Cell culture insert for generation and hydrogel-encapsulation of 3D biological samples

Abstract

A hanging drop cell culture insert is described comprising an upper retaining piece and a lower culture piece. The upper retaining piece is designed so as to retain the insert in a culture well. The lower culture piece comprises a plate portion having a plurality of holes extending therethrough, each of the holes being configured to accommodate a hanging drop of liquid formed by application of liquid to the hole. The upper retaining piece and the lower culture piece comprise cooperating portions configured so as to retain the upper piece and lower piece together, and to allow relative movement of the upper piece and lower piece along a longitudinal axis so as to alter the separation between the upper piece and the plate portion of the lower piece.

Description

[0001] Cell culture insert for generation and hydrogel-encapsulation of 3D biological samples

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a culture well insert for cultivation of biological material in a three-dimensional environment. Aspects of the invention further relate to methods for cultivation and, optionally, encapsulation of biological material.

[0004] BACKGROUND TO THE INVENTION

[0005] Many biological protocols, for example, drug discovery, involve culturing and observing cells grown in tissue culture. Cell culture in flat plates has the disadvantage that cells which are constrained to an essentially two-dimensional environment may not exhibit behaviour or phenotype which accurately reproduces that of an in vivo three- dimensional environment.

[0006] In order to obtain a three-dimensional cell culture, various techniques can be used. One such technique is so-called “hanging drop” tissue culture, in which a small volume of growth medium is suspended from a surface so as to allow gravity and surface tension to maintain the medium in a droplet. Culturing cells within this hanging drop allows growth in a three-dimensional environment, so potentially allowing observation of behaviour and phenotype without the constraints of two-dimensional cell culture. One use of hanging drop culture in particular is generation of spheroids, three- dimensional aggregates of multiple cells which are considered to more accurately model tissues and tumours, given the presence of both surface and internal cells, which may have different proliferation rates.

[0007] Clearly the traditional method of hanging drop culture - in which single drops are formed manually - is ill-suited to higher throughput screening techniques. Some protocols and equipment have been developed for larger-scale generation of multiple hanging drop cultures. For example, US 8,906,685 B2 to Takayama et al describes a multiwell (eg, 96-well) array plate having an insert provided with multiple holes so as to form hanging drops. Similarly, WO 2010 / 031194 A1 describes another multiwell device having conduits of a specific geometry formed through a plate so as to allow addition of medium from above, and formation of a hanging drop below. This format of device is considered compatible with automated processes for loading with medium and cells, and is said to result in homogeneous formation of spheroids in multiple wells.

[0008] However, such known techniques still have several disadvantages. One particular disadvantage is that, in order to further use the generated spheroids in, for example, drug discovery, the spheroids must be removed from the initial culture plate and transferred to a new cell culture environment. Often this will require collection and transfer of spheroids; when doing so, the cells will come into contact with the materials being used to perform the transfer which can affect their behaviour. Further, many drug discovery protocols will involve encapsulating the spheroids in a 3D hydrogel; for example, Matrigel™, which replicates the extracellular matrix; or other hydrogels including naturally derived materials (collagen, fibrin, alginate), synthetic materials (polyacrylamide, polyethylene glycol), or hybrid materials (hyaluronic acid, polypeptides). The known techniques as described do not lend themselves to such encapsulation without first transferring the spheroids from the multiwell plate.

[0009] The present invention is intended to address this need for an improved device and method for hanging drop cell culture.

