Apparatus, kits and methods for the production of biomimetic constructs

Abstract

This invention relates to apparatus, kits and methods for the production of biomimetic constructs by plastically compressing a gel, such as a collagen gel, in a well using a plunger, which may be porous. The apparatus, kits and methods allow biomimetic constructs to be produced in a controlled and reproducible manner and are suitable for the production of multilayered constructs.

Claims

1. A method of producing a biomimetic construct comprising: (i) introducing a gel solution to a well having an opening, (ii) incubating the gel solution to form a gel, (iii) introducing a porous plunger to the well, (iv) compressing the gel with the porous plunger such that liquid is expelled from the gel through a surface of the gel that is contacted by the porous plunger, the liquid, prevented from being expelled through the well bottom, entering the porous plunger and the volume of the gel being reduced, thereby producing the biomimetic construct, wherein a volume of the liquid expelled from the gel into the plunger per unit of surface area of the plunger that contacts the gel is about 2 mm to about 14 mm, and (v) removing the porous plunger containing expelled liquid to leave said biomimetic construct in the well.

2. A method according to claim 1 wherein the gel solution is seeded with cells.

3. A method according to claim 1 further comprising: (vi) introducing a further gel solution onto the biomimetic construct in the well, (vii) incubating the further gel solution to form a further gel, (viii) introducing a porous plunger to the well, (ix) compressing the further gel with the porous plunger such that liquid is expelled from the gel into the porous plunger and the volume of the gel is reduced, (x) removing the porous plunger to leave a biomimetic construct comprising multiple layers of compressed gel in the well, optionally repeating steps (vi) to (x) one or more times to produce a biomimetic construct comprising multiple layers of compressed gel.

4. A method according to claim 1 wherein the porous plunger comprises one or more projections which emboss recesses, optionally grooves, into the surface of the compressed gel.

5. A method according to claim 1 further comprising seeding the surface of the compressed gel with cells.

6. A method according to claim 1 wherein biomimetic constructs are produced in an array of wells simultaneously.

7. A method according to claim 1 wherein the gel solution is a collagen solution and the gel is a collagen gel.

8. A method of producing a biomimetic construct comprising a roofed channel comprising: (i) providing a compressed gel with one or more grooves on the surface thereof, (ii) introducing a further gel solution onto the surface of the compressed gel, (iii) setting the further gel solution to form a further gel on the surface of the compressed gel, and; (iv) compressing the further gel with a porous plunger such that liquid is expelled from the further gel through a surface of the further gel that is contacted by the porous plunger, the liquid, prevented from being expelled through a well bottom, entering the porous plunger and the volume of the further gel being reduced to produce a further compressed gel, wherein a volume of the liquid expelled from the further gel into the plunger per unit of surface area of the plunger that contacts the gel is about 2 mm to about 14 mm, wherein said further compressed gel covering the one or more grooves to produce a biomimetic construct containing a roofed channel.

9. The method of claim 1 wherein compressing the gel with the porous plunger includes expelling at least 95% of the liquid from the gel into the porous plunger.

10. The method of claim 1 wherein a bottom of the well is impermeable or mounted on an impermeable support.

Description

(1) Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described below.

(2) FIG. 1 shows a schematic of an example of a method of the invention.

(3) FIG. 2 shows a schematic of another example of a method of the invention.

(4) FIG. 3 shows an array of plungers which may be used in kits of the invention in a disposable tray.

(5) FIG. 4 shows a support containing integral wells which may be used in kits of the invention, along with a lid for covering the wells of the support and a guide plate for guiding plungers to the wells.

(6) FIG. 5 shows individual wells inserted into a mounting plate with a lid, which may be used in kits of the invention,

(7) FIGS. 6a to 6g show a schematic of an apparatus performing a method of the invention.

(8) FIG. 7 shows a cross-section of the plunger head of an apparatus according to an embodiment of the invention.

(9) FIG. 8 shows an apparatus according to an embodiment of the invention which comprises a linear set of processing stations. Station 1 is a plate stack station to present new multiwell plates to the system. Station 2 is a dispensing station which adds liquid reagents to wells in the plates. For example, the station may dispense collagen solution, buffer, nutrients, and cells to the wells, or a cell suspension, for example to seed the surface of a construct in the well. Station 3 is an incubation station which maintains the wells in the plate at a raised temperature (e.g. 37 C.) to set the collagen solution in the wells or to incubate cells in or on the constructs. Station 4 is a plunger loading station where fresh plungers are loading onto the plunger holders of an apparatus for compression. Station 5 is a compression station where the plungers loaded at station 4 are used to compress the gel which was set in the wells of the plates at station 3. Station 6 is a plunger removal station where used plungers are unloaded.

