Methods for attaching mesh inserts to the interior region of a roller bottle for improved performance

Abstract

Bioreactor vessels that solve existing problems with currently available high-density cell culture vessels are provided. The bioreactor vessels include a housing and an insert. Cell cultures may adhere to the interior surface of the housing and adhere to the insert surface, and both surfaces may optionally be treated to increase the adherence of cells. The inserts may be seated in the housing and an element on the housing, the insert, or a cap that covers a necked port on the housing may seat the insert. The insert may have a single layer or may have multiple layers. The bioreactor vessels improve the amount of viable cell yield compared to currently available standard and high-density cell culture vessels.

Description

METHODS FOR ATTACHING MESH INSERTS TO THE INTERIOR REGION OF A ROLLER BOTTLE FOR IMPROVED PERFORMANCECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority under 35 U.S.C. §119 of U.S. Provisional Application Serial No. 63 / 684, 154 filed on August 16, 2024, and U.S. Provisional Application Serial No. 63 / 566,678 filed on March 18, 2024, the content of which are relied upon and incorporated herein by reference in their entirety.FIELD

[0002] The present disclosure relates to cell culture systems for cell production. More specifically, the present disclosure relates to bioreactor vessels with inserts that improve cell culture production.BACKGROUND

[0003] The culturing of cells is used in a variety of laboratory and manufacturing processes such as vaccines, therapeutic proteins, antibodies, and cell therapy products. Various vessels and techniques for culturing cells have been developed over the years, but in practice, specific vessel types work better for certain types of applications. For example, roller bottles have been the gold standard for applications like the manufacturing of vaccines. As another example, single-use or stainless steel stirred tank bioreactors are used for monoclonal antibody production. However, despite the continued development of apparatuses to improve cell culture, in practice, performance in these improved apparatuses is less than optimal, particularly in commercial manufacturing processes. Therefore, a need still exists to provide a cell culture apparatus that improves cell homogeneity in tandem with generating a high yield of viable cells.

[0004] The present disclosure solves the deficiencies in existing high-density roller bottle designs by providing a roller bottle apparatus with cost-effective inserts designed to culture cells to a high density efficiently and homogenously.SUMMARY

[0005] According a first aspect of the present disclosure, a bioreactor vessel is provided that comprises a housing that comprises an exterior surface, an interior surface atop end and a bottom end, a cell culture chamber inside the housing, an insert, and an element on the insert that prevents the external side of the insert from contacting the interior surface of the housing. The insert comprises a top edge, a bottom edge, a first end, a second end, a length, an external side, and an internal side. The insert is configured for adherent cells to attach.

[0006] In some embodiments of the first aspect, the element of the bioreactor vessel comprises a band that runs along the length of the insert. Both the insert and the band have thicknesses, and the thickness of the band is greater than the thickness of the insert. In one further embodiment, the element of the bioreactor comprises two or more bands. In a different further embodiment, the band has a thickness from about 1.5 mm to about 71 mm. In another different further embodiment, the band comprises a first end and a second end, and the first end and second end are configured to attach together. The first and second ends may attach with a configuration selected from the group consisting of: a snap, magnets, interlocking tabs, hooks, a sealed joint, and a combination thereof.

[0007] In other embodiments of the first aspect, the element of the bioreactor vessel comprises one or more gaskets or o-rings around the external side of the insert. In other embodiments, the element comprises one or more clips on a top surface of the insert, on a bottom surface of the insert, or a combination thereof. In some other embodiments, the element comprises one or more protrusions from the top edge of the insert, the bottom edge of the insert, or a combination thereof, and the protrusions extend into the top end of the housing, the bottom end of the housing, or a combination thereof. In yet other embodiments, the element is an adhesive between the exterior side of the insert and the interior side of the housing. In another other embodiment, the element is formed from one or more locations on the exterior side of the insert and protrudes towards the interior surface of the housing.

[0008] In some embodiments of the first aspect, the insert of the bioreactor vessel comprises a modification for cell attachment.

[0009] In some embodiments of the first aspect, the insert of the bioreactor vessel is a mesh or is a polymer film with a pattern of pores in the film. In one further embodiment, the mesh comprises polyethylene terephthalate and the polymer film comprises polystyrene.

[0010] According to a second aspect of the present disclosure, a bioreactor vessel is provided that comprises a housing, a cell culture chamber inside the housing, an insert in the cell culture chamber, a necked access port on the housing, and a cap that attaches to the necked access port. The housing comprises an exterior surface and an interior surface. The insert is configured for adherent cells to attach. The insert is seated by at least one element on the housing or the cap.

[0011] In some embodiments of the second aspect, the at least one element comprises a plurality of notches on the housing that extend inwardly from a bottom interior surface of the housing or from a top interior surface of the housing, or both from the top and bottom interior surfaces of the housing. In other embodiments, the at least one element comprises a ring that extends inwardly from a bottom interior surface of the housing or from a top interior surface of the housing, or both from the top and bottom interior surface of the housing. In yet other embodiments, the at least one element comprises a flexible or foldable support structure. The support structure may comprise a shaft and at least two extendable arms connected to the shaft. The support structure seats the insert when the support structure is in its extended form, and the shaft attaches to the cap.

[0012] In some embodiments of the second aspect, the at least one element comprises at least one outwardly extending notch from a bottom interior surface of the housing that extends around the bottom surface of the housing. In one further embodiment, the at least one element further comprises at least one inwardly extending notch from a top interior surface of the housing that extends around the top surface of the housing. In a different further embodiment, the at least one element further comprises at least one outwardly extending notch from atop interior surface of the housing that extends around the top surface of the housing.

[0013] In some embodiments of the second aspect, the at least one element comprises a plurality of bumps extending inwardly from the interior surface of the housing. In other embodiments, the at least one element comprises grooves or ridges on a bottom interior surface of the housing. In yet other embodiments, the at least one element comprises sidewalls of thehousing, and the insert comprises an outermost rolled insert layer having a diameter that is between about 0.25% and about 10% greater than an interior surface diameter of the housing.

[0014] In another embodiment of the second aspect, the at least one element comprises protrusions from the interior side of the housing that seat the insert in a main body of the housing. The protrusions extend between 1 mm and 10 mm from the interior surface of the housing.

[0015] In some other embodiments of the second aspect, the at least one element is on the cap of the bioreactor. In one further embodiment, an interior diameter of the cap is at least 40% of an interior diameter of the housing.

[0016] In a third aspect of the present disclosure, a bioreactor vessel is provided that comprises a housing, a cell culture chamber inside the housing, and an insert. The insert comprises a top edge, a bottom edge, a length, an external side, and an internal side, and the insert is configured in an accordion shape along the top edge and bottom edge of the insert. The housing has an exterior surface and an interior surface. The insert is configured for adherent cells to attach. In some embodiments, the cell culture chamber comprises a circumference and the length of the insert is from about 1 to 3 times the circumference of the cell culture chamber. In other embodiments, the insert is configured to adhere 1 x 104cells / cm2to about 1 x 106cells / cm2.

[0017] In a fourth aspect of the present disclosure, a bioreactor vessel is provided that comprises a housing, a cell culture chamber, and an insert in the cell culture chamber comprising a first height, an external side, and an internal side. The insert is configured for adherent cells to attach. The housing comprises an exterior surface and an interior surface. The cell culture chamber comprises a second height, which is the height that the insert in the cell culture chamber can lie flush to the interior surface of the housing. The first height is smaller than the second height. In some embodiments of this aspect, the first height is between about 75% to about 98% of the second height.

[0018] In a fifth aspect of the present disclosure, a method of culturing cells is provided that comprises the steps of providing any of the bioreactor vessels of the preceding aspects and their embodiments, providing cells of a cell type, and culturing the cells in the bioreactor. At least 90% of the cells are viable cells after culturing, as measured by a trypan blue exclusionassay. In some embodiments of this aspect, the number of cells per unit surface area cultured are from about 1 x 104cells / cm2to about 1 x 106cells / cm2.

[0019] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from that description or recognized by practicing the embodiments as described herein, including the detailed description which follows, the claims, as well as the appended drawings.

[0020] It is to be understood that both the foregoing general description and the following detailed description are merely exemplary and are intended to provide an overview or framework to understanding the nature and character of the claims. The accompanying drawings are included to provide a further understanding and are incorporated in and constitute a part of this specification. The drawings illustrate one or more embodiment(s), and together with the description serve to explain principles and operation of the various embodiments.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The following is a description of the figures in the accompanying drawings, given purely by way of non-limiting example. The figures are not necessarily to scale, and certain features and certain views of the figures may be shown exaggerated in scale or in schematic in the interest of clarity and conciseness.

