Sample holder, mould and methods for preparing a sample holder and tissue samples for histological analysis

Abstract

A sample holder (1) for preparing tissue samples (2), preferably 3D model systems, for histological analysis, comprises a base body (3) with at least one recess (4), preferably a plurality of recesses (4), for receiving a tissue sample (2), wherein each of said at least one recess (4) comprises at least one tapered receiving section (5) with a taper (6') extending along a central taper axis (7'), wherein each of said at least one tapered receiving section (5) extends laterally, preferably from the associated recess (4), with regard to an extension direction (4') of the associated recess (4), wherein said central taper axis (7') extends in a plane (8), preferably a common plane (8) for a plurality of tapered receiving sections (5), preferably being at least essentially parallel to a cutting plane (9, 9a, 9b). Furthermore, the invention relates to a mould (16) for a sample holder (1), a method for preparing a sample holder (1) and a method for preparing tissue samples (2).

Description

[0001] Sample holder, mould and methods for preparing a sample holder and tissue samples for histological analysis

[0002] The present invention relates to a sample holder and a method for preparing tissue samples, preferably 3D model systems, for histological analysis.

[0003] Further, the present invention relates to a mould and a method for preparing a sample holder.

[0004] The histological analysis of fixed tissue samples typically involves embedding the tissue samples in a paraffin block that is then cut in sections and stained. However, when preparing a large number of tissue samples, creating individual blocks of paraffin for each tissue sample and subsequently staining each section individually is a highly labour intensive task with very limited scalability.

[0005] In order to tackle this problem, it has become known to prepare tissue microarrays, where tissue samples, typically biopsy samples, are cut as cylindrical cores and embedded in paraffin in an array configuration (see, for example, Nature Medicine, 1998, 4:844-847). Further related publications comprise: Molecular Pathology, 2003, 56(4): 198-204; Scientific Data, 2017, 4, 170170; Human Molecular Genetics, 2001 , 10(7):657-662.

[0006] For small tissue samples, for example organoids or spheroids, such cylindrical cuts / punch outs are not possible. In such cases, methods have become known, wherein a solid structure containing mini wells with a cylindrical shape to accommodate the small tissue samples in an array are prepared and subsequently embedded in paraffin. These, so prepared sample blocks are then sectioned into slices, wherein each slice comprises cross-sections of several embedded tissue samples due to the arrangement of the tissue samples in the array (see for example: Scientific Reports, 2022, 12:9991 ; Scientific Reports, 2019, 9:16287; Biotechniques, 2015, 59.5:279-286; Journal of Biological Engineering, 2020, 14, 7).

[0007] Irrespective of the well geometry and arrangement, the aforementioned systems can present two fundamental problems. Firstly, since the base of each well is arranged on the same level, also the specimens sit on the well base at this same level irrespective of their size. Consequently, the centres of specimens with differing sizes are not arranged coplanarly. Thus, a slice is not representative of the position within the specimens and requires other slices to understand its positioning. Furthermore, a slice cut in the centre of most specimens could entirely miss the presence of a small specimen. Secondly, when hydrogel is poured on top of specimens placed in the array for fully embedding them, the specimens may float and shift their position due to buoyancy and flow drag. As a result, even uniform populations of species can end up not being arranged coplanarly. These problems negatively affect the quality of the histological analysis and might result in additional use of reagents, in particular expensive antibodies, and working expenditure, since the user will have to analyze several slides to capture all the organoids in the desired central section.

[0008] One of the objectives of the present invention is to improve and further develop a sample holder and a method for preparing tissue samples, preferably 3D model systems, for histological analysis, in particular in a labour-efficient manner and with easy means.

[0009] A further objective of the present invention is to improve and further develop a mould and a method for preparing a sample holder, in particular in a labour-efficient manner and with easy means.

[0010] In a first aspect, the present invention provides a sample holder for preparing tissue samples, preferably 3D model systems, for histological analysis, comprising a base body with at least one recess, preferably a plurality of recesses, for receiving a tissue sample, characterized in that each of said at least one recess comprises at least one tapered receiving section with a taper extending along a central taper axis, wherein each of said at least one tapered receiving section extends laterally, preferably from the associated recess, with regard to an extension direction of the associated recess, wherein said central taper axis extends in a plane, preferably a common plane for a plurality of tapered receiving sections, preferably being at least essentially parallel to a cutting plane.

[0011] In a second aspect, the present invention provides a mould according to the first aspect of the invention comprising a base mould with a bottom section, a side wall section and an opening opposite to said bottom section, wherein said bottom section and said side wall section delimit an inner space for receiving a casting material, and at least one first protrusion, preferably a plurality of first protrusions, each comprising at least one tapered protrusion section corresponding to said at least one tapered receiving section of said sample holder.

[0012] In a third aspect, the present invention provides a method for preparing a sample holder with a mould according to the second aspect of the invention, comprising the steps of:

[0013] Providing said mould, filling a casting material inside said mould, preferably by means of gravitational force, solidifying said casting material, and at least partially demoulding said sample holder, preferably comprising removing a first and / or a second lid of said mould.

[0014] In a fourth aspect, the present invention provides a method for preparing tissue samples, preferably 3D model systems, for histological analysis with a sample holder according to the first aspect of the invention, comprising the steps of:

[0015] Providing said sample holder, placing at least one tissue sample inside said at least one recess, preferably using a spatula and / or a pipetter, embedding said at least one tissue sample to form a sample block comprising said sample holder, such that said at least one tissue sample is positioned in said sample block, and sectioning said sample block along said cutting plane to form at least one sample block slice to be analysed.

