Biomimetic bioreactor for biomimetic stimulation, method for forming tissue in such a biomimetic bioreactor and sample holder suitable for use in such a reactor

Abstract

The present invention relates to a sample holder for use in a biomimetic bioreactor. The sample holder comprises a top wall having an outer circumferential edge and in inner circumferential edge, a circumferential outer wall, extending downwards over a first distance d1 and having a first cross section, an upper circumferential inner wall, extending downwards over a second distance d2 smaller than the first distance d1 and having a second cross section, smaller than the first cross section of the circumferential outer wall, and a support surface, a configured as a surface extending inwards from a bottom edge of the upper circumferential inner wall. The invention also is related to a biomimetic bioreactor comprising the sample holder and to a method of forming or maturing a tissue using the sample holder within the biomimetic bioreactor.

Description

[0001] Title: Biomimetic bioreactor for biomimetic stimulation, method for forming tissue in such a biomimetic bioreactor and sample holder suitable for use in such a reactor

[0002] Description

[0003] The present invention is related to the field of tissue formation and maturation in general, and especially, however not exclusively, to stimulation of tissue engineered constructs such as membranes cultured with cells.

[0004] Bioreactors and methods for stimulating tissue engineered constructs are known for stimulating engineered tympanic membranes. A tympanic membrane, commonly known as eardrum, is a thin, concave tissue of the middle ear that captures sound pressure waves from the environment and transmits them as mechanical vibrations to the inner ear. Surgical placement of autologous tissue graft has been the “gold standard” for treating damaged tympanic membranes. Given the lack of available tissues for transplantations, regenerative medicine has emerged as a promising alternative.

[0005] An important aspect of the tympanic membrane is its acoustic performance when stimulated with sound waves. The acoustic response of tissue engineered constructs, in the form of resonant frequency and displacement, must match that of the human tympanic membrane to truly function as a clinically relevant eardrum replacement. Furthermore, in humans, the stem cells reside within a complex tissue environment and respond to diverse mechano-acoustic or biophysical cues. Such cues are known to have a profound impact on stem cell differentiation and, therefore, are crucial for developing and culturing artificial tympanic membranes.

[0006] An artificial membrane as referred to in this document, is a membrane that will replace the native tympanic membrane like an implant. The membrane may be a combination of scaffold (a better and broader alternative to membrane) with cells. A scaffold can have multiple formats, an example here can be additive-manufactured fibers patterned on top of the membranes. In that case that combined “membrane” is better called scaffold. Whereas the present invention is especially related to artificial tympanic membranes, the invention also applies to various other cell types and the invention is not limited in its application to artificial tympanic membranes. The invention is related to tissue formation and maturation in general, and especially membranes. It is to be noted that the invention is not limited to artificial tissues or membranes. It’s also within the scope of the present invention to stimulate a cadaveric human tissue that is extracted from a donor human being or body or from an animal. After stimulation, the tissue can be returned to its original position or transplanted into another human being. Further, in this document, the term “sample” is meant to include tissues, constructs, cells, organoids, etcetera.

[0007] Although the importance of mimicking the complex natural cell environment is highlighted in copiousness of research papers, using air-liquid interface or acoustical stimulation, current tissue models remain simplistic and, surprisingly, no advanced “plug-and-play” bioreactor is available. The simple in vitro 2D (monolayer) and 3D models presently used lack true physiological properties and hinder the advancements in a regenerative solution towards hearing loss.

[0008] In known bioreactors and methods for stimulation of tissues, a generic culture well plate, like a so-called 6 (12, 24,...) well plate, a petri-dish, or other container may be used to hold organic culturing cells or organoids in the bioreactor. A 6 well plate comprises 6 wells that can each hold a liquid substrate. A membrane is submerged in the substrate of every well.

[0009] It’s also known to use an acoustic source to stimulate cells or a tissue lying at the bottom of a well and being submerged in a substrate. The acoustic source generates acoustic waves that propagate to the membranes submerged in the wells Sound signals generated by a sound source that is positioned above the well will have to cross through the entire substrate to reach the cells or the tissue. Alternatively, a sound source may be positioned beneath the well, wherein sound waves generated by the sound source generate turbulence in substrate present in the well. If the sound source is placed beneath the well, the sound signals have to travel through an additional medium like the wall of a container or reservoir, which results in significant signal dampening. The known containers are limited to liquid-liquid acoustical stimulation, in which a sample is fully submerged in the substrate.

