METHODS

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Patent Application No. 62/557,808, filed September 13, 2017, and titled SUSPENSION CULTURE DEVICE FABRICATED FROM BREATHABLE MATERIALS AND METHODS OF MAKING THE SAME, the content of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

[0002] The presently disclosed subject matter relates generally to devices for biologic studies. Particularly, the presently disclosed subject matter relates to suspension culture devices and systems.

BACKGROUND

[0003] To have useful predictive capability for biologic studies, in vitro cell culture models must reproduce as many features as possible of the in vivo cells or tissues that they are representing. As an example, using screening drugs for hepatoxicity using cultured liver cells is useful to the extent that those cells display the characteristics of cells in the body's liver. However, most, if not all, differentiated cells derived from tissues, lose their specialized features and de-differentiate when grown under traditional two-dimensional "petri dish" cell culture conditions. Suspension culture, where cells float in the media without adhering to plastic dishes, is the most popular technique of minimizing this problem and maintaining the cell's specialized features. One approach to suspension cultures is to stir the media to prevent the cells from sedimenting on the floor of the vessel. Several recent reviews have summarized the use of suspension culture, scale-up of suspension micro carriers, and automation of roller-bottle forms of suspension culture. A major drawback of these approaches is the resulting turbulences and impact against the stirrers damages the cells.

[0004] Another option is to rotate the vessel to keep the cells in suspension. The rotating wall vessel is designed to keep cells in suspension while exposing them to a controlled physiological or pathological amounts of shear stress. There is a rich and diverse array of available culture vessels for suspension culture and there is a great deal of information on the optimization of suspension culture by utilizing the engineering principles of fluid physics to minimize shear and turbulence. Cells grown in a rotating wall vessel recapitulate many of features of in vivo tissues and do so better and longer than cells in two-dimensional (2D) cultures. Rotating cell culture devices can also colocalize cells of diverse size and density.

[0005] Heptocytes grown in a rotating wall vessel form three-dimensional (3D) organoids that maintain their function for at least several weeks. Currently available rotating wall vessels have a large capacity (i.e., 10 to 500 ml) and are well adapted for uses such as generation of cell-derived products. However, the vessels can be costly, require expensive electrical hardware to rotate them, are labor-intensive to load, maintain, and harvest, and are poorly amenable to automation.

[0006] Furthermore, the large numbers of cells and large volume required per vessel can be prohibitively expensive for use in application, such as drug screening, where thousands of individual cultures are needed to assay the multiple drugs, each at varying doses and times of exposure. For at least these reasons, there is a need for a low- cost, rotating wall vessel with small capacity that can be manipulated robotically and adapted to large-scale, robotic screening approaches.

[0007] There are other approaches to improve the differentiation and viability of cultured cells, including growing them in hanging drops, sandwiching them between layers of gelled extracellular proteins, adding feeder cells such as mouse fibroblasts, and bioprinting them in a variety of organ-on-a-chip platforms. However, none of these techniques have been shown to combine exposure of the cultured cells to controlled physiological or pathological levels of shear stress at low cost, with simplicity, and facile with automation. Physiological stress is a stimulus that is key for cell differentiation and that is not provided by other culture strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Drawings, which are not necessarily drawn to scale, and wherein:

[0009] FIG. 1 is a top perspective view of an example suspension culture device 100 in accordance with embodiments of the present disclosure;

[0010] FIG. 2 is an exploded view of the base including the silicone rubber material for the ports of the suspension culture device shown in FIG. 1;

[0011] FIGs. 3 and 4 illustrate a side view and a top view, respectively, of the top component shown in FIG. 2;

[0012] FIGs. 5 and 6 illustrates a side view and a top view, respectively, of the bottom component shown in FIG. 2;

[0013] FIGs. 7 and 8 illustrates a side view and a top view, respectively, of the middle component shown in FIG. 2;

[0014] FIG. 9 is a side view of a person's hand holding the device shown in

FIG. 1;

[0015] FIG. 10 is a perspective view of a bottle roller rotating multiple suspension culture devices in accordance with embodiments of the present disclosure;

[0016] FIGs. 11 and 12 illustrate different views of another example suspension culture device in accordance with embodiments of the present disclosure;

[0017] FIG. 13 is an exploded view of another example suspension culture device in accordance with embodiments of the present disclosure;

[0018] FIGs. 14 and 15 are perspective views of a top component and a bottom component, respectively, that can be assembled together to form an example suspension culture device in accordance with embodiments of the present disclosure; and

[0019] FIG. 16 is a perspective view of a bottom side of the bottom component shown in FIG. 15.