[0016] The sample holder according to the first aspect of the invention may be used in a method for preparing tissue samples, preferably 3D model systems, for histological analysis comprising the step of sectioning said samples arranged in said sample holder along the cutting plane, for example to obtain a slice comprising sectioned samples to be analysed. In particular, the sample holder according to the first aspect of the invention may be used in a method according to the second aspect of the invention. It has been recognized, that the tapered receiving section has a self-centering effect on a tissue sample arranged therein. This means that the centre of the tissue sample will substantially lie on the central taper axis of this tapered receiving section. Secondly, the taper can effectively prevent the tissue sample from slipping out of this self-centered position, for example due to its buoyancy or due to fluidic forces, when the tissue sample is embedded in the tapered receiving section in order to be positioned in place. Thirdly, by providing a laterally extending tapered receiving section, in particular by providing a parallel alignment of the central taper axis and the cutting plane, the position of the centre of this tissue sample in relation to the cutting plane is known. In other words, each tapered receiving section extends laterally with regard to the extension direction and / or a longitudinal axis of the associated recess, wherein the tapered receiving section may be arranged on a lateral side of the recess or may be arranged underneath the recess. Furthermore, the tip of the tapered receiving section may laterally extend beyond the recess or the tip of the tapered receiving section may line up with a side wall of the recess. This way, the cutting plane can, for example, be chosen in a way that it is coplanar to the central taper axis and consequently coplanar to the centre position of the tissue sample. In other words, the central taper axis and the centre position lie in the cutting plane. It is noted that alternative cutting planes may be chosen in order to obtain a sample block slice with one or more tissue samples with desired cross sections. In the case of a coplanar alignment of the tissue sample centre and the cutting plane, the obtained sample block slice comprises a high amount of tissue that can be analysed, since the centre of the tissue sample usually corresponds to a large or even a maximum cross sectional area. By analysing a large cross section of the tissue sample, the quality of the histological analysis can be improved.

[0017] Furthermore, a plurality of tapered receiving sections with a tissue sample arranged in each of them can be provided. When the central taper axes extend in a common plane for these tapered receiving sections, preferably wherein the common plane is parallel to the cutting plane, a sample block slice comprising a cross-section of every tissue sample irrespective of their individual sizes can be obtained, in particular with a coplanar alignment of the tissue sample centres and the cutting plane. This increases the efficiency of the histological analysis, since it is not necessary to prepare several sample block slices as long until at least one cross-section of every tissue sample is captured. Furthermore, when preparing a plurality of tissue samples it can be ensured that one sample block slice comprises comparable cross-sections of each tissue sample embedded therein. Therefore, a sectioning, staining and imaging workflow with a uniform staining and imaging exposure may achieve uniform results across a single focal plane. Comparability between the analysed samples may be ensured. In addition, a high-throughput embedding with a significant reduction of labour intensive and tedious manual procedures, in particular with regard to sectioning and staining, may be achieved. This may facilitate a high tissue sample density per sample block, decreasing reagent expenditure.

[0018] The tissue samples prepared according to the method according to the fourth aspect of the invention may be analysed comprising imaging of the tissue samples. Especially, when preparing a plurality of tissue samples, these tissue samples are arranged on a single slice. Therefore, staining and imaging variability can be mitigated.

[0019] With regard to the mould according to the second aspect of the invention and the method according to the third aspect of the invention, it has been recognized that the sample holder can be manufactured by means of casting. The casting material can be easily introduced into the base mould, for example with the help of gravitational force. The preparation of the sample holder is simplified, i.e. making it easier and more efficient compared to alternative methods, like, for example, machining said sample holder from a solid material. Furthermore, a consistent quality may be achieved by using the mould. The mould may also be reusable.

[0020] The term "tissue sample" is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to samples of biological tissue, in particular of in vitro and / or ex vivo models. In vitro models may comprise organoids, in particular 3D model systems. Such 3D model systems may comprise in vitro derived 3D cell aggregates derived from primary tissue or stem cells and / or 3D monolayer cultures, in particular 3D epithelial monolayer cultures, extracted from a thin hydrogel / matrix (e.g. with imprinted topography) using a puncher, and / or 3D monolayers, in particular 3D epithelial monolayers, co-cultured with immune cells and extracted from a thin hydrogel / matrix (e.g. with imprinted topography). The term "cutting plane" is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a flat plane along which a cutting tool, in particular a blade of a microtome, moves to produce a slice of one or more tissue samples for further analysis, for example for microscopic analysis. The slice might additionally comprise material used to embed the one or more tissue samples. The cutting plane may be chosen in a way that it is essentially parallel to a top side of the sample holder, from which the at least one recess extends, and / or to a bottom side of the sample holder being opposite to the top side. Furthermore, the cutting plane may be chosen to be positioned between the top and bottom side.

[0021] The term "taper" is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a reduction in at least one dimension of the cross-section of the tapered receiving section along the central taper axis, preferably wherein the reduction in the at least one dimension maintains a bilateral symmetry with respect to the central taper axis. The at least one dimension may comprise a diameter and / or a width and / or a height of the cross-section.

[0022] Further features, advantages and preferred embodiments are disclosed or may become apparent in the following.

[0023] According to a further embodiment of the invention, said taper comprises a conical, a pyramidal, a hemispherical or a hemiellipsoidal shape or a segmented shape thereof. The taper shape can be chosen depending on the shape of the at least one tissue sample in order to ensure improved positioning and securing of the at least one tissue sample. Furthermore, such shapes can provide a high inherent stiffness of the tapered receiving sections and can be easily manufactured.

[0024] According to a further embodiment of the invention, said taper of each of said at least one tapered receiving section extends along said respective central taper axis with a common orientation. This way, the positioning of tissue samples in the tapered receiving sections can be facilitated. For placing the tissue samples inside the recesses, the sample holder can be arranged in a single position in a way that gravitational force can be used to self-centre each tissue sample in the respective tapered receiving section. According to a further embodiment of the invention, each of said at least one recess comprises a plurality of tapered receiving sections, preferably three tapered receiving sections. Thereby, the amount of recesses can be reduced which can simplify the design of the sample holder and can enhance the inherent stiffness and strength of the sample holder. Furthermore, the size of each recess can be designed larger providing more space for placing tissue samples inside using for example a pipette.