[0010] Therefore, according to a first aspect, the present invention aims to provide a platform that is suitable for, however not limited to, air-liquid acoustic stimulation of a membrane designed for tympanic membrane regeneration or repair.

[0011] According to the first aspect, the present invention is related to a biomimetic bioreactor. The invention aims to solve the abovementioned problems and therefore provides a biomimetic bioreactor according to claim 1. In use, a sample, for example a membrane or a tissue or a construct is supported by the support surface.

[0012] The sound waves acoustically stimulate tissue of the sample, for example a tissue engineered membrane like an artificial tympanic membrane. The acoustic nature of the membrane and its response to it is an important aspect of the tympanic tissue. The response to acoustic input, such as surface motion and velocity, most match human tympanic tissue to truly function as eardrum replacement.

[0013] The biomimetic bioreactor according to the invention not only allows liquidliquid and air-air acoustical simulation. In addition, the biomimetic bioreactor also allows air-liquid acoustical simulation. The liquid substrate that contacts the lower side of a sample supported by the support surface promotes formation and maturation of cells in the sample. The air contact with the upper side of a sample supported by the support surface allows natural acoustical stimulation, because the sound waves will not be hindered by liquid being present between the sound source and the sample.

[0014] In a preferred embodiment, the sound source is provided with a hole, preferably a central hole. Such a hole enables a user to have a look at the sample during the process of stimulating the sample without having to remove the sound source.

[0015] In an optional embodiment of the biomimetic bioreactor, the substrate reservoir and the holder are integrated, in that the holder is provided with a bottom, closing the opening defined by the lower edge of the circumferential outer wall. The majority of the substrate can be poured in said biomimetic bioreactor through the opening defined by the inner edge of the support surface before the sample is positioned, which sample closes said opening. Then, a last bit of the substrate can be added to the substrate through the trough hole that is preferably located in the upper wall of the holder, until the substrate level is raised to reach the bottom of the sample.

[0016] In a preferred embodiment of the biomimetic bioreactor, the substrate reservoir is a container of a generic culture well plate, preferably a 6, 12, 24, ... well plate and wherein preferably a sample holder is positioned in each of the containers of the generic culture well plate. If the sample holder is designed with dimensions to fit in a well of a generic culture well plate, a user only has to invest in sample holders, which can be placed in generic culture wells that are already present. This makes the improved biomimetic bioreactor more accessible, even if large quantities of biomimetic bioreactors are needed. Alternatively, the substrate reservoir may be a (known) petri-dish, which may also already be present at the site where the biomimetic bioreactor is (to be) used.

[0017] The sample holder may be press fitted in the substrate reservoir. The reservoir may contain a substrate to a level of or Oust) above the level of the sample to enable air-liquid stimulation or liquid-liquid stimulation. However, preferably the substrate is held only in the part of the substrate reservoir that is surrounded by the circumferential outer wall. If an air-air stimulation is envisaged, no substrate needs to be contained in the substrate reservoir. Cells would still require the media to be delivered to them, so the current bioreactor would need appropriate modifications to achieve that.

[0018] According to a second aspect, the present invention relates to a method for forming, maturing or stimulating a tissue. Known methods suffer from the same problems as already mentioned hereinabove with respect to known bioreactors. The present invent has as an objective to provide a method that can take away, or at least alleviate, said problems, and therefore provides a method for forming or maturing a tissue, the method comprising the steps of: providing a biomimetic bioreactor according to the first aspect of the present invention; placing a sample on the support surface of the sample holder; and placing the sample holder in the substrate reservoir.

[0019] The steps of the method don’t have to be performed in the order in which they are presented. For example, the holder may be placed in the substrate reservoir before a sample is placed on the support surface. And whereas the reservoir is called a substrate reservoir, the method is also applicable for an air-air situation, in which no substrate is present in the substrate reservoir.

