Oxygenating reservoirs for microfluidic applications and methods of using thereof

Abstract

Oxygenating reservoirs and methods of using thereof are described herein. The Oxygenating reservoirs can provide improved oxygenation and mixing of culture media flowed therethrough. The oxygenating reservoirs can be used for various microfluidic applications when incorporated into microfluidic systems or platforms.

Description

[0001] OXYGENATING RESERVOIRS FOR MICROFLUIDIC APPLICATIONS AND METHODS OF USING THEREOF CROSS-REFERENCED TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 710,402, filed October 22, 2024, which is hereby incorporated herein by reference in its entirety.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with government support under grant numbers EB029132 and HD 110335 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0004] FIELD OF THE INVENTION

[0005] This invention is generally in the field of oxygenating reservoirs for culture media which can be used in microfluidic applications.

[0006] BACKGROUND OF THE INVENTION

[0007] Currently, reservoirs for culture media used in microfluidic applications typically feature a media inlet and outlet at the bottom of the reservoir, but this configuration minimizes mixing of the culture media. Although the culture media in contact with air is oxygenated, that culture media does not move to the bottom of the reservoir to the outlet. This causes only a fraction of the culture media to circulate, with the majority of the oxygenated media remaining stagnant in the reservoir. Such a lack of oxygenation in the culture media can have deleterious effects, such as inhibiting cell and tissue culture.

[0008] Therefore, there remains a need for reservoirs which can address and overcome the issues of currently used reservoirs.

[0009] Therefore, it is the object of the present invention to provide improved reservoirs which can provide improved oxygenation and mixing of culture media.

[0010] It is a further object of the present invention to provide methods of using such reservoirs for microfluidic applications.

[0011] SUMMARY OF THE INVENTION

[0012] Described herein are oxygenating reservoirs, or oxygenators, that can be integrated with a microfluidic device, such as a microfluidic chip, or in a microphysiological system. Such oxygenating reservoirs can replace existing culture media reservoirs to improve passive oxygenation of recirculating culture media in the reservoir. In one non- limiting instance, an oxygenating reservoir includes:

[0013] a culture medium spreader that spreads at least one culture medium into a layer for oxygenating the at least one culture medium; and

[0014] wherein recirculation or pumping of the at least one culture medium on the culture medium spreader results in an increase in oxygen content of the at least one culture medium, as compared to prior to the recirculation or pumping.

[0015] The oxygenating reservoir includes a culture medium spreader feature which can take various designs or forms, as described herein. The design of the culture medium spreader is to spread the culture media being recirculated or pumped into a thin layer with increased surface area, allowing for improved oxygenation and mixing of the culture media. In some instances, the culture medium spreader is a fountain where the least one culture medium flows through a vertical riser. In some other instances, the culture medium spreader is defined by a stepped spiral including spiral steps. In yet other instances, the culture medium spreader includes a plurality of cut-outs on an inner wall of the oxygenating reservoir. In yet other instances, the culture medium spreader includes a waterfall feature including a plurality of vertical flutes. In some other instances, the culture medium spreader includes a circular waterfall feature including a plurality of vertical flutes on an inner wall of the oxygenating reservoir

[0016] In some instances, the oxygenating reservoir further includes a spreading feature that interfaces the vertical riser and the culture medium spreader and is for flowing the at least one culture medium from the vertical riser into the culture medium spreader.

[0017] In some instances, the oxygenating reservoir can include a built-in pressure drop feature. Such a pressure drop can have applications in microfluidic experiments in which, for example, perfusable vessel formation is of interest, as a pressure drop is a way to encourage flow across tissue culture.

[0018] In some instances, the oxygenating reservoir further includes a moat or moat-like feature including a sacrificial fluid that is adjacent to or at least partially surrounds the oxygenating reservoir to reduce or prevent evaporation of the at least one culture medium.

[0019] The oxygenating reservoirs described herein can be integrated into a microfluidic device or microphysiological system either as an integral built-in component or as a removable slot-in module. The oxygenating reservoirs serve for oxygenating and mixing culture media. In one nonlimiting instance, a method of oxygenating a culture medium includes the steps of: (a) recirculating or pumping at least one culture medium in an oxygenating reservoir described herein.

[0020] In some instances, a method of oxygenating a culture medium includes the steps of:

[0021] (a’) recirculating or pumping at least one culture medium in an oxygenating reservoir forming part of a device, such as a microfluidic device or microphysiological system.

[0022] In some instances of the above methods, the oxygen content of the culture medium is increased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, as compared to the oxygen content of the culture medium prior to step (a) or (a’). In some instances of the above methods, the oxygen content of the culture medium is increased by an amount in a range from between about 1% to 95%, or higher, as compared to the oxygen content of the culture medium prior to step (a) or (a’), or individual values or sub-ranges contained within the aforementioned range.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Non- limiting embodiments are described by way of example with reference to the accompanying Figures.

[0025] Figure 1 A shows a non-limiting cross-sectional view of an oxygenating reservoir design 100 having a fountain-type culture medium spreader feature where a fluid to be oxygenated, such as a culture media, ascends / flows through a vertical riser or pillar 102 that has an inlet hole 104 for flowing the culture media out and down vertical scratches / flutes 106, gathering in the bottom of the oxygenating reservoir, then exiting via an outlet hole 108. This illustration shows a riser or pillar having a concave profile.

[0026] Figure IB shows a non-limiting representation of a riser or pillar 102 that has an inlet hole 104 and which is a cylinder of constant diameter with vertical scratches 106 thereon.

[0027] Figure 1C shows a non-limiting representation of a riser or pillar 102 having a geometry that is a tapered cylinder with vertical scratches 106 thereon and having a plurality of inlet holes 104 at the top from which culture media flows out and down the sides.

[0028] Figure 2A shows a non- limiting three-dimensional view illustrating an oxygenating reservoir 200 including a stepped spiral culture medium spreader feature 202 in which the radius decreases with each downward turn of the spiral and a vertical riser 204 for flowing fluid, such as culture media, to the top of the spiral that flow downs edges of the spiral to the bottom of the spiral where an outlet hole 206 is present.

[0029] Figure 2B shows a non-limiting cross-sectional view of spiral culture medium spreader feature 202 having a stepped spiral design that decreases in radius with each downward turn of the spiral down to the bottom of the oxygenating reservoir and shows the vertical riser 204 through which fluid flows up to the top of the stepped spiral and flows down edges (arrows) of the spiral to the bottom of the spiral where an outlet hole 206 is present.

[0030] Figure 2C shows an expanded view of an edge of a stepped spiral having a fluid 208 thereon, where the edge has an angle 210, such as of 90 degrees, that encourages the fluid to follow down the stepped spiral.

[0031] Figure 2D shows a non-limiting cross-sectional view of an oxygenating reservoir 300 having a spiral culture medium spreader feature 302 defined by cuts 304 present in the wall of the oxygenating reservoir, where fluid flows down (arrows) from the top to the bottom of the reservoir from a vertical riser (not shown) to the fluid outlet (not shown) at the bottom.

[0032] Figure 2E shows a non- limiting three-dimensional view of spiral culture medium spreader feature 302 defined by the spiral that is cut into the wall of the oxygenating reservoir and shows a vertical riser 306 and a fluid outlet 308 at the bottom.