[0025] According to a further embodiment of the invention, a plurality of tapered receiving sections is provided, wherein said tapered receiving sections are arranged in an array with at least two tapered receiving sections being aligned in a first array dimension and / or at least two tapered receiving sections being aligned in a second array dimension. This way, multiple tissue samples can be analysed at the same time, improving the efficiency of the preparation. When arranged in the array, the tapered receiving sections may be equidistantly spaced apart from each other and preferably equally oriented. Additionally or alternatively, the first and second array dimension may be perpendicular to each other.

[0026] According to a further embodiment of the invention, each of said at least one tapered receiving section comprises a cross-section, being perpendicular to its central taper axis, with a maximum width and a depth extending along its central taper axis, wherein a ratio of said maximum width to said depth is at least 0.1 and less than or equal to 10, preferably at least 0.5 and less than or equal to 3, more preferably equal to 1 .33. A tapered receiving section, which is dimensioned in this way, provides an exceptionally well self-centering effect facilitating the positioning of a tissue sample inside the tapered receiving section.

[0027] According to a further embodiment of the invention, each of said at least one tapered receiving section comprises a cross-section, being perpendicular to its central taper axis, with a maximum width of at least 0.5 mm and less than or equal to 5 mm, preferably at least 2 mm and less than or equal to 3 mm, more preferably of 1 .2 mm or 2.4 mm. Such dimensions are suitable for accommodating a wide range of tissue samples, in particular organoids and / or 3D model systems that are commonly histologically analysed for diagnostic and research purposes. According to a further embodiment of the invention, each of said at least one tapered receiving section comprises a depth extending along its central taper axis of at least 0.5 mm and less than or equal to 5 mm, preferably at least 1 mm and less than or equal to 2.5 mm, more preferably of 0.9 mm or 1 .8 mm. Such dimensions are suitable for accommodating a wide range of tissue samples, in particular organoids and / or 3D model systems, that are commonly histologically analysed for diagnostic and research purposes.

[0028] According to a further embodiment of the invention, each of said at least one recess comprises a first section extending along a longitudinal axis, preferably being aligned with said extension direction of each of said at least one recess, wherein the associated tapered receiving section extends laterally from said first section with regard to said longitudinal axis. Such a first section facilitates the introduction of a pipetter tip for placing and positioning a tissue sample inside the recess.

[0029] According to a further embodiment of the invention, said longitudinal axis and / or said extension direction of said at least one recess is inclined to said central taper axis at an angle of at least 90° and less than or equal to 150°, preferably at an angle of 90°. Consequently, when releasing the tissue sample in the recess, the tissue sample can reach the tapered receiving section and centre itself under the influence of gravitational force. This results in a time and labour efficient preparation of tissue sample for histological analysis.

[0030] According to a further embodiment of the invention, said base body comprises a top side, from which said at least one recess extends, wherein said top side is parallel to said at least one central taper axis. The top side usually faces the histology cassette when being attached thereto. When the histology cassette and the sample holder are consequently attached to a microtome for sectioning, the top side and at least one central taper axis can be automatically aligned parallel to the cutting plane.

[0031] According to a further embodiment of the invention, said longitudinal axis is inclined to said top side at an angle of at least 30° and less than or equal to 90°, preferably at an angle of 90°. During placing of a tissue sample inside a recess, it is conceivable that the sample holder is arranged or tilted so that the longitudinal axis is positioned parallel with the vertical. This enables the tissue sample to position itself in the tapered receiving section under the influence of gravitational force.

[0032] According to a further embodiment of the invention, a material of said sample holder comprises a curable gel, preferably a hydrogel, and / or a polymer. The curing may comprise gelation and / or polymerisation of the gel. The curable gel may comprise a polymer network with a dispersion medium. The curable hydrogel may comprise a polymer network with water as the dispersion medium. Preferably, the hydrogel comprises HistoGel®. Additionally or alternatively, the material of the sample holder may comprise at least one of the following: agarose gel; paraffin wax; Optimal Cutting Temperature (OCT) compound; gelatin; methylcellulose; or any material with a similar composition and / or similar properties as the aforementioned materials.

[0033] According to a further embodiment of the invention, said at least one first protrusion forms an integral part of said base mould and / or forms part of a separate element of said mould, preferably wherein said separate element is a first lid which can be arranged on said base mould to form an assembled state of said mould. The integral part can ensure the correct positioning of the at least one first protrusion when the casting material is filled inside the mould. The separate part can provide a mould that is easy to manufacture and easy to handle. Furthermore, the separate part, preferably the first lid, can be removed after solidification of the casting material, without the need to fully demould the sample holder from the base mould.

[0034] According to a further embodiment of the invention, said mould comprises a second lid with at least one second protrusion, preferably a plurality of second protrusions, wherein said second lid can be arranged on said first lid and / or on said base mould to form an assembled state of said mould, wherein, in said assembled state, each first and second protrusions form a protrusion pair associated with one of said at least one recess of said sample holder, wherein for said protrusion pair said second protrusion is arranged on a side of said first protrusion opposite to a protrusion direction of said at least one tapered protrusion section. This enables the first and second lid to be assembled and disassembled stepwise in a way that the risk of damaging the sample holder during demoulding is minimized. This process can comprise removing the second lid, preferably comprising pulling the at least one second protrusion out of the at least one recess of the sample holder and the at least one aperture, moving the first lid in a direction opposite to a protrusion direction of the at least one tapered protrusion section, preferably into a space exposed after removing the second lid, and removing the first lid, preferably comprising pulling the at least one first protrusion out of the at least one recess of the sample holder.

[0035] According to a further embodiment of the invention, said first lid comprises at least one aperture, preferably a plurality of apertures, associated with said at least one first protrusion, wherein, in an assembled state, said at least one second protrusion extends through said at least one aperture. Thereby, the second lid can be arranged on top of the first lid and is stabilized against a lateral movement in at least one lateral direction by the at least one aperture.

[0036] According to a further embodiment of the invention, said base mould and / or said first lid and / or said second lid form an injection port and / or wherein said first lid comprises at least one stiffening element, for example a wall, arranged between at least two of said tapered protrusion sections. The injection port facilitates the filling of the casting material inside the mould. By arranging at least one stiffening element the deformation of the mould, in particular of the tapered protrusion sections, resulting from the moulds own weight and / or from the influence of pressure forces from the casting material can be minimized. Thereby, the sample holder prepared with the mould comprises a high dimensional accuracy.