[0020] In a preferred embodiment, the method further comprises one or more of the steps of: placing a securing element on top of the sample, securing the sample present on the support surface in the holder; placing a sealing element on top of the sample, the sealing element sealingly in contact with the upper circumferential inner wall; and filling a substrate in the reservoir to a level equal to or higher than the support surface when the sample holder is present in the substrate reservoir. Again, the order in which the steps are presented is not limiting the invention. For example, the steps of placing a securing element and placing a sealing element may coincide, by placing an element that both secures the sample and seals the upper circumferential inner wall against leakage of substrate from beneath the sample to above the sample. An O-ring, for example, is perfectly suitable for performing both the securing and sealing functions.

[0021] Preferably, the method further comprises arranging a sound source, for example a piezoelectric bender, above the sample holder, the sound source in use generating sound waves that propagate to the sample in the sample holder. As mentioned hereinabove sound waves can stimulate a tissue. The normal audible spectrum spans from 20 Hz to 20000 Hz, although the majority of human hearing occurs below the frequency of 4000 Hz. Therefore, preferably a frequency range of 1 Hz to 10000 Hz, more preferably in the range of 100 Hz to 5000 Hz, most preferably of 2900 Hz to 3000 Hz, especially of 2950 Hz, may be applied for a biomimetic stimulation of the tympanic membrane constructs.

[0022] According to a third aspect, the invention provides a sample, construct or tissue holder for use in a biomimetic reactor or a method according to the first and second aspect, respectively, and hereinafter called sample holder, the sample holder comprising: a top wall having an outer circumferential edge and in inner circumferential edge; a circumferential outer wall, depending on the outer circumferential edge of the top wall, extending downwards over a first distance d1 and having a first cross section; an upper circumferential inner wall, depending on the inner circumferential edge of the top wall, extending downwards over a second distance d2 smaller than the first distance d1 of the circumferential outer wall and having a second cross section, smaller than the first cross section of the circumferential outer wall; and a support surface, configured as a surface extending inwards along a bottom edge of the upper circumferential inner wall, from the upper circumferential inner wall to an inner edge of the support surface. While the term wall may suggest a continuous wall, this isn’t necessarily the case for the current invention. The term top wall may be interpreted broadly and comprises an edge from which both the circumferential outer wall and the upper circumferential inner wall extend downwardly, i.e. the inner and outer edge of top wall coincide. The support surface of the sample holder can support tissue, monolayer, or n-layer of cells and / or organoids, with and without a supporting material, commonly known as a scaffold, composed of natural materials, such as polysaccharides, and / or synthetic materials such as polymers, and / or inorganic materials.

[0023] The circumferential outer wall extends downwardly to beyond the circumferential inner wall and is thus able to place the sample holder in a stable manner at an underground in a substrate reservoir of a biomimetic bioreactor. The reservoir may be a known culture tissue container, like a generic culture 6 well plate, however, other suitable containers may also be considered. A tissue, for example a membrane, or a scaffold supporting a tissue, can be placed upon the support surface. In use, the lower side of the tissue may be contacted by a liquid substate being present in the biomimetic reactor, while the upper side of the tissue can be free of liquid substrate. If a sound source positioned above the sample holder, or at least above the tissue, generates sound waves that propagate to the tissue, the tissue will be stimulated by the sound waves. Thus, the objective of the present invention is met.

[0024] It is to be mentioned that the sample holder is not limited to use in combination with the biomimetic bioreactor according to claim 1 , of the method according to claim 2, for example to use of air-liquid acoustical stimulation or to stimulation of a tympanic membrane. It is possible to submerge a tissue present at the support surface for application in liquid-liquid stimulation or to omit the substrate for application in air-air acoustical stimulation. Also, the sample holder is not limited to acoustical stimulation; it can be used for stimulating, differentiation and proliferation of various kinds of tissues. Advantageously, the sample holder according to the present invention can be designed to be used in any cell / organoid / tissue / construct culturing medium container. A commercial advantageous application of the sample holder according to the invention is in known generic (6) well-plates that are widely used. If the dimensions of the sample holder are selected well, it is possible to perform air-liquid acoustic simulation without the necessity to replace existing substrate reservoir equipment and to invest in new equipment. In a preferred embodiment, the sample holder comprises a lower circumferential inner wall, depending on the inner edge of the support surface, and extending downwards over a fourth distance d4, wherein d1> d2+d4, and having a third cross section, smaller than the second cross section of the circumferential outer wall. The lower circumferential wall contributes to the rigidity of the sample holder, especially of the support surface, and prevents a sharp edge of the support surface that may damage a sample supported by the sample holder. It also has a dampening effect on possible movements in the liquid substrate at the level of the sample present on the support surface. Further, because d1 >d2+d4, in other words, because the circumferential outer wall extends further than the joint circumferential inner walls, the outer wall will contact a surface, a bottom of a substrate reservoir, for example, while the lower inner wall is free of contact with said surface.