(10) FIG. 9 shows the results of haematoxylin and eosin (H&F) histological staining of a biomimetic construct (cross-section) produced as described herein with the apparatus shown in figures and 7. Cross sections of biomimetic constructs produced in two separate wells simultaneously by the apparatus are shown.

(11) FIG. 10 shows representative images of roofed micro-channels in double-layered PC collagen constructs embossed using projections of different dimensions. Scale bar 20 m. Arrows show the opening of the channels.

(12) A method of producing a biomimetic construct according to an embodiment of the invention is shown in FIG. 1. The production of a construct in a single well is described, but constructs ay be produced in an array of wells simultaneously in the same way.

(13) A porous plunger 1 with an impermeable connector 2 on top is provided in a tray 3 that can hold 24 plungers (FIG. 1a #1).

(14) The tray is placed onto the collagen compression apparatus (not shown) and the aperture 4 of a plunger holder 5 is forced into contact with the plunger connector 2 (FIG. 1a #2).

(15) The plunger head 6, which contains the entire mechanism holding the plunger, is then lifted, lifting the plunger 1 held in the plunger holder 5 from the tray 3 (FIG. 1a #3). The tray 3 is then discarded.

(16) A well (which may also be referred to as an insert) is placed into position on a mounting plate 8. The bottom of the well 7 may be porous. The well has flanges 10 that rest on top of the mounting plate 8. The mounting plate is placed onto a support 9. The support 9 may be a multiwell plate. The support 9 is shown in FIG. 1a #4 with cross-hatching; the mounting plate 8 has vertical hatching; the well 7 has a solid outline.

(17) The well 7 is then filled with collagen solution 11 FIG. 1a #5. Optionally, the collagen solution 11 may be seeded with cells before or after introduction to the well 7.

(18) The mounting plate 8 containing the well 7 is then covered with a lid 12 and the collagen solution 11 is incubated to form a gel (FIG. 1a #6) The lid 12 is shown in FIG. 1a #6 as a densely dotted line.

(19) The lid 12 is then removed and the plunger head 6 is moved over the mounting plate 8 (FIG. 1b #7). The head 6 is lowered so that the mounting plate 8 is secured into position with the outer resilient guide 13. The plunger 1 is held in the plunger holder 5 above the collagen gel 14 in the well 7.

(20) The well 7 is then secured in position with inner resilient guides 15 that force the well to the bottom of the support 9, thereby preventing expulsion of liquid from the bottom of the well 7, when a well 7 with a permeable bottom is employed. The plunger 1 is made to move toward the bottom of the well 7 through the release of the plunger holder 5, which grips the plunger 1 in its aperture 4. The plunger holder 5 is movably held within the plunger head 6 by the stop 18, which limits the movement of the plunger holder 5 (FIG. 1b #7) until it is released (FIG. 1b #8)

(21) The downward movement of he plunger holder 5 moves he plunger towards the bottom of its well 7, compressing the collagen gel 14 in the well 7 and absorbing liquid expelled from it FIG. 1b #9.

(22) After compression, the plunger head 6 is raised, lifting the plunger 1 held in the plunger holder 5 out of the well 7. The mounting plate 8 is freed, and the head 6 is moved away from the well 7 containing compressed collagen 16 (FIG. 1b #10).

(23) The lid 12 then placed back over the well 7 to keep the compressed collagen 16 clean and facilitate incubation.

(24) The plunger 1 is then released from the plunger head 6 by probe 17 which through the aperture 4 of the plunger holder 5 and dislodges the connector 2 of the plunger 1.

(25) The plunger 1, which contains the liquid expelled from the gel 14 during compression, is then discarded.

(26) Another embodiment of a method of producing a biomimetic construct is shown in FIGS. 2a and 2b. The method is similar to that shown in FIGS. 1a and 1b except the wells 7 are integral to the support 9 (FIG. 2a #4). Unlike FIGS. 1a and 1b, separate wells and mounting plates are not required. A guide plate may optionally be used to facilitate proper positioning of the plungers into their respective wells. In this embodiment, the plunger 1 moves down directly into the well 7 in the support 9 and may therefore display a slightly larger diameter than that used with a separate insert well.

(27) Collagen solution 11 is dispensed directly into the well 7 in the support 9 (FIG. 2a #5)

(28) A lid 12 is placed directly over the support 9 to incubate the collagen solution 11 in the well 7 and cause it to set to produce a collagen gel (FIG. 2a #6).

(29) The lid 12 is then removed and the plunger head 6 is moved over the support 9 (FIG. 2b #7). The plunger 1 is held in the plunger head 5 above the collagen gel 14 in the well 7.

(30) The well 7 is then secured in position with the inner resilient guides 15 which are connected to the plunger stop 18. The contact between the inner resilient guides 15 and the support 9 disengages the plunger holder 5 from the plunger stop 18 and causes the plunger 1 to move towards the bottom of the well 7. The plunger holder 5 is movably held within the plunger head 6 by the stop 18, which defines the extent of the upward and downward movement of the plunger holder 5 (FIG. 2b #8).