[0033] Figure 2F shows an expanded partial view of the top portion of spiral culture medium spreader feature 302 having an interface between the vertical riser and the beginning of the spiral 310, and where the spreading feature allows fluid to travel from the top of the vertical riser 306 to the beginning of the spiral culture medium spreader feature.

[0034] Figure 3A shows a non-limiting overhead (top-down) view of an oxygenating reservoir with a circular, ring-shaped culture medium spreader feature 400 that forms a downward spiral and showing an outlet of a vertical riser 402 for fluid to flow into and down the spiral to be oxygenated.

[0035] Figure 3B shows a non-limiting overhead (top-down) view of an oxygenating reservoir with a culture medium spreader feature 400’ that forms a downward spiral having a non-symmetrical shape resembling an egg and showing an outlet of a vertical riser 402’ for fluid to flow into and down the spiral to be oxygenated.

[0036] Figure 4A shows a non-limiting cross-sectional view of an oxygenating reservoir 500 with a culture medium spreader feature which is defined by a multi -start spiral formed of multiple individual paths 501 for culture media to enter and flow down (arrows) the paths from the top to the bottom of the reservoir from a vertical riser (not shown) to a fluid outlet (not shown) at the bottom.

[0037] Figure 4B shows a non-limiting three-dimensional view of oxygenating reservoir 500 with culture medium spreader feature defined by a multi-start spiral that further illustrates vertical riser 502, where at the bottom of the oxygenating reservoir the oxygenated culture media 504 gathers to return to the tissue culture via a fluid outlet 508.

[0038] Figure 5A shows a non-limiting three-dimensional view of an oxygenating reservoir 600 with a culture medium spreader feature 602 that forms a wall with a waterfall design featuring vertical flutes or scratches 604 on the top surface and on one face of the wall 602. The wall sits in the oxygenating reservoir, dividing it into an inlet side having a fluid inlet 606 and an outlet side having a fluid outlet 608.

[0039] Figure 5B shows a non-limiting cross-sectional side view of a culture medium spreader with a waterfall design where the wall 602 sits in the oxygenating reservoir, dividing it into an inlet side having fluid inlet 604 and an outlet side having fluid outlet 608 and where the fluid shown on the outlet side has flowed down the vertical flutes / scratches has been oxygenated.

[0040] Figure 5C shows a non-limiting three-dimensional view of an oxygenating reservoir 700 with a culture medium spreader featuring a circular waterfall design 702 featuring vertical flutes or scratches 704 going down toward the bottom and where culture media flows from a vertical riser 706 down the inside walls of the spreader and out of a fluid outlet (not shown) at the bottom of the oxygenating reservoir.

[0041] Figure 5D shows a non- limiting partial views of segments of vertical scratch profiles with various profile options, where the profiles can be a semi-circular profile 708 or with a triangular profile 708’, respectively.

[0042] Figure 6A shows a non-limiting view of a partial section of an oxygenating reservoir 800 with a spreading feature has a chamfered edge 802 at the interface between the spreading feature and flutes / scratches 804; and fluid to be oxygenated flows out from a vertical riser 806 and down the flutes / scratches to be oxygenated.

[0043] Figure 6B shows a non-limiting view of a partial section of an oxygenating reservoir 800’ where a spreading feature includes a fluid guiding feature 802’ , such as a cut out, around the vertical riser inlet hole 804’ .

[0044] Figure 6C shows a non-limiting view of a partial section of an oxygenating reservoir 800” which shows a spreading feature having an edge 802” that can be at an angle, such as 45 degrees, facing inward toward the center-point of the oxygenating reservoir; and has a vertical riser inlet hole 804”.

[0045] Figure 7A shows a non-limiting cross-sectional side view of a concentric oxygenating reservoir design 900, where the concentric reservoir involves at least one fluid inlet 902, marked with an upward arrow, in the outer ring, and at least one fluid outlet 904 on bottom of the inner ring, marked with an upward arrow, where fluid from the inlet flows over the walls of the inner ring, which include flutes / scratches on the inner surfaces, and oxygenated fluid collects and can flow out of the outlet.

[0046] Figure 7B shows a non-limiting top view of the concentric oxygenating reservoir 900 with a spoke design which illustrates how the reservoir can be manufactured as a single piece. Figure 7C shows a non-limiting three-dimensional view of the concentric oxygenating reservoir 900, showing vertical flutes or scratches 906 on the inside of the internal reservoir.

[0047] Figure 8 shows a non-limiting cross-sectional view of oxygenating reservoir 1000 having a built-in pressure drop feature involving a first inlet 1002 at the bottom of the reservoir and at the same height as an outlet 1004, as well as another inlet at the top of the reservoir in the form of a vertical riser 1006, and wherein fluid flows out from the vertical riser and down the wall having vertical flutes or scratches (not shown) to oxygenate the fluid. The pressure change is given by AP = pgAh where AP is the change in pressure, p (rho) is the fluid density, g is the acceleration due to gravity, and Ah is the change in height.

[0048] Figure 9A shows a non-limiting cross-sectional view of an integrated moat or moat-like feature 1102, such as an annular sacrificial fluid moat, surrounding an oxygenating reservoir 1100, where the height difference between the walls of the sacrificial fluid-filled moat and the oxygenating reservoir prevents excess evaporation of the culture media therein. A lid 1104, such as a standard petri dish lid, is optional and can be used to protect against contamination.

[0049] Figure 9B shows a non-limiting three-dimensional representation of an oxygenating reservoir 1100 having an annular sacrificial fluid moat 1102 surrounding the reservoir, excluding the petri dish lid.

[0050] Figure 9C shows a non-limiting oxygenating reservoir-moat configuration 1102’ with a partially-surrounding moat design.

[0051] Figure 9D shows a non-limiting oxygenating reservoir-moat configuration with an independent moat 1102”, which can be rectangular shaped, placed adjacent to the oxygenating reservoir.

[0052] Figure 10A shows a non- limiting cross-sectional representation of oxygenating reservoir 1200 sumounded by a fluid filled an annular moat 1202 and having a lid 1204, such as made of a material like as PDMS, where the lid can be secured by a clamping mechanism (not shown).

[0053] Figure 10B shows two non-limiting examples of a lid with different shapes, which can be varied to match the reservoir and moat feature geometry, such as a circular lid and an arch-shaped lid, respectively.

[0054] Figure 11A shows a non- limiting three-dimensional representation of an oxygenating reservoir 1300 having a plurality of fluid inlets 1302, which form part of vertical risers 1304, and fluid outlets (not shown) at the bottom, where the fluid flows from the inlets over a spreader feature and down the sidewalls having vertical flutes or scratches 1306 thereon, and which can be useful in applications, such as multi-organ platforms. The arrows of the bottom half show the flow of the fluid into and out of the reservoir.

[0055] Figure 11B shows a non-limiting top-down view and cross-sectional side view of oxygenating reservoir 1300 having a plurality of fluid inlets 1302 and outlets 1314. Flows of fluid to be oxygenated up through vertical risers and down the sides of the reservoir and out of the fluid outlets are depicted by arrows.

[0056] Figure 12A shows a non- limiting representation of a segment of an oxygenating reservoir with a pressure drop feature 1402 where a sphere 1404 of known mass is placed over the entry of the vertical riser 1406 and a cage-like feature restrains the sphere but allows culture medium fluid to flow. The arrows show the flow path of the fluid.