[0025] If the upper circumferential inner wall of the sample holder is non permeable for liquid, the substrate reservoir of the biomimetic bioreactor can be filled with a substrate to a level above the support surface without the substrate flooding a sample present on the support surface. In use, the sample may seal a space defined by the inner edge of the support surface. Thus, the bioreactor may be filled with a substrate to above the level of the support surface of the sample holder, still enabling an airliquid stimulation, i.e. the lower surface of the sample being contacted by the substrate to promote culturing of cells, the upper surface of the sample being contacted by air, such that sound waves generated by a sound source will propagate to the sample through the air, not obstructed by substrate.

[0026] In a preferred embodiment, the top wall of the sample holder comprises a through hole. Alternatively, the through hole may be provided in the upper circumferential inner wall, or in the circumferential outer wall, above the level of the support surface. The through hole provides an opening for (re)filling substrate in the substrate reservoir.

[0027] While the sample holder may have any cross-sectional form, the circumferential inner and / or outer walls preferably have a circular cross-section.

[0028] In a preferred embodiment the circumferential walls have a continuous cross section over the entire length (height) of the wall. If the cross section of the upper circumferential inner wall is continuous, substrate is prevented from entering the inner space and flooding the sample, which would disturb an air-liquid stimulation. Preferably, also the outer wall is impermeable to liquid. In use, the sample holder, especially the lower edge of the circumferential outer wall thereof, is press fitted in a substrate reservoir and then the majority of the substrate is poured in said biomimetic bioreactor through the opening defined by the inner edge of the support surface before the sample is positioned, where the sample closes said opening. Then, a last bit of the substrate can be added to the substrate through the through hole that is preferably located in the upper wall of the holder, until the substrate level is raised to reach the bottom of the sample. Some substrate may undesirably, however not fatally, leak out the holder along the press fit. However, if the circumferential outer wall would be permeable for the substrate, substrate would pass through the circumferential outer wall, unnecessarily requiring an excessive refill of the substrate through the through hole in the sample holder.

[0029] The sample holder is especially suitable for holding a sample in a bioreactor if the circumferential outer wall has a diameter in the range of 1 mm to 300mm, preferably in the range of 3 mm to 250 mm. If the circumferential outer wall has a diameter in the range of 5 mm to 200 mm, preferably of 10 mm - 100 mm and most preferably in the range of 15-35 mm, the sample holder is very well applicable in combination with widely used 6 wells or other commercial wells.

[0030] The sample holder is also especially suitable for holding a sample in a bioreactor if d1 is in the range of 1 mm to 50 mm, preferably in the range of 5 mm to 35 mm, more preferably in the range of 10 mm to 25 mm and most preferably is 15 mm, the sample holder is very well applicable in combination with widely used culture well plates or petri-dishes The sample holder is further especially suitable for holding a sample in a bioreactor if d2 is in the range of 0.01 mm to 25 mm, preferably in the range of 0.1 mm to 15 mm, more preferably in the range of 1 mm to 10 mm If d2 is in the range of 3 mm to 7 mm, the sample holder is very well applicable in combination with widely used 6 wells or other wells.

[0031] The sample holder is further especially suitable for holding a sample in a bioreactor if a space defined by the circumferential outer wall, the upper wall, the circumferential inner walls, the support ring forming the support surface and a face in which a lower edge of the circumferential outer wall extends has a volume of 1 to 100 ml, preferably 2 to 50 ml more preferably 5 ml to 20 or 10 ml and most preferably of around 7 ml. In a preferred embodiment, the support surface extends substantially horizontal to provide the best support for a sample during formation or maturation.

