Perfusion bioreactor, scaffold cassette, and method for culturing cells

Abstract

Provided herein is a perfusion bioreactor having a flow chamber including a flow chamber top, a flow chamber bottom, and an interior space defined between the flow chamber top and the flow chamber bottom. Along the length of the flow chamber, the interior space includes an inlet portion, an outlet portion, and a central portion arranged therebetween. A sealing member is arranged between the flow chamber top and the flow chamber bottom. Further, the inlet portion includes at least one funnel element having a diameter in the transverse direction that increases over a respective length of the inlet portion in the longitudinal direction. A method for culturing cells is also provided herein.

Description

Perfusion Bioreactor, Scaffold Cassette, and Method for Culturing CellsField of the invention

[0001] The present disclosure relates to a perfusion bioreactor, particularly suitable for culturing collagen sheets, and which is configured to enable dual-sided perfusion of culture medium to both the top and bottom surfaces of a scaffold arranged therein. The disclosure further relates to a scaffold cassette configured for securing a scaffold and a method for culturing cells.Background

[0002] Bioreactors are specialized vessels that can be designed for growth and cultivation of cells, tissue(s), and microorganisms. Bioreactors come in various shapes and sizes, ranging from small benchtop units to large industrial-scale vessels. Some bioreactors are single-use while others are designed for multiple uses. The type and / or design of a bioreactor thus depends on factors like the specific application(s), type of cell(s) being cultured, and the level of production required.

[0003] One particular type of bioreactor suitable for adherent cultures is a perfusion bioreactor. This type of bioreactor employs a perfusion process that involves a continuous culturing method in which cells are retained in the bioreactor and typically cultured over what are often relatively long periods of time. In particular, cells are usually kept in a cell retention compartment inside of the bioreactor while culture medium is continuously exchanged. This way, fresh medium replenishes the nutrient(s) delivered to the cells while cellular waste and medium that has been depleted of nutrients are removed. The exchange is typically performed at the same rate for the introduction of fresh media and the removal of waste product(s). By continuously removing spent media and replacing it with new or reconditioned media, nutrient levels are maintained for improved growing conditions and cell waste product is removed to avoid toxicity.

[0004] A persistent challenge in these bioreactor systems is achieving efficient perfusion of nutrients and removal of waste products. Bioreactor designs often fail to adequately address this issue, which may in turn lead to non-uniform cell growth, non-uniform tissue formation, and / or variable regions within the layer of cultured cells.

[0005] Single-sided perfusion systems, while effective for relatively thin scaffolds, are insufficient for thicker constructs due to limited nutrient diffusion including oxygen diffusion and poor waste removal from deep within the scaffold. Various designs have attempted to enhance perfusion through different mechanisms, such as central channel perfusion and multi-channel systems. However, these approaches still face significant limitations in uniformly distributing nutrients across the entire thickness of the scaffold, especially in more complex tissue constructs.

[0006] Designs incorporating rotating bioreactors and disposable bioreactor systems have introduced new methods to enhance perfusion. Rotating bioreactors aim to improve nutrient distribution through mechanical agitation. However, this can induce uneven shear stress distribution along the scaffold surface(s), which may reduce the homogeneity of the culture conditions and even cell distribution. Not to mention, the setup may also be more prone to mechanical failure. Disposablebioreactor systems, utilizing flexible containers, offer scalability and ease of use but do not fundamentally solve the challenge of uniform perfusion in thicker scaffolds.

[0007] Accordingly, even with the multitude of bioreactor systems that exist, there still remains a need to provide a bioreactor system that is enabled to reliably produce cultured cell layers with an increased relative thickness and an improved homogeneity across the cultured layer(s), wherein the system is capable of being easily scaled-up. Such bioreactors can be particularly useful for leather production.Summary

[0008] The present disclosure relates to a bioreactor system that is particularly suitable for culturing homogeneous layers of cells and designed especially for scale-up on the meter-scale. A useful application of this is the culturing of fibroblasts, which can lead to the production of homogeneous collagen sheets.

[0009] Cultured collagen sheets in accordance with the present disclosure are particularly desirable for use in the in vitro production of leather. It is, therefore, highly desirable that the cultured sheets have a thickness of around 1.5 mm as this is particularly suitable for subsequent tanning process(es) and end product manufacturing.

[0010] An objective of the present disclosure is thus to enable perfusion of medium to both the top and bottom surfaces of a scaffold arranged in the bioreactor. Dual-sided perfusion of the scaffold is highly desirable because it can enable cell viability and enhance collagen secretion throughout the entire thickness of the scaffold, even scaffolds with relative thicknesses above 0.5 mm.

[0011] Another objective is to improve the fluid / mass flow characteristics within the bioreactor. In this respect, it can be desirable to improve the shear stress experienced at the scaffold surface(s), particularly by making the shear stress relatively (more) uniform. Such a uniform shear also suggests a uniform distribution of solutes such as oxygen or nutrients, as well as uniformity of waste removal.

[0012] A further objective for improving the fluid / mass flow characteristics is to disrupt (incoming) high-velocity flow at the inlet portion such that the center of the scaffold does not experience the same high-velocity flow observed at fluid entry point(s) into the bioreactor.

[0013] Yet another objective for improving the fluid / mass flow characteristics within the bioreactor is the provision of a homogenous or essentially homogenous flow pattern across both the top and bottom scaffold surfaces.

[0014] In addition, it is further desirable still, particularly with relatively larger-size (e.g., meterscale) cultured layers or sheets, that sagging of the scaffold(s) be minimized, if not all together avoided.

[0015] The objectives set forth herein are achieved by the present perfusion bioreactor as defined in the appended claims.

[0016] Accordingly, in a first aspect there is provided a perfusion bioreactor comprising a flow chamber and a sealing member. The flow chamber has a width in a transverse direction along a transverse axis (x) and a length in a longitudinal direction along a longitudinal axis (y) and comprisesa flow chamber top, a flow chamber bottom, and an interior space defined between the flow chamber top and the flow chamber bottom. Along the length of the flow chamber, the interior space comprises an inlet portion, an outlet portion, and a central portion arranged between the inlet and outlet portions. The sealing member is arranged between the flow chamber top and the flow chamber bottom. Further, the inlet portion comprises at least one funnel element having a diameter in the transverse direction (x) that increases over a respective length of the inlet portion in the longitudinal direction (y) from a minimum diameter at a first end to a maximum diameter at a second end, the second end arranged proximal to the central portion.

[0017] Having at least one funnel element facilitates the provision of homogeneous or essentially homogenous supply of oxygen and nutrients over the full width of the scaffold in the transverse direction (x). A maximum reduction of oxygen concentration on the scaffold surface(s) of not more than 55% may also be realized.

[0018] A scaffold cassette may be further provided in certain exemplary non-limiting embodiments of the perfusion bioreactors described herein. In such examples, the scaffold cassette may comprise a first support member and a second support member which are configured to secure a scaffold therebetween in the interior space. It may be further preferred, in certain non-limiting examples, that the scaffold cassette is arranged in the central portion with the scaffold spaced a respective distance apart in a z-direction along a z-axis from the flow chamber top and the flow chamber bottom. Not only does securing the scaffold with the scaffold cassette ensure that the scaffold is sufficiently positioned and held in place in the bioreactor, but it also assists to minimize, if not all together prevent, the scaffold from sagging.

[0019] Further, the scaffold cassette enables the scaffold to be arranged with spacing in the z- direction from the flow chamber top and the flow chamber bottom. Stated in different terms, with the scaffold cassette, the scaffold can be arranged so that it does not directly contact the interior surface(s) of the flow chamber. In this sense, the top surface of the scaffold can be spaced a distance apart in the z-direction from the flow chamber top, and similarly, the bottom surface of the scaffold can be spaced a distance apart in the z-direction from the flow chamber bottom. By arranging the scaffold with this spacing from the interior surface(s) of the flow chamber, it is possible to advantageously perfuse media such as culture media to both the top and bottom surfaces of the scaffold. As a result of this dual surface perfusion, cell viability and performance across the entire scaffold can be improved, and by extension, may contribute to improving the overall quality and / or increased thickness of the cultured cell layers or collagen sheets.

[0020] The inlet portion may comprise at least one wedge element. This may contribute to breaking up the incoming, high-velocity flow, which reduces, if not entirely prevents, high fluid velocity in the center of the scaffold. By extension, this may facilitate uniformity in the shear stress at the scaffold surface(s), and may promote uniformity in the distribution of solutes and removal of waste. The wedge element may also contribute to enabling the provision of a relatively homogenous flow pattern across the scaffold surface(s).

[0021] It may be preferable, in certain non-limiting embodiments, that the wedge element has a diamond shape. However, it is to be understood that a diamond shape is exemplary and that oneof ordinary skill in the art may select other suitable shape(s) for the wedge element provided that the selected shape also contributes to the reduction and / or prevention of high fluid velocity in the center of the scaffold. The wedge element may also contribute to ensuring that the flow distribution in the transverse direction (x) over the top and bottom scaffold surfaces is homogeneous or essentially homogenous.

[0022] The wedge element may be arranged proximal to the first end of the inlet portion. In other words, the wedge element may be positioned along the longitudinal axis (y) such that it is relatively closer to the minimum diameter of the funnel element than to its maximum diameter. Arrangement of the wedge element in this manner may ensure a homogenous flow of fluid over the scaffold surface(s) in the central portion of the interior space of the flow chamber and may further contribute to interrupting the high-velocity flow between the inlet portion the central portion.

[0023] It is to be understood that formation of the wedge element is not intended to be particularly limited. In certain non-limiting embodiments, the wedge element may be formed integrally with the flow chamber bottom. However, it is contemplated that in other non-limiting embodiments, the wedge element may be formed separately from the flow chamber and subsequently added thereto.

[0024] To further facilitate perfusion to both the top and bottom surfaces ofthe scaffold, the scaffold cassette may be arranged such that the scaffold is spaced a respective distance apart in the z- direction from the interior surfaces of the flow chamber top and the flow chamber bottom. In exemplary non-limiting embodiments, the respective distances between the scaffold and each of the flow chamber bottom and the flow chamber top in the z-direction are each in a range of 0 to 5, preferably 0.5 to 5 mm. In certain non-limiting embodiments, it may be preferred that the respective distances between the scaffold and each of the flow chamber top and the flow chamber bottom are in a range of 1 to 5 mm, preferably 2 to 4 mm, and more preferably at least 3 mm. In any case, this spacing contributes to ensuring that cells throughout the full thickness of the scaffold can be provided with necessary nutrients and oxygen via the perfused culture medium.