[0022] FIGS. 1A-1B illustrate examples of the bioreactor vessel, according to embodiments of the present disclosure. FIG. 1A is a schematic of a bioreactor vessel without an insert, according to embodiments. FIG. IB is a schematic of a housing with a straight necked port and an inwardly flared portion at the top end of the housing, according to embodiments.

[0023] FIGS. 2A-2C are schematic drawings of two different types of film inserts, according to embodiments. FIG. 2A illustrates a side view of a film having a pattern of round pores, while FIG. 2B illustrates a side view of a film having a pattern of elongated line pores positioned horizontally. FIG. 2C is a three-dimensional perspective view of an example mesh insert.

[0024] FIG. 3 is a schematic from a top view of a bioreactor vessel with a two-layer insert inside the housing. The insert is rolled in a clockwise direction while the bioreactor vessel is rotated during cell culture in a counterclockwise direction.

[0025] FIGS. 4A-4C are schematics of bioreactor vessels having different heights of inserts in the bioreactor vessels, according to embodiments. FIG. 4A illustrates a side view of a bioreactor vessel with an insert at its maximum height in a bioreactor vessel having inwardly flared top and bottom ends. FIG. 4B illustrates a side view of a bioreactor vessel with an insert at its maximum height in a bioreactor vessel having an inwardly flared top end and a flat bottom. FIG. 4C illustrates a side view of a bioreactor vessel with an insert having a shortened height in a bioreactor vessel having inwardly flared top and bottom ends.

[0026] FIG. 5 is a schematic from a top view of a bioreactor vessel having a coarse mesh layered around an insert inside the housing of the bioreactor vessel, according to embodiments.

[0027] FIG. 6 is a schematic showing a side view of an insert having bands at top and bottom portions of the insert, according to embodiments.

[0028] FIG. 7 is a schematic of a bioreactor vessel from a side view with a rolled insert having thin strips of material on the exterior surface of the insert, according to embodiments.

[0029] FIGS. 8A-8B are schematics showing side views of a mesh insert having unjoined ends (FIG. 8A), that are then attached together in a sealing j oint (FIG. 8B), according to embodiments .

[0030] FIGS. 9A-9B are schematics of mesh inserts in their extended form with tabs and receptacles for tabs, according to embodiments. FIG. 9A illustrates a side view of a mesh insert with rectangular tabs and double-slotted receptacles, while FIG. 9B illustrates a side view of a mesh insert with T-shape tabs and single-slotted receptacles.

[0031] FIG. 10 is a schematic illustrating a top-view of an insert having an accordion configuration, according to embodiments.

[0032] FIG. 11 is a schematic illustrating a side view of a bioreactor vessel with an insert and an element having a foldable support structure seating the insert in the bioreactor vessel, according to embodiments.

[0033] FIG. 12 is a schematic illustrating a side view of a bioreactor vessel with an insert seated in the bioreactor vessel by an element having inwardly extending discontinuous notches on the top end the housing and an outwardly extending continuous notch around the bottom end of the housing, according to embodiments.

[0034] FIG. 13 is a schematic illustrating a side view of a bioreactor system during formation of the bioreactor vessel and after formation of the bioreactor vessel, according to embodiments.

[0035] FIGS. 14A-14B are photographs illustrating the uniformity results of Crystal violet-staining mesh inserts post-cell culture, where the mesh inserts have a maximum height in a roller bottle (FIG. 14A) and a shortened height in a roller bottle (FIG. 14B), according to embodiments. The dark portions of the photographs represent areas where cell growth occurred.

[0036] FIG. 15 is a graph of the viable cell yield results from culturing cells in standard roller bottles, a high-density roller bottle having pleats on the housing of a bioreactor vessel to expand the surface area, a standard roller bottle having a mesh insert with a single layer, and a standard roller bottle having a mesh insert with two layers, according to embodiments.

[0037] FIG. 16 is a schematic showing a side view of a bioreactor vessel with an insert seated in the bioreactor vessel by o-rings on the external side of the insert that are positioned towards the ends of the height of the insert, according to embodiments.

[0038] FIG. 17 is a schematic illustrating a side view of a bioreactor vessel with an insert seated in the bioreactor vessel by small portions of the exterior side of the insert being melted to the interior side of the housing of the bioreactor vessel, according to embodiments.

[0039] FIG. 18 is a schematic illustrating a side view of a bioreactor vessel with an insert seated in the bioreactor vessel with adhesive attaching small portions of the exterior side of the insert to the interior side of the housing, according to embodiments.

[0040] FIG. 19 is a schematic illustrating a side view of a bioreactor vessel with an insert seated in the bioreactor vessel using clips that attach to the top side of the insert using the clip portion and that attach to the inside surface of the housing using the exterior facing sides of the clips, according to embodiments.

[0041] FIG. 20 is a schematic illustrating a side view of a bioreactor vessel with an insert seated in the bioreactor vessel by protrusions that extend outwardly from the top and bottom sides of the insert, according to embodiments.

[0042] FIG. 21 is a schematic illustrating a side view of a bioreactor vessel with an insert seated in the bioreactor vessel by protrusions that extend outwardly from the interior surface of the housing in the endcap portions of the housing and secure the insert in the main body of the housing, according to embodiments.DETAILED DESCRIPTION

[0043] The various aspects and embodiments will now be fully described herein. These aspects and embodiments may, however, be embodied in many different forms and should not be construed as limiting; rather, these embodiments are provided so the disclosure will be thorough and complete, and will fully convey the scope of the present subject matter to those skilled in the art. All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0044] Modifications of the disclosure will occur to those skilled in the art and to those who make or use the disclosure. Therefore, it is understood that the embodiments shown in the drawings and described above are merely for illustrative purposes and not intended to limit the scope of the disclosure, which is defined by the following claims, as interpreted according to the principles of patent law, including the doctrine of equivalents.A. Definitions

[0045] Unless defined otherwise, all terms and phrases used herein include the meanings that the terms and phrases have attained in the art, unless the contrary is clearly indicated or clearly apparent from the context in which the term or phrase is used. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, particular methods and materials are now described.

[0046] As used herein the terms “the,” “a,” or “an,” mean “at least one,” and should not be limited to “only one” unless explicitly indicated to the contrary. Thus, for example, reference to “a component” includes embodiments having two or more such components unless the context clearly indicates otherwise.

[0047] Unless otherwise stated, the use of individual numerical values are stated as approximations as though the values were preceded by the word “about” or “approximately.”Similarly, the numerical values in the various ranges specified in this application, unless expressly indicated otherwise, are stated as approximations as though the minimum and maximum values within the stated ranges were both preceded by the word “about” or “approximately.” In this manner, variations above and below the stated ranges can be used to achieve substantially the same results as values within the ranges. As used herein, the terms “about” and “approximately” when referring to a numerical value shall have their plain and ordinary meanings to a person of ordinary skill in the art to which the disclosed subject matter is most closely related or the art relevant to the range or element at issue. The amount of broadening from the strict numerical boundary depends upon many factors. For example, some of the factors which may be considered include the criticality of the element and / or the effect a given amount of variation will have on the performance of the claimed subject matter, as well as other considerations known to those of skill in the art. As used herein, the use of differing amounts of significant digits for different numerical values is not meant to limit how the use of the words “about” or “approximately” will serve to broaden a particular numerical value or range. Thus, as a general matter, “about” or “approximately” broaden the numerical value. Also, the disclosure of ranges is intended as a continuous range including every value between the minimum and maximum values plus the broadening of the range afforded by the use of the term “about” or “approximately.” Consequently, recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein.

[0048] As used herein, the term “and / or,” when used in a list of two or more items, means that any one of the listed items can be employed by itself, or any combination of two or more of the listed items can be employed. For example, if a composition is described as containing components A, B, and / or C, the composition can contain A alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.

[0049] As used herein, “have,” “having,” “include,” “including,” “comprise,” “comprising” or the like are used in their open ended sense, and generally mean “including, but not limited to.”

[0050] “Optional” or “optionally” means that the subsequently described element, component or circumstance may or may not occur, so that the description includes instances where the element, component, or circumstance occurs and instances where it does not.

[0051] “Viable cell” means a live cell that is capable of excluding trypan blue.

[0052] In this document, relational terms, such as first and second, top and bottom, and the like, are used solely to distinguish one entity or action from another entity or action, without necessarily requiring or implying any actual such relationship or order between such entities or actions.

[0053] All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.B. Introduction

[0054] The present disclosure provides cell culturing vessels for high-density cell culture production that are improved over existing culturing vessels having a configuration of a roller bottle. Currently available high-density roller bottles create a high surface area in the roller bottle to culture cells using pleats or ribs molded into the surface of the bottle. These features can double the surface area of the bottle and so should theoretically double cell yields. However, both pleat and rib roller bottle designs can cause reduced performance, and the surface area utilization is poor. Therefore, with currently available high-density roller bottle designs, the productivity is not double that of a comparable smooth-walled bottle. These designs also result in challenges with harvesting the cells due to non-uniform cell growth and heterogenous cell populations that occur in the pleat and rib roller bottle designs for high- density cell culture.

