CELL CULTURE DEVICE COMPRISING AT LEAST ONE NOTCH FORMED IN A SIDE WALL FOR EXCHANGE OF FLUID MEDIA - Patent application

Abstract

Embodiments of the present disclosure relate to a device for three-dimensional cell growth. The device includes a substrate having an ultra-low attachment surface and a plurality of cavities for growing cell cultures. The device further includes at least one sidewall surrounding the substrate. At least one notch is formed in the at least one sidewall, the at least one notch being configured to accommodate a serological pipette for exchange of fluid media.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119 to U.S. Provisional Application No. 63 / 428,142, filed November 28, 2022, the contents of which are relied upon and incorporated herein by reference in their entirety. [Background technology]

[0002] The present disclosure relates generally to cell culture devices, and more particularly to cell culture devices that include a microcavity substrate bottom that includes features that limit disruption of cell cultures during fluid exchange.

[0003] Three-dimensional (3D) cell culture models have attracted attention compared to two-dimensional (2D) monolayer cell culture methods because they better mimic the complex microenvironment of cells and tissues that occurs in vivo. Advances in biological research, cancer research, and the development of regenerative therapies have led to an increasing demand for generating larger quantities of 3D cell cultures, including cell aggregates, tumor spheroids, embryoid bodies, and organoids. Cell culture devices are available that enable the generation of multiple 3D cell cultures within the same analytical plate. Indeed, certain microplates incorporate microcavity substrates with ultra-low attachment surfaces (ULA) within an open frame, enabling the formation of thousands of 3D cell cultures of reproducible sizes and shapes within a conventional analytical plate footprint. Maintaining 3D cell cultures over the days and weeks required for the formation of multicellular 3D models and organoids requires multiple fluid medium changes while retaining cells within the microcavity structures with minimal disturbance. For some cell culture devices, medium changes can be performed without disturbing the 3D culture because only small volumes (in the μL to mL range) of liquid are required to fill the cavity and support the culture. However, for certain high-density cell culture devices, it is necessary to move larger volumes of liquid (e.g., 25 mL) to support the bulk 3D cell culture volume present throughout the device. Moving such relatively large volumes of fluid can undesirably disturb the 3D cell culture. Summary of the Invention

[0004] According to aspect (1), there is provided a device for three-dimensional cell growth, the device comprising: a substrate having an ultra-low attachment surface and a plurality of cavities for growing cell cultures; and at least one sidewall surrounding the substrate, wherein at least one notch is formed in the at least one sidewall, the at least one notch being configured to accommodate a serological pipette for exchange of fluid media.

[0005] According to aspect (2), there is provided the device of aspect (1), wherein the at least one sidewall comprises a first short sidewall, a second short sidewall, a first long sidewall, and a second long sidewall, wherein the first short sidewall and the second short sidewall each have a first length, and the first long sidewall and the second long sidewall each have a second length, the second length being greater than the first length.

[0006] According to aspect (3), there is provided the device of aspect (2), wherein the at least one notch comprises a first notch formed on the first short sidewall.

[0007] According to aspect (4), there is provided the device according to aspect (3), wherein the first notch is located at a midpoint of the first short sidewall.

[0008] According to aspect (5), there is provided the device of aspect (3) or (4), wherein the at least one notch comprises a second notch formed in a corner between the first short sidewall and the first long sidewall.

[0009] According to aspect (6), there is provided the device of aspect (2), wherein the at least one notch comprises a first notch formed in a first corner between the first short sidewall and the first long sidewall.

[0010] According to aspect (7), there is provided the device of aspect (6), wherein the at least one notch comprises a second notch formed in a second corner between the first short sidewall and the second long sidewall.

[0011] According to aspect (8), there is provided the device of any one of the preceding aspects, wherein the at least one notch comprises a substantially planar notch wall.

[0012] According to an aspect (9), there is provided the device according to any one of aspects (1) to (7), wherein the at least one notch comprises a curved notch wall.

[0013] According to aspect (10), there is provided the device of any one of the preceding aspects, wherein the at least one notch comprises a floor, the floor being angled relative to a plane defined by the substrate.

[0014] According to an aspect (11), there is provided the device according to aspect (10), wherein the floor is at a different height than the plane defined by the substrate.

[0015] According to an aspect (12), there is provided a device according to any one of the preceding aspects, wherein the plurality of cavities are formed within 1 cm of the substrate. 2 At least five cavities per cavity.