[0025] In addition to the inlet portion having at least one funnel element, the outlet portion may also, in exemplary non-limiting embodiment, comprise at least one funnel element having a diameter in the transverse direction (x) that tapers over a respective length of the outlet portion in the longitudinal direction (y) from a maximum diameter at a first end to a minimum diameter at a second end, the first end of the outlet portion arranged proximal to the central portion of the interior space. By providing a funnel element in the outlet portion, and with a taper that mirrors the taper of the funnel element in the inlet portion at least about the transverse axis (x) of the flow chamber, it is further envisioned that, in some non-limiting embodiments, the flow in the bioreactor can be reversed, i.e. from entering at the inlet and exiting at the outlet to instead entering at the outlet and exiting at the inlet.

[0026] In certain non-limiting embodiments, at least one of the inlet portion and the outlet portion may comprise a plurality of funnel elements. This may facilitate scale-up of the bioreactor system, for example in the transverse and / or longitudinal directions, in order to be able to produce relatively larger sized cultured cell layers or collagen sheets. In this regard, incoming high-velocity flow can be more easily broken-up over a relatively longer dimension (e.g., a transverse and / or longitudinallength). Further, it may be preferable, in such non-limiting embodiments, that the plurality of funnel elements is arranged in parallel in the transverse direction (x).

[0027] In order to introduce and remove culture medium into and out of the flow chamber, the inlet portion may comprise at least one inlet port and the outlet portion may comprise at least one outlet port. One or both of the at least one inlet port and the at least one outlet port may be adapted to connect to a medium reservoir.

[0028] It is further contemplated that the medium can be introduced into the flow chamber either from a lateral side or from the top. In this regard, it is preferable in certain non-limiting embodiments that the at least one inlet port is arranged in either an xy-plane (e.g., lateral side) or an xz-plane (e.g. top and / or bottom) of the flow chamber.

[0029] Like the at least one inlet port, it may also be preferred that the at least one outlet port is arranged in either an xy-plane (e.g., lateral side) or an xz-plane (e.g. top and / or bottom) of the flow chamber. The provision and arrangement of the at least one outlet port may, in certain non-limiting embodiments, facilitate effectuation of a reversal of the flow through the flow chamber. In other words, each of the at least one inlet port and the at least one outlet port may function as either an inlet port or an outlet. This interchangeability of the inlet and outlet ports allows the medium to be introduced into the flow chamber through the at least one outlet port in the outlet portion and exit out of the flow chamber through the at least one inlet port in the inlet portion.

[0030] In certain non-limiting embodiments, a mesh layer may be arranged between the scaffold and the flow chamber bottom. The mesh layer may support the scaffold, and by extension, contribute to the reduction and / or prevention of scaffold from sagging while arranged in the bioreactor system.

[0031] It is further contemplated that one or both of scaffold cassette and the mesh layer may also comprise a coating. For example, an anti-fouling coating may be applied to any one or more of the first support member, the second support member, and the mesh layer to reduce, if not eliminate, cells and / or the scaffold from sticking to the scaffold cassette and / or the mesh layer. This may, by extension, further facilitate the ease of which the scaffold may be extracted from the bioreactor system at the end of culturing.

[0032] In certain exemplary non-limiting embodiments, it is further contemplated that one or more interior surfaces of the flow chamber may also comprise a coating, such as an anti-fouling coating. The coating applied on the interior surface(s) of the flow chamber may be the same coating applied on the scaffold cassette and / or the mesh layer, a similar type of a coating (e.g., a similar anti-fouling coating), a different coating, or combinations thereof.

[0033] To facilitate securement of the scaffold, the first support member and the second support member may each have a respective perimeter that comprises a respective scaffold contacting surface. It is to be understood that the scaffold may be secured either in partially or fully along the respective scaffold contacting surfaces.

[0034] In some non-limiting embodiments, the first support member and the second support member may each have a respective scaffold contacting surface that is flat or essentially flat. Further, it is to be understood that a respective scaffold contacting surface that is flat or essentiallyflat may provide a larger surface area over which the scaffold may be clamped relative to a scaffold contacting surface that is not flat, as discussed hereafter. However, the comparatively larger surface area may be offset by clamping the scaffold only partially around a perimeter thereof.

[0035] In other exemplary embodiments, one or both of the respective scaffold contacting surfaces may comprise a curvature. Such curvature(s) may be arranged either partly or fully around the respective perimeter and may advantageously decrease a surface area over which the scaffold may be clamped, and extension, further ensure securement of the scaffold in the bioreactor system. In this regard, only an apex or top of each of the first and second support members may be in contact with the secured scaffold.

[0036] The respective perimeters of the first and second support members may also be provided in the form of any shape suitable for placement and securement of the scaffold in the interior space, including but not limited to, circle, oval, ellipse, triangle, square, rectangle, or other polygon. However, it may be preferable, in some non-limiting embodiments, that the first and second support members are rectangular, and more preferably, square-shaped.

[0037] Due at least in part to the provision of a funnel element in the inlet portion, the second end of the inlet portion, which is adjacent to the central portion, may form an inner angle therewith and relative to the transverse axis (x). This inner angle (a) may, in some non-limiting embodiments, fall within a range of from 35 to 55 degrees. Further still, it may be preferred that the inner angle (a) is within a range of from 40 to 50 degrees. Arranging the inlet and central portions with this inner angle therebetween may further contribute to the provision of a homogenous supply of nutrients and oxygen over the full scaffold width.

[0038] In some non-limiting embodiments, the inlet portion and the outlet portion may be at least mirror symmetric about the transverse axis (x). Optionally, the inlet and outlet portions may be additionally mirror symmetric about the longitudinal axis (y). By having the inlet and outlet portions be mirror symmetric at least about the transverse axis (x) and optionally about the longitudinal axis (y), it is possible that the flow of medium through the bioreactor system can easily be reversed (i.e., flow entering into the bioreactor system from the outlet portion and exiting through the inlet portion).

[0039] In other non-limiting embodiments, the inlet portion and the outlet portion may be asymmetrical about at least the transverse axis (x). For example, it may be preferred, in some nonlimiting embodiments, that the inlet portion extends over about 30% of a total length of the interior space in the longitudinal direction (y), the central portion extends over about 60% of the total length, and the outlet portion extends over about 10% of the total length.

[0040] Further, is it to be understood that the flow chamber may be constructed or formed from one or more of a number of suitable source materials. For example, source material for the flow chamber may be appropriately selected by one or ordinary skill in the art from any one or more of an epoxy, polycarbonate (PC), polypropylene (PP), polystyrene (PS), stainless steel, anodized aluminium, plexiglass, glass, polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), and polyether ether ketone (PEEK).

[0041] In another aspect of the present disclosure, there is provided a scaffold cassette which is configured to secure a scaffold and to minimize, if not all together prevent, sagging of the securedscaffold, particularly in a thickness dimension perpendicular (e.g., in a z-direction along a z-axis) to the width and length dimensions (e.g., transverse and longitudinal directions along x- and y- directions, respectively) of the scaffold. The scaffold cassette comprises a first support member and a second support member which are configured to secure a scaffold therebetween. In addition, the scaffold cassette may optionally comprise a mesh layer to further contribute to minimizing and / or preventing sagging of the scaffold.

[0042] In yet another aspect of the present disclosure, there is provided a method for culturing, the method comprising the steps of: i) providing a scaffold; ii) placing the scaffold in a perfusion bioreactor as described above; seeding cells onto the scaffold in the bioreactor; and culturing the cells. Preferably the cells are fibroblasts, more preferably dermal fibroblasts, even more preferably bovine dermal fibroblasts. Preferably the culturing of step iv) is under conditions conducive to collagen production to form a collagen sheet; and the method optionally further comprises the step of: v) isolating the collagen sheet. Preferably the scaffold is a glass fibre sheet, wherein the glass fibre sheet more preferably is a chopped strand mat. In preferred embodiments culturing is performed for about 2 to 9 weeks, preferably 3 to 8 weeks, more preferably for about 4 to 7 weeks or for about 6 to 7 weeks.

[0043] Further objectives, features and advantages of the present absorbent article are described in the detailed description below with reference to the appended drawings.Brief description of the drawings

[0044] The present invention will be discussed in more detail below, with reference to the attached drawings, in which:

[0045] Figure 1 shows an exploded view of an exemplary perfusion bioreactor according to the present disclosure;

[0046] Figure 2A shows a top view of an exemplary flow chamber bottom with asymmetrical inlet and outlet portions;

[0047] Figure 2B shows a cross section of the flow chamber bottom of Fig. 2A along the line II;

[0048] Figure 3A shows an exemplary support member with a flat scaffold contacting surface and Figure 3B shows another exemplary support member with a curved scaffold contacting surface;

[0049] Figure 4A shows an exemplary flow chamber top with symmetrical inlet and outlet portions and Figure 4B shows an exemplary flow chamber bottom that corresponds with the flow chamber top of Fig. 4A;

[0050] Figure 5 shows a schematic of an exemplary perfusion bioreactor system according to the present disclosure;

[0051] Figures 6A-6C show the modelled oxygen concentration on the scaffold surface in exemplary perfusion bioreactors of the present disclosure;

[0052] Figures 7A-7C show the modelled respective distributions of shear stress over the scaffold surface area in the exemplary perfusion bioreactors of Figs. 6A-6C;

[0053] Figure 8 shows an isolated collagen sheet produced using an exemplary bioreactor in accordance with the present disclosure, and

[0054] Figure 9 shows the isolated collagen sheet after application of a tanning process thereto.Detailed description

[0055] As a general reference, more than one term may be used to refer to the same axis and / or reference conventions of the bioreactors and scaffold cassettes described herein. For example, the terms width (dimension), transverse direction, and x-direction may be used interchangeably herein to refer to the x-axis; the terms length (dimension), longitudinal direction, and y-direction may be used interchangeably herein to refer to the y-axis; and likewise, the terms thickness (dimension) and z-direction may be used herein to refer to the z-axis.

[0056] In addition, it is to be generally understood herein that a scaffold is preferably a substantially planar, substantially rectangular scaffold having a width in a transverse direction along a transverse axis (x) and a length in a longitudinal direction along a longitudinal axis (y).

[0057] Figure 1 shows an exploded view of an exemplary bioreactor. The bioreactor has a flow chamber that includes a flow chamber top 21 and a flow chamber bottom 22. The flow chamber top and the flow chamber bottom can be secured together by any suitable securement means. For example, it is contemplated that the securement means may comprise any one or more of bolt(s), clamp(s), screw(s), nail(s), or the like. In any case, the securement means should permit the flow chamber top and the flow chamber bottom to be at least partially separable from one another when the bioreactor system is not in use to allow access, for example, to facilitate insertion and / or removal of a scaffold 5 from the bioreactor. In addition, the securement means assist to prevent leaks from the assembled bioreactor and further facilitate maintenance of the sterility of the culture inside the bioreactor.

