Perfusable Bioreactor

Abstract

Provided herein are injection-molded perfusable bioreactors and systems including the bioreactor. The bioreactor can include a lid, a frame with an array of sample wells, a membrane attached to the bottom of the frame below the array of sample wells, and a skirt attached to the bottom of the frame such that the membrane is positioned between the frame and the skirt. The skirt can include multiple channels corresponding to the sample wells. When the sample wells are filled with a 3D cell culture support matrix, pressure above the 3D cell culture support matrix is ​​atmospheric pressure and pressure below the membrane is less than atmospheric pressure, thereby perfusing fluid along a vertical fluid flow path from the sample wells through the 3D cell culture support matrix, through the membrane, and along the channels to a reservoir.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 047,673, entitled "Clear Single-use Passive Perfusion Enabled 3D Bio-reactor Well Plate for Cell Culture and Experimentation," filed July 2, 2020, and U.S. Provisional Application No. 63 / 161,704, entitled "PERFUSION-ENABLED BIOREACTOR," filed March 16, 2021, the disclosures of each of which are incorporated herein by reference in their entireties. [Background technology]

[0002] 3D culture environments are necessary for laboratory research that does not entirely rely on the use of animals or highly complex bioreactors. Animal care is often quite costly, and the results of animal research cannot always be adequately translated to human biology. Furthermore, existing bioreactors for creating 3D culture environments are often complex, involving computer-controlled pumps and custom-made components, and may require highly trained personnel to operate them. In short, both existing paths to 3D culture tend to have high barriers to entry in terms of cost and skill. [Brief explanation of the drawings]

[0003] Further aspects of the present disclosure will be more readily understood by reviewing the following detailed description of various embodiments thereof when taken in conjunction with the accompanying drawings.

[0004] [Figure 1] FIG. 1 illustrates an exploded view of a perfusable bioreactor according to an embodiment of the present disclosure.

[0005] 2A-2B show the bioreactor 100 from FIG. 1 in a closed and assembled position according to an embodiment of the present disclosure. [Figure 2A] FIG. 2A is a perspective view. [Figure 2B] FIG. 2B is a bottom view.

[0006] [Figure 3] FIG. 3 shows a cross-sectional view of the perfusable bioreactor shown in FIG. 2 according to an embodiment of the present disclosure.

[0007] 4A-4F provide diagrams showing various views of a perfusable bioreactor according to an embodiment of the present disclosure. [Figure 4A] FIG. 4A is a multi-sectional view in which the bioreactor is divided into quadrants to show the interior at various depths. [Figure 4B] FIG. 4B is a top view of the bioreactor. [Figure 4C] 4C and 4D are end and side views, respectively. [Figure 4D] 4C and 4D are end and side views, respectively. [Figure 4E] 4E and 4F are cross-sectional side views taken along the cross section shown in FIG. 4D. [Figure 4F] 4E and 4F are cross-sectional side views taken along the cross section shown in FIG. 4D.

[0008] 5A-5F provide views of the frame from various angles. [Figure 5A] FIG. 5A shows a top perspective view. [Figure 5B] FIG. 5B shows a bottom perspective view. [Figure 5C] FIG. 5C shows a bottom view. [Figure 5D] FIG. 5D shows a top view. [Figure 5E] FIG. 5E shows a side view. [Figure 5F]FIG. 5F shows an end view.

[0009] 6A-6F provide views of the base from various angles. [Figure 6A] FIG. 6A shows a top perspective view. [Figure 6B] FIG. 6B shows a bottom perspective view. [Figure 6C] FIG. 6C shows a top view. [Figure 6D] FIG. 6D shows a bottom view. [Figure 6E] FIG. 6E shows a side view. [Figure 6F] FIG. 6F shows an end view.

[0010] 7A-7C provide views of the lid from various angles. [Figure 7A] FIG. 7A shows a bottom perspective view. [Figure 7B] FIG. 7B shows a top perspective view. [Figure 7C] FIG. 7C shows a bottom view (underside of the lid).