[0016] According to aspect (13), there is provided the device of any one of the preceding aspects, wherein the at least one notch is configured to accommodate a tip of a 25 mL serological pipette oriented perpendicular to the substrate within the at least one notch.

[0017] According to an aspect (14), there is provided the device of any one of the preceding aspects, wherein the device has dimensions in accordance with ANSI / SLAS1-2004.

[0018] According to an aspect (15), there is provided the device according to aspect (1), wherein the at least one sidewall is a single circular sidewall.

[0019] According to aspect (16), there is provided a method, the method including: inserting a pipette into a first notch formed in at least one sidewall, the at least one sidewall surrounding a substrate of a cell culture device, the substrate comprising an ultra-low attachment surface and a plurality of cavities for growing cell cultures; and adding a fluid into the first notch such that the fluid covers the substrate within the at least one sidewall.

[0020] According to aspect (17), there is provided a method according to aspect (16), wherein the at least one side wall comprises a first short side wall, a second short side wall, a first long side wall, and a second long side wall, wherein the first short side wall and the second short side wall each comprise a first length, and the first long side wall and the second long side wall each comprise a second length, and the second length is greater than the first length.

[0021] According to aspect (18), there is provided the method of aspect (17), wherein the inserting further comprises inserting the pipette into a first notch, the first notch being formed on the first short sidewall.

[0022] According to aspect (19), there is provided the method of aspect (18), further comprising removing fluid from above the substrate using a pipette inserted into the first notch.

[0023] According to aspect (20), there is provided the method of aspect (19), wherein before removing, the method further includes raising the second short sidewall, the second short sidewall facing the first short sidewall such that the fluid flows toward the first notch formed on the first short sidewall.

[0024] According to an aspect (21), there is provided the method of any one of aspects (18) to (20), wherein the first notch is located at a midpoint of the first short sidewall.

[0025] According to aspect (22), there is provided the method of aspect (17), wherein the cell culture device further comprises a second notch, wherein one of the first notch or the second notch is formed on the first short side wall, and the other of the first notch or the second notch is formed at a corner between the first short side wall and the first long side wall.

[0026] According to aspect (23), there is provided the method of aspect (22), further comprising removing fluid from above the substrate using a pipette inserted into the second notch.

[0027] According to aspect (24), there is provided the method of aspect (22), further comprising raising the second short sidewall, the second short sidewall facing the first short sidewall such that fluid flows toward at least one of the first notch or the second notch.

[0028] According to aspect (25), there is provided the method of aspect (17), wherein the inserting further comprises inserting the pipette into a first notch, the first notch being formed at a first corner between the first short sidewall and the first long sidewall.

[0029] According to aspect (26), there is provided a method according to aspect (25), wherein the cell culture device further comprises a second notch formed at a second corner between the first short sidewall and the second long sidewall, and the method further comprises removing fluid from above the substrate using a pipette inserted into the second notch.

[0030] According to aspect (27), the method of any one of aspects (16) to (26), wherein at least one notch has a substantially planar notch wall.

[0031] According to aspect (28), the method of any one of aspects (16) to (26), wherein at least one notch has a curved notch wall.

[0032] According to aspect (29), the method of any one of aspects (16) to (28) is characterized in that at least one notch includes a floor, the floor being angled relative to a plane defined by the substrate.

[0033] According to an aspect (30), there is provided the method of aspect (29), wherein the floor is at a different height than the plane defined by the substrate.

[0034] According to aspect (31), the plurality of cavities are formed within 1 cm of the substrate. 2 The method of any one of aspects (16) to (30), wherein there are at least five cavities per cavity.

[0035] According to aspect (32), the method described in any one of aspects (16) to (31) is configured to accommodate a 25 mL serological pipette oriented perpendicular to the substrate within the at least one notch.

[0036] According to aspect (33), the method according to any one of aspects (16) to (32), wherein the cell culture device has dimensions conforming to ANSI / SLAS1-2004.

[0037] According to an aspect (34), there is provided the method according to aspect (16), wherein the at least one side wall is a single circular side wall.

[0038] Additional features and advantages will be set forth in the detailed description that follows, and in part will become readily apparent to those skilled in the art from the description, or may be learned by practicing the embodiments as described in the description and claims herein, and as illustrated in the accompanying drawings.

[0039] It is to be understood that both the foregoing general description and the following detailed description are exemplary only and are intended to provide an overview or framework for understanding the nature and character of the claims.

