pat. No. 4,948,442, entitled “Method of Making a Multi Well Test Plate”. 
         [0009]    The above prior art patents disclose filtration devices which have some disadvantages. These prior art patents disclose methods of making microtiter filtration plates by sealing a sheet of filter material between thermoplastic trays or require cutting the filter material from the microtiter plates into discrete discs for analysis. If the filter material is left as an uncut sheet, wicking and cross-contamination occur because the filter material has a pore structure that runs laterally. 
         [0010]    Accordingly, first off, these prior art devices have great difficulty solving the problem of cross contamination and wicking. Secondly, because of the problem arising if the sheet is uncut, these prior art designs necessitate the use of many discrete components. Thirdly, in so doing, they are not easily fabricated. Fourthly, they do not provide for the maximum surface area of a permeable support or for miniaturization to allow for increased well density formats. 
         [0011]    Another piece of prior art entitled, “Reversible Membrane Insert for Growing Tissue Cultures”, U.S. Pat. No. 5,759,851, assigned to the assignee hereof, discloses a well with a membrane inside and at the bottom of it, where the well is then placed in a separate reservoir well. In this type of device, two separate containers are necessary: a membrane-containing well and a reservoir well, much like Transwells which are known in the art. Cells can be seeded in the membrane-containing well on the membrane&#39;s upward facing side where, as mentioned above, the membrane is located at the bottom of the well. After these cells become adherent, the membrane-containing well can be removed from the reservoir, and using a jig tool, the membrane itself can then be moved to the opposite end of the membrane-containing well. The membrane-containing well is placed inverted into the reservoir thereby allowing cells to seed on the other side of the membrane, the side without cells already seeded. Accordingly, this enables seeding cells on both sides of the membrane. 
         [0012]    One of the major disadvantages with this design is that separate, detached wells or containers are needed, one membrane-containing well and one reservoir well. Furthermore, an additional device, such as a jig, is required to move the membrane from one end of the well to the other prior to inverting and seeding with cells. 
         [0013]    Accordingly, a new apparatus and method of manufacture is needed that preferably overcomes the disadvantages of any of the prior art solutions above-mentioned that provides a resolution to the problem of cross-contamination and wicking, maximizes the surface area of the permeable supports, allows for miniaturization to increased well density formats, reduces the number of necessary components and simplifies manufacturing/fabrication while also enabling the seeding of cells on both sides of the membrane if necessary. 
       SUMMARY OF THE INVENTION 
       [0014]    A class of designs is provided for a double-sided multiwell plate where the design principle includes opposite facing wells on either side of a permeable support layer or membrane, where each side is accessible by inversion of the double-sided multiwell plate. Well fluid is held in place by capillary forces in the case of aligned upper and lower array wells or multiwell plates or by surface tension on patterned well regions of a permeable membrane. 
         [0015]    One embodiment of the present invention relates to a plurality of first wells forming a first array, a plurality of second wells aligned with the first array of first wells, forming a second array, and bottom portions of the first and second arrays of wells coupled together and having a permeable membrane at their interface. 
         [0016]    Additionally, each well of the first array has a respective well opening at the top of each well, these respective well openings facing up; and each well of the second array has a respective well opening at the top of each well, these respective well openings facing down. 
         [0017]    Another embodiment of the present invention relates to the well openings of the second array being accessible by flipping the multiwell plate upside-down, thereby positioning the second array such that the respective well openings at the top of each well are facing up and positioning the first array such that the respective well openings at the top of each well are facing down. Fluid in the wells of both the first and second arrays is retained within the wells due to surface tension or capillary forces. 
         [0018]    One embodiment of the present invention relates to bonding of the permeable membrane midway through the first and second arrays of wells and wherein the permeable membrane is preferably a track-etch membrane. 
         [0019]    Another aspect of the embodiment of the present invention relates to a rigid lid for covering the well openings of the multiwell plate, where the lid is compatible for robotic handling. Yet another aspect is that the lid is a gas permeable lid on at least a portion of the lid and including thin polymer sheets or discs that allow for gas exchange. The lid further includes an elastomeric gasket that fits into a recess on the multiwell plate. 