[0011] 8A-8E provide views of the skirt from various angles. [Figure 8A] FIG. 8A shows a top perspective view. [Figure 8B] FIG. 8B shows a bottom perspective view. [Figure 8C] FIG. 8C shows a top view. [Figure 8D] FIG. 8D shows a bottom view. [Figure 8E] FIG. 8E shows a side view.

[0012] [Figure 9A] 1 provides an exploded view of a bioreactor molded from a transparent material according to an embodiment of the present disclosure. [Figure 9B] 1 provides an assembly drawing of a bioreactor molded from a transparent material according to an embodiment of the present disclosure.

[0013] The drawings depict only exemplary embodiments and, therefore, should not be considered limiting of the scope described herein, as other equally effective embodiments are within the scope and spirit of the present disclosure. The elements and features shown in the drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of the embodiments. Additionally, certain dimensions may be exaggerated to help visually convey certain principles. In the drawings, like reference numbers between the figures designate similar or corresponding, but not necessarily identical, elements. DETAILED DESCRIPTION OF THE INVENTION

[0014] Before describing the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which will be limited by the appended claims.

[0015] Where a range of values ​​is provided, unless the context clearly dictates otherwise, it is understood that each intervening value between the upper and lower limit of that range, to the tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0016] Unless otherwise defined, 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 disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this disclosure, the preferred methods and materials are now described.

[0017] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0018] Embodiments of the present disclosure employ techniques of biology, materials science, mechanical engineering, and the like, which are within the skill of the art, unless otherwise indicated.

[0019] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of the methods and uses of the materials and devices disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperatures, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperatures are in °C, and pressures are at or near atmospheric. Ambient temperature and pressure are defined as 20°C and 1 atmosphere.

[0020] Before describing embodiments of the present disclosure in more detail, it is to be understood that this disclosure is not limited to particular materials, reagents, reaction materials, or manufacturing processes, etc., unless otherwise indicated, as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It is also possible that steps in the present disclosure can be executed in differing order, where this is logically possible.

[0021] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0022] As used herein, the following terms have the meanings ascribed to them unless otherwise specified. In this disclosure, "consisting essentially of" or "consisting essentially of," and the like, when applied to methods and compositions encompassed by this disclosure, refers to compositions such as those disclosed herein, but which may contain additional structural groups, composition components, or method steps (or analogs or derivatives thereof). However, such additional structural groups, composition components, method steps, and the like do not materially affect the basic and novel characteristics of the composition or method as compared to those of the corresponding composition or method disclosed herein. When applied to methods and compositions encompassed by this disclosure, "consisting essentially of" or "consisting essentially of" and the like have the meaning ascribed to them in U.S. patent law, and the terms are open-ended, allowing for the presence of more than those recited, but excluding prior art embodiments, so long as the basic or novel characteristics of the recited items are not altered by the presence of more than those recited.

[0023] Overall Considerations In accordance with the purposes of this disclosure, as embodied and broadly described herein, embodiments of the present disclosure relate in some aspects to perfusable bioreactors, systems including perfusable bioreactors, and methods for 3D cell culture using perfusable bioreactors.

[0024] The perfusable bioreactors described herein are designed as part of a system for efficient and effective culturing of cells or other biological materials in three dimensions (3D). The perfusable bioreactors described herein can be used in conjunction with a gel-like support matrix seeded with cells or biological materials. The system uses a low-pressure gradient to drive the flow of liquid culture medium over the cells, allowing the liquid to perfuse through the support matrix and an underlying microporous membrane into a collection reservoir. The constant flow of fresh culture medium throughout the culture maintains viability in the culture by flushing out metabolic waste products and providing fresh nutrients and other factors from the fresh medium. Advantageously, in the bioreactors and systems described herein, the perfusion flow can flow consistently for an extended period of time before the liquid medium at the top of the bioreactor (also referred to as a plate or perfusion plate) is replenished. In some embodiments, the perfusion flow can flow consistently for approximately 24 hours before the liquid is replenished. The flow rate can also be adjusted by adjusting one or more of the membrane porosity and gel structure. In some embodiments, the perfusion flow can be consistent for more than 24 hours before the fluid is replenished.

