Stackable coverslip holder
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2026-02-03
- Publication Date
- 2026-08-13
Smart Images
Figure SE2026010038_13082026_PF_FP_ABST
Abstract
Description
[0001] Stackable coverslip holder
[0002] Technical field
[0003]
[0001] The present disclosure relates to the field of laboratory equipment, such as single-use articles for cell culture, and in particular a coverslip holder that enables convenient and repeatable co-culturing of cells.
[0004] Background
[0005]
[0002] In vitro cell culture methods are used for studying physiological systems in a controlled laboratory environment. Such methods are essential for research and development in several fields, such as pharmacology, drug development, regenerative medicine, toxicology and biomaterial development.
[0006]
[0003] In a cell culture, isolated cells are incubated under controlled conditions. A common and well-established method is to grow a single layer of cells on a flat surface, providing a physiological media with sufficient nutrients, growth factors etc., and controlling relevant environmental factors such as the temperature and sterilization. The containers used for cell culture include single-use flasks or plates such as Petri dishes or multi-well plates. The cell culture conditions are of high importance for achieving sufficient cell viability and proliferation.
[0007]
[0004] In the above-mentioned fields of research and development, there is a transition from using laboratory animals (animal models) to the use of in vitro models, e.g., cell cultures. However, modelling a part of a full system e.g., a tissue, an organ or even an organism understandably requires more complex cell culture systems. One important aspect is that such systems need to contain several cell types and consequently involve cellular communication through paracrine signaling. More complex cell culture systems, often referred to as co-culture systems, make it possible to culture several cell types simultaneously and thereby incorporate cellular signaling.
[0005] For example, the international patent application WO 2023 / 218188 discloses a cell culture system that allows the combination of up to four different cell types in one co-culture.
[0008]
[0006] There is however always a need for improved products and supplies for cell culture, and with an increasing demand for in vitro testing, and in particular the transition towards non-animal testing methods in drug-development and medical research, there remains a need for new, improved devices and methods for coculturing cells.
[0009] Summary
[0010]
[0007] A first aspect of the present invention relates to a stackable coverslip holder comprising an upper portion having an outer diameter (DI) and a height (Hl), said upper portion forming a rim defining a first space for receiving a coverslip; a lower portion forming a skirt defining a second space having an inner diameter (D2) and a height (H2); wherein the outer diameter (DI) of said upper portion is smaller than the inner diameter (D2) of said second space making one coverslip holder stackable on another coverslip holder; and wherein the height (Hl) of said upper portion is the same or smaller than the height (H2) of said second space; and wherein said skirt has multiple openings extending from the outer periphery of the skirt to the second space.
[0011]
[0008] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, the first space comprises a circumferential abutment for supporting a coverslip.
[0012]
[0009] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said rim further has a circumferential projection which together with the abutment defines a space for receiving a coverslip and, optionally, a locking element.
[0010] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, the first space comprises a second circumferential abutment for supporting the locking element.
[0013] [Oil] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said rim comprises an indentation or cutout reaching from an outer periphery to an inner periphery of said rim and forming a discontinuation in said circumferential abutment.
[0014]
[0012] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said skirt comprises two vertical cutouts defining a protrusion.
[0015]
[0013] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said upper portion and the second space both have a shape chosen from a truncated cone, a cube, or a truncated pyramid, wherein the upper cross-section of the upper portion is smaller than the bottom cross-section of the second space.
[0016]
[0014] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said first space is dimensioned for receiving a round coverslip with a diameter chosen from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 30, 32, 35, 40, or 50 mm.
[0017]
[0015] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, the coverslip is a round coverslip with a diameter of 18 mm.
[0018]
[0016] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said first space is dimensioned for receiving a square coverslip having a size chosen from 15 x 15 mm, 18 x 18 mm, 20 x 20 mm, or 22 x 22 mm.
[0019]
[0017] A second aspect of the present invention relates to a coverslip holder system comprising a coverslip holder according to the first aspect and any embodimentsthereof, said system comprising a coverslip and a locking element, and one or more elements chosen from annular elements, and / or membranes.
[0020]
[0018] According to one embodiment of said second aspect, freely combinable with other embodiments disclosed herein, wherein in the coverslip holder an area available for cell growth on the coverslip in said coverslip holder is defined by an annular element placed onto the coverslip, said annular element having an aperture with an area (a) smaller than the area of the coverslip.
[0021]
[0019] According to one embodiment of said second aspect, freely combinable with other embodiments disclosed herein, wherein in the coverslip holder a volume available for cell growth on the coverslip held in said coverslip holder, is defined horizontally by an annular element having an aperture with an area (a) and a height (h), and vertically by a permeable membrane.
[0022]
[0020] According to one embodiment of said second aspect, freely combinable with other embodiments disclosed herein, wherein in the coverslip holder a volume available for cell growth on the coverslip held in said coverslip holder, is defined horizontally by a first annular element and a second annular element, and vertically by a permeable membrane attached to said second annular element.
