Improvements in or relating to celluar assays
The cell culture retaining insert with a movably coupled retention element addresses variable retention forces by using mass and fluid displacement to maintain consistent force, ensuring secure and stable sample retention with fluid exchange across varying dimensions and spacings.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- 3BRAIN AG
- Filing Date
- 2025-12-09
- Publication Date
- 2026-06-25
AI Technical Summary
Existing cell culture retention systems apply variable retention forces based on sample size and spacing, leading to inadequate retention or sample damage, particularly in three-dimensional biological samples with varying dimensions and manufacturing tolerances.
A cell culture retaining insert with a movably coupled retention element that applies a force determined by the difference between its mass and displaced fluid volume, independent of sample size or spacing, using a planar retention surface and fluid conduits for stable sample retention.
The insert maintains consistent retention force across varying sample sizes and spacings, ensuring secure sample retention while allowing fluid exchange, accommodating intersample and intra-sample variability, and preventing damage.
Smart Images
Figure EP2025086177_25062026_PF_FP_ABST
Abstract
Description
[0001] IMPROVEMENTS IN OR RELATING TO CELLUAR ASSAYS
[0002] This invention relates to a cell culture retaining insert, a cell culture retaining insert assembly, and a cellular assay including such and insert.
[0003] Cell-based assays, i.e. cellular assays, are important experimental tools in life science research and biomanufacturing. They may be based on cell culture methods, where live cells are grown in vitro and used as model systems to assess the biochemistry and physiology of both healthy and diseased cells. Such three-dimensional biological samples may also contain one or more non-living cells, or be or include a micro-tissue, a spheroid, an organoid, a tumoroid, an assembloid or a biopsy. They may also include a gel bead which contains a single cell or multiple cells within its volume.
[0004] According to a first aspect of the invention there is provided a cell culture retaining insert, for retaining a three-dimensional biological sample in a cell culture vessel, the cell culture retaining insert comprising: an insert body having a positioning formation to in-use locate the cell culture retaining insert relative to a cell culture vessel, and a retention element receiving formation; and a retention element movably coupled with the receiving formation of the insert body, whereby a distal retention end of the retention element is freely movable away from and towards the insert body, and whereby in-use the weight of the retention element less a buoyant force equal to the weight of any fluid the retention element displaces gives rise to the retention end exerting a retention force on or more said biological samples.
[0005] The inclusion of such a retention element that is movably coupled with the receiving formation of an insert body, so as to have a retention end that is freely movable away from and towards the said insert body, desirably provides a retaining insert that is configured to, in use, position the retention end proximate to a well bottom in a cell culture vessel, and thus proximate also to a three-dimensional biological sample lying in the said well bottom.
[0006] Moreover, having such a freely movable retention end, and thereby one which, in use, exerts a retention force based on the difference between the mass of the retention element and the volume of any fluid it may displace, advantageously maintains an essentially identical retention force irrespective of the positioning, i.e. spacing, of the retention end relative to the insert body, and thus irrespective of the size of the biological sample, i.e. the height of the biological sample above the well bottom, or the spacing of the insert body from the well bottom.
[0007] In other words, the retention force applied by the retention end, which in-use (and allowing for any frictional losses) is essentially determined by the weight of the retention element acting towards the well bottom, less an opposite buoyant force acting away from the well bottom generated by the mass of any fluid in the well that might be displaced by the retention element, is unaffected by the dimensional configuration of either the biological sample or the insert body.
[0008] This contrasts with other types of retention element, such as a resiliently deformable or similarly sprung element, that might exert a force on a biological sample which varies (e.g. according to Hooke's Law) linearly with the amount of displacement, and thus can give rise to the application of only a relatively low force, e.g. upon a relatively small biological sample causing little displacement, but a relatively high force, e.g. upon a larger biological sample which causes greater displacement. Such large variations in the applied force are undesirable since at the low end they can be insufficient to correctly retain the biological sample, e.g. while liquid is flowing during experimentation on the sample, whereas at the high end the force can damage the biological sample.
[0009] The retaining insert of the invention is therefore able to accommodate both intersample variability (which might arise between spheroids that can be as small as 100, 150 or 200pm in diameter or thickness and organoids that can be 3-5mm in diameter), and / ntra-sample variability (e.g. in a batch of organoids of nominal 4mm diameter, where the difference between different samples can be 500pm or more), while applying essentially the same retention force via the retention end of the retention element.
[0010] Additionally, the retaining insert of the invention is also able to accommodate, in use, differences in the spacing of the distal end of the insert body from a well bottom of a cell culture vessel, e.g. as might arise from the manufacturing tolerance associated with the insert body, while similarly ensuring an essentially identical retention force is applied to any biological sample irrespective of such manufacturing tolerance differences in spacing.
[0011] Preferably the retention element includes a proximal lip formation. The inclusion of such a lip formation limits the extent of movement of the retention end away from the distal end of the insert body, as well as beneficially helping to prevent separation of the retention element from the insert body.
[0012] Optionally the retention element includes at least one protrusion formation adjacent to the distal retention end thereof.
[0013] Having one or more such protrusion formations desirably limits the extent of movement of the retention end towards the distal end of the insert body, and additionally helps to prevent separation of the retention element from the insert body.
[0014] The distal retention end may define a planar retention surface.
[0015] The inclusion of a planar retention surface provides the retention end with a degree of structural integrity to desirably continue to apply a suitable, essentially fixed retention force to any given biological sample.
[0016] Moreover, such a planar retention surface, i.e. an essentially solid distal retention end, can be manufactured relatively straightforwardly, e.g. a single-piece, retention element.
[0017] Preferably the planar retention surface includes one or more fluid transmission conduits passing therethrough.
