Cell culture device comprising at least one recess formed in sidewall for exchanging fluid medium

Abstract

Embodiments of the present disclosure relate to an apparatus 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 groove is formed in the at least one sidewall, and the at least one groove is configured to receive a serum pipette for exchanging a fluid medium.

Description

[0001] Cross - reference to related applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 428,142, filed on November 28, 2022, under 35 U.S.C. § 119, the content of which is hereby incorporated by reference in its entirety. Background of the Invention

[0003] The present disclosure generally relates to a cell culture device, and more particularly, to a cell culture device having a bottom of a micro - cavity substrate that includes features that limit disruption to a cell culture during fluid exchange processes.

[0004] Compared with two - dimensional (2D) monolayer cell culture methods, three - dimensional (3D) cell culture models can better simulate the complex microenvironment of cells and tissues present in vivo, and thus have received much attention. With the development of biological research, cancer research, and regenerative therapies, there is an increasing demand for generating larger quantities of 3D cell cultures, including cell aggregates, tumor spheroids, embryoid bodies, and organoids. Cell culture devices capable of generating multiple 3D cell cultures in the same assay plate are available. In fact, certain microtiter plates can form thousands of 3D cell cultures with repeatable sizes and shapes within the space occupied by a traditional assay plate by incorporating a micro - cavity substrate with an ultra - low attachment surface (ULA) in an open - frame. To maintain 3D cell cultures over the days and weeks required to form multicellular 3D models and organoids, it is necessary to perform multiple rounds of fluid medium exchange while retaining the cells within the micro - cavity structure and minimizing disruption. For some cell culture devices, since only a small amount (in the μL - mL range) of liquid is required to fill the cavities and support the cultures, medium exchange can be performed without disturbing the 3D cultures. However, for certain high - density cell culture devices, a larger volume of liquid (e.g., 25 mL) needs to be moved to support a large number of 3D cell cultures throughout the device. This relatively large movement of fluid can cause adverse interference to the 3D cell cultures. Summary of the Invention

[0005] According to aspect (1), there is provided a device for three - dimensional cell growth. The device comprises: a substrate comprising an ultra - low attachment surface and a plurality of cavities for growing a cell culture; and at least one sidewall surrounding the substrate; wherein at least one groove is formed in the at least one sidewall, and the at least one groove is configured to receive a serum pipette for exchanging a fluid medium.

[0006] According to aspect (2), there is provided the apparatus 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 comprise a first length, and wherein the first long sidewall and the second long sidewall each comprise a second length, the second length being greater than the first length.

[0007] According to aspect (3), there is provided the apparatus of aspect (2), wherein the at least one groove comprises a first groove formed on the first short sidewall.

[0008] According to aspect (4), there is provided the apparatus of aspect (3), wherein the first groove is located at the midpoint of the first short sidewall.

[0009] According to aspect (5), there is provided the apparatus of aspect (3) or (4), wherein the at least one groove comprises a second groove formed at a corner between the first short sidewall and the first long sidewall.

[0010] According to aspect (6), there is provided the apparatus of aspect (2), wherein the at least one groove comprises a first groove formed at a first corner between the first short sidewall and the first long sidewall.

[0011] According to aspect (7), there is provided the apparatus of aspect (6), wherein the at least one groove comprises a second groove formed at a second corner between the first short sidewall and the second long sidewall.

[0012] According to aspect (8), there is provided the apparatus of any of the foregoing aspects, wherein the at least one groove comprises a generally flat groove wall.

[0013] According to aspect (9), there is provided the apparatus of any of aspects (1) to (7), wherein the at least one groove comprises a rounded groove wall.

[0014] According to aspect (10), there is provided the apparatus of any of the foregoing aspects, wherein the at least one groove comprises a bottom plate, wherein the bottom plate is angled relative to the plane defined by the substrate.

[0015] According to aspect (11), there is provided the apparatus of aspect (10), wherein the height of the bottom plate is different from the height of the plane defined by the substrate.

[0016] According to aspect (12), there is provided the apparatus of any of the foregoing aspects, wherein the plurality of cavities is at least 5 cavities per square centimeter of the substrate.