[0040] 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 embodiments and, together with the description, serve to explain the principles and operation of various embodiments. [Brief explanation of the drawings]

[0041] [Figure 1A] 1 depicts an open-well cell culture device including two notches formed in the corners of the sidewalls of the device, according to an exemplary embodiment. [Figure 1B] 1B depicts a detailed view of a cavity of the cell culture device of FIG. 1A according to an exemplary embodiment. [Figure 2] 1 depicts a cell culture device including a fluid medium, according to an exemplary embodiment. [Figure 3] 1 depicts a cell culture device having a lid and a tray, according to an exemplary embodiment. [Figure 4] 1 depicts a cell culture device having a notch formed in a sidewall of the device, according to an exemplary embodiment. [Figure 5A] 1 depicts a cell culture device having one notch formed in a sidewall of the device and another notch formed at a corner of the sidewall of the device, according to an exemplary embodiment. [Figure 5B] 5B depicts a detailed view of a corner notch, such as the corner notch shown in FIG. 5A, according to an exemplary embodiment. [Figure 6] 1 depicts a notch with curved walls in accordance with an exemplary embodiment; [Figure 7] 1 depicts a notch with planar walls according to an exemplary embodiment; [Figure 8] 1 depicts a cross-sectional view of a notch and substrate showing the angle of the notch floor relative to the plane of the substrate according to an exemplary embodiment; [Figure 9] 1 depicts an embodiment of a multi-well cell culture device having a notch provided in each well, according to an exemplary embodiment. [Figure 10] 1 depicts a process flow diagram of a method for adding and removing fluid media from a cell culture device, according to an illustrative embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0042] Generally, with reference to the following description and accompanying drawings, various embodiments of a cell culture device having at least one notch formed in a sidewall are provided. As described more fully below, the notch formed in the sidewall of the cell culture device accommodates a pipette for adding and removing fluids (e.g., medium, etc.) from the cell culture device. Such devices can be used to grow scaffold-free three-dimensional cell cultures (e.g., cell aggregates, spheroids, organoids, etc.) within cavities in the substrate of the cell culture device. Advantageously, the notch allows for fluid exchange while reducing disturbance to the growing cell culture. That is, fluid exchange for nutrient replenishment and waste removal is typically associated with some level of cell culture loss as a result of fluid flow over the substrate. By providing a notch through which fluids can be added to and removed from the microplate, cell culture loss during fluid exchange is reduced. These and other aspects and advantages of the disclosed cell culture device and methods of use thereof are described herein and with reference to the drawings. Such exemplary embodiments are provided for purposes of illustration and not limitation.

[0043] FIG. 1A depicts one embodiment of a cell culture device, particularly a plate 100, e.g., an open-well plate, configured for three-dimensional cell growth. Plate 100 may be a microplate. Open-well plate 100 comprises a substrate 102 having a plurality of cavities 104 (as shown in FIG. 1B) for growing, among other things, cell cultures, e.g., cell aggregates, spheroids, and organoids. For example, in one or more embodiments, the plurality of cavities 104 may extend over a 1 cm area of ​​the substrate. 21B provides a close-up view of the substrate 102 depicting the cavities 104 containing cell cultures, particularly spheroids 105, within each cavity 104. In the particular embodiment shown in FIG. 1B, the cavities 104 have a diameter of approximately 800 μm. In one or more embodiments, the substrate 102, and particularly the cavities 104, are coated with an ultra-low attachment (ULA) coating, which refers to a class of coatings known in the art for forcing cells into a suspended state.

[0044] 1A , the open well plate 100 further includes a plurality of sidewalls 106 that form a perimeter surrounding the substrate 102. At least one notch 108 is formed in the plurality of sidewalls 106. In one or more embodiments, the at least one notch 108 is configured to accommodate a pipette, such as a serological pipette, for fluid exchange. For example, in one or more embodiments, the at least one notch 108 is configured to accommodate a 25 mL serological pipette oriented perpendicular to the substrate 102 within the at least one notch 108.