[0058] When assembled, an interior space is defined in between the flow chamber top and the flow chamber bottom. Along the length of the bioreactor in the longitudinal direction (y), the interior space is divided into three sections - an inlet portion, an outlet portion, and a central portion that that is arranged between the inlet and outlet portions. These sections will be discussed in further detail hereafter.

[0059] A sealing member s is provided between the flow chambertop and the flow chamber bottom to ensure sealing engagement therebetween. Without the sealing member, there is a risk that the bioreactor may leak and / or the environmental condition(s) (e.g., temperature, pressure, etc.) in the interior space of the flow chamber may be negatively affected. Adequate sealing of the bioreactor also assists with ensuring that the sterility of the culture be maintained.

[0060] In certain non-limiting exemplary embodiments of the bioreactors described herein, a scaffold cassette 4 which is configured to secure a scaffold therewith may be provided in the interior space of the bioreactor. The scaffold cassette includes a first support member 41 and a second support member 42. In the non-limiting example of Fig. 1 , the first support member and the second support member are stacked in the z-direction with the scaffold sandwiched therebetween. In this sense and relative to the scaffold, the first support member may be seen as a top support member and the second support member may be seen as a bottom support member. Here also, both the first support member and the second support member are frame-like elements. Each of the firstsupport member and the second support member has a respective perimeter that comprises a respective scaffold contacting surface. With this particular exemplary scaffold cassette, the scaffold is secured only along its length in the longitudinal (y) direction and not its entire perimeter (i.e., also along its width in the transverse direction (x). Because of this configuration, the majority of a surface area of the scaffold is not obscured or obstructive from direct contact with perfused culture medium. However, it is to be understood, that for other non-limiting embodiments, the scaffold may also be secured at least partially along the frame-like support elements of the scaffold cassette arranged in the transverse (x) direction and / or around an entire perimeter thereof.

[0061] Further, the first support member and the second support member may be similarly attached and removably secured together by cassette securement means. Similar to the securement means used to attach and secure the flow chamber top and the flow chamber bottom together, the cassette securement means may comprise any one or more of bolt(s), clamp(s), screw(s), nail(s), or the like.

[0062] In some exemplary, non-limiting embodiments, the scaffold cassette may further comprise a mesh layer (not shown). In such embodiments, it is typically preferable that the mesh layer is arranged between the scaffold and the flow chamber bottom. This helps to further support the scaffold and to prevent against sagging due to gravity. In terms of materials, the ordinarily skilled person may readily select any one or more materials suitable for use in a culturing environment to form the mesh layer. For example, non-exhaustive exemplary materials may include stainless steel, inert and / or biocompatible polymer(s). The mesh can generally have the same shape as the scaffold that it is envisioned to support.

[0063] In addition, it is contemplated that the scaffold cassette, the mesh layer, any one or more of the interior surfaces of the flow chamber, or combinations thereof, may comprise a coating, such as an anti-fouling coating. Such coating(s) may be applied to any one or more of the first support member, the second support member, the mesh layer, and the interior surface(s) of the flow chamber, such as those of the flow chamber top and the flow chamber bottom.

[0064] In certain non-limiting exemplary embodiments, one or more support elements 241 , such as studs, projections, protuberances, or the like, may be provided in the flow chamber bottom. These one or more support elements may be arranged to provide additional support for the scaffold cassette and / or mesh layer in the bioreactor. Further, the one or more support elements may assist with ensuring that the scaffold is spaced a distance apart from the flow chamber bottom in the z- direction, and thereby enabling perfusion of medium over the bottom surface of the scaffold.

[0065] An exemplary flow chamber bottom is depicted in Figure 2A. As can be seen in the figure, the length of the flow chamber is divisible into three distinct portions - an inlet portion 23, an outlet 25, and a central portion 24 arranged between the inlet and outlet portions. In this non-limiting example, the inlet and outlets are symmetrical in the longitudinal direction about the longitudinal axis (y) and asymmetric in the transverse direction about the transverse axis (x). Here, the inlet portion comprises funnel element 26. The funnel element has a diameter in the transverse direction (x) that increases over the respective length of the inlet portion in the longitudinal direction (y) from minimum diameter at a first end to a maximum diameter at a second end. Here, the maximumdiameter of the funnel element is arranged proximal, or directly adjacent, to the central portion whereas the first end with the minimum diameter is arranged farthest from the central portion in the longitudinal direction (y). The inlet portion and / or the funnel element may be provided with rounded corners. This ensures sufficient distribution of flow, particularly without the need for any recirculation zone(s).

[0066] The inlet portion is arranged adjacent to the central portion at a first of two opposing side edges in the longitudinal direction. An inner angle (a) is formed relative to the transverse axis (x) where the two portions meet. The inner angle (a) is an acute angle, which is preferably in a range of from 35 to 55 degrees, and even more preferably in a range of from 40 to 50 degrees. Further, structuring the inlet portion and the central portion to form such an inner angle therebetween may facilitate the provision of a homogenous supply (flow) of nutrients and oxygen across the full scaffold.

[0067] As can be seen in Fig. 2A, the inlet portion further comprises a wedge element 27, which in this particular example, is arranged proximal, or nearest, to the first end of the inlet portion. The wedge element is provided in the inlet portion primarily to disrupt the incoming high-velocity flow. By interrupting the flow at this point, it is possible to reduce, if not prevent, high fluid velocity in the center of the scaffold. This configuration may also facilitate homogeneity in the fluid flow over the scaffold surface, and by extension, provide uniformity in shear stress at the scaffold surface(s).

[0068] Here, the wedge element is shown as having a diamond-shape. It is to be understood, however, that this shape is but one example. It is contemplated that one of ordinary skill in the art may readily select other shape(s) for the wedge element provided that the selection is also suitable to assist with breaking-up the incoming flow such that high fluid velocity in the center of the scaffold may be minimized, if not all together avoided.

[0069] The inlet portion also comprises at least one inlet port which is adapted to connect to a medium reservoir. The exemplary flow chamber bottom of Fig. 2A is provided with one inlet port 231 , which is shown in Fig. 2B. In this instance, the inlet port is arranged an xy-plane of the flow chamber. By way of this arrangement, fluid can be introduced into and / or removed from the chamber from the side (laterally).

[0070] The central portion of the exemplary flow chamber depicted in Fig. 2A is the portion having the largest dimensions both in the transverse and longitudinal directions (x, y) and relative to the inlet and outlet portions. During operation of the bioreactor, a scaffold is arranged in the scaffold area 245 of the central portion. It may be preferable that the central portion is rectangular or essentially rectangular. Because the flow chamber is typically also rectangular in shape, having a rectangular or essentially rectangular central portion enables the surface area coverage of the scaffold arranged in the bioreactor to be maximized in both the transverse and longitudinal directions (x, y), thereby making full use of the available interior space.

[0071] Adjacent to the second opposing side edge of the central portion in the longitudinal direction (y) is the third of the three distinct portions, the outlet portion. In the example shown in Fig. 2A, the outlet portion also comprises a funnel element 26. On this side, the funnel element has a diameter in the transverse direction (x) that tapers over a respective length of outlet portion in the longitudinaldirection (y) from a maximum diameter at a first end to a minimum diameter at a second end, and the first end of the outlet portion is arranged proximal, or directly adjacent, to the central portion. Like the funnel element in the inlet portion, the funnel element in outlet portion may also be provided with rounded corners. Again, this can ensure sufficient distribution of flow, particularly without the need for any recirculation zone(s).

[0072] An inner angle (p) is formed relative to the transverse axis (x) where the central portion and the outlet portion meet. Like the inner angle (a) formed between the inlet portion and the central portion, the inner angle formed between the central portion and the outlet portion is also an acute angle. In certain non-limiting embodiments, the inner angle (p) may be in a range of from 10 to 55 degrees.

[0073] In addition, when the inlet and outlet portions are not mirror symmetric about both the transverse and longitudinal axes (x, y), it may be preferable that the inner angle (p) is smaller than the inner angle (a). For example, it may be preferable that the inner angle (p) is in a range of from 10 to 30 degrees.

[0074] The outlet portion, too, comprises at least one outlet port, which is adapted to connect to a reservoir and / or other fluid discharge means. In Fig. 2B, the exemplary flow chamber bottom is provided with one outlet port 251 . In this non-limiting example, the outlet port is arranged an xy- plane of the flow chamber. By way of this arrangement, fluid can be discharged laterally from the side of the chamber.

[0075] As indicated above, the scaffold may be secured in position, about the x, y, and z axes, in the flow chamber with a scaffold cassette by the first and second support members thereof. Exemplary support members are shown in each of Figs. 3A and 3B. Each support member 41 is a rectangular frame-like element with two transverse frame members and two longitudinal frame members. In these examples, the scaffold is secured in the longitudinal direction (y) along the scaffold contacting surfaces 410A, 410B of the longitudinal frame members. The scaffold is not secured in the transverse direction (x) along the non-contact surfaces 411 A, 411 B of the transverse frame members.

[0076] For the avoidance of the doubt, it is to be understood that the scaffold may be secured between first and second support members, though only one support member 41 is shown in each of Figs. 3A and 3B. It should also be understood that it is typically preferred that the second support member is mirror symmetric with the first support member, in the non-limiting embodiments, such that the scaffold may effectively be sandwiched in place therebetween.

[0077] In addition, the scaffold contacting surfaces 410A, 410B of the support members may, in certain non-limiting embodiments be planar or flat, such as those shown in Fig. 3A; alternatively, in other non-limiting embodiments, the scaffold contacting surfaces 410A, 410B may be curved or comprise a curvature, such as those shown in Fig. 3B.

[0078] Figures 4A and 4B depict the flow chamber top and corresponding flow chamber bottom, respectively, of another exemplary flow chamber in accordance with the present disclosure. Different from the exemplary flow chamber of Fig. 2A, the flow chamber of Fig. 4A has inlet and outlet portions that are mirror symmetric about both of the transverse and longitudinal axes (x, y) ofthe bioreactor. As mentioned above, by having inlet and outlet portions that are symmetrical, it is possible to wholly reverse the direction of flow within the bioreactor; in other words, fluid can either be directed to enter the bioreactor via the inlet portion and exit through the outlet portion, or vice versa, with fluid directed to enter the bioreactor via the outlet portion and exit through the inlet portion.