[0025] Advantageously, the perfusable bioreactors and systems described herein allow for constant perfusion flow throughout the culture without complex pumps / machines or modifications to existing culture equipment used in conventional culture.

[0026] The perfusable bioreactors and systems described herein can be used for a wide range of 3D culture experiments, some of which may not yet be envisioned. Some applications include cell culture, including cancer and other diseases, and the study of drug effects on cell-cell interactions (e.g., immune cell-tumor interactions) in 3D, in vitro, and in silico cell motility, as well as multi-organoid co-culture studies.

[0027] The present disclosure includes a perfusable bioreactor including a lid, a frame including a sample well or an array of sample wells, and a base. The base may include at least one reservoir. The perfusable bioreactor may include a membrane located below the frame. The membrane may be adhered to the bottom of the sample well. The base may be wider than the frame and the lid. A locking mechanism may seal the frame to the base. At least the base and frame may be injection molded. The lid may also be injection molded.

[0028] In some embodiments, the perfusable bioreactor can include a skirt attached to the bottom of the frame, with the membrane sandwiched between the frame and the skirt. The skirt can have one or more channels, each channel correlating with a sample well and leading to one or more reservoirs in the base. Advantageously, the skirt can direct droplets formed from each well to each of the quadrants and prevent wicking across to other wells. The skirt can also enhance adhesion of the membrane to the frame. The skirt can also be injection molded.

[0029] In some embodiments, the perfusable bioreactor can be disposable (e.g., for single use or as a consumable item). Advantageously, disposable perfusable bioreactors allow for sterilization and packaging before use in a study and minimal cleanup thereafter without having to consider resterilization for reuse. Furthermore, the easily reproducible complex geometries that can be produced by injection molding mean that the device can incorporate a wide range of features while still being rapidly mass-producible. These features can include visual feedback using scales in the collection reservoir, pressure port geometries for connecting negative pressure tubing, glue removal channels for attaching membranes without clogging the wells, and elastomeric ring locking mechanisms that slightly clamp the assembly to ensure a proper seal.

[0030] The frame may include an array of sample wells. The frame may include a recess, where a well or group of wells resides in the bottom of the recess. In certain embodiments, the frame may include 24 individual wells, which may be divided into four quadrants, each quadrant having a recess with six wells. Corresponding dividers may be present in the base. In some embodiments, the base may include multiple separate reservoirs. The base may be divided into quadrants so that there are four reservoirs (e.g., collection wells), one reservoir in each quadrant. A skirt is adhered to the bottom of the frame, such that the microporous membrane is sandwiched between the skirt and the frame. The membrane is heat-sealed or glued to the bottom of the wells in the frame, and the skirt is adhered (e.g., with an adhesive) to the frame. The skirt provides channels leading directly from each individual well into the collection reservoir for the corresponding quadrant of the plate. Thus, the base is divided into multiple reservoirs, with each channel directing fluid from the sample well into a specific reservoir. Advantageously, this arrangement ensures that the effluent from each well falls into the appropriate reservoir without wicking into other areas where experimental conditions may be different.

[0031] In some embodiments, the base may contain a single reservoir so that all effluent from all wells is collected in one large reservoir. In such embodiments, the skirt may be omitted.

[0032] In still other embodiments, different array patterns or number of wells can be envisioned by those skilled in the art. For example, the frame can have 24 wells divided into 6 groups, 36 wells divided into 6 groups, 12 wells divided into 4 groups, etc. Each group can reside in a recess. The base can have corresponding dividers to create reservoirs for each group of wells in the frame.

[0033] In some embodiments, each well group may include a recessed fill port used when filling the plate to prevent the gel in each well from fracturing. Advantageously, the fill port prevents a direct jet of fluid from pushing the gel against the surrounding area or crushing the seeded cells. The fill port may be a hemispherical recess. The fill port may accommodate an 18-gauge needle and syringe, or the like. The recess above the well may be filled by inserting a needle into the fill port and slowly depressing the plunger on the syringe. In this way, liquid (e.g., liquid culture medium) may be added to the recess above the gel in each well. The fill port allows turbulence from the liquid flow from the needle to be directed upward, not into or around the surrounding wells filled with gel. Without the fill port, the gel may fractured when liquid is added during initial experiment setup or liquid refilling.