[0023]
[0021] According to one embodiment of said second aspect, freely combinable with other embodiments disclosed herein, wherein in the coverslip holder comprises a circumferential projection which together with the abutment defines a space for receiving a coverslip, and one or more elements chosen from annular elements, and / or membranes, wherein a second circumferential abutment supports the locking element.
[0024]
[0022] A third aspect of the present invention relates to a co-culture arrangement comprising at least two, preferably three or more coverslip holders according to the first aspect and any embodiments thereof, and / or at least one coverslip holder system according to the second aspect and any embodiments thereof.
[0025]
[0023] A fourth aspect of the present invention relates to a cell culture method wherein at least two different cell types are cultured simultaneously in one culturemedium, wherein each cell type is cultured on a coverslip placed in a coverslip holder according to the first aspect and any embodiments thereof, and at least two coverslip holders housing different cell types are placed in a culture medium, preferably stacked.
[0026]
[0024] According to one embodiment of said fourth aspect, wherein at least two, preferably three or more different cell types are cultured simultaneously in the same culture medium.
[0027]
[0025] A fifth aspect of the present invention relates to a cell culture method using a coverslip holder according to the first aspect and any embodiments thereof.
[0028]
[0026] A sixth aspect of the present invention relates to a cell culture method using a coverslip holder system according to the second aspect and any embodiments thereof.
[0029]
[0027] A seventh aspect of the present invention relates to a cell culture method using a co-culture arrangement according to the third aspect and any embodiments thereof.
[0030]
[0028] An eighth aspect of the present invention relates to an element for use in a cell culture system according to the second aspect and any embodiments thereof, comprising an annular element with an inner aperture, having a height and inner diameter defining an inner volume, and a membrane attached thereto, said membrane being permeable to water and macromolecules but impervious to cells.
[0031]
[0029] Further aspects and embodiments, as well as their advantages, will be apparent from the attached drawings and the detailed description.
[0032] Short description of the drawings
[0033]
[0030] The invention is now described, by way of example, with reference to the accompanying drawings, in which:
[0031] Fig. 1 shows an example of a cell culture system known in the prior art, the Nanostacks™ system described in WO 2023 / 218188, which allows the combination of up to four different cell types, cultured on permeable membranes (2) integrated in stackable, concave receptacles (1).
[0034]
[0032] Fig. 2A shows a perspective view of a coverslip holder (100') according to an embodiment of the present disclosure, seen from above showing inter alia an upper part forming a rim (111), and a lower portion (131), forming a skirt.
[0035]
[0033] Fig. 2B shows a perspective view of a coverslip holder (100") showing the above features and additionally illustrating an embodiment where a cutout (112) forms a discontinuity in the rim, freely combinable with another embodiment, where two cutouts define a protrusion (135) in the skirt.
[0036]
[0034] Fig. 3A shows a cross-section of a coverslip holder (100') according to an embodiment of the present disclosure, said coverslip holder comprising an upper part forming a rim (111), and a lower portion (130), forming a skirt (131), and a circumferential abutment (113) for receiving a coverslip (200).
[0037]
[0035] Fig. 3B shows a cross-section of a coverslip holder (100') according to an embodiment of the present disclosure, said coverslip holder comprising the above features, and additionally a second circumferential abutment (1130), for supporting an additional element, for example a locking element.
[0038]
[0036] Fig. 4A shows a view of a coverslip holder (100') from above, illustrating the space defined by the upper portion (120).
[0039]
[0037] Fig. 4B shows a view of a coverslip holder (100") from above, illustrating the space defined by the upper portion (120), and showing a cutout in the rim (112), freely combinable with two cutouts (133, 134) in the skirt, defining a projection or handle (135).
[0040]
[0038] Fig. 5A shows a view of a coverslip holder (100') from below, showing the lower portion (130) defining a second space (140) and a skirt (131) with multiple openings (132).
[0039] Fig. 5B shows a view of a coverslip holder (100") from below with the above features and further illustrates an embodiment with a discontinuation (114) in the abutment (113), freely combinable with two cutouts (133, 134) in the skirt defining a projection or handle (135).
[0041]
[0040] Fig. 6 shows an exploded view of a coverslip holder (100"), with a coverslip (200) and a locking element (300).
[0042]
[0041] Fig. 7A shows an exploded view of an embodiment of a coverslip holder (100") where a coverslip (200), a first annular element (401), a second annular element (402) and a permeable membrane (500), and optionally a locking element (300), form a cage limiting a volume defined by the coverslip, annular elements, and the membrane.
[0043]
[0042] Fig. 7B shows an exploded view of an annular element (402) and a membrane (500) according to an embodiment of the present disclosure.
[0044]
[0043] Fig. 8A shows a perspective view with a cross-section of a coverslip holder (100") according to an embodiment of the present disclosure, with a coverslip (200), an annular element (401) and a locking element (300).