[0055] Previous roller bottle designs have focused on modifying the bottle itself. In these designs, the surface area of the bottle is increased by patterning the surface of the roller bottle with pleats or ribs. The pleats refer to vertically oriented features on the roller bottle’s interior surface, while ribs refer to horizontally oriented features on the roller bottle’s interior surface. However, roller bottles with pleats and ribs negatively impact the flow dynamics in the roller bottle and cell attachment to the roller bottle surface. In practice, with a horizontal(rib) orientation, the cells tend to fall into the grooves and attach only to the bottom portion of the horizontal features. With a vertical (pleat) orientation, the cells tend to only seed on one side of the vertical features due to the direction of rotation unless rotation is alternated to counteract this effect, which is problematic. Another problem with these designs is that the cells form heterogenous populations, which are highly undesirable for transfection and harvesting.

[0056] There is therefore a need for cell culture vessels that have high-density cell productions that are more efficient and that create more homogenous cell populations than existing options. The cell culture vessels of the present disclosure provide a cost-effective solution to achieve higher density cell productions by increasing the surface area available for cell growth in roller bottles while maintaining a more homogenous cell production. This is achieved by employing inserts in the roller bottle, as described herein, which increases the surface area for cell attachment and improves uniform culturing conditions.

[0057] Additional features and advantages will be set forth in the detailed description which follows and will be apparent to those skilled in the art from the description or recognized by practicing the embodiments as described in the following description, together with the claims and appended drawings.C. Bioreactor Vessel for Cell Production

[0058] In some aspects, the present disclosure provides a bioreactor vessel configured as a roller bottle. Referring to FIG. 1A, the bioreactor vessel 1 comprises a housing 10 having an exterior surface and an interior surface, a cell culture chamber 20 inside the housing 10, and cap 30 that goes over a necked port (not shown) on the top side of housing 10. The interior surface of housing 10 is the outer part of the cell culture chamber 20. The interior surface of housing 10 can attach cells (25a, 25b) in some embodiments. Cells that do not attach to any surface of the bioreactor vessel remain in suspension 28. A longitudinal axis and a latitudinal axis extend from the center point of the cell culture chamber. The housing is capable of containing an insert for cell attachment (a “substrate”) inside the cell culture chamber, according to one or more of the embodiments described in this disclosure. The top portion of the housing vessel 10 may have a flat top or the top portion may be inwardly flared. When the top portion is inwardly flared, the flare may extend only inwardly from the cylindrical portion of the sidewalls of the housing towards the cap (as shown in FIG. 1 A), or the flare may extendoutwardly from the cylindrical portion of the sidewalls of the housing before then continuously extending inwardly toward the cap (for example, FIG. IB). Similarly, the bottom portion of the housing vessel 10 may have a flat bottom or the bottom portion may be inwardly flared. Where the bottom portion is inwardly flared, the flare may extend only inwardly from the cylindrical portion of the sidewalls of the housing towards the of the bottom of the housing (as shown in FIG. 1A), or the flare may extend outwardly from the cylindrical portion of the sidewalls of the housing before then extending inwardly towards the bottom of the housing (for example, FIG. IB). Both the top and bottom portions may be inwardly flared, both may be flat, or one may be inwardly flared and the other may be flat.

[0059] In the roller bottle configuration, the bioreactor vessel may be operably attached to a means for moving the bioreactor vessel about the central longitudinal axis of the cell culture chamber. For example, the bioreactor vessel may be rotated about the central longitudinal axis. The rotation may be continuous (e.g., continuing in one direction) or discontinuous (e.g., an intermittent rotation in a single direction or alternating directions, or oscillating in back and forth rotational directions). In operation, the rotation of the bioreactor vessel causes movement of cells and / or liquid (fluid) within the chamber. This movement can be considered relative with respect to the interior surface of the chamber. For example, as the bioreactor vessel rotates about the central longitudinal axis, gravity may cause the liquid, culture media, and / or unadhered cells to remain toward a lower portion of the cell culture chamber.

[0060] The bioreactor vessel may have one or more ports. In some embodiments, the one or more ports may be necked (an angled neck or a straight neck). In some embodiments, the one or more ports are on a top side of the housing (such as in FIGS. 1A-1B), a bottom side of the housing, or on both the top and bottom side of the housing. FIG. IB illustrates an example of a straight necked port 35 extending out of an inwardly flared portion 15 of housing 10, on the top side of a bioreactor vessel. Through the one or more ports, cells or liquids may be added, removed, or exchanged in the cell culture chamber. A single port in the vessel may act as both an inlet and outlet (for example, the port shown in FIG. IB). Alternatively, multiple ports may be provided for dedicated inlets and outlets. The bioreactor may have one or more caps (an exemplary cap 30 is shown in FIG. 1A) that may be attached to one or more ports on the housing. In some embodiments, the cap may be removeable and may be vented or unvented, may be gas permeable but liquid impermeable, or a combination thereof. In someembodiments, the cap may be permanent (not intentionally removable) and may be vented or unvented, may be gas permeable but liquid impermeable, or a combination thereof.

[0061] The interior surface of the housing is smooth (i.e., it contains no features that expand the surface area for cells to attach to), or the interior surface may have a feature on it, such as plurality of ribs, a plurality of pleats, or a combination of at least one rib and at least one pleat. When the interior surface has at least feature, it may have a single feature or a combination of features. Although ribs have a horizontal orientation and pleats have a vertical orientation, these features do not need to be in line-type configurations along the length or width of the vessel. For example, a rib or pleat may have a wave, zig-zag, or other configuration. In some embodiments, the feature (s) are oriented with at least one of a longitudinal axis and a latitudinal axis extending from the center point of the cell culture chamber. In other embodiments, the interior surface has at least one feature that increases the surface area for cell attachment and at least one feature is angled from the longitudinal axis, the latitudinal axis, or both axes.

[0062] The housing material may be fabricated with polymeric materials compatible in cell culture applications, including, for example, polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, and polypropylene oxide, or a combination thereof. In one embodiment, the housing comprises polystyrene or polyethylene terephthalate.

[0063] In one aspect of the disclosure, the interior surface of the housing is a substrate for adherent cells to attach to. In some embodiments, cells attach directly to the interior surface of the housing without any modifications to the surface. In other embodiments, the interior surface of the housing is modified to improve cell adherence. The modification to improve cell adherence may be a physical treatment rendering the interior surface with cell adhesive properties through treatment of the interior surface with one or more of plasma, process gases, and / or chemicals known in the industry, including for example, vacuum-gas plasma treatment and corona-gas treatment. The modification to improve cell adherence may be a chemical treatment of the interior surface, including by treatment of the interior surface with poly-D- lysine, among others known to those of skill in the art. The modification to improve cell adherence may be performed by grafting cell adhesion molecules onto the interior surface itself. Grafting of cell adhesion molecules may be done by any method known to those of skill in the art, such as adsorption of extracellular matrix proteins or mimetics onto the surface.Examples of cell adhesion molecules for grafting include collagen, laminin, and fibronectin, among other known to those of ordinary skill in the art. The modification to improve cell adherence of the interior surface of the housing may be performed by coating the interior surface with a material that improves cell adhesion. For example, the interior surface can be coated with a thin layer of a biocompatible hydrogel, such as collagen or other extracellular matrices (e.g., Coming® Matrigel®), among other known to those of ordinary skill in the art.

[0064] To add additional cell culturing surface area to the bioreactor vessel, an insert is provided to the interior of the cell culture chamber, which creates additional surface area for adhering cells. Like the housing, the insert may be made with polymeric materials suitable for culturing cells. For example, the insert may comprise polystyrene, polyethylene terephthalate, polycarbonate, polyvinylpyrrolidone, polybutadiene, polyvinylchloride, polyethylene oxide, polypyrroles, or polypropylene oxide, or a combination thereof. These are merely examples of materials that work for the insert, it should be understood that any material known to those of ordinary skill in the art that is non-toxic and suitable for culturing cells may be used. In one specific embodiment, the insert comprises polyethylene terephthalate. In another specific embodiment, the insert comprises polystyrene.