[0017] According to aspect (13), there is provided an apparatus of any of the foregoing aspects, wherein the at least one groove is configured to receive the tip of a 25 mL serum pipette oriented perpendicular to the substrate within the at least one groove.

[0018] According to aspect (14), there is provided an apparatus of any of the foregoing aspects, wherein the apparatus has dimensions compliant with ANSI / SLAS 1-2004.

[0019] According to aspect (15), there is provided the apparatus of aspect (1), wherein the at least one sidewall is a single circular sidewall.

[0020] According to aspect (16), there is provided a method. The method includes: inserting a pipette into a first groove formed in at least one sidewall surrounding a substrate of a cell culture apparatus, the substrate including an ultra-low attachment surface and a plurality of cavities for growing a cell culture; and adding a fluid to the first groove such that the fluid covers the substrate within the at least one sidewall.

[0021] According to aspect (17), there is provided the method of aspect (16), wherein the at least one sidewall includes 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 include a first length, and wherein the first long sidewall and the second long sidewall each include a second length, the second length being greater than the first length.

[0022] According to aspect (18), there is provided the method of aspect (17), wherein inserting further includes inserting the pipette into the first groove formed in the first short sidewall.

[0023] According to aspect (19), there is provided the method of aspect (18), which further includes removing the fluid from above the substrate using the pipette inserted into the first groove.

[0024] According to aspect (20), there is provided the method of aspect (19), wherein prior to removal, the method further includes raising the second short sidewall, which is opposite the first short sidewall, such that the fluid flows towards the first groove formed in the first short sidewall.

[0025] According to aspect (21), there is provided the method of any of aspects (18) to (20), wherein the first groove is located at the midpoint of the first short sidewall.

[0026] According to aspect (22), there is provided the method of aspect (17), wherein the cell culture device further comprises a second groove, and wherein one of the first groove or the second groove is formed on the first short sidewall, and the other of the first groove or the second groove is formed at a corner between the first short sidewall and the first long sidewall.

[0027] According to aspect (23), there is provided the method of aspect (22), which further comprises removing the fluid from above the substrate using a pipette inserted into the second groove.

[0028] According to aspect (24), there is provided the method of aspect (22), the method further comprising raising the second short sidewall, which is opposite to the first short sidewall, such that the fluid flows towards at least one of the first groove or the second groove.

[0029] According to aspect (25), there is provided the method of aspect (17), wherein the insertion further comprises inserting the pipette into the first groove, which is formed at a first corner between the first short sidewall and the first long sidewall.

[0030] According to aspect (26), there is provided the method of aspect (25), wherein the cell culture device further comprises a second groove formed at a second corner between the first short sidewall and the second long sidewall, and wherein the method further comprises removing the fluid from above the substrate using a pipette inserted into the second groove.

[0031] According to aspect (27), for the method of any one of aspects (16) to (26), wherein the at least one groove comprises a generally flat groove wall.

[0032] According to aspect (28), for the method of any one of aspects (16) to (26), wherein the at least one groove comprises a rounded groove wall.

[0033] According to aspect (29), for the method of any one of aspects (16) to (28), wherein the at least one groove comprises a bottom plate, wherein the bottom plate is angled relative to the plane defined by the substrate.

[0034] According to aspect (30), there is provided the method of aspect (29), wherein the height of the bottom plate is different from the height of the plane defined by the substrate.

[0035] According to aspect (31), for the method of any one of aspects (16) to (30), wherein the plurality of cavities are at least 5 cavities per square centimeter of the substrate.

[0036] According to aspect (32), a method according to any one of aspects (16) to (31), wherein the at least one groove is configured to receive a 25 mL serum pipette oriented perpendicular to the substrate within the at least one groove.

[0037] According to aspect (33), a method according to any one of aspects (16) to (32), wherein the cell culture device has dimensions compliant with ANSI / SLAS 1-2004.

[0038] According to aspect (34), there is provided a method according to aspect (16), wherein the at least one sidewall is a single circular sidewall.

[0039] Additional features and advantages will be set forth in the detailed description which follows, and in part will be readily apparent to those skilled in the art from the description, or recognized by practicing the embodiments described in the written description and the claims and the accompanying drawings.