[0057] Figure 12B shows a non-limiting cross-sectional side view of a segment of pressure drop feature 1402 where a sphere 1404 of known mass is placed over the outlet of the vertical riser 1406 and a cage-like feature restrains the sphere. Culture media flows up through the vertical riser only when its pressure is greater than the pressure exerted by the sphere. The arrow shows the flow path of the fluid.

[0058] Figure 12C shows a non-limiting cross-sectional side view of a segment of the pressure drop feature 1402 where a sphere 1404 of known mass is placed over the outlet of the vertical riser and a cage-like feature restrains the sphere, as well as showing an exemplary design for the cage-like feature having fluid paths (arrows) exiting out of the cage.

[0059] Figure 13A shows a non- limiting segment view of a spreader feature 1502 which is a welllike spreader with a rounded wall over which fluid can flow and down a side-wall having vertical flutes or scratches 1506 thereon.

[0060] Figure 13B shows a non-limiting cross-sectional side view of a spreader feature 1502 which is a well-like spreader with a fluid 1508, such as culture media, in a well and the fluid can flow over the rounded wall (arrow) and down the side-wall having vertical flutes or scratches 1506 thereon (not shown).

[0061] Figure 14 shows a non- limiting example of an acrylic platform block 1602, elastomeric membrane 1604, an aluminum base plate 1606, and a nitrogen deoxygenation system 1612 is used to measure the efficacy of oxygenating reservoir designs and together form a fluidic validation platform 1600, as used in Example 1. This platform interfaces with an interchangeable oxygenating reservoir 1608 on a base 1610, which together form an interchangeable oxygenator patch.

[0062] Figure 15 shows a graph of three oxygen probes used in evaluation of an oxygenating reservoir in Example 1. For times prior to ~75 minutes, all the oxygen probes were placed in ambient conditions. For times after ~75 minutes, the oxygen probes were placed in the positions indicated in the graph.

[0063] Figure 16 shows a bar graph of the difference in oxygen partial pressure of fluid measured at the reservoir inlet and reservoir outlet, as well as the ambient pressure marked, for the oxygenating reservoir evaluated in Example 1.

[0064] DETAILED DESCRIPTION OF THE INVENTION

[0065] Oxygenating reservoirs for microfluidic applications that provide improved oxygenation of culture media and methods of using thereof are described herein.

[0066] I. Definitions

[0067] It is to be understood that the disclosed oxygenating reservoirs, and methods of making thereof, are not limited to specific manufacturing methods, specific materials, or to particular dimensions or shapes unless otherwise specified, and, as such, may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular forms and embodiments only and is not intended to be limiting.

[0068] “Oxygen content,” as used herein refers to the dissolved oxygen level or concentration, which can be expressed as a molar concentration of oxygen or as an oxygen partial pressure (kPa), in a fluid, such as a culture medium. The dissolved oxygen level or concentration can be measured by known techniques, such as but not limited to commercially available oxygen sensor probes, electrodes, meters, or gas analyzers. Oxygen level or concentration measurements can be determined at different time points and used to monitor and compare differences / changes in the oxygen content of a given fluid between the different time points.

[0069] “Oxygenation,” “reoxygenation,” “oxygenating,” or “reoxygenating” as used herein refer to increasing the level of dissolved oxygen in a fluid, such as a culture medium.

[0070] The term “media” or “medium,” refers to a culture fluid that is used for cell culture and contains nutrients, growth factors, or other biomolecules that are included to grow and proliferate cells.

[0071] As used herein, the term “biodegradable”, refers to a material that will degrade or erode by enzymatic action and / or hydrolysis under physiologic conditions to smaller units or chemical species that are capable of being metabolized and / or eliminated.

[0072] The teim “microfluidic” typically refers to a system that involves the control and manipulation of small fluid volumes in channels with dimensions on the order of a few micrometers up to a few millimeters and total system volumes on the scale of nanoliters to a few milliliters. The numerical ranges disclose individually each possible number that such a range could reasonably encompass, as well as any sub-ranges and combinations of sub-ranges encompassed therein. Tor example, in a given range of flow rates from 0.1 to 1 ul / second the range also discloses 0.2, 7, 0.45, and 0.99 pl / second, as well as any subrange between these numbers (for example, 0.3 to 0.7 pl / second), and any possible combination of ranges possible between these values.

[0073] Use of the term "about" is intended to describe values either above or below the stated value, which the term “about” modifies, to be within a range of approximately + / - 10%. When the term "about" is used before a range of numbers (i.e., about 1-5) or before a series of numbers (z'.e., about 1, 2, 3, 4, etc.) it is intended to modify both ends of the range of numbers and / or each of the numbers recited in the entire series, unless specified otherwise.

[0074] IL Oxygenating Reservoirs

[0075] Described herein are oxygenating reservoirs, or oxygenators, that can be integrated with a microfluidic device, such as a microfluidic chip, or in a microphysiological system. Such oxygenating reservoirs can replace existing culture media reservoirs, such those used in a microfluidic chip, to improve passive oxygenation of recirculating culture media in the reservoir. Fundamentally, the oxygenating reservoirs described herein differ from a typical, non-oxygenating reservoir because it spreads the culture media into a thin layer with larger surface area that allows for increased passive diffusion of oxygen into the media and also enables mixing of the culture media within the reservoir. Various oxygenating reservoirs are described below.

[0076] In one non-limiting instance, an oxygenating reservoir includes:

[0077] a culture medium spreader that spreads at least one culture medium into a layer for oxygenating the at least one culture medium; and

[0078] wherein recirculation or pumping of the at least one culture medium on the culture medium spreader results in an increase in oxygen content of the at least one culture medium, as compared to prior to the recirculation or pumping.

[0079] The recirculation or pumping of the at least one culture medium in the oxygenating reservoir can also provide mixing of the at least one culture medium. Any suitable culture medium or culture media can be used with the oxygenating reservoir, such as those known in the art of microfluidics and microphysiological systems.

[0080] In some instances, the oxygenating reservoir includes one or more fluid inlets and / or one or more fluid outlets. See Figures 11 A and 11B. Such inlets / outlets may come from different sources. As discussed below, such inlets / outlets can be useful in applications, such as multi-organ platforms. In some instances, the various culture medium spreaders described herein can have surface(s) which have been roughened, such as by machining, or plasma-treatment, using art known techniques.

[0081] a. Culture Medium Spreader Feature

[0082] The oxygenating reservoir includes a culture medium spreader feature which can take various designs or forms, as described below. The design of the culture medium spreader is to spread the culture media being recirculated or pumped into a thin layer with increased surface area, allowing for improved oxygenation and mixing of the culture media.

[0083] 1. Fountain Design

[0084] In some instances, the culture medium spreader is a fountain where the least one culture medium flows up through a vertical riser. See Figures 1A-1C. In some instances, the vertical riser is inside a pillar which is optionally located at the oxygenating reservoir’s center. In some instances, the vertical riser and / or pillar each include at least one hole which can act as an inlet; and the vertical riser and / or pillar each independently include scratches, such as vertical scratches, or flutes, such vertical flutes, on their surface. In some instances, the pillar has a concave or tapered profile, fhe culture medium fluid flows down the length of the pillar and gathers at the bottom before exiting the oxygenating reservoir.