[0034] In some embodiments, the frame contains pressure ports. In some embodiments, there may be a pressure port for each reservoir. The pressure ports may function as both media collection ports and ports for tubing. The pressure ports are designed so that tubing can be pressurized into the pressure port, and the pressurization action seals the tubing. Advantageously, any mechanism for actuating low pressure may be used in conjunction with this tubing (e.g., valves, pumps, syringes, etc.). The amount of pressure used to drive the perfusion flow is variable and can be changed based on experimental needs.

[0035] Advantageously, the reservoirs in the base can be individually accessed, such as via a syringe needle through the port used to actuate the pressure change. Effluent medium can be collected individually from each of the reservoirs without the need to disassemble the plate. The base dividers can be positioned so that liquid from each of the reservoirs can be accessed through a single port.

[0036] In some embodiments, the frame may include an interference fit pressure port that allows for the use of any vacuum mechanism with a compatible tubing diameter.

[0037] In some embodiments, the pressure port may be located in the base.

[0038] The lid can sit atop the plate to protect the wells from atmospheric contaminants that could interfere with the experiment. The lid may include a downward 1-2 mm air gap to prevent contaminants from falling into the wells while allowing gas exchange. The lid is designed to sit flush with the frame. As mentioned above, the widest and largest part of the plate is the base. With a flush lid, there is a lower risk that the user will accidentally lift the lid instead of the entire unit, thereby exposing the plate to contaminants.

[0039] In some embodiments, the lid may include a protrusion on the underside to center the lid on the frame and provide sufficient clearance for gas exchange. In some embodiments, the protrusion corresponds to the gap separating groups of sample wells in the frame. In some embodiments, an air filter may be placed in the gap to further reduce the ingress of contaminants. The lid may also include a gas port to allow desired gases to flow directly onto the medium in the bioreactor.

[0040] One or more of the lid, frame, base, and skirt may be injection molded. The injection-molded parts may be made from any injection-moldable plastic or polymer that is stable up to physiological temperatures (e.g., about 37°C). Suitable materials include, but are not limited to, polystyrene, nylon, acrylic, polycarbonate, polyoxymethylene, acrylonitrile butadiene styrene (ABS), polypropylene, and polyethylene.

[0041] In some embodiments, the injection molded parts can be optically clear, allowing for monitoring of the liquid medium level at the top and bottom of the plate without the need for disassembly. Advantageously, the use of fastener-free, thin-walled, clear plastic parts increases the overall allowable volume of liquid culture medium without increasing the footprint of the assembly and provides useful visual feedback on the perfusion rate.

[0042] The frame can be secured onto the base by a locking mechanism. In some embodiments, the frame is pressed onto the base via elastic bands (e.g., O-rings or gaskets) on each end of the plate, which function as the locking mechanism. The assembled bioreactor can include grooves on each end to prevent the elastic bands from sliding out of place. The base and frame can be designed to mate and form a seal (e.g., a labyrinth seal or a compression seal). For example, the base can include a channel around its top edge filled with a soft elastomeric material (e.g., a gasket or silicone-based material). The channel can receive a protruding ridge on the bottom of the frame. The clamping force applied by the elastic bands around each end of the assembled plates can press the frame down onto the base, thus forcing the protruding ridge on the frame into the elastomer-filled seal channel on the base. The frame and bottom can form a seal along this edge due to compression of the elastomer in the seal channel. In other embodiments, the seal channel can be in the frame and the protrusion can be in the base. The seal creates an airspace in the assembled plate that is only accessible through a pressure port or through a microporous membrane at the bottom of each of the wells. In some embodiments, the channel can be filled with an elastomeric material that is cured to form a seal. In other embodiments, a gasket, O-ring, or other resilient seal can be attached to the channel. Alternatively, the locking mechanism can be a clamp, clip, latch, snap closure, or other suitable closure sufficient to apply force so that the base and frame are sealably mated.