         [0020]    Another aspect of the present invention relates to a sleeve-type lid covering the well openings of the first and second arrays of the multiwell plate, where the sleeve-type lid is thermoformed or molded from a polymer and includes means for accessing the multiwell plate with gripper cut-out areas on top and bottom sides of the sleeve-type lid. In yet another aspect of the present invention, the sleeve-type lid is rigid and gas-permeable on at least a portion of the lid. 
         [0021]    Another embodiment of the present invention relates to wells of first and second arrays being patterned onto the permeable membrane to enable cell binding wherein the patterning may be a hydrophilic interaction, specific molecular interaction, coating with a lipid solution, or lamination, onto the permeable membrane. 
         [0022]    A still further aspect of the present invention relates to the permeable membrane including non-well regions patterned around the first and second well arrays onto the permeable membrane to prevent cell binding or coating with lipid solutions wherein the patterning may be a coating or lamination with a hydrophobic material. Another aspect of the present invention relates to the patterned permeable membrane being substantially flat. 
         [0023]    Additional features and advantages of the invention will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from the description or recognized by practicing the invention as described in the written description and claims hereof, as well as the appended drawings. 
         [0024]    It is to be understood that both the foregoing general description and the following detailed description are merely exemplary of the invention, and are intended to provide an overview or framework to understanding the nature and character of the invention as it is claimed. 
         [0025]    The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s) of the invention, and together with the description serve to explain the principles and operation of the invention. 
     
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0026]    The invention is further illustrated with reference to the following drawings in which: 
           [0027]      FIG. 1  is a perspective view of a multiwell plate format in accordance with a preferred embodiment of the present invention. 
           [0028]      FIGS. 2 and 3  are cross-sectional views of  FIG. 1 . 
           [0029]      FIG. 4  is a cross-sectional view of one well of  FIG. 2  showing the separation by a permeable membrane in accordance with a preferred embodiment of the present invention. 
           [0030]      FIG. 5  is a multiwell plate in accordance with an alternate preferred embodiment of the present invention. 
           [0031]      FIG. 6  is a cross-sectional view of one well region of  FIG. 5 . 
           [0032]      FIG. 7  is a multiwell plate lid in accordance with a preferred embodiment of the present invention. 
           [0033]      FIG. 8  is the underside view of the multiwell plate lid shown in  FIG. 7 . 
           [0034]      FIG. 9  is a gas permeable multiwell plate lid in accordance with a preferred embodiment of the present invention. 
           [0035]      FIG. 10  is the underside view of the multiwell plate lid shown in  FIG. 9 . 
           [0036]      FIGS. 11 and 12  show a sleeve type multiwell plate lid covering both upper and lower well openings in accordance with a preferred embodiment of the present invention. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0037]    Reference will now be made in detail to the present preferred embodiments of the invention, examples of which are illustrated in the accompanying drawings. Wherever possible, the same reference numbers will be used throughout the drawings to refer to the same or like parts. 
         [0038]    The terms “well” and “well region” as used throughout are intended to signify areas in the novel multiwell plates herein described in which seeding of cells is enabled. Accordingly, these terms are thought to be equivalent and may be used interchangeably throughout. 
         [0039]    Referring to  FIG. 1 , a novel double-sided multiwell plate  100  is shown in accordance with a preferred embodiment of the present invention. The standard format of multiwell plate  100  is preferably a high density well format, such as a 384 or 1536 well plate, to increase throughput. The double-sided multi-well plate  100  is preferably one plate that can be utilized on both sides. It is preferably formed from arrays of upper and lowers wells, which may be one plate having upper and lower well arrays or two multiwell plates assembled together into one plate and in either case with a permeable support layer disposed in between (as will be discussed below). For clarity and to distinguish between the upper and lower sides of the double-sided multiwell plate  100 , the terms upper and lower multiwell plates will be used herein. Accordingly, multiwell plate wells  110  form an array of wells  115  on an upper multiwell plate  120 . Additionally, multiwell plate  100  includes wells  130  found on a lower multiwell plate  150  which form an array  140  (shown with dashed circles) aligned with the wells on the upper multiwell plate. For simplicity, a fewer total number of wells  110 ,  115 ,  130 ,  140  are shown on upper and lower multiwell plates  120  and  150  than are actually physically possible in a standard format of a multiwell plate  100  (e.g. 384 well plate). 