[0045]
[0044] Fig. 8B shows a perspective view with a cross-section of a coverslip holder (100") according to an embodiment of the present disclosure, with a coverslip (200), a first annular element (401), a second annular element (402), a membrane (500) and a locking element (300), wherein said coverslip, annular elements and membrane define a volume (150) available for cell culture.
[0046]
[0045] Fig. 9A shows how several coverslip holders (101a, 102a, 103a) are stacked, forming a co-culture arrangement.
[0047]
[0046] Fig. 9B shows how several coverslip holders (101b, 102b, 103b) are stacked, forming a co-culture arrangement, wherein each coverslip (200), annular elements (401, 402) and membrane (500) define a volume (150) available for cell culture.
[0047] Fig. 10 shows schematically a stack of coverslip holders (101, 102, 103), here shown as three but not limited to any specific number, immersed in a nutrient solution for co-culturing different cells separately, while under the same conditions.
[0048]
[0048] Fig. 11 illustrates how the cutout (112) facilitates the insertion and removal of the coverslip (200), and how the protrusion (135) forms a handle that facilitates handling of individual coverslip holders (100"), here shown with a schematical illustration of tweezers
[0049]
[0049] Fig. 12 schematically shows different geometries of the coverslip holder, chosen from a truncated cone (701), a box-like structure or cube (702), and a truncated pyramid (703) with a square base.
[0050]
[0050] Fig. 13 shows the results of a three-day end-point viability quantification of different cell types; A) MC3T3-E1, B) RAW 264.7 and C) DICERlf / f cultured on a variety of surfaces: tissue culture plastic (TCP), glass coverslip (Glass) and CoInserts.
[0051]
[0051] Fig. 14 shows the results of a seven-day end-point viability quantification of the pre-osteoblastic cell line MC3T3-E1 via: A) PrestoBlue™, B) LDH activity after full cell lysis, and C) protein content detection.
[0052]
[0052] Fig. 15 is a bar diagram showing acid phosphatase activity (ACP) normalized with PrestoBlue™ viability measurements after seven and twenty-one days of culture for both tri- and monoculture.
[0053] Detailed description
[0054]
[0053] Before the present invention is described, it is to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims and equivalents thereof.
[0055]
[0054] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.
[0055] In the present specification and the appended claims, the term "about" is used to indicate a range of ±10% of the specified value, unless otherwise indicated in the context of the specific disclosure.
[0056]
[0056] A coverslip, also known as a cover glass or cover slip, is a thin and flat piece of transparent material typically made of glass or plastic. Coverslips have been used since more than a century in microscopy to cover a specimen mounted on a microscope slide. Coverslips are available in different materials, such as glass and plastics, and are commercially available in various sizes and thicknesses.
[0057] Traditionally coverslips have been made of glass, and this is still the most widely used material. Coverslips can be made of float glass, borosilicate, quartz, fused silica, sapphire and calcium fluoride, and the choice of material is dictated by the intended application. Each material offers different properties, such as refractive index, chromatic aberration, degree of enhanced UV transparency, and the ability to work at high temperatures. In cell culture applications, cell adherence is often decisive. However, when cell staining and labelling techniques are used, the optical qualities are very important. Plastic coverslips are also available in different materials, such as vinyl and copolymers, and with coated or uncoated surfaces.
[0058]
[0057] An important advantage of the inventive coverslip holder is that the user is free to choose any coverslip material as desired, depending on the cell type or types or the experimental set-up in question.
[0059]
[0058] A first aspect of the present invention relates to a stackable coverslip holder (100', 100", 101a, 101b, 102a, 102b, 103a, and 103b) comprising an upper portion (110) having an outer diameter (DI) and a height (Hl), said upper portion forming a rim (111) defining a first space (120) for receiving a coverslip (200); a lower portion (130) forming a skirt (131) defining a second space (140) having an inner diameter (D2) and a height (H2); wherein the outer diameter (DI) of said upper portion (110) is smaller than the inner diameter (D2) of said second space (140) making one coverslip holder stackable on another coverslip holder; and wherein the height (Hl) of said upper portion (110) is the same or smaller than the height (H2) of said second space (140); and wherein said skirt (131) has multiple openings(132) extending from the outer periphery of the skirt (131) to the second space (140).
[0060]
[0059] This is illustrated in Fig. 2A showing a perspective view of a coverslip holder (100'), Fig. 3A showing a cross-section of a coverslip holder (100'), Fig. 4A showing a coverslip holder (100') from above, and Fig. 5A showing a coverslip holder (100') from below. The coverslip holder (100') comprises an upper portion (110) with a rim (111) defining a space (120), and a lower portion (130) comprising a skirt (131) defining a second space (140), wherein said rim (111) has a height (Hl) which is smaller than the height (H2) of said second space (140) making the coverslip holder stackable. Similarly, the outer diameter (DI) of the upper portion (110) is smaller than the inner diameter (D2) of the space (140) defined by the lower portion (130) and in particular the skirt (131), making the coverslip holder stackable. This design results in a detachable but nevertheless stable connection between the individual coverslip holders and allows the arrangement of multiple coverslip holders on top of each other, stabilized in vertical and horizontal direction.