[0037] According to a further embodiment of the invention, said side wall section forms at least one restraint element, for example at least one contact edge, for preventing a lateral displacement of said first and / or second lid in at least one lateral direction in an assembled state, preferably wherein said at least one lateral direction comprises a lateral direction corresponding to a protrusion direction of said at least one tapered protrusion section and / or one or both lateral directions being perpendicular to said protrusion direction of said at least one tapered protrusion section. Thereby, the position of the first and / or second lid can be secured during the filling of the casting material inside the mould and during the solidification of the casting material. In particular, a shifting of the second lid in the wrong direction can be prevented, in case the second lid needs to be removed laterally for demoulding. This ensures a high quality and dimensional accuracy of the sample holder prepared with the mould. According to a further embodiment of the invention, said bottom section and / or said side wall section comprises a plurality of grooves, preferably parallel grooves, on a side facing said inner space. The grooves can comprise indentations and / or elevations and can prevent the casted sample holder from moving during demoulding, in particular during the removement of the first and / or second lid. This is achieved by a form-fit interconnection of the casting material with the indentations and / or elevations of the grooves.

[0038] According to a further embodiment of the invention, said side wall section comprises at least one undercut, preferably a circumferential undercut, preferably wherein said at least one undercut extends laterally at a level of said bottom section. The at least one undercut can prevent the casted sample holder from moving during demoulding, in particular during removing of the first and / or second lid. This is achieved by a formfit interconnection of the casting material with the at least one undercut.

[0039] According to a further embodiment of the invention, said base mould and / or said first lid and / or said second lid is fabricated by means of additive manufacturing, preferably by means of 3D-printing. The base mould and / or the first lid and / or the second lid mould may thus be easily manufactured with a high design complexity. Furthermore, additive manufacturing enables the production of custom-made base mould and / or first lid and / or second lid in a short period of time.

[0040] According to a further embodiment of the invention, said base mould and / or said first lid and / or said second lid comprises a hydrophobic material. The hydrophobic properties of the base mould and / or said first lid and / or said second lid can improve the casting process by enhancing casting material flow and reducing air bubbles, in particular since the casting material is prevented from sticking to the mould during filling. Furthermore, the hydrophobic material can simplify demoulding and help to achieve a smoother finish of the sample holder. Additionally or alternatively, said base mould and / or said first lid and / or said second lid may comprise a hydrophilic material. This way, it can be ensured that also filigree portions, for example filigree cavities, of the mould are properly wetted by casting material

[0041] According to a further embodiment of the invention, said at least one tapered protrusion section comprises a taper extending along a central taper axis, wherein said central taper axis extends in a plane, preferably a common plane for a plurality of tapered protrusion sections.

[0042] According to a further embodiment of the invention, said taper of said at least one tapered protrusion section comprises a conical, a pyramidal, a hemispherical or a hemiellipsoidal shape or a segmented shape thereof.

[0043] According to a further embodiment of the invention, said taper of each of said at least one tapered protrusion section extends along said respective central taper axis with a common orientation.

[0044] According to a further embodiment of the invention, each of said at least one first protrusion comprises a plurality of tapered protrusion sections, preferably three tapered protrusion sections.

[0045] According to a further embodiment of the invention, a plurality of tapered protrusion sections is provided, wherein said tapered protrusion sections are arranged in an array with at least two tapered protrusion sections being aligned in a first array dimension and / or at least two tapered protrusion sections being aligned in a second array dimension.

[0046] According to a further embodiment of the invention, each of said at least one tapered protrusion section comprises a cross-section, being perpendicular to a protrusion axis of said at least one tapered protrusion section, with a maximum width and a length extending along said protrusion axis, wherein a ratio of said maximum width to said length is at least 0.1 and less than or equal to 10, preferably at least 0.5 and less than or equal to 3, more preferably equal to 1 .33.

[0047] According to a further embodiment of the invention, each of said at least one tapered protrusion section comprises a cross-section, being perpendicular to a protrusion axis of said at least one tapered protrusion section, with a maximum width of at least 0.5 mm and less than or equal to 5 mm, preferably at least 2 mm and less than or equal to 3 mm, more preferably of 1 .2 mm or 2.4 mm. According to a further embodiment of the invention, each of said at least one tapered protrusion section comprises a length extending along a protrusion axis of said at least one tapered protrusion section of at least 0.5 mm and less than or equal to 5 mm, preferably at least 1 mm and less than or equal to 2.5 mm, more preferably of 0.9 mm or 1 .8 mm.

[0048] According to a further embodiment of the invention, said at least one tapered receiving section and / or said tapered protrusion section comprises a rounded tip, preferably wherein a radius of said rounded tip substantially corresponds to a radius of said tissue samples or is smaller than said radius of said tissue samples. The radius of the rounded tip may additionally or alternatively depend on the geometry of the tapered receiving section, in particular on an angle formed by the tapered receiving section and / or on a height and / or a width and / or a depth of the tapered receiving section. A rounded tip can facilitate the additive manufacturing process of the sample holder and / or the mould. Furthermore, with a radius substantially corresponding to the radius of the tissue sample an entrapment of air bubbles can be minimized when arranging the tissue sample in the tapered receiving section.

[0049] According to a further embodiment of the invention, said at least one first protrusion comprises a longitudinal section, wherein said tapered protrusion section is arranged on a lateral side and / or on a front end of said longitudinal section. An arrangement on the lateral side enables a high degree of design freedom for the geometry of the tapered protrusion section. An arrangement on the front end can facilitate the assembly and disassembly of the mould, in particular when removing the first protrusion.