         [0040]    Referring now to  FIG. 2 , a cross-sectional side-view segment of  FIG. 1 , multiwell plate  100  is shown to include an upper multiwell plate  120  and lower multiwell plate  150 , each with an array of wells  115  and  140 , respectively. The bottom portions  205 ,  206  of the wells of the upper and lower multiwell plate arrays are joined such that the bottom portion of each of the upper wells  205  is aligned with the bottom portion of each of the lower wells  206 . Accordingly, the openings  210  of the wells  110  in the upper array  115  are facing up and the well openings  230  of the wells  130  in the lower array  140  are facing down. The well openings  230  of the lower array are accessible by flipping the multiwell plate  100  upside-down, thereby positioning the lower array such that the top of each well  230  in the lower array  140  is now facing up and positioning the upper array  115  such that the top of each well  210  in the upper array is now facing down. 
         [0041]    In accordance with a preferred embodiment of the present invention, a permeable support  250  layer, preferably a permeable or porous membrane such as a track-etch membrane, (for example, consistent with supports found in Corning® Transwells® Permeable Supports), is attached or bonded, preferably midway between upper and lower multiwell plates  120  and  150  or at the interface of the bottom portions  205 ,  206  of upper and lower well arrays. The permeable support layer or membrane  250  may also be treated or coated. Coating materials may include biological materials such as a collagen or various lipids in organic solvents (for PAMPA or IAM applications). Track-etch membranes may be a desirable design choice for those of skill in the art because their size, shape, and density of pores may present advantageous characteristics over other membrane materials for cell transport and migration assays. The pore size of the permeable support layer  250  is typically 1 micron pore size, but can range from 0.1 to 12 microns depending on its end use. The permeable support layer  250  or membrane is stationary, and as such, no extra device is required to move the membrane. Further, the instant invention allows cell seeding to occur on both sides of the permeable membrane  250 , with the mere inversion of multiwell plate  100 . 
         [0042]    Upper and lower multiwell plates  120  and  150  may be joined together via molded fittings as part of the mold design or via known assembly methods such as the use of adhesive, overmolding, solvent bonding, and welding (ultrasonic, RF, laser, platen, radiation, etc.). When joined together, the thickness or height of the multiwell plate  100  may be slightly taller than the traditional multiwell plate, but preferably falls within the range of 14 to 22 millimeters. 
         [0043]    Referring to  FIG. 3 , another cross-section of multiwell plate  100  is shown where the upper and lower multiwell plates  120  and  150 , respectively, are shown as being capable of retaining fluid within the wells  110  and  130  due to surface tension or capillary forces. The upper well array  115  has upper fluid compartments  310  within each upper well  110  and the lower well array  140  has lower fluid compartments  320  within each lower well. The compartments  310  and  320  are divided by the permeable support layer  250 . Communication between fluids retained in upper and lower fluid compartments  310  and  320  occur through the permeable support  250 . The upper fluid compartments have walls  315  and the lower fluid compartments have walls  325 . The compartment walls  315  and  325  may also be treated, coated, or textured in such a way to facilitate retention of fluid in the areas immediately adjacent to the permeable support or membrane  250 . 
         [0044]    Referring now to  FIG. 4 , a close up view of one upper well  110  and one lower well  130  is shown. The multiwell plate wells  110 ,  130  are divided in half by a permeable support layer  250  or membrane as discussed above. The permeable support layer  250  is attached or bonded at the interface of the bottom portions  205 ,  206  of the upper well  110  and lower well  130 , respectively. Upper fluid  410  in upper fluid compartment  310  and lower fluid  420  in lower fluid compartment  320  stay within wells  110  and  130 , respectively, due to capillary forces. No additional reservoir is needed in the instant invention, as in the prior art, since upper and lower fluids  410  and  420  will be held next to the membrane  250  on both sides. As discussed above, well openings  210  and  230  can be accessed by flipping the multiwell plate  100  upside-down or inverting it to the opposite side. 