[0010] SUMMARY OF THE INVENTION

[0011] According to the present invention, there is provided a hanging drop cell culture insert comprising an upper retaining piece and a lower culture piece, wherein: the upper retaining piece is sized and shaped to be retained at an upper opening of a well in a cell culture plate; the lower culture piece comprises a plate portion separated from said upper retaining piece and having an upper and lower surface, and a plurality of holes extending through the plate portion, each of the holes being configured to accommodate a hanging drop of liquid formed by application of liquid to the hole; the insert defining a longitudinal axis between the upper retaining piece and the plate portion; and the upper retaining piece and the lower culture piece comprise cooperating portions configured so as to retain the upper piece and lower piece together, and to allow relative movement of the upper piece and lower piece along the longitudinal axis so as to alter the separation between the upper piece and the plate portion of the lower piece. An insert according to the invention provides a number of advantages for hanging drop cell culture, and in particular generation of spheroids for use in drug discovery. The upper retaining piece allows the insert to be located within a well of a multiwell plate (typically a 24-well plate), so avoiding the need for specialist culture plates in spheroid generation. The presence of multiple holes in the plate portion (such holes also referred to herein as “drop formation sites”) means that multiple hanging drops may be formed on a single insert, with the consequence that multiple spheroids may be formed independently within a single culture well. This permits replicates of a particular experiment to be carried out within a single well, ensuring that each replicate is exposed to the same conditions within the well. Multiple wells allow for further replicates, or for different conditions in each well.

[0012] Further, the ability to adjust the separation between the upper retaining piece and the plate means that the location of the hanging drop within the well may be adjusted; for example, to bring the hanging drop into contact with culture medium within the well once spheroid formation is complete, or to alter the location of the drop within the well to bring it to the correct focal distance for automated or other imaging of the spheroid.

[0013] Finally, the construction of the insert with drop formation sites formed by holes extending through a plate allows for culture medium to be added or removed from above the drop without disturbing any cultured cells, as well as allowing encapsulation material (such as a hydrogel) to be added from above the drop formation site. This latter feature in particular allows spheroids to be grown and encapsulated on a single piece of equipment without the need to transfer the spheroids from a growth site to an encapsulation site. This means that the insert of the invention allows truly contact-free formation and encapsulation of spheroids.

[0014] In some embodiments, the upper retaining piece is generally annular in form. The piece may be retained at an upper opening of a well in a cell culture plate by placing the annular piece on the upper surface of the cell culture plate, around the well opening.

[0015] The lower culture piece may extend beyond the upper retaining piece, and in preferred embodiments the plate is separated from and extends beyond the upper retaining piece. The lower culture piece may be in the form of a generally tapered truncated cone; and optionally may include the plate portion as the truncated face of the cone, while the sides of the cone comprise at least a part of the cooperating portions which retain the upper and lower pieces together. The remaining part of the cooperating portions may be included on the upper piece.

[0016] The holes of the plate portion may be generally circular in form. The lower surface of the plate portion - that is, the side at which hanging drops are formed - may include a raised annulus protruding from the surface around each hole. This raised annulus serves to assist in retaining a hanging drop on the hole by surface tension. In a preferred embodiment, in addition to the holes which are involved in hanging drop formation, the plate portion comprises a vent (for example, in the form of a further aperture) extending between the upper and lower surfaces. This vent is not intended to be involved in hanging drop formation, and so may take a different form from that of the holes; in particular, it need not be generally circular (although it may be, or at least may be radially symmetrical), and it need not include a raised annulus. Preferably the vent is in a generally central location with respect to the holes, with the plurality of holes arranged around the aperture. In other embodiments, the insert may include multiple vents (for example, two, three, four, or more); where multiple vents are present, these will generally be arranged non-centrally. The vent allows, in some embodiments, encapsulating material such as hydrogel to be added from the upper surface of the plate and to make its way to the lower surface, so as to encapsulate spheroids formed within the hanging drops. Likewise, in some embodiments, the initial application of culture medium to form hanging drops may be made through the vent . In some embodiments, the vent may provide a convenient location which will align with a tool - for example, a pipette tip - which can be used to manipulate the insert, as will be described.

[0017] In a preferred embodiment, the plate portion includes at least two holes, and preferably at least three holes, and more preferably at least six, and most preferably six, holes. In some embodiments the number of holes is a multiple of three and / or a multiple of six. The holes are preferably arranged around a central vent. In embodiments, the size and shape of the insert and holes is so as to promote formation of hanging drops of around 12 pl; for example 5-20 pl, 7-18 pl, 10-15 pl. In other embodiments, larger drops may be used, for example around 20-30 pl, or up to 40 pl, or up to 50pl. It has been determined that this configuration, with the combination of number of holes and droplet size, lends itself well to use of the insert in 24-well plates, while providing six droplets per well allows for a reasonable number of replicates in each well. Hence, the insert as a whole is sized to be compatible with a 24-well culture plate; but of course the invention is not limited to such plates.