[0065] The cells may adhere to the insert, while movement of the bioreactor vessel (e.g., rotation around a central axis for a roller bottle configuration) allows the cells to be exposed to both the cell culture media or liquid, and to oxygen or other gases within the cell culture chamber. The insert may take on a variety of configurations, such as mesh (e.g., as shown in FIGS. 4A-4C) and patterned film embodiments (e.g., film 43 in FIGS. 2A-2B) as described below, or it may be in other configurations that can be affixed or attached to the bioreactor vessel (as described herein). Like the interior surface of the housing, the insert may be modified to improve cell adhesion (z.e., a modification for cell attachment). The modifications to the insert to improve cell adhesion may be any of those described above for the interior surface of the housing.

[0066] In some embodiments, the insert has a mesh configuration. In a mesh configuration, fibers of substrate (e.g., polyethylene terephthalate, polypropylene, etc.) are layered or woven with each other, creating geometric pores (e.g., square, rectangle, and hexagon shapes) for fluid to flow through. Cells can bind to the substrate portion of the mesh insert. The mesh configurations include any of those described in commonly owned U.S. Pat. No. 11,111,470, which is incorporated herein by reference.

[0067] In some embodiments, the insert has a film configuration. In a film configuration, the insert comprises polymer film (e.g., a thin film of polystyrene of other polymer), which is used as a substrate for the cells to adhere to. In one specific embodiment, the film comprises pores. The pores may be in certain sections of the film or throughout the film and may be patterned in the film or not patterned in the film. In one embodiment, the insert is a film configuration and comprises at least one pattern of pores. The pores may be any shape, such as round, square, rectangle, or any other shape. The pores may be spaced into a pattern. Examples of inserts having a patterned film configuration are illustrated in FIGS. 2A-2B. FIG. 2A depicts an insert 40 made from film 43 with round pores 46 in a pattern across the film 43. FIG. 2B depicts an insert 40 made from film 43 with elongated line pores 48 across the film 43.

[0068] Referring now to FIG. 2C, a three-dimensional perspective view of a schematic of an exemplary insert 40 is shown. The insert 40 is shown in an extended, or unrolled format, and is shown having a mesh configuration. The insert 40 has a top edge 50 and a bottom edge 55 along the length (L) of the insert, and a first side 60 and a second side 65. The top edge 50 is opposite the bottom edge 55, and the first side 60 is opposite the second side 65. Further, the insert 40 has an interior side 70 that faces inwardly towards the center of the cell culture chamber, and it has an exterior side (not shown) opposite the interior side 70, which faces outwardly towards the housing of the bioreactor vessel. The insert 40 also has a thickness t along the length of the insert and a height htacross the insert, as is shown in FIG. 2C. When the insert is in a film configuration or other type of configuration, it likewise has a top edge, a bottom edge, a first side, a second side, an interior side, an exterior side, a thickness t along the length of the insert and a height htacross the insert.

[0069] In some embodiments, the insert is a single layer that tracks the interior surface of the housing. This layer may be rolled to be cylindrical or cylindrical-like in form. In some embodiments, the insert has more than one layer that spirals toward a central longitudinal axis in the cell culture chamber. In some embodiments, the insert has more than one layer and the layers are concentric in form. In some embodiments, the insert may have from about 1 to about 2 layers, from about 1 to about 3 layers, from about 1 to about 4 layers, or more than about 4 layers. In some further embodiments, when two or more layers of insert are used and all of the layers are formed from a continuous length of insert, it is advantageous for the insert to be in the housing rolled in a direction opposite that of any rolling of the bioreactor vessel during cellculture to prevent the insert from collapsing on itself during the cell culture process. FIG. 3 illustrates such an embodiment. FIG. 3 is a top view of a bioreactor vessel with housing 310, cap 330, and an insert comprising two layers (340a, 340b) formed from a single, continuous layer of insert. The insert is rolled in a clockwise direction while the direction of rolling the bioreactor vessel is in the counterclockwise direction. However, it should be understood that in other embodiments the insert could be rolled in a counterclockwise direction and the bioreactor vessel could be rolled in a clockwise direction.

[0070] Regarding height of the insert, in one aspect of the disclosure, the height of the insert is approximately the maximum height a single layer of the insert can be parallel against the interior surface of the housing. For a roller bottle configuration, the maximum height of the insert is the height of the cylindrical sidewalls of the housing. Referring to FIG. 4A, bioreactor vessel 400 has an inwardly flared top portion 412 and an inwardly flared bottom portion 417, with cylindrical sidewalls having height hs. Insert 440 is positioned parallel to the cylindrical sidewall portion of bioreactor vessel 400. If insert 440 was to extend into the inwardly flared top portion 412 or the inwardly flared bottom portion 417, the insert 440 would be forced to bend inwards by the sidewalls of the top and bottom portions and no longer be able to maintain its parallel configuration. In a slightly different configuration shown in FIG. 4B, bioreactor vessel 400 has an inwardly flared top portion 412 and cylindrical sidewalls having height hs, but the bottom of the bioreactor vessel is flat and so insert 440 may extend down to or near the bottom of the housing.

[0071] In another aspect of the disclosure, the height of the insert ( / ?,) is less than the maximum height a single layer of the insert can be parallel against the interior surface of the housing. FIG. 4C illustrates an example of this type of embodiment. Like FIG. 4A, the bioreactor vessel 400 of FIG. 4C has an inwardly flared top portion 412, an inwardly flared bottom portion 417, but the height hsof the cylindrical sidewalls is greater than the height ht of insert 440. In one specific embodiment, the height htof the insert is from about 70% to about 99% of height hs.

[0072] In some embodiments, it is advantageous to have a height of the insert be less than or equal to the maximum height the insert ( / ?,) can be parallel against the interior surface of the housing. The shorter mesh (compared to a mesh having height extending into the endcap), may have improved uniformity of cell attachment and growth on the insert, as measured by visual observation with a crystal -violet staining test. This may be particularly truefor portions of the housing having an inwardly flared portion. In other words, it may be particularly advantageous to have a mesh shorter than the maximum height the insert can sit parallel to the sidewalls of the housing at locations where the housing has an inwardly flared portion. The highest level of improvement may be seen when the inwardly flared portion has a flare that extends outwards from the cylindrical wall of the housing before extending inwardly.

[0073] The shorter mesh (compared to a mesh having height extending into the endcap), may have improved yields of viable cells, as measured by an automated cell counter (e.g., Beckman Coulter Vi-Cell analyzer), which counts the viable cells using a Trypan Blue dye exclusion assay that infdtrates the walls of nonviable cells. In some embodiments, the top quarter and bottom quarter of an insert having a height htless than or equal to hsstains at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the mesh, with crystalviolet staining as measured by visual observation. In some embodiments, a bioreactor vessel with an insert having a height htless than or equal to height hshas at least a 2-fold increase, a 3 -fold increase, a 4-fold increase, a 5 -fold increase, or more in viable cell yield, compared to the same type of bioreactor vessel without the insert, as measured by a Trypan Blue dye exclusion assay using an automated cell counter (e.g., Beckman Coulter Vi-CELL analyzer). In some embodiments, a bioreactor vessel with an insert having a height ht less than or equal to height hshas between about a 1.5-fold increase and about a 10-fold increase in viable cell yield, compared to the same type of bioreactor vessel without the insert, as measured by a Trypan Blue dye exclusion assay using an automated cell counter (e.g., Beckman Coulter Vi- CELL analyzer). In some embodiments, a bioreactor vessel with an insert having a height htless than or equal to height hscultures adherent cells to a final surface area utilization of substrate of between about 1 x 103cells / cm2and about 1 x 107cells / cm2, or between about 1 x 104cells / cm2and about 1 x 106cells / cm2.

[0074] In some aspects of the present disclosure, the insert is prevented from contacting the interior surface of the housing. As used herein, “contacting the housing” means when more than 15% of the exterior side of the insert contacts the interior surface of the housing at one time during cell culturing. When too much of the surface area of the exterior side of the insert contacts the interior surface of the housing, cell yields may decrease due to there being less surface area for cells to adhere at and near the contact points, due to less media, nutrients, and oxygen being able to reach cells at or near the contact points, and due to variability in cell culturing conditions caused by the contact points.

[0075] In some embodiments, the insert is prevented from contacting the interior surface of the housing by using a layer of coarse mesh. In some embodiments, the coarse mesh may be non-binding to cells, meaning that all or substantially all of the cells in the bioreactor will not bind to the coarse mesh. As used herein, “substantially all” means at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater. To achieve non-binding, the coarse mesh may be made of a material that does not adhere to cells or may be coated with a material that prevents adherence of cells. The coarse mesh has larger pore sizes than the insert and is next to the exterior side of the insert such than the interior side of the coarse mesh contacts various points of the exterior side of the insert, while the exterior side of the mesh contacts various points on the interior surface of the housing. When multiple layers of insert are used, the coarse mesh is further used to separate the layers of insert from contacting itself. One such embodiment is shown in FIG. 5. FIG. 5 is a top view of bioreactor vessel 500 comprising housing 510, a cap 530, and a layer of a coarse mesh 590 between insert 540 and the interior surface of housing 510. The insert 540 has more than one layer (it has two layers) and the coarse mesh 590 separates the inner layer of the insert 540 from contacting the outer layer.

