Patent Publication Number: US-2023134238-A1

Title: Well plate and petri dish fluid exchange plug

Description:
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT 
     This invention was made with government support under Grant No. 449041-AK-79854 awarded by the National Cancer Institute of the National Institutes of Health. The government has certain rights in the invention. 
    
    
     BACKGROUND OF THE INVENTION 
     Existing well plates and petri dishes require systems for improved environmental control over internal conditions without the need for specialized equipment, engineering, or manufacturing. 
     Thus, there is a need in the art for improved systems for establishing fluidic exchange systems in multi-well plates and petri dishes. This invention satisfies this unmet need. 
     SUMMARY OF THE INVENTION 
     In one aspect, the present invention provides a fluid exchange plug device configured for insertion into two or more wells of a multi-well plate, comprising: a plug body having an upper surface and a lower surface; two or more sockets positioned within the plug body, each comprising a first opening, a second opening and a lumen therebetween, wherein two or more sockets, are accessible by the first opening on the upper surface of the plug body; two or more stoppers extending from the lower surface of the plug body having a first end and a second end, wherein the second end comprises at least one opening; wherein each stopper is positioned below each socket and is sized to fit within a well of a multi-well plate, wherein the second opening is fluidly connected to the at least one opening of the second end by at least one socket lumen extending through the plug body, and wherein the second end of a first stopper is fluidly connected to the second end of at least one second stopper by a stopper lumen extending through the plug body. In one embodiment, the two or more stoppers further comprise a middle section positioned anywhere along the length of each stopper, wherein the middle section has a diameter smaller than a diameter of the two or more stoppers. In one embodiment, the two or more stoppers each comprise a gasket or O-ring positioned on the middle section. In one embodiment, the two or more stoppers each extend from the lower surface of the plug body by a height ranging between about 1-25 mm. In one embodiment, the two or more sockets each comprise a threaded interior. In one embodiment, the two or more sockets each are sized to receive fluidic or microfluidic tubing. In one embodiment, the two or more sockets are each adjacent to each other and the two or more stoppers are each adjacent to each other. In one embodiment, the plug is configured for insertion into a multi-well plate selected from the group consisting of: a 6 well plate, a 12 well plate, a 24 well plate, a 48 well plate, a 96 well plate, a 384 well plate, and a 1536 well plate. In one embodiment, the device is a single use device. In one embodiment, the device is a multiple use device. 
     In another aspect, the present invention provides a fluid exchange plug device configured for insertion into a petri dish, comprising: a plug body having an upper surface and a lower surface; two or more sockets positioned within the plug body, each socket having a first opening, a second opening and a lumen therebetween, wherein two or more sockets are accessible by the first opening on the upper surface of the plug body; a stopper extending from the lower surface of the plug body having a first end and a second end, wherein the second end comprises at least one opening and wherein the stopper is sized to fit within a petri dish; and wherein the second opening of each socket is fluidly connected to the at least one opening of the second end by at least one socket lumen extending through the plug body. In one embodiment, the two or more stoppers further comprise a middle section positioned anywhere along the length of the stopper, wherein the middle section has a diameter smaller than a diameter of the stopper. In one embodiment, the stopper comprises a gasket or O-ring positioned on the middle section. In one embodiment, the stopper extends from the lower surface of the plug body by a height ranging between about 1-40 mm. In one embodiment, the device further comprising at least one channel embedded in the second end of the stopper. In one embodiment, the stopper is sized to fit flush against a lower surface of a petri dish. In one embodiment, the two or more sockets each comprise a threaded interior. In one embodiment, the two or more sockets each are sized to receive fluidic or microfluidic tubing. In one embodiment, the device is a single use device. In one embodiment, the device is a multiple use device. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The following detailed description of invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. 
         FIG.  1    depicts a side view of an exemplary fluid exchange plug device of the present invention. 
         FIG.  2    depicts a top view of an exemplary fluid exchange plug device of the present invention. 
         FIG.  3    depicts a top view of an exemplary fluid exchange plug device of the present invention. 
         FIG.  4    depicts a top view of an exemplary fluid exchange plug device of the present invention. 