[0061]
[0060] Furthermore, when stacking several coverslip holders on top of each other, the multiple openings (132) in said skirt (131) allow for surrounding liquid, e.g. a cell culture medium, to reach the cell culture area. Consequently, the cells can receive sufficient oxygen, nutrients and growth factors from the cell medium.
[0062] Examples of these multiple openings are shown in Figs. 2A and 2B showing perspective views of coverslip holders (100', 100"), or Figs. 5A and 5B showing coverslip holders (100' and 100") from below, illustrating the lower portion (130) and the skirt (131) with multiple openings (132).
[0063]
[0061] In a coverslip holder according to the present disclosure, the coverslip (200) is received and supported in the first space (120). Several embodiments are envisaged such as a circumferential abutment (113) as shown inter alia in Figs. 2A, 3A and 3B, several smaller protruding parts (not shown), a receiving groove (not shown), or protruding hooks (not shown).
[0064]
[0062] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, the first space (120) comprises a circumferential abutment (113) for supporting a coverslip. The circumferentialabutment (113) is illustrated in Fig. 2A and in the cross-section of the coverslip holder (100') in Fig. 3A. One advantage thereof is that said circumferential abutment (113) increases the stability for the coverslip (200) when placed in the holder. Preferably the coverslip also forms a tight seal against said abutment.
[0065]
[0063] The stackable coverslip holder has several design features that make it suitable for cell cultures. Firstly, the holder forms a stable platform for culturing cells on a coverslip and also simplifies the handling of coverslips. For example, at the end of an experiment, or during longitudinal studies, a coverslip can be removed, the cells analysed and optionally put back onto the holder. Common methods for analysis, such as colorimetric assays, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR.), optical microscopy, fluorescence microscopy etc., can be applied for analysing the cells directly on the coverslip.
[0066]
[0064] Importantly, as mentioned above, the coverslip holders are stackable.
[0067] Stacking multiple holders on top of each other saves space and simplifies handling when several cell culturing layers are used in the same experiment, immersed in the same culture medium. There are several additional advantages of this feature, including the capability to culture several cell types under the same conditions i.e., the same temperature, pH, in the same media etc. This may be an advantage since local exposures can be tested on different levels, e.g. different coverslip coatings or different coverslip materials.
[0068]
[0065] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said rim (111) further has a circumferential projection (115) which together with the abutment (113) defines a space for receiving a coverslip (200) and, optionally, a locking element. The circumferential projection (115) is also illustrated in Fig. 3A. The said circumferential projection (115) together with the abutment (113) increases the stability of the coverslip (200) when placed in the coverslip holder. Furthermore, the circumferential projection (115) makes it possible to include a locking element (300). Examples of locking elements are so called retaining rings, also referred to as "snap rings", made of a resilient material. The locking element preferably has a cut-out or discontinuity which makes it possible to compress the ring, temporarily reducing itsradius. This allows the locking element to be inserted, whereafter it returns to its original shape and / or radius, and "locks" in position. Conversely, the locking element can be removed, making it possible to access and handle the coverslip with cells growing on its surface, for example for staining and microscopy. The locking element (300) can be used for locking the coverslip in the holder, further increasing the stability. The locking element (300) can be elastically deformed when it is inserted into the space between the circumferential projection and the abutment. Locking elements (300) are illustrated e.g. in Fig. 6 and 7A showing exploded views of a coverslip holders, coverslips (200) and locking elements (300).
[0069]
[0066] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, the first space (120) comprises a second circumferential abutment (1130) for supporting the locking element. The embodiment is illustrated in the cross-section of a coverslip holder (100') in Fig. 3B. An advantage of the second circumferential abutment (1130) is that it helps aligning the elements arranged above the coverslip, such as first, second and further annular elements, as well as the locking element (300), and furthermore gives separate support to the locking element (300), which further increases the stability of the arrangement.
[0070]
[0067] Fig. 8A and 8B show perspective views of coverslip holders with crosssections where coverslips (200) rest on the abutment (113) and are held in place by a locking element (300) that rests on a second circumferential abutment (1130) in the first space (120). The locking element (300) may be inserted into the coverslip holder by being elastically deformed into the space between the circumferential projection (115) and the second circumferential abutment (1130).
[0071]
[0068] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said rim comprises an indentation or cutout reaching from an outer periphery to an inner periphery of said rim and forming a discontinuation in said circumferential abutment. This is illustrated inter alia in Fig.