[0085] 2. Spiral Design

[0086] In some other instances, wherein the culture medium spreader is defined by a stepped spiral including spiral steps;

[0087] where the spiral steps each have a radius that decreases in a downward direction towards the bottom of the stepped spiral;

[0088] where each of the spiral steps is angled to cause downward flow of the at least one culture medium due to gravity;

[0089] where each of the spiral steps include an upward facing feature which is angled to prevent or reduce rolling or overflow of the at least one culture medium off of the spiral steps;

[0090] where the at least one culture medium flows into the stepped spiral via a vertical riser and flows down the spiral steps to the bottom of the stepped spiral, which includes at least one outlet hole.

[0091] Figures 2A-2B show a non-limiting example of a culture medium spreader based on a stepped spiral motif in which the spiral steps shows a radius decreases as it goes down toward the bottom of the reservoir. Figure 2C shows an edge of a stepped spiral and the upward facing feature has an angle of 90 degrees and has fluid thereon. In yet other instances, the culture medium spreader includes a plurality of cut-outs on an inner wall of the oxygenating reservoir;

[0092] where the plurality of cut-outs on the inner wall defines a spiral feature, such as threads of a screw;

[0093] where the spiral feature is angled to cause downward flow of the at least one culture medium due to gravity;

[0094] where the spiral feature includes an upward facing feature which is angled to prevent or reduce rolling or overflow of the at least one culture medium off of the spiral feature;

[0095] where the at least one culture medium flows into the spiral feature via a vertical riser and flows down the spiral feature to the bottom of the oxygenating reservoir, which includes at least one outlet hole.

[0096] Figure 2D shows a non- limiting example of a culture medium spreader having a spiral feature cut into the wall of the oxygenating reservoir. Figures 2E-2F show three-dimensional views of the same spiral-based culture medium spreader and an interface between the vertical riser and the spiral that allows fluid to travel from the top of the vertical riser to the start of the spiral.

[0097] For the non-limiting spiral-based designs, such as shown in Figures 2A-2F, the culture medium fluid flows upward through a vertical riser located in the outer wall of the oxygenating reservoir before entering the downward spiral. The spiral is angled such that gravity motivates fluid movement. The surface over which the fluid flows as it descends through the spiral can be angled such that the fluid is encouraged to follow the path rather than “rolling” off of the spiral (see Figure 2C). Fluid continues down one defined spiraling path from the top of the oxygenating reservoir to the bottom, where it is oxygenated, gathers, and exits the oxygenating reservoir.

[0098] In still other instances, the culture medium spreader includes a plurality of cut-outs on an inner wall of the oxygenating reservoir;

[0099] wherein the plurality of cut-outs on the inner wall defines a plurality of individual fluid paths each independently forming a spiral and each has an inlet hole;

[0100] wherein the plurality of individual fluid paths is angled to cause downward flow of the at least one culture medium due to gravity;

[0101] wherein the at least one culture medium flows into the plurality of individual fluid paths via a vertical riser and flows down the plurality of individual fluid paths to the bottom of the oxygenating reservoir, which includes at least one outlet hole.

[0102] The design discussed above can be considered a multi-start spiral that features a plurality of paths for culture medium fluid to enter the spiral, as shown in Figures 4A and 4B. For instance, the fluid travels upward through a vertical riser, distributes over a spreading feature, and then flows down the plurality of individual fluid paths which form, for example, individual spiral flutes, and at the bottom of the reservoir, the oxygenated fluid gathers and exits the oxygenating reservoir from a fluid outlet, as shown in Figure 4B.

[0103] 3. Waterfall Design

[0104] In yet other instances, the culture medium spreader includes a waterfall feature including a plurality of vertical scratches or flutes;

[0105] where the waterfall feature is defined by a wall that divides the oxygenating reservoir into an inlet side including at least one inlet hole and an outlet side including at least one outlet hole; where the recirculation or pumping of the at least one culture medium causes the at least one culture medium to flow from the inlet side over the waterfall feature into the outlet side.

[0106] In some other instances, the culture medium spreader includes a circular waterfall feature including a plurality of vertical flutes on an inner wall of the oxygenating reservoir;

[0107] where the at least one culture medium flows into the plurality of vertical flutes via a vertical riser and flows down the plurality of vertical flutes to the bottom of the oxygenating reservoir, which includes at least one outlet hole.

[0108] In the first waterfall design, a wall in the oxygenating reservoir divides it into an inlet side and an outlet side. Culture medium fluid fills the inlet side until it flows over the wall. The side of the wall facing the outlet have scratches or flutes, encouraging the fluid to flow in a thin sheet before exiting the reservoir, as illustrated in Figures 5 A and 5B. In the second circular waterfall design, the culture medium fluid flows down the inside walls of the oxygenating reservoir, as shown in Figure 5C. More particularly, the fluid travels upward through a vertical riser, distributes over the spreading feature, and then flows down the internal walls of the oxygenating reservoir along vertical scratches or flutes.

[0109] The vertical flutes of these designs are typically straight, scratch features which serve to spread and guide culture medium fluid over a surface in a thin, evenly-distributed sheet or layer. The vertical scratches / flutes can take on various profiles without limitation. In some instances, the vertical flutes of the plurality each have a semi-circular profile, triangular profile, or combinations thereof. For example, they can be cut with a drill (semi-circular profile) or have a serrated broach (triangular profile), as illustrated in Figure 5D.

[0110] b. Spreading Feature

[0111] In some instances, the oxygenating reservoir further includes a spreading feature that interfaces the vertical riser and the culture medium spreader and is for flowing the at least one culture medium from the vertical riser into the culture medium spreader. This feature can effectively increase surface area of the culture medium fluid, where the media can be spread evenly after exiting the vertical riser, which acts as an inlet for the fluid.

[0112] In some instances, the spreading feature includes at least one hole from which the at least one culture medium flows out from the vertical riser; and optionally the spreading feature has a circular ring shape or non-symmetrical shape, such as an egg-shape. For example, a spreading feature can have a circular ring shape, as represented in Figure 3A. However, there is no particular limit on the shape of the spreading feature. It could have an alternative shape, for example, resembling an egg, as shown in Figure 3B.

[0113] In some instances, the spreading feature is angled or slanted down towards a center-point of the oxygenating reservoir to prevent the at least one culture medium from gathering on the spreading feature when flowed thereon. In some instances, the spreading feature further has a chamfered edge at interfaces between the spreading feature and the culture medium spreader, and / or features thereof, to prevent the at least one culture medium from gathering on the spreading feature when flowed thereon. In some instances, the spreading feature is well spreading feature includes a rounded wall.