         FIG.  5    comprising  FIG.  5 A  through  FIG.  5 E  depicts an overviews of the fluid exchange plug device.  FIG.  5 A  depicts a close-up view of a plug designed to connect two, neighboring wells in a commercially available 96 well plate. The holes and slits are specialized features required to fill the well due to the small size of this particular size (plug diameter 7 mm, channel diameter 0.5 mm).  FIG.  5 B  depicts an overview of a large plug (size of a lid) designed to connect adjacent wells across an entire 96 well-plate.  FIG.  5 C  depicts a reverse side of the device showing threaded holes used to attach microfluidic tubing.  FIG.  5 D  depicts a fluid exchange plug (lid size) applied to a standard 96 well plate with a quarter section of the bottom quarter showing the microfluidic channels embedded in the device to connect arbitrary (in this case adjacent) wells.  FIG.  5 E  depicts a close up section of the at least one socket lumen and at least one stopper lumen through the fluidic exchange plug (lid size). 
         FIG.  6    comprising  FIG.  6 A  through  FIG.  6 B  depicts a fluid exchange plug designed for a 100 mm Petri dish with two sockets.  FIG.  6 A  depicts an isometric view showing the plug and a rubber gasket (black).  FIG.  6 B  depicts a cross-sectional view showing the presence of fluidic channels within the device, as well as two sockets (an inlet and outlet port). 
         FIG.  7    comprising  FIG.  7 A  through  FIG.  7 C  depicts  FIG.  1 C  depicts a fluid exchange plug designed for a 100 mm Petri dish with two sockets.  FIG.  7 A  depicts a cross sectional view of plug applied to a commercial 100 mm well plate. The void in the plate would be filled with fluid and flow can be directed across the plate.  FIG.  7 B  depicts a sectional view of a fluid exchange plug in a petri dish. The petri dish is shown in light blue. The plug has multiple inlets and outlets. Also, a channel geometry is manufactured into the bottom of the plug to create a parallel plate flow chamber (red) with additional ports for fluid monitoring.  FIG.  7 C  depicts a perspective view of a half-section showing the location of the channels relative to the bottom of the petri dish. 
         FIG.  8    depicts multiple views of an exemplary fluid exchange plug device of the present invention. 
         FIG.  9    depicts a side view of an exemplary well turned into perfusion bioreactors. 
         FIG.  10    depicts an exemplary implementation of the fluid exchange plug device of the present invention for the co-culture of patient-derived cell types including tumor cells and peripheral blood mononuclear cells (PBMCs). In this exemplary implementation, the fluid exchange plug device of the present invention is applied to a 96 well-plate filled with tumor spheroids. The invention allows non-invasive monitoring of the biological samples by imaging on an inverted microscope and analysis of the effluent from the well-plate chamber. 
         FIG.  11    depicts a top view and a side view of an exemplary fluid exchange plug device of the present invention. 
         FIG.  12    depicts a side view of an exemplary fluid exchange plug device of the present invention with added tubing as inlet and an outlet. 
         FIG.  13    depicts a top view and a perspective view of an exemplary fluid exchange plug device of the present invention. 
         FIG.  14    depicts a top view of an exemplary fluid exchange plug device of the present invention with added tubing as inlet and an outlet. 
         FIG.  15    depicts a close up view of an exemplary fluid exchange plug device of the present invention with added tubing as inlet and an outlet. 
     
    
    
     DETAILED DESCRIPTION 
     Definitions 
     It is to be understood that the figures and descriptions of the present invention have been simplified to illustrate elements that are relevant for a clear understanding of the present invention, while eliminating, for the purpose of clarity, many other elements typically found in the art. Those of ordinary skill in the art may recognize that other elements and/or steps are desirable and/or required in implementing the present invention. However, because such elements and steps are well known in the art, and because they do not facilitate a better understanding of the present invention, a discussion of such elements and steps is not provided herein. The disclosure herein is directed to all such variations and modifications to such elements and methods known to those skilled in the art. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. 
     As used herein, each of the following terms has the meaning associated with it in this section. 
     The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. 
     “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods. 
     Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range. 