[0079] In the exemplary flow chamber of Fig. 4A, the inlet portion comprises two inlet ports 231 A, 231 B and the outlet portion comprises two outlet ports 251 A, 251 B. Each of the inlet and outlet ports is adapted to connect to at least one reservoir. Depending on the configuration of the bioreactor system, the at least one reservoir may contain new, recycled, used, or discharged medium.

[0080] In this particular non-limiting example, the inlet and outlet ports are all arranged in a xz- plane of the flow chamber. This enables the fluid to enter into the flow chamber from above, rather than laterally from the side. Having the inlet and outlet ports arranged in this manner also means that the overall size of the bioreactor can be made relatively smaller, which provides material usage and cost savings. Not to mention, the relatively smaller-sized bioreactor is also lighter and thus easier to handle, which may be desirable for everyday use.

[0081] Unlike the inlet portion shown in Fig. 2A, the inlet portion of Fig. 4A includes a plurality of funnel elements. In this specific example, there are four funnel elements 26A-D that are arranged in parallel in the transverse direction (x). All of the funnel elements of the plurality are identical. Each funnel element has a diameter in the transverse direction (x) that increases over a length of the inlet portion in the longitudinal direction (y) from minimum diameter at a first end to a maximum diameter at a second end which is arranged proximal to the central portion. In this non-limiting example, each inlet port feeds incoming fluid flow to a pair of adjacent funnel elements. More specifically, inlet port 231 A feeds fluid flow to funnel elements 26A and 26B while inlet port 231 B feeds fluid flow to funnel elements 26C and 26D.

[0082] Because the inlet portion and the outlet portion are mirror symmetric along both the transverse and longitudinal axes (x, y), it further follows that the outlet portion also includes a plurality of funnel elements 26E-H, specifically four, that are arranged in parallel in the transverse direction (x). Likewise, the funnel elements arranged in the outlet portion are identical to one another, and each funnel element has a diameter in the transverse direction (x) that tapers over a length of outlet portion in the longitudinal direction (y) from a maximum diameter at a first end to a minimum diameter at a second end. Further, fluid flow through each outlet port is fed by a pair of adjacent funnel elements. More specifically, fluid flow from funnel elements 26E and 26F is fed to outlet port 251 A and fluid flow from funnel elements 26G and 26H is fed to outlet port 251 B.

[0083] In addition, the inlet portion comprises a plurality of respective wedge elements that correspond with the plurality of funnel elements. With reference to Fig. 4A, it is noted that only the respective wedge elements 27 of funnel elements 26A and 26E are specifically labelled with a reference numeral in the figure; the respective wedge elements of the other funnel elements, though not specifically indicated with reference numerals, are also shown. Each wedge element is arranged in the longitudinal direction (y) in the first end of a corresponding funnel element. As such, theplurality of wedge elements are arranged nearer to the minimum diameter of the funnel element than to the maximum diameter. The outlet portion also comprises a plurality of wedge elements that correspond with its own plurality of funnel elements. Similar to the wedge elements in the inlet portion, each wedge element in the outlet portion is arranged nearer to the minimum diameter of the funnel element than to the maximum diameter.

[0084] Also different from the exemplary flow chamber of Figs. 1 , 2A, and 2B is the scaffold cassette of Fig. 4A. Here in this example embodiment, the scaffold cassette also includes first support member 41 and second support member 42, however, these first and second support members are merely longitudinal support members. There are no transverse members and no perimeter is formed around the scaffold by the first support member and / or the second support member. Rather, each support member is arranged on only one of the opposing sides of a scaffold in the longitudinal direction (y). The scaffold may be secured with the first support member and the second support member via any suitable securement means such one or more scaffold clamps (not shown).

[0085] The corresponding flow chamber bottom of the exemplary flow chamber top of Fig. 4A is shown in Fig. 4B. As can be seen, the flow chamber bottom is relatively planar, particularly in the inlet and outlet portions. The central portion is also relatively planar, apart from the provision of the first and second support members in the longitudinal direction for securing the scaffold. In addition, the central portion is provided with a plurality of support elements 241 arranged in the scaffold area that contribute to keeping the scaffold out of direct contact with the surface of the flow chamber bottom. By extension, this helps to ensure that perfusion of medium to the bottom surface of the scaffold arranged in the bioreactor is not inhibited.

[0086] Figure 5 shows a schematic overview of a bioreactor system including an exemplary perfusion bioreactor as described herein. In this non-limiting example, the bioreactor 100 has four access points that include two inlets and two outlets for delivering medium to and removing medium from the bioreactor. This is performed in a closed loop configuration, which includes an upper loop and a lower loop. For the avoidance of the doubt, it is to be understood that the upper and lower loops as described and illustrated herein may be arranged in the same horizontal plane such that the loops are next to each other.

[0087] The upper loop is configured as follows: the bioreactor is connected with medium reservoir 9A on the lefthand side by line 10Lupand on the righthand side by line 10Rup. Similarly, in the lower loop, the bioreactor is connected with medium reservoir 9A on the lefthand side by line 10Liow and on the righthand side by line 10Riow. Because of the closed loop configuration, the direction of flow through the bioreactor can be driven from left to right (10Lup / iow to 100 to 10Rup / ow to 9A and back to 1 0Lup / iow) , or inversely, the direction of flow can be driven from right to left (10Rup / iowto 100 to 1 0Lup / iow to 9A and back to 10Rupiow). A peristaltic pump 8 is also provided on each of the respective tubing lines to control the flow of fluid from the medium reservoir through the lines and into the bioreactor. In Fig. 5, the pumps are shown arranged on the lefthand side of the exemplary bioreactor system. However, it is to be understood that each pump may, in other non-limiting embodiments, additionallyand / or alternatively be arranged on the righthand side of the system. One or more additional reservoirs 9B may also be provided.

[0088] While the invention has been described herein by reference to certain embodiments, it is to be understood that modifications in addition to those described herein may be made to the structures and techniques described herein without departing from the spirit and scope of the invention. Accordingly, although specific embodiments have been described, they are examples only and are not limiting upon the scope of the invention.

[0089] Also provided is a method for culturing cells, the method comprising the steps of:

[0090] i) providing a scaffold;

[0091] ii) placing the scaffold in a perfusion bioreactor as described herein;

[0092] iii) seeding cells onto the scaffold in the bioreactor;

[0093] iv) culturing the cells.

[0094] Also provided is a method for providing a collagen sheet, the method preferably comprising the steps of: i) providing a scaffold such as a glass fibre sheet preferably comprising solid fibres; ii) placing the scaffold such as the glass fibre sheet in a perfusion bioreactor as described herein; iii) seeding cells such as fibroblasts onto the scaffold in the bioreactor; iv) culturing the cells, preferably under conditions conducive to collagen production to form a collagen sheet; and optionally v) isolating the collagen sheet.

[0095] Such methods can be referred to herein as a method according to the invention. The collagen sheets produced by this method have a good collagen content, and can be tanned to produce in vitro leather of good quality. In preferred embodiments the steps are performed in numerical order. In preferred embodiments step v) is not optional. In other embodiments, step v) is not performed.

[0096] In step i) a scaffold is provided. Several types of scaffold can be used, a preferred type being a fibrous scaffold. A fibrous synthetic scaffold may comprise one or more of a carbon fibre scaffold, an aramid fibre scaffold such as a Kevlar scaffold, or a glass fibre scaffold such as a fiberglass scaffold. Further suitable scaffolds can be scaffolds comprising polyhydroxyalkanoate (PHA), polyhydroxybutyrate (PHB), polylactic acid (PLA), polyethylene terephthalate (PET), polybutylene succinate (PBS), silk, viscose, cellulose, or blends of these materials. One particular example of a fibrous scaffold that may be used as described herein is silk. Silk (e.g., organic and / or synthetic silk) may be formed (e.g., spun) to a predetermine fiber thickness and used in a woven and / or non-woven sheet forming the scaffold onto which ECM-releasing cells may be cultured.

[0097] Most preferably as a scaffold a glass fibre sheet is provided, and said sheet preferably comprises solid fibres. In preferred embodiments substantially all fibres of the glass fibre sheet are solid fibres. The glass fibre sheet preferably does not comprise sol-gel fibres, or fibres derived from a sol-gel process. The fibres are preferably melt-derived, wherein for example the required elements (such as silica, boron, aluminium, calcium, magnesium) are heated (1100°C < AT < 1500°C) until molten, followed by extrusion such as pressure extrusion in single fibres. A suitable feed format can be 1 ,5m x 20m (w x I). Preferred solid fibres are fused silica glass fibres. Preferably, the glass fibre comprises glass containing 5-70% SiO, 5-40% BO, and 1-50% CaO in mass-% ofoxide conversion as a glass composition. The glass fibres can comprise at least Si, B, and Ca, preferably at least Si, B, Ca, and Mg, or Si, B, Ca, and Al, even more preferably Si, B, Ca, Al, and Mg. Optionally P is also present.

[0098] In some embodiments, silica, boron, and calcium may each be present in the compositions in an amount of about 1 % to about 99%, based on the weight of the glass. In further embodiments, silica, boron, and calcium may each be present in the composition in about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In certain embodiments, silica, boron, and calcium may each be present in the composition in about 5 to about 10%, about 10 to about 15%, about 15 to about 20%, about 20 to about 25%, about 25 to about30%, about 30 to about 35%, about 35 to about 40%, about 40 to about 45%, about 45 to about50%, about 50 to about 55%, about 55 to about 60%, about 60 to about 65%, about 65 to about70%, about 70 to about 75%, about 75 to about 80%, about 80 to about 85%, about 85 to about90%, about 90 to about 95%, or about 95 to about 99%. Some embodiments may contain substantially each of silica, boron, calcium, aluminium, and magnesium, with only traces of other elements if any, preferably no other elements (outside of any required elements such as H or O) are present.

[0099] The glass fibres may further comprise one or more of a silicate, borosilicate, borate, or calcium, including CaO, P2O5, SiC>2, and B2O3. Suitable glass types are widely known. Examples are A-glass, C-glass, D-glass, E-glass, R-glass, and S-glass. Most preferably the glass is E-glass, which is alumino-borosilicate glass with less than 1 % (w / w) of alkali oxides. The glass is preferably alkali-free, which in this context refers to an alkali oxide content of less than 1 % (w / w). The glass is preferably acid / base resistant.