[0018] Including one or more fluid transmission conduits in the planar retention surface safely facilitates the selective introduction and removal of a liquid to / from contact with a given sample, while ensuring the sample remains retained in position within a well of a cell culture vessel.
[0019] The retention element may include at least one fluid conduit arranged in fluid communication between the or each fluid transmission conduit and a proximal end of the retention element.
[0020] Such a fluid conduit (or plurality of fluid conduits) permits the transmission of a fluid, e.g. an aqueous-based buffer, cell culture medium, hydrogel, or a bioactive compound, to at least a part of a biological sample held, in-use, by the retention end of the retention element, e.g. via one or more of the fluid transmission conduits in the said retention end. In a preferred embodiment of the invention a body of the retention element extending between its distal retention end and proximal end includes at least one opening arranged in fluid communication with one or more of the said fluid conduits.
[0021] The addition of one or more such openings advantageously permits the ingress of fluid from a side of the retention element also (and thus subsequent transmission to a biological sample), as well as via the proximal end of the retention element.
[0022] The planar retention surface may be or include a porous membrane.
[0023] Such a porous membrane similarly safely facilitates the selective introduction and removal of a liquid to / from contact with a given sample, while ensuring the sample remains retained in position.
[0024] In a preferred embodiment of the invention the planar retention surface is resiliently deformable.
[0025] Having a distal retention end with a planar retention surface that is resiliently deformable further assists the retention element in accommodating minor variations in the shape of one or more three-dimensional biological samples while continuing to apply and essentially fixed retention force to the or each sample in a well bottom.
[0026] Preferably a resiliently deformable portion of the distal retention end is selectively separable from the remainder of the retention element.
[0027] Providing a selectively removable, i.e. when a user or other research operative desires, resiliently deformable portion of the distal retention end allows such a portion to be readily made from a different, e.g. less rigid, material than the remainder of the retention element such that each separate part can have desirable performance characteristics, e.g. rigid and readily slidable relative to the insert body in the case of the remainder of the retention element, and softer and more pliable, but possibly grippier, in the case of the resiliently deformable portion.
[0028] Optionally the receiving formation of the insert body has an internal cross-sectional profile shape which is complementary to at least a part of the external cross-sectional profile shape of the retention element. Such a complementary relationship desirably ensures a sufficient degree of coupling between the insert body and the retention element, while nevertheless allowing the required free movement of the retention end away from and towards the insert body.
[0029] Preferably the receiving formation has an at least partially circular internal cross- sectional shape and the retention element has a complementary at least partially circular external cross-sectional shape.
[0030] Circular internal and external cross-sectional shapes are readily manufacturable, while allowing the required free movement of the retention element away from and towards the insert body, as well as additionally rotational movement of the retention element within the insert body, which further adds to the degrees of freedom of the retention element relative to the insert body, and thus further helps the retention end of the retention element to accommodate different sizes and shapes of biological sample.
[0031] Optionally the complementary portions of the external and internal cross-sectional profiles cooperate with one another to inhibit rotation of the retention element about its elongate axis relative to the insert body.
[0032] Such cooperation is beneficial in circumstances where a certain orientation of the retention element relative to the insert body is required, e.g. because of the shape and configuration of a corresponding cell culture vessel, and / or the positioning of the fluid transmission conduits in the planar retention surface of the retention element.
[0033] The complementary portion of at least one of the external or internal cross-sectional profile may be configured to reduce the friction therebetween.
[0034] In another preferred embodiment of the invention configuring the or each complementary portion to reduce the friction takes the form or one or more of: forming it from a low-friction material; applying a surface finishing to it; and / or shaping it to reduce the overall contact area.
[0035] Such steps, i.e. applying incorporating such features, in the or each complementary portion advantageously further helps to ensure that only the mass and volume of the retention element influences the retention force it applies, in use. According to a second aspect of the invention there is provided a cell culture retaining insert assembly comprising a plurality of cell culture retaining inserts as described hereinabove fixedly secured to one another.
[0036] Such a cell culture positioning insert assembly is particularly well adapted to retaining one or more respective three-dimensional biological samples as desired in each of a plurality of wells within a cell culture vessel.
[0037] According to a third aspect of the invention there is provided a cellular assay comprising a cell culture vessel having at least one well, the or each well having associated therewith a cell culture retaining insert as described hereinabove.
[0038] Accordingly, there may be provided a cellular assay comprising: one or more cell culture retaining inserts according to the first aspect; and a cell culture vessel having at least one well, the or each well having associated therewith a respective one of the one or more cell culture retaining inserts.
[0039] Optionally, the or each well may be associated with a respective one of the one or more cell culture retaining inserts by way of the cell culture retaining insert being provided for insertion into a respective well, such as by being configured for removable engagement with the respective well of the cell culture vessel. Alternatively, the or each well may be associated with a respective one of the one or more cell culture retaining inserts by way of a cell culture retaining inserts being fixedly secured in or to the well. Thus, in one or more examples, the one or more cell culture retaining inserts may be affixed to respective wells of the cell culture vessel, such as by adhesive, welding or by being, at least in part, integrally moulded therewith.
[0040] Optionally, in cellular assays including a cell culture vessel with a plurality of wells, the said plurality of wells has a least one cell culture retaining insert assembly as described hereinabove associated therewith.
[0041] Providing such cellular assays extends the benefits associated with the cell culture retaining insert of the invention to cellular assays more generally.
[0042] In one or more examples, the positioning formation of the or each respective cell culture retaining insert is configured to, in use, sit on the cell culture vessel over the opening of the respective well or lie within the respective well. In one or more examples, the cellular assay includes at least one cell culture retaining insert assembly according to the second aspect and wherein the cell culture vessel comprises a plurality of wells, wherein the said plurality of wells has the at least one cell culture retaining insert assembly associated therewith.