     Well Plate and Petri Dish Fluid Exchange Plug 
     The present invention provides a well plate and petri dish fluid exchange plug device configured to extend the utility of current standard well plates and petri dishes to allow improved environmental control over the internal conditions. In one embodiment, the fluid exchange plug of the present invention is configured to retrofit standard cell/tissue culture well-plates including but not limited to 6, 12, 24, 48, 96, 384, 1536 well-plate, and the like and petri dishes. In one embodiment, the fluid exchange plug of the present invention may be used for transfer of both air and liquid. In one embodiment, the fluid exchange plug device of the present invention houses fluidic channels that can be used to add, remove, and exchange fluid from well-plate and petri dish configurations in order to alter the physical, chemical, or biological conditions within the well-plate/petri dish without requiring the design of custom chemical or biological reactors. In one embodiment, the fluid exchange plug device of the present invention facilitates intermittent and continuous fluid exchange and/or perfusion through both individual wells and connected series of wells/dishes that can be used to expand the utility of current commercial well-plates and petri dishes by introducing a novel dimension of experimental conditions including but not limited to shear stress, nutrient concentration, cell-based stimuli, gas compositions and etc. In one embodiment, the device of the present invention allows for imaging during use, eliminating the need to remove the cells/samples from the environmental conditions created within the sealed compartments for imaging. In some embodiments, the device of present invention enables perfusion of biological cellular aggregates in commercially available plates. In one embodiment, the device of present invention maintains the physical form and layout of the commercially available plastics, while also adding the capabilities of custom bioreactors and fluidic systems. In one embodiment, the fluid exchange plug device of the present invention may be used to gently perfuse cell culture media into a well-plate to nourish, but not disrupt, fragile cell aggregates. In one embodiment, the fluid exchange plug device of the present invention may be used with imaging systems including but not limited to a microscope, a plate reader, a cytometer, etc. 
     Referring now to  FIG.  1   , an exemplary fluid exchange plug device  100  of the present invention is shown. Fluid exchange plug  100  comprises a plug body  102  having an upper surface  104 , a lower surface  106  and two or more sockets  108  positioned within plug body  102 , and two or more stoppers  110 . 
     In one embodiment, plug body  102  may have a height ranging between about 5-25 mm. In one embodiment, plug body  102  may be any shape including but not limited to circular, oval, square, rectangular, etc. In one embodiment, plug body  102  may be made of any material known to one skilled in the art including but not limited to plastics, metals, etc. In one embodiment, plug body  102  may be made of plastics such as PMMA, Acetal, polystyrene, etc. In one embodiment, plug body  102  may be made with any other plastic known to one skilled in the art. In one embodiment, plug body  102  may be made with any process known to one skilled in the art including but not limited to injection molding, milling, three-dimensional printing, etc. 
     Two or more sockets  108 , each comprise a first opening  112 , a second opening  114  and a lumen  116  therebetween. First opening  112  is positioned on upper surface  104 . In one embodiment, first opening  112  has a diameter ranging between about 2-10 mm. In one embodiment, lumen  116  has a height ranging between about 2-25 mm. In one embodiment, lumen  116  may comprise a threaded interior. In one embodiment, lumen  116  may be compatible with luer locks. In one embodiment, lumen  116  may be compatible with press-in adapters. In one embodiment, two or more sockets  108  are configured to receive microfluidic tubing. In one embodiment, the two or more sockets  108  are each adjacent to each other. In one embodiment, the distance between two sockets  108  may be ranging between about 5-200 mm. 
     Two or more stoppers  110  are positioned in a downward direction from lower surface  106  and comprise a first end  117  and a second end  118 . In one embodiment, each stopper  110  is positioned beneath each socket  108 . In one embodiment, two or more stoppers  110  have a diameter sized to fit within a well of a multi-well plate. In one embodiment, multi-well plate may be selected from one including but not limited to a 6 well-plate, a 12 well-plate, a 24 well-plate, a 48 well-plate, a 96 well-plate, a 384 well-plate, a 1536 well-plate, etc. In one embodiment, two or more stoppers  110  have a height ranging between about 1-25 mm. It is understood that the dimensions of two more stoppers  110  may vary upon the dimension of the well to be sealed, and that stoppers  110  of different heights may be provided. In one embodiment, two or more stoppers  110  may enter the wells to an arbitrary depth that can be modified to be tailored to the application. 