[0045] In one or more embodiments, the plurality of sidewalls 106 includes a first short sidewall 110a, a second short sidewall 110b, a first long sidewall 112a, and a second long sidewall 112b. The first short sidewall 110a and the second short sidewall 110b each have a first length L1, and the first long sidewall 112a and the second long sidewall 112b each have a second length L2. In one or more embodiments, the second length L2 is greater than the first length L1. In one or more embodiments, the plurality of sidewalls 106 define a rounded rectangular perimeter of the substrate 102. In one or more embodiments, the dimensions of the open well plate 100 comply with ANSI / SLAS1-2004. While specific embodiments and shapes of the open well plate 100 are described and depicted herein, other shapes and sizes are encompassed by the present disclosure.

[0046] In one or more embodiments, the at least one notch 108 includes a first notch 114 and a second notch 116. In one or more embodiments, the first notch 114 is formed at the corner between the first short sidewall 110a and the first long sidewall 112a. In one or more embodiments, the second notch 116 is formed at the corner between the first short sidewall 110a and the second long sidewall 112b. As previously described, the notch 108 is provided to facilitate the exchange of fluids in the substrate 102. For example, the fluid may contain nutrients for growing cell cultures; therefore, as the nutrients become depleted, the fluid may be replenished. Additionally, old fluid may be removed to remove waste products from the cell cultures in the fluid.

[0047] FIG. 2 depicts an example of an open well plate 100 in which the substrate 102 is covered with a fluid 118. While cell cultures are growing within the cavities 104 of the substrate 102, the open well plate 100 may be configured for storage or handling. Accordingly, as shown in FIG. 3, the open well plate 100 is provided with a lid 120 and a tray 122. In one or more embodiments, the lid 120 is provided to prevent contamination and spills. In one or more embodiments, such as the embodiment shown in FIG. 3, the lid 120 is made of a transparent material so that the contents of the open well plate 100 can be viewed through the lid 120. In one or more embodiments, the tray 122 provides a flat, stable surface for the open well plate 100.

[0048] FIG. 4 depicts another embodiment of the open well plate 100. In one or more embodiments, the at least one notch 108 of the open well plate 100 is a first notch 114 formed on the first short sidewall 110a. In one or more embodiments, including the embodiment depicted in FIG. 4, the first notch 114 is located at approximately a midpoint 124 of the first short sidewall 110a. In one or more embodiments, the midpoint 124, about which the first notch 114 is centered, is a point between 40% and 60% of the first length L1 of the first short sidewall 110a (i.e., between 0.4L1 and 0.6L1).

[0049] 5A depicts another embodiment of an open well plate 100 having two notches 108. As with the previous embodiment, a first notch 114 is provided near the midpoint 124 of the first short sidewall 110a. A second notch 116 is provided at the corner between the first short sidewall 110a and the second long sidewall 112b.

[0050] FIG. 6 depicts an embodiment of a notch 108 having curved notch walls 126. In one or more embodiments, the curved notch walls 126 are curved with a radius of curvature between 3.0 mm and 4.2 mm. Furthermore, the notch 108 has a width W and a depth (defined by the radius of curvature) configured to accommodate a pipette, such as a 25 ml serological pipette. In one or more embodiments, the width W is between 5 mm and 15 mm, and more particularly, between 8 mm and 11 mm. The notch 108 also has a notch floor 128. In one or more embodiments, the notch floor 128 is at a different height than the substrate 102. In one or more embodiments, the notch floor 128 is spaced a distance D above the substrate 102. In one or more embodiments, the distance D is between 0.5 mm and 1.5 mm. In one or more embodiments, the notch 108 having the curved notch wall 126 may be either or both of the first notch 114 and the second notch 116 of any of the embodiments of the open well plate 100 described herein.

[0051] FIG. 7 depicts an embodiment of a notch 108 including a plurality of substantially planar notch walls 126. In one or more embodiments, the notch 108 has three planar notch walls 126 that define a rectangular notch 108. However, in one or more other embodiments, the notch 108 may have two planar notch walls 126 or three or more planar notch walls 126. In one or more embodiments, the notch 108 has a width W and a depth configured to accommodate a pipette, such as a 25 ml serological pipette. In one or more embodiments, the width W is between 5 mm and 15 mm, particularly between 8 mm and 11 mm. The notch 108 also has a notch floor 128. In one or more embodiments, the notch floor 128 is spaced a distance D above the substrate 102, for example, a distance D of between 0.5 mm and 1.5 mm. In one or more embodiments, the notch 108 having a substantially planar notch wall 126 may be either or both of the first notch 114 and the second notch 116 of any of the embodiments of the open well plate 100 described herein.