[0043] Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, and the claims and / or the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and all features of any embodiment can be combined in any way and / or combination, unless such features are incompatible. The applicant reserves the right to change any originally filed claim or file any new claim accordingly, including the right to amend any originally filed claim to depend from and / or incorporate any feature of any other claim although not originally claimed in that manner.
[0044] There now follows a brief description of preferred embodiments of the invention, by way of non-limiting examples, with reference being made to the following figures in which:
[0045] Figure 1(a) shows a cell culture retaining insert according to a first embodiment of the invention;
[0046] Figure 1(b) shows a partially sectioned view of the cell culture retaining insert shown in Figure 1(a)
[0047] Figure 1(c) shows an insert body which forms part of the cell culture retaining insert shown in Figure 1(a);
[0048] Figure 1(d) shows an enlarged view of a first retention element which forms another part of the cell culture retaining insert shown in Figure 1(a);
[0049] Figure 2 shows an enlarged view of a second retention element which forms a part of a cell culture retaining insert according to a second embodiment of the invention;
[0050] Figure 3(a) shows a partial view of a cell culture retaining insert according to a third embodiment of the invention;
[0051] Figure 3(b) shows an exploded view of a third retention element forming a part of the cell culture retaining insert shown in Figure 3(a);
[0052] Figure 4 shows a partial view of a cell culture retaining insert according to a fourth embodiment of the invention;
[0053] Figure 5(a) shows a cell culture retaining insert according to a fifth embodiment of the invention; Figure 5(b) shows an enlarged view of a third resiliently deformable element that forms a part of a fifth retention element within the cell culture retaining insert shown in Figure 5(a);
[0054] Figure 5(c) shows the cell culture retaining insert shown in Figure 5(a) lying within a portion of a well within a cell culture vessel;
[0055] Figure 6 shows an exploded view of a cellular assay according to sixth embodiment of the invention, which includes a cell culture retaining insert assembly according to a further, seventh embodiment of the invention;
[0056] Figure 7A-7C shows a further example embodiment of a cell culture retaining insert, which may be inserted or integrated with a well;
[0057] Figure 8 shows a section through a well in which the cell culture retaining insert is integrally moulded with the well;
[0058] Figure 9A shows a cell culture retaining insert according to a still further embodiment of the invention;
[0059] Figure 9B shows the retention element of Figure 9(a); and
[0060] Figure 9C shows the cell culture retaining insert shown in Figure 9A lying within a portion of a well within a cell culture vessel.
[0061] A cell culture retaining insert, for retaining a three-dimensional biological sample in a cell culture vessel (not shown), according to a first embodiment of the invention is designated generally by reference numeral 10, e.g. as shown in Figure 1.
[0062] The first cell culture retaining insert 10 includes an insert body 12 which has a positioning formation 16 to in-use locate the cell culture retaining insert 10 relative to a cell culture vessel (not shown in Figure 1). In the embodiment shown, the positioning formation 16 is located at a proximal end 14 of the insert body 12, although this need not necessarily be the case.
[0063] In the embodiment shown, the positioning formation 16 takes the form of an annular positioning ring 18, which is complementary in shape to the opening in a corresponding cell culture vessel with which the first retaining insert 10 is intended to be used, and is configured, in use, to sit on the cell culture vessel over the opening of a corresponding well. Other forms of positioning formation are, however, also possible, particularly in light of the shape of whatever corresponding cell culture vessel the retaining insert is intended for use with, and further positioning formations may also be configured to lie within a well of an associated cell culture vessel. Additionally, the insert body 12 includes a retention element receiving formation 20 which, in the embodiment shown is at a distal end 22 thereof, opposite the proximal end 14, although again this need not necessarily be the case.
[0064] Remaining with the embodiment shown, the retention element receiving formation 20 takes the form of a hollow, tubular conduit 24 which depends from the positioning formation 16 via a support ring 26 and four leg members 28, although other configurations are also possible, e.g. fewer than or more than four leg members, and the receiving formation need not necessarily have a circular inner cross-sectional shape, i.e. other cross-sectional shapes are also possible.
[0065] In any event, the insert body 12 preferably is formed from a rigid material, i.e. a non- resiliently deformable material, and preferably is also integrally formed as a single element.
[0066] In addition to the foregoing, the first retaining insert 10 also includes a first retention element 30 that is movably coupled with the receiving formation 20 of the insert body 12, and more particularly in the embodiment shown is slidably received within the tubular conduit 24 of the receiving formation 20, although other coupling arrangements are also possible.
[0067] In any event, such movable coupling of the first retention element 30 with the receiving formation 20 allows a distal retention end 32 of the retention element 30 to freely move away from and towards the insert body 12, i.e. away from and towards the distal end 22 of the insert body 12.
[0068] Additionally, the retention element 30 includes a proximal lip formation 34 which is intended to prevent separation of the retention element 30 from the insert body 12, at least in so far as movement of the retention element 30 in a distal direction DD relative to the insert body 12.
[0069] In the embodiment shown, the proximal lip formation 34 is defined by an annular lip ring 36, although other embodiments may include a different type of proximal lip formation, but which nonetheless continues to inhibit separation of the retention element 30 from the insert body 12.
[0070] The retention element 30 also includes a protrusion formation 38 adjacent to the distal retention end 32 thereof. The protrusion formation 38 is similarly intended to prevent separation of the retention element 30 from the insert body 12, but in this instance so far as movement of the retention element 30 in a proximal direction DP relative to the insert body 12.