(31) The downward movement of the plunger holder 5 moves the plunger 1 towards the bottom of the well 7, compressing the collagen gel 14 in the well 7 and absorbing liquid expelled from it FIG. 2b #9.

(32) After compression, the plunger head 6 is raised, lifting the plunger 1 held in the plunger holder 5 out of the well 7. The support 9 is freed, and the head 6 is moved away from the well 7 containing compressed collagen 16 (FIG. 2b #10).

(33) The well 7 is then covered by the lid 12 to keep the compressed collagen 16 clean and facilitate incubation (FIG. 2b #11).

(34) The plunger 1 is then released from the plunger head 6 by probe 17 which through the aperture 4 of the plunger holder 5 and dislodges the connector 2 of the plunger 1 (FIG. 2b #12).

(35) The plunger 1, which contains the liquid previously expelled from the gel during compression, is then discarded (FIG. 2b #13).

(36) An array of plungers 1 for compressing gels in an array of wells is shown in FIG. 3. The plungers 1 are mounted with their connectors 2 uppermost in a disposable presentation tray 3 which positions the plungers 1 for loading onto the plunger holders of a collagen compression apparatus.

(37) An impermeable support 9 is shown in FIG. 2. An array of wells 7 is integral to the support A lid 12 is used to cover the wells 7 during incubations and storage. A guide plate 28 may be used to cover the wells 7 during compression. The guide plate 28 contains an array of apertures 29 which correspond to the wells 7 in the support 9. The apertures 29 are tapered i.e. the internal diameter of the apertures at the upper surface 30 of the guide plate is greater than the internal diameter at the lower surface of the guide plate. At the lower surface of the guide plate, the apertures 29 have the same internal diameter as the wells 7 in the support 9. The guide plate 28 guides plungers as they move downwards into the wells 7 of the support 9.

(38) An array of individual wells 7 is shown in FIG. 5. The wells 7 are tapered so that the internal diameter at the opening 32 is greater than the internal diameter at the bottom of the well 33. The wells 7 are mounted in apertures 31 in the mounting plate 8. The flanges 10 of the wells 7 rest on the mounting plate 8 and prevent the wells 7 from falling through the apertures 31. A lid 12 is used to cover the wells 7 in the mounting plate during incubation and storage.

(39) The operation of an apparatus for simultaneously producing biomimetic constructs in an array of wells is shown in FIG. 6.

(40) Initially, the apparatus 19 is in a resting state with the plunger head raised and no consumables, such as plungers or wells, loaded (FIG. 6a). The apparatus 19 comprises a plunger head 6 pivotally mounted on a post 25 and movable between a compression station 26 and an unloading station 21. The head 6 contains an array of plunger holders not visible and resilient inner and outer guides 15 and 13.

(41) The compression station 26 comprises a mount 20 for accommodating wells which can be covered by a lid 12. The mount 20 rests on a heated plate not shown to incubate wells accommodated in the mount 20. The apparatus contains a display 22 which indicates the temperature and duration of incubation of wells positioned in the mount and controls 23 to allow these parameters to be adjusted. A removable waste tray 21 s positioned in an unloading station 21.

(42) In a first stage, the plunger holders 5 within the plunger head 6 are loaded with plungers 1. A tray 3 containing an array of plungers 1 is positioned on the mount 20 and the connectors 2 of the plungers 1 are introduced to the apertures of the array of plunger holders not visible mounted in the plunger head 6. The engagement of the connector 2 with the aperture holds each plunger 1 in its corresponding plunger holder 5, so that the array of plungers 1 can be lifted from the tray 3 by the plunger head 6 (FIG. 6b). This stage is also shown schematically in steps 1 and 2 of FIGS. 1 and 2.

(43) The plunger head 6 loaded with the array of plungers 1 is then moved aside by pivoting it around the post 25 to be positioned over the waste tray 21. The wells 7 are positioned in a mounting plate 8 on the support 9 in a 24 well array and then introduced to the mount 20. With the plunger head 6 positioned over the waste tray 21, the wells 7 are accessible for the introduction of collagen solution; suspensions of cells; and/or other reagents (FIG. 6c). This stage is also shown schematically in steps 3, 4 and 5 of FIGS. 1 and 2.

(44) Whilst the loaded plunger head 6 is positioned over the waste tray 21, the wells 7 in the mounting tray 8 are filled with collagen solution (not visible), optionally seeded with cells, and covered with a lid 12. The wells are then heated in the covered mount 20 by the heater (not shown) and incubated at 37 C. to cause the collagen solution to set into a gel (FIG. 6d). This stage is also shown schematically in step 6 of FIGS. 1 and 2.