[0040] It is to be understood that both the foregoing general description and the following detailed description are exemplary and intended to provide an overview or framework for understanding the nature and characteristics of the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] 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 operations of the various embodiments. In the drawings:

[0042] Figure 1A depicts an open-cell culture device according to an exemplary embodiment, the device including two grooves formed at the corners of the device sidewalls;

[0043] Figure 1B depicts according to an exemplary embodiment Figure 1A a detailed view of the cavity of a cell culture device;

[0044] Figure 2 depicts a cell culture device containing a fluid medium according to an exemplary embodiment;

[0045] Figure 3 depicts a cell culture device having a lid and a tray according to an exemplary embodiment;

[0046] Figure 4 depicts a cell culture device according to an exemplary embodiment, wherein one groove is formed in the sidewall of the device;

[0047] Figure 5ADepict a cell culture device according to an exemplary embodiment, wherein one groove is formed in the side wall of the device, and another groove is formed at the corner of the side wall of the device;

[0048] Figure 5B Depict a detailed view of a corner groove (such as the corner groove shown as Figure 5A shown) according to an exemplary embodiment;

[0049] Figure 6 Depict a groove with rounded walls according to an exemplary embodiment;

[0050] Figure 7 Depict a groove with flat walls according to an exemplary embodiment;

[0051] Figure 8 Depict a cross-sectional view of a groove and a substrate according to an exemplary embodiment, showing the angle of the groove bottom plate relative to the substrate plane;

[0052] Figure 9 Depict an embodiment of a porous cell culture device according to an exemplary embodiment, wherein each well of the device is provided with a groove; and

[0053] Figure 10 Depict a flowchart of a method for adding and removing a fluid medium to and from a cell culture device according to an exemplary embodiment. Detailed Description

[0054] Generally referring to the following description and drawings, various embodiments of a cell culture device having at least one groove formed in a side wall are provided. As will be described more fully below, the groove formed in the side wall of the cell culture device houses a pipette for adding and removing a fluid (such as a culture medium, etc.) to and from the cell culture device. Such devices can be used to grow scaffold-free three-dimensional cell cultures (such as cell aggregates, spheroids, organoids, etc.) in the cavity of the substrate of the cell culture device. Advantageously, the groove can exchange fluids while reducing interference with the growing cell culture. That is, fluid exchange for supplementing nutrients and removing waste typically causes a certain degree of loss of the cell culture due to the flow of the fluid above the substrate. By providing a groove through which a fluid can be added to and removed from a microplate, the loss of the cell culture during the fluid exchange process is reduced. These and other aspects and advantages of the disclosed cell culture device and its use method will be described herein and in conjunction with the drawings. Such exemplary embodiments are provided by way of illustration and not limitation.

[0055] Figure 1ADepicts an embodiment of a cell culture device, particularly a plate 100 configured for three-dimensional cell growth, such as an open-well plate. The plate 100 can be a microplate. The open-well plate 100 includes a substrate 102 having a plurality of cavities 104 (such as Figure 1B shown) for growing cell cultures, such as cell aggregates, spheroids, and organoids. For example, in one or more embodiments, the plurality of cavities 104 is at least 5 cavities per square centimeter of the substrate. Figure 1B Provides an enlarged view of the substrate 102 depicting the cavities 104, each containing a cell culture, particularly a spheroid 105. In Figure 1B the specific embodiment shown, the diameter of the cavity 104 is about 800 μm. In one or more embodiments, the substrate 102, particularly the cavities 104, is coated with an ultra-low attachment surface (ULA), which refers to a class of coatings known in the art for forcing cells into a suspended state.

[0056] Returning to Figure 1A , the open-well plate 100 further includes a plurality of sidewalls 106 that form a perimeter around the substrate 102. At least one groove 108 is formed in the plurality of sidewalls 106. In one or more embodiments, the at least one groove 108 is configured to accommodate a pipette for fluid exchange, such as a serum pipette. For example, in one or more embodiments, the at least one groove 108 is configured to accommodate a 25 mL serum pipette oriented perpendicular to the substrate 102 within the at least one groove 108.