[0114] Various non-limiting examples of spreading features are shown in Figures 6A-6C and 13A-13B. For example, Figure 6A shows a partial segment of an oxygenating reservoir with spreading feature which has a chamfered edge at the interface between the spreader and flutes / scratches, which can help prevent culture media from gathering on the spreading feature without flowing down the flutes / scratches. “Chamfered” refers to an edge has been beveled or cut at an angle, usually at 45 degrees, instead of being left sharp or square in shape. The gathering effect can occur as a result of surface tension, causing the culture media to form a bubble-like mass that rolls down into the reservoir, preventing optimal oxygenation. Figure 6B shows a partial segment of an oxygenating reservoir where the spreading feature includes a fluid guiding feature, such as a cut out, around the vertical riser inlet hole that can help prevent culture media from gathering on the spreading feature without flowing down the flutes / scratches. Figure 6C shows a partial segment of an oxygenating reservoir which shows that the spreading feature can be at an angle, facing inward toward the center-point of the oxygenating reservoir, which reduces the likelihood of the culture media fluid becoming trapped in the corner and can encourage it to spread out and flow through the oxygenating reservoir. In some instances, the angle is about 45 degrees but other angles are possible. Figures 13A and 13B show a rounded wall variation.

[0115] In still other instances, the spreading feature is not required and the oxygenating reservoir is alternatively a concentric oxygenating reservoir. For instance, in one non-limiting instance, such a concentric oxygenating reservoir includes: at least a first reservoir and a second reservoir;

[0116] where the second reservoir is smaller than the first reservoir and is within the first reservoir; where the first reservoir includes an inlet region including at least one inlet hole and the second reservoir includes an outlet region including at least one outlet hole;

[0117] wherein recirculation or pumping of at least one culture medium causes the at least one culture medium to flow over from the inlet region, down one or more side-wall(s) of the second reservoir, and into the outlet region of the second reservoir; and

[0118] wherein the recirculation or pumping of the at least one culture medium results in an increase in oxygen content of the at least one culture medium, as compared to prior to the recirculation or pumping.

[0119] In some instances, the one or more side-wall(s) of the second reservoir includes scratches, such as vertical scratches, or flutes, such as vertical flutes, on their surface.

[0120] Designs based on two concentric reservoirs can act as an alternative to a spreading feature and rely on the culture media filling the outer annulus of concentric reservoirs until it reaches the height of the inner, shorter reservoir therein. As the interior of the inner reservoir can have vertical scratches / flutes, the culture media flows in a layer or sheet down the inside wall of the inner reservoir. The oxygenated fluid / media then gathers at the bottom of the inner reservoir before exiting the reservoir, as illustrated in Figures 7A through 7C. Such concentric reservoirs could be connected with a UV-curing optical adhesive or a threaded connector, or they can be machined as a single piece.

[0121] c. Pressure Drop Feature

[0122] In some instances, the oxygenating reservoir can include a built-in pressure drop feature. Such a pressure drop can have applications in microfluidic experiments in which, for example, perfusable vessel formation is of interest, as a pressure drop is a way to encourage flow across a (micro) vascular tissue culture.

[0123] In one instance, a pressure drop feature can be based on a design that includes at least two inlets for the culture media to enter the oxygenating reservoir: one at the bottom of the reservoir, at the same height as the outlet, and one al the top of the reservoir formed by a vertical riser. The difference in height between the top of the vertical riser and the surface of the culture media that is gathered in the oxygenating reservoir defines and controls the pressure drop. For instance, the two inlets in Figure 8, would enable the controlled creation of a pressure difference across a tissue culture if these two inlets are connected to culture media channels on opposite sides of a tissue culture. In another instance, a pressure drop feature involves placing a sphere (of mass m) over the entry / inlet hole of the vertical riser, as shown in Figure 12A. The sphere should be larger than the inlet hole, and the mass of the sphere determines the pressure drop produced. Culture media flows through the vertical riser only when its pressure is greater than the pressure exerted by the sphere, as shown in Figure 12B. The top of the vertical riser has a radius to ensure a defined contact point between the sphere and the vertical riser inlet, as shown in Figure 12C. As shown in Figures 12B and 12C, a cage-like feature at the top of the vertical riser prevents the sphere from traveling too far during culture medium fluid flow.

[0124] d. Moat and Lid Feature

[0125] In some instances, the oxygenating reservoir further includes a moat or moat-like feature including a sacrificial fluid that is adjacent to or at least partially surrounds the oxygenating reservoir to reduce or prevent evaporation of the at least one culture medium.

[0126] Such a sacrificial fluid filled moat can help manage unwanted evaporation of culture media. Without limitation, in one instance, a buffer, such as a phosphate buffered saline (PBS) fills the moat, and the difference in the height of the moat and the oxygenating reservoir walls helps to control and limit evaporation of the fluid or culture medium collected in the oxygenating reservoir, as shown in Figure 9A. In some instances, the moat-like feature is an annular moat filled with the sacrificial fluid. Figure 9B shows an example of a moat or moat-like feature which is annular and surrounds the oxygenator completely. However, the moat or moat-like feature can have a different geometry, for example, it could partially surround the oxygenating reservoir, as shown in Figure 9C, or could sit next to the oxygenating reservoir, as shown in Figure 9D. The moat or moat-like feature can be attached to a device, such as a microfluidic device or microphysiological system itself, or could be attached to a nearby surface, such as the wall of a device / system, or component thereof. The shape of the moat or moat-like feature is not particularly restricted and can be annular, semi-annular, or rectangular.

[0127] In some instances, a lid is placed over the moat or moat-like feature. Thus, in some instances, the moat or moat-like feature and the oxygenating reservoir can be covered by a lid, such as standard petri dish lid, with the height of the moat walls slightly being higher than those of the oxygenating reservoir, as illustrated in Figure 9 A. Alternatively, a piece

[0128] of oxygen-permeable material, such as polydimethylsiloxane (PDMS), could be fabricated in a thin (—1 / 8”) sheet and secured to the top of the reservoir to serve as the lid, as shown in Figure 10A. Thus, in some instances, the lid is oxygen-permeable, such as made of an oxygen-permeable material like PDMS. In some instances, the lid is secured to the moat or moat-like feature, such as by a clamping mechanism, such as a quarter-turn thread. The moat or moat- like feature and the lid can have any suitable shape which can vary to match the particular geometry of the oxygenating reservoir, as shown in Figure 10B. In some instances, the lid has a circular- shape or arch-shape.

[0129] e. Microfluidic Devices or Systems including an Oxygenating Reservoir Therein

[0130] The various oxygenating reservoirs described herein can be integrated into a microfluidic device or microphysiological system either as an integral built-in component or as a removable slotin module. Any suitable microfluidic device or microphysiological system can be used. In some instances, the oxygenating reservoirs described herein can be used in commercially available microfluidic devices or microphysiological systems, such CN Bio’s PhysioMimix® OOC Microphysiological Systems, including single-organ and multi-organ systems.

[0131] In some instances, the oxygenating reservoir, of any variation described herein, can include a base which optionally includes one or more attachments, such as magnetic attachments, to allow for connection to a microfluidic device or microphysiological system. The base can be used to couple the oxygenating reservoir to a microfluidic device or microphysiological system. Without limitation, Figure 14 shows an exemplary oxygenating reservoir which includes a rectangular base, fhe shape and dimensions of the base, if present, can be any suitable dimensions.

[0132] f. Manufacturing Oxygenating Reservoirs

[0133] The oxygenating reservoirs and components thereof, as described herein, can be manufactured according to suitable methods and using suitable materials known to the person of ordinary skill in the field of microfluidics. For instance, suitable manufacturing techniques can include without limitation, soft lithography, 3D printing, micro-machining, and injection molding. Suitable materials can include without limitation, PDMS (Polydimethylsiloxane), PMMA (Polymethyl Methacrylate), biodegradable polymers (such as PLA (Polylactic Acid) or PCL (Polycaprolactone) suitable for biocompatible reservoirs). Other fabrication methods and materials are possible.