[0076] In some aspects of the present disclosure, the exterior side of the insert is prevented from contacting the interior surface of the housing by an element attached to the insert. As used herein, an “element” is a feature of the bioreactor vessel that alters the ability of the insert to fully contact (i.e ., 100% of the contact points between two surfaces) itself or the interior surface of the housing.

[0077] In one aspect, the element comprises at least one band. A band is an addition to the insert that runs along the length of the insert, and it may be made of the same material as the insert or the housing, or it may be a different material that is nontoxic to cells. The band may contact the interior surface of the housing while simultaneously preventing the exterior surface of the insert from contacting the interior surface of the housing. In this way, the band acts a physical buffer between the interior surface of the housing and the exterior surface of the insert. In some embodiments, the stiffness k of the insert with the band(s) is greater than the stiffness k of the insert without the band(s).

[0078] In some embodiments the band may cover the top edge, the bottom edge, or the top and bottom edges of the insert. In these embodiments, the thickness of the band is thicker than the thickness of the insert. In some embodiments, the thickness of the band is from about 1.5 mm to about 76 mm, or any value between. In another embodiment, the thickness of theband is from about 6 mm to about 51 mm, or any value between. In one embodiment, the band may have a groove on one side of the band sized to attach to the insert by snapping the groove over the edge of the insert. In another embodiment, the band may be attached over the edge of the insert with an adhesive. Any adhesive known to those of skill in the art may be used. Other methods of attaching the band over the edge of the insert known to those of skill in art may also be used. Alternatively, the band(s) may be molded as part of the insert itself during formation of the insert.

[0079] In some embodiments, the band may be a thin strip of material that covers a top portion of the exterior side of the insert near the top edge, a bottom portion of the exterior side of the insert near the bottom edge, or some other portion of the exterior side of the insert between the top and bottom edges. In some embodiments, the height of the thin strip may be from about 1.5 mm to about 20 mm, or any value between. In one embodiment, the height of the thin strip may be from about 3 mm to about 13 mm, or any value between. The thin strip of material may be attached to the insert in any way known to those of ordinary skill art, for example, with adhesive, or through bonding or welding. Alternatively, the thin strip of material may be molded as part of the insert itself during the formation of the insert. In one embodiment, the insert comprises at least two bands having thin strips of a material that covers two separate portions of the insert. FIG. 6 illustrates a two-dimensional side view of an insert 640 in its extended form having bands 642a and 6426 near the top and bottom edges of the exterior side 675 of the insert 640.

[0080] In another aspect, the element may be at least one thin strip of material positioned on only the exterior side of the insert, or on only the interior side of the insert. In such embodiments, one or more thin strips of material may be positioned horizontally, vertically, or at some angled or patterned position on the insert. Further, the thin strip of material may be continuous or disjointed across the length or height of the insert. The thin strip of material may be the same as the insert material or the housing material, or it may be some other material that is non-toxic to cells known to those of ordinary skill in the art. FIG. 7 illustrates an example of a schematic of a bioreactor vessel 700 having a housing 710, a cap 730 covering a necked access port (not shown), and an insert 740 inside the cell culture chamber of the housing. The insert 740 has thin strips of material (744a, 7446) on the exterior side 775 of the insert. The insert 740 is rolled to conform to the shape of the sidewalls of the housing 710.

[0081] In another aspect, the element may be at least one gasket or o-ring positioned on the exterior side of the insert. In such embodiments, the gasket or o-ring may be removable or may be permanently affixed to the exterior side of the insert. In embodiments where more than one gasket or o-ring is used, the gaskets or o-rings may be all removable or may all be permanently affixed or may be a combination of removable and permanently affixed. The placement of the gasket or o-ring along the exterior surface of the insert is not limited. In some embodiments, the gasket(s) or o-ring(s) may be towards or at the center of the height of the exterior surface of the insert. In other embodiments, the gasket(s) or o-ring(s) may be towards one or both ends of the height of the exterior surface of the insert. In yet other embodiments, the gasket(s) or o-ring(s) may be between the center and ends of the height of the exterior surface of the insert. The gasket(s) or o-ring(s) contact the interior surface of the housing, which helps minimize sliding of the insert towards the ends of the housing.

[0082] FIG. 16 illustrates an exemplary schematic of a bioreactor vessel 1600 having a housing 1610, a cap 1630 that covers a necked access port (not shown), and an insert 1640 inside the cell culture chamber of the housing. The insert 1640 has o-rings 1622 and 1623 near the ends of the insert 1640, on the exterior surface of the insert 1640.

[0083] In yet another aspect, the element may physically attach certain small portions of the exterior side of the insert to the interior side of the housing. The physical attachment may come directly from the exterior side of the insert or the interior surface of the housing using a targeted melting of the insert and / or housing to each other. Alternatively, the physical attachment may come from using an adhesive that secures the exterior surface of the insert to the interior surface of the housing. Suitable adhesives that are biocompatible and non-cytotoxic are known to those of ordinary skill in the art, and include UV curable, silicone and epoxy based adhesives, for example Master Bond LED405Med, Master Bond EP62-1HT Med, Master Sil 912Med. There may be one or more physical attachment locations to secure the insert to the housing. In one embodiment, the protrusions are formed from the exterior surface of the insert itself. The protrusions may be formed using melting of the exterior surface of the insert, in a mold used to make the insert, or through any other means known to those of ordinary skill in the art. The protrusions may then be attached or affixed to locations on the exterior surface of the housing that are at locations complementary to the location of the protrusions when the insert is in the vessel (z.e., at complimentary locations on the housing). Except for the portion attaching to the housing, the remainder of the insert does not contact the housing.

[0084] FIGS. 17-18 depict schematics of a bioreactor vessel having a housing, a cap that covers a necked access port, and an insert inside the cell culture chamber of the housing. In FIG. 17, vessel 1700 contains an insert 1740 and small portions of the exterior surface of insert 1740 at locations 171 la-c / has been melted to attach to the interior surface of housing 1710. In FIG. 18, vessel 1800 contains an insert 1840 and an adhesive at locations 1817a and 18176 secures small portions of the exterior surface of insert 1840 to the interior surface of housing 1810.

[0085] In other aspect, the element may be one or more clips that attach to the top and / or bottom sides of the insert that are then attached or affixed to the interior side of the housing. The clip(s) have an exterior-facing side that is outwardly facing. The exterior-facing side of the clip can be attached or affixed to the interior side of the housing by any means known to those of ordinary skill in the art, including magnets, epoxy, adhesives, melting to affix, and welding. The clip(s) may be on a top side of the insert, a bottom side of the insert, or both. FIG. 19 illustrates an exemplary schematic of a bioreactor vessel 1900 with a housing and an insert 1940 inside the cell culture chamber of the housing. The insert 1940 has a top side 1915 and a bottom side 1916, and clips 1922a and 19226 that attach to the top side 1915 of the insert 1940. The clips 1922a and 19226 are affixed to the interior side of the housing to secure the insert 1940 in place.

[0086] In another aspect, the element may be one or more protrusions on the top and bottom sides of the insert, where the protrusions extend into the ends of the housing and act to prevent the main body of the insert from shifting deeply into the ends to the of the housing. The main body of the insert may shift slightly into the ends of the housing, which is acceptable so long as no visible bubbles are being formed when the vessel is in use. The protrusions may be affixed to the main body of the insert, or they may extend from the main body of the insert. The protrusions may be any shape that keeps the main body of the insert from shifting into the ends of the housing such that visible bubbles are formed during use of the vessel. The protrusions may be the same material as the insert or may be a different material.

[0087] FIG. 20 illustrates an exemplary schematic of a bioreactor vessel 2000 having a housing and an insert 2040 inside the cell culture chamber of the housing. The insert 2040 has a top side 2015 and a bottom side 2016, protrusions 203 la and 20316 that extend from the top side 2015 of insert 2040, and protrusions 2031c and 203 Id that extend from the bottom side

[0088] When the elements preventing the exterior side of the insert from contacting the interior surface of the housing are attached to the insert, it may further be advantageous for the insert to also attach to itself to aid in holding the insert in a configured form, such as a rolled form, while in the cell culture chamber of the housing. In one embodiment, the insert may attach to itself at the two ends along the length of the insert (e.g., the first end 60 and second end 65 in FIG. 2C), creating a sealed joint. This type of attachment can be used when the insert is configured with a single layer. As illustrated in FIGS. 8A-8B, an insert 840 having two ends (a first end and a second end) with exterior surface 875 may start unjoined (FIG. 8A), and then the first end and second end may be joined to create sealed joint 863 (FIG. 8B). The two ends may be attached by bonding, welding, melding, or melting the two ends together, for either part or all of the joint between the two ends. Alternatively, the two ends may be attached with a grooved or notched band that secures both ends to the grooves of the band.