[0100] The form or shape of the scaffold such as the sheet is not essential. It can be convenient to have a scaffold such as a glass fibre sheet that is roughly the same shape as the interior space that is available in the intended bioreactor. Most scaffolds such as glass fibre sheets can be cut to size using scissors or knives. A skilled person can select an appropriate size. Preferred scaffolds such as glass fibre sheets are rectangular or substantially rectangular, such as square. Preferably, the sides of the scaffold such as the glass fibre sheet are about 0.1-50 cm, more preferably about 1-40 cm, still more preferably about 5-35, still more preferably about 5-30 cm, still more preferably 10-25 cm. For the eventual production of in vitro leather, areas of 100 cm2(10x10 cm) or 625 cm2(25x25 cm) or preferably 900 cm2(30x30 cm) or more preferably 1225 cm2(35x35 cm) can be suitable. Other preferred scaffold sizes are 4x4 and 5x5 cm and 10x10 cm. Alternately, for use in larger bioreactors, areas of over 10000 cm2can be considered, such as 20000 cm2(100x200 cm). For sheets having areas of over 10000 cm2it is preferred that the thickness of the sheet is at least 1500 pm.

[0101] The glass fibre sheet can be of any suitable type. Examples of glass fibre sheets are woven fabrics, knit fabrics, unidirectional fabrics, or chopped strand mats. It was found that chopped strand mats offer an attractive spatial distribution of fibres for culturing, and accordingly the glass fibre sheet is preferably a chopped strand mat. In chopped strand mats glass fibres lay randomly across each other. Chopped strand mats can further comprise a binder, which in preferred embodimentsis washed out of the sheet prior to cell culture. Chopped strand mats are preferably isotropic. In general, fibres in chopped strand mats are substantially of equal length. Chopped strand mats are widely available from commercial sources (see for instance the examples). Suitable commercially sourced mats are chopped strand mats, such as chopped strand mats for marine use, for roofing, or for use as battery separator, more preferably comprising GRP resin (which is a commonly used polyester resin).

[0102] For reference, chopped strand mats are often found in fibreglass that can be used for repair works, for instance on boat hulls. Such mats generally consist of glass fibres and a binder. Mats are typically processed using a hand lay-up technique, where sheets of material are placed on a mold and brushed with resin. The material conforms to different shapes when wetted, and the binder conveniently dissolves in the resin, leaving the glass fibres. After the resin cures, a hardened product remains. This makes commercially available fibreglass chopped strand mats convenient sources of glass fibre sheets for use in the invention - the non-glass component can be easily washed away and a glass fibre sheet remains. In preferred embodiments the glass fibre sheet is a washed fibreglass sheet. In preferred embodiments the glass fibre sheet essentially consists or consists of glass fibres.

[0103] It was found that cell seeding and proliferation was optimal for glass fibres of certain diameters, at certain density and with certain inter-fibre distances. In preferred embodiments is provided the method according to the invention, wherein i) the glass fibres have an average diameter of from about 1 to about 25 pm, preferably of from about 5 to about 15 pm; ii) the fibre density in the glass fibre sheet is from about 1 to about 30 mg / cm2, preferably from about 1 .5 to about 25 mg / cm2, more preferably from about 2 to about 20 mg / cm2; in some embodiments the density is about 20-25 mg / cm2. In some embodiments the density is about 1 .5 to about 8 mg / cm2; iii) the gap size between fibres in the glass fibre sheet is from about 10 to about 140 pm, preferably from about 25 to about 125 pm; and / or iv) the scaffolds such as the glass fibre sheets have a thickness of about 10-3000, preferably 15-2000, more preferably 20-1000 pm, or optionally the scaffolds such as the glass fibre sheets have a thickness of about 10-5000, preferably 1000-4000, more preferably 1500-3500 pm. More preferably all four of the above apply. Glass fibre diameter is preferably at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 pm. Glass fibre diameter is preferably at most 25, 24, 23, 22 ,21 , 20, 19, 18, 17, 16, or 15 pm. Preferably the diameter is from about 3 to about 20 pm, more preferably from about 7 to about 18 pm, still more preferably from about 10 to about 14 pm. Fibre diameter can be measured using any known technique, a suitable example is by scanning electron microscopy. The fibre diameter is preferably the radius of the cylinder that best represents the fibre - it is generally the smallest dimension of the fibre.

[0104] Fibre density is preferably at least 1 .1 , 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1 .9, 2, 2.2, 2.4, 2.6, 2.8, 3, 3.2, 3.4, 3.6, 3.8, 4, 4.2, 4.4, 4.6, 4.8, 5, 5.2, 5.4, 5.5, 5.6, 5.7, or 5.8 mg / cm2. Fibre density is preferably at most 10, 9.5, 8, 7.5, 7, 6.5, 6.4, 6.3, 6.2, 6.1 , 6, 5.9, or 5.8 mg / cm2. A thicker sheet generally has a higher density in mg / cm2. Further preferred densities are about 2 to about 7 mg / cm2, or about 3.5 to about 6.5 mg / cm2. Fibre density can be determined by conventional weighing of a fragment of known dimensions. In some embodiments the density is higher, for instance at most 30mg / cm2, such as about 15-30 mg / cm2, preferably 17-27 mg / cm2, more preferably about 20-25 mg / cm2.

[0105] The gap size between fibres can influence the dimensions available to cells when they are mobile, or during proliferation. It is known that the dimensions of a surrounding matrix can influence the behaviour of cells, and thus can impact collagen output. Good results were obtained with scaffolds such as glass fibre sheets having a gap size between fibres from about 10 to about 140 pm, preferably about 15 to about 130 pm, more preferably about 20 to about 120 pm, more preferably about 30 to about 110 pm, still more preferably about 40 to about 100 pm, more preferably about 50 to about 90 pm, more preferably about 60 to about 80 pm. Gap size can be determined by scanning electron microscopy.

[0106] The scaffold, such as the glass fibre sheet, preferably has a thickness of not more than 5000, more preferably not more than 4000, even more preferably not more than 3500, still more preferably not more than 3000 pm. When thicker scaffolds are desired, multiple sheets can be placed on top of one another. For mechanical robustness, the sheets preferably do not have a thickness of less than 20 pm. The scaffolds such as the sheets can have a thickness of 30-2000 pm, preferably 40-1700 pm, preferably 45-1500 pm, more preferably 50-800 pm, still more preferably 100-750 pm, still more preferably 150-700, 200-650, 250-600, 300-550, 350-500, or most preferably about 400-450 pm. In preferred embodiments the thickness is 1500 pm. Optionally the scaffolds such as the glass fibre sheets have a thickness of about 10-5000, preferably 1000-4000, more preferably 1500-3500 pm. Thickness can be determined by scanning electron microscopy or by using high precision callipers.

[0107] Increased cell seeding and collagen content were obtained with glass fibre sheets having a coating. In preferred embodiments the scaffolds such as the glass fibre sheet has been treated with a coating agent that introduces an organic coating on the glass fibres. Coating agents are a well-known class of compounds, and coating chemistry, particularly for glass material, is an established field.

[0108] It is not preferred that an organic coating refers to a proteinaceous coating or to an opsonisation or a deposition of extracellular material. An organic coating in this context is a coating of organic moieties, preferably of small organic moieties such as short alkyl chains bearing at least one heteroatom. The coating is preferably homogeneous, as can be studied using microscopy or FTIR techniques. The coating preferably consists of only a single type of moiety. The organic coating is preferably covalently linked to the glass fibres. Covalent linkage can be confirmed using ATR techniques such as ATR-FTIR.

[0109] In preferred embodiments the organic coating comprises amines or thiols. Thiols are generally of formula -SH and can also be referred to as mercaptans. Thiols are preferably free thiols. Amines are preferably of general formula -NH2 and can also be quaternary amines as later described herein. Glass fibres coated with primary amines are particularly useful as a material that can be converted into glass fibres coated with quaternary amines.

[0110] It was found that the invention could be advantageously practiced when the coating agent is a small molecule. Such a small molecule is preferably organic and preferably has a molecular weight of no more than 1000 Dalton, more preferably of no more than 500 Dalton.

[0111] Silanization is the attachment of an organosilyl group to chemical species such as glass. Silanization often refers to conversion of a silanol-terminated surface such as glass to an alkylsiloxy- terminated surface. This conversion can introduce new chemical moieties to the treated surface, and is often used to modify the surface properties of glass. Silanizing agents often have the formula (RO)3-n(CH3)nSiR' or R'SiCh, where R is an alkyl chain, preferably a short alkyl chain such as methyl or ethyl, n is 1 or 2, and R' is an alkyl chain or a functionalized alkyl group. Preferred agents are of the formula (RO)3SiR', where R is methyl, ethyl, or propyl, preferably methyl or ethyl, and R' is - CH2(CH2)m-X, wherein m is 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or 1 1 , preferably 1 , 2, 3, 4, 5, 6, or 7, more preferably 1 , 2, or 3, most preferably 2, and X is -SH or -NH2 or -N+(CH3)3, preferably -SH or -NH2. In preferred embodiments the coating agent is a silanization agent, preferably a trialkoxysilane such as a triethoxysilane or a trimethoxysilane. A preferred silane is a triethoxysilane or a trimethoxysilane. A preferred aminoalkylsilane is a trimethoxysilane or a triethoxysilane, preferably a triethoxysilane. A preferred mercaptoalkylsilane is a trimethoxysilane or a trmethoxysilane, preferably a trimethoxysilane. The term silane generally refers to the -Si(RO)3 moiety as comprised in (RO)3SiR' defined above.

[0112] In further preferred embodiments the coating agent is an aminoalkylsilane such as a (3- aminopropyl)silane, a mercaptoalkylsilane such as a (3-mercaptopropyl)silane, a glycidyloxyalkylsilane such as a (3-glycidyloxypropyl)silane, a cyanoalkylsilane such as a 2- (cyanoethyl)silane, or an isocyanatoalkylsilane such as a (3-isocyanatopropyl)silane; preferably an aminoalkylsilane or a mercaptoalkylsilane; more preferably a (3-aminopropyl)silane or a (3- mercaptopropyl)silane; most preferably a (3-aminopropyl)silane such as (3- aminopropyl)triethoxysilane. A highly preferred coating agent is (3-mercaptopropyl)trimethoxysilane (which can also be referred to as MPTES).

[0113] The coating agent preferably introduces thiols (-SH) or amines. Amines can be primary amines or can be further substituted amines. Preferred amines are primary amines (-NH2) and quaternary amines (-N+(alkyl)3), which are herein sometimes referred to as QA. The alkyl moieties in quaternary amines are preferably identical, and are preferably methyl, ethyl, or propyl, most preferably methyl. Quaternary amines are charged and can have any suitable counterion, preferably a physiologically acceptable counterion such as chloride. The selection of a suitable counterion is trivial and can be performed by any skilled person.