[0134] III. Methods of Using Oxygenating Reservoirs

[0135] The oxygenating reservoirs described herein can be used for various microfluidic applications. For instance, the oxygenating reservoirs can form part of a device, such as a microfluidic device or microphysiological system. Such devices can be used for tissue culture or as multi-organ platforms, where tissue is cultured in separate chambers but circulating media needs to be mixed. Thus, in some instances, for a multi-inlet / multi-outlet oxygenating reservoir see Figures 11 A and 1 IB), the circulating media from various tissue cultures can be both reoxygenated and mixed in one reservoir. As noted, the oxygenating reservoirs serve for oxygenating and mixing culture media. In one non-limiting instance, a method of oxygenating a culture medium includes the steps of:

[0136] (a) recirculating or pumping at least one culture medium in an oxygenating reservoir described herein.

[0137] In some instances, a method of oxygenating a culture medium includes the steps of:

[0138] (a’) recirculating or pumping at least one culture medium in an oxygenating reservoir forming part of a device, such as a microfluidic device or microphysiological system.

[0139] In some instances of the above methods, the oxygen content of the culture medium is increased by at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, as compared to the oxygen content of the culture medium prior to step (a) or (a’). In some instances of the above methods, the oxygen content of the culture medium is increased by an amount in a range from between about 1% to 95%, or higher, as compared to the oxygen content of the culture medium prior to step (a) or (a’), or individual values or sub-ranges contained within the aforementioned range.

[0140] In some instances, the efficacy of the oxygenating reservoir can be measured by including a nitrogen deoxygenation system where nitrogen bubbling deoxygenates fluid as it flows through the circuit and can be used to evaluate the oxygenation efficiency of the oxygenating reservoir for the fluid being flowed through the reservoir.

[0141] The disclosed oxygenating reservoirs and methods of using thereof can be further understood through the following numbered paragraphs:

[0142] Paragraph 1. An oxygenating reservoir comprising:

[0143] a culture medium spreader that spreads at least one culture medium into a layer for oxygenating the at least one culture medium; and

[0144] wherein recirculation or pumping of the at least one culture medium on the culture medium spreader results in an increase in oxygen content of the at least one culture medium, as compared to prior to the recirculation or pumping.

[0145] Paragraph 2. The oxygenating reservoir of paragraph 1 , wherein the culture medium spreader is a fountain wherein the least one culture medium flows through a vertical riser.

[0146] Paragraph 3. The oxygenating reservoir of paragraph 2, wherein the vertical riser is inside a pillar which is optionally located at the oxygenating reservoir’s center.

[0147] Paragraph 4. The oxygenating reservoir of paragraph 3, wherein the vertical riser and / or pillar each comprise at least one hole which can act as an inlet or outlet; and the vertical riser and / or pillar each independently comprise scratches, such as vertical scratches, on their surface. Paragraph 5. The oxygenating reservoir of any one of paragraphs 3-4, wherein the pillar has a concave or tapered profile.

[0148] Paragraph 6. The oxygenating reservoir of paragraph 1, wherein the culture medium spreader is a stepped spiral comprising spiral steps;

[0149] wherein the spiral steps each have a radius that decreases in a downward direction towards the bottom of the stepped spiral;

[0150] wherein each of the spiral steps is angled to cause downward flow of the at least one culture medium due to gravity;

[0151] wherein each of the spiral steps comprise an upward facing feature which is angled to prevent or reduce rolling or overflow of the at least one culture medium off of the spiral steps; wherein the at least one culture medium flows into the stepped spiral via a vertical riser and flows down the spiral steps to the bottom of the stepped spiral, which comprises at least one outlet hole.

[0152] Paragraph 7. The oxygenating reservoir of paragraph 1, wherein the culture medium spreader comprises a plurality of cut-outs on an inner wall of the oxygenating reservoir;

[0153] wherein the plurality of cut-outs on the inner wall defines a spiral feature, such as threads of a screw;

[0154] wherein the spiral feature is angled to cause downward flow of the at least one culture medium due to gravity;

[0155] wherein the spiral feature comprises an upward facing feature which is angled to prevent or reduce rolling or overflow of the at least one culture medium off of the spiral feature;

[0156] wherein the at least one culture medium flows into the spiral feature via a vertical riser and flows down the spiral feature to the bottom of the oxygenating reservoir, which comprises at least one outlet hole.

[0157] Paragraph 8. The oxygenating reservoir of paragraph 1, wherein the culture medium spreader comprises a plurality of cut-outs on an inner wall of the oxygenating reservoir;

[0158] wherein the plurality of cut-outs on the inner wall defines a plurality of individual fluid paths each independently forming a spiral and each has an inlet hole;

[0159] wherein the plurality of individual fluid paths is angled to cause downward flow of the at least one culture medium due to gravity;

[0160] wherein the at least one culture medium flows into the plurality of individual fluid paths via a vertical riser and flows down the plurality of individual fluid paths to the bottom of the oxygenating reservoir, which comprises at least one outlet hole. Paragraph 9. The oxygenating reservoir of paragraph 1, wherein the culture medium spreader comprises a waterfall feature comprising a plurality of vertical flutes:

[0161] wherein the waterfall feature is defined by a wall that divides the oxygenating reservoir into an inlet side comprising at least one inlet hole and an outlet side comprising at least one outlet hole;

[0162] wherein the recirculation or pumping of the at least one culture medium causes the at least one culture medium to flow from the inlet side over the waterfall feature into the outlet side.

[0163] Paragraph 10. The oxygenating reservoir of paragraph 1. wherein the culture medium spreader comprises a circular waterfall feature comprising a plurality of vertical flutes on an inner wall of the oxygenating reservoir;

[0164] wherein the at least one culture medium flows into the plurality of vertical flutes via a vertical riser and flows down the plurality of vertical flutes to the bottom of the oxygenating reservoir, which comprises at least one outlet hole.

[0165] Paragraph 11. The oxygenating reservoir of any one of paragraphs 9-10, wherein the vertical flutes of the plurality each have a semi-circular profile, triangular profile, or combinations thereof.

[0166] Paragraph 12. The oxygenating reservoir of any one of paragraphs 2-8 or 10-11, wherein the oxygenating reservoir further comprises a spreading feature that interfaces the vertical riser and the culture medium spreader and is for flowing the at least one culture medium from the vertical riser into the culture medium spreader.

[0167] Paragraph 13. The oxygenating reservoir of paragraph 12, wherein the spreading feature comprises at least one hole from which the at least one culture medium flows out from the vertical riser; and optionally wherein the spreading feature has a circular ring shape or non-symmetrical shape, such as an egg-shape.

[0168] Paragraph 14. The oxygenating reservoir of any one of paragraphs 12-13, wherein the spreading feature is angled or slanted down towards a center-point of the oxygenating reservoir to prevent the at least one culture medium from gathering on the spreading feature when flowed thereon.

[0169] Paragraph 15. The oxygenating reservoir of any one of paragraphs 12-14, wherein the spreading feature further is a chamfered edge at interfaces between the spreading feature and the culture medium spreader, and / or features thereof, to prevent the at least one culture medium from gathering on the spreading feature when flowed thereon.