[0071] The protrusion formation 38 takes the form of an annular protrusion ring 40, although because of some of the further features described below of the retention element 30, the protrusion formation 38 in effect takes the form of four annular protrusion ring portions 42. Other embodiments of the invention may, however, include a different type of protrusion formation, so long as it functions to inhibit separation of the retention element 30 from the insert body 12 in the manner described, i.e. when the retention element 30 is moving in a proximal direction DP relative to the insert body 12.
[0072] In any event, however, the protrusion formation 38, i.e. the annular protrusion ring 40, preferably is shaped and configured to allow the retention element 30 to be inserted into and through the receiving formation 20, e.g. in a distal direction DD, during initial assembly of the first retaining insert 10.
[0073] Meanwhile, the distal retention end defines a planar retention surface 44 which includes a plurality of fluid transmission conduits 46 passing therethrough. More particularly, in the embodiment shown the plurality of fluid transmission conduits 46 take the form of a grid 48.
[0074] Other configurations are, however, also possible such as different sizes, e.g. different diameters, of fluid transmission conduit. In particular, the fluid transmission conduits preferably are adapted to the nature of the biological sample to be retained. For example, the cross-sectional area of the remaining planar retention surface that will be in contact with the biological sample desirably is considered, with a goal being to have a readily manufacturable distal retention end geometry that maximizes the cross- sectional area of the fluid transmission conduits (i.e. the sum of the cross-sectional area of each fluid transmission conduit), so as to provide an advantageously high exposure of the biological sample to fluidics, which are often essential to maintaining the viability of the said sample. At the same time, it is necessary to ensure the sample is still retained in the well bottom of a cell culture vessel (e.g. if spheroids of 300pm and organoids of 3mm are to be retained, any individual fluid transmission conduit should not be larger than 300pm).
[0075] The first retention element 30 shown still further includes a fluid conduit 50 that extends between the distal retention end 32 and a proximal end 52 thereof, i.e. includes a fluid conduit 44 that is arranged in fluid communication between the plurality of fluid transmission conduits 46 and the proximal end 52 of the first retention element 30. In other embodiments of the invention (not shown) the retention element may include a plurality of such lengthwise extending fluid conduits.
[0076] In addition, a body 54 of the retention element 30 that extends between its distal retention end 32 and its proximal end 52 includes four openings 56, each of which is arranged in fluid communication with the aforementioned fluid conduit 50.
[0077] Fewer than or more than four openings are possible in other embodiments of the invention, while in the embodiment shown the openings 50 act to divide the aforementioned annular protrusion ring 40 into the said four annular protrusion ring portions 42, although fewer than or more than four openings in other embodiments of the invention would of course have a different effect on such an annular protrusion ring, or indeed any protrusion formation extending around the entire periphery of the retention element body 54.
[0078] As shown, the receiving formation 20, i.e. the tubular conduit 24, of the insert body 12 has a circular internal cross-sectional profile shape which is complementary to the circular external cross-sectional profile shape of the first retention element 30.
[0079] In other embodiments of the invention (not shown), the complementary cross- sectional profile shapes need not be circular, and in still further embodiments of the invention, the whatever internal cross-sectional profile shape of the receiving formation need only be complementary to just a part (or parts) of the external cross-sectional profile shape of the retention element. Preferably, however, the overall cross-sectional area of the complementary part (or parts) is such that essentially linear movement of the retention element 30 relative to the insert body 12 in the distal and proximal directions DD, DP is maintained, i.e. pivoting of the retention element 30 along its own elongate axis AE relative to the insert body 12 is desirably avoided.
[0080] In addition to the foregoing, embodiments of the invention may include a complementary portion of at least one of the external or internal cross-sectional profiles that is configured to reduce the friction between the said profiles.
[0081] Examples of how the or each such complementary portion may be so configured include the following, either individually or in combination: forming it from a low-friction material, such as polytetrafluoroethylene; applying a surface finishing to it, e.g. polishing; and / or shaping it to reduce the overall contact area, which might include removing some material, e.g. by forming one or more channels, to reduce the contact area.
[0082] Additionally, the retention element 30 shown is formed from a rigid material, i.e. a non-resiliently deformable material, and preferably is also integrally formed as a single element. Other embodiments of the invention (not shown) may, however, have a retention element integrally formed from a resiliently deformable material.
[0083] In use, the inclusion in the first retaining insert 10 of a first retention element 30 that is movably coupled with the receiving formation 20 of the insert body 12, i.e. slidably received within the tubular conduit 24 of the receiving formation 20, means that a retention force exerted by the retention end 32 of the retention element 30, e.g. on a three-dimensional biological sample trapped between the retention end 32 and a well bottom of a cell culture vessel, is essentially determined (accounting for any losses that may arise) by the difference between the mass of the retention element 30 and the volume of any liquid, e.g. an aqueous-based buffer, cell culture medium, or hydrogel, it might displace.
[0084] In other words, in use, the retention force applied by the retention end 32 is determined by the weight of the retention element 30 acting towards the well bottom, less an opposite buoyant force acting away from the well bottom generated by the mass of any liquid in the well displaced by the retention element 30, and this is unaffected by any displacement of the retention element 30 relative to the insert body 12 or well bottom, such as may be caused by the dimensional configuration of either the biological sample or the insert body 12.
[0085] Figure 2 shows a second retention element 60 according to a second embodiment of the invention.
[0086] The second retention element 60 is almost identical to the first retention element 30 but has a distal retention end 32 with a planar retention surface 44 that omits any fluid transmission conduits, i.e. has a solid planar retention surface 44.
[0087] The second retention element 60 is wholly interchangeable with the first retention element 30, so as combine with the aforementioned insert body 12 to form a further cell culture retaining insert (not shown) according to another embodiment of the invention. A second cell culture retaining insert according to a third embodiment of the invention is designated generally by reference numeral 70, e.g. as shown in Figure 3(a).