[0057] 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 defines a rounded rectangular perimeter of the substrate 102. In one or more embodiments, the dimensions of the open-well plate 100 conform to ANSI / SLAS1-2004. Although specific embodiments and shapes of the open-well plate 100 are described and depicted herein, the present disclosure also encompasses other shapes and sizes.

[0058] In one or more embodiments, the at least one groove 108 includes a first groove 114 and a second groove 116. In one or more embodiments, the first groove 114 is formed at a corner between the first short sidewall 110a and the first long sidewall 112a. In one or more embodiments, the second groove 116 is formed at a corner between the first short sidewall 110a and the second long sidewall 112b. As mentioned, the groove 108 is provided to facilitate fluid exchange of the substrate 102. For example, the fluid may contain nutrients for growing a cell culture, so when the nutrients are depleted, the fluid can be replenished. In addition, the old fluid can be removed to remove the waste of the cell culture in the fluid.

[0059] Figure 2 An example of the perforated plate 100 is depicted, where the substrate 102 is covered by the fluid 118. When a cell culture grows in the cavity 104 of the substrate 102, the perforated plate 100 can be configured for storage or processing. Thus, as Figure 3 shown, the perforated 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 spillage. In one or more embodiments, as Figure 3 shown in the embodiment, the lid 120 is made of a transparent material such that the contents of the perforated plate 100 can be observed through the lid 120. In one or more embodiments, the tray 122 provides a flat and stable surface for the perforated plate 100.

[0060] Figure 4 Another embodiment of the perforated plate 100 is depicted. In one or more embodiments, the at least one groove 108 of the perforated plate 100 is only the first groove 114 formed on the first short sidewall 110a. In one or more embodiments, including Figure 4 the embodiment depicted in, the first groove 114 is approximately located at the midpoint 124 of the first short sidewall 110a. In one or more embodiments, the midpoint 124 where the first groove 114 is centered is a point between 40% and 60% (i.e., between 0.4L1 and 0.6L1) of the first length L1 of the first short sidewall 110a.

[0061] Figure 5A Another embodiment of the perforated plate 100 having two grooves 108 is depicted. As in the previous embodiment, the first groove 114 is approximately provided at the midpoint 124 of the first short sidewall 110a. The second groove 116 is provided at a corner between the first short sidewall 110a and the second long sidewall 112b.

[0062] Figure 6Depict an embodiment of a recess 108 having rounded recess walls 126. In one or more embodiments, the rounded recess walls 126 are curved with a radius of curvature of 3.0 mm to 4.2 mm. Additionally, the width W and depth (defined by the radius of curvature) of the recess 108 are configured to accommodate a pipette, such as a 25 ml serum pipette. In one or more embodiments, the width W is 5 mm to 15 mm, particularly 8 mm to 11 mm. The recess 108 also has a recess bottom plate 128. In one or more embodiments, the height of the recess bottom plate 128 is different from the height of the substrate 102. In one or more embodiments, the recess bottom plate 128 is spaced a distance D above the substrate 102. In one or more embodiments, the distance D is 0.5 mm to 1.5 mm. In one or more embodiments, the recess 108 having rounded recess walls 126 can be either or both of the first recess 114 and the second recess 116 of any embodiment of the open plate 100 described herein.

[0063] Figure 7 Depict an embodiment of a recess 108 including a plurality of generally flat recess walls 126. In one or more embodiments, the recess 108 has three flat recess walls 126, thereby defining a rectangular recess 108. However, in one or more other embodiments, the recess 108 can have two flat recess walls 126 or more than three flat recess walls 126. In one or more embodiments, the width W and depth of the recess 108 are configured to accommodate a pipette, such as a 25 ml serum pipette. In one or more embodiments, the width W is 5 mm to 15 mm, particularly 8 mm to 11 mm. The recess 108 also has a recess bottom plate 128. In one or more embodiments, the recess bottom plate 128 is spaced a distance D above the substrate 102, such as a distance D of 0.5 mm to 1.5 mm. In one or more embodiments, the recess 108 having generally flat recess walls 126 can be either or both of the first recess 114 and the second recess 116 of any embodiment of the open plate 100 described herein.