[0018] The cooperating portions of the upper and lower pieces preferably permit movement between two distinct positions; a first position with a lesser separation between the upper portion and the plate, and a second position with a greater separation between the upper portion and the plate. This arrangement allows the drop to be moved along a vertical axis, for example so as to locate the drop (and the cell culture) in a suitable focal plane to improve imaging of the drop via microscopy techniques. In preferred embodiments, the cooperating portions are configured to preferentially move from the first to the second position, but not vice versa. For example, in some embodiments the lower piece may comprise one or more flexible arms which engage with a lip or ridge on an inner face of the upper piece in the first position. In other embodiments, the arm and lip or ridge may be swapped, such that an arm is on the upper piece which engages with a lip or ridge on the lower piece. The one or more flexible arms may include a chamfered edge which will allow movement of the arm inwards to disengage from the lip or ridge when the lower piece is pressed downwards; the lower piece may then move downwards as a whole to reengage with a second lip or ridge to hold the lower piece in the second position. Where the plate comprises a vent or aperture in addition to holes, as described above, a user may apply force to the plate via placing a pipette tip in the vent and gently pushing. This will cause the insert to move from the first to the second position. In embodiments the lower piece comprises three flexible arms.

[0019] After use, the flexible arms may be squeezed by a user to separate the upper and lower pieces; these may then be washed and reused once reset into the first position, or may be recycled.

[0020] Alternatively, the upper piece may comprise one or more flexible arms having two separated detents or stops, which engage with the lower piece which may be essentially only the plate. The plate may then be moved between first and second positions defined by the stops on the flexible arms of the upper piece. The insert is preferably made of a biocompatible material, preferably a biocompatible polymer material - for example, cyclic olefin copolymer (COC), polycarbonate (PC), polyetherimide (PEI), polyvinylchloride (PVC), polyethersulfone (PES), polyethylene (PE), polyetheretherketone (PEEK) or polypropylene (PP). A preferred material is polystyrene. The insert is preferably formed of an optically transparent material.

[0021] The insert may, in some embodiments, be provided as a kit including one or more inserts and a cell culture plate.

[0022] Also provided is a kit of parts comprising, separately, the upper retaining piece and the lower culture piece as described herein. The kit may further comprise a cell culture plate.

[0023] The invention further provides a method of preparing encapsulated spheroids, the method comprising: adding culture medium comprising one or more cells to an insert as described herein located within a well of a culture plate, to thereby form a plurality of hanging drops comprising cells on the insert; allowing the cells to grow within the hanging drops to form a plurality of spheroids; adding an encapsulation medium to the insert, and allowing the encapsulation medium to gelify; to thereby form encapsulated spheroids on the insert.

[0024] The method may further comprise exposing the encapsulated spheroids to an environmental condition, and monitoring the exposed spheroids. The environmental condition may include, for example, exposure to a test compound, growth under particular conditions, and so forth.

[0025] BRIEF DESCRIPTION OF THE DRAWINGS

[0026] These and other aspects of the invention will now be described by way of example only and with reference to the accompanying drawings, in which:

[0027] Figure 1 shows a perspective view of a hanging drop insert in accordance with an embodiment of the invention in a first position; Figure 2 shows a perspective view of a hanging drop insert in accordance with an embodiment of the invention in a second position;

[0028] Figure 3 shows a top view and a sectional view of a hanging drop insert in accordance with an embodiment of the invention in a first position;

[0029] Figure 4 shows a top view and a sectional view of a hanging drop insert in accordance with an embodiment of the invention in a second position; and

[0030] Figure 5 shows a 24-well plate with multiple hanging drop inserts of the invention.

[0031] DETAILED DESCRIPTION OF THE INVENTION

[0032] Described herein is a hanging drop insert which is intended to be placed in a well of a multiwell plate (eg, 24-well plate), to allow generation of multiple spheroids in hanging drops in a single well. The same hanging drop insert is able to further be used to encapsulate the formed spheroids into a biocompatible hydrogel. The hanging drop insert thus provides multiple separated encapsulated spheroids in a single well. This increases experimental throughput, allows multiple timepoint sample collection, and avoids any contact of the biological model with plastic.