[0088] The second retaining insert 70 is very similar to the first retaining insert 10, and like features share the same reference numerals.
[0089] In particular, the insert body 12 of the second retaining insert 60 is identical to the insert body 12 in the first retaining insert 10.
[0090] The second retaining insert 70 does, however, include a third retention element 72 which while very similar to the first retention element 30 of the first retaining insert 10, and so in effect includes the same rigid, unitary body 54 and lip and protrusion formations 34, 38 as the first retention element 30, instead has a distal retention end 32 which includes a single, large fluid transmission conduit 46.
[0091] Additionally, the distal retention end 32 is resiliently deformable.
[0092] In some embodiments of the third retaining insert (not shown), a portion of the unitary body of the retention element, i.e. at or immediately adjacent to the retention end may be formed from a resiliently deformable material, e.g. by two-shot moulding or overmoulding.
[0093] However, in the embodiment shown, the third retention element 72 includes a separate, resiliently deformable first element 74, which combines with the unitary body 54 to define the distal retention end 32 of the third retention element 72 while being selectively separable, i.e. removable if desired, from the remainder of the third retention element 72.
[0094] More particularly, the resiliently deformable first element 64 defines the planar retention surface 44 and similarly includes a plurality of fluid transmission conduits 46 passing therethrough, which together give rise to a resiliently deformable grid 48.
[0095] The resiliently deformable first element 74 and the third retention element 72 include mutually cooperable formations, which in the embodiment shown are an annular securing ring (not shown) on a proximal end 76 of the first element 74 and complementary depression portions 78 in the third retention element 72 (although other mutually cooperable formations are possible), that permit the selective securing of the first element 74 to the third retention element 72 and removal therefrom.
[0096] In use, the resiliently deformable first element 74 advantageously assists the third retention element 72 to accommodate minor variations in the shape of one or more three-dimensional biological samples it holds against the well bottom of a cell culture vessel, while nevertheless continuing to apply an essentially fixed retention force to the or each said sample.
[0097] Meanwhile, the provision of a porous, grid 48 (whether resiliently deformable or not) safely facilitates the selective introduction and removal of a liquid to / from contact with a given sample, while ensuring the sample remains retained in position within the well bottom.
[0098] A third cell culture retaining insert according to a fourth embodiment of the invention is designated generally by reference numeral 90, e.g. as shown in Figure 4.
[0099] The third retaining insert 90 is very similar to the second retaining insert 70, and like features again share the same reference numerals.
[0100] The insert body 12 of the third retaining insert 90 is again identical to the insert body 12 in the first retaining insert 10, while the third retaining insert 90 includes a fourth retention element 92 that is similar to the third retention element 72.
[0101] To that end, the fourth retention element 92 also includes the same rigid, unitary body 54 and lip and protrusion formations 34, 38 as the first retention element 30, but instead includes a resiliently deformable second element 94, which differs from the resiliently deformable first element 74, but nevertheless still defines a planar retention surface 44 which forms the distal retention end 32 of the fourth retention element 92.
[0102] The resiliently deformable second element 94 takes the form of a porous membrane 96, such as a hollow fibre membrane.
[0103] In the embodiment shown, the resiliently deformable second element 94, i.e. the porous membrane 96, is fixedly secured, e.g. adhered, to the fourth retention element 92, although in other embodiments the second element 94 may be selectively separable from the fourth retention element 92. In still further embodiments of the invention (not shown) the porous membrane may be substantially rigid, i.e. not resiliently deformable.
[0104] In use of the embodiment shown, the resiliently deformable second element 94 similarly advantageously assists the fourth retention element 92 to accommodate minor variations in the shape of one or more three-dimensional biological samples it holds against the well bottom of a cell culture vessel, and facilitates the selective introduction and removal of a liquid to / from contact with a given sample, while nevertheless continuing to apply an essentially fixed retention force to the or each said sample.
[0105] A fourth cell culture retaining insert according to a fifth embodiment of the invention is designated generally by reference numeral 100, e.g. as shown in Figure 5(a).
[0106] The fourth cell culture retaining insert 100 includes many of the same features as the first, second and third retaining inserts 10; 70; 80, and these share the same reference numerals even though the individual elements may be shaped, configured or arranged differently.
[0107] To that end, the fourth retaining insert 100 similarly includes an insert body 104 which has a positioning formation 16 to in-use locate the fourth retaining insert 100 relative to a cell culture vessel 102. However, as shown, the positioning formation 16 extends over most of the second insert body 104 and is located at its distal end 22, while being spaced inboard of the proximal end 14 of the second insert body 104.
[0108] A further difference in the second insert body 104 is that the positioning formation 16 takes the form of first and second opposed annular segments 106, although more than two such segments may be included and they need not necessarily be opposed to one another, which are complementary in shape to an internal opening 108 in a corresponding cell culture vessel 102 with which the fourth retaining insert 100 is intended to be used, e.g. as shown in Figure 5(b).
[0109] More particularly, the positioning formation 16, i.e. the first and second opposed annular segments 106, are configured, in use, to lie within a well 112 (only a part of which is shown in Figure 5(b) of the cell culture vessel 102.
[0110] The second insert body 104 similarly includes a retention element receiving formation 20 which, in the embodiment shown, lies between the proximal and distal ends 14, 22 of the second insert body 104, albeit closer to the proximal end 14, although again this need not necessarily be the case.
[0111] Remaining with the embodiment shown, the retention element receiving formation 20 takes the form of a hollow conduit 24 which has a partially circular inner cross-sectional shape, and in particular in the embodiment shown has a rounded square cross- sectional shape, although other cross-sectional shapes are also possible.