     In one embodiment, two or more stoppers  110  may further have a middle section  120 . In one embodiment, middle section  120  may be positioned anywhere along the length of each stopper  110 . In one embodiment, middle section  120  may have a diameter smaller than the diameter of stopper  110 . In one embodiment, middle section  120  is configured to receive a gasket or an O-ring  122 . In one embodiment, gasket or O-ring  122  are configured to facilitate a watertight seal between fluid two or more stoppers  110  and the multi-well plates. In another embodiment, gasket or O-ring  122  may facilitate the pressurization of each well, allowing it to be used as a reservoir for downstream fluidic and microfluidic applications. In one embodiment, middle section  120  may be positioned anywhere along the length of each stopper  110 . In one embodiment, gasket or O-ring  122  may facilitate the pressurization of each well, allowing it to be used for experiments requiring pressurization of a reaction or biological sample including but not limited to flowing inert gas over a chemical reaction or cyclically applying pressure to lung organoids. In one embodiment, gasket or O-ring  122  may be made from any material including but not limited to rubber. 
     Referring now to  FIG.  2   , second end  118  comprises at least one opening  124 . In one embodiment, at least one opening  124  may have any shape known to one in the art including but not limited to a circular, oval, square, rectangular, etc. In one embodiment, at least one opening  124  may be circular. In one embodiment, at least one opening  124  may have a diameter ranging between about 50 μm-1 cm. In one embodiment, at least one opening  124  may be a slit. 
     Second opening  114  is fluidly connected to at least one opening  124  through at least one socket lumen  126 . In one embodiment, at least one socket lumen  126  has a diameter ranging between about 50 μm-1 cm. In one embodiment, at least one socket lumen  126  may be used to connect to fluidic tubing and external pumps and reservoir. In one embodiment, at least one socket lumen  126  may act as an inlet port configured to permit adding or supplementing culture medium to the wells. In one embodiment, at least one socket lumen  126  may act as an outlet port configured to permit removal or aspiration of culture medium from the wells. In one embodiment, fluid exchange plug device  100  may comprise at least one inlet port and at least one outlet port. 
     Second end  118  of two adjacent stoppers  110  may be fluidly connected to each other through a stopper lumen  128 . Stopper lumen  128  extends through plug body  102 , connecting two adjacent stoppers  110 . In one embodiment, stopper lumen  128  may extend through plug body  102  and connect at least two stoppers  110  ( FIG.  3    and  FIG.  4   ). In one embodiment, stopper lumen  128  may connect any two stoppers together. In one embodiment, stopper lumen  128  may have a diameter ranging between about 5 μm-5 mm. 
     In one embodiment, fluid exchange plug device  100  may be a single use device. In one embodiment, fluid exchange plug device  100  may be a multiple use device. In one embodiment, the single or multiple use of device  100  may be configured based on the composing material. 
     Referring now to  FIG.  5 A  through  FIG.  5 E , in one embodiment, plug body  102  may comprise any number of sockets  108  and stoppers  110 , enough to fill all wells of a multi-well plate. 
     Referring now to  FIG.  6 A  through  FIG.  6 B , an exemplary fluid exchange plug device  200  of the present invention is shown. Fluid exchange plug device  200  comprises a plug body  202  having an upper surface  204 , a lower surface  206  and two or more sockets  208  positioned within plug body  202 , and a stopper  210 . 
     In one embodiment, plug body  202  may have a height ranging between about 5-25 mm. In one embodiment, plug body  202  may be any shape including but not limited to circular, oval, square, rectangular, etc. In one embodiment, plug body  202  may be made of any material known to one skilled in the art including but not limited to plastics, metals, etc. In one embodiment, metals may be used as to facilitate use with harsh chemicals with glass well-plates. In one embodiment, plug body  202  may be made of plastics such as PMMA, Acetal, polystyrene, etc. In one embodiment, plug body  202  may be made with any other plastic known to one skilled in the art. In one embodiment, plug body  202  may be made with any process known to one skilled in the art including but not limited to injection molding, milling, three-dimensional printing, etc. 