[0032] Preferably, the sample holder comprises a securing element to secure a sample supported by the support surface of the sample holder. This prevents a sample in use dropping or slipping through the opening defined by the inner edge of the support surface, and in use in an air-liquid situation, to float on the substrate if the level of the substrate is higher than the support surface.

[0033] The securing element preferably has a cross section the vertical projection of which fits substantially between edges of the support surface, so that the securing element is (indirectly) supported by the support surface when the securing element is placed on a sample present on the support surface. Securing may be provided by gravity, for example. The securing element may be configured as an O-ring.

[0034] The sample holder preferably has a sealing element that in use provides a fluid tight seal in sealing contact with the upper circumferential wall of the sample holder. This prevents a substrate flooding a sample present on the support surface if the level of the substrate is higher than the level of the support surface.

[0035] Advantageously, the sealing element is made of a flexible material and has an outer circumference that generally corresponds to the inner circumference of the upper circumferential inner wall. For providing a reliable seal, corresponding to the inner circumference of the upper circumferential inner wall in this respect means that the outer circumference of the sealing element is equal to, or slightly larger than the inner circumference of the upper circumferential inner wall.

[0036] In a preferred embodiment, the securing element and the sealing element are configured as one integral element, preferably an O-ring, for example made of rubber or thermoplastics.

[0037] Preferably, the sample holder is made of plastics, for example of polycarbonate, polymethyl methacrylate (PMMA), polystyrene, or polyvinyl chloride (PVC), metal or alloy such as stainless steel, or glass. These are materials that are easy to process for making the sample holder.

[0038] If the sample holder is made of a transparent material, this allows a user to easily monitor the level of substrate in the sample holder from outside the sample holder. The invention will now be explained referring to the appended drawing, in which:

[0039] Fig. 1 shows a schematic perspective view from above of a sample holder according to the present invention;

[0040] Fig. 2 shows a schematic perspective view from below of the sample holder of Fig. 1 ;

[0041] Fig 3a shows a schematic perspective view of an arrangement of a sample holder according to the present invention in a well and holding a sample, a sound source being arranged above the sample holder;

[0042] Fig 3b, shows a vertical cross sectional view through the central axis of the arrangement of Fig. 3a, however without the well;

[0043] Fig. 4 shows a schematic perspective view from above of a biomimetic acoustical bioreactor according to the present invention;

[0044] Fig. 5 shows a view from above at a biomimetic acoustical bioreactor according to the present invention; and

[0045] Fig.6 shows a diagram showing the steps of a method for stimulating a tissue sample according to the present invention.

[0046] Now looking to the figures in more detail, Figs. 1 and 2 show a schematic perspective view from above and from below, respectively, of a sample holder 1 according to the present invention. The sample 1 holder is made of polycarbonate and has an annular upper wall 2 with an inner diameter of 8 mm and an outer diameter of 20 mm. A circumferential outer wall 3 depends from an outer edge of the upper wall 2 and extends 15 mm downwards. An upper circumferential inner wall 4 depends from an inner edge of the upper wall 2 and extends 5 mm downwards. A support surface 5 projects inwards, i.e. to an imaginary central axis of the sample holder 1 from the lower edge of the upper circumferential inner wall 4, over a distance of 2.5 mm. A lower circumferential inner wall 6 extends 1 mm downwards from an inner edge of the support surface 5 to provide the holder and the support surface sufficient mechanical rigidity. A through hole 7 is provided in the upper wall 2. In use, the lower edge 8 of the circumferential outer wall 2 provides a basis on which the sample holder 1 rests on a surface, a substrate reservoir of a bioreactor, for example.