[0043] An embodiment of the present disclosure includes a system including a perfusable bioreactor as described above, wherein the base of the well plate is configured to sealably mate with the frame, such that, when mated, the sample wells are in fluid communication with the reservoirs through the channels in the skirt. When the sample wells are filled with a 3D cell culture support matrix, the system is configured such that the pressure above the 3D cell culture support matrix is ​​atmospheric and the pressure below the membrane is negative (e.g., subatmospheric). The 3D cell culture support matrix contains seeded cells and sits atop the microporous membrane. Liquid cell culture medium is flowed onto the top of the 3D cell culture support matrix. The negative pressure perfuses the liquid cell culture medium along a vertical fluid flow path from the sample wells through the 3D cell culture support matrix, through the membrane, and along the channels to the reservoirs. The perfused fluid can then be collected from the reservoirs through one or more ports, such as with a syringe.

[0044] The microporous membrane may be constructed of polycarbonate, cellulose, nylon, PEEK, polypropylene, or a combination thereof, for example.

[0045] Referring now to the figures, FIG. 1 provides an exploded view of an exemplary perfusable bioreactor 100. This example includes a frame 110, a portion of which is divided into four quadrants. Each quadrant has a recess 114. A membrane 160 seals to the bottom of the frame 110. A skirt 140 adheres to the bottom quadrant of the frame 110, sandwiching the membrane 160. The skirt 140 contains channels 142 that correspond to sample wells 180 located in the frame 110 (sample wells 180 are not visible in this view). The frame 110 has a protruding ridge 116 that seats within a sealing channel 122 in the base 120. The sealing channel 122 contains an elastomeric material 123 (not shown). The locking mechanism 150 is a gasket that compresses the frame 110 and base 120 together to form a seal. Frame 110 and base 120 have grooves 112 to hold locking mechanism 150 in place. Lid 130 sits atop the frame with a small amount of internal clearance (e.g., about 1-2 mm) to allow for gas exchange. Reservoir 124 is formed within base 120 by partition 126 and one or more curved partitions 128, which are shown in more detail in Figures 6A-6F.

[0046] Figure 2A shows a perspective view of the bioreactor 100 from Figure 1 in the closed and assembled position. The pressure port 170 is shown on the frame 110. Figure 2B is a perspective view of the unit turned upside down to show the base 120.

[0047] Figure 3 provides a cross-sectional view of the bioreactor 100 shown in Figures 1-2E. The membrane 160 is not shown in this view. Each of the quadrants in the frame 110 contains six sample wells 180, as can be seen from the corresponding channels 142 in the skirt 140. The grouped sample wells 180 flow through the channels into separate reservoirs 124 formed in the base 120.

[0048] Figures 4A-4F provide various views of another example of a perfusable bioreactor. Figure 4A is a multi-section view from the top, with the bioreactor divided into quadrants to show the interior at various depths. The upper left shows the frame 110 in which the sample wells 180 are grouped together. The sample wells 180 and fill port 190 are visible in this section. The upper right shows the lid 130. The lower left shows the reservoir 124 in the base 120, which corresponds to the shape of the group of sample wells 180 seated in from the frame 110 described above. The lower right quadrant shows a skirt with six channels to accommodate the six sample wells in the frame. As can be seen from this view, the reservoir 124 in the base 120 (lower left quadrant of the figure) can be accessed through a pressure port 170 in the frame 110 (upper left quadrant). Reservoir 124 is separated into quadrants by dividers 126 and 128 (see the description of Figures 6A-6F for a detailed description). Figure 4B is a top view of the bioreactor. Figures 4C and 4D are end and side views, respectively, of the assembled bioreactor. Exemplary dimensions of the bioreactor are provided. In this example, the entire unit has a height of approximately 60 mm, a width of approximately 50 mm, and a length of approximately 100 mm. This unit can be scaled as envisioned by those skilled in the art. Figures 4E and 4F are side cross-sectional views taken along sections AA and BB, respectively, as shown in Figure 4F. In Figure 4E, membrane 160 is shown seated between sample well 180 and skirt 140. Figure 4F shows the mating of sample well 180 and skirt channel 142.