[0112] The second insert body 104 is again preferably formed from a rigid material, i.e. a non- resiliently deformable material, and preferably is also integrally formed as a single element.
[0113] In addition to the foregoing, the fourth retaining insert 100 also similarly includes a fifth retention element 114 that again is movably coupled with the receiving formation 20 of the second insert body 104, and more particularly in similarly slidably received within the rounded square conduit 24 of the receiving formation 20, although other coupling arrangements are again also possible.
[0114] In any event, such movable coupling of the fifth retention element 114 with the receiving formation 20 similarly allows the distal retention end 32 of the fifth retention element 114 to freely move away from and towards the second insert body 104.
[0115] Additionally, the fifth retention element 114 also similarly includes a proximal lip formation 34 which is intended to prevent separation of the fifth retention element 114 from the second insert body 104, again at least in so far as movement of the fifth retention element 114 in a distal direction DD relative to the second insert body 104.
[0116] In the embodiment shown, the proximal lip formation 34 is again defined by an annular, rounded square, lip ring 36, although other embodiments may include a different type of proximal lip formation, but which nonetheless continues to inhibit separation of the fifth retention element 114 from the second insert body 104.
[0117] The firth retention element 114 also similarly includes a protrusion formation 38 adjacent to the distal retention end 32 thereof. The protrusion formation 38 is similarly intended to prevent separation of the fifth retention element 114 from the second insert body 104, but in this second instance so far as movement of the fifth retention element 114 in a proximal direction DP relative to the second insert body 104. In the fifth retention element 114, the protrusion formation 38 instead takes the form of an annular chamfered ring 116, although because of some of the further features described below of the fifth retention element 114, the protrusion formation 38 again in effect takes the form of four chamfer portions 118. Other embodiments of the invention may, however, include a different type of protrusion formation, so long as it functions to inhibit separation of the fifth retention element 114 from the second insert body 114 in the manner described, i.e. when the fifth retention element 114 is moving in a proximal direction DP relative to the second insert body 104.
[0118] In any event, however, the protrusion formation 38, i.e. each of the respective chamfer portions 118, preferably is shaped and configured to allow the fifth retention element 114 to be inserted into and through the receiving formation 20, e.g. in a distal direction DD, during initial assembly of the fourth retaining insert 100.
[0119] Meanwhile, the distal retention end 32 similarly defines a planar retention surface 44 in the form of a separate resiliently deformable third element 120 that is similar to the resiliently deformable first element 74. To that end, the resiliently deformable third element 120 similarly combines with the unitary body 54 to define the distal retention end 32 of the fifth retention element 114 while being selectively separable, i.e. removable if desired, from the remainder of the fifth retention element 114.
[0120] More particularly, the resiliently deformable third element 120 defines the planar retention surface 44 and similarly includes a plurality of fluid transmission conduits 46 passing therethrough, which together give rise to a resiliently deformable grid 48.
[0121] The resiliently deformable third element 120 and the fifth retention element 114 also similarly include mutually cooperable formations, which in the embodiment shown are respective securing notches 122 lying inboard of each chamfer portion 118 and a complementary annular overhang 124 on the resiliently deformable third element 120 (although, likewise, other mutually cooperable formations are possible), that permit the selective securing of the third element 120 to the fifth retention element 114 and removal therefrom.
[0122] Other types of planar retention surface are also possible, e.g. such as those described hereinabove in connection with the retention elements 30; 60; 92 of the other embodiments of the invention. The fifth retention element 114 shown still further includes a fluid conduit 50 that extends between the distal retention end 32 and a proximal end 52 thereof, i.e. includes a fluid conduit 50 that is arranged in fluid communication between the porous membrane 96 and the proximal end 52 of the fifth retention element 114. In other embodiments of the invention (not shown) the fifth retention element may similarly include a plurality of such lengthwise extending fluid conduits.
[0123] In addition, a body 54 of the fifth retention element 30 that extends between its distal retention end 32 and its proximal end 52 includes four openings 56, each of which is arranged in fluid communication with the aforementioned fluid conduit 50.
[0124] Fewer than or more than four openings are possible in other embodiments of the invention, and the openings need not necessarily be located at the rounded corners of the fifth retention element 114. Meanwhile in the embodiment shown, the openings 50 act to divide the aforementioned annular chamfering ring 116 into the said four chamfer portions 118, although fewer than or more than four openings in other embodiments of the invention would of course have a different effect on such an annular chamfering ring, or indeed any protrusion formation extending around the entire periphery of the retention element body 54.
[0125] As shown, the receiving formation 20, i.e. the rounded square conduit 24, of the second insert body 104 has a partially circular internal cross-sectional profile shape which is complementary to the partially circular external cross-sectional profile shape, i.e. the rounded square external cross-sectional profile shape, of the fifth retention element 114.
[0126] Additionally, the internal cross-sectional profile shape of the receiving formation 20, i.e. the rounded square conduit 24 of the second insert body 104, which is complementary to the rounded square external cross-sectional profile shape of the fifth retention element 114 cooperate with one another to inhibit rotation of the fifth retention element 114 about its elongate axis AE relative to the second insert body 104.
[0127] Preferably the fifth retention element 114 is similarly formed from a rigid material, i.e. a non-resiliently deformable material, and preferably is also integrally formed as a single element. Other embodiments of the invention (not shown) may, however, have a retention element integrally formed from a resiliently deformable material. In use, the inclusion in the fourth retaining insert 100 of a fifth retention element 114 that is again movably coupled with the receiving formation 20 of the second insert body 104, i.e. slidably received within the rounded square conduit 24 of the receiving formation 20, means that the retention force applied by the distal retention end 32 is similarly determined by the weight of the fifth retention element 114 acting towards a well bottom, less an opposite buoyant force acting away from the well bottom generated by the mass of any liquid in the well displaced by the fifth retention element 114, and this is similarly unaffected by any displacement of the fifth retention element 114 relative to the second insert body 104 or well bottom, such as may be caused by the dimensional configuration of either the biological sample or the second insert body 104.