[0052] FIG. 5B depicts a particular embodiment of a corner notch 108 having curved notch walls 126. For purposes of discussion, reference is made to the second notch 116 in FIG. 5A, but the corner notch 108 can be located at any corner. As can be seen in FIG. 5B, the second notch 116 is formed at the intersection of the first short sidewall 110a and the second long sidewall 112b. Furthermore, in one or more embodiments, the second notch 116 opens at an angle θ relative to the second long sidewall 112b, for example, between 20° and 40°, and particularly about 34°. Due to the angled opening of the second notch 116, the edge of the second notch 116 relative to the second long sidewall 112b is longer than the edge of the second notch 116 provided on the first short sidewall 110a. 5B is sloped toward the substrate 102. Due to the slope of the notch floor 128 and the longer extension of one edge of the second notch 116, fluid added to the second notch 116 flows, under the influence of surface tension, toward the lowest point of the second notch 116, toward the edge formed by the second long sidewall 112b. In this manner, fluid added to the second notch 116 is directed away from the substrate and toward the second long sidewall 112b. Advantageously, this helps to reduce the disturbance that the addition of liquid has on cells growing within the substrate 102.

[0053] FIG. 8 depicts a cross-sectional view of the notch 108 relative to the substrate 102. As can be seen in FIG. 8, the substrate 102 defines a plane 130. In one or more embodiments, the notch floor 128 forms an angle α with the plane 130. In one or more embodiments, the angle α is at most 15°, specifically at most 11°. Advantageously, the angled notch floor 128 helps to promote the flow of fluid medium from the notch 108 into the region above the substrate 102. Particularly when combined with the angled opening of the corner notch (as described in connection with FIG. 5B ), the fluid is first drawn toward the sidewall 106 and then flows indirectly into the region above the substrate 102.

[0054] While the above-described embodiments have focused on an open-well plate 100 having sidewalls 106 defining a rectangular perimeter of a single substrate 102, other embodiments of the device may include multiple isolated compartments (i.e., microwells, multiple wells, alternate dish, or reservoir shapes) each with its own unique microcavity substrate bottom. According to the exemplary embodiment shown in Figure 9, the plate 100 includes multiple wells 150a-f, each with a corresponding substrate 102a-f. Each well 150a-f is defined by a single corresponding sidewall 106a-f (circular sidewall), and each sidewall 106a-f includes at least one notch for adding and removing fluids from the wells 150a-f. In one or more embodiments, each well 150a-f includes two notches: a first notch for adding fluid medium and a second notch for removing fluid medium (for ease of illustration, only notch 108a, including first notch 114a and second notch 116a of well 150a, is labeled). Wells 150a-f can be used to grow different cell cultures in each well and / or to grow the same cell culture under different conditions in each well, among other possibilities.

[0055] Although FIG. 9 depicts a plate 100 having multiple substrates 102, other embodiments may have a single substrate 102 and a single sidewall 106 (eg, a circular dish), among other possibilities.

[0056]

[0013] Embodiments of the present disclosure also relate to methods for adding and removing fluids from plates, such as microplates. Figure 10 provides a process flow diagram of method 200, which will be described with reference to the embodiment of plate 100 depicted in Figures 1A-5A (although method 200 applies to any cell culture device having a notch for adding or removing fluids). In one or more embodiments, method 200 includes a first step 201 of inserting a pipette into a notch 108 formed in a peripheral sidewall 106 of open-well plate 100.

[0057] In one or more embodiments, the method 200 further includes a second step 202 of adding a fluid into the notch 108 such that the fluid covers the substrate 102 within the plurality of sidewalls 106. In one or more embodiments, the pipette is positioned perpendicular to the substrate 102 such that the fluid is directed substantially toward the notch floor 128. However, in one or more other embodiments, the pipette is positioned transverse to the plane of the substrate 102 such that the fluid is directed at least partially toward the notch walls 126. Once the fluid covers the substrate 102, the open well plate 100 can be stored in a controlled environment that allows for the growth of cell cultures.

[0058] After the desired length of time has elapsed and the fluid medium needs to be replaced, the plate 100 can be retrieved for removal of the fluid medium. To facilitate removal of the fluid medium, in one or more embodiments, the method 200 further includes an optional third step 203 of elevating the side wall 106 of the plate 100 opposite the notch 108 (either the first notch 114 or the second notch 116) formed in the peripheral side wall 106 of the plate 100, so that the fluid medium flows toward the notch 108. In experiments, the inventors have found that elevating one side of the plate 100 facilitates removal of the fluid medium by allowing the fluid medium to drain to the opposite side of the device. However, the inventors have also found that removal of more than 50% of the fluid medium is possible even when the notch 108 is not elevated on one side to drain the fluid to the opposite side.