SUMMARY

[0020] The presently disclosed subject matter relates to suspension culture devices and systems and related methods. According to an aspect, a suspension culture device includes a rotatable base having an exterior surface. The base defines an interior space for holding a culture. All or at least a portion of the base may be made of a breathable material extending between the interior space and outside the rotatable base. The suspension culture device includes multiple self sealing ports that each permit fluid communication between the interior space and outside the rotatable base.

[0021] According to another aspect, a suspension culture system includes one or more rollers and a mechanism configured to turn the roller(s). Further, the system includes a suspension culture device. The suspension culture device includes a rotatable base having an exterior surface that engages the roller(s) for rotation of the rotatable base when the roller(s) are turning. The base defines an interior space for holding liquid, cells, biologicals, micro-organisms and matrices including beads. All or at least a portion of the base may be made of a breathable material extending between the interior space and outside the rotatable base. The suspension culture device includes multiple ports that each permit fluid communication between the interior space and outside the rotatable base.

DETAILED DESCRIPTION

[0022] The following detailed description is made with reference to the figures. Exemplary embodiments are described to illustrate the disclosure, not to limit its scope, which is defined by the claims. Those of ordinary skill in the art will recognize a number of equivalent variations in the description that follows.

[0023] For the purposes of promoting an understanding of the principles of the present disclosure, reference will now be made to various embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended, such alteration and further modifications of the disclosure as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the disclosure relates.

[0024] Articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "an element" means at least one element and can include more than one element. [0025] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0026] The present disclosure relates, in part, to a suspension culture device. This device may be a 3D vessel that comprises a breathable material with a large ratio of breathable surface to media volume that can be spun or rotated by one or more rollers. The device may be made of biocompatible material. In some embodiments, the suspension culture device may define an interior space sized and shaped to hold volumes as low as 0.1 ml.

[0027] FIG. 1 illustrates a top perspective view of an example suspension culture device 100 in accordance with embodiments of the present disclosure. In this example, the device 100 includes a rotatable base 102 having an exterior surface 104. The rotatable base 102 may be substantially shaped as a disk or any other suitable shape such that it can suitably engage with one or more rollers for rotation of the rotatable base 102. In this example, the rotatable base 102 includes an axis of rotation, which is represented by broken line 106, around which the rotatable base 102 can rotate when moved by the rollers as described in further detail herein. The rotatable base 102 may define an interior space (not shown in FIG. 1) for holding a culture. When the rotatable base 102 is rotated, the culture can be kept in suspension such that it does not settle at the bottom of the interior space. The culture may be a cell culture medium. Also, the interior space may hold, for example, one or more of support structures, beads, test substances, drugs, peptides, and viruses.

[0028] In accordance with embodiments of the present disclosure, some or the entirety of the rotatable base 102 may be made of a breathable material that extends between the interior space and outside the rotatable base 102. As a result, oxygen or other gas from outside the rotatable base 102 may enter into the interior space to thereby allow cells in the culture to maintain their metabolism and differentiation. Further, gases such as carbon dioxide, produced by cell metabolis, can escape the interior space. The breathable material is selected for differential gas exchange such that water is retained orders of magnitude better, than oxygen and carbon dioxide are diffused. In some embodiments, the rotatable base 102 has one or more portions that are thinner than other portions to provide an easier pathway for oxygen from the outside into the interior space. These portions of the rotatable base 102 can be any suitable size, shape, and provide any suitable thickness between the outside and the interior space. An example of these portions is 0.001" thick FEP (fluorinated ethylene propylene). In examples, these portions can take the form of divots, indentations, and the like in the base 102. Moreover, the rotatable base may be designed for controlling an amount of oxygenation, carbon dioxide removal, and water retention, desired within the interior space where the culture is located.

[0029] The breathable material of the base 102 may be any suitable material that permits gas to pass through it. Example breathable material includes, but is not limited to, fluoroplastic, fluorinated ethylene propylene (FEP), PerFluoroAlkoxy (PFA), polytetrafluoroetylene (PTFE), the like, and combinations thereof.

[0030] The device 100 shown in FIG. 1 includes multiple ports 108 (only one of which is shown in FIG. 1) that each permit fluid communication between the interior space and outside the rotatable base 102. In particular, a port 108 may be used for introducing culture into the interior space of the rotatable base 102. A port 108 may also be used for removing air, another gas, or liquid from the interior space of the rotatable base 102.