[0047] The sample holder 1 provides a support for a tissue sample to be formed or matured (not shown in Figs. 1 and 2). In use, the outer wall 2 carries the sample holder 1. A sample (not shown in Figs. 1 and 2) is supported by the support surface 5. If the sample holder 1 is used for air / liquid stimulation of a sample, a liquid culturing medium or substrate may be filled, possibly through the through hole 8 in the space defined by the circumferential outer wall 3, the upper wall 2, the circumferential inner walls 4, 6, and the support ring forming the support surface 5 (see Fig. 2, showing the inner space of the sample holder 1 from below). At least the upper circumferential inner wall 4 and the support surface 5 are impermeable to the liquid substrate. The sample may be lightly permeable to the liquid substrate in such a way that the liquid substrate may be absorbed by the sample, however, can not pass through it. If the sample is fixed at the support surface 5, the bottom of the sample will be exposed to the liquid substrate for culturing cells of the sample, while the top of the sample will be exposed to air.

[0048] Fig. 3a shows a schematic perspective view of an arrangement of a sample holder 21 according to the present invention in a well 30 and holding a sample 31 , a sound source 32 being arranged above the sample holder 21. The sample holder 21 may be identical to the sample holder 1 of Figs. 1 and 2, however this is not necessarily the case. To prevent unnecessary introduction of elements, elements of sample holder 21 that correspond with those of sample holder 1 are provided with a reference sign that is 20 higher than the corresponding element of sample holder 1. Fig. 3b shows a vertical cross sectional view through the central axis of the arrangement of Fig. 3a, however without the well 30 and including an O-ring 34.

[0049] Shown is a well 30, for example as part of a conventional 6-well plate, partly filled with culture medium (not visible in Fig 3a) as the liquid substrate, at the bottom of which well 30 the sample holder 21 is positioned. A sample 31 , in this example a tissue scaffold 31 is positioned at a support surface 25 of the sample holder 21 , which support surface is not visible in Fig. 3a. As can be seen in Fig 3b, the tissue scaffold 25 is secured in position by a rubber O-ring 34 (not shown in Fig 3a), which is also in sealing contact with the upper circumferential inner wall 24. The O-ring 34 is thus both a securing element as a sealing element. The sound source 32 is arranged above the sample holder 21.

[0050] The level of the culture medium 33 in the tissue holder 21 is above the level of the support surface 25 and the O-ring 34. However, in the middle of the sample holder 21 , the tissue scaffold 25 together with the O-ring 34 form a barrier for the culture medium 33, preventing the culture medium 33 to level with the culture medium 33 present between the outer and upper circumferential inner walls 23, 24. As a result, the space above the tissue scaffold 31 enclosed by the upper circumferential inner wall 24 is an empty space, i.e. filled with environmental air. When the sound source 32 generates sound waves 35, sound waves 35 will be able to propagate to the tissue scaffold 31 (see Fig 3b) to stimulate the tissue scaffold 31.

[0051] Fig 4. shows a schematic perspective view from above of a biomimetic acoustical bioreactor 40 according to the present invention. Again, to prevent unnecessary introduction of elements, elements shown in Fig. 4 that correspond with those shown in Figs 3a, 3b are provided with a reference sign that is 20 higher than the corresponding element of Figs 3a, 3b.

[0052] The biomimetic bioreactor 40 is a combination of a conventional 6-well plate 56 and six sample holders 41 according to the third aspect of the invention. Thus, the biomimetic bioreactor 40 combines a conventional 6-well plate 56 with innovative sample holders 41. The sample holders 41 are received in the wells of the well-plate 56.

[0053] As shown in Fig. 5, the biomimetic bioreactor 40 can be easily upgraded to a biomimetic acoustical bioreactor, by arranging sound sources 52, 6 in Fig 5, one above each sample holder 41 in a well of the 6-well plate 56. Sound waves generated by the sound sources 52 can propagate to the sample 51 for stimulating formation and maturation.

[0054] The invention also relates to a method of formation and / or maturation of a tissue sample in a (biomimetic) bioreactor according to the invention. The diagram of Fig, 6 shows an exemplary and non-limiting embodiment of a method according to the invention.

[0055] In a first step, a biomimetic bioreactor according to the first aspect of the invention is provided. The biomimetic bioreactor comprises a 6 well-plate and six sample holders according to the third aspect of the invention.