[0114] Quaternary amines can be conveniently obtained from primary amines using haloalkyl. In preferred embodiments, the coating agent introduces organic amines, wherein the organic amines are subsequently quaternized, preferably using a halomethane such as iodomethane. An amine- functionalized scaffold such as a glass fibre sheet, such as one formed by APTES treatment of glass fibre, can be contacted with a solution of haloalkyl in a suitable organic solvent, such as in a lower alcohol such as methanol, ethanol, or propanol, preferably ethanol. A convenient way of contacting is to submerge the glass fibre in the organic solvent comprising the haloalkyl. Thecontacting is preferably performed in the absence of light, and the contacting can be of any suitable duration, such as from 1-96 hours, or 12-84 hours, preferably 18-76 hours. After this contacting the glass fibre is preferably washed such as by using PBS, for instance washed three times.

[0115] In step i) a scaffold such as a glass fibre sheet that has been treated with a coating agent that introduces an organic coating on the glass fibres is provided. In other words, in step i) a scaffold such as a glass fibre sheet having an organic coating on the glass fibres is provided. The organic coating is introduced by a coating agent, and this can be part of step i) but this can also have occurred prior to the method. Introducing an organic coating on the scaffold such as on the glass fibres can be achieved by treating the scaffold such as the glass fibre sheet with a coating agent. In preferred embodiments this comprises contacting the scaffold such as the glass fibre sheet with a solution comprising 0.05-5 vol.-% of the coating agent, preferably 0.1-1 .5 vol.-%, more preferably about 0.5-1 .2 vol.-%. In general, as used herein, when a percentage is mentioned without further specification, it is intended to be vol.-%.

[0116] The solution is preferably an organic solution, more preferably a protic organic solution. In preferred embodiments the solution consists or essentially consists of the coating agent and an organic solvent. Preferred organic solvents are lower alcohols such as methanol, ethanol, propanol, butanol, and isomers thereof, preferably methanol or ethanol, most preferably ethanol. In some embodiments the solution comprises 0.05-10 vol.-% of the coating agent, preferably 0.1-7.5 vol.-%, more preferably about 0.2-5 vol.-% , more preferably about 0.3-4 vol.-% , more preferably about 0.4-3 vol.-%, still more preferably about 0.5-2.5 vol.-%, most preferably about 0.75-1 .25 vol.-%.

[0117] Treating the scaffold such as the glass fibre sheet with a coating agent is preferably performed in a fume hood. Preferably, the scaffold such as the glass fibre sheet to be treated is a dry glass fibre sheet. A scaffold such as a glass fibre sheet can be conveniently dried by dehydration in acetone, for instance at about 20 °C, for instance for about 2-12 minutes such as 5 minutes. The acetone can be evaporated at room temperature. The scaffold such as a glass fibre sheet is then contacted with the solution comprising the coating agent, wherein the contacting is preferably submerging of the scaffold such as a glass fibre sheet. The contacting can be for any suitable amount of time, such as 1-120 minutes, preferably 5-60, more preferably 10-30 minutes, most preferably 15-25 minutes such as about 20 minutes. The contacting can be at any suitable temperature such as about 20 °C. The contacting can also be at a temperature above ambient temperature, for instance at a temperature of about 40-80 °C, preferably about 50-70 °C, such as about 65 to 68 °C. After this contacting the scaffold such as a glass fibre sheet is a coated scaffold such as a coated glass fibre sheet, or a scaffold such as a glass fibre sheet that has an organic coating. This coated scaffold such as a glass fibre sheet can be washed, for instance washed 3 to 5 times such as 4 times, in an aqueous solution such as a buffer, for instance PBS. Optionally prior to washing or as part of the first wash, coated scaffold such as a glass fibre sheets can be incubated in an aqueous solution such as PBS to allow unused coating agent to hydrolyse. Coated sheets can be kept in a desiccator until their further use.

[0118] The scaffold such as a glass fibre sheet can be additionally pre-treated before cells such as fibroblasts are seeded on it. This pre-treatment can be prior to placement in the bioreactor, or itcan be performed in the bioreactor. The latter is convenient when the pre-treatment comprises washing steps because a bioreactor can often conveniently be filled with or drained of various liquids. Pre-treatment before placement in the bioreactor is convenient when it comprises steps such as heating the sheet, or autoclaving it. Preferred scaffolds such as a glass fibre sheets are pre-treated.

[0119] Suitable pre-treatments can comprise steps of washing, heating, drying, or prewetting. Washings can be 1 , 2, 3, 4, 5, or more washes. Washes can be performed for 1 , 5, 10, 15, 20, 25, 30 minutes or more. Washings can be performed in any suitable medium, such as water, saline buffer, or organic solvents. Water is preferably ultrapure water. Saline buffer is preferably phosphate buffered saline. Organic solvents are preferably C1-C4 alcohols such as methanol, ethanol, and isopropyl alcohol. Organic solvents can be mixed with water. A preferred washing medium is 70% C1-C4 alcohol in water, more preferably ethanol. Washing medium can be decanted, drained, or aspirated, preferably aspirated.

[0120] Heating can be to 37 °C, to 50 or 55 °C, to 80 °C, to 100 °C, to 120 °C, or more. Drying can be in a vacuum or preferably under an air flow, for instance under a nitrogen flow. Drying can be performed for 15, 30, 45, 60, 90, 120, 150, 180, 210 minutes or more. Drying under air flow can conveniently be performed in a flow cabinet. Drying is preferably performed when all washing steps have concluded. Autoclaving can be performed under any suitable condition, for instance using PBS (preferably at a pH of about 7-8 such as about 7.2). Good results were obtained when the scaffold such as a glass fibre sheet was autoclaved as part of pre-treatment, accordingly in preferred embodiments step i) comprises autoclaving the scaffold such as a glass fibre sheet. Preferably this is autoclaving in PBS, more preferably at a pH of 7-8, even more preferably of about 7.2.

[0121] Prewetting was found to improve performance of the scaffold such as a glass fibre sheet, particularly when prewetting was performed with a medium comprising proteins. This makes cell culture media excellent prewetting media. Prewetting is preferably performed using a cell culture medium, such as DMEM or SF medium. The medium can be complete medium (for instance DMEM with FBS such as 10% FBS and optionally with antibiotics such as Penicillin-Streptomycin- Amphotericin B, also known as P / S / A). Prewetting can be for about 0.5-48 hours, preferably about 1-36 hours, more preferably about 2-24 hours, more preferably about 4-20 hours, more preferably about 6-18 hours, more preferably about 8-16 hours, more preferably about 10-14 hours, such as about 12 hours. Prewetting is concluded by removal of the prewetting medium, optionally followed by washing steps as described above. Improved cell seeding was observed when prewetting was performed using cell culture medium, preferably complete cell culture medium.

[0122] An example of a pre-treatment is three washes with 70% C1-C4 alcohol (preferably ethanol) in water, followed by drying. A more preferred example is autoclaving of the scaffold such as a glass fibre sheet followed by three washes with 70% C1-C4 alcohol (preferably ethanol) in water, followed by drying. These examples are preferably followed by prewetting in cell culture medium.

[0123] In step ii) the scaffold such as a glass fibre sheet is placed in a bioreactor as described herein. Bioreactors are preferably sterilised before the scaffold is placed. After the scaffold isplaced, the scaffold inside the bioreactor can be sterilised, for instance by an ethanol wash such as 70% ethanol incubation for 10-40 minutes, preferably 20-30 minutes. Prior to seeding of cells, the bioreactor and / or the scaffold are preferably washed with saline such as with PBS. Alternately, the scaffold is placed inside the bioreactor after which both are sterilised simultaneously. Alternately, the scaffold and the bioreactor are sterilised separately before the scaffold is placed inside the bioreactor.

[0124] In some embodiments in step ii) multiple scaffolds such as glass fibre sheets are placed in the bioreactor to form a multi-layered scaffold. Preferably when multiple scaffolds are used, they are of substantially equal size, or are of equal size. In some embodiments two are used. In some embodiments three are used. In some embodiments, 4, 5, 6, 7, or 8 scaffolds are used. Optionally even more are used. As discussed above, the scaffold sheet can be placed in a bioreactor prior to any pre-treatment. Steps i) and ii) can be performed simultaneously, for instance through the provision of a bioreactor that already comprises a scaffold such as a glass fibre sheet.

[0125] In preferred embodiments the bioreactor is sterilised prior to step iii). Sterilisation can be performed using any known suitable method, such as rinsing with organic solvents such as ethanol, or heat treatment. Heat treatment is preferred, and can comprise passing steam through the bioreactor, or autoclaving the bioreactor. Advantageously, the bioreactor can be heat treated in its entirety, either in its empty state, or while already comprising a scaffold. It was found that certain materials such as stainless steel or PEEK were particularly suitable for forming part or all of the bioreactor, as these materials were particularly suitable for heat treatment.

[0126] In preferred embodiments of the method, the cells are fibroblasts, preferably dermal fibroblasts, more preferably bovine dermal fibroblasts. In steps iii) and iv), cells are cultured on the scaffold in the bioreactor. The cells excrete collagen, forming a collagen sheet suitable for the production of in vitro leather, for instance through tanning. Conventional animal hide is formed primarily of collagen, a fibrous protein. Collagen is a generic term for a family of at least 28 distinct collagen types; animal skin is typically type 1 collagen, and thus a preferred collagen is type 1 collagen. Other types of collagen do exist and may also be used in forming leather. Collagens are characterized by a repeating triplet of amino acids, -(Gly-X-Y)n-, so that approximately one-third of the amino acid residues in collagen are glycine. X is often proline and Y is often hydroxyproline. Structurally, collagen may consist of twined triple units of peptide chains of differing lengths. Different animals and thus different cells may produce different amino acid compositions of the collagen, which may result in different properties. Collagen fibre monomers may be produced from alpha-chains of about 1050 amino acids long, so that the triple helix takes the form of a rod of about 300 nm long, with a diameter of 1.5 nm. In the production of extracellular matrix by cells such as fibroblasts, triple helix monomers may be synthesized and the monomers may self-assemble into a fibrous form. These triple helices may be held together by salt links, hydrogen bonding, hydrophobic bonding, and covalent bonding. Triple helices can be bound together in bundles called fibrils, and fibril bundles come together to create fibres. Fibres typically divide and join with each other throughout a layer of skin. Variations of the crosslinking or linking may provide strength to the material. In step iii), cells are seeded onto the scaffold. The cells are preferably animal cells, morepreferably mammalian or reptile cells, most preferably mammalian cells such as from cows (bovine), sheep, goats, equines, buffalo, pigs, and aquatic mammals such as seals and alligators. Good results were obtained using bovine cells. The cell is preferably a collagen-releasing cell, more preferably a fibroblast. A mixture of different cells may also be used. A fibroblast is a type of cell that synthesizes the extracellular matrix and collagen, and it produces the structural framework (stroma) for animal tissues. Fibroblasts are the most common cells of connective tissue in animals. Preferred fibroblasts are dermal fibroblasts, more preferably bovine dermal fibroblasts.