[0031] In this example, a port 108 is made of a silicone rubber material that is positioned within a hole defined in the base 102. The hole provides a passageway that extends from outside the base 102 to the interior space. A blunt or sharp (sharp needle hole can seal better) needle (e.g., 18 to 26 gauge blunt or sharp needle) or other suitable instrument may penetrate the rubber material of the port 108 such that liquids can be introduced into the interior space. Once the needle is removed, the rubber material may reseal the port 108. Air may be bled from the interior space by use of another needle at another port.

[0032] As shown in FIG. 1, the ports 108 are positioned at an outer edge of the base 102. Particularly, the ports 108 are positioned at the exterior surface 104. Alternatively, the ports 108 may be positioned along any other suitable area of the base 102.

[0033] The device 102 also includes multiple windows 110 attached to the base 102 for permitting viewing into the interior space. For example, cells in the interior space may be stained with fluorescent dyes and imaged by inverted fluorescent microscopy. In this example, the base 102 defines multiple holes where the windows 110 are positioned. The windows 110 are sealed such that liquid cannot escape from the interior space. Further, the windows 110 may be made of transparent, semi-transparent, or substantially transparent such that a person or instrument may see into the interior space. In an example, the windows 110 may be made of FEP and have a thickness of between about 0.0005" and 0.05". As in described herein, example breathable material includes, but is not limited to, fluoroplastic, fluorinated ethylene propylene (FEP), PerFluoroAlkoxy (PFA), polytetrafluoroetylene (PTFE), the like, and combinations thereof. .

[0034] Alternatively, the material of the base 102 may be partially or entirely transparent such that the contents of the interior space can be viewed from the outside.

[0035] It is noted that the components may be made by 3D printing or any other suitable technique, such as injection molding. Examples include, but are not limited to, FEP and PFA techniques.

[0036] FIG. 2 illustrates an exploded view of the base 102 including the silicone rubber material for the ports 108 of the suspension culture device 100 shown in FIG. 1. Now referring to FIG. 2, the base 102 includes three (3) pieces that can be assembled together as shown in FIG. 1. Particularly, these pieces include a top component 200, a middle component 202, and a bottom component 204. In this example, the bottom component 202 and the middle component 204 can be fitted together such that protrusions 206 extending from a base 208 of the bottom component 204 fit into respective notches 210 defined in a base 212 of the middle component 202. In addition, a suitable adhesive may be used to further secure the bottom component 202 to the middle component 204. As a result, the bottom component 202 and the middle component 204 can be sealed together to avoid leakage from the interior space 214 defined with the components 200, 202, and 204 are arranged as shown in FIG. 1. As shown in FIG. 2, the ports are positioned at substantially opposing portions of the outer edge of the bottom component 202. Although only two ports are shown in this example, it should be understood that the rotatable base may include any suitable number of ports (e.g., 3, 4 or more ports).

[0037] The top component 200 may include protrusions 216 that extend from a base 218 of the top component 200 to engage with respective notches 220 defined in the middle component 202. The top component 200 and the middle component 202 may sealingly engage one another where they contact in order to prevent leakage from the interior space 214. The top component 200 and the middle component may be sealed together by glue. Alternatively, components may be configured such that they can be opened and closed while still retaining a seal when closed.

[0038] With continuing reference to FIG. 2, the middle component 202 defines holes or apertures 222 that extend from the interior space 214 to outside the device 102. Rubber material 224 may be inserted into respective apertures 222 to form the ports. The middle component 202 may also define notches 226 within the apertures 222 to grasp the rubber material 224 so that the rubber material 224 may be further secured inside the apertures 222. The rubber material 224 may be tightly fitted in the apertures 222 such that there is no leakage from the interior space 214, or movement of the silicone rubber port during needle insertion and removal.

[0039] The interior space 214 is substantially shaped as a disk. Although, it should be understood that the interior space may be any suitable shape or size. For example, the interior space may have a volume between about 0.3 milliliters and about 5.0 milliliters.

[0040] Other views of the components 200, 202, and 204 are shown in FIGs. 3 - 8. Particularly, FIGs. 3 and 4 illustrate a side view and a top view, respectively, of the top component 200 shown in FIG. 2. FIGs. 5 and 6 illustrates a side view and a top view, respectively, of the bottom component 204 shown in FIG. 2. FIGs. 7 and 8 illustrates a side view and a top view, respectively, of the middle component 202 shown in FIG. 2.

[0041] FIG. 9 illustrates a side view of a person's hand 900 holding the device 100 shown in FIG. 1. Referring to FIG. 9, the person's other hand 902 is shown holding a hypodermic needle 904 that is inserted into a port of the device 100 for injecting culture into the interior space of the device 100. Also, an apparatus 906 is interfaced at another port of the device 100 for removing or "bleeding" air from the interior space. As shown, the port receiving the culture is positioned at the bottom, and the port where the air exits is at the top to more easily receive the culture and remove the air. The air may be removed by a suitable needle, such as a 26 gauge needle.