[0049] Figures 5A-5F provide views of an embodiment of frame 110 from various angles. Figure 5A shows a top perspective view in which the frame is divided into quadrants of sample wells 180. Recesses 114 and pressure ports 170 are visible. Figure 5B shows a bottom perspective view in which six sample wells per quadrant recess 114 are visible. Figure 5C shows a bottom view, Figure 5D shows a top view in which fill ports 190 are visible, Figure 5E shows a side view, and Figure 5F shows an end view.

[0050] FIGS. 6A-6F provide views of the base 120 from various angles. The base is configured to receive the quadrant-divided frame 110, as shown in FIGS. 5A-5F. FIG. 6A shows a top perspective view, FIG. 6B shows a bottom perspective view, FIG. 6C shows a top view, FIG. 6D shows a bottom view, FIG. 6E shows a side view, and FIG. 6F shows an end view. As can be seen from the figures, particularly FIGS. 6A and 6C, the base is divided into four reservoirs 124 by dividers. In the illustrated embodiment, there is one long divider 126 that runs the length of the plate. Then, on either side of the long divider 126, there are two mirror-image "curved" dividers 128. These curved dividers 128 act to extend the reservoirs on the side of the plate farthest from the pressure ports (the two reservoirs on the right in FIG. 6C). At the ends of the curved dividers 128, there is a point where the dividers meet, forming a plus (+) shape. This point 129 is located directly below pressure ports 170 (also referred to as media collection ports), through which each reservoir can be reached with a needle and individually collected without removing the frame 110 from the base 120.

[0051] Liquid medium perfuses through the gel layer in each well and then drops down through the skirt 140 into its collection reservoir 124. When a reservoir 124 is full and visible through the clear base, it needs to be emptied so that perfusion can continue without overfilling the base 120. This medium removal can be done without separating the frame 110 from the base 120 by withdrawing the medium in the reservoir 124 through the medium collection port (pressure port 170). A plus (+) shaped area 129 of the divider in the base 120 allows medium from each reservoir 124 to be accessed with a needle through the medium collection (pressure) port 170.

[0052] 7A-7C provide views of the lid 130 from various angles. FIG. 7A shows a bottom perspective view (underside of the lid). The lid 130 includes dividers 132 that correspond to recesses 114 in the frame 110 and / or provide structural support to the lid 130. Curved corner supports 134 in the corners of the lid seat on the frame 110. The curved corner supports 134 allow the lid 130 to self-center on the frame 110. This creates a consistent gap between the edge of the lid 130 and the top of the frame 110 all the way around the bioreactor 100. FIG. 7B shows a top perspective view, and FIG. 7C shows a bottom view (underside of the lid). As shown in FIG. 7C, in some embodiments, the corner supports 134 may be straight.

[0053] Figures 8A-8E provide views of skirt 140 from various angles. Figure 8A shows a top perspective view, Figure 8B shows a bottom perspective view, Figure 8C shows a top view, Figure 8D shows a bottom view, and Figure 8E shows a side view. Skirt 140 includes a lip 144 to provide a seat for frame 110. In this example, channel 142 corresponds to a 24-well array such as that shown in Figures 5A-5F.

[0054] 9A and 9B provide exploded and assembled views, respectively, of a bioreactor 100 molded from a transparent material.

[0055] Aspects of the Disclosure The present disclosure is better understood by reading the following numbered aspects, which should not be confused with the claims: Any of the following numbered aspects may, in some instances, be combined with aspects described elsewhere in this disclosure, and such combinations are intended to form part of this disclosure.

[0056] Aspect 1. A perfusable bioreactor comprising: a lid; a frame having at least one sample well in a recess and at least one fill port; a membrane located below the frame, the membrane being adhered to the bottom of the at least one sample well; a base having at least one reservoir, the base being wider than the frame and the lid; and a locking mechanism sealing the frame to the base, wherein at least the frame and base are injection molded.