[0072] 2B showing a perspective view of a coverslip holder (100"), where said upper annular portion (110) and rim (111) have a cutout (112) forming a discontinuity(114) in the abutment (113). The cutout (112) and the discontinuity (114) facilitate removal of the coverslip, e.g., using a tool as illustrated in Fig. 11. As explained above, in connection with the locking element, it becomes possible to remove the coverslip, for example, for microscopic examination or other analysis. Fig. 4B shows a coverslip holder (100") from above, and Fig. 5B the same from below, where the indentation or cutout (112), as well as the discontinuation (114) in the abutment (113), are clearly visible.
[0073]
[0069] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said skirt comprises two vertical cutouts defining a protrusion. This is illustrated inter alia in Fig. 2B, 4B, and 5B. Said cutouts (133, 134) in the skirt (131) define a protrusion (135) which can function as a handle in the lower portion (130). Such handle facilitates handling of the coverslip holder, e.g., using a tool, such as tweezers, to move one or more coverslip holders for inspection or analysis. This is illustrated in Fig. 11, where it is shown how a coverslip holder (100") can be manipulated by engaging the protrusion (135) as a handle using a tool. The protrusion (135) can also function as an indication of the orientation of the coverslip holder, for example in applications where it is important that a measurement or an image is taken in the same direction or orientation. The protrusion (135) can furthermore be used for recording the position of the coverslip holder, or for securing it to a secondary structure (not shown).
[0074]
[0070] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said upper portion and the second space both have a shape chosen from a truncated cone, a box-like structure or cube, or a truncated pyramid, wherein the upper cross-section of the upper portion is smaller than the bottom cross-section of the second space. Examples of three alternative coverslip holder designs are shown in Fig. 12 by, from top to bottom, a truncated cone (701), a box-like structure or cube (702), or a truncated pyramid (703). Other geometries are also possible, with the proviso that the coverslip holder remains stackable.
[0071] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said first space is dimensioned for receiving a round coverslip with a diameter chosen from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 28, 30, 32, 35, 40, or 50 mm.
[0075]
[0072] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, the coverslip is a round coverslip with a diameter of 18 mm.
[0076]
[0073] According to one embodiment of said first aspect, freely combinable with other embodiments disclosed herein, said first space is dimensioned for receiving a square coverslip having a size chosen from 15 x 15 mm, 18 x 18 mm, 20 x 20 mm, or 22 x 22 mm.
[0077]
[0074] As shown by the embodiments above, the design of the stackable coverslip holder is possible to adapt for receiving coverslips of different shapes, sizes and thicknesses. An advantage of this feature includes the possibility of using well-established protocols with several types of standard coverslips, already available as standard laboratory equipment.
[0078]
[0075] Additionally, the coverslip holder makes it possible to use coverslips made of different materials, such as glass or plastic, or the use of suitable membranes.
[0079]
[0076] A second aspect of the present invention relates to a coverslip holder system comprising a coverslip holder according to the first aspect and any embodiments thereof, said system comprising a coverslip and a locking element, and one or more elements chosen from annular elements, and / or membranes. One example of elements in a coverslip holder system is shown in Fig. 7A. In this example, a coverslip (200) is placed in a coverslip holder (100"), two annular elements (401, 402) are placed on top, with a membrane (500), and a locking element (300). A perspective view with a cross-section of this coverslip holder system is shown in Fig. 8B. Another example on how to arrange the elements in a coverslip holder system is illustrated in Fig. 8A, showing a perspective view with a cross-section of a coverslip holder (100") with a coverslip (200), an annular element (401) and a locking element (300).
[0077] An advantage of the annular elements (401, 402) may be the possibility to alter the geometry of the culture area on the coverslip. Furthermore, the annular elements (401, 402) may have a sealing function, wherein the annular element(s) (401, 402) provides a tight seal against the coverslip holder, the locking element and the coverslip holder. Other examples for arranging elements in a coverslip holder system is also envisaged, for example, a coverslip (200) placed in a coverslip holder (100") with a first annular element (401), a membrane (500), a second annular element (402), and a locking element (300) (not shown in the figures).
[0080]
[0078] According to one embodiment of said second aspect, freely combinable with other embodiments disclosed herein, wherein an area available for cell growth on the coverslip in said coverslip holder is defined by an annular element placed onto the coverslip, said annular element having an aperture with an area (a) smaller than the area of the coverslip. Examples of such annular elements (401, 402) are shown in Figs. 7A and 7B. Said area can have any shape, round, square or other, and it may also be subdivided, to define multiple areas available for cell growth, such as a grid or other pattern. The annular elements may also be attached to the coverslip, for example, using plasma-activated bonding.
[0081]
[0079] An annular element can be used to limit the cell culture area, in particular the area available on individual coverslips in place in a coverslip holder. This can be done in order to adapt the cell type ratio in a co-culture to mimic the natural cell ratio in tissues or organs. For example, in one such co-culture with three different cell types, cultured in three different stackable coverslip holders, the first cell type can constitute 10%, the second cell type can constitute 30%, and a third cell type can constitute 60% of the total cell count.