[0064] Figure 5B Depict a specific embodiment of a corner recess 108 having rounded recess walls 126. For purposes of discussion, reference will be made to Figure 5A the second recess 116 in Figure 5BAs shown, the second groove 116 is formed at the intersection of the first short side wall 110a and the second long side wall 112b. Additionally, in one or more embodiments, the opening angle θ of the second groove 116 with respect to the second long side wall 112b is from 20° to 40°, particularly about 34°. Due to the angled opening of the second groove 116, the edge of the second groove 116 abutting against the second long side wall 112b is longer than the edge of the second groove 116 disposed on the first short side wall 110a. Additionally, as described more fully in conjunction with Figure 8 the groove bottom plate 128 of the second groove 116 in Figure 5B is inclined towards the substrate 102. Due to the slope of the groove bottom plate 128 and the longer extension of one edge of the second groove 116, the fluid added at the second groove 112 will flow towards the lowest point of the second groove 116 under the influence of surface tension, i.e., towards the edge formed with the second long side wall 112b. In this way, the fluid added to the second groove 116 is guided away from the substrate and towards the second long side wall 112b. Advantageously, this helps to reduce the interference caused by the added liquid to the cells growing in the substrate 102.

[0065] Figure 8 A cross-sectional view depicting the groove 108 with respect to the substrate 102. As Figure 8 shown, the substrate 102 defines a plane 130. In one or more embodiments, the groove bottom plate 128 is angled α with respect to the plane 130. In one or more embodiments, the angle α is at most 15°, particularly at most 11°. Advantageously, the angled groove bottom plate 128 helps to promote the flow of the fluid medium from the groove 108 into the region above the substrate 102. In particular, when combined with the angled opening of the corner groove (as described in conjunction with Figure 5B ), the fluid indirectly flows into the region above the substrate 102 after first being drawn towards the side wall 106.

[0066] Although the foregoing embodiments have focused on the side wall 106 perforated plate 100 having a rectangular perimeter defining a single substrate 102, other embodiments of the device may include multiple isolated compartments (i.e., micro-wells, multiple holes, alternative dish or reservoir geometries), each compartment having its own microchamber substrate bottom. According to Figure 9In an example embodiment shown, the plate 100 includes a plurality of holes 150a-f, each hole having a corresponding substrate 102a-f. Each hole 150a-f is defined by a single corresponding sidewall 106a-f (circular sidewall), and each sidewall 106a-f includes at least one groove for adding and removing fluid to and from the hole 150a-f. In one or more embodiments, each hole 150a-f includes two grooves, namely a first groove for adding a fluid medium and a second groove for removing the fluid medium (for ease of illustration, only the grooves 108a of hole 150a are labeled, including a first groove 114a and a second groove 116a). The holes 150a-f can be used to grow different cell cultures in each hole and / or to grow the same cell culture in each hole under different conditions, among other possibilities.

[0067] Figure 9 Figure 100 depicts a plate 100 having a plurality of substrates 102, but other embodiments can have a single substrate 102 and a single sidewall 106 (e.g., a circular disk), among other possibilities.

[0068] Embodiments of the present disclosure also relate to a method of adding and removing fluid to and from a plate (such as a microplate). Figure 10 A process flow diagram of method 200 is provided, which will be described in connection with Figure 1A - 5A the embodiment of the plate 100 depicted in Figure 100 (although method 200 is applicable to any cell culture device having grooves for adding or removing fluid). In one or more embodiments, method 200 includes a first step 201 of inserting a pipette into a groove 108 formed in the peripheral sidewall 106 of the open-well plate 100.

[0069] In one or more embodiments, method 200 further includes a second step 202 of adding fluid to the groove 108 such that the fluid covers the substrates 102 within the plurality of sidewalls 106. In one or more embodiments, the pipette is arranged perpendicular to the substrate 102 such that the fluid is directed generally onto the bottom plate 128 of the groove. However, in one or more other embodiments, the pipette is arranged transverse to the plane of the substrate 102 such that the fluid is directed at least partially against the groove wall 126. Once the fluid covers the substrate 102, the open-well plate 100 can be stored in a controlled environment to allow cell culture growth.