[0050] According to a further embodiment of the invention, said casting material is a curable gel, in particular a hydrogel, wherein said gel solidifies by curing, preferably wherein said curing is achieved by cooling said gel at a temperature between 0 °C and 10 °C, more preferably between 4 and 5 °C, in particular for a duration of at least 20 min. The curing may comprise gelation and / or polymerisation of the gel. Before curing, the gel may be present in a liquid form. For providing this liquid form, the gel may be warmed, for example to a temperature of about 65°C. It is also conceivable, that the gel comprises a liquid form at room temperature and does not have to be warmed beforehand. Hence, when filling the gel in a liquid form into the mould for the sample holder, it may be ensured that all cavities are filled and a forming of air pockets may be prevented. The curable gel may comprise a polymer network with a dispersion medium. The curable hydrogel may comprise a polymer network with water as the dispersion medium. Preferably, the hydrogel comprises HistoGel®. Additionally or alternatively, the casting material may comprise at least one of the following: agarose gel; paraffin wax; Optimal Cutting Temperature (OCT) compound; gelatin; methylcellulose; or any material with a similar composition and / or similar properties as the aforementioned materials. The cured gel may be flexible and therefore facilitate demoulding of the cured gel. It is pointed out that the skilled person can easily define the curing duration for a particular material.

[0051] According to a further embodiment of the invention, said at least partially demoulding of said sample holder comprises the steps of:

[0052] Removing said second lid, preferably comprising pulling said at least one second protrusion out of said at least one recess of said sample holder and said at least one aperture, moving said first lid in a direction opposite to a protrusion direction of said at least one tapered protrusion section, preferably into a space exposed after removing said second lid, and removing said first lid, preferably comprising pulling said at least one first protrusion out of said at least one recess of said sample holder.

[0053] This process can ensure that the fine details of the sample holder, in particular the at least one tapered receiving section, are not damaged during the demoulding of the sample holder.

[0054] According to a further embodiment of the invention, said sample holder is prepared by means of casting, preferably using a method according to claim 11 or 12. Casting simplifies the preparation of the sample holder, making it easier and more efficient compared to alternative methods, like, for example, machining the sample holder from a solid material. Furthermore, a consistent quality may be achieved by casting. Overall, the process of preparing the at least one tissue sample for histological analysis can be greatly improved.

[0055] According to a further embodiment of the invention, said at least one tissue sample is embedded by means of casting using a sample block mould, wherein said sample holder at least partially forms said sample block mould. By using the sample holder, at least partially, as a mould the at least one tissue sample may be easily and time- effectively embedded without having to install a complex embedding arrangement.

[0056] According to a further embodiment of the invention, said sample block mould comprises a surrounding enclosure, preferably wherein an upper edge of said surrounding enclosure extends beyond said at least one tissue sample. Hence, a full embedment of the at least one tissue sample may be achieved when filling the sample block mould with casting material until it reaches the upper edge of the surrounding enclosure. This may result in a sample block with a flat top surface, facilitating the handling and further processing of said sample block.

[0057] According to a further embodiment of the invention, said surrounding enclosure is integrally formed with said sample holder or is formed by a separate part. An integrally formed surrounding enclosure reduces the manufacturing effort. With a separate part of the surrounding enclosure, the sample block mould may be easily customized depending on various boundary conditions.

[0058] According to a further embodiment of the invention, said casting using said sample block mould comprises the steps of:

[0059] Filling a casting material inside said sample block mould, preferably by means of gravitational force, solidifying said casting material, and demoulding said sample block,

[0060] These simple steps may be performed without the need of special equipment and provide a low risk for an incorrect execution by the user. Generally, demoulding may be achieved by gravity, inverting the mould, and, if necessary, with the help of a spatula. Alternatively or additionally, demoulding may be achieved by bending the mould and, if necessary, pressing the solidified casting material out of the mould by hand. In the solidified state, the casting material may be rigid or flexible. A flexible casting material may facilitate demoulding by bending it and, if necessary, pressing the solidified casting material out of the mould by hand. A flexible casting material may be particularly useful for complex geometries, e.g. dense arrays. According to a further embodiment of the invention, said casting material is a curable gel, in particular a hydrogel, wherein said gel solidifies by curing, preferably wherein said curing is achieved by cooling said gel at a temperature between 0 °C and 10 °C, more preferably between 4 and 5 °C, in particular for a duration of at least 20 min. The curing may comprise gelation and / or polymerisation of the gel. Before curing, the gel may be present in a liquid form. For providing this liquid form, the gel may be warmed, for example to a temperature of about 65°C. It is also conceivable, that the gel comprises a liquid form at room temperature and does not have to be warmed beforehand. Hence, when filling the gel in a liquid form into the sample block mould, it may be ensured that all cavities are filled and a forming of air pockets may be prevented. After solidification of the gel, the at least one tissue sample is positioned in the sample block. The curable gel may comprise a polymer network with a dispersion medium. The curable hydrogel may comprise a polymer network with water as the dispersion medium. Preferably, the hydrogel comprises HistoGel®. Additionally or alternatively, the casting material may comprise at least one of the following: agarose gel; paraffin wax; Optimal Cutting Temperature (OCT) compound; gelatin; methylcellulose; or any material with a similar composition and / or similar properties as the aforementioned materials. The cured gel may be flexible and therefore facilitate demoulding of the cured gel. Furthermore, it is pointed out that the skilled person can easily define the curing duration for a particular material. Preferably, for casting the sample holder and the sample block the same casting material is used for ensuring sufficient bonding and therefore a high strength of the sample block.

[0061] According to a further embodiment of the invention, said sample holder is arranged in a way that said central taper axis of said at least one tapered receiving section is inclined to the vertical at an angle of at least 90° and less than or equal to 180°, preferably at an angle of at least 120° and less than or equal to 180°, more preferably at an angle of 135°, in particular during placing said at least one tissue sample inside said at least one recess and / or during embedding said at least one tissue sample. This enables the tissue sample to position itself in the tapered receiving section under the influence of gravitational force, resulting in an accurate positioning of the tissue sample inside the respective tapered receiving section and in a time and labour efficient preparation of tissue sample for histological analysis. It is conceivable that the sample holder is arranged on and / or in a device, for example a tiltable table, which is suitable to arrange the sample holder in the aforementioned way. Alternatively, the sample holder itself may be designed to be positioned in the aforementioned way.