[0082]
[0080] According to one embodiment of said second aspect, freely combinable with other embodiments disclosed herein, a volume available for cell growth on the coverslip held in said coverslip holder, is defined horizontally by an annular element having an aperture with an area (a) and a height (h), and vertically by a permeable membrane. Examples of such annular elements (401, 402) are shown in Figs. 7A and 7B. The membrane (500) can be placed on the upper edge of any type of annular element having an aperture with an area (a) and a height (h). The volumeavailable for cell growth can be further increased by using an annular element with an increased height, such as the annular element (402), shown in more detail in the exploded view in Fig. 7B. In this example, the annular element (402) has a lower part (1402) and an upper part (1404) with an upper edge (1406), defining a volume (1408) available for cell growth. The membrane (500) may be attached to the upper edge (1406) in any suitable manner, for example thermally bonded, such as welded, chemically bonded, such as glued, or mechanically bonded, such as riveted, to the annular element.
[0083]
[0081] According to one embodiment of said second aspect, freely combinable with other embodiments disclosed herein, wherein a volume available for cell growth on the coverslip held in said coverslip holder, is defined horizontally by a first annular element and a second annular element, and vertically by a permeable membrane attached to said second annular element. The term permeable is to be understood as relating to the physical and chemical properties of the membrane, such as pore size and hydrophobicity / hydrophilicity, which determine whether said membrane is permeable to a culture medium but non-permeable to the cells to be cultured within said volume.
[0084]
[0082] This is illustrated in Fig. 8B showings a cross-section of a coverslip holder system with a coverslip holder (100") according to another embodiment of the present disclosure, where a first annular element (401) and a second annular element (402) are placed on the coverslip (200) creating a distance between said coverslip (200) and a membrane (500), wherein said coverslip, annular elements and membrane define a volume available for cell culture (150). The annular element is held in place by a locking element (300). The height of the volume (150) available for cell growth increases when several annular elements (401, 402, and so on, not shown) are placed on top of each other. The use of a defined cell growth volume is advantageous when culturing semi-adherent cells and also when culturing cells in a suspension culture, such as non-adherent, semi-adherent and / or floating cells that are detached from the substrate and remain suspended in the medium. For such cultures, the membrane should be permeable to the culture medium butnon-permeable to the cells to be cultured within said volume. Nevertheless, other types of permeable membranes can also be used.
[0085]
[0083] A third aspect of the present invention relates to a co-culture arrangement comprising at least two, preferably three or more coverslip holders according to the first aspect and any embodiments thereof and / or at least one coverslip holder system according to the second aspect and any embodiments thereof. Examples of such co-culture arrangements are illustrated in Figs. 9A and 9B. Fig. 9A shows a coculture arrangement with three coverslip holder systems (101a, 102a, 103a), each including a coverslip holder (100"), a coverslip (200), an annular element (401) and a locking element (300). Fig. 9B shows a co-culture arrangement with three coverslip holder systems (101b, 102b, 103b), each including a coverslip holder (100"), a coverslip (200), a first annular element (401), a second annular element (402), a membrane (500) and a locking element (300). Furthermore, Fig. 10 illustrates a co-culture arrangement with three coverslip holder systems (101, 102, 103), immersed in a nutrient solution for co-culturing different cells separately, while under the same culture conditions and with the possibility of signalling.
[0086]
[0084] The above examples of co-culture arrangements all use three coverslip holder systems, nonetheless stackable coverslip holders and coverslip holder systems can be used in co-culture arrangement with at least two coverslip holders and / or coverslip holder systems. An advantage of using the stackable coverslip holders in a co-culture includes the possibility to study the interaction between different cell types. In this modular co-culture system, one or several coverslips can be removed for analysis, and optionally put back into the co-culture after the analysis. Common analytical methods, such as colorimetric assays, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), optical microscopy, fluorescence microscopy etc., can be applied directly to the cells on the coverslips.
[0087]
[0085] A fourth aspect of the present invention relates to a cell culture method wherein at least two different cell types are cultured simultaneously in one culture medium, wherein each cell type is cultured on a coverslip placed in a coverslipholder according to the first aspect and any embodiments thereof, and at least two coverslip holders housing different cell types are placed in a culture medium, preferably stacked. This is schematically illustrated in Fig. 10 showings a stack of three coverslip holders (101, 102, and 103) immersed in a nutrient solution for coculturing different cells under the same conditions. Any combination of individual coverslip holders in parallel, alone or together with stacked coverslip holders is also possible.
[0088]
[0086] A fifth aspect of the present invention relates to a cell culture method using a coverslip holder according to the first aspect and any embodiments thereof.
[0089]
[0087] A sixth aspect of the present invention relates to a cell culture method using a co-culture system according to the second aspect and any embodiments thereof.
[0090]
[0088] A seventh aspect of the present invention relates to a cell culture method using a co-culture arrangement according to the third aspect and any embodiments thereof.