[0042] FIG. 10 illustrates a perspective view of a bottle roller 1000 rotating multiple suspension culture devices 100 in accordance with embodiments of the present disclosure. Referring to FIG. 10, the bottle roller 1000 includes multiple rollers 1002 that may be operatively connected to a computer-controlled mechanism for turning the rollers 1002 to thereby rotate the devices 100. The rollers 1002 may be controlled to rotate the devices 100 at any suitable rotation speed. As can be seen in the figures, the outer edge of the rotatable base of the devices 100 is round for contact with the rollers for rotating the rotatable base.

[0043] In an example use of one of the culture devices 100 and the bottle roller 1000 shown in FIG. 10, the use may begin with inserting a cell sample into a device 100 in accordance with embodiments of the present disclosure. Further, the device 100 may be placed on the rollers as shown in FIG. 10. Subsequently, the roller 1000 may be controlled to rotate the device 100 at a speed sufficient to allow the cells to settle at their terminal velocity and aggregate in an annulus around the axis of rotation. Further, the device may be maintained at a biologically appropriate temperature.

[0044] The rotating wall vessel form of suspension culture can deliver controlled levels of physiological and pathophysiologically relevant shear. This is achieved by maintaining laminar flow. Laminar flow is maintained by completely filling the vessel, without air bubbles. If there are bubbles, the flow becomes turbulent and shear increases dramatically, often causing severe cell damage. Delivered physiological and pathophysiological shear levels in the rotating wall vessel can be modulated by changing cell/particle size such as bead size, gravity, the difference in density between the cells/particles and fluid medium, and viscosity. Low shear mimics physiological flow in organs such as liver, kidney and brain. Increased shear to pathophysiological levels mimics conditions such as chronic kidney disease and hepatic cirrhosis.

[0045] In embodiments of the present disclosure, robots can be used to recognize defined structural features, allowing part selection, orientation, and vessel assembly. There features include, but are not limited to, elements shown in FIG. 1 as reference numerals 102 and 108, FIG. 2 as reference numerals 200, 204, 206, 216, 220, and 222, FIG. 3 as reference numerals 200 and 216, FIG. 5 as reference numerals 204 and 206, FIG. 6 as reference numerals 204 and 206, FIG. 7 as reference numerals 202, 210, 220, and 222, FIG. 8 as reference numeral 220, FIG. 12 as reference numerals 1108, 1110, and 1112, FIG. 13 as reference numerals 1302, 1306, and 1308, FIG. 14 as reference numeral 1404, and FIG. 15 as reference numeral 1502.

[0046] In accordance with embodiments, the presently disclosed subject matter may be used for screening drugs for hepatoxicity and nephrotoxicity. The suspension holder device when suitably rotated may maintain target cells in a state of differentiation that improves acute toxicity testing. Further, the device may maintain viable and functional cells for long time periods to allow for evaluation of chronic toxicity. Further, the device may maintain induce pathological states such as shear elevated to a few to several times physiological to mimic pathological flows, such as increased renal tubular flow in many types of chronic kidney disease. The device also requires only small amounts of cells and reagents, including drugs. Further, the devices may be used for robotic handling in some embodiments. The devices and systems disclosed herein may be applicable to a broad range of cells and tissues. In example, the devices and systems may be used to study kidney metabolism, antibody production, cartilage implants, cosmetic components, pancreatic islets, and stem cells. In examples, the devices and systems may be used to study human, veterinary, animal husbandry, and agriculturally derived cells. In other examples, the devices and systems may be used to optimize production of biofuels, production of cosmetic components, and cell-cell interactions such as viral reactivation, microbial invasion assays, and the value of co- cultures of different cell types. These devices and systems are beneficial, for example, due to low cost, simplicity, ease of automation, and the lack of a need for cell feeder layers, size constraints of hanging drop cultures, or multiple complex biological preparative steps.

[0047] In accordance with embodiments, the presently disclosed subject matter of suspension culture devices may be used in various technology spaces including, but not limited to, human cell cultures, veterinary cultures, cosmetic components, microorganisms, and agricultural initiatives. The devices and systems disclosed herein may be used as a platform technology for improving cost efficiency, simplicity, and ease of automation in these various biotechnology areas.