[0070] After the required amount of time has elapsed and there is a need to exchange the fluid medium, the plate 100 can be retrieved to remove the fluid medium. To facilitate the removal of the fluid medium, in one or more embodiments, method 200 further includes an optional third step 203 of raising the sidewall 106 of the plate 100 opposite the recess 108 such that the fluid medium flows towards the recess 108 (the first recess 114 or the second recess 116) formed in the peripheral sidewall 106 of the plate 100. In experiments, the inventors found that raising one side of the plate 100 can cause the fluid medium to drain to the opposite side of the device, thereby facilitating the removal of the fluid medium. However, the inventors also found that even when one side is not raised to drain the fluid to the opposite side, the recess 108 allows for the removal of more than 50% of the fluid medium.

[0071] In one or more embodiments, method 200 further includes a fourth step 204 of removing the fluid medium from the recess 108 (the first recess 114 or the second recess 116) formed in the peripheral sidewall 106 of the plate 100. Method 200 can be repeated until the cell culture has grown to the desired level.

[0072] Advantageously, embodiments of the device provide a convenient way to exchange the fluid medium without causing significant interference to the cell culture. In this regard, the inventors envision that the fluid medium can be exchanged while retaining ≥90% of the cell yield of the device. In the absence of the recess 108 or recesses 108 formed in the sidewall 106, the addition or removal of the fluid medium would occur directly above the cell culture bottom 102 or against one of the sidewalls 106, resulting in significant cell interference and displacement. The inventors observed that when using a conventional cell culture device for the exchange, the cell culture yield is typically less than 90%, often far less than 90%. However, at least one recess 108 can keep the placement of the pipette used for fluid medium exchange consistent. In fact, random placement of the pipette during the fluid medium exchange would result in inconsistent culturing and results. Additionally, having multiple recesses 108 can accommodate left-handed and right-handed operators while providing consistent pipette placement during the fluid medium exchange.

[0073] Various embodiments of the plate 100 were tested to determine the ease of exchanging the fluid medium and the degree of interference to the cell culture.

[0074] In the first experiment, the following perforated plates 100 were prepared: (1) having two grooves 108, wherein a first groove 114 is located at a first corner on one side and a second groove 116 is located at a second corner on said side; (2) having a single groove 108 at the midpoint on one side; (3) having two grooves 108, wherein a first groove 114 is located at the midpoint on one side and a second groove 116 is located at a corner on said side; and (4) having a single groove 108 at one corner.

[0075] Regarding the first perforated plate 100, 13 mL of fluid was added at the first groove 114, and during fluid removal, 11 mL of fluid could be removed while the perforated plate 100 remained flat. It was observed that the two grooves 108 were beneficial for both left-handed and right-handed operators, especially when accessing the front or rear of the perforated plate as needed.

[0076] Regarding the second perforated plate 100, 13 mL of fluid was added at the single-sided groove 108, and during removal, 7.5 mL of fluid could be removed while the perforated plate 100 remained flat.

[0077] Regarding the third perforated plate 100, 13 mL of fluid was added at the second groove 116 located at the corner, and during removal, only a few milliliters could be removed from either the first groove 114 or the second groove 116. When the side opposite the groove was raised by approximately 3°, 10.2 mL could be removed from the second groove 116.

[0078] Regarding the fourth perforated plate 100, 13 mL of fluid was added at the single corner groove 108, and during removal, only 5 mL of fluid could be removed while the plate 100 remained flat. It was observed that the rounded groove walls 126 located at the corner could easily accommodate the pipette tip.

[0079] In a second experiment, a microwell plate was prepared having three grooves 108 formed in the sidewalls 106. In particular, the first short sidewall 110a included a groove 108 at each corner and a groove 108 at the midpoint 124. The plate 100 was prepared by providing a ULA surface. The trapped air in the microcavities was removed by centrifugation. Subsequently, pre-formed spheres were filled in the base cavities near the first short sidewall 110a. The plate was imaged before adding or removing the fluid medium. To evaluate sphere disruption, 10 mL of fluid medium was added and then removed. During the removal process, the second short sidewall 110b was raised by approximately 3°. Three fluid exchange conditions were considered: (1) adding and removing fluid from the same corner groove 108, (2) adding fluid at the midpoint groove 108 and removing fluid from the corner groove 108, and (3) adding fluid at one corner groove 108 and removing fluid from another corner groove 108. After each fluid addition and each fluid removal, the plate 100 was imaged using a Nikon NEXIV Visual Measuring System (VMR).