[0127] In some preferred embodiments, cells are primary cells, preferably fibroblasts are primary fibroblasts. Cells may be derived from tissue extracts / explants, or manipulated (transgenic) cell lines, or any variation thereof. In some variations, the cell may be grown to complete confluence (e.g., 100% confluence), in which cells are inhibited from further growth but may continue to produce or be stimulated to produce and release collagen. In some variations, the cells may not be grown to complete confluence, (e.g., approximately 99% confluence, 95 % confluence, 90% confluence, 85% confluence, 80% confluence, etc.). Cells may be cultured until greater than 80% confluence, greater than 85% confluence, greater than 90% confluence, greater than 95% confluence and / or just under full (100%) confluence. Primary fibroblasts can be used after initial expansion, preferably after about 3 to 10 passages, more preferably about 4 to 9 passages, even more preferably about 5 to 8 passages, such as after 6 passages (thus using P6 cells).

[0128] Before seeding, cells can be proliferated in any suitable culturing medium. Preferred media are DMEM or SF media. Media can be complete (generally comprising 10% serum), or serum-free, or comprise 5% serum. Preferably, media for proliferation prior to seeding are serum-free or comprise 5% serum. A preferred medium for proliferation prior to seeding is 50% DMEM such as DMEM-F12 + 10% serum such as FBS + 1 % antibiotics such as P / S / A, combined with 50% serum- free proliferation media based on DMEM such as DMEM-F12, supplemented with 1-5 such as about 2.5 mg / mL protein such as albumin, 1-10 such as 5 pg / mL hormone such as insulin, 2-10 such as about 5.5 pg / mL iron source such as transferrin, 2-12 such as 6.7 pg / mL trace elements such as selenium, 100-500 such as about 250 nM vitamin such as vitamin C, preferably 2-phospho-L- ascorbic acid, and 1-100 ng / mL growth factors such as 10 ng / mL basic fibroblast growth factor (bFGF) and 50 ng / mL platelet-derived growth factor (PDGF), and 0.02-0.5 mM such as 0.1 mM amino acid such as glutamine. This serum-free proliferation medium can also be used by itself. Another preferred medium for proliferation prior to seeding is FBS medium. Another preferred medium for proliferation prior to seeding is SF medium. In any medium described herein, as an antibiotic, gentamicin can be used, for instance 50 pg / mL gentamicin.

[0129] Cells can be seeded at any suitable density. Good results were achieved with 1 x 106cells I cm2. Accordingly, cells are preferably seeded at a density from about 1x104to about 1x108cells per cm2of scaffold such as a glass fibre sheet, preferably from about 1x105to about 1x107cells per cm2, more preferably from about 5x105to about 5x106cells per cm2. In preferred embodiments is provided the method, wherein in step iii) from about 1x104to about 1x108fibroblasts are seeded per cm2of scaffold such as a glass fibre sheet, preferably from about 1x105to about 1x107fibroblasts per cm2, more preferably from about 5x105to about 5x106fibroblasts per cm2. In otherembodiments seeding density is from about 5x105to about 1x107, most preferably from about 1x106to about 5x106cells per cm2. Alternately, the same good results were achieved with 4.5x104cells / mm3. Accordingly, cells are preferably seeded at a density from about 1x104to about 1x105cells per mm3of scaffold such as a glass fibre sheet, preferably from about 2.5x104to about 4.5x104cells per mm3.

[0130] Other preferred seeding densities were identified using a model square scaffold having an area of 2.5 cm2(being 25 mm3of glass fibre in this screening test). Preferred densities were found to be 2.5x107cells / cm3to 1x108cells / cm3, preferably 3.5x107to 6x107cells / cm3, such as about 4.5x107cells / cm3, which gave the best results, particularly for seeding on a scaffold where only a single seeding was performed. Good results were also obtained for a larger scaffold (15 cm3), where multiple seedings such as 4 seedings were performed. Good cell density was found to be in line with the screening test described above.

[0131] Cells can be seeded from a concentrated suspension. Cells can be seeded by dropping a volume of cell suspension in the centre of the scaffold. Cells can also be seeded via multiple drops, which can be spatially distributed over the scaffold. After seeding, the cells can be allowed to attach to the scaffold such as a glass fibre sheet prior to the addition of additional culturing medium. This can be for 1-3 hours, preferably for 1 .5-2 hours. In preferred embodiments cells can be seeded by perfusion of cell suspensions in the bioreactor, preferably of a volume that matches the volume of the scaffold, or that matches the volume of the bioreactor chamber that holds the scaffold.

[0132] Multiple seedings can be performed to improve cell density. In some embodiments 2, 3, 4, 5, or 6 seedings are performed, preferably 2, 3, 4, or 5 seedings, more preferably 3 or 4 seedings such as 4 seedings. Further seedings can be performed identically to the first seeding. Seedings are preferably separated by about 1-4 days of culturing, more preferably by 2-3 days of culturing. This allows the original cells to attach. The ratio between attached cells and suspended cells can be referred to as the seeding efficiency. This is preferably determined 24h after seeding. During the seeding process, culturing is preferably under static conditions - not under gentle agitation.

[0133] In step iv) the cells are cultured under conditions conducive to collagen production to form a collagen sheet. A skilled person knows how to culture cells such as fibroblasts. Generally, cells are cultured at 35-40 °C, preferably at about 37 °C. Generally, cells are cultured under a controlled CO2 atmosphere, such as 3-8% CO2, preferably at about 5% CO2. Preferably cells are cultured in a humidified environment. Conventional cell incubators can be used to provide such conditions. Media are preferably changed every 2-3 days. More preferably, media are perfused to provide cells inside the bioreactor with fresh medium. Perfusion can be facilitated by any suitable means, such as by a pump, preferably a peristaltic pump. The design of the bioreactor advantageously allows the provision of media under laminar flow with remarkable homogeneity. It was found that flow rates were robustly tolerated by cells inside the bioreactor, as both conventional flow rates and doubled flow rates supported cell adhesion and allowed for good proliferation and full confluence.

[0134] In preferred embodiments, the culturing is performed for about 1 to 6 or 2 to 6 weeks, preferably for about 2 to 5 weeks, more preferably for about 3 to 5 weeks. It was found that after about 35 days the collagen sheet was suitable for isolation, and accordingly culturing can be forabout 30 to about 40 days. In some embodiments, the culturing is performed for about 2 to 3 weeks, which was found suitable for development purposes.

[0135] In preferred embodiments of the method, the culturing is performed for about 2 to 9 weeks, preferably 3 to 8 weeks, more preferably for about 4 to 7 weeks or for about 6 to 7 weeks.

[0136] Cells can be cultured in any suitable medium. When scaffolds such as a glass fibre sheets are prewetted, the cells are preferably cultured in the same medium as was used for prewetting. DMEM and SF were found to be very suitable for production of collagen, of which DMEM was preferred. Preferably the culturing in step iv) is performed in DMEM / F-12 medium that optionally comprises 2-20% serum such as 10% fetal bovine serum, and that optionally comprises 2-phospho- L-ascorbic acid. In some embodiments the culturing in step iv) is performed in medium that comprises 2-20% serum such as 10% fetal bovine serum, and that optionally comprises 2-phospho- L-ascorbic acid. In some embodiments the culturing in step iv) is performed in medium that comprises 2-phospho-L-ascorbic acid.

[0137] When medium comprises 2-20% serum, it preferably comprises 4-18%, more preferably 5- 16%, more preferably 7-14%, still more preferably 8-12%, still more preferably 9-11 %, most preferably 10% serum. A skilled person can select a suitable serum, examples are platelet lysate such as human platelet lysate, fetal calf serum, horse serum, and newborn calf serum. Fetal bovine serum is most preferred.

[0138] 2-phospho-L-ascorbic acid was found to promote collagen production. When medium comprises 2-phospho-L-ascorbic acid, it preferably comprises 20-2000 pM, more preferably 50- 1500 pM, still more preferably 100-1250 pM, still more preferably 150-1000 pM. Good results were obtained with 200-750 pM, for instance with 250-500 pM.

[0139] In preferred embodiments of the method, the culturing of step iv) is under conditions conducive to collagen production to form a collagen sheet; and wherein the method optionally further comprises the step of: v) isolating the collagen sheet. In other preferred embodiments the culturing of step iv) is under conditions conducive to collagen production to form a collagen sheet; and wherein the method further comprises the step of: v) isolating the collagen sheet

[0140] In preferred embodiments the culturing of step iv) is performed while the bioreactor is oriented horizontally. A bioreactor that is oriented horizontally can generally be seen as being positioned on its largest plane, such as lying on its largest plane, for instance while lying flat on an incubator shelve.

[0141] In step v) the collagen sheet that has formed is isolated. The sheet was found to retain its shape, while collagen sheets cultured without scaffolds such as a glass fibre sheets were found to contract when removed from the bioreactor. Thus the invention provides a means for obtaining form-fast collagen sheets by using a readily available material as a scaffold. The collagen sheet can be fixed before or after its isolation. Means for fixing a tissue are well-known, and a skilled person can select a suitable method. For instance, the collagen sheet can be fixed using formaldehyde such as 4% paraformaldehyde in water. The collagen sheet can be isolated using any suitable means, such as manually, with a spatula, with a scraper, or preferably using tweezers.

[0142] Hydroxyproline content is a good measure for collagen content. In preferred embodiments the collagen sheet comprises at least 1 pg / mg hydroxyproline, more preferably at least 2, still more preferably at least 3, more preferably at least 4 pg / mg, still more preferably at least 8 pg / mg, still more preferably at least 12 pg / mg, still more preferably at least 20 pg / mg, still more preferably at least 30 pg / mg, most preferably at least 50 pg / mg. Hydroxyproline content can be assayed using known techniques, for instance by measuring absorbance in a chromogenic assay, conveniently by using commercially available kits such as described in the examples.

[0143] In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition the verb “to consist” may be replaced by “to consist essentially of’ meaning that a product or a composition or a nucleic acid molecule or a peptide or polypeptide of a nucleic acid construct or vector or cell as defined herein may comprise additional component(s) than the ones specifically identified; said additional component(s) not altering the unique characteristic of the invention. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 10% of the value.