[0056] In a second step, a sample is placed on the support surface of each of the sample holders. A rubber O-ring is placed on top of each of the samples, securing the sample at the support surface of the sample holder.

[0057] In a third step, the sample holders are placed in each of the wells of the 6-well plate. A culture medium is filled in the wells of the 6-well plate through the through holes in the top walls of the sample holders. In a fourth step a piezoelectric sound source is positioned above each of the sample holders in the wells and the piezoelectric sound sources are actuated to generate sound waves. In this regard, a resonant frequency of the used piezoelectric electric was 2950 Hz.

[0058] The invention is not limited to the use to study and / or produce tympanic membranes; it should be noted that other stem cells are also affected by acoustic simulation. For example, ultrasound techniques, like ultra-sound driven stimulation of peripheral nerves based on implantable piezoelectric thin film nanogenerators, are showing great promise in musculoskeletal tissue repair. Another application is the use of soundwaves to promote cell agglomeration, such as cancerous cells, which is of great interest for tissue engineering and drug testing because of their physiological relevance in comparison to monolayer cultures. Furthermore, it has been demonstrated that mimicking the high frequency vibration patterns of the human vocal cord can be used to produce engineered extracellular matrix when exciting human laryngeal fibroblasts (cells of the vocal cords). These are only a few applications of the invention.

[0059] In the described example, the resonant frequency of the used piezoelectric electric was 2950 Hz. However, it should be noted that it is possible to apply other frequency ranges guided by the respective sound sources for diverse biomedical applications.

[0060] The present invention is able to be applied on a relatively small scale, for R&D purposes as well as on a relatively high scale, for production purposes on a commercial scale.

[0061] While the description and the drawings describe and show a sample holder having a circular cross section, the sample holder can have any cross section, like polygonal with any number of identical or different corners, oval or otherwise.

[0062] While the description only describes use of the biomimetic bioreactor for airliquid stimulation, the skilled person will understand that the holder and the biomimetic bioreactor can be used for air-air stimulation, by simply not filling the bioreactor with a liquid, like the culturing medium. Similarly, the holder and the biomimetic bioreactor can easily be used for liquid-liquid stimulation by applying a liquid, like the culturing medium, within the space defined by the upper circumferential inner wall and the sample. The description describes only a few embodiments of aspects of the present invention. However many alternatives or changes that are or are not obvious to the skilled person are possible within the scope of the present invention, that is limited only by the appended claims.

[0063] Reference numeral list

[0064] 1 , 21 , 41 sample holder

[0065] 2, 22, 42 upper wall

[0066] 3, 23 circumferential outer wall

[0067] 4, 24 upper circumferential inner wall

[0068] 5, 25 support surface

[0069] 6, 26 lower circumferential inner wall

[0070] 7, 27, 47 through hole

[0071] 8, 28 lower edge of circumferential outer wall

[0072] 30, 50 well

[0073] 31 , 51 sample

[0074] 32, 52 sound source

[0075] 33 culture medium

[0076] 34, 54 O-ring

[0077] 35 sound wave

[0078] 40 biomimetic bioreactor

[0079] 56 6-well plate

Claims

CLAIMS1. Biomimetic bioreactor comprising: at least one substrate reservoir configured to receive a substrate and a sample holder; and a sample holder comprising: a top wall having an outer circumferential edge and in inner circumferential edge; a circumferential outer wall, depending from the outer circumferential edge of the top wall, extending downwards over a first distance d1 and having a first cross section; an upper circumferential inner wall, depending from the inner circumferential edge of the top wall, extending downwards over a second distance d2 smaller than the first distance d1 of the circumferential outer wall and having a second cross section, smaller than the first cross section of the circumferential outer wall; and a support surface, configured as a surface extending inwards from a bottom edge of the upper circumferential inner wall to an inner edge of the support surface wherein the upper circumferential inner wall of the sample holder defines an upper inner space, the biomimetic bioreactor comprising a sound source being present above the upper inner space, the sound source in use of the bioreactor generating sound waves that in use propagate in the direction of a sample supported by the support surface of the sample holder.

2. Biomimetic bioreactor according to claim 1 , wherein the substrate reservoir is a container of a generic culture well plate, preferably a 6, 12 ,24, 48, or 96 well plate and wherein preferably a sample holder is positioned in each of the containers of the well plate; or wherein the substrate reservoir is a petri-dish.