[0080] Five different operators performed the above-described fluid addition and removal steps, and it was observed that each operator experienced at least some sphere loss when using each of the three addition and removal techniques. However, it was generally also observed that fluid addition had a greater impact on sphere loss than fluid removal. In addition, it was also observed that the sphere loss from the midpoint groove 108 was greater than the sphere loss from the corner groove 108.

[0081] In a third experiment, the sphere retention rate was again evaluated. 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 seeded at 12 × 10 per plate 6Cells (about 1,000 cells per basal cavity) were seeded. 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 VMR to provide a control image. The other twelve plates had their media exchanged and incubation continued. Four media exchange methods were evaluated and each method was performed by three operators. The four methods involved: (1) using a pipette at a lateral angle to the plane of the substrate 102 to remove and add fluid media at the corner recess 108; (2) using a pipette at a perpendicular angle to the plane of the substrate 102 to remove and add fluid media at the corner recess 108; (3) using a pipette at a lateral angle to the plane of the substrate 102 to remove fluid media from the corner recess 108 and add fluid media at the midpoint recess 108; and (4) using a pipette at a perpendicular angle to the plane of the substrate 102 to remove fluid media from the corner recess 108 and add fluid media at the midpoint recess 108. The three operators each used these methods to perform three more fluid exchanges over the next eight days. After the fourth media exchange on the tenth day, the twelve plates were imaged using VMR and analyzed using Halcon to quantify sphere loss.

[0082] From this experiment, it was determined that most sphere loss occurred in the cavities within the first 30% of the region adjacent to the sidewall where the liquid was added. In particular, according to the first method, i.e., using an angled pipette to remove and add fluid media from the corner recess 108, a total of 5% of the spheres were lost (average between three microplates), and 73% (average) of the sphere loss occurred within the first 30% of the substrate 102. According to the second method, i.e., using a perpendicular pipette to remove and add fluid media from the corner recess 108, a total of 4% of the spheres were lost, and 46% of the sphere loss occurred within the first 30% of the substrate 102. According to the third method, i.e., using an angled pipette to remove fluid media from the corner recess 108 and add fluid media at the midpoint recess 108, a total of 6% of the spheres were lost, and 68% of the sphere loss occurred within the first 30% of the substrate 102. The results obtained from the fourth method were the same as the third method. The inventors observed that this experiment confirmed the previous observation that fluid addition has a greater impact on sphere loss than fluid removal, and that sphere loss is greater when adding fluid media at the midpoint recess 108. It was further determined that the angle of the pipette had little effect at the midpoint recess 108, but when the pipette was used at a perpendicular angle for removal / addition at the corner recess 108, the sphere loss was reduced by approximately 1%. Nevertheless, it was observed that more than 90% of the spheres were not disturbed by the fluid media exchange performed according to any method, indicating that the recesses (wherever placed) are beneficial for increasing the yield of 3D cell cultures in open-well microplates.

[0083] Unless otherwise expressly stated, it is not intended that any method described herein be construed as requiring that its steps be performed in a particular order. Thus, in the event that a method claim does not actually recite the order in which its steps are to be followed or in the absence of another specific statement in the claims or specification that the steps are limited to a particular order, it is not intended to infer any particular order. Furthermore, the article "a" as used herein is intended to include one or more than one component or element and is not intended to be construed to mean only one.

[0084] It is obvious to those skilled in the art that various modifications and changes may be made without departing from the spirit or scope of the disclosed embodiments. Since those skilled in the art may conceive of modified combinations, sub-combinations, and changes of the disclosed embodiments that incorporate the spirit and substance of the embodiments, the disclosed embodiments should be interpreted as including all contents within the scope of the attached claims and their equivalents.