[0062] According to a further embodiment of the invention, said sample holder is arranged in a way that said longitudinal axis of said at least one recess is inclined to the vertical at an angle of at least 0° and less than or equal to 45°, preferably at an angle of 30°, in particular during placing said at least one tissue sample inside said at least one recess and / or during embedding said at least one tissue sample. This way, the introduction of a pipette tip for placing and positioning a tissue sample inside the recess can be facilitated, which consequently improves the efficiency of the preparation of tissue sample for histological analysis. Furthermore, when using a pipetting robot, the accessibility of the at least one recess for the pipetter tip can be ensured.

[0063] According to a further embodiment of the invention, prior to sectioning, said sample block is attached to a histology cassette using a fixing matrix, preferably wherein said fixing matrix comprises a paraffin and / or a resin. The histology cassette may facilitate the handling of the sample block for further steps of the histological analysis. Attaching the sample block may comprise the following steps. The sample block may be distributed into the histology cassette and dehydrated. Subsequently, the sample block may be embedded in liquid paraffin in a metallic mould and capped with the histology cassette. After polymerization of the paraffin, preferably at -20 °C, the metallic mould may be removed and the sample block with the attached histology cassette may be stored at -20 °C until sectioning.

[0064] According to a further embodiment of the invention, said sectioning is performed by means of a microtome. With the microtome, extremely thin slices of the sample block may be produced for observation under transmitted light or laser light. In particular, brightf ield and / or confocal imaging may be used for analysing the slices of the sample block. A thickness of the slices may range from 3 pm to 15 pm.

[0065] According to a further preferred embodiment of the invention, said in said at least one sample block slice is stained, preferably by means of haematoxylin and eosin staining and / or multiplex staining. Staining may provide a high-contrast coloration of the tissue for a better differentiation of tissue types, of cellular structures and / or of the distribution of chemical substances. Multiplex staining may comprise fluorescent or chromogenic staining for detecting multiple biomarkers, for example protein and / or nucleic acid markers.

[0066] According to a further embodiment of the invention, at least one of said steps forms part of an automated process, preferably wherein said placing and / or embedding of said at least one tissue sample is performed using a pipetting robot. Automating the process, particularly the placement and embedding of the at least one tissue sample with a pipetting robot, enhances precision and repeatability, reducing human error and variability. Additionally, the automated process can increase efficiency and throughput. Therefore, more samples can be processed consistently.

[0067] According to a further embodiment of the invention, a pipette tip of said pipetter and / or of said pipetting robot is coated with a coating agent by contacting said pipette tip with a buffer comprising said coating agent, preferably wherein said coating agent is a protein, for example 10 % BSA (Bovine Serum Albumin) in a PBS (Phosphate Buffered Saline) solution. Coating the pipette tip with a protein-based agent, such as BSA in PBS, can reduce sample adhesion to the tip, thereby improving accuracy and consistency in sample handling. The coating can furthermore minimize crosscontamination and sample loss, leading to more reliable and reproducible results, in particular in an automated pipetting processes.

[0068] According to a further preferred embodiment of the invention, a pipette tip of said pipetter and / or of said pipetting robot is positioned at a distance of a maximum of 0.6 mm, preferably 0.6 mm, to said at least one tissue sample for aspirating said at least one tissue sample, preferably wherein at least 10 pL and a maximum of 15 pL of a solution comprising said at least one tissue sample is aspirated. Positioning the pipette tip at a precise maximum distance of 0.6 mm from the at least one tissue sample can enhance the accuracy of the sample aspiration and consequently minimize sample loss.

[0069] There are several ways how to design and further develop the teaching of the present invention in an advantageous way. To this end, it is to be referred to the patent claims subordinate to the patent claims directed to the first, second, third and fourth aspect of the invention on the one hand and to the following explanation of preferred examples of embodiments of the invention, illustrated by the drawing on the other hand. In connection with the explanation of the preferred embodiments of the invention by the aid of the drawing, generally preferred embodiments and further developments of the teaching will be explained.

[0070] In the drawing

[0071] Fig. 1 a shows a sample holder according to an embodiment of the present invention in a three-dimensional view,

[0072] Fig. 1 b shows the sample holder according to the embodiment of Fig. 1 a in a top view,

[0073] Fig. 2 shows a detail view of a cross-section of the sample holder according to the embodiment of Fig. 1 a with tissue samples arranged therein and a detail view of a cross-section of a sample block prepared from this sample holder,

[0074] Fig. 3a-3c show a mould according to an embodiment of the invention in different views, comprising a base mould (Fig. 3a), a first lid (Fig. 3b) and a second lid (Fig. 3c),

[0075] Fig. 4a-4c show a mould according to a further embodiment of the invention in different views, comprising a base mould (Fig. 4a), a first lid (Fig. 4b) and a second lid (Fig. 4c),

[0076] Fig. 5 shows a mould comprising a base mould, a first lid and a second lid according to a further embodiment of the invention in an unassembled and an assembled state in a side view,

[0077] Fig. 6a-6c show the mould according to the embodiment of Fig. 5, wherein the first and second lids are shown in an assembled state in a bottom view (Fig. 6a) and wherein a three-dimensional view (Fig. 6b) and a top view (Fig. 6c) of the fully assembled mould are shown, Fig. 7a-7c show base moulds according to three different embodiments of the invention,

[0078] Fig. 8a-8d show pairs of first and second lids of moulds according to four further embodiments of the invention in bottom views,

[0079] Fig. 9 shows a visualization of steps of demoulding a sample holder from the mould according the embodiment of Fig. 5,

[0080] Fig. 10a-b show two different embodiments of a first protrusion of a first lid of the mould according to a further embodiment of the invention in a bottom view (left) and in a 3D-view from the bottom (right),

[0081] Fig. 11 a shows steps of a method for preparing tissue sample for histological analysis according to an embodiment of the present invention,

[0082] Fig. 11 b shows a visualization of steps of the method according to the embodiment of Fig. 11 a,