[0057] Aspect 2. A perfusable bioreactor as described in aspect 1, further comprising a skirt adhered to the bottom of the frame, the membrane being positioned between the frame and the skirt, and the skirt comprising channels leading from each sample well to the reservoir.

[0058] Aspect 3. A perfusable bioreactor according to any of the preceding aspects, wherein the frame comprises an array of sample wells, the base is divided into a plurality of reservoirs, and each of the channels directs fluid from the sample well into a particular reservoir.

[0059] Embodiment 4. The perfusable bioreactor of embodiment 3, wherein the frame comprises a medium collection port capable of collecting fluid from each reservoir.

[0060] Embodiment 5. The perfusable bioreactor of any preceding embodiment, wherein the locking mechanism comprises a pair of gaskets at each end of the bioreactor that clamp the frame to the base.

[0061] Embodiment 6. The perfusable bioreactor of any preceding embodiment, wherein the base includes a channel around an upper edge for receiving a lower edge of the frame such that the base and frame are sealably mated.

[0062] Embodiment 7. The perfusable bioreactor of any preceding embodiment, wherein the bottom of the lid sits flush with the frame, and the lid comprises a raised central portion that provides about 1-2 mm of clearance from the frame.

[0063] Embodiment 8. The perfusable bioreactor of any preceding embodiment, wherein the membrane is adhered to the bottom of the plurality of wells by heat sealing.

[0064] Aspect 9. The perfusable bioreactor of any preceding aspect, wherein the membrane is constructed of polycarbonate, cellulose, nylon, PEEK, polypropylene, or a combination thereof.

[0065] Aspect 10. The perfusable bioreactor of any preceding aspect, wherein one or more of the lid, frame, base, and skirt are transparent.

[0066] Embodiment 11. The perfusable bioreactor of any preceding embodiment, wherein the frame comprises a plurality of recesses, each recess containing a plurality of sample wells and at least one fill port.

[0067] Embodiment 12. A system for 3D cell culture, comprising: an injection molded perfusable bioreactor; the perfusable bioreactor comprising: a lid; a frame including at least one recess with an array of sample wells and at least one fill port; a membrane adhered to a bottom of the frame below the array of sample wells; a skirt adhered to the bottom of the frame such that the membrane is located between the frame and the skirt, the skirt comprising a plurality of channels; a base comprising at least one reservoir, the base being wider than the frame and the lid; and a locking mechanism sealing the frame to the base; the base is configured to be sealably mated with the frame such that, when mated, the sample wells are in fluid communication with the reservoirs through the channels in the skirt; the frame includes medium collection ports through which medium can be collected from each reservoir in the base; and when the sample wells are filled with the 3D cell culture support matrix, the system is configured such that pressure above the 3D cell culture support matrix is ​​atmospheric pressure and pressure below the membrane is negative pressure, and the negative pressure perfuses fluid along a vertical fluid flow path from the sample wells through the 3D cell culture support matrix, through the membrane, and along the channel to the reservoirs.

[0068] Embodiment 13. The system of embodiment 12, wherein the port is configured to allow a sample of the perfused fluid to be collected from the reservoir using a syringe.

[0069] Embodiment 14. The system of embodiment 12, wherein the negative pressure is drawn by a tube connected to the port.

[0070] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed in a range format herein. Such a range format is used for convenience and brevity, and therefore should be understood to be interpreted in a flexible manner to include not only the numerical values ​​explicitly recited as the limits of the range, but also all individual numerical values ​​or subranges subsumed within that range, as if each numerical value and subrange were explicitly recited. By way of example, a concentration range of "about 0.1% to about 5%" should be interpreted not only to include the explicitly recited concentration of about 0.1% by weight to about 5% by weight, but also to include individual concentrations (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the stated range. In one embodiment, "about 0" can refer to 0, 0.001, 0.01, or 0.1. In one embodiment, the term "about" can include conventional rounding according to the significance of numerical values. Furthermore, the phrase "about 'x' to 'y'" includes "about 'x' to about 'y'".