[0091]
[0089] An eighth aspect of the present invention relates to an element for use in a cell culture system according to the second aspect and any embodiments thereof, comprising an annular element (402) with an inner aperture (1408), having a height and inner diameter defining an inner volume, and a membrane (500) attached thereto, said membrane being permeable to culture media, such as water, nutrients, and macromolecules but impervious to cells. An example of one such element is shown in Fig. 7A. In the example, the annular element (402) has a lower part (1402) and an upper part (1404) with an upper edge (1406). The membrane (500) may be attached to the upper edge (1406). For example, the membrane (500) may be fastened to the upper edge (1406) of the annular element in a suitable manner, for example but not limited to thermal bonding (such as welding), adhesive bonding or mechanical fastening means (such as stapling).
[0092] Examples
[0093] Example 1: Monocultures on Co-Inserts
[0090] Material & Methods: Three cell types were used for this experiment, preosteoblasts (MC3T3-E1 subclone 4), macrophages (RAW 264.7) and mesenchymal cells (Dicerlf / f). All cell types were mouse derived and bought from the same source (ATCC®). These cells were studied in three separate experiments, and cultured on tissue culture plastic (TCP), glass coverslip (glass) and a stackable coverslip holder as disclosed in the present description (referred to as "Co-Insert" in the following). First, cells were seeded to create a fully confluent layer for all groups. The cells in the TCP culture were seeded in a standard 12-well plate, while cells in both the glass and Co-Insert culture were seeded on sterilized, uncoated glass coverslips. All the cells were left to adhere and stabilize overnight. After 24h, the coverslips for the stackable coverslip holder group (Co-Insert) were inserted in the stackable coverslip holders and the media was changed for all cultures. After three days, the viability of all cells was assessed via PrestoBlue™ assay and DAPI staining with subsequent cell counting through imaging. A one-way ANOVA test with Tukey post hoc correction was performed for all datasets (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
[0094]
[0091] Results: All the cell types in all three cultures were viable in every experiment. Fig. 13 shows the three-day viability quantifications for all cell types: A) MC3T3-E1, B) RAW 264.7 and C) DICERlf / f, and the three cultures. Only significant values are shown in Fig. 13, n.s. have been omitted. For the pre-osteoblastic cell line (Fig. 13A), no significant differences were observed between groups. With regard to macrophages (Fig. 13B), the analysis on cell number shows no significant variation between the different cultures, and the analysis on metabolic activity shows a significant difference only between cells cultured on TCP and on glass. For mesenchymal cells (Fig. 13C), none of the assays showed a significant variation in cell viability between the tested groups. A higher variation in cell count was observed in this cell type than in the other two, although it was not a statistically significant difference. Importantly, no significant differences were observed for the Co-Insert culture vs. TCP or glass. Consequently, the Co-Insert can be used interchangeably, as compared to glass or TCP, as a culturing surface in in vitro experiments using all three cell types.Example 2: Position effect on monoculture on three layers
[0095]
[0092] Material & Methods: All the coverslips were first sterilized and coated with collagen I and then 150,000 pre-osteoblast (MC3T3-E1 subclone 4) were seeded on top and left to adhere overnight. The next day, the coverslips with the cells were moved into the stackable coverslip holders (Co-Inserts). Three groups were investigated in this experiment: collagen coating only, one monoculture using one Co-Insert, and one tri-culture with three Co-Inserts stacked on top of each other. The cells were grown for seven days. Media was changed every 2-3 days. On the last day, after a short optical microscopy session, the cells were exposed to a PrestoBlue™ solution, which was then collected after 2h. After a brief wash with PBS, the cells were completely lysed with a R.IPA buffer. The lysate was then used to react with a LDH substrate solution as well as with a protein quantification kit. Fluorescent and absorbance measures were then performed. A one-way ANOVA test with Tukey post hoc correction was performed for all of the datasets (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
[0096]
[0093] Results: The results from the viability quantification of the pre-osteoblastic cells are shown in Fig. 14: PrestoBlue™ (A), LDH activity after full cell lysis (B) and protein content detection (C). All the performed assays indicate that, after seven days in culture, the cells in each of the layers of the stacked tri-culture were not different in number and in metabolism compared to the same cells cultured in a monoculture Co-Insert. An optical microscopy analysis of pre-osteoblasts after seven days of culture showed cells both in the middle and on the edges of the coverslips in the Co-Inserts, for the monoculture group and for all layers of the tri-culture group. This is evidence that the cells in the different layers have access to the same conditions within the culture vessel (e.g., nutrients, oxygen, etc.).