[0170] Paragraph 16. The oxygenating reservoir of any one of paragraphs 12-13, wherein the spreading feature is well spreading feature comprising a rounded wall. Paragraph 17. An oxygenating reservoir comprising:

[0171] at least a first reservoir and a second reservoir;

[0172] wherein the second reservoir is smaller than the first reservoir and is within the first reservoir;

[0173] wherein the first reservoir comprises an inlet region comprising at least one inlet hole and the second reservoir comprises an outlet region comprising at least one outlet hole;

[0174] wherein recirculation or pumping of at least one culture medium causes the at least one culture medium to flow over from the inlet region, down one or more side-wall(s) of the second reservoir, and into the outlet region of the second reservoir; and

[0175] wherein the recirculation or pumping of the at least one culture medium results in an increase in oxygen content of the at least one culture medium, as compared to prior to the recirculation or pumping.

[0176] Paragraph 18. The oxygenating reservoir of paragraph 17, wherein the one or more sidewalks) of the second reservoir comprise scratches, such as vertical scratches, on their surface.

[0177] Paragraph 19. The oxygenating reservoir of any one of paragraphs 1-18, wherein the oxygenating reservoir further comprises a pressure drop feature.

[0178] Paragraph 20. The oxygenating reservoir of any one of paragraphs 1-19, further comprising a moat-like feature comprising a sacrificial fluid that is adjacent to or at least partially surrounds the oxygenating reservoir to reduce or prevent evaporation of the at least one culture medium.

[0179] Paragraph 21. The oxygenating reservoir of paragraph 20, wherein the moat-like feature is an annular moat filled with the sacrificial fluid.

[0180] Paragraph 22. The oxygenating reservoir of paragraph 21, wherein a height difference is present between walls of the moat- like feature and the oxygenating reservoir that prevents or reduces the evaporation of the at least one culture medium.

[0181] Paragraph 23. The oxygenating reservoir of any one of paragraphs 20-22, wherein a lid is placed over the moat-like feature.

[0182] Paragraph 24. The oxygenating reservoir of paragraph 23, wherein the lid is oxygen-permeable, such as made of an oxygen-permeable material like PDMS.

[0183] Paragraph 25. The oxygenating reservoir of any one of paragraphs 23-24, wherein the lid is secured to the moat-like feature, such as by a clamping mechanism.

[0184] Paragraph 26. The oxygenating reservoir of any one of paragraphs 23-25, wherein the lid has a circular- shape or arch- shape. Paragraph 27. The oxygenating reservoir of any one of paragraphs 1-26, wherein the oxygenating reservoir comprises one or more fluid inlets, one or more fluid outlets, or combinations thereof.

[0185] Paragraph 28. A device comprising the oxygenating reservoir of any one of paragraphs 1-27.

[0186] Paragraph 29. The device of paragraph 28, wherein the device is a microfluidic device. Paragraph 30. The device of paragraph 28, wherein the device is a microphysiological system.

[0187] Paragraph 31. A method of oxygenating a culture medium comprising the steps of:

[0188] (a) recirculating or pumping at least one culture medium in the oxygenating reservoir of any one of paragraphs 1-27.

[0189] Paragraph 32. A method of oxygenating a culture medium comprising the steps of:

[0190] (a’) recirculating or pumping at least one culture medium in the oxygenating reservoir of the device of any one of paragraphs 28-30.

[0191] The present invention will be further understood by reference to the following non-limiting examples.

[0192] EXAMPLES

[0193] Example 1: Oxygenating Reservoir

[0194] Materials and Methods:

[0195] The setup for a study of an oxygenating reservoir as shown in Figure 14. The oxygenating reservoir is in a patch format, meaning that it has a rectangular base allowing it to interface with the acrylic platform block shown. The acrylic platform block acts as an interface for the oxygenator, pump connections, and probes. The appearance of the oxygenator patch will vary depending on which of the oxygenator designs is being tested. This particular example includes an oxygenating resevoir with vertical scratches, similar to the representation shown in Figure 7A. The differences between the oxygenator used in the instant example and the oxygenator shown in Figure 7 A are: (1) that the oxygenator used in instant example includes a patch to interface with the rest of the setup, such as shown in Figure 14; and (2) the oxygenator used in the instant example is not perfectly concentric.

[0196] The acrylic platform block is aluminum base plate, with the elastomeric membrane clamped in between that seals the fluidic channels. The elastomeric membrane acts as a boundary between fluidic channels and the aluminum base plate, which acts as a support to seal the fluidic channels. The assembly of the acrylic platform block, elastomeric membrane, and aluminum base plate are referred to as the fluidic validation platform. The fluidic validation platform includes a fluid inlet and outlet, which connect to pumps. These pumps move fluid from a glass flask, in which nitrogen gas is bubbling to deoxygenate the fluid.

[0197] In this example, water was used in place of cell culture media for evaluation of oxygenation efficacy by the oxygenating reservoir. When the pumps were turned on, deoxygenated water flowed through the circuit, entered the oxygenator, flowed through the oxygenator, exited the oxygenator, and returned to the flask.

[0198] In this example, optical dissolved oxygen content measurement probes from Lucid Scientific were used to measure the oxygen in the water. One probe was placed at the inlet of the oxygenator, and one was placed at the outlet of the oxygenator. A third oxygen probe was used to measure the ambient oxygen content.

[0199] Results and Discussion:

[0200] The graph in Figure 15 shows the collected data from the experiment. The three oxygen probes are labeled in Figure 15 on the right-side of the graph. All probes were kept at ambient conditions for the first -75 minutes, as shown in the graph in Figure 15, to equilibriate. At a time of -75 minutes, they were moved to the locations indicated in the legend: one was placed at the reservoir inlet, and another was placed at the reservoir outlet. The reservoir inlet measurement indicated the oxygenation of the media prior to entering the oxygenating reservoir. The reservoir outlet measurement indicated the oxygenation of the media after flowing through the oxygenating reservoir.

[0201] It is worthwhile noting that the reservoir inlet reading was less than 1 kPa of oxygen pressure, which is not likely to be correct. Accordingly, the key takeaway from the graph in Figure 15 is not the absolute values of the oxygen pressure, but rather the difference in oxygen pressure between the probes. While the negative reading indicated that the oxygen probes were likely to be slightly miscalibrated, the first 75 minutes of the experiment showed that the readings of the oxygen probes relative to eachother were reliable, as they all read extremely similar values over an extended period of time in the same environment.

[0202] Figure 16 shows the difference in oxygenation before and after the water passed through the oxygenator. Although a comparison to a non-oxygenating reservoir was not made, it is known from prior experiments and simulations that very little media mixing occurs in non-oxygenating reservoirs, so little that cells relying on such media as their sole oxygen source cannot survive. With the amount of oxygen supplied by the oxygenating reservoir, cells relying on media as their sole oxygen source should be able to survive. The instant example demonstrated that the oxygenating reservoir as described herein was able to oxygenate a fluid flowed through the reservoir.

[0203] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0204] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific instances of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMSWe claim:

1. An oxygenating reservoir comprising:a culture medium spreader that spreads at least one culture medium into a layer for oxygenating the at least one culture medium; andwherein recirculation or pumping of the at least one culture medium on the culture medium spreader results in an increase in oxygen content of the at least one culture medium, as compared to prior to the recirculation or pumping.