         [0045]    As miniaturization of multiwell plate wells increases in the industry, in particular to provide higher density formats, the total membrane area for cell growth is decreased since the wells may become too small in size to be of utility and further as mentioned above, the close proximity of the sidewalls in the multiple components can lead to wicking or cross-contamination problems. Accordingly, an alternate preferred embodiment of the instant invention provides for a permeable membrane layer or sheet, patterned to retain fluids, attached in a rigid frame in an alternate multiwell plate  500 , as shown in  FIGS. 5 and 6 . 
         [0046]    Following the same principles as described supra in previous embodiments, a permeable or porous membrane  510  is held at, preferably, the midpoint of a rigid frame  520  dividing the plate  500  into upper and lower multiwell “plates” (or sides or arrays),  515 , and  516 , respectively. The membrane  510  is preferably a track-etch membrane. The two sides of the permeable membrane  510  accommodate both upper and lower multiwell plates,  515  and  516  respectively. The permeable membrane  510  is modified or patterned such that a pattern of well regions (or wells)  530  are created that permit cell binding for drug transport and cell migration assays and non-well regions  540  (or non-wells) that prevent cell binding on both upper (shown) and lower sides (not shown) of the permeable membrane  510 ; non-well regions  540  are disposed around well regions  530 . 
         [0047]    Preferably, the modification or patterning of the permeable membrane is accomplished with an array of hydrophilic well regions  530  with a hydrophobic grid surrounding them. For PAMPA and/or IAM applications, this can be achieved by a simple hydrophobic membrane spotted with a lipid or organic solvent. The lipid produces hydrophilic spots or well regions. In cell-based testing, where lipids are not spotted or printed, a hydrophilic membrane may be used initially with a hydrophobic material, such as a punched sheet of polyethylene or polypropylene, laminated to the hydrophilic membrane material to create the well regions  530  and non-well regions  540 , respectively. 
         [0048]    Accordingly, the patterning of the membrane  510  may be completed by a hydrophobic or hydrophilic interaction, a specific molecular interaction, coating or spotting with a lipid or organic solvent, or lamination on top of the permeable membrane or in any other plausible manner. 
         [0049]    The well regions  530  that permit cell binding and/or are coated with lipid solution are preferably aligned such that they are located in the same place on either side of the permeable membrane  510 , and are preferably in the same location as the wells found on any format of an industry standard multiwell plate for automation compatibility purposes, though the number of wells per plate can vary widely and with any spacing desired. 
         [0050]    As mentioned, the non-well regions  540  of the permeable membrane  510  may be modified or patterned by coating or lamination of another material to the permeable membrane, to prevent cell binding and/or coating with lipid solutions. This modification to prevent cell binding and/or coating with lipid solution may also generate demarcations of additional material  550  outlining the perimeter of well regions  530 , particularly if lamination is the method of making these well regions, so that the delineation between regions  530  and  540  of the permeable membrane  510  are readily apparent. 
         [0051]    One further aspect of the alternate preferred embodiment of the present invention is that despite being modified or patterned, the permeable membrane  510  is substantially flat. 
         [0052]    In  FIG. 6 , a cross-section of the alternate multiwell plate  500  of  FIG. 5  is shown depicting a single well region  530  located between non-well regions  540  where a porous or permeable membrane  510  is shown disposed at the midpoint of the multiwell plate  500 , held by a rigid frame  550 . As mentioned, regions  530  and  540  are patterned to enable or prevent cell binding, respectively, and/or coated with lipid solutions in accordance with preferred aspects of the present invention. 
         [0053]    As with embodiments described above, fluid  560  on both sides of the permeable membrane  510  is held in place by surface tension. The rigid frame  550  of alternative multiwell plate  500  preferably has the size and footprint of an industry standard multiwell plate and as with multiwell plate  100 , can be inverted for accessing the well regions of the permeable membrane  510  in the lower array  516 . 