[0059] In one or more embodiments, method 200 further includes a fourth step 204 of removing the fluid medium from the notches 108 (either from the first notch 114 or the second notch 116) formed in the peripheral sidewall 106 of the plate 100. Method 200 can be repeated until the cell culture has grown to a desired level.

[0060] Advantageously, device embodiments provide a convenient method for exchanging fluid media without significantly disturbing the cell culture. In this regard, the inventors envision that fluid media exchanges can be performed while maintaining greater than 90% cell yield from the device. Without the notches 108 formed in the sidewalls 106, or without the notches 108, fluid media would be added or removed directly onto the cell culture substrate 102 or one of the sidewalls 106, resulting in extensive cell disturbance and displacement. The inventors have observed that cell culture yields below 90%, and often below 90%, are typical of exchanges using conventional cell culture devices. However, at least one notch 108 allows for consistent pipette placement during fluid media exchanges. Indeed, random pipette placement during fluid media exchanges can result in inconsistent cultures and results. Furthermore, having multiple notches 108 accommodates both left-handed and right-handed operators while providing consistent pipette placement during fluid media exchanges.

[0061] Various embodiments of plate 100 were tested to determine the ease with which the fluid medium could be changed and the degree to which the cell culture was disturbed.

[0062] In a first experiment, open well plates 100 were prepared having: (1) two notches 108 with a first notch 114 at a first corner of the side and a second notch 116 at a second corner of the side; (2) a single notch 108 located at the midpoint of the side; (3) two notches 108 with a first notch 114 at the midpoint of the side and a second notch 116 located at a corner of the side; and (4) a single notch 108 located at one corner.

[0063] For the first open well plate 100, 13 mL of fluid was added to the first notch 114, and 11 mL of fluid could be removed while the open well plate 100 remained flat during fluid removal. The two notches 108 were observed to be particularly beneficial for left- and right-handed operators to access the front or rear of the open well plate as desired.

[0064] For the second open well plate 100, 13 mL of fluid was added to the single side notch 108, and 7.5 mL of fluid could be removed while the open well plate 100 remained flat during removal.

[0065] For the third open well plate 100, 13 mL of fluid was added to the second notch 116 located in the corner, and during removal, only a few milliliters could be removed from either the first notch 114 or the second notch 116. When the opposing faces of the notches were raised by approximately 3°, 10.2 mL could be removed from the second notch 116.

[0066] For the fourth open-well plate 100, 13 mL of fluid was added to the single corner notch 108, and only 5 mL of fluid could be removed during removal, with the plate 100 remaining flat. It was observed that the curved notch wall 126 located within the corner was able to comfortably accommodate a pipette tip.

[0067] In a second experiment, a microplate was prepared with three notches 108 formed in the sidewall 106. Specifically, the first short sidewall 110a included a notch 108 at each corner and a notch 108 at the midpoint 124. The plate 100 was prepared by providing a ULA surface. Trapped air was removed from the microcavities by centrifugation. The cavities in the substrate near the first short sidewall 110a were then filled with preformed spheroids. The plate was imaged before the addition or removal of fluid medium. To assess spheroid disruption, 10 mL of fluid medium was added and then removed. During removal, the second short sidewall 110b was elevated by approximately 3°. Three fluid exchange conditions were investigated. (1) adding and removing fluid to the same corner notch 108, (2) adding fluid at the midpoint notch 108 and removing fluid from the corner notch 108, and (3) adding fluid at one corner notch 108 and removing fluid from the other corner notch 108. After each fluid addition and each fluid removal, the plate 100 was imaged using a Nikon NEXIV Vision Measurement System (VMR).

[0068] Five different operators performed the above fluid addition and removal steps, and each operator was observed to experience at least some spheroid loss using each of the three addition and removal techniques. However, it was also generally observed that fluid addition had a higher impact on spheroid loss than fluid removal. Furthermore, it was observed that spheroid loss from the midpoint notch 108 was greater than from the corner notch 108.