[0048] FIGs. 11 and 12 illustrate different views of another example suspension culture device 1100 in accordance with embodiments of the present disclosure. In particular, FIG. 11 illustrates a perspective view of the device 1100. The device 1100 includes a rotatable base 1102 having an exterior surface 1104. In this example, the rotatable base 1102 is substantially shaped as a disk, although it may be any other suitable shape such that it can suitably engage with one or more rollers for rotation of the base 1102. In this example, the rotatable base 1102 includes an axis of rotation, which is represented by broken line 1106, around which the rotatable base 102 can rotate when moved by the rollers as described in further detail herein. The rotatable base 1102 may define an interior space (not shown in FIG. 11) for holding a culture. When the rotatable base 1102 is rotated, the culture can be kept in suspension such that it does not settle at the bottom of the interior space nor hit the walls, rather the cells and other contents travel in an annulus between the outer wall and center point of the interior space axis or rotation. In accordance with embodiments of the present disclosure, some or the entirety of the rotatable base 1102 may be made of a breathable material that extends between the interior space and outside the rotatable base 1102. Various portions of the rotatable base 1102 may have a suitable size, shape, and provide any suitable thickness between the outside and the interior space for controlling oxygenation, carbon dioxide removal, and water retention within the interior space where the culture is located.

[0049] With continuing reference to FIG. 11, the rotatable base 1102 include two (2) components, a top component 1108 and a bottom component 1110. Components 1108 and 1110 may be fitted together as shown to form the base 1102. Also, the components 1108 and 1110 may be sealingly fitted together such that the contents of the interior space do not leak to the outside.

[0050] FIG. 12 illustrates an exploded view of the base 1102 shown in FIG. 11. Referring to FIG. 12, the device 1100 includes ports 1112 that each permit fluid communication between the interior space 1114 and outside the base 1102. The ports 1112 may be used for introducing culture or other matter into the interior space 1114. Further, the ports 1114 may be used for removing air, another gas, or liquid from the interior space 1114.

[0051] In this example, a port 1114 is made of a silicone rubber material that is positioned within a hole defined in the top component 1108. The hole provides a passageway that extends from outside the base 102 to the interior space 1114. A hypodermic needle or other suitable instrument may penetrate the rubber material of the port 1114 such that liquids can be introduced into the interior space. All or a portion of the base 1102 may be made of transparent or semi-transparent material such that the contents of the interior space 1114 may be viewed.

[0052] FIG. 13 illustrates an exploded view of another example suspension culture device 1300 in accordance with embodiments of the present disclosure. Referring to FIG. 13, the device 1300 include a rotatable base 1302 having a top component 1304 and a bottom component 1306 that may be sealingly attached to one another for forming an interior space. In this example, the device 1300 includes four (4) ports 1308.

[0053] In accordance with embodiments, a system may be provided for maintaining the culture within a suspension culture device as described herein at a physiological appropriate temperature. An example appropriate temperature is about 37 degrees Celsius, although, the appropriate temperature range may be determined by the biological system being studied. Other example temperatures are in the range of between about 22 degrees and about 37 degrees Celsius. It is noted that FEP has a continuous service temperature range from -240 to 205 °C (-400 to 400°F).

[0054] FIGs. 14 and 15 illustrate perspective views of a top component 1400 and a bottom component 1500, respectively, that can be assembled together to form an example suspension culture device in accordance with embodiments of the present disclosure. Particularly, FIG. 14 is a perspective view of an underside of the top component 1400. An O-ring 1402 is affixed to the underside of the top component 1400 as shown for forming a sealed interior space between the top component 1400 and the bottom component 1500 when they are positioned together.

[0055] The bottom component 1400 includes multiple protrusions 1404 arranged as shown for engaging respective notches 1502 shown in the bottom component 1500 shown in FIG. 15. In this way, the top component 1400 and the bottom component 1500 are fitted together to thereby seal an interior space. The top component 1400 has a port 1406 by which the interior space can be accessed. In this example, the port 1406 is made of a silicone rubber material as described herein.

[0056] FIG. 15 shows a top side of the bottom component 1500, and FIG. 16 shows a bottom side of the bottom component 1500. Referring to FIGs. 15 and 16, the bottom component 1500 has multiple breathable areas 1600 that extend between the interior space and the outside. In this example, the bottom component 1500 is made of a breathable material and the thickness of the breathable areas 1600 is thin enough to provide a pathway for oxygen from the outside into the interior space.

[0057] While the embodiments have been described in connection with the various embodiments of the various figures, it is to be understood that other similar embodiments may be used, or modifications and additions may be made to the described embodiment for performing the same function without deviating therefrom. Therefore, the disclosed embodiments should not be limited to any single embodiment, but rather should be construed in breadth and scope in accordance with the appended claims.