3. Biomimetic bioreactor according to claim 1 , wherein the sound source is provided with a hole, preferably a central hole.

4. Method of forming or maturing a tissue, the method comprising the steps of: providing a biomimetic bioreactor according to one or more of claims 1-3; placing a sample on the support surface of the sample holder; and placing the sample holder in the substrate reservoir.

5. Method according to claim 4, the method further comprising one or more of the steps of: placing a securing element on top of the sample, securing the sample present on the support surface in the holder; placing a sealing element on top of the sample, the sealing element sealingly in contact with the upper circumferential inner wall; filling a substrate in the reservoir, preferably through a through hole present in the upper wall of the sample holder, to a level equal to or higher than the support surface when the sample holder is present in the reservoir.

6. Method according to claim 4 or 5, the method further comprising arranging a sound source above the sample holder, the sound source in use generating sound waves that propagate to the sample in the sample holder.

7. Sample holder suitable for use in a biomimetic bioreactor according to one or more of claims 1-3, the sample holder comprising: a top wall having an outer circumferential edge and in inner circumferential edge; a circumferential outer wall, depending from the outer circumferential edge of the top wall, extending downwards over a first distance d1 and having a first cross section; an upper circumferential inner wall, depending from the inner circumferential edge of the top wall, extending downwards over a second distance d2 smaller than the first distance d1 of the circumferential outer wall and having a second cross section, smaller than the first cross section of the circumferential outer wall; anda support surface, configured as a surface extending inwards from a bottom edge of the upper circumferential inner wall to an inner edge of the support surface.

8. Sample holder according to claim 7, comprising a lower circumferential inner wall, depending from the inner edge of the support surface, and extending downwards over a fourth distance d4, wherein d1 > d2+d4, and having a third cross section, smaller than the second cross section of the circumferential outer wall.

9. Sample holder according to claim 7 or 8, wherein the upper circumferential inner wall is non permeable for liquid.

10. Sample holder according to one or more of claims 7-9, wherein the top wall comprises a through hole.

11. Sample holder according to one or more of claims 7-10, wherein the circumferential inner and / or outer walls have a circular cross section, and / or wherein the circumferential walls have a constant cross section, and / or wherein the circumferential outer wall preferably has a diameter in the range of 1 mm to 100 mm, preferably in the range of 5 mm to 75 mm, more preferably in the range of 10 mm to 50 mm and most preferably of 15 mm - 35 mm.

12. Sample holder according to one or more of claims 7-11 , wherein d1 is in the range of 1 mm to 50 mm, preferably in the range of 5 mm to 35 mm, more preferably in the range of 10 mm to 25 mm and most preferably is 15 mm, and / or wherein d2 is in the range of 0.01 mm to 25 mm, preferably in the range of 0.1 mm to 15 mm, more preferably in the range of 1 mm to 10 mm and most preferably is 5 mm, and / or wherein a space defined by the circumferential outer wall, the upper wall, the circumferential inner walls, a support ring forming the support surface and a face in which a lower edge of the circumferential outer wall extends has a volume of 1 to 100 ml, preferably 2 to 50 ml more preferably 5 to 20 or 10ml and most preferably of 7 ml, and / orwherein the support surface extends substantially horizontal.

13. Sample holder according to one or more of claims 7-12, further comprising a securing element to secure a sample supported by the support surface of the sample holder, the securing element preferably having a cross section the vertical projection of which fits substantially between edges of the support surface, and / or a sealing element that in use provides a fluid tight seal in sealing contact with the upper circumferential inner wall of the sample holder, preferably made of a flexible material and having an outer circumference that generally corresponds to the inner circumference of the upper circumferential inner wall, wherein the securing element and the sealing element preferably are configured as one integral element, preferably an O-ring.

14. Sample holder according to one or more of claims 7-13, wherein the sample holder is made of plastics, for example of polycarbonate, polymethyl methacrylate polystyrene, or PVC, metal, for example stainless steel or glass, and / or wherein the sample holder is made of a transparent material.

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