[0128] Figure 6 shows a cellular assay 130 according to a further, sixth embodiment of the invention.
[0129] The cellular assay 130 includes a cell culture vessel 132 with a plurality of wells 134, e.g. twenty-four in the embodiment shown, along with a cell culture retaining insert assembly 136 according to a seventh embodiment of the invention which includes a matrix of a corresponding plurality, i.e. twenty-four, of e.g. first retaining inserts 10 as described hereinabove that are fixedly secured to one another.
[0130] Other embodiments of cellular assay (not shown) may instead include one or more individual cell culture retaining insert assemblies, each of which in turn may include a plurality of either first, second, third, or fourth retaining inserts alone, or any combination of first, second, third, and / or fourth retaining inserts.
[0131] Figures 7A-7C show a further example embodiment. Figure 7A shows a further example embodiment of an insert body of a cell culture retaining insert. Figure 7B shows a further example embodiment of a retention element. Figure 7C shows the cell culture retaining insert with the insert body and the retention element slidably coupled together. It will be appreciated that the cell culture retaining insert may be provided separate from a well for insertion and securement therewith. Alternatively, the cell culture retaining insert may be affixed or integrated, such as by integrally moulding or 3D printing, within a well. The same reference numerals have been used for like parts.
[0132] The cell culture retaining insert 10 includes the insert body 12 comprising the retention element receiving formation 20 which depends from the positioning formation 16 by two leg members 28. In this example, the retention element receiving formation 20 takes the form of a hollow, conduit 24 having a rectangular or square cross-section.
[0133] Such a cross-section may inhibit rotation of the retention element.
[0134] Further, the retention element receiving formation 20 includes guide projections 140 that extend from a periphery of the conduit 24 towards the positioning formation 16 to provide further longitudinal guidance to the retention element 30 when it is slidably received within the conduit and between the one or more guide projections 140.
[0135] In this example, the retention element 30 does not have a protrusion ring 42. Further, the distal retention end forms the planar retention surface 44, which in this embodiment is smaller than the previous embodiments and includes a plurality of fluid transmission conduits 46 passing therethrough. The planar retention surface may be about 1.2mm by 1.2mm or, even smaller at 0.5mm x 0.5mm. It will be appreciated by those skilled in the art that the size of the retention surface and cell culture retaining insert may depend on the multi-well cell culture vessel with which it engages. The multi-well cell culture vessels can have, for instance, 24 wells, 96 wells, 384 wells or even more. The external dimensions of these plates are standardised (e.g. by the ANSI SLAS 1-2004 international standard ANSI / SLAS Microplate Standards'), and therefore the higher the number of wells, the smaller their surface at the bottom (where the sample is) and the smaller the distal part of the retention element 30.
[0136] The retention element 30 includes a generally conical lip ring 36 connected to the distal retention end by the retention element body 54. The conical lip ring 36, or more generally a proximal mass, provides the retention element 30 with more weight. The larger volume conical lip ring may be advantageous when the conical lip ring is of low- density materials (e.g. plastics) and more weight is required. The size of the conical lip ring 36 can be reduced if the ring 36 is of higher density materials (e.g. metal alloys).
[0137] The increase in weight due to the presence of the proximal mass, results in a greater retention force (weight of the retention element less a buoyant force equal to the weight of any fluid the retention element displaces) on the sample. The retention element 30 may be of plastics. Also, the density of the retention element 30 may be selected to control the desired retention force.
[0138] The retention element body 54 includes a plurality of openings, such as four openings 56. The conical lip ring 36 includes cut-outs 141 and 142 configured in longitudinal alignment with the leg members 28, such that when the retention element 30 is in an extended configuration with the retention element extended in the distal direction DD, the legs 28 may be received within the cut-outs 141, 142.
[0139] The retention element 30 is also provided with one or more holes or tool receiving formations 143 (only one of two visible) to facilitate easy removal from the cell culture vessel using a tweezer tool.
[0140] Figure 8 shows an example in which the cell culture retaining insert 210 is integrally moulded with a well 212. In Figure 8 a positioning formation 214, comprising a pair of ridges, is formed in the wall of the well 212 which locates a retention element receiving formation 220 towards a base of the well. A retention element (not shown in Figure 8) which may take the form of any previous example, is slidably received within the retention element receiving formation 220. Operation is similar to previous examples, except that as the positioning formation is integrally moulded with the well 212, there is no need to place the cell culture retaining insert 210 into the well 212.
[0141] The one well shown in figure 8 may be part of a cellular assay comprising a plurality of wells. Thus, there may be further wells wherein one or more of the wells have an integrally formed cell culture retaining insert.
[0142] In general, we therefore disclose a cellular assay comprising a cell culture vessel having one or more wells, wherein at least one of the one or more wells has a cell culture retaining inserts integrally formed therein. The cell culture retaining insert thus retains one or more three-dimensional biological samples in the cell culture vessel, the cell culture retaining insert comprising: an insert body integrally formed relative to a cell culture vessel, the insert body comprising a retention element receiving formation; and a retention element movably coupled with the retention element receiving formation of the insert body, whereby a distal retention end of the retention element is freely movable away from and towards the insert body, and whereby in-use the weight of the retention element less a buoyant force equal to the weight of any fluid the retention element displaces gives rise to the retention end exerting a retention force on or more said biological samples.