[0033] Referring to Figures 1 and 2, these show a hanging drop insert 100 according to an embodiment of the invention in first (Figure 1) and second (Figure 2) positions. The hanging drop insert 100 is formed of two separate parts: an upper ring 102 and a lower hanging tripod 104, including a plate 106 which has six hanging drop formation sites in the form of through holes 108, and three flexible arms 110. The plate 106 also includes a central aperture 120 around which the holes 108 are arranged. The upper ring 102 is configured with three receiving portions 112 each of which engages with one of the flexible arms 110. The upper ring 102 also includes notches and ridges 114, 116 which engage together to allow multiple inserts 100 to be connected together on a single plate (see, for example, Figure 5).

[0034] As shown more clearly in the top and sectional views of the insert illustrated in Figures 3 and 4, the receiving portions 112 of the upper ring and the flexible arms 110 include cooperating elements which allow relative movement of the two pieces 102, 104. The receiving portion 112 of the upper ring includes first 114 and second 116 stepped rings, with the second 116 being of lesser radius than the first 114. The flexible arm 110 includes a chamfered lip 118, which rests on either of the stepped rings to retain the lower tripod 104 in position with respect to the upper ring 102.

[0035] In the first position (Figures 1, 3) the chamfered lip 118 rests on the first 114 stepped ring, holding the plate 104 closer to the upper ring 102; this in turn will maintain the plate 104 separated from the lower walls of the well within which the insert is located. To move the insert into the second position, the user may exert a gentle downward force on the plate, for example, by placing a pipette tip into the aperture 120 and gently pushing. This causes the chamfered lip 118 and flexible arm 110 to be urged inwardly, away from the first stepped ring 114, and allowing the hanging tripod 104 to move downward. The lip 118 is then urged against the second stepped ring 116, which retains the insert in the second position (Figures 2, 4), in which the plate 104 is closer to the lower wall of the well.

[0036] As can also be most clearly seen in the sectional views of Figures 3 and 4, each of the holes 108 is shaped with a raised annulus 120 surrounding the lower opening, which tapers toward the interior of the hole, and including an upper recess 122. Together these features improve formation of a hanging drop, as the upper recess 122 guides liquid (eg, culture media) into the hole, while the annulus 120 aids in ensuring surface tension is sufficient to retain the hanging drop on the plate 104.

[0037] In use, the hanging drop insert may be used as follows. The insert is placed in a well of a multiwell plate - see Figure 5 for an illustration of multiple inserts in a 24-well plate. The insert is initially arranged such that the ring and hanging tripod are retained in the upper (first) position, as shown in Figures 1 and 3. While in this position, a mixture of culture media with cells can be dispensed into the hanging tripod; the medium will pass from the upper to the lower surface of the plate, through the holes, to form six individual droplets on the lower surface of the tripod. The geometry of the holes is such as to assist the formation of hanging drops. Furthermore, as the insert is in the upper position, with the plate being relatively far from the lower surface of the well, the droplets will not touch the plate well bottom.

[0038] The cells are then cultured for 24-48 hours under suitable conditions, to allow 3D cell spheroids to form. (Suitable culture conditions and reagents will be known to the skilled person). After this time, up to 80% of the media in the droplets is aspirated from the top of the insert and replaced with a lower volume of encapsulating hydrogel. Any suitable hydrogel material may be used; again, the skilled person will be aware of such materials. Following hydrogel polymerization to encapsulate the spheroids, the tripod is moved to the lower position by gently pressing on the plate with a pipette tip. The lower position can be selected so as to provide an appropriate focal plane for imaging the spheroid, for example via microscopy. No hydrogel droplet touches the plastic well bottom, and the spheroid is fully encapsulated in hydrogel, conferring a more physiological context as avoiding any contact with plastic. Once lowered into the well, experiment reagents (eg, growth medium, test compounds, etc) can be added directly to the well; as the encapsulated spheroids are in the lower position, these will be immersed in the reagents and their growth and behaviour monitored.