[0097] Example 3: Tri-culture on Co-Inserts
[0098]
[0094] Material & Methods: Three cell types were used for this experiment with the purpose to mimic bone tissue, pre-osteoblasts (MC3T3-E1, subclone 4), mesenchymal cells (Dicerlf / f) and macrophages (RAW 264.7). All cells were grown on stackable coverslip holders (Co-Inserts) in a tri-culture. Macrophages wereplaced at the bottom, mesenchymal in the middle and pre-osteoblasts at the top. A monoculture for each cell type was also cultured separately as a control. After seven and twenty-one days of culture, cell viability and differentiation were quantified. For viability, PrestoBlue™, LDH after complete lysis and a total protein detection kit were used. For differentiation, an acid phosphatase activity (ACP) kit was used as a measure of osteoclastic differentiation. A two-way ANOVA statistical test was performed for the dataset (* p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001).
[0099]
[0095] Results: Fig. 15 shows the acid phosphatase activity (ACP) normalized with PrestoBlue™ viability measurements after seven and twenty-one days of culture for both tri- and monoculture. ACP activity is a marker for osteoclastic differentiation, showing that the cells were communicating via paracrine signaling through the RANK / RANKL pathway.
[0100]
[0096] Without further elaboration, it is believed that a person skilled in the art can, using the present description, including the examples, utilize the present invention to its fullest extent. Also, although the invention has been described herein with regard to its preferred embodiments, which constitute the best mode presently known to the inventors, it should be understood that various changes and modifications as would be obvious to one having the ordinary skill in this art may be made without departing from the scope of the invention which is set forth in the claims appended hereto.
[0101]
[0097] Thus, while various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for purposes of illustration and are not intended to be limiting, with the true scope and spirit being indicated by the following claims.
Claims
Claims1. A stackable coverslip holder (100', 100") comprising an upper portion (110) having an outer diameter (DI) and a height (Hl), said upper portion forming a rim (111) defining a first space (120) for receiving a coverslip (200); a lower portion (130) forming a skirt (131) defining a second space (140) having an inner diameter (D2) and a height (H2); wherein the outer diameter (DI) of said upper portion (110) is smaller than the inner diameter (D2) of said second space (140) making one coverslip holder stackable on another coverslip holder; wherein the height (Hl) of said upper portion (110) is the same or smaller than the height (H2) of said second space (140); and wherein said skirt (131) has multiple openings (132) extending from the outer periphery of the skirt (131) to the second space (140).
2. The coverslip holder according to claim 1, wherein the first space (120) comprises a circumferential abutment (113) for supporting a coverslip (200).
3. The coverslip holder according to claim 2, wherein said rim (111) further has a circumferential projection (115) which together with the abutment (113) defines a space for receiving a coverslip and, optionally, a locking element (300).
4. The coverslip holder according to claim 2 or 3, wherein the first space (120) comprises a second circumferential abutment (1130) for supporting the locking element (300).
5. The coverslip holder according to any one of claims 2 - 4, wherein said rim (111) comprises an indentation or cutout (112) reaching from an outer periphery to an inner periphery of said rim (111) and forming a discontinuation (114) in said circumferential abutment (113).
6. The coverslip holder according to any one of claims 1 - 5, wherein said skirt (131) comprises two vertical cutouts (133, 134) defining a protrusion (135).
7. A coverslip holder system comprising a coverslip holder according to any one of claims 1 - 6, said system comprising a coverslip (200) and a locking element (300), and one or more elements chosen from annular elements (401, 402), and / or membranes (500).
8. The coverslip holder system according to claim 7, wherein in the coverslip holder (100', 100") an area available for cell growth on the coverslip (200) in said coverslip holder (100', 100") is defined by an annular element (401) placed onto the coverslip (200), said annular element having an aperture with an area (a) smaller than the area of the coverslip (200).
9. The coverslip holder system according to claim 8, wherein in the coverslip holder (100', 100") a volume available for cell growth on the coverslip (200) held in said coverslip holder (100', 100") is defined horizontally by an annular element (401, 402) having an aperture with an area (a) and a height (h), and vertically by a permeable membrane (500).
10. The coverslip holder system according to claim 9, wherein in the coverslip holder (100', 100") a volume available for cell growth on the coverslip (200) held in said coverslip holder (100', 100") is defined horizontally by a first annular element (401) and a second annular element (402), and vertically by a permeable membrane (500) attached to said second annular element.
11. A co-culture arrangement comprising at least two coverslip holders (100', 100") according to any one of claims 1 - 6 and / or at least one coverslip holder system according to any one of claims 7 - 10.
12. A cell culture method wherein at least two different cell types are cultured simultaneously in one culture medium, wherein each cell type is cultured on a coverslip (200) placed in a coverslip holder (100', 100") according to any one of claims 1 - 6.
13. A cell culture method wherein a coverslip holder according to any one of claims 1 - 6, a coverslip holder system according to any of claims 7 - 10 or a coculture arrangement according to claim 11 is used.
14. An element for use in a cell culture system according to any one of claims 7 -10, comprising an annular element (402) with an inner aperture, having a height and inner diameter defining an inner volume (1408), and a membrane (500) attached thereto, said membrane (500) being permeable to water and macromolecules but impervious to cells.