[0089] In other embodiments, the two ends of the insert may be attached by snaps, magnets, interlocking tabs (e.g., FIG. 9A-9B), hooks, or other fastener types that are on the insert, configured at or near the first and second ends of the insert. Regarding tabs, FIG. 9A illustrates an example of a tab structure that may be used. In FIG. 9A, the exterior side 975 of a mesh insert 940 having tabs 962 extending outwardly from a first side of the insert and receptacles 964 for the tabs near the second end of the insert. In this specific embodiment, the tabs 962 are rectangular in shape and the receptacles 964 are double slotted such that, when joined, each rectangular tab weaves through the corresponding two slots to secure the ends of the insert together. In FIG. 9B, the exterior side 975 of a mesh insert 940 having tabs 967 extending outwardly from a first side of the insert and receptacles 969 for the tabs near the second end of the insert. In this specific embodiment, the tabs 967 are in a T-shape and the receptacles 969 are single slots such that, when joined, each T-shape tab attaches through the corresponding single slot, the two ends of the insert are secured together.

[0090] In yet another embodiment, the insert may attach to itself at one or more locations around the insert, including at locations on the insert other than the two ends. The insert may attach at these locations in the same manner as described above (e.g., by a sealed joint or with magnets, snaps, interlocking tabs, hooks, or other fastener types). This type of attachment location is particularly advantageous when the insert is configured with more than one layer. This type of joining location allows the layers to be attached to each other whilemaintaining at least partial separation from each other. In some embodiments, layers are fully separated even with the layers being attached together.

[0091] In another aspect of the present disclosure, the insert does not contain an element to prevent contact between the housing and the insert but rather has the insert configured to minimize the contact between the housing and the insert while simultaneously maximizing the additional surface area for culturing cells in the cell culture chamber provided by the insert. In this aspect, the insert is configured in an accordion shape, which can also be attached to itself in the manners just described for inserts with attached elements to prevent contact between the housing and the insert. An example of an insert with an accordion configuration illustrated from a top view is shown in FIG. 10. The insert 1040 has a first end 1060 and a second end 1065, an interior surface 1070, and an exterior surface 1075. In FIG. 10, the points of the accordion that extend outwardly (e.g., 1087a, 10876) from the exterior surface 1075 of the insert 1040 may contact the interior surface of the housing, while the points of the accordion that extend inwardly toward the center of the cell culture chamber do not contact the interior surface of the housing. In this way, the surface area of the insert that contacts the interior surface of the housing is minimized while leaving the remainder of the surface area open for cell culturing.

[0092] In yet another aspect of the present disclosure, the insert is seated in the bioreactor vessel by at least one element on the housing or a cap that is attached to a necked access port on the housing. Seating the insert in the bioreactor vessel secures the insert inside the vessel so it is does not freely shift around in the vessel during use. Further, in some embodiments, seating the insert in a bioreactor vessel decreases or eliminates air bubbles from forming in the bottle during the cell culturing process. In one embodiment, the seating of an insert in the bioreactor vessel decreases at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or more, of the air bubbles that would form if the insert was not seated in the bioreactor vessel. In another embodiment, a post-cultured bio reactor vessel with an insert seated on a housing or a cap of the bioreactor vessel is free of or substantially free of air bubbles. As used herein, “substantially free of air bubbles” means at least 85%, at least 87%, at least 90%, at least 95%, at least 97%, or more, of the cell culture chamber is free of air bubbles.

[0093] In some embodiments, the element that seats the insert comprises a plurality of notches on the housing. In one specific embodiment, the notch may be a ridge (a notch havinga narrowed top portion of the notch). The notches may seat a single layer of insert, or multiple layers of insert. The notches may be created as part of the interior surface itself, formed as part of the housing during the formation of the housing. Alternatively, the notches may be separately formed and attached to the interior surface of the housing. The attachment may be by bonding, welding, thermal melting, or other technique known to those of ordinary skill in the art. In one specific embodiment, the notches extend inwardly from the bottom interior surface of the housing. In another specific embodiment, the notches extend inwardly from a top interior surface of the housing. In yet another specific embodiment, notches extend from both the top and bottom interior surfaces of the housing towards the center point of the housing.

[0094] In some embodiments, the element that seats the insert comprises a plurality of bumps on the housing. The bumps may seat a single layer of insert, or multiple layers of insert. The bumps may be created as part of the interior surface itself, formed as part of the housing during the formation of the housing. Alternatively, the bumps may be separately formed and attached to the interior surface of the housing. The attachment may be by bonding, welding, thermal melting, or other technique known to those of ordinary skill in the art. In one specific embodiment, the bumps extend inwardly from the bottom interior surface of the housing. In another specific embodiment, the bumps extend inwardly from a top interior surface of the housing. In yet another specific embodiment, bumps extend from both the top and bottom interior surfaces of the housing towards the center point of the housing.

[0095] In one embodiment, the element that seats the insert comprises one or more protrusions on the interior side of the housing. The protrusions extend between 1 mm and 10 mm, or between 1mm and 5mm, from the interior surface of the housing into the interior of the vessel. The protrusions are located above and below where the insert is desired to sit in the vessel to secure the insert in place. The protrusions may be any shape and have any number that restrains the insert in the main body of the housing. The protrusions may be part of the mold of the housing, or the protrusions may be formed separately and then permanently or removably attached to the interior surface of the housing. The protrusions may be in the main body (cylindrical body) of the housing, or they may be in the top or bottom endcaps of the housing.

[0096] FIG. 21 illustrates a side view of an exemplary schematic of a bioreactor vessel 2100 with a housing 2110 and an insert 2140 inside the cell culture chamber of the housing. The interior surface of the housing 2110 has a main body 2105, a top endcap 2103, a bottomendcap 2107, protrusions 2124a and 21246 that extend from the bottom of the top endcap 2103 of the interior surface of the housing 2110, and protrusions 2124c and 2124 that extend from the top of the bottom endcap 2107 ofthe interior surface of the housing 2110. Insert 2140 sits between the protrusions.

[0097] In some other embodiments, the element that seats the insert comprises at least one ring on the housing. The rings may seat a single layer of insert, or multiple rings may seat multiple layers of insert. In one embodiment, the ring is a series of connected notches extended around the bottom interior surface of the housing, the top interior surface of the housing, or both the top and bottom interior surfaces of the housing. In another embodiment, the ring is shaped as a continuous notch that extends around the top interior surface of the housing, the bottom interior surface of the housing, or the top and bottom surfaces of the housing. Like notches, the rings may be part of the interior surface of the housing itself or may be formed separately and then attached to the interior surface of the housing. Also like the notches, rings may extend inwardly from the bottom interior surface of the housing, from the top interior surface of the housing, or from both the top and bottom interior surfaces of the housing. Alternatively, the rings may extend outwardly from the bottom interior surface of the housing, from the top interior surface of the housing, or from both the top and bottom interior surfaces of the housing. In one specific embodiment, the ring may be a groove.

[0098] In other embodiments, the element that seats the insert comprises a flexible or foldable support structure comprising a shaft and least two extendable arms connected to the shaft. The extendable arms may attach to the insert through hooks, magnets, bonding, welding, melding, or other attachment mechanisms known to those of ordinary skill in the art. However, in some embodiments the extendable arms do not attach to the insert and instead use tension against the insert. In embodiments having the extendible arms attaching to the insert, the attachment between the extendable arms and the insert may be a reversible attachment or an irreversible attachment. When the support structure is a flexible support structure, the extendible arms may extend continuously from the shaft. Further, the extendible arms may fold against the shaft when pressure is exerted on the extendible arms in the direction of the shaft and then extend outwards automatically when the pressure is released. When the support structure is a foldable support structure, the extendible arms may reversibly or irreversibly attach to the shaft. Further, the extendible arms may fold against the shaft when pressure is exerted on the extendible arms in the direction of the shaft and may either automatically extendoutwards when the pressure is released or may instead be pulled on to extend outwards when the pressure is released. With either the flexible support structure or the foldable support structure, the shaft may be attached to a cap of the bioreactor vessel. When attached, the shaft is in a fixed position. When the shaft attaches to the cap, it may be attached reversibly or irreversibly. One example of a reversible attachment of the shaft to the cap includes a threaded receptacle on the cap and threads matching the receptacle threads on shaft, or vice versa. Another example of a reversible attachment of the shaft to the cap includes a pin and a receptacle for a pin. However, other reversible fasteners known to those of ordinary skill in the art may be used. Alternatively, the attachment of the cap to the shaft may be irreversible (permanent), which may include bonding, welding, melting, or other techniques for permanent attachment known to those of ordinary skill in the art. In any of these embodiments having a support structure, the support structure may rest on or attach to an end support on the bottom end of the housing. The end support assists in keeping the shaft on a vertical line in the bioreactor vessel. The end support may be a raised portion of the bottom of the housing or an indented portion of the bottom of the housing. It may be integral with the housing or separate from the housing and attached to the housing during formation of the bioreactor vessel.

[0099] Referring now to FIG. 11, a side view of a schematic depicting an embodiment of a bioreactor vessel having a foldable support structure is shown. Bioreactor vessel 1100 comprises a housing 1110, a cap 1130 to prevent contents in the bioreactor vessel 1100 from escaping, an insert 1140, an end support 1116, and a flexible support structure. The flexible support structure comprises a shaft 1112 and extendible arms 1114a and 11146 that extend from the shaft 1112 out to the insert 1140. The end support 1116 is a raised portion on the bottom of the housing 1110 and assists in keeping the bottom end of the foldable support structure in a vertical position. The shaft 1112 attaches to the inside of the cap 1130.

[0100] In some embodiments, the element that seats the insert is on a cap of the bioreactor vessel. In one embodiment, the element on the cap is plurality of notches, bumps, or rings that seats the insert. The notches, bumps, or rings may be of the type described above for the housing. In another embodiment, the element on the cap may be a physical attachment of the cap to the insert. The attachment may be reversible or irreversible, and may include hooks, magnets, threads, bonding, welding, melting, or other attachment mechanisms known to those of ordinary skill in the art. Further, in some embodiments, the size of the interior diameter ofthe cap may be at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or more, of the size of the interior diameter of the housing.

[0101] In one particular embodiment, the element that seats the insert comprises the sidewalls of the housing vessel itself. In this embodiment, the insert is a rolled insert of at least one layer. The outermost layer of the rolled insert has a diameter. The interior surface of the housing also has a diameter. The diameter of the outermost layer is between about 0.25% and about 10% greater than diameter of the interior surface of the housing . Despite the insert having a larger diameter than the housing, the insert still fits inside of the housing, maintaining its rolled shape. The tension created when the insert is put into the housing prevents the insert from sliding relative to the interior surface of the housing.

[0102] In some aspects of the present disclosure, two or more of the aforementioned aspects for seating the insert in the housing may be combined together. For example, in some embodiments, a housing may seat an insert by having notches on one end of the housing and rings on the other end of the housing. FIG. 12 illustrates one such embodiment. FIG. 12 illustrates a cross-sectional schematic from a side view of a bioreactor vessel 1200 comprising a housing 1210, with a necked port (not shown), insert 1240, and cap 1230 over the necked port. The insert 1240 is seated on the housing 1210 with notches 1246a and 12466 that extend inwardly from the top end of the housing 1210 and with an inwardly extending notch 1216 that goes continuously around a circumference on the bottom end of the housing 1210. The insert is seated between the sidewalls of the housing 1210 and either the discrete notches 1246a and 12466 on the top end of the housing 1210 and the continuous notch 1216 that forms a ring on the bottom end of the housing 1210. As another example, in some embodiments a housing may seat an insert by having bumps on one end of the housing and the insert reversibly attaching to itself with snaps. These are merely exemplary embodiments, any of the elements and other ways of seating the insert described in the aspects of the paragraphs above may be combined together.

[0103] In some aspects of the present disclosure, an insert is formed and put into a partially formed housing before the housing is secured together. In other aspects of the present disclosure, a housing is formed and then an insert is added into the housing and secured. FIG. 13 illustrates an example of an insert being put into a partially formed housing, followed by securing the housing together. In FIG. 13, bioreactor vessel 1300 is formed by providing a housing base 1310a comprising a outwardly extending notch 1344 that goes around the interiorsurface of the bottom of the housing, providing a housing top 13106 that has inwardly facing notches 1346 and a necked port 1335, providing an insert 1340 that increases the surface area for culturing cells in the bioreactor vessel 1300, then inserting the insert 1340 into the housing base, and then attaching the housing top 13106 to the housing base 1310a to form the full housing 1310 with insert 1340 fully enclosed by the housing.

[0104] Bioreactor vessels with inserts as described above may be cultured with adherent cells of a cell type. Examples of cell types that may be cultured in the bioreactor vessels include chicken embryo fibroblasts, HEK-293, HEK-293T, Vero, HeLa, CHO, PER.C6, among others known to those of ordinary skill in the art. However, it should be understood these are merely examples, any cell type known to those of ordinary skill in the art may be cultured with the bioreactor vessels. The exact cell densities that may be achieved depend at least in part on the cell line cultured and the volume of the media used for the cell culture, as well as the seeding density, duration of culture, and nutrient concentrations. In one embodiment, the surface area utilization of cells adhering to the surface of the interior surface of the housing and the insert may be from about 1 x 103cells / cm2to about 1 x 107cells / cm2, or any value in between. In one embodiment, the surface area utilization may be from about 1 x 104cells / cm2to about 1 x 106cells / cm2, or any value in between. In one embodiment, the surface area utilization may be at least 1 x 103cells / cm2, 1 x 104cells / cm2, 1 x 105cells / cm2, 1 x 106cells / cm2, or greater. Further yet, in some embodiments, the actual yield of cells grown in a vessel is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the theoretical yield of cells able to be grown on the surface areas of the vessel and the insert are achieved. Theoretical yield refers to the maximum yield of cells that could attach to the interior surface of the housing and the surface of the insert.D. ExamplesExample 1

[0105] The effect of mesh insert height on cell cultures was investigated. A first polyethylene terephthalate (“PET”) mesh insert was put into a standard Coming® 850 cm2polystyrene roller bottle having a tissue culture treated interior surface. The height of the insert was the maximum the mesh could sit flush in roller bottle. The PET mesh insert was a single layer of mesh, 23.25 cm in height. It was rolled and inserted into the access port. The mesh wasnot sealed and was not attached to the bottle. The PET mesh was 23.25 cm x 35.25 cm. The roller bottle had an inwardly flared top end and a flat bottom end.

[0106] A second PET mesh insert that was 2 cm shorter in height than the first PET mesh insert (but having the same characteristics otherwise) was put inside a second roller bottle (Coming® 850 cm2polystyrene roller bottle) in a similar way to the first. The second roller bottle had the same characteristics as the first roller bottle. The shorter mesh was not attached to the bottle. After inserting the mesh, the bottle was manually tapped until the mesh was located within the cylinder region (not within the end portions).

[0107] Cell culture media was added to the bottles. The bottles were placed on a roller bottle apparatus that was located in a cell culture incubator (37°C, 5% CO2, humidified). The bottles were rotated at 0.33-4 rpm for 1 hour to equilibrate the media. The bottles were then each inoculated with 10,000 - 20,000 cells / cm2. The bottles were then incubated on the roller bottle apparatus at 0.33-4 rpm for 5 days. After 5 days, the cells from the bottle were either harvested for quantitation or stained with crystal violet to evaluate uniformity. In the bottle with the first mesh insert (the taller one), bubbles appeared when the roller bottle had liquid added, was placed on its side in the roller apparatus, and when rotated.

[0108] Once the cultures had finished culturing, both meshes were stained with Crystal violet staining (Sigma Aldrich #65092A-95) and then removed from the roller bottles and observed. FIGS. 14A-14B show the results of the staining. FIG. 14A shows the staining observed for the first PET mesh insert with the maximum height the mesh would sit flush in the roller bottle while FIG. 14B shows the staining observed for the second PET mesh insert that had a shorter height. The dark areas on the photographs are the stained area and represent the areas where cells attached to the mesh. The staining on the second mesh having shorter height (FIG. 14B) was more uniform and had more coverage than the staining of the first mesh (FIG. 14A). Uniformity was determined by visual observation of the mesh after staining. With the first mesh (maximum height mesh), non-uniform growth was observed on the top and bottom portions of the mesh with more uniform growth along the middle portion of the mesh, as can be seen in FIG. 14A. With the second mesh (shorter height mesh), more uniform growth was observed throughout the mesh. The cell yield and cell viability data with each insert is summarized in Table 1 below.Table 1.It was unexpected that decreasing the size of the mesh would result in higher uniformity and higher viable cell yields.Example 2

[0109] The effect of different high-surface density surfaces on cell cultures was investigated. Viable cell yields with a standard Coming® 850 cm2polystyrene smooth-walled roller bottle (Coming® Prod. No. #430849) was used as a control, and was compared to (1) a 1700 cm2expanded surface roller bottle having pleats on the interior surface of the housing to increase the surface area (Coming® Prod. No. #431191), (2) an 850 cm2smooth-walled roller bottle (Coming® Prod. No. #430849) with a single layer PET mesh insert having 2627 cm2total surface area on the mesh, and (3) an 850 cm2smooth-walled roller bottle (Coming® Prod. No. #430849) with a two-layer PET mesh insert having 4403 cm2total surface area on the mesh. The PET mesh inserts were the same height as the shorter mesh used in Example 1. Mesh inserts were manually rolled and inserted into the port of the bottle. The mesh was manually localized to the cylinder region of the bottle. The bottle with 2-layer mesh was concentric and was not separated from the wall of the bottle. The mesh was rolled in a direction counter to the direction of rolling during the cell culture process. All bottles were rotated in the counterclockwise direction during the culture process.

[0110] Cell culture media was added to the bottles. The bottles were placed on a roller bottle apparatus located in a cell culture incubator (37°C, 5% CO2, humidified). The bottles were rotated at 0.33 rpm for 1 hour to equilibrate the media. The bottles were then inoculated at 10,000 cells / cm2. The bottles were then incubated on the roller bottle apparatus at 0.33 rpm for 4 days. After 4 days, the cells from the bottles were harvested for quantitation. Cells were quantified with an automated cell counter (Beckman Coulter Vi-CELL). Cell yield was determined by multiplying the total harvest volume by the cell concentration (determined by the cell counter). Each experiment was mn twice. The results demonstrated that the bottles having the mesh inserts provided higher viable cell yields than the high-density roller bottle having the expanded surface area on the housing of the bottle itself in the form of pleats, andeven higher yields compared to the standard smooth-walled roller bottle without an insert. Further, the two-layer mesh provided significantly higher viable cell yields over the others tested. The results are shown in FIG. 15 and demonstrate that incorporating the two-layer PET mesh insert provided about 4.4 times the number of viable cells than the standard roller bottle, incorporating the one-layer PET mesh insert provided about 3.0 times the number of viable cells than the standard roller bottle, and the pleated roller bottle provided about 1.8 times the number of viable cells than the standard roller bottle.

[0111] While the present disclosure includes a limited number of embodiments, those skilled in the art, having benefit of this disclosure, will appreciate that other embodiments can be devised which do not depart from the scope of the present disclosure.

[0112] All reference, patents, and / or patent applications cited herein are incorporated by reference in their entireties to the extent permitted by law.

Claims

CLAIMSWhat Is Claimed Is:

1. A bioreactor vessel, comprising: a housing comprising an exterior surface, an interior surface, a top end and a bottom end; a cell culture chamber inside the housing; an insert comprising a top edge, a bottom edge, a first end, a second end, a length, an external side, and an internal side; and an element on the insert that prevents the external side of the insert from contacting the interior surface of the housing; wherein the insert is configured for adherent cells to attach; and wherein the insert is in the cell culture chamber.

2. The bioreactor vessel of claim 1, wherein the element comprises a band and the band runs along the length of the insert, wherein the insert has a thickness and the band has thickness, and wherein the thickness of the band is greater than the thickness of the insert.

3. The bioreactor vessel of claim 2, wherein the element comprises two or more bands.

4. The bioreactor vessel of 2, wherein the band has a thickness from about 1.5 mm to about 71 mm.

5. The bioreactor vessel of claim 2, wherein the band comprises a first end and a second end, and the first end and second end are configured to attach together.

6. The bioreactor vessel of claim 5, wherein the first and second ends attach with a configuration selected from the group consisting of: a snap, magnets, interlocking tabs, hooks, a sealed joint, and a combination thereof.

7. The bioreactor vessel of claim 1, wherein the insert further comprises a modification for cell attachment.

8. The bioreactor vessel of claim 1, wherein the insert is a mesh or is a polymer film with a pattern of pores in the film.

9. The bioreactor vessel of claim 8, wherein the mesh comprises polyethylene terephthalate and the polymer film comprises polystyrene.

10. The bioreactor vessel of claim 1, wherein the element comprises one or more gaskets or o-rings around the external side of the insert.

11. The bioreactor vessel of claim 1, wherein the element comprises one or more clips on a top surface of the insert, on a bottom surface of the insert, or a combination thereof.

12. The bioreactor vessel of claim 1, wherein the element comprises one or more protrusions from the top edge of the insert, the bottom edge of the insert, or a combination thereof, and wherein the protrusions extend into the top end of the housing, the bottom end of the housing, or a combination thereof.

13. The bioreactor vessel of claim 1, wherein the element is an adhesive between the exterior side of the insert and the interior side of the housing.

14. The bioreactor vessel of claim 1 , wherein the element is formed from one or more locations on the exterior side of the insert and protrudes towards the interior side of the housing.

15. A bioreactor vessel, comprising: a housing comprising an exterior surface and an interior surface; a cell culture chamber inside the housing; an insert in the cell culture chamber comprising mesh, or a film with a pattern of pores; a necked access port on the housing; and a cap that attaches to the necked access port; wherein the insert is seated by at least one element on the housing or the cap; and wherein the insert is configured for adherent cells to attach.

16. The bioreactor vessel of claim 15, wherein the at least one element comprises a plurality of notches on the housing that extend inwardly from a bottom interior surface of the housing or from a top interior surface of the housing, or both from the top and bottom interior surfaces of the housing.

17. The bioreactor of claim 15, wherein the at least one element comprises a ring that extends inwardly from a bottom interior surface of the housing or from a top interior surface of the housing, or both from the top and bottom interior surfaces of the housing.

18. The bioreactor vessel of claim 15, wherein the at least one element comprises a flexible or foldable support structure comprising a shaft and at least two extendable arms connected to the shaft, and the support structure in its extended form seats the insert, and wherein the shaft attaches to the cap.

19. The bioreactor vessel of claim 15, wherein the at least one element comprises at least one outwardly extending notch from a bottom interior surface of the housing that extends around the bottom surface of the housing.

20. The bioreactor vessel of claim 19, wherein the at least one element further comprises at least one inwardly extending notch from a top interior surface of the housing that extends around the top surface of the housing.

21. The bioreactor vessel of claim 19, wherein the at least one element further comprises at least one outwardly extending notch from a top interior surface of the housing that extends around the top surface of the housing.

22. The bioreactor vessel of claim 15, wherein the at least one element comprises a plurality of bumps extending inwardly from the interior surface of the housing.

23. The bioreactor vessel of claim 15, wherein the at least one element comprises grooves or ridges on a bottom interior surface of the housing.

24. The bioreactor vessel of claim 15, wherein the at least one element is on the cap of the bioreactor.

25. The bioreactor vessel of claim 24, wherein an interior diameter of the cap is at least 40% of an interior diameter of the housing.

26. The bioreactor vessel of claim 15, wherein the at least one element comprises sidewalls of the housing, and wherein the insert comprises an outermost rolled insert layer comprising a diameter that is between 0.25% and 10% greater than an interior surface diameter of the housing.

27. The bioreactor vessel of claim 15, wherein the at least one element comprises protrusions from the interior side of the housing that seat the insert in a main body of the housing, and wherein the protrusions extend between 1 mm and 10 mm from the interior surface of the housing.

28. A bioreactor vessel, comprising: a housing comprising an exterior surface and an interior surface; a cell culture chamber inside the housing; and an insert comprising a top edge, a bottom edge, a length, an external side, and an internal side; wherein the insert is configured for adherent cells to attach; and wherein the insert is configured in an accordion shape along the top edge and bottom edge of the insert.

29. A bioreactor vessel, comprising: a housing comprising an exterior surface and an interior surface; a cell culture chamber inside the housing; and an insert in the cell culture chamber comprising a first height, an external side, and an internal side; wherein the insert is configured for adherent cells to attach; wherein the cell culture chamber comprises a second height, which is the height that the insert in the cell culture chamber can lie flush to the interior surface of the housing; and wherein the first height is less than or equal to the second height.

30. The bioreactor vessel of claim 29, wherein the first height is between 75% to 99% of the second height.

31. The bioreactor vessel of claim 29, wherein the cell culture chamber comprises a circumference and the length of the insert is between 1 and 3 times the circumference of the cell culture chamber.

32. The bioreactor vessel of claim 29, wherein the insert is configured to adhere 1 x 104cells / cm2to about 1 x 106cells / cm2.

33. A method of culturing cells, comprising the steps of: providing the bioreactor of any of claims 1-32; providing cells of a cell type; culturing the cells in the bioreactor; wherein at least 90% of the cells are viable cells after culturing, as measured by a trypan blue exclusion assay.

34. The method of claim 33, wherein the number of cells per unit surface area cultured are from about 1 x 104cells / cm2to about 1 x 106cells / cm2.

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