[0083] Fig. 12a shows steps of a method for preparing tissue samples for histological analysis according to a further embodiment of the present invention,

[0084] Fig. 12b shows a visualization of steps of the method according to the embodiment of Fig. 12a,

[0085] Fig. 13 shows a sample holder with retinal organoids of different sizes placed in recesses in the form of cylindrical wells with flat bottoms, a sample block formed with this sample holder after embedding with hydrogel as well as four H&E stained sample block slices obtained from sectioning this sample block at different cutting planes,

[0086] Fig. 14 shows a sample block formed with a sample holder according to an embodiment of the invention with retinal organoids of different sizes placed in the recesses after embedding with hydrogel as well as three H&E stained sample block slices obtained from sectioning this sample block at different cutting planes, and

[0087] Fig. 15 shows (a) thresholding of an original H&E stained sample block slice, wherein only the outer rim of the organoids is considered to calculate their area, irrespective of inner cavities, (b) values of the organoid areas per sample block slice obtained from the sample block of Fig. 12 (“Control”) and obtained from the sample block of Fig. 13 (“3D histomold”), normalized to the maximum organoid area within those sample block slices, wherein colour gradients between red and green indicate proximity to the respective maximum area, and (c) percentage of organoids in each sample block slice that reach at least 80% of their maximum area.

[0088] Fig. 1 a shows a sample holder according to an embodiment of the present invention in a three-dimensional view.

[0089] Fig. 1 b shows the sample holder according to the embodiment of Fig. 1 a in a top view.

[0090] The sample holder 1 for preparing tissue samples 2, preferably 3D model systems, for histological analysis comprises a base body 3 with a plurality of recesses 4 for receiving tissue samples 2. Each of the recesses 4 comprises one tapered receiving section 5 with a taper 6' extending along a central taper axis 7', wherein each of the tapered receiving sections 5 extends laterally from the associated recess 4 with regard to an extension direction 4' of the associated recess 4, wherein the central taper axis 7' extends in a common plane 8 for the tapered receiving sections 5. The common plane 8 is parallel to a cutting plane 9. The base body 3 comprises a top side 10, from which the recesses 4 extend, wherein the top side 10 is parallel to the central taper axis 7.

[0091] Each taper 6' comprises a conical shape and extends along its respective central taper axis 7' with a common orientation. The tapered receiving sections 5 are arranged in an array 11 ' with five tapered receiving sections 5 being aligned in a first array dimension and four tapered receiving sections 5 being aligned in a second array dimension.

[0092] Each of the recesses 4 comprises a first section 12 extending along a longitudinal axis 13, wherein the associated tapered receiving section 5 extends laterally from the first section 12 with regard to the longitudinal axis 13. Each longitudinal axis 13 is aligned with the extension direction 4' of the associated recesses. Each first section 12 comprises a rectangular cross-section. The longitudinal axis 13 and the extension direction 4' are inclined to the central taper axis 5 associated with each recess 3 at an angle 14a of 90°. Furthermore, the longitudinal axis 13 is inclined to the top side 10 of the base body 3 at an angle 14b of 90°. The sample holder 1 is made of a curable gel, in particular a hydrogel.

[0093] Fig. 2 shows a detail view of a cross-section of the sample holder according to the embodiment of Fig. 1 a with tissue samples arranged therein and a detail view of a cross-section of a sample block prepared from this sample holder. It is noted, that the recesses of the sample holder shown in Fig. 2 are closed at the bottom of the recess, which is not shown in the detailed views.

[0094] As can be seen from Fig. 2, the tissue samples 2 have different sizes and shapes. Due to the self-centering effect of the tapered receiving sections 5, each tissue sample 2 is arranged in a way that the centre 15 of the tissue sample 2 substantially lies on the central taper axis 7' of the respective tapered receiving section 5 and therefore in the common plane 8. This position is maintained during embedding of the tissue sample 2. Consequently, each centre 15 of the tissue samples 2 is fixedly arranged in the common plane 8. A cutting plane 9a can be chosen in a way that it is coplanar with the common plane 8.

[0095] It is noted that alternatively a cutting plane 9b can be chosen in a way that it is arranged parallel to and offset from the common plane 8.

[0096] Fig. 3a-3c show a mould according to an embodiment of the invention in different views, comprising a base mould (Fig. 3a), a first lid (Fig. 3b) and a second lid (Fig. 3c). The mould 16 comprises ten first protrusions 17, wherein each of the first protrusions 17 comprises three tapered protrusion sections 18. The first protrusions 17 form part of the first lid 19 shown in Fig. 3b, wherein the first protrusions 17 extend perpendicularly from a first lid plate 20. The first lid 19 can be arranged on the base mould 21. Additionally, the mould 16 comprises a second lid 22 with ten second protrusions 23, wherein the second lid 22 can be arranged on the first lid 19 and therefore indirectly on the base mould 21 to form an assembled state of said mould

[0097] 16. The second lid 22 is shown in Fig. 3c. As can be seen, the second protrusions 23 extend perpendicularly from a second lid plate 24.

[0098] Each tapered protrusion section 18 comprises a taper 6” with a conical shape extending along a central taper axis 7”, wherein the central taper axes 7” of all tapered protrusion sections 18 extend in a common plane 8. The tapers 6” extend along the respective central taper axis 7” with a common orientation. The tapered protrusion sections 18 are arranged in an array 11 ” with five tapered protrusion sections 18 being aligned in a first array dimension and six tapered protrusion sections 18 being aligned in a second array dimension.

[0099] Furthermore, each tapered protrusion section 18 comprises a length 25 extending along a protrusion axis 26 of the tapered protrusion section 18 of 1.8 mm. A crosssection of each tapered protrusion section 18, being perpendicular to the protrusion axis 26, comprises a maximum width 27 of 2.4 mm. The maximum width 27 corresponds to the maximum diameter of the conical shape. Consequently, a ratio of the maximum width 27 to the length 25 is equal to 1.33. For a sample holder 1 prepared with this mould 16, the dimensions of the tapered receiving sections 5 correspond to the dimensions of the tapered protrusion sections 18.