     Two or more sockets  208 , each comprise a first opening  212 , a second opening  214  and a lumen  216  therebetween. First opening  212  is positioned on upper surface  204 . In one embodiment, first opening  212  has a diameter ranging between about 2-15 mm. In one embodiment, lumen  216  has a height ranging between about 2-25 mm. In one embodiment, lumen  216  may comprise a threaded interior. In one embodiment, lumen  216  may be compatible with luer locks. In one embodiment, lumen  216  may be compatible with press-in adapters. In one embodiment, two or more sockets  208  are configured to receive microfluidic tubing. In one embodiment, the two or more sockets  208  may be placed anywhere on lower surface  206 . In one embodiment, the distance between two sockets  208  may be ranging between about 1-20 cm. 
     Stopper  210  is positioned in a downward direction from lower surface  206  and comprises a first end  217  and a second end  218 . In one embodiment, stopper  210  is positioned beneath plug body  202 . In one embodiment, stopper  210  has a diameter sized to fit within a petri dish. In one embodiment, petri dish may be selected from one including but not limited to a petri dish having a diameter of 60 mm, 100 mm, 150 mm, etc. In one embodiment, stopper  210  may have a height ranging between about 1-20 mm. It is understood that the dimensions of stopper  210  may vary upon the dimension of the petri dish to be sealed, and that stopper  210  of different heights may be provided. 
     In one embodiment, stopper  210  may further have a middle section  220 . In one embodiment, middle section  220  may be positioned anywhere along the length of each stopper  210 . In one embodiment, middle section  220  may have a diameter smaller than the diameter of stopper  210 . In one embodiment, middle section  220  may have a diameter ranging between about 15-100 mm. In one embodiment, middle section  220  is configured to receive a gasket or an O-ring  222 . In one embodiment, gasket or O-ring  222  are configured to facilitate a watertight seal between stopper  210  and the petri dish. In another embodiment, gasket or O-ring  222  may facilitate the pressurization of a petri dish, allowing it to be used as a reservoir for downstream fluidic and microfluidic applications. In one embodiment, gasket or O-ring  222  may facilitate the pressurization of a petri dish, allowing it to be used for experiments requiring pressurization of a reaction or biological sample including but not limited to flowing inert gas over a chemical reaction or cyclically applying pressure to lung organoids. In one embodiment, gasket or O-ring  222  may be made from any material including but not limited to rubber. 
     Second end  218  comprises at least one opening  224 . In one embodiment, at least one opening  224  may have any shape known to one in the art including but not limited to a circular, oval, square, rectangular, etc. In one embodiment, at least one opening  224  may be circular. In one embodiment, at least one opening  224  may have a diameter ranging between about 10 μm-1 cm. In one embodiment, at least one opening  224  may be a slit. 
     Second opening  214  is fluidly connected to at least one opening  224  through at least one socket lumen  226 . In one embodiment, at least one socket lumen  226  has a diameter ranging between about 10 μm-1 cm. In one embodiment, at least one socket lumen  226  may be used to connect to fluidic tubing and external pumps and reservoir. In one embodiment, at least one socket lumen  226  may act as an inlet port configured to permit adding or supplementing culture medium to the wells. In one embodiment, at least one socket lumen  226  may act as an outlet port configured to permit removal or aspiration of culture medium from the wells. In one embodiment, fluid exchange plug device  200  may comprise at least one inlet port and at least one outlet port. 
     Referring now to  FIG.  7 A  through  FIG.  7 C , in one embodiment, stopper  210  may comprise at least one channel  228  embedded in second end  218 , wherein stopper  210  is sized to fit flush against a lower surface of a petri dish. In one embodiment, stopper  210  may have a height of ranging between about 1-40 mm to fit flush against the lower surface of the petri dish. In one embodiment, at least one channel  228  may be any shape known to one skilled in the art. In one embodiment, the geometry of at least one channel  228  may be modified to made it to adapt for the creation of fluidic and microfluidic circuits and linear flow chambers within the petri dish. In one embodiment, two or more sockets  208  may be positioned within at least one channel  228 . 
     In one embodiment, fluid exchange plug device  200  may be a single use device. In one embodiment, fluid exchange plug device  200  may be a multiple use device. In one embodiment, the single or multiple use of device  200  may be configured based on the composing material. 
     The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.