(45) The lid (not shown) is then removed from the mount 20 and the plunger head 6 is pivoted around the post 25 into a position over the wells 7, so that the plungers 1 attached to the plunger holders (not visible) are ready to insert into the wells 7 of the plate (FIG. 6e). This stage is also shown schematically in step 7 of FIGS. 1 and 2.

(46) The plunger head 6 is then latched down so that the plungers 1, which are weighted by the plunger heads (not visible), rest on the gel (not visible) in each well 7. The plungers 1 compress the collagen gel not visible and absorb the liquid expelled from the gel by the compression (FIG. 6f). This stage is also shown schematically in steps 8 and 9 of FIGS. 1 and 2.

(47) The plunger head 6 is then lifted, so that the plungers 1, which now contain expelled liquid from the compressed gel in the wells 7, are removed from the wells 7 in the mount 20. This stage is also shown schematically in step 10 of FIGS. 1 and 2.

(48) The plunger head 6 containing the wet plungers 1 is then pivoted around the post 25 and positioned over the waste tray 21 in the unloading station 24. The wet plungers 1 are then ejected into the waste tray 21. The wells 7 of compressed collagen remain in position in the mounting plate 8 in the mount 20, without being covered by the lid (FIG. 6g). This corresponds to steps 11 to 13 of FIGS. 1 and 2.

(49) In some embodiments, the waste tray 21 may be removed and the plunge holders 1 in the plunger head 6 may be loaded with fresh plungers 1. Additional collagen solution, optionally seeded with cells, may be added to the compressed collagen in the wells 7. The steps shown in FIGS. 6c to 6g may then be repeated to produce a multilayered collagen construct in the wells 7.

(50) The method may be repeated until the constructs in the wells possess the desired number of layers of compressed collagen.

(51) A cross section of the plunger head 6 of the apparatus 19 for producing biomimetic constructs of FIG. 6 is shown in FIG. 7. An array of plungers 1 is held in an array of plunger holders 5 by the frictional engagement of connectors 2 with apertures 4 within the plunger holders 5. The plungers 1 are shown touching the bottoms of an array of wells 7 which are accommodated in a mounting plate 8 on a support 9 positioned on a heating base plate 27. The wells 7 do not contain collagen.

(52) The plunger head is aligned on the mount and the mounting plate 8 is secured by the outer guides 13. A latch 34 secures the head an position. The wells 7 have flanges 10 at their openings which rest on the mounting plate 8. During compression, inner guides 15 secure the wells 7 on the mounting plate 8 and drive the bottom of the wells 7 against the support 9. The individual plunger holders 5 are released from the head 6 and exert a gravitational force on the plungers 1 in the wells 7.

(53) A probe 17 is engaged in the aperture 4 of each plunger holder 5 and can be moved downwards to dislodge the connector 2 and eject the plunger 1 from the plunger holder 5 after use.

(54) FIG. 8 shows a multistation apparatus of the invention. Plates containing wells may move through one or more of the stations several times. For example, after one pass through each station, a plate may pass for a second time through the dispensing station for an intermediate layer of cells, followed by the dispensing station again for a new collagen layer, followed by incubation, plunger addition, compression, and plunger removal.

(55) Cross sections of biomimetic constructs produced with the apparatus shown in FIGS. 6 and 7 were stained with haematoxylin and eosin H&E). The results are shown in FIG. 9. A dense collagenous structure was observed with human dermal fibroblast cells (initial density 100,000 cells/ml) dispersed throughout the structure, akin to the anatomy of the dermis layer of skin. This demonstrates that the constructs produced by the apparatus described herein are highly biomimetic.

(56) Two-part micro-channels were created by template micro-moulding using unidirectional fluid flow in a multi-well format. In first stage, grooves were embossed in the surface of one layer. In the next stage, a layer of fresh collagen solution was set over the top of these grooves and compressed onto the first to act as a micro-channel roof. This technique (producing roofed -channels) allowed for simple and rapid fabrication of controlled thickness constructs with pre-formed channels. The dimensions of the channels were found to be a predictable proportion of stamp or template used for moulding. Under the conditions used, the proportion was between 20% and 50% and for 75 m and 125 m deep stamps, channels were typically formed as 25 m and 50 to 100 m wide and up to 30 m deep (FIG. 10). The compressed collagen matrix was made with viable cells in place which grew and remodelled the matrix for some weeks, though the channels remained. Channel walls were made from compacted, orientated collagen fibrils which accumulated around the mould during compression, providing durability in culture. Detailed study of the relationship between template dimensions and the channels ultimately produced has shown that moulding fidelity is sensitive to stamp shape and collagen density. These results demonstrate that plastic compression with moulding and multi-layering can be used to make highly predictable -channelled, living 3D constructs with good perfusion.