         [0054]    In accordance with preferred embodiments of the present invention,  FIGS. 7-12  show different types of lids for covering the upper and lower arrays of multiwell plate  100  (and/or alternate multiwell plate  500 ) and are capable of fitting a multiwell plate format with any number of wells, though for demonstration and clarity purposes, fewer wells than an industry standard plate are shown Additionally, since multiwell plate  100  and alternate multiwell plate  500  of the instant invention are “open” on both sides, these lids are contemplated to be used to cover both sides of the multiwell plates disclosed herein. 
         [0055]      FIG. 7  shows a lid  710  covering the upper array of multiwell plate  100  or the multiwell plate  500  of  FIG. 5 . Lid  710  is preferably a common rigid multiwell plate lid compatible for robotic handling. In order to prevent evaporation, preferably two such lids  710  will be needed in the instant invention, one for covering each side of either the double-sided multiwell plate  100  or  500 .  FIG. 8  shows the inverted (or underside view) of lid  710 . Also shown in accordance with a preferred aspect of the present invention is an elastomeric gasket  820  on the inside or around the inside perimeter of lid  710 , allowing the lid  710  to fit into a recess on the multiwell plate  100 . The gasket  820  is preferably made of a thermoplastic elastomer material. 
         [0056]    In an alternate preferred embodiment of the present invention, a gas permeable multiwell plate lid  900  for cell based assays is shown in  FIG. 9  having a rigid lid portion  910  with holes overlying the wells or well regions and discs  920  made of gas permeable material covering those wells or well regions and allowing for gas exchange. A gas exchange environment may also be provided by a thin polymer sheet (not shown) across the surface of the lid  900  in lieu of discs  920 .  FIG. 10  shows an inverted view of lid  900  in  FIG. 9 , where the inverted rigid lid  910  has discs  920  and also shows an elastomeric gasket  1010  around the inside perimeter of lid  910 , allowing the lid  910  to fit into a recess on the multiwell plate  100  (or alternate multiwell plate  500 ). Gasket  1010  is similar in construction to gasket  820  of  FIG. 8 . 
         [0057]    Referring now to  FIG. 11 , in accordance with another alternative preferred embodiment of the present invention, a sleeve type lid  1100  is shown. The multiwell plate  100  (or alternate multiwell plate  500 ) can slide into the sleeve type lid  1100  (as shown in  FIG. 12 ), thereby both upper and lower well arrays are covered with just one lid, rather than two. Also contemplated are gripper cut-out areas  1120  and  1130 , located anywhere on lid  1100 , but preferably (as shown) at the ends of the top and bottom portions of the sleeve type lid  1100 , respectively, to provide easy access to the multiwell plate  100  housed inside the sleeve type lid  1100 , as can be seen in  FIG. 12 . Alternate multiwell plate  500  can also use this sleeve type lid, though not shown in  FIG. 12 . 
         [0058]    The sleeve type lid  1100  may be thermoformed or molded from a clear or opaque polymer. The polymer on the top and bottom portions of the sleeve type lid  1100  preferably has a gas permeable sheet to allow for gas exchange of the wells and may be supported by a rigid frame  1110  found along the perimeter and on sides of lid  1100 . 
         [0059]    The various multiwell plates and/or lids described herein may have identifiers or tags associated with them, such that a user can track which of the upper or lower well arrays has been previously utilized, seeded, etc. These identifiers may be of any type desired, but some examples are labels with numerals (e.g. No. 1 and No. 2) placed on each of the upper and lower sides of the plate, or visible etchings anywhere or on the sides of the multiwell plate. 
         [0060]    It should be noted that all figures described supra are not of actual size but represent accurate renditions and structural block diagrams of the preferred embodiments of the present invention. 
         [0061]    Several commercial applications are contemplated for use with the embodiments of the present invention such as, but not limited to, for instance, applications involving PAMPA and IAM as previously mentioned. 
         [0062]    Having described various preferred embodiments of the present invention, it will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit and scope of the invention. Thus, it is intended that the present invention covers the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents.