Cell culture articles having textured surfaces and uses thereof in the culture of islet cells and beta cells

Abstract

This disclosure relates generally to cell culture articles (e.g., in the form of bags) having textured surfaces for growth of cells, e.g., in the form of organoids or spheroids. In one aspect, the disclosure provides a cell culture article having a first polymer film as a first boundary of a cell culture internal volume, the first polymer film having a plurality of pyramidal voids extending from a pyramid base at the inner surface or the outer surface of the first polymer film, to a pyramid tip, each of the pyramidal voids having a first base lateral dimension in the range of 150-2500 microns, a first tip lateral dimension up to 100 microns, and a depth in the range of 40-1000 microns, wherein the plurality of pyramidal voids forms a plurality of cell culture wells at the inner surface of the first polymer film.

Description

CELL CULTURE ARTICLES HAVING TEXTURED SURFACES AND USES THEREOF IN THE CULTURE OF ISLET CELLS AND BETA CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application no. 63 / 687,144, which is hereby incorporated herein by reference in its entirety.BACKGROUND OF THE DISCLOSUREField of the Disclosure

[0002] This disclosure relates generally to cell culture articles (e.g., in the form of bags) having textured surfaces for growth of cells, e.g., in the form of organoids or spheroids. More particularly, the present disclosure relates to cell culture articles having a textured surface, and to methods for culturing biological agents like islet cells and beta cells using such containers and substrates.Technical Background

[0003] Cell culture and cell isolation are important processes in a number of applications. For example, various cells for use in therapeutic applications (e.g., immunotherapy, regenerative medicine, etc.) are typically isolated and cultured in vitro. For example, cells such as progenitor cells and mesenchymal stem cells, and monocytes and other immune cells are present in blood in relatively low concentrations, and accordingly are typically isolated from blood and cultured in vitro. Similarly, neuronal cells, cardiomyocytes, epithelial cells, and other cells for regenerative medicine (e.g., bone repair, skin repair, pancreatic islets regeneration, etc.) can be cultured in vitro.

[0004] Polymer bags are commonly used for cell cultures. Such bags are typically inexpensive, disposable, portable and easy to use. However, many polymer surfaces are poor-adhesion substrates for anchorage-dependent cells, which require an environment comparable to their natural cell niche to survive. Moreover, differentiation of various stem cells can be dependent on the cell’s microenvironment, including the adhesion substrate.

[0005] Islet cells, also known as pancreatic islets or islets of Langerhans, are clusters of cells in the pancreas that produce hormones that regulate bodily functions. These functions include blood sugar levels and stomach acid production. It can be desirable to culture islet cells for use in diabetes therapy; this is often done via differentiation from stem cells. But culture of islet cells provides a number of problems. Islet cells are best cultured under conditions in which they can adhere to a surface, which can prove problematic. And islet cells have requirements for a high degree of gas exchange (e.g., oxygen and carbondioxide). This can be difficult to provide in a sterile environment. Pancreatic beta cells suffer from similar issues.

[0006] Accordingly, there remains a need for cell culture articles, especially for the culture of islet cells, beta cells and organoids.SUMMARY OF THE DISCLOSURE

[0007] In one aspect, a cell culture article having a cell culture volume, the cell culture article having a first polymer film as a first boundary of the cell culture internal volume, the first polymer film having an inner surface facing the cell culture volume and an outer surface facing away from the cell culture internal volume, the first polymer film having a plurality of pyramidal voids extending from a pyramid base at the inner surface or the outer surface of the first polymer film, to a pyramid tip, each of the pyramidal voids having a first base lateral dimension in the range of 150-2500 microns, a first tip lateral dimension up to 100 microns, and a depth in the range of 40-1000 microns, wherein the plurality of pyramidal voids forms a plurality of cell culture wells at the inner surface of the first polymer film.

[0008] In various embodiments, the pyramid base of each pyramidal void is at the outer surface of the first polymer film, with the tip of each pyramidal void extending toward the cell culture volume, the article further comprising a plurality of humps at the inner surface of the first polymer film in registration with the pyramidal voids, the humps defining a plurality cell culture wells formed from of inter-hump spaces at the inner surface of the first polymer film.

[0009] In other embodiments, the pyramid base of each pyramidal void is at the inner surface of the first polymer film, with the tip of each pyramidal void extending away from cell culture volume of the container, wherein the pyramidal voids themselves form cell culture wells.

[0010] In another aspect, the disclosure provides a method for cultivating a plurality of cells, comprising incubating the plurality of cells in an cell culture medium in cell culture volume of the article as described herein. In some embodiments, the cells undergo an aggregation phase during the incubation.

[0011] Other aspects of the disclosure will be apparent to the person of ordinary skill in the art in view of the disclosure herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 provides a schematic top-down (top) and cross-sectional (bottom) view of a cell culture article in the form of a bag, according to one embodiment of the disclosure.

[0013] FIG. 2 provides a schematic top-down views of cell culture articles in the form of bags, according to various embodiments of the disclosure.

[0014] FIG. 3 provides partial schematic plan view, and FIG. 4 provides a partial schematic cross-sectional view of an article according to one embodiment of the disclosure.

[0015] FIG. 5 provides partial schematic plan view, and FIG. 6 provides a partial schematic cross-sectional view of an article according to another embodiment of the disclosure.

[0016] FIG. 7 provides partial schematic plan view of an article according to another embodiment of the disclosure.

[0017] FIG. 8 is schematic view of stages of culturing pancreatic beta cells from human pluripotent stem cells.

[0018] FIGS. 9, 10 and 11 provide views of MIN6 aggregates as they are cultured in cell culture articles of the disclosure, and FIG. 12 provides graphs of summary data.DETAILED DESCRIPTION

[0019] In various aspects, the disclosure provides a cell culture article (e.g., in the form of a bag) having an internal volume, the container having a first polymer film as a first boundary of the container, the first polymer film having an inner surface facing the internal volume, the first polymer film having a plurality of pyramidal voids extending from a pyramid base at the inner surface of the first polymer film, to a pyramid tip, each of the pyramidal voids having a first base lateral dimension in the range of 150-2500 microns, a first tip lateral dimension up to 100 microns, and a depth in the range of 40-1000 microns.

[0020] The containers of the disclosure can be provided in a number of forms. One especially convenient form is a bag, e.g., formed from one or more films as described herein. The person of ordinary skill in the art will be familiar with bag structures such as those used in cell culture, and will be able to adapt conventional bag structures for use in bags and methods of the disclosure based on the description herein Of course, the person of ordinary skill in the art will appreciate that the containers of the disclosure can be provided in a number of other forms, e.g., flasks, tubes, dishes.

[0021] Accordingly, one aspect of the disclosure is a cell culture article having a cell culture volume. The cell culture articles of the disclosure can take many forms. In various embodiments, the cell culture article is a bag. An embodiment of such a bag is shown in schematic top-down view (top) and cross-sectional view (bottom) in Fig. 1. Bag 100 of Fig. 1 includes a first polymer film 110 having an outer surface 114 and an inner surface 112, and further includes ports 130 and 140, located at opposite ends of the bag for adding orremoving medium (e.g., feed medium or waste medium, respectively) to or from the bag. The person of ordinary skill in the art will appreciate that the number and location of ports of the containers of the disclosure are not particularly limited, and accordingly can be positioned, for example, for convenience of use or manufacture. Bag 100 can be the product of bonding two polymeric films (e.g., a first polymer film as described herein and another film) together at their edges (e.g., by laser welding, corona discharge, radiation, heat or melt lamination, etching, plasma treatment, wetting, adhesives, or combinations thereof) to form cell culture volume 120. As an alternative, a single film can be folded over and sealed at the folded-over edges to form cell culture volume 120. In either event, Ports 130 and 140 can be sealable to provide a sealed compartment for bounding the cell culture volume 120.

[0022] The person of ordinary skill in the art is familiar with the design of cell culture containers such as bags, and can apply conventional design factors to provide desirable containers. In various embodiments, the internal volume is in the range of 100 mL to 10 L.

[0023] Fig. 2 shows several exemplary embodiments of configurations for culture bags suitable for use in the bags and methods of the disclosure. Bag 200a has only a single port 230a, providing access to compartment 220a. Bag 200b is in the so-called “serpentine” configuration, in which a longer path length through the system can be provided; ports 230b and 240b are connected by a serpentine path formed by serpentine-shaped compartment 220b formed by appropriate welding of the sheets forming the bag. And bag 200c has a non-rectangular shape, with a corresponding non-rectangular compartment 220c between ports 230c and 240c.

[0024] Of course, a variety of other configurations are possible. For example, in some embodiments, the cell culture article is a cassette including a plurality of first polymer films held between a plurality of frames in a stacked configuration. The person of ordinary skill in the art will identify other suitable configurations.

[0025] As described in more detail below, when the cell culture volume is a substantially closed volume, as is the case for a bag, one or more of the walls defining the cell culture volume (e.g., the first polymer film and / or a second polymer film) can be permeable to gases produced and consumed in a cell culture (e.g., O2, CO2) but impermeable to liquids (e.g., water). This can allow for passive exchange of gases across the container walls with the atmosphere to allow for respiration of cells in the container.

[0026] In desirable embodiments, the cell culture articles of the disclosure are desirably formed such that there is substantially no contamination of a fluid in the cell culture volume. Accordingly, it is desirable for the inner surface of the article to be formed from materials thatwill not leach organics into the fluid. For example, in various embodiments as otherwise described herein, an inner surface (e.g., the inner surface of the first polymer film) of the cell culture volume is formed of a polymer (e.g., a fluoropolymer such as fluorinated ethylene propylene) having a total organic carbon (TOC) in water of less than 0.1 mg / cm2(e.g., less than 0.05 mg / cm2, or less than 0.05 mg / cm2). Such articles are described, e.g., in U.S. Patent Application Publications nos. 2016 / 0178490 and 2016 / 0178491 , each of which is hereby incorporated herein by reference in its entirety; the person of ordinary skill in the art can, based on the description herein, adapt such articles for use in the articles and methods of the present disclosure.

[0027] As used herein, TOC is measured for an article, for example by extraction from an internal surface area of the article (with results reflected as mg / cm2are for the TOC per square centimeter of the internal area). TOC is measured according to US Pharmacopeia (USP) 643 and with equipment that utilizes a high temperature wet oxidation reaction of UV- promoted chemical oxidation (Ultra-Clean Technology Handbook: Volume 1 : Ultra-Pure Water, Ohmi, Tadahiro; CRC Press, 1993, pp. 497-517). Purified water is placed in contact with the polymer for 24 hours at 70° C., for example at a ratio of 3 cm2of article surface area to 1 mL of water. The water is removed from contact with the polymer and tested in a TOC analyzer. A suitable piece of equipment is a TEKMAR DOHRMANN Model Phoenix 8000 TOC analyzer.

[0028] Particular materials that are especially suitable for construction of articles, and especially for the first polymer film thereof, are described in more detail, below.

[0029] As noted above, a cell culture article has a first polymer film as a first boundary of the article, as a first boundary of the cell culture volume. The first polymer film has an inner surface facing the cell culture volume, and an outer surface. Notably, the first polymer film has a plurality of pyramidal voids extending from a pyramid base at the inner surface or the outer surface of the polymer film, to a pyramid tip. Each of the pyramidal voids having a first base lateral dimension in the range of 150-2500 microns, a first tip lateral dimension up to 100 microns, and a depth in the range of 40-1000 microns.

[0030] In some embodiments, the pyramid base of each pyramidal void is at the outer surface of the first polymer film, with the tip of each pyramidal void extending toward the cell culture volume. In such embodiments, the pyramidal voids can show through to the inner surface as humps in registration with the pyramidal voids. The humps can define a plurality of inter-hump spaces to form cell culture wells at the inner surface of the first polymer film.

[0031] FIG. 3 provides a schematic plan view of one example of a plurality of pyramidal voids 350 of a first polymer film, while FIG. 4 provides a cross-sectional view along thediagonal line 351 . In this example, the tip 353 of each of the pyramidal voids 150 extends from the pyramid base 352 at the outer surface 314 of the first polymer film toward the cell culture volume 320.

[0032] Here, while pyramidal voids 350 are formed in the outer surface 314 of the polymer film 310, humps 360 show through to the inner surface 312. These humps will have the same general pattern as the pyramidal voids, and will define inter-hump spaces 163 at the inner surface 312 of the first polymer film 310. The present inventors have determined that these inter-hump spaces 363, and particularly intersections thereof, can form cell culture wells 165 at the inner surface of the first polymer film.

[0033] Such polymer films can be made by a molding process, for example, by embossing or otherwise molding the surface of the polymer film that is to face away from the cell culture volume. The person of ordinary skill in the art can, based on the present disclosure, determine sizes, shapes and arrangement of pyramidal voids to provide desired cell culture wells at the inner surface of the first polymer film. Notably, when the first polymer film is formed of a flexible material, there may be some sagging of the material in use that reduces the spacing of the pyramidal features and thus reduces the sizes of the inter-hump spaces and of the cell culture wells; this sagging can in some embodiments play an important role in definition of the cell culture wells. The person of ordinary skill in the art can determine the extent of such sagging and can adjust the design of the molding tool accordingly,

[0034] In other embodiments, the pyramid base of each pyramidal void is at the inner surface of the first polymer film, with the tip of each pyramidal void extending away from cell culture volume of the container. In such embodiments, the pyramidal voids themselves form cell culture wells. .

[0035] For example, FIG. 5 provides a schematic plan view of one example of a plurality of pyramidal voids 550 of a first polymer film, while FIG. 6 provides a cross-sectional view along the diagonal line 551 . In this example, the tip 553 of each of the pyramidal voids 550 extends from the pyramid base 552 at the inner surface 512 of the first polymer film toward the cell culture volume 520 of the container.

[0036] Here, pyramidal voids 550 are formed in the inner surface 512 of the polymer film 510. These pyramidal voids can form cell culture wells 165 at the inner surface of the first polymer film.

[0037] Such polymer films can be made by molding, for example, by embossing or otherwise molding the surface of the polymer film that is to face toward the cell culture volume. The person of ordinary skill in the art can, based on the present disclosure,determine sizes, shapes and arrangement of pyramidal voids to provide desired cell culture wells at the inner surface of the first polymer film. Notably, when the first polymer film is formed of a flexible material, there may be some sagging of the material in use that reduces the size and spacing of the pyramidal voids. The person of ordinary skill in the art can determine the extent of such sagging and can adjust the design of the molding tool accordingly,

[0038] The pyramidal voids, whether formed at the inner surface or the outer surface of the first polymer film, can have a variety of shapes. For example, in various embodiments, and shown in FIG. 3, each of the plurality of pyramidal voids has a base shape that is rectangular, e.g., square. But the person of ordinary skill in the art will appreciate that other shapes are possible. For example, in various embodiments, each of the plurality of pyramidal voids has a base shape that is polygonal, e.g., triangular, or pentagonal or hexagonal. In other embodiments, each of the plurality of pyramidal voids has a base shape that is round, e.g., circular or elliptical or oval (i.e., for the purposes of this disclosure, a cone is a type of pyramid).

[0039] Another embodiment is shown in plan view in FIG. 7. Here, the pyramidal voids 750 are formed as triangular prisms. In this embodiment, the pyramidal voids are formed in the outer surface of the first polymer film, with humps showing through at the inner surface. This arrangement can provide cell culture wells 765 at the inner surface of the first polymer film.

[0040] Of course, in other embodiments, the triangular pyramidal voids themselves can be the cell culture wells, as described above with respect to FIGS. 4 and 5.

[0041] The present inventors have determined particular size ranges for the pyramidal voids that can provide good performance in the growth of islets and pancreatic beta cells. For example, it is desirable for each of the pyramidal voids to have a first base lateral dimension (555 in FIG. 6) in the range of 150-2500 microns. As used herein, the first base lateral dimension is the maximum dimension of a feature in the plane of the first polymer film. For example, in the square pyramidal voids shown in FIGS. 3-6, the “first base lateral dimension” is the diagonal of the square. In a triangular pyramidal void, the “first base lateral dimension” is generally the longest side of the triangle. In various embodiments, in each of the pyramidal voids the first base lateral dimension is in the range of 150-1750 microns, e.g., 150-1500 microns, or 150-1250 microns. In various embodiments, in each of the pyramidal voids the first base lateral dimension is in the range of 150-1000 microns, e.g., 150-850 microns, or 150-700 microns, or 150-600 microns. In various embodiments, in each of the pyramidal voids the first base lateral dimension is in the range of 300-2000 microns, e.g.,300-1750 microns, or 300-1500 microns, or 150-1250 microns. In various embodiments, in each of the pyramidal voids the first base lateral dimension is in the range of 300-1000 microns, e.g., 300-850 microns, or 150-700 microns, or 300-600 microns. In various embodiments, in each of the pyramidal voids the first base lateral dimension is in the range of 700-2500 microns, e.g., 700-2000 microns, or 700-1750 microns, or 700-1500 microns, or 700-1250 microns. In various embodiments, in each of the pyramidal voids the first base lateral dimension is in the range of 1000-2500 microns, e.g., 100-2000 microns, or 1000- 1750 microns, or 1000-1500 microns, or 1500-2500 microns, or 1500-2000 microns.

[0042] Each of the pyramidal voids also has a second base lateral dimension, defined as the largest lateral dimension at the base of the pyramidal void in a direction perpendicular to the dimension of the first base lateral dimension (e.g., a dimension perpendicular to the page in FIG. 4). By definition, the second base lateral definition can be no longer than the first base lateral dimension. In various desirable embodiments, each of the plurality of pyramidal voids has a second base lateral dimension that is a substantial fraction of the first base lateral dimension. For example, in various embodiments, each of the plurality of pyramidal voids has a second base lateral dimension that is at least 50% of the first base lateral dimension. In various embodiments, each of the plurality of pyramidal voids has a second base lateral dimension that is at least 75% of the first base lateral dimension. In various embodiments, each of the plurality of pyramidal voids has a second base lateral dimension that is at least 90% of the first base lateral dimension.

[0043] The pyramidal voids can vary in area,, i.e., within the boundaries of the first base lateral dimension as described herein. For example, in various embodiments, each of the plurality of pyramidal voids has a base area in the range of 10000-6250000 square microns, e.g., 10000-1000000 square microns. In various embodiments, each of the plurality of pyramidal voids has a base area in the range of 90000-490000 square microns, e.g., 90000- 250000 square microns. In various embodiments, each of the plurality of pyramidal voids has a base area in the range of 90000-4000000 square microns, e.g., 90000-1000000 square microns. In various embodiments, each of the plurality of pyramidal voids has a base area in the range of 90000-490000 square microns, e.g., 90000-250000 square microns. In various embodiments, each of the plurality of pyramidal voids has a base area in the range of 490000-3062500 square microns, e.g., 1000000-3062500 square microns, or 1650000- 3062500 square microns. In various embodiments, each of the plurality of pyramidal voids has a base area in the range of 490000-6250000 square microns, e.g., 1000000-6250000 square microns, or 1650000-6250000 square microns.

[0044] The pyramid tips can come essentially to a point, or can have a mesa at the top thereof, e.g., up to 100 microns in first tip lateral dimension (556 in FIG. 6). For example, invarious embodiments, each of the plurality of pyramidal voids has a first tip lateral dimension up to 70 microns, e.g., up to 50 microns. In various embodiments, each of the plurality of pyramidal voids has a first tip lateral dimension up to 30 microns, e.g., up to 20 microns, or up to 15 microns.

[0045] The depths of the pyramidal voids can also be an important feature. The present inventors have noted that diffusion of gases like oxygen and carbon dioxide through the first polymer film can be an important consideration for cell culture, and that deeply-formed pyramids can provide areas of the inner surface that have a relatively short distance through the first polymer film to the container exterior, and thus a higher rate of transmission of gases like oxygen and carbon dioxide. For example, in systems where the pyramidal voids are formed in the outer surface of the first polymer, as in FIGS. 3 and 4, the spaces between pyramids at the inner surface of the film can have a relatively thin layer of polymer material separating them from the outside, and as such can be good areas for cell clustering and growth. Similarly, as described above with respect to FIGS. 5 and 6, when the pyramidal voids are formed in the inner surface of the first polymer film and themselves provide cell culture wells, the tips of the pyramids can have a relatively thin layer of polymer material separating them from the outside, and as such can be good areas for cell clustering and growth.

[0046] Accordingly, each of the plurality of pyramidal voids has a depth (measured from the pyramidal base at the relevant surface of polymer film to the tip, as indicated by item 557 in FIG. 6) in the range of 40-1000 microns. For example, in various embodiments, each of the plurality of pyramidal voids has a depth in the range of 40-800 microns, e.g., 40-600 microns, or 40-500 microns, or 40-400 microns. In various embodiments, each of the plurality of pyramidal voids has a depth in the range of 40-300 microns, e.g., 40-150 microns, or 40-100 microns. In various embodiments, each of the plurality of pyramidal voids has a depth in the range of 100-1000 microns, e.g., 100-800 microns, or 100-600 microns, or 100-500 microns, or 100-400 microns. In various embodiments, each of the plurality of pyramidal voids has a depth in the range of 100-300 microns, e.g., 100-200 microns. In various embodiments each of the plurality of pyramidal voids has a depth in the range of 250-1000 microns, e.g., 250-800 microns, or 250-600. microns, or 250-500 microns, or 250-400 microns. The person of ordinary skill in the art can select a desired pyramidal void depth, especially in conjunction with the overall thickness of the first polymer film.

[0047] In various embodiments, and as described above with respect to FIGS. 3 and 4, the pyramidal voids are formed in the outer surface of the first polymer film, and humps at the first polymer film in registration with the pyramidal voids define inter-hump spaces thatprovide cell culture wells. Accordingly, in such embodiments it is the spaces between the pyramidal voids that provide sites for cell culture. The person of ordinary skill in the art can provide space the pyramidal voids so as to provide desired inter-hump spacings to provide a desired cell culture well size, to provide right-sized areas for cellular clustering and growth. For example, in various embodiments, each of the pyramidal voids has an average nearest- neighbor base-to-base distance up to 2000 microns, e.g., up to 1500 microns, or up to 1000 microns. The average nearest-neighbor base-to-base distance is the average of the base- to-base distances from the base of the pyramidal void to the bases of its three nearest neighbor pyramidal voids. In various embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance up to 800 microns, e.g., up to 600 microns, or up to 400 microns. In various embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance up to 300 microns, e.g., up to 200 microns, or up to 100 microns. Indeed, even when there is substantially no space between bases of pyramidal features, suitable cell culture wells may be formed at the inner surface. In various embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance of at least 20 microns, e.g., at least 40 microns, or at least 70 microns. In various embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance of at least 100 microns, e.g., at least 300 microns, or at least 500 microns. In various embodiments, each of the pyramidal voids has an average nearest-neighbor base- to-base distance of at least 800 microns, e.g., at least 1000 microns, or at least 1300 microns. For example, in some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance in the range of 20-2000 microns, e.g., 20-1500 microns, or 20-1000 microns, or 20-800 microns, or 20-600 microns, or 20-400 microns, or 20-300 microns, or 20-200 microns, or 20-100 microns. In some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance in the range of 40- 2000 microns, e.g., 40-1500 microns, or 40-1000 microns, or 40-800 microns, or 40-600 microns, or 40-400 microns, or 40-300 microns, or 40-200 microns, or 40-100 microns. In some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to- base distance in the range of 70-2000 microns, e.g., 70-1500 microns, or 70-1000 microns, or 70-800 microns, or 70-600 microns, or 70-400 microns, or 70-300 microns, or 70-200 microns. In some embodiments, each of the pyramidal voids has an average nearest- neighbor base-to-base distance in the range of 100-2000 microns, e.g., 100-1500 microns, or 100-1000 microns, or 100-800 microns, or 100-600 microns, or 100-400 microns, or 100- 300 microns, or 100-200 microns. In some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance in the range of 300-2000 microns, e.g., 300-1500 microns, or 300-1000 microns, or 300-800 microns, or 300-600 microns, or 300- 400 microns. In some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance in the range of 500-2000 microns, e.g., 500-1500 microns, or 500-1000 microns, or 500-800 microns. In some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance in the range of 800-2000 microns, e.g., 800-1500 microns, or 800-1000 microns. In some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance in the range of 1000-2000 microns, e.g., 1000-1800 microns, or 1000-1500 microns. In some embodiments, each of the pyramidal voids has an average nearest-neighbor base-to-base distance in the range of 1300-2000 microns, e.g., 1300-1800 microns, or 1300-1500 microns. The person of ordinary skill in the art can provide a desirable average nearest- neighbor base-to-base distance to provide a desired cell culture well size for cell clustering and growth in spaces between pyramidal voids.

[0048] In embodiments in which the pyramidal voids are formed in the inner surface of the first polymer film as described with respect to FIGS. 5 and 6, the pyramidal voids themselves act as cell culture wells. Accordingly, the spacings of the pyramidal voids do not generally define the sizes of the cell culture wells. The person of ordinary skill in the art will select a suitable spacing, in order to maintain film strength and provide a desired density of cell culture wells at the inner surface. In various embodiments, the spacings are as described in any embodiment in the preceding paragraph. And in many embodiments, the pyramidal voids can have very little, or even substantially no spacing therebetween.

[0049] The pyramidal voids can be arranged in any desirable fashion. It can be convenient to array them in a grid pattern, e.g., with perpendicular rows / columns or otherwise (such as a triangular grid or a hexagonal grid). In embodiments in which the pyramidal voids are formed in the outer surface of the first polymer film, the arrangement of the pyramidal voids will be important to define desirably-sized cell culture wells. In such cases, the intersections of the linear spaces between the pyramidal voids can provide especially suitable positions for cell clustering and growth. But other arrangements of the pyramidal voids, are possible. Even irregular arrangements, at appropriate densities, can provide suitable cell culture wells.

[0050] Here, too, in embodiments in which the pyramidal voids are formed in the inner surface of the first polymer film as described with respect to FIGS. 5 and 6, the pyramidal voids themselves act as cell culture wells. Accordingly, the arrangement of the pyramidal voids do not generally define the sizes of the cell culture wells. The person of ordinary skill in the art will select a suitable arrangement, in order to maintain film strength and provide a desired density of cell culture wells at the inner surface. In various embodiments, the arrangement is as described in any embodiment in the preceding paragraph.

[0051] As noted above, the first polymer film has cell culture wells at the inner surface thereof, whether pyramidal voids are formed in the inner surface (in which case they themselves are the cell culture wells), or alternatively whether pyramidal voids are formed in the outer surface (in which case humps in registration with the pyramidal voids define cell culture wells). The cell culture wells desirably have a longest dimension at half-maximum depth in the range of 100-2000 microns, e.g., in the range of 100-1500 microns, or 100-1000 microns, or 100-500 microns. In various embodiments, cell culture wells have a longest dimension at half-maximum depth in the range of 200-2000 microns, e.g., in the range of 200-1500 microns, or 200-1000 microns, or 200-500 microns. In various embodiments, cell culture wells have a longest dimension at half-maximum depth in the range of 500-2000 microns, e.g., in the range of 500-1500 microns, or 500-1000 microns. In various embodiments, cell culture wells have a longest dimension at half-maximum depth in the range of 1000-2000 microns, e.g., in the range of 1000-1500 microns, or 1000-1000 microns, or 200-500 microns.

[0052] The first polymer film desirably has a maximum thickness that is sufficient to provide sufficient mechanical robustness for ease of handling and processing. The minimum thickness desirably provides locations of relatively thinner first polymer film in the cell culture wells, at which cells can cluster and grow to take advantage of increased gas diffusion. For example, in various embodiments, the first polymer film has a maximum thickness (reference number 516 on FIG. 6) of at least 25 microns, e.g., at least 100 microns, or at least 200 microns, or at least 300 microns. In various embodiments, the first polymer film has a maximum thickness in the range of 25-700 microns, e.g., 25-600 microns, or 25-500 microns, or 100-700 microns, or 100-600 microns, or 100-500 microns. In various embodiments, the first polymer film has a minimum thickness in the cell culture wells (e.g., reference number 368 on FIG. 4, and 568 on FIG. 6) of at least 20 microns, e.g., at least 50 microns, or at least 750 microns. In various embodiments, the first polymer film has a minimum thickness in the cell culture wells in the range of 20-200 microns, e.g., 20-150 microns, or 20-125 microns, or 50-200 microns, or 50-150 microns, or 50-125 microns, or 75-200 microns, or 75-175 microns, or 75-150 microns. In various embodiments, the first polymer film has a ratio of minimum thickness in the cell culture wells to maximum thickness in the range of 15-85%, e.g., in the range of 15-60%, or 15-50%, or 15-35%. In various embodiments, the first polymer film has a ratio of minimum thickness in the cell culture wells to maximum thickness in the range of 25-85%, e.g., in the range of 25-60%, or 25-50%, or 25-35%. In various embodiments, the first polymer film has a ratio of minimum thickness in the cell culture wells to maximum thickness in the range of 35-85%, e.g., in the range of 35- 60%, or 35-50%.

[0053] A variety of materials are suitable for use in the first polymer film. For example, in various embodiments, the first polymer film has a polymer surface that is a fluoropolymer surface disposed at the inner surface thereof. Fluoropolymers can provide very low levels of leachables as described above. The entire first polymer film can be a fluoropolymer film, or, in other embodiments, wherein the first polymer film is a fluoropolymer laminate having a fluoropolymer layer proximal the interior volume of the container, and a layer of another non- fluoropolymer (e.g., a silicone layer) distal the interior volume of the container. In various embodiments as otherwise described herein the outer surface of first polymer film comprises a material other than a fluoropolymer. For example, in various such embodiments, the material at the outer surface of the first polymer film comprises a thermoplastic polymer, a thermoplastic elastomer, a silicone, a rubber, or any combination thereof.

[0054] Various suitable materials are described, e.g., in U.S. Patent Application Publications nos. 2016 / 0177247, 2016 / 0178490 and 2016 / 0178491 , each of which is hereby incorporated herein by reference in its entirety. But materials other than those can be suitable in some embodiments.

[0055] A variety of fluoropolymers can be suitable for use. For example, in various desirable embodiments, the fluoropolymer is fluorinated ethylene propylene. In various embodiments, the fluoropolymer is polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), ethylene tetrafluoroethylene (ETFE), polyvinylidene fluoride (PVDF), polychlorotrifluoroethylene (PCTFE), ethylene chlorotrifluoroethylene (ECTFE), fluorinated ethylene propylene (FEP), ethylene fluorinated ethylene propylene (EFEP), perfluoropolyether (PFPE), modified polytetrafluoroethylene (TFM), polyvinyl fluoride (PVF), or any mixture thereof.

[0056] In various embodiments, the fluoropolymer at the inner surface of the first polymer film has a thickness of at least 5 microns, e.g., at least 10 microns, or at least 25 microns, or at least 50 microns. For example, in various such embodiments, fluoropolymer at the inner surface of the first polymer film has a thickness in the range 5-500 microns, e.g., 5-200 microns, or 5-100 microns, or 10-500 microns, or 10-200 microns, or 10-100 microns, or 50-500 microns, or 50-200 microns.

[0057] In various other embodiments, the first polymer film has a polymer surface that is a silicone surface disposed at the inner surface thereof. The first polymer film can, e.g., be formed substantially of silicone. A variety of silicone materials can be suitable; in many embodiments, the silicone is a polymer or copolymer of dimethylsiloxane, e.g., having at least 75 wt% or at least 90 wt% dimethylsiloxane residues. Silicone materials canconveniently be cast against a molding tool bearing pyramidal features to form the pyramidal voids.

[0058] But other polymers can be useful. For example, in various embodiments, the first polymer film has a polymer surface disposed at the inner surface thereof, which can be poly(ethylene-co-vinyl acetate) or a polyolefin, e.g., polyethylene or polypropylene.

[0059] The person of ordinary skill in the art can select suitable materials based on the present disclosure.

[0060] In various embodiments, the polymer surface is not a polystyrene surface. Such surfaces are typically rigid and difficult to process, and incompatible with the bag and cassette systems that are highly desirable in cell culture.

[0061] The surfaces described herein can be made by a variety of methods familiar to the person of ordinary skill in the art. For example, embossing or otherwise molding a polymer film can be used to provide the pyramidal voids.

[0062] Especially when the first polymer film has a polymer surface that is a fluoropolymer surface or a silicone surface disposed at the inner surface thereof, it can be desirable to provide a plurality of functional groups at the inner surface of the first polymer film, for example, to provide a surface that is more conducive to cell adhesion. For example, the fluoropolymer of the inner surface may be functionalized with a carboxyl group, hydroxyl group, aldehyde group, carbonyl group, amine group, imine group, amide group, ester group, anhydride group, thiol group, disulfide, phenol, guanidine, thioether, indole, imidazole, or diazonium group. In various embodiments as otherwise described herein, the functional groups include hydrophilic functional groups such as hydroxyl groups, amino groups, carbonyl groups, carboxyl groups, and phosphate groups. For example, in various such embodiments, the functional groups include aldehyde groups. In various embodiments as otherwise described herein, the functional groups include nitrogen-containing groups. For example, in various such embodiments, the inner surface of the container comprises a plurality of amino functional groups.

[0063] Though the number of functional groups per unit of inner surface (i.e., functional group density) is not particularly limited, the person of ordinary skill in the art will appreciate that the functional group density can, typically, be greater than the number of biological agents (e.g., cells) that can adhere to the surface per unit of substrate surface (e.g., adhered agent density). Accordingly, in various embodiments, the functional group density of the inner surface is greater than the adhered agent density of the inner surface (i.e., when in use). The functional group density can, for example, be selected to provide a desired watercontact angle, to provide a desired surface energy for adhesion of a desired cell type to the inner surface of the container.

[0064] In various embodiments as otherwise described herein, the functional groups at the inner surface of the first polymer film are the product of etching of the fluoropolymer. For example, in various such embodiments, the etching comprises chemical etching, physicalmechanical etching, or plasma etching. For example, in various embodiments, the functional groups at the inner surface are the product of chemical etching of the fluoropolymer. In various such embodiments, the chemical etching comprises etching with sodium ammonia or sodium naphthalene. In another example, in various embodiments, the functional groups at the inner surface are the product of physical-mechanical etching. In various such embodiments, the physical-mechanical etching comprises sandblasting or air abrasion with silica. In another example, the functional groups at the inner surface are the product of plasma etching. In various such embodiments, the plasma etching comprises etching with reactive plasmas such as hydrogen, oxygen, acetylene, methane, and mixtures thereof with nitrogen, argon, and helium.

[0065] In various embodiments as otherwise described herein, the functional groups at the inner surface of the container are the product of activation of the fluoropolymer in the presence of a reactive species. For example, in various such embodiments, the activation is plasma activation. In various embodiments, plasma activation includes formation of reactive species on the fluoropolymer by treatment with gases such as, for example, argon, hydrogen, nitrogen, carbon dioxide, oxygen and mixtures thereof. In various embodiments, plasma activation generates radicals and / or peroxides on a fluoropolymer. Plasma activation can, in various embodiments, be performed at a pressure within the range of 0.1 Torr to 0.6 Torr, or within the range of 700 Torr to 760 Torr. In another example, in various such embodiments, the activation is corona activation. In various embodiments, corona activation includes activation of the fluoropolymer under gases such as, for example, argon, nitrogen, hydrogen, and mixtures thereof to form active sites on the fluoropolymer (e.g., susceptible to a reactive species or subsequent chemical treatment). In various embodiments, the activation (e.g., plasma activation or corona activation) includes a reactive hydrocarbon vapor such as, for example, ketones, alcohols, p-chlorostyrene, acrylonitrile, propylene diamine, anhydrous ammonia, styrene sulfonic acid, carbon tetrachloride, tetraethylene pentamine, cyclohexyl amine, tetra isopropyl titanate, decyl amine, tetrahydrofuran, diethyl triamine, tertiary butyl amine, ethylene diamine, toluene-2,4- diisocyanate, glycidyl methacrylate, triethylene tetramine, hexane, triethyl amine, methyl alcohol, vinyl acetate, methylisopropyl amine, vinyl butyl ether, methyl methacrylate, 2-vinyl pyrrolidone, methylvinylketone, xylene, or mixtures thereof. In various embodiments asotherwise described herein, activation (e.g., plasma activation) including a polymerizable hydrocarbon vapor selected from, for example, butylene, ethylene, glutaraldehyde, etc., provides a polymer (i.e. , comprising a functional group as otherwise described herein) coated onto the fluoropolymer. The person of ordinary skill in the art will appreciate that, in various embodiments, plasma activation including a polymerizable hydrocarbon vapor (i.e., plasma polymerization) can provide a relatively disorganized, highly cross-linked polymer coating.

[0066] In various embodiments as otherwise described herein, the functional groups at the inner surface of the container are the product of chemically treating an activated fluoropolymer. For example, in various such embodiments, the activated fluoropolymer is the product of plasma activation or corona activation of the fluoropolymer. In various such embodiments, the chemical treatment is a chemical reaction such as, for example, grafting polymerization, coupling, click chemistry, condensation, or addition. In various embodiments, the chemical treatment is grafting polymerization in solution, comprising polymerizing vinyl monomers via radical polymerization (e.g., initiated by radicals generated through plasma activation of the fluoropolymer). In various such embodiments, the vinyl monomers are selected from, for example, acrylic acid, (meth)acrylates, (meth)alkylacrylates, styrenes, dienes, alpha-olefins, halogenated alkenes, (meth)acrylonitriles, acrylamides, N-vinyl carbazoles, N-vinyl pyrrolidones, and maleic anhydride. For example, radical polymerization of acrylic acid monomers on the fluoropolymer can, in various embodiments, provide a dense surface of carboxyl groups. In various embodiments, such polymerized products can be relatively organized (e.g., as compared to plasma-polymerized products).

[0067] In various embodiments as otherwise described herein, the functional groups at the inner surface of the container are the product of coating an activated fluoropolymer. For example, in various such embodiments, the activated fluoropolymer is the product of plasma activation or corona activation of the fluoropolymer. In various such embodiments, the coating is wet coating, powder coating, or chemical vapor deposition. In various embodiments, the coating is plasma-enhanced chemical vapor deposition or initiated chemical vapor deposition. In various embodiments the coating is wet coating, for example, of one or more extracellular matrix compounds.

[0068] Additional methods for providing suitable surfaces for cell growth, especially on fluorinated materials, is described in U.S. Patent Application Publication no. 2024 / 0166997, which is hereby incorporated herein by reference in its entirety.

[0069] In various embodiments as otherwise described herein, the textured inner surface comprises one or more extracellular matrix (ECM) compounds attached to the fluoropolymer. For example, in various such embodiments, the extracellular matrix compounds are selected from collagen I, poly-L-lysine, fibronectin, retronectin, hyaluronic acid, and polydopamine. Other examples include Laminin, Collagen IV, heparan sulfate proteoglycans, entactin / nidogen, and various growth factors; Coring Matrigel® can be used to provide such materials, e.g., as a deposit in the cell culture wells. In various embodiments as otherwise described herein, one or more extracellular matrix compounds are covalently linked to a functional group of the inner polymer surface. For example, in various such embodiments, the inner surface comprises one or more extracellular matrix proteins, attached through a peptide linkage to a carboxyl group of the fluoropolymer. In another example, the inner surface comprises one or more amine-terminated extracellular matrix proteins, attached through an imine linkage to an aldehyde group of the fluoropolymer.

[0070] The first polymer film can be used in a cell culture article in a variety of manners. The person of ordinary skill in the art will appreciate that one convenient form of cell culture article is a bag. A bag can be formed, e.g., by sealing the first polymer film to a second polymer film to form the cell culture volume of the bag. The second polymer film need not be patterned as described herein; as the second polymer film would form the top of the bag during incubation, there would be relatively little cell growth thereon. The person of ordinary skill in the art can select a suitable material for the second polymer film, based on the state of the art of cell culture bags.

[0071] In other embodiments, the article is in the form of a cassette, e.g., as described above. Of course, the person of ordinary kill in the art will appreciate that other forms of cell culture articles can be provided with the first polymer film as described herein.

[0072] The cell culture article can desirably have an aqueous medium, such as a cell culture medium, disposed in the cell culture volume. The person of ordinary skill in the art will identify appropriate cell culture media and other aqueous media for use in various process steps in the culture of cells.

[0073] In various embodiments (especially when a cell culture medium is present in the cell culture volume), the article has a plurality of cells disposed therein. As shown in the Examples below, cells can be advantageously cultured in the articles of the disclosure. In some embodiments, the cells are stem cells, especially human pluripotent stem cells that can differentiate into pancreatic cells like islet cells and pancreatic beta cells. In other embodiments, the cells are islet cells or pancreatic beta cells (i.e., which can be differentiated from stem cells).

[0074] In various embodiments, the cells, after growth, can be desirably at least partially present (e.g., at least 50%) in clusters, such as spheroids or organoids. The present inventors have found that the patterned surfaces described here can maintain clusters of a desirably small size, which can maintain a high degree of health of the clusters, without as much necrosis as can be present when clusters grow too large. In various embodiments, clusters have a number-average longest dimension in the range of 50-2000 microns. In some embodiments, the clusters have a number-average longest dimension in the range of 50-1500 microns, or 50-1000 microns, or 50-500 microns, or 50-200 microns. In some embodiments, the clusters have a number-average longest dimension in the range of 100- 2000 microns, e.g., 100-1500 microns, or 100-1000 microns, or 100-500 microns, or 100- 200 microns. In some embodiments, the clusters have a number-average longest dimension in the range of 500-2000 microns, e.g., 500-1500 microns, or 500-1000 microns. In some embodiments, the clusters have a number-average longest dimension in the range of 500- 2000 microns, e.g., 500-1500 microns, or 500-1000 microns.

[0075] The present inventors have noted that the size of the cell culture wells can help to limit the size of the clusters. This can be important, as clusters that grow too large can suffer from necrosis. In various embodiments, a number-average longest dimension of the clusters is no more than 150% of the longest dimension at half-maximum depth of the cell culture wells in which they are disposed, e.g., no more than 120%, or no more than 100%. In various embodiments, In various embodiments, a number-average longest dimension of the clusters is no more than the first base lateral dimension of the pyramidal voids.

[0076] In various embodiments, the cells are substantially adhered at the inner surface of the first polymer film. As used herein, cells adhered to a surface include cells associated with a surface sufficiently to avoid deleterious effects on cell function. The adhered cell can be relatively weakly associated with a surface (e.g., retaining a spherical shape) or strongly associated with a surface (e.g., forming a pancake-like shape on the surface). The cell can adhere directly to the fluoropolymer (e.g., comprising hydrophilic and / or nitrogen-containing functional groups) of the textured inner surface described herein (e.g., having a surface roughness within the range of 1 nm to 500 nm and / or comprising a pattern of one or more regularly spaced features, each feature having a spacing within the range of 0.1), or can interact with a protein environment (e.g., including extracellular matrix compounds) of the textured inner surface.

[0077] Notably, in various desirable embodiments, especially when cells are present in the cell culture volume, the inner surface of the first polymer film faces upward.

[0078] Another aspect of the disclosure is a method for cultivating a plurality of cells, the method includes incubating the plurality of cells in an aqueous medium (such as a cell culture medium) in the cell culture volume of an article as described herein. The cells can be as described above, for example, stem cells (e.g., which can be differentiated), or islet cells or pancreatic beta cells (e.g., which have been differentiated from stem cells). In especially desirable embodiments, cells are introduced as stem cells, and differentiate into islet cells or pancreatic beta cells, during the incubation. The cells can be grown in clusters as described above, and can adhere to the inner surface of the first polymer film as described above.

[0079] The person of ordinary skill in the art is familiar with a variety of manners for culture of cells. FIG. 8 provides a schematic view of stages of culturing pancreatic beta cells from human pluripotent stem cells. There are three main phases: adherent planar culture (S0-S3); aggregation (S4); and suspension 3D aggregate culture (S5-S7). The systems and methods of the disclosure can be especially useful in the aggregation phase.

[0080] One example of a protocol for generating clusters of pancreatic progenitor cells is provided here:• Following culture in flasks or other relevant adherent surface, cells from differentiated human pluripotent stem cells (hPSCs) or from relevant cell line models (such as MIN6) are dissociated using enzymatic digestion (TrpLE Express or Accumax) at a concentration of ~0.1 mL / cm2.• Cells are enumerated and subsequently seeded in the article at a rate of -500 cells / cell culture well.• The aggregates are cultured for 24 hours in an incubator at 37 °C and 5% CO2.• The aggregates are collected by washing the cell culture volume withCa+7Mg++phosphate-buffered saline, followed by enzymatic digestion at 37 °C for 10 minutes.• The dissociated cells are centrifuged at 300g for 5 minutes and enumerated.• The dissociated cells are fixed and stained with relevant antibodies for analysis by flow cytometry.But the person of ordinary skill in the art will appreciate that a wide variety of cell culture methodologies can be used.

[0081] The systems and methods of the present disclosure can result in a number of advantages over conventional methodologies. At the aggregation stage (S4), the formation of 3D cell aggregates in the claimed containers can help to control the aggregate sizes, which desirably can provide uniformity of size and sphericity and improve the viability of the aggregates, as well as provide good cell health due to increased oxygen permeability.These two effects can decrease the occurrence of the necrotic center in aggregates, thereby ensuring healthier cell cultures and higher chance of differentiation into mature islet cells. Moreover, the articles described herein can be conveniently provided as closed systems, which can provide advantages with respect to contamination over conventional open systems.

[0082] A patterned film was constructed by embossing FEP film (on the order of 5-10 mils in thickness) with a tool bearing pyramids in a square grid pattern. On the tool, the pyramids had square bases about 500 microns on a side, heights in the range of about 140- 220 microns, tip widths of about 5-10 microns, and about 60 micron spacing between pyramids. However, during embossing, the tool was not pressed into the film to its full depth; on the surface of the film opposite the embossed surface, the resulting humps had heights of about 70-80 microns, lateral dimensions of about 350-450 microns, and interhump spacings of about 300 microns. The film was formed into bags with an unpatterned sheets of FEP. In each, the side of the film opposite the embossed surface was used as an inner surface of the bag.

[0083] At the beginning of the Aggregation phase (S4), MIN6 cells (mouse pancreatic beta cells) in a cell culture medium were seeded into the bags described above at 200 cells / well and 400 cells / well. AggreWell plates (open containers with a patterned polystyrene surface) were used as a comparative example. The cells were incubated at 37 °C for 72 hours, after which the resulting clusters were harvested as described in the general procedure above. FIGS. 9, 10 and 11 provide images at 24, 48 and 72 hours, and FIG. 12 provides data collected on the resulting MIN6 islet aggregates. Notably, the clusters are substantially located in cell culture wells positioned at grid intersections. The data demonstrate that the cell culture articles of the disclosure can provide good results, especially with respect to cluster circularity and roundness.

[0084] Additional aspects of the disclosure are provided by the enumerated embodiments listed below, which can be combined in any number and in any fashion that is not technically or logically inconsistent.Embodiment 1 . A cell culture article having a cell culture volume, the cell culture article having a first polymer film as a first boundary of the cell culture internal volume, the first polymer film having an inner surface facing the cell culture volume and an outer surface facing away from the cell culture internal volume, the first polymer film having a plurality of pyramidal voids extending from a pyramid base at the inner surface or the outer surface of the first polymer film, to a pyramid tip, each of the pyramidal voids having a first base lateral dimension in the range of 150-2500 microns, a first tip lateral dimension up to 100 microns,and a depth in the range of 40-1000 microns, wherein the plurality of pyramidal voids forms a plurality of cell culture wells at the inner surface of the first polymer film.Embodiment 2. The article of embodiment 1 , wherein the pyramid base of each pyramidal void is at the outer surface of the first polymer film, with the tip of each pyramidal void extending toward the cell culture volume, the article further comprising a plurality of humps at the inner surface of the first polymer film in registration with the pyramidal voids, the humps defining a plurality cell culture wells formed from of inter-hump spaces at the inner surface of the first polymer film.Embodiment 3. The article of embodiment 2, wherein the cell culture wells are formed at intersections of inter-hump spaces.Embodiment 4. The article of embodiment 1 , wherein the pyramid base of each pyramidal void is at the inner surface of the first polymer film, with the tip of each pyramidal void extending away from cell culture volume of the container, wherein the pyramidal voids themselves form cell culture wells.Embodiment 5. The article of any of embodiments 1 -4, wherein each of the plurality of pyramidal voids has a base shape that is rectangular, e.g., square.Embodiment 6. The article of any of embodiments 1 -4, wherein each of the plurality of pyramidal voids has a base shape that is polygonal, e.g., triangular or pentagonal or hexagonal.Embodiment 7. The article of any of embodiments 1 -4, wherein each of the plurality of pyramidal voids has a base shape that is round, e.g., circular or elliptical or oval.Embodiment 8. The article of any of embodiments 1-7, wherein in each of the pyramidal voids the first base lateral dimension is in the range of 150-1750 microns, e.g., 150-1500 microns, or 150-1250 microns.Embodiment 9. The article of any of embodiment 1-7, wherein in each of the pyramidal voids the first base lateral dimension is in the range of 150-1000 microns, e.g., 150-850 microns, or 150-700 microns, or 150-600 microns.Embodiment 10. The article of any of embodiments 1 -7, wherein in each of the pyramidal voids the first base lateral dimension is in the range of 300-2000 microns, e.g., 300-1750 microns, or 300-1500 microns, or 300-1250 microns.Embodiment 11. The article of any of embodiment 1 -7, wherein in each of the pyramidal voids the first base lateral dimension is in the range of 300-1000 microns, e.g., 300-850 microns, or 150-700 microns, or 300-600 microns.Embodiment 12. The article of any of embodiments 1 -7, wherein in each of the pyramidal voids the first base lateral dimension is in the range of 700-2500 microns, e.g., 700-2000 microns, or 700-1750 microns, or 700-1500 microns, or 700-1250 microns.Embodiment 13. The article of any of embodiments 1 -7, wherein in each of the pyramidal voids the first base lateral dimension is in the range of 1000-2500 microns, e.g., 100-2000 microns, or 1000-1750 microns, or 1000-1500 microns, or 1500-2500 microns, or 1500-2000 microns.Embodiment 14. The article of any of embodiments 1-13, wherein each of the plurality of pyramidal voids has a second base lateral dimension that is at least 50% of the first base lateral dimension.Embodiment 15. The article of any of embodiments 1 -13, wherein each of the plurality of pyramidal voids has a second base lateral dimension that is at least 75% of the first base lateral dimension.Embodiment 16. The article of any of embodiments 1 -13, wherein each of the plurality of pyramidal voids has a second base lateral dimension that is at least 90% of the first base lateral dimension.Embodiment 17. The article of any of embodiments 1-16, wherein each of the plurality of pyramidal voids has a base area in the range of 10000-6250000 square microns, e.g., 10000-1000000 square microns.Embodiment 18. The article of any of embodiments 1-16, wherein each of the plurality of pyramidal voids has a base area in the range of 90000-490000 square microns, e.g., 90000-250000 square microns.Embodiment 19. The article of any of embodiments 1-16, wherein each of the plurality of pyramidal voids has a base area in the range of 90000-6250000 square microns, e.g., 90000-1000000 square microns.Embodiment 20. The article of any of embodiments 1-16, wherein each of the plurality of pyramidal voids has a base area in the range of 90000-490000 square microns, e.g., 90000-250000 square microns.Embodiment 21. The article of any of embodiments 1-16, wherein each of the plurality of pyramidal voids has a base area in the range of 490000-3062500 square microns, e.g., 1000000-3062500 square microns, or 1650000-3062500 square microns.Embodiment 22. The article of any of embodiments 1-16, wherein each of the plurality of pyramidal voids has a base area in the range of 490000-6250000 square microns, e.g., 1000000-6250000 square microns, or 1650000-6250000 square microns.Embodiment 23. The article of any of embodiments 1-22, wherein each of the plurality of pyramidal voids has a first tip lateral dimension up to 70 microns, e.g., up to 50 microns.Embodiment 24. The article of any of embodiments 1 -22, wherein each of the plurality of pyramidal voids has a first tip lateral dimension up to 30 microns, e.g., up to 20 microns, or up to 15 microns.Embodiment 25. The article of any of embodiments 1 -24, wherein each of the plurality of pyramidal voids has a depth in the range of 40-800 microns, e.g., 40-600 microns, or 40- 500 microns, or 40-400 microns.Embodiment 26. The article of any of embodiments 1 -24, wherein each of the plurality of pyramidal voids has a depth in the range of 40-300 microns, e.g., 40-150 microns, or 40- 100 microns.Embodiment 27. The article of any of embodiments 1 -24, wherein each of the plurality of pyramidal voids has a depth in the range of 100-1000 microns, e.g., 100-800 microns, or 100-600. microns, or 100-500 microns, or 100-400 microns.Embodiment 28. The article of any of embodiments 1 -24, wherein each of the plurality of pyramidal voids has a depth in the range of 100-300 microns, e.g., 100-200 microns.Embodiment 29. The article of any of embodiments 1 -24, wherein each of the plurality of pyramidal voids has a depth in the range of 250-1000 microns, e.g., 250-800 microns, or 250-600. microns, or 250-500 microns, or 250-400 microns.Embodiment 30. The article of any of embodiments 1-29, wherein each of the pyramidal voids has an average nearest-neighbor base-to-base distance up to 2000 microns, e.g., up to 1500 microns, or up to 1000 microns.Embodiment 31. The article of any of embodiments 1 -29, wherein each of the pyramidal voids has an average nearest-neighbor base-to-base distance up to 800 microns, e.g., up to 600 microns, or up to 400 microns.Embodiment 32. The article of any of embodiments 1 -29, wherein each of the pyramidal voids has an average nearest-neighbor base-to-base distance up to 300 microns, e.g., up to 200 microns, or up to 100 microns.Embodiment 33. The article of any of embodiments 1 -32, wherein each of the pyramidal voids has an average nearest-neighbor base-to-base distance of at least 20 microns, e.g., at least 40 microns, or at least 70 microns.Embodiment 34. The article of any of embodiments 1 -32, wherein each of the pyramidal voids has an average nearest-neighbor base-to-base distance of at least 100 microns, e.g., at least 300 microns, or at least 500 microns.Embodiment 35. The article of any of embodiments 1 -32, wherein each of the pyramidal voids has an average nearest-neighbor base-to-base distance of at least 800 microns, e.g., at least 1000 microns, or at least 1300 microns.Embodiment 36. The article of any of embodiments 1-35, wherein the plurality of pyramidal voids is arranged in a grid pattern.Embodiment 37. The article of any of embodiments 1-35, wherein the plurality of pyramidal voids have triangular bases, and are arranged in a triangular pattern.Embodiment 38. The article of any of embodiments 1 -37, wherein the first polymer film has a maximum thickness of at least 25 microns, e.g., at least 100 microns, or at least 200 microns, or at least 300 microns.Embodiment 39. The article of any of embodiments 1 -37, wherein the first polymer film has a maximum thickness in the range of 25-700 microns, e.g., 25-600 microns, or 25-500 microns, or 100-700 microns, or 100-600 microns, or 100-500 microns.Embodiment 40. The article of any of embodiments 1 -39, wherein the first polymer film has a minimum thickness in the cell culture wells of at least 20 microns, e.g., at least 50 microns, or at least 75 microns.Embodiment 41. The article of any of embodiments 1-40, wherein the first polymer film has a minimum thickness in the cell culture wells in the range of 20-200 microns, e.g., 20- 150 microns, or 20-125 microns.Embodiment 42. The article of any of embodiments 1 -40, wherein the first polymer film has a minimum thickness in the cell culture wells in the range of 50-200 microns, e.g., 50- 150 microns, or 50-125 microns, or 75-200 microns, or 75-175 microns, or 75-150 microns.Embodiment 43. The article of any of embodiments 1 -42, wherein the first polymer film has a ratio of minimum thickness in the cell culture wells to maximum thickness in the range of 15-85%, e.g., in the range of 15-60%, or 15-50%, or 15-35%.Embodiment 44. The article of any of embodiments 1 -42, wherein the first polymer film has a ratio of minimum thickness in the cell culture wells to maximum thickness in the range of 25-85%, e.g., in the range of 25-60%, or 25-50%, or 25-35%.Embodiment 45. The article of any of embodiments 1 -42, wherein the first polymer film has a ratio of minimum thickness in the cell culture wells to maximum thickness in the range of 35-85%, e.g., in the range of 35-60%, or 35-50%.Embodiment 46. The article of any of embodiments 1 -45, wherein the first polymer film has a polymer surface that is a fluoropolymer surface disposed at the inner surface thereof.Embodiment 47. The article of embodiment 46, wherein the first polymer film is a fluoropolymer film.Embodiment 48. The article of embodiment 46, wherein the first polymer film is a fluoropolymer laminate having a fluoropolymer layer proximal the interior volume of the container, and a silicone layer distal the interior volume of the container.Embodiment 49. The article of embodiment 46, wherein the fluoropolymer is fluorinated ethylene propylene copolymer.Embodiment 50. The article of any of embodiment 47 or embodiment 48, wherein the fluoropolymer is polytetrafluoroethylene (PTFE), perfluoroalkoxy (PFA), ethylene tetrafluoroethylene (ETFE), polyvinylidene fluoride (PVDF), polychlorotrifluoroethylene (PCTFE), ethylene chlorotrifluoroethylene (ECTFE), fluorinated ethylene propylene (FEP), ethylene fluorinated ethylene propylene (EFEP), perfluoropolyether (PFPE), modified polytetrafluoroethylene (TFM), polyvinyl fluoride (PVF), or any mixture thereof.Embodiment 51. The article of any of embodiments 1 -45, wherein the first polymer film has a polymer surface that is a silicone surface disposed at the inner surface thereof.Embodiment 52. The article of embodiment 51 , wherein the first polymer film is a silicone film.Embodiment 53. The article of embodiment 51 or embodiment 52, wherein the silicone is a polymer or copolymer of dimethylsiloxane, e.g., having at least 75 wt% or 90 wt% dimethylsiloxane units.Embodiment 54. The article of embodiments 1-53, wherein the polymer surface has a plurality of functional groups attached thereto.Embodiment 55. The article of embodiment 54, wherein the functional groups include hydrophilic functional groups (e.g., hydroxyl groups, amino groups, carbonyl groups, carboxyl groups, and phosphate groups).Embodiment 56. The article of embodiment 54, wherein the functional groups include nitrogen-containing groups (e.g., amino groups).Embodiment 57. The article of any of embodiments 54-56, wherein the functional groups are the product of etching of the polymer surface.Embodiment 58. The article of embodiment 57, wherein the chemical modification comprises chemical etching, physical-mechanical etching, or plasma etching.Embodiment 59. The article of any of embodiments 54-56, wherein the functional groups are the product of activation of the polymer surface in the presence of a reactive species.Embodiment 60. The article of embodiment 59, wherein the activation is plasma activation or corona activation, and the reactive species comprises one or more reactive hydrocarbon vapors.Embodiment 61. The article of embodiment 59, wherein the activation is plasma activation, and the reactive species comprises one or more polymerizable hydrocarbon vapors (e.g., selected from butylene, ethylene, and glutaraldehyde).Embodiment 62. The article of any of embodiments 54-56, wherein the functional groups are the product of chemically treating an activated polymer surface.Embodiment 63. The article of embodiment 62, wherein the activated polymer surface is the product of plasma activation or corona activation, and the chemical treatment is grafting polymerization, coupling, click chemistry, condensation, or addition.Embodiment 64. The article of any of embodiments 54-57, wherein the functional groups are the product of coating an activated polymer surface.Embodiment 65. The article of embodiment 64, wherein the activated polymer surface is the product of plasma activation or corona activation, and the coating is wet coating, powder coating, or chemical vapor deposition.Embodiment 66. The article of any of embodiments 1-65, comprising one or more extracellular matrix (ECM) compounds attached to the polymer surface.Embodiment 67. The article of embodiment 66, wherein the extracellular matrix compounds are selected from laminin, collagen IV, heparin sulfate proteoglycans, entactin / nidogen, growth factors, collagen I, poly-L-lysine, fibronectin, retronectin, hyaluronic acid, and polydopamine.Embodiment 68. The article of embodiment 66 or embodiment 67, wherein one or more extracellular matrix compounds are covalently linked to a functional group of the polymer surface.Embodiment 69. The article of any of embodiments 1-68, in the form of a bag.Embodiment 70. The article of embodiment 69, further comprising a second polymer film, the second polymer film being sealed to the first polymer film to form the internal volume of the container.Embodiment 71. The article of any of embodiments 1 -68, in the form of a cassette.Embodiment 72. The article of any of embodiments 1-71 , having an aqueous medium, such as a cell culture medium, disposed in the cell culture volume.Embodiment 73. The article of any of embodiments 1-72, having a plurality of cells disposed in the cell culture volume.Embodiment 74. The article of embodiment 73, wherein the plurality of cells are stem cells.Embodiment 75. The article of embodiment 73, wherein the plurality of cells are islet cells or beta cells.Embodiment 76. The article of any of embodiments 73-75, wherein the cells, after growth, are at least partially (e.g., at least 50%) present as clusters, e.g., spheroids or organoids.Embodiment 77. The article of embodiment 76, wherein the clusters have a numberaverage longest dimension in the range of 50-2000 microns, e.g., in the range of 50-1500 microns, or 50-1000 microns, or 50-500 microns, or 50-200 microns.Embodiment 78. The article of embodiment 76, wherein the clusters have a numberaverage longest dimension in the range of 100-2000 microns, e.g., 100-1500 microns, or 100-1000 microns, or 100-500 microns, or 100-200 microns.Embodiment 79. The article of embodiment 76, wherein the clusters have a numberaverage longest dimension in the range of 500-2000 microns, e.g., 500-1500 microns, or 500-1000 microns.Embodiment 80. The article of embodiment 76, wherein the clusters have a numberaverage longest dimension in the range of 500-2000 microns, e.g., 500-1500 microns, or 500-1000 microns.Embodiment 81. The article of any of embodiments 76-80, wherein a number-average longest dimension of the clusters is no more than 150% of the longest dimension at halfmaximum depth of the cell culture wells in which they are disposed, e.g., no more than 120%, or no more than 100%.Embodiment 82. The article of any of embodiments 76-80, wherein a number-average longest dimension of the clusters is no more than the first base lateral dimension of the pyramidal voids.Embodiment 83. The article of any of embodiments 73-82, wherein the cells are substantially adhered at the inner surface of the first polymer film.Embodiment 84. The article of any of embodiments 1-83, wherein the inner surface of the first polymer film faces upward.Embodiment 85. A method for cultivating a plurality of cells, comprising incubating the plurality of cells in an cell culture medium in cell culture volume of the article of any of embodiments 1-84.Embodiment 86. The method of embodiment 85, wherein the plurality of cells as described above with respect to any of embodiments 73-83.Embodiment 87. The method of any of embodiments 85 and 86, wherein cells are introduced as stem cells, and differentiate into islet cells or beta cells during the incubation.Embodiment 88. The method of any of embodiments 85 and 86, wherein the cells undergo an aggregation phase during the incubation.

[0085] The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present invention only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the invention. In this regard, no attempt is made to show structural details of the invention in more detail than is necessary for the fundamental understanding of the invention, the description taken with the drawings and / or examples making apparent to those skilled in the art how the several forms of the invention may be embodied in practice. Thus, before the disclosed processes and devices are described, it is to be understood that the aspects described herein are not limited to specific embodiments, apparati, or configurations, and as such can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and, unless specifically defined herein, is not intended to be limiting.

[0086] The terms “a,” “an,” “the” and similar referents used in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Ranges can be expressed herein as from “about” one particular value, and / or to “about” another particular value. When such a range is expressed, another aspect includes from the one particular value and / or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.

[0087] All methods described herein can be performed in any suitable order of steps unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0088] Unless the context clearly requires otherwise, throughout the description and the claims, the words ‘comprise’, ‘comprising’, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. Words using the singular or plural number also include the plural and singular number, respectively. Additionally, the words “herein,” “above,” and “below” and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of the application.

[0089] As will be understood by one of ordinary skill in the art, each embodiment disclosed herein can comprise, consist essentially of or consist of its particular stated element, step, ingredient or component. As used herein, the transition term “comprise” or “comprises” means includes, but is not limited to, and allows for the inclusion of unspecified elements, steps, ingredients, or components, even in major amounts. The transitional phrase “consisting of” excludes any element, step, ingredient or component not specified. The transition phrase “consisting essentially of’ limits the scope of the embodiment to the specified elements, steps, ingredients or components and to those that do not materially affect the embodiment.

[0090] Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. When further clarity is required, the term “about” has the meaning reasonably ascribed to it by a person skilled in the art when used in conjunction with a stated numerical value or range, i.e. denoting somewhat more or somewhat less than the stated value or range, with a precision that is typical in the art.

[0091] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains various errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0092] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included in, or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims.

[0093] Some embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on these described embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventor expects skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0094] Numerous references have been made to patents and printed publications throughout this specification. Each of the cited references and printed publications are individually incorporated herein by reference in their entirety.

[0095] Furthermore, it is to be understood that the embodiments of the invention disclosed herein are illustrative of the principles of the present invention. Other modifications that may be employed are within the scope of the invention. Thus, by way of example, but not of limitation, alternative configurations of the present invention may be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims

What is claimed is:

1. A cell culture article having a cell culture volume, the cell culture article having a first polymer film as a first boundary of the cell culture internal volume, the first polymer film having an inner surface facing the cell culture volume and an outer surface facing away from the cell culture internal volume, the first polymer film having a plurality of pyramidal voids extending from a pyramid base at the inner surface or the outer surface of the first polymer film, to a pyramid tip, each of the pyramidal voids having a first base lateral dimension in the range of 150-2500 microns, a first tip lateral dimension up to 100 microns, and a depth in the range of 40-1000 microns, wherein the plurality of pyramidal voids forms a plurality of cell culture wells at the inner surface of the first polymer film.

2. The article of claim 1 , wherein the pyramid base of each pyramidal void is at the outer surface of the first polymer film, with the tip of each pyramidal void extending toward the cell culture volume, the article further comprising a plurality of humps at the inner surface of the first polymer film in registration with the pyramidal voids, the humps defining a plurality cell culture wells formed from of inter-hump spaces at the inner surface of the first polymer film.

3. The article of claim 2, wherein the cell culture wells are formed at intersections of inter-hump spaces.

4. The article of claim 1 , wherein the pyramid base of each pyramidal void is at the inner surface of the first polymer film, with the tip of each pyramidal void extending away from cell culture volume of the container, wherein the pyramidal voids themselves form cell culture wells.

5. The article of claim 1 , wherein each of the plurality of pyramidal voids has a base shape that is rectangular, e.g., square.

6. The article of claim 1 , wherein in each of the pyramidal voids the first base lateral dimension is in the range of 300-2000 microns.

7. The article of claim 1 , wherein each of the plurality of pyramidal voids has a second base lateral dimension that is at least 50% of the first base lateral dimension.

8. The article of claim 1 , wherein each of the plurality of pyramidal voids has a base area in the range of 10000-6250000 square microns.

9. The article of claim 1 , wherein each of the plurality of pyramidal voids has a first tip lateral dimension up to 70 microns.

10. The article of claim 1 , wherein each of the plurality of pyramidal voids has a depth in the range of 250-800 microns.11 . The article of claim 1 , wherein each of the pyramidal voids has an average nearest- neighbor base-to-base distance up to 800 microns.

12. The article of claim 1 , wherein each of the pyramidal voids has an average nearest- neighbor base-to-base distance of at least 20 microns.

13. The article of claim 1 , wherein the plurality of pyramidal voids is arranged in a grid pattern.

14. The article of claim 1 , wherein the first polymer film has a maximum thickness in the range of 25-700 microns.

15. The article of claim 1 , wherein the first polymer film has a ratio of minimum thickness in the cell culture wells to maximum thickness in the range of 15-85%.

16. The article of claim 1 , wherein the first polymer film has a polymer surface that is a fluoropolymer surface disposed at the inner surface thereof.

17. The article of claim 1 , wherein the first polymer film has a polymer surface that is a silicone surface disposed at the inner surface thereof.

18. The article of claim 1 ,, comprising one or more extracellular matrix (ECM) compounds attached to the polymer surface, wherein the extracellular matrix compounds are selected from laminin, collagen IV, heparin sulfate proteoglycans, entactin / nidogen, growth factors, collagen I, poly-L-lysine, fibronectin, retronectin, hyaluronic acid, and polydopamine.

19. The article of claim 1 ,, in the form of a bag, the article further comprising a second polymer film, the second polymer film being sealed to the first polymer film to form the internal volume of the container.

20. The article of claim 1 ,, having a cell culture medium and a plurality of cells disposed in the cell culture volume.21 . The article of claim 20, wherein the cells, after growth, are at least partially (e.g., at least 50%) present as clusters, e.g., spheroids or organoids, having a number-average longest dimension in the range of 50-500 microns.

22. The article of claim 20, wherein a number-average longest dimension of the clusters is no more than 150% of the longest dimension at half-maximum depth of the cell culture wells in which they are disposed.

23. The article of claim 20, wherein the inner surface of the first polymer film faces upward.

24. A method for cultivating a plurality of cells, comprising incubating the plurality of cells in an cell culture medium in cell culture volume of the article of any of claims 1-23.

25. The method of claim 24, wherein cells are introduced as stem cells, and differentiate into islet cells or beta cells during the incubation.

88. The method of claim 24, wherein the cells undergo an aggregation phase during the incubation.

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