2. The oxygenating reservoir of claim 1, wherein the culture medium spreader is a fountain wherein the least one culture medium flows through a vertical riser.

3. The oxygenating reservoir of claim 2, wherein the vertical riser is inside a pillar which is optionally located at the oxygenating reservoir’s center.

4. The oxygenating reservoir of claim 3, wherein the vertical riser and / or pillar each comprise at least one hole which can act as an inlet or outlet; and the vertical riser and / or pillar each independently comprise scratches, such as vertical scratches, on their surface.

5. The oxygenating reservoir of any one of claims 3-4, wherein the pillar has a concave or tapered profile.

6. The oxygenating reservoir of claim 1, wherein the culture medium spreader is a stepped spiral comprising spiral steps;wherein the spiral steps each have a radius that decreases in a downward direction towards the bottom of the stepped spiral;wherein each of the spiral steps is angled to cause downward flow of the at least one culture medium due to gravity;wherein each of the spiral steps comprise an upward facing feature which is angled to prevent or reduce rolling or overflow of the at least one culture medium off of the spiral steps; wherein the at least one culture medium flows into the stepped spiral via a vertical riser and flows down the spiral steps to the bottom of the stepped spiral, which comprises at least one outlet hole.

7. The oxygenating reservoir of claim 1, wherein the culture medium spreader comprises a plurality of cut-outs on an inner wall of the oxygenating reservoir;wherein the plurality of cut-outs on the inner wall defines a spiral feature, such as threads of a screw;wherein the spiral feature is angled to cause downward flow of the at least one culture medium due to gravity;wherein the spiral feature comprises an upward facing feature which is angled to prevent or reduce rolling or overflow of the at least one culture medium off of the spiral feature;wherein the at least one culture medium flows into the spiral feature via a vertical riser and flows down the spiral feature to the bottom of the oxygenating reservoir, which comprises at least one outlet hole.

8. The oxygenating reservoir of claim 1, wherein the culture medium spreader comprises a plurality of cut-outs on an inner wall of the oxygenating reservoir;wherein the plurality of cut-outs on the inner wall defines a plurality of individual fluid paths each independently forming a spiral and each has an inlet hole;wherein the plurality of individual fluid paths is angled to cause downward flow of the at least one culture medium due to gravity;wherein the at least one culture medium flows into the plurality of individual fluid paths via a vertical riser and flows down the plurality of individual fluid paths to the bottom of the oxygenating reservoir, which comprises at least one outlet hole.

9. The oxygenating reservoir of claim 1, wherein the culture medium spreader comprises a waterfall feature comprising a plurality of vertical flutes;wherein the waterfall feature is defined by a wall that divides the oxygenating reservoir into an inlet side comprising at least one inlet hole and an outlet side comprising at least one outlet hole;wherein the recirculation or pumping of the at least one culture medium causes the at least one culture medium to flow from the inlet side over the waterfall feature into the outlet side.

10. The oxygenating reservoir of claim 1, wherein the culture medium spreader comprises a circular waterfall feature comprising a plurality of vertical flutes on an inner wall of the oxygenating reservoir;wherein the at least one culture medium flows into the plurality of vertical flutes via a vertical riser and flows down the plurality of vertical flutes to the bottom of the oxygenating reservoir, which comprises at least one outlet hole.

11. The oxygenating reservoir of any one of claims 9-10, wherein the vertical flutes of the plurality each have a semi-circular profile, triangular profile, or combinations thereof.

12. The oxygenating reservoir of any one of claims 2-8 or 10-11, wherein the oxygenating reservoir further comprises a spreading feature that interfaces the vertical riser and the culture medium spreader and is for flowing the at least one culture medium from the vertical riser into the culture medium spreader.

13. The oxygenating reservoir of claim 12, wherein the spreading feature comprises at least one hole from which the at least one culture medium flows out from the vertical riser; and optionally wherein the spreading feature has a circular ring shape or non-symmetrical shape, such as an eggshape.

14. The oxygenating reservoir of any one of claims 12-13, wherein the spreading feature is angled or slanted down towards a center-point of the oxygenating reservoir to prevent the at least one culture medium from gathering on the spreading feature when flowed thereon.

15. The oxygenating reservoir of any one of claims 12-14, wherein the spreading feature further is a chamfered edge at interfaces between the spreading feature and the culture medium spreader, and / or features thereof, to prevent the at least one culture medium from gathering on the spreading feature when flowed thereon.

16. The oxygenating reservoir of any one of claims 12-13, wherein the spreading feature is well spreading feature comprising a rounded wall.

17. An oxygenating reservoir comprising:at least a first reservoir and a second reservoir;wherein the second reservoir is smaller than the first reservoir and is within the first reservoir;wherein the first reservoir comprises an inlet region comprising at least one inlet hole and the second reservoir comprises an outlet region comprising at least one outlet hole;wherein recirculation or pumping of at least one culture medium causes the at least one culture medium to flow over from the inlet region, down one or more side-wall(s) of the second reservoir, and into the outlet region of the second reservoir; andwherein the recirculation or pumping of the at least one culture medium results in an increase in oxygen content of the at least one culture medium, as compared to prior to the recirculation or pumping.

18. The oxygenating reservoir of claim 17, wherein the one or more side-wall(s) of the second reservoir comprise scratches, such as vertical scratches, on their surface.

19. The oxygenating reservoir of any one of claims 1-18, wherein the oxygenating reservoir further comprises a pressure drop feature.

20. The oxygenating reservoir of any one of claims 1-19, further comprising a moat- like feature comprising a sacrificial fluid that is adjacent to or at least partially surrounds the oxygenating reservoir to reduce or prevent evaporation of the at least one culture medium.

21. The oxygenating reservoir of claim 20, wherein the moat-like feature is an annular moat filled with the sacrificial fluid.

22. fhe oxygenating reservoir of claim 21, wherein a height difference is present between walls of the moat-like feature and the oxygenating reservoir that prevents or reduces the evaporation of the at least one culture medium.

23. The oxygenating reservoir of any one of claims 20-22, wherein a lid is placed over the moatlike feature.

24. The oxygenating reservoir of claim 23, wherein the lid is oxygen-permeable, such as made of an oxygen-permeable material like PDMS.

25. The oxygenating reservoir of any one of claims 23-24, wherein the lid is secured to the moat-like feature, such as by a clamping mechanism.

26. The oxygenating reservoir of any one of claims 23-25, wherein the lid has a circular-shape or arch-shape.

27. The oxygenating reservoir of any one of claims 1-26, wherein the oxygenating reservoir comprises one or more fluid inlets, one or more fluid outlets, or combinations thereof.

28. A device comprising the oxygenating reservoir of any one of claims 1-26.

29. The device of claim 28, wherein the device is a microfluidic device.

30. The device of claim 28, wherein the device is a microphysiological system.

31. A method of oxygenating a culture medium comprising the steps of:(a) recirculating or pumping at least one culture medium in the oxygenating reservoir of any one of claims 1-27.

32. A method of oxygenating a culture medium comprising the steps of:(a’) recirculating or pumping at least one culture medium in the oxygenating reservoir of the device of any one of claims 28-30.

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