[0039] Thus, the present invention allows for spheroid formation and encapsulation using the same device, under conditions which ensure full spheroid encapsulation in hydrogel with no contact with plastic, enhancing physiological microenvironment and relevance of models. Importantly, no spheroid transfer is required either for subsequent encapsulation or for subsequent experimental manipulation. Further, the multiple drops per insert can increase experimental throughput per well and maintain multiple separate samples in the same well. Finally, the invention also allows timepoint sample collection from same insert, and reduces the volume of reagents to be used for endpoint assays. The encapsulated spheroids can be used in static and / or microfluidic environments based on 24 well plates, and the plate format offers the possibility to automate liquid dispensing in the insert, for ease of use and throughput increase.

Claims

CLAIMS:

1. A hanging drop cell culture insert comprising an upper retaining piece and a lower culture piece, wherein: the upper retaining piece is sized and shaped to be retained at an upper opening of a well in a cell culture plate; the lower culture piece comprises a plate portion separated from said upper retaining piece and having an upper and lower surface, and a plurality of holes extending through the plate portion, each of the holes being configured to accommodate a hanging drop of liquid formed by application of liquid to the hole; the insert defining a longitudinal axis between the upper retaining piece and the plate portion; and the upper retaining piece and the lower culture piece comprise cooperating portions configured so as to retain the upper piece and lower piece together, and to allow relative movement of the upper piece and lower piece along the longitudinal axis so as to alter the separation between the upper piece and the plate portion of the lower piece.

2. An insert according to claim 1 , wherein the upper retaining piece is generally annular in form.

3. An insert according to any preceding claim, wherein the lower culture piece extends beyond the upper retaining piece.

4. An insert according to any preceding claim, wherein the lower culture piece is in the form of a generally tapered truncated cone; and optionally the plate portion forms the truncated face of the cone.

5. An insert according to any preceding claim, wherein the holes of the plate portion are generally circular in form.

6. An insert according to any preceding claim, wherein the lower surface of the plate portion includes a raised annulus protruding from the surface around each hole.

7. An insert according to any preceding claim, wherein the plate portion comprises a vent extending between the upper and lower surfaces.

8. An insert according to claim 7, wherein the vent is in a generally central location with respect to the holes, with the plurality of holes arranged around the aperture.

9. An insert according to any preceding claim, wherein the plate portion includes at least two, more preferably at least three, and most preferably at least six, holes.

10. An insert according to any preceding claim, wherein the cooperating portions of the upper and lower pieces permit movement between a first position with a lesser separation between the upper portion and the plate, and a second position with a greater separation between the upper portion and the plate.

11. An insert according to claim 10, wherein the cooperating portions are configured to preferentially move from the first to the second position, but not vice versa.

12. An insert according to any preceding claim, wherein one or the upper or the lower piece comprises one or more flexible arms which engage with a lip or ridge on an inner face of the other of the lower or the upper piece.

13. An insert according to claim 12, wherein the one or more flexible arms include a chamfered edge which will allow movement of the arm inwards to disengage from the lip or ridge when the lower piece is pressed downwards.

14. An insert according to any preceding claim, wherein the insert is made of a biocompatible material, preferably a biocompatible polymer material, and more preferably polystyrene.

15. An insert according to any preceding claim, wherein the insert is formed of an optically transparent material.

16. An insert according to any preceding claim, further comprising a cell culture plate.

17. A kit of parts comprising, separately, the upper retaining piece and the lower culture piece as recited in any preceding claim.

18. A kit of parts according to claim 17, further comprising a cell culture plate.

19. A method of preparing encapsulated spheroids, the method comprising: adding culture medium comprising one or more cells to an insert as described in any of claims 1 to 15 located within a well of a culture plate, to thereby form a plurality of hanging drops comprising cells on the insert; allowing the cells to grow within the hanging drops to form a plurality of spheroids; adding an encapsulation medium to the insert, and allowing the encapsulation medium to gelify; to thereby form encapsulated spheroids on the insert.

20. A method according to claim 19, further comprising the step of removing a portion of the culture medium prior to the step of adding the encapsulation medium,21. A method according to claim 19 or 20, further comprising exposing the encapsulated spheroids to an environmental condition, and monitoring the exposed spheroids.

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