[0069] In a third experiment, spheroid retention was assessed again. Thirteen ULA-coated plates were pre-wetted with 10 mL of 0.2 μm-filtered 35% ethanol, 15 mL of water, and 2 × 15 mL of DPBS. HT29 / GFP cells were filtered through a 70 μm cell strainer and plated at 12 × 10 cells per plate. 6Cells were seeded at 1000 cells per cavity of the substrate. The plates were incubated in a humidified incubator at 37°C and 5% CO2. On the third day of incubation, one plate was imaged using a VMR to provide a control image. The other 12 plates underwent a medium change and were returned to incubation. Four medium change methods were evaluated, with each method performed by three operators. Four methods were included. (1) Removal and addition of fluid medium at the corner notch 108 using a pipette angled transverse to the plane of the substrate 102, (2) Removal and addition of fluid medium at the corner notch 108 using a pipette angled perpendicular to the plane of the substrate 102, (3) Removal of fluid medium from the corner notch 108 and addition of fluid medium at the midpoint notch 108 using a pipette angled perpendicular to the plane of the substrate 102, and (4) Removal of fluid medium from the corner notch 108 and addition of fluid medium at the midpoint notch 108 using a pipette angled perpendicular to the plane of the substrate 102. Fluid was exchanged three more times over the next eight days using these methods by each of three operators. After the fourth medium exchange on day 10, 12 plates were imaged using VMR and analyzed using Halcon to quantify spheroid loss.

[0070] From this experiment, it was determined that most spheroid loss occurred in cavities within the first 30% of the area adjacent to the sidewall where liquid was added. Specifically, the first method, using an angled pipette to remove and add fluid medium to the corner notch 108, resulted in a total loss of 5% of the spheroids (average across three microplates), with 73% (on average) of the lost spheroids located within the first 30% of the substrate 102. The second method, using a vertical pipette to remove and add fluid medium to the corner notch 108, resulted in a total loss of 4% of the spheroids, with 46% located within the first 30% of the substrate 102. The third method, using an angled pipette to remove fluid medium from the corner notch 108 and add fluid medium to the midpoint notch 108, resulted in a total loss of 6% of the spheroids, with 68% of the lost spheroids located within the first 30% of the substrate 102. Results from the fourth method were similar to those from the third method. The inventors observed that this experiment confirmed previous observations that fluid addition had a greater impact on spheroid loss than fluid removal, resulting in greater spheroid loss when adding fluid medium at the midpoint notch 108. Furthermore, it was determined that the angle of the pipette had little effect at the midpoint notch 108, but that spheroid loss was reduced by approximately 1% when the pipette was at a perpendicular angle for removal / addition at the corner notch 108. Nevertheless, it was observed that over 90% of the spheroids were not disturbed by exchange of fluid medium by either method, thereby indicating that notches (wherever placed) are beneficial for improving the yield of 3D cell cultures in open-well microplates.

[0071] Unless otherwise expressly stated, any method set forth herein is in no way intended to be construed as requiring that its steps be performed in a particular order. Thus, unless a method claim actually recites the order in which its steps are to be followed, or the claim or description specifically states that the steps are to be limited to a particular order, no order is intended to be inferred in any respect. Additionally, as used herein, the article "a" is intended to include one or more components or elements, and is not intended to be construed as meaning only one.

[0072] It will be apparent to those skilled in the art that various modifications and variations can be made without departing from the scope or spirit of the disclosed embodiments. Since combinations, subcombinations, and variations of modifications of the disclosed embodiments that incorporate the spirit and essence of the embodiments may occur to those skilled in the art, the disclosed embodiments should be construed as including all within the scope of the appended claims and their equivalents.

Claims

1. 1. A device for three-dimensional cell growth, comprising: a substrate comprising an ultra-low attachment surface and a plurality of cavities for growing cell cultures; at least one sidewall surrounding the substrate; The device, wherein at least one notch is formed in the at least one side wall, the at least one notch being configured to accommodate a serological pipette for exchange of fluid media.

2. 2. The device of claim 1, wherein the at least one sidewall comprises a first short sidewall, a second short sidewall, a first long sidewall, and a second long sidewall, the first short sidewall and the second short sidewall each comprising a first length, and the first long sidewall and the second long sidewall each comprising a second length, the second length being greater than the first length.

3. The device of claim 2 , wherein the at least one notch comprises a first notch formed on the first short sidewall.

4. The device of claim 3 , wherein the first notch is located at a midpoint of the first short sidewall.

5. 5. The device of claim 3 or 4, wherein the at least one notch comprises a second notch formed in a corner between the first short sidewall and the first long sidewall.