Claims

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; and at least one sidewall surrounding the substrate; wherein at least one groove is formed in the at least one sidewall, and the at least one groove is configured to receive a serum pipette for exchanging a fluid culture medium.

2. The device according to 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, wherein the first short sidewall and the second short sidewall each comprise a first length, and wherein the first long sidewall and the second long sidewall each comprise a second length, the second length being greater than the first length.

3. The device according to claim 2, wherein the at least one groove comprises a first groove formed in the first short sidewall.

4. The device according to claim 3, wherein the first groove is located at the midpoint of the first short sidewall.

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

6. The device according to claim 2, wherein the at least one groove comprises a first groove formed at a first corner between the first short sidewall and the first long sidewall.

7. The device according to claim 6, wherein the at least one groove comprises a second groove formed at a second corner between the first short sidewall and the second long sidewall.

8. The device according to any one of the preceding claims, wherein the at least one groove comprises a generally flat groove wall.

9. The device according to any one of claims 1 to 7, wherein the at least one groove comprises a rounded groove wall.

10. The device according to any one of the preceding claims, wherein the at least one groove comprises a bottom plate, and the bottom plate is angled relative to a plane defined by the substrate.

11. The device according to claim 10, wherein the height of the bottom plate is different from the height of the plane defined by the substrate.

12. The device according to any one of the preceding claims, wherein the plurality of cavities are at least 5 cavities per square centimeter of the substrate.

13. The device according to any one of the preceding claims, wherein the at least one groove is configured to receive the tip of a 25 mL serum pipette oriented perpendicular to the substrate within the at least one groove.

14. The device according to any one of the preceding claims, wherein the device has dimensions compliant with ANSI / SLAS1-2004.

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

16. A method, comprising: inserting a pipette into a first groove formed in 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 Add fluid to the first recess such that the fluid covers the substrate within the at least one sidewall.

17. The method according to claim 16, wherein the at least one sidewall includes 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 include a first length, and wherein the first long sidewall and the second long sidewall each include a second length, the second length being greater than the first length.

18. The method according to claim 17, wherein the inserting further includes inserting the pipette into the first recess formed in the first short sidewall.

19. The method according to claim 18, further comprising removing the fluid from above the substrate using the pipette inserted into the first recess.

20. The method according to claim 19, wherein prior to the removing, the method further comprises raising the second short sidewall opposite the first short sidewall such that the fluid flows towards the first recess formed in the first short sidewall.

21. The method according to any one of claims 18 to 20, wherein the first recess is located at the midpoint of the first short sidewall.

22. The method according to claim 17, wherein the cell culture device further includes a second recess, and wherein one of the first recess or the second recess is formed in the first short sidewall, and the other of the first recess or the second recess is formed at a corner between the first short sidewall and the first long sidewall.

23. The method according to claim 22, further comprising removing the fluid from above the substrate using the pipette inserted into the second recess.

24. The method according to claim 22, wherein prior to the removing, the method further comprises raising the second short sidewall opposite the first short sidewall such that the fluid flows towards at least one of the first recess or the second recess.

25. The method according to claim 17, wherein the inserting further includes inserting the pipette into the first recess formed at a first corner between the first short sidewall and the first long sidewall.

26. The method according to claim 25, wherein the cell culture device further includes a second recess formed at a second corner between the first short sidewall and the second long sidewall, and wherein the method further comprises removing the fluid from above the substrate using the pipette inserted into the second recess.

27. The method according to any one of claims 16 to 26, wherein the at least one recess includes a generally flat recess wall.

28. The method according to any one of claims 16 to 26, wherein the at least one recess includes a rounded recess wall.

29. The method according to any one of claims 16 to 28, wherein the at least one recess includes a bottom plate, wherein the bottom plate is angled relative to the plane defined by the substrate.

30. The method according to claim 29, wherein the height of the bottom plate is different from the height of the plane defined by the substrate.

31. The method according to any one of claims 16 to 30, wherein the plurality of cavities are at least 5 cavities per square centimeter of the substrate.

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

33. The method according to any one of claims 16 to 32, wherein the cell culture device has dimensions compliant with ANSI / SLAS 1-2004.

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

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