[0071] It should be emphasized that the above-described embodiments of the present disclosure are merely possible examples of implementations, and are set forth solely for a clear understanding of the principles of the present disclosure. Many variations and modifications may be made to the above-described embodiments of the present disclosure without substantially departing from the spirit and principles of the present disclosure. All such modifications and variations are intended to be included herein within the scope of the present disclosure.

Claims

1. 1. A perfusable bioreactor comprising: The lid and a frame comprising at least one sample well within a recess and at least one fill port having a hemispherical recess configured to prevent turbulence generated during filling of a liquid from being directed into or around the surrounding sample well; a membrane located below the frame, the membrane being adhered to the bottom of the at least one sample well; a base comprising at least one reservoir, said base being wider than said frame and said lid; a locking mechanism that seals the frame to the base; A perfusable bioreactor, wherein at least the frame and the base are injection molded.

2. 2. The perfusable bioreactor of claim 1, further comprising a skirt adhered to the bottom of the frame, the membrane being positioned between the frame and the skirt, the skirt comprising channels leading from each sample well to the reservoir.

3. 3. The perfusable bioreactor of claim 2, wherein the frame comprises an array of sample wells, the base is divided into a plurality of reservoirs, and each of the channels directs fluid from a sample well into a particular reservoir.

4. 4. The perfusable bioreactor of claim 3, wherein the frame includes a medium collection port capable of collecting fluid from each reservoir.

5. 5. The perfusable bioreactor of claim 1, wherein the locking mechanism comprises a pair of gaskets at each end of the bioreactor that clamp the frame to the base.

6. 6. The perfusable bioreactor of claim 1, wherein the base includes a channel around an upper edge for receiving a lower edge of the frame such that the base and frame are sealably mated.

7. 7. The perfusable bioreactor of any one of claims 1 to 6, wherein the bottom of the lid sits flush with the frame and the lid comprises a raised central portion that provides 1 to 2 mm of clearance from the frame.

8. 8. The perfusable bioreactor of claim 1, wherein the membrane is adhered to the bottom of the plurality of wells by heat sealing.

9. 9. The perfusable bioreactor of claim 1, wherein the membrane is constructed of polycarbonate, cellulose, nylon, PEEK, polypropylene, or a combination thereof.

10. 10. The perfusable bioreactor of any one of claims 1 to 9, wherein one or more of the lid, the frame, and the base are transparent to the extent that it is possible to monitor the level of liquid medium at the top and bottom of the bioreactor.

11. 11. The perfusable bioreactor of claim 1, wherein the frame comprises a plurality of recesses, each recess containing a plurality of sample wells and at least one fill port.

12. 1. A system for 3D cell culture, comprising: an injection molded perfusable bioreactor, the perfusable bioreactor comprising: The lid and a frame including at least one recess having an array of sample wells and at least one fill port having a hemispherical recess configured to prevent turbulence generated during filling of a liquid from being directed into or around the surrounding sample wells; a membrane adhered to the bottom of the frame beneath an array of sample wells; a skirt adhered to the bottom of the frame, the membrane being positioned between the frame and the skirt, the skirt comprising a plurality of channels; a base comprising at least one reservoir, said base being wider than said frame and said lid; a locking mechanism that seals the frame to the base; a well plate base configured to sealably mate with the frame such that, when mated, the sample wells are in fluid communication with the reservoirs through the channels in the skirt; the frame includes a medium collection port through which medium can be collected from each reservoir in the base; 10. The system of claim 1, wherein when the sample well is filled with a 3D cell culture support matrix, the system is configured such that there is atmospheric pressure above the 3D cell culture support matrix and negative pressure below the membrane, the negative pressure perfusing fluid along a vertical fluid flow path from the sample well through the 3D cell culture support matrix, through the membrane, and along the channel to the reservoir.

13. 13. The system of claim 12, wherein the medium collection port is configured to allow a sample of the perfused fluid to be collected from the reservoir using a syringe.

14. 13. The system of claim 12, wherein the negative pressure is drawn by a tube connected to the medium collection port.

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