[0143] Figures 9A-9C show a further example embodiment. Figure 9A shows a further example embodiment of a retention element of a cell culture retaining insert. Figure 9B shows the planar retention surface shown in figure 9A. Figure 9C shows the cell culture retaining insert with the insert body and the retention element slidably coupled together and lying within a portion of a well within a cell culture vessel.
[0144] The cell culture retaining insert 300 comprises an insert body 301 having a positioning formation, which in this embodiment comprises two arms 320 and 321 to engage the interior surface of the well of the cell culture vessel 307. The retention element 330 comprises an enlarged lip ring 336 that comprises four wings 302-305 that extend from a central hub formed by the retention element receiving formation 306. As shown in Figure 9C, the arms 320 and 321 locate the cell culture retaining insert within the cell culture vessel 307 and the wings 302-305 are slidable relative thereto. The retention element receiving formation 306 takes the form of a hollow, tubular conduit within the central hub. Thus, in this example, the retention element receiving formation 306 lies in the same plane as the lip 336 or wings 302-305.
[0145] The retention element 330 comprises the planar retention surface 310 and a body comprising two or more legs 311, 312 extending therefrom. The legs 311, 312 are configured to the central hub from which the wings 302-305 project and is configured to move up or down with the wings 302-305. The hub is configured with grooves in its side wall configured and arranged complementary to the legs 311, 312. Thus, the planar retention surface 310 and the wings 302-305 are movably coupled within the receiving formation 306 of the insert body 301.
Claims
23CLAIMS:
1. A cell culture retaining insert, for retaining one or more three-dimensional biological samples in a cell culture vessel, the cell culture retaining insert comprising: an insert body having a positioning formation to in-use locate the cell culture retaining insert relative to a cell culture vessel, and a retention element receiving formation; and a retention element movably coupled with the receiving formation of the insert body, whereby a distal retention end of the retention element is freely movable away from and towards the insert body, and whereby in-use the weight of the retention element less a buoyant force equal to the weight of any fluid the retention element displaces gives rise to the retention end exerting a retention force on one or more said biological samples.
2. A cell culture retaining insert according to Claim 1 wherein the retention element includes a proximal lip formation.
3. A cell culture retaining insert according to Claim 1 or Claim 2 wherein the retention element includes at least one protrusion formation adjacent to the distal retention end thereof.
4. A cell culture retaining insert according to any preceding claim wherein the distal retention end defines a planar retention surface.
5. A cell culture retaining insert according to Claim 4 wherein the planar retention surface includes one or more fluid transmission conduits passing therethrough.
6. A cell culture retaining insert according to Claim 5 wherein the retention element includes at least one fluid conduit arranged in fluid communication between the or each fluid transmission conduit and a proximal end of the retention element.
7. A cell culture retaining insert according to Claim 6 wherein a body of the retention element extending between its distal retention and proximal ends includes at least one opening arranged in fluid communication with one or more of the said fluid conduits.
8. A cell culture retaining insert according to any of Claims 5 to 7 wherein the planar retention surface is or includes a porous membrane.
9. A cell culture retaining insert according to any of Claims 4 to 7 wherein the planar retention surface is resiliently deformable.
10. A cell culture retaining insert according to Claim 9 wherein a resiliently deformable portion of the distal retention end is selectively separable from the remainder of the retention element.
11. A cell culture retaining insert according to any preceding claim wherein the receiving formation of the insert body has an internal cross-sectional profile shape which is complementary to at least a part of the external cross-sectional profile shape of the retention element.
12. A cell culture retaining insert according to Claim 11 wherein the receiving formation has an at least partially circular internal cross-sectional shape and the retention element has a complementary at least partially circular external cross- sectional shape.
13. A cell culture retaining insert according to Claim 11 or Claim 12 wherein the complementary portions of the external and internal cross-sectional profiles cooperate with one another to inhibit rotation of the retention element about its elongate axis relative to the insert body.
14. A cell culture retaining insert according to any of Claims 11 to 13 wherein the complementary portion of at least one of the external or internal cross-sectional profile is configured to reduce the friction therebetween.
15. A cell culture retaining insert according to Claim 14 wherein configuring the or each complementary portion to reduce the friction takes the form or one or more of: forming it from a low-friction material; applying a surface finishing to it; and / or shaping it to reduce the overall contact area.
16. A cell culture retaining insert assembly comprising a plurality of cell culture retaining inserts according to any preceding claim fixedly secured to one another.
17. A cellular assay comprising:one or more cell culture retaining inserts according to any of Claims 1 to 15; and a cell culture vessel having at least one well, the or each well having associated therewith a respective one of the one or more cell culture retaining inserts.
18. A cellular assay according to claim 17, wherein the positioning formation of the or each respective cell culture retaining insert being configured to, in use, sit on the cell culture vessel over the opening of the respective well or lie within the respective well.
19. A cellular assay according to Claim 17 or 18 including at least one cell culture retaining insert assembly according to Claim 16 and wherein the cell culture vessel comprises a plurality of wells, wherein the said plurality of wells has the at least one cell culture retaining insert assembly associated therewith.
20. A cellular assay comprising a cell culture vessel having one or more wells, wherein at least one of the one or more wells has a cell culture retaining insert integrally formed therein, the cell culture retaining insert configured to retain one or more three-dimensional biological samples in the cell culture vessel, the cell culture retaining insert comprising: an insert body integrally formed with the cell culture vessel, the insert body comprising a retention element receiving formation; and a retention element movably coupled with the retention element receiving formation of the insert body, whereby a distal retention end of the retention element is freely movable away from and towards the insert body, and whereby in-use the weight of the retention element less a buoyant force equal to the weight of any fluid the retention element displaces gives rise to the retention end exerting a retention force on or more said biological samples.