[0144] Whenever a parameter or a substance is discussed in the context of this invention, it is assumed that unless otherwise specified, the parameter is determined, measured, or manifested under physiological conditions. Physiological conditions are known to a person skilled in the art, and comprise aqueous solvent systems, atmospheric pressure, pH-values between 6 and 8, a temperature ranging from room temperature to about 37 °C (from about 20 °C to about 40 °C), and a suitable concentration of buffer salts or other components.

[0145] AII patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Unless otherwise indicated each embodiment as described herein may be combined with another embodiment as described herein. The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.ExamplesExample 1 - description of the modelling outcome

[0146] Using computational fluid dynamics, performance of the perfusion bioreactor was analysed with three different configurations: (1) divergent inlet section; (2) stretched inlet and outlet sections; and (3) stretched inlet and outlet sections plus inlet section wedge element.Figs. 6A-6C show the relative distribution of oxygen concentrations on the scaffold surface (cf. rectangle part) alongside the flow pattern of culture medium in the horizontal direction across the bioreactor starting from the point of entry at the inlet section to exit at the outlet section in the threeconfigurations. Figs. 7A-7C show the respective distributions of shear stress over the scaffold surface area.

[0147] As can be seen in Fig. 6A, recirculation in the inlet section occurred in the first configuration of the bioreactor with a divergent inlet section. The flow (indicated by the substantially horizontal lines) appears concentrated in the inlet section with few lines extending across the scaffold area in the horizontal direction to the outlet section. This indicates that the homogeneity of the flow was effectively reduced, and particularly in the width direction of the scaffold (denoted by the z-axis). Also, in Fig. 7A, it is apparent that the distribution shear stress over the scaffold surface area has some uniformity near the center of the scaffold area.

[0148] Relative to the first configuration of the bioreactor, the inlet and outlet sections were stretched in the longitudinal direction (y). Specifically, the inlet section was stretched by a factor of 1 .5 and the outlet section was stretched by a factor of 2.5. Stretching of the inlet and outlet sections exhibited a higher level of homogeneity in the flow across the bioreactor than in the first configuration. An indicator of this relatively higher level of homogeneity in the flow pattern is the increased number of lines that extend across the scaffold area in the horizontal direction toward the outlet section. As can be seen in Fig. 6B, there is also an increase in the straightness of iso-contours (the substantially vertical lines), which are orthogonal to the flow, over the entire transverse width of the bioreactor. In addition, the distribution of shear stress over the scaffold surface area in Fig. 7B has more uniformity than the first configuration.

[0149] For the third configuration, the stretched inlet and outlet sections of the second configuration were again used but in combination with a wedge element arranged in the inlet section. Here, and as shown in Fig. 6C, there was an even further increase relative to the first and second configurations in the straightness of the iso-contours across the entire transverse width of the bioreactor as well as a further increase in the number of lines extending across the scaffold area in the horizontal direction toward the outlet section indicating an even higher level of homogeneity in the flow across the bioreactor. Likewise, the distribution of shear stress over the scaffold surface area shown in Fig. 7C has even more uniformity than the second configuration.Example 2 - use of the bioreactor in the provision of collagen sheets

[0150] In this example a collagen sheet is produced using a bioreactor as described herein, after which the collagen sheet is tanned to provide leather. Using an autoclave, metal clamps, stainless steel mesh, tweezers, and a salinized petri dish were sterilized. Then a glass fibre scaffold (100 mm x 50 mm x 1.5 mm, chopped strand mat, MPTES-coated, Alkali-free, E-type, acid / base resistant, melting point: 1100 °C < AT < 1500 °C, diameter 10 ± 1.1 pm, (mean ± SD); thickness 1.5 mm (mean); gap size 50 pm < gaps < 100 pm; fibre density 3 mg / cm2) was autoclaved, then sterilized using 70% EtOH and it was washed multiple times with PBS. Using tweezers, the sterile scaffold was placed on top of the mesh and in between the stainless steel parts. All layers were tightened using the hex key and bolts, after which the scaffold with clamp was placed in a petri dish. A cell suspension (bovine fibroblasts) was pipetted on top of the scaffold, after which the volume was adjusted to the volume of the scaffold (100 mm x 100 mm x 1.5 mm = 15 mL). It was thenincubated for 1 hour in an incubator (37°C, 5% CO2), before adding growth medium (DMEM with 10% FBS and supplemental amino acids, vitamins, and growth factors) in the petri dish. Then, it was placed in the incubator for static culture. For some embodiments this cell seeding process was repeated after 1 week. Medium was changed about two times per week.

[0151] After 13 days of static culture and 3 seedings, an epoxy tissue bioreactor was sterilized using 70% EtOH and placed in a laminar flow cabinet where the EtOH was left to evaporate until all parts were completely dry. All parts were exposed to UV-light for additional sterilization, and a silicon gasket (2.0 mm thickness), tubing, connectors, tweezers, a hex key, a screw cap with tubing connectors and a glass bottle were all autoclaved. Using the tweezers, the clamp with mesh, scaffold and adhered cells was placed in the bottom part of the tissue bioreactor, after which the gasket was placed on top of the bottom part. The top epoxy part was aligned on top of the bottom part and screwed together using the large and small bolts and a hex key, after which the connectors and tubing was attached. The glass bottle was filled with culture medium after which the system was closed and placed in an incubator. The tubing was inserted in a pump head via a hole in the back of the incubator and the peristaltic pump was started. This culture was continued for 18 more days, with ~2-3 medium changes per week. The collagen sheet was isolated from the bioreactor and tanned, producing a shiny leather with a good homogeneous density and appearance. Fig. 8 shows the isolated collagen sheet, and Fig. 9 shows the tanned leather.

[0152] In a separate experiment two bioreactors were cultured inside an incubator - one positioned horizontally (lying on its largest side) and the other positioned vertically (standing on its long edge). Both setups supported cell adhesion, collagen formation, and gap closure. Both orientations allowed the provision of a good collagen sheet. It was found that of the two, the horizontally positioned bioreactor eventually yielded the best piece of leather.

Claims

-28-Claims1 . A perfusion bioreactor comprising a flow chamber having a width in a transverse direction along a transverse axis (x) and a length in a longitudinal direction along a longitudinal axis (y), the flow chamber comprising a flow chamber top, a flow chamber bottom, and an interior space defined between the flow chamber top and the flow chamber bottom, wherein along the length of the flow chamber the interior space comprises an inlet portion, an outlet portion, and a central portion arranged between the inlet and outlet portions, and a sealing member arranged between the flow chamber top and the flow chamber bottom, wherein the inlet portion comprises at least one funnel element having a diameter in the transverse direction (x) that increases over a respective length of the inlet portion in the longitudinal direction (y) from a minimum diameter at a first end to a maximum diameter at a second end, the second end arranged proximal to the central portion.

2. The perfusion bioreactor according to claim 1 , wherein the inlet portion comprises at least one wedge element that is preferably diamond-shaped.

3. The perfusion bioreactor according to claim 2, wherein the at least one wedge element is arranged proximal to the first end of the inlet portion, and / or wherein the at least one wedge element is formed integrally with the flow chamber bottom.

4. The perfusion bioreactor according to any one of the preceding claims, wherein the outlet portion comprises at least one funnel element having a diameter in the transverse direction (x) that tapers over a respective length of the outlet portion in the longitudinal direction (y) from a maximum diameter at a first end to a minimum diameter at a second end, the first end of the outlet portion arranged proximal to the central portion.

5. The perfusion bioreactor according to any one of the preceding claims, wherein at least one of the inlet portion and the outlet portion comprises a plurality of funnel elements, the plurality of funnel elements preferably arranged in parallel in the transverse direction (x).

6. The perfusion bioreactor according to any one of the preceding claims, wherein the inlet portion comprises at least one inlet port and the outlet portion comprises at least one outlet port, one or both of the at least one inlet port and the at least one outlet port is / are adapted to connect to a medium reservoir, and preferably wherein each of the at least one inlet port and the at least one outlet port is arranged in either in an xy-plane of the flow chamber or in a xz-plane of the flow chamber.

7. The perfusion bioreactor according to any one of the preceding claims, further comprising a mesh layer arranged between the scaffold and the flow chamber bottom, and preferably wherein at least one or both of an inner surface of the flow chamber and the mesh layer comprises an anti-fouling coating.

8. The perfusion bioreactor according to any one of the preceding claims, further comprising a scaffold cassette comprising a first support member and a second support member which are configured to secure a scaffold there between in the interior space, the scaffold cassette preferably arranged in the central portion with the scaffold spaced a respective distance apart in a z-direction along a z-axis from the flow chamber top and the flow chamber bottom.

9. The perfusion bioreactor according to claim 8, wherein the first support member and the second support member each have a respective perimeter comprising a respective scaffold contacting surface.

10. The perfusion bioreactor according to claim 9, wherein one or both of the respective scaffold contacting surfaces comprises a curvature that is arranged either partly or fully around the respective perimeter.11 . The perfusion bioreactor according to any one of claims 8-10, wherein the respective distances between the scaffold and the flow chamber bottom and the flow chamber top in the z-direction are each in a range of 0 to 5 mm, preferably of 0.5 to 4 mm.

12. The perfusion bioreactor according to any one of the preceding claims, wherein the second end of the inlet portion meets the central portion and forms an inner angle therewith in a range of 35 to 55 degrees, preferably 40 to 50 degrees.

13. The perfusion bioreactor according to any one of the preceding claims, wherein the inlet portion and the outlet portion are mirror symmetric at least about the transverse axis (x), and optionally mirror symmetric about the longitudinal axis (y).

14. The perfusion bioreactor according to any one of claims 1 to 12, wherein the inlet portion and the outlet portion are asymmetrical about the transverse axis (x), and preferably wherein the inlet portion extends over about 30% of a total length of the interior space in the longitudinal direction (y), the central portion extends over about 60% of the total length, and the outlet portion extends over about 10% of the total length.

15. The perfusion bioreactor according to any one of the preceding claims, wherein the flow chamber is formed of material selected from any one or more of an epoxy, polycarbonate (PC), polypropylene (PP), polystyrene (PS), stainless steel, anodized aluminium, plexiglass (PMMA),glass, polyvinylidene difluoride (PVDF), polytetrafluoroethylene (PTFE), and polyether ether ketone (PEEK).

16. Method for culturing cells, the method comprising the steps of: i) providing a scaffold; ii) placing the scaffold in a perfusion bioreactor as described in any one of claims 1- 15; iii) seeding cells onto the scaffold in the bioreactor; iv) culturing the cells.

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