6. The device of claim 2 , wherein the at least one notch comprises a first notch formed in a first corner between the first short sidewall and the first long sidewall.

7. 7. The device of claim 6, wherein the at least one notch comprises a second notch formed in a second corner between the first short sidewall and the second long sidewall.

8. 10. A device according to any one of the preceding claims, wherein the at least one notch comprises a substantially planar notch wall.

9. The device of any one of claims 1 to 7, wherein the at least one notch comprises a curved notch wall.

10. 10. A device according to any one of the preceding claims, wherein the at least one notch comprises a floor, the floor being angled relative to a plane defined by the substrate.

11. The device of claim 10 , wherein the floor is at a different elevation than the plane defined by the substrate.

12. The cavities are formed within 1 cm of the substrate. ２ 10. A device according to any one of the preceding claims, with at least five cavities per cavity.

13. 10. The device of any one of the preceding claims, wherein the at least one notch is configured to accommodate the tip of a 25 mL serological pipette oriented perpendicular to the substrate within the at least one notch.

14. 10. A device according to any one of the preceding claims, wherein the device has dimensions in accordance with ANSI / SLAS1-2004.

15. The device of claim 1 , wherein the at least one sidewall is a single circular sidewall.

16. 1. A method comprising: inserting a pipette into a first notch formed in at least one sidewall, the at least one sidewall surrounding a substrate of a cell culture device, the substrate comprising an ultra-low attachment surface and a plurality of cavities for growing cell cultures; adding a fluid into the first notch such that the fluid covers the substrate within the at least one sidewall.

17. 17. The method of claim 16, wherein the at least one sidewall comprises a first short sidewall, a second short sidewall, a first long sidewall, and a second long sidewall, the first short sidewall and the second short sidewall each comprising a first length, and the first long sidewall and the second long sidewall each comprising a second length, the second length being greater than the first length.

18. 18. The method of claim 17, wherein inserting further comprises inserting the pipette into the first notch, the first notch being formed on the first short sidewall.

19. 20. The method of claim 18, further comprising removing the fluid from above the substrate using a pipette inserted into the first notch.

20. 20. The method of claim 19, wherein prior to removing, the method further comprises raising the second short sidewall, the second short sidewall facing the first short sidewall such that the fluid flows toward the first notch formed on the first short sidewall.

21. The method of any one of claims 18 to 20, wherein the first notch is located at the midpoint of the first short side wall.

22. 18. The method of claim 17, wherein the cell culture device further comprises a second notch, wherein one of the first notch or the second notch is formed on the first short side wall and the other of the first notch or the second notch is formed in a corner between the first short side wall and the first long side wall.

23. 23. The method of claim 22, further comprising removing the fluid from above the substrate using a pipette inserted into the second notch.

24. 23. The method of claim 22, wherein prior to removing, the method further comprises raising the second short sidewall, the second short sidewall opposing the first short sidewall such that the fluid flows toward at least one of the first notch or the second notch.

25. 18. The method of claim 17, wherein inserting further comprises inserting the pipette into the first notch, the first notch being formed at a first corner between the first short sidewall and the first long sidewall.

26. 26. The method of claim 25, wherein the cell culture device further comprises a second notch formed in a second corner between the first short sidewall and the second long sidewall, the method further comprising removing the fluid from above the substrate using a pipette inserted into the second notch.

27. A method according to any one of claims 16 to 26, wherein the at least one notch comprises a substantially planar notch wall.

28. The method of any one of claims 16 to 26, wherein the at least one notch comprises a curved notch wall.

29. A method according to any one of claims 16 to 28, wherein the at least one notch comprises a floor, the floor being angled relative to a plane defined by the substrate.

30. 30. The method of claim 29, wherein the floor is at a different elevation than the plane defined by the substrate.

31. The cavities are formed within 1 cm of the substrate. ２ 31. The method of any one of claims 16 to 30, wherein there are at least 5 cavities per cavity.

32. 32. The method of any one of claims 16 to 31, wherein the at least one notch is configured to accommodate a 25 mL serological pipette oriented perpendicular to the substrate within the at least one notch.

33. The method of any one of claims 16 to 32, wherein the cell culture device has dimensions according to ANSI / SLAS1-2004.

34. The method of claim 16 , wherein the at least one side wall is a single circular side wall.

Images