[0100] The first lid 19 additionally comprises five apertures 28 associated with the ten first protrusions 17, wherein, in an assembled state, the second protrusions 23 extend through the apertures 28. In this assembled state, each first and second protrusions

[0101] 17, 23 form a protrusion pair associated with one of the recesses 3 of a sample holder 1 prepared with the mould 16. For each protrusion pair the respective second protrusion 23 is arranged on a side 29 of the first protrusion 17 opposite to a protrusion direction 30 of the tapered protrusion section 18. The base mould 21 shown in Fig. 3a comprises a bottom section 31 , a side wall section 32 and an opening opposite to the bottom section 31 , wherein the bottom section 31 and the side wall section 32 delimit an inner space 33 for receiving a casting material 34. The side wall section 32 forms a restraint element 35 in the form of a contact edge for preventing a lateral displacement of the first lid 19 in multiple lateral directions 36 in an assembled state. These lateral directions 35 comprise a lateral direction 36a corresponding to the protrusion direction 30 of the tapered protrusion sections 18 and both lateral directions 36b, 36c being perpendicular to the protrusion direction 30 of the tapered protrusion sections 18. The first lid 19 comprises a section with a shape corresponding to the restraint element 35. Furthermore, the bottom section 31 comprises a plurality of parallel grooves 37 on a side facing the inner space 33.

[0102] Fig. 4a-4c show a mould according to a further embodiment of the invention in different views, comprising a base mould (Fig. 4a), a first lid (Fig. 4b) and a second lid (Fig. 4c).

[0103] Compared to the mould 16 shown in Fig. 3a-3c, the first lid 19 comprises forty first protrusions 17 with one tapered protrusion section 18 each and five apertures 28. The second lid 22 comprises forty second protrusions 23. Each tapered protrusion section 18 comprises a taper 6" with a conical shape and a length 25 extending along a protrusion axis 26 of the tapered protrusion section 18 of 0.9 mm. A cross-section of each tapered protrusion section 18, being perpendicular to the protrusion axis 26, comprises a maximum width 27 of 1.2 mm. The maximum width 27 corresponds to the maximum diameter of the conical shape. Consequently, a ratio of the maximum width 27 to the length 25 is equal to 1 .33.

[0104] The base mould 21 shown in Fig. 4a comprises a side wall section 32 forming two restraint elements 35 with a contact edge. The restraint elements 35 prevent a lateral displacement of the first lid 19 in a lateral direction 36a corresponding to the protrusion direction 30 of the tapered protrusion sections 18 and in both lateral directions 36b, 36c being perpendicular to the protrusion direction 30 of the tapered protrusion sections 18. The first lid 19 comprises sections with shapes corresponding to the restraint elements 35. Fig. 5 shows a mould comprising a base mould, a first lid and a second lid according to a further embodiment of the invention in an unassembled and an assembled state in a side view.

[0105] Fig. 6a-6c show the mould according to the embodiment of Fig. 5, wherein the first and second lids are shown in an assembled state in a bottom view (Fig. 6a) and wherein a three-dimensional view (Fig. 6b) and a top view (Fig. 6c) of the fully assembled mould are shown.

[0106] The design of the mould 16 shown on Fig. 5 and 6a-6c corresponds to the design of the mould 16 shown in Fig. 4a-4c, with the exception that the first lid 19 comprises twenty first protrusions 17 with one tapered protrusion section 18 each and twenty apertures 28. The second lid 22 comprises twenty second protrusions 23.

[0107] In order to assemble the mould 16, the first lid 19 is placed on the base mould 21 so that an edge 38 of the first lid 19 contacts the two restraint elements 35. Subsequently, the second lid 22 is placed on top of the first lid 19 with the second protrusions 23 being inserted through the apertures 28 of the first lid 19. In the assembled state, the first lid 19 is arranged on the base mould 21 and the second lid 22 is arranged on the first lid 19, wherein the second protrusions 23 extend through the apertures 28 of the first lid 19 adjacent to the first protrusions 17. As is apparent from Fig. 6b, the first and second lids 19, 22 each comprise handling sections 39 extending laterally to facilitate the handling during the assembly or disassembly of the mould 16. The base mould 21 , the first lid 19 and the second lid 22 form an injection port 40 shown in Fig. 6c.

[0108] The first and second protrusions 17, 23 partially extend inside the inner space 33 of the base mould 21. Each first and second protrusions 17, 23 form a protrusion pair associated with one of the recesses 3 of a sample holder 1 prepared with this mould 16. For each protrusion pair the respective second protrusion 23 is arranged on a side 29 of the first protrusion 17 opposite to the protrusion direction 30 of the tapered protrusion sections 18.

[0109] Fig. 7a-7c show base moulds according to three different embodiments of the invention. Each one of the base moulds 21 shown in Fig. 7a-7c comprises a bottom section 31 , a side wall section 32 and an opening opposite to the bottom section 31 , wherein the bottom section 31 and the side wall section 32 delimit an inner space 33 for receiving a casting material 34. Furthermore, the side wall section 32 of each base mould 21 forms a restraint element 35 with a contact edge.

[0110] The bottom section 31 of the base mould 21 shown in Fig. 7b comprises a plurality of parallel grooves 37 on a side facing the inner space 33. The side wall section 32 of the base mould 21 shown in Fig. 7c comprises a circumferential undercut 41 , wherein the circumferential undercut 41 extends laterally at a level of the bottom section 31 .

[0111] Fig. 8a-8d show pairs of first and second lids of moulds according to four further embodiments of the invention in bottom views.

[0112] As can be seen from Fig. 8a, the first lid 19 comprises five apertures 28 and five first protrusions 17, each comprising five tapered protrusion sections 18. The second lid 22 comprises five second protrusions 23 extending through the apertures 28 of the first lid 19. The tapered protrusion sections 18 are arranged in an array 11 " with five tapered protrusion sections 18 being aligned in a first array dimension and five tapered protrusion sections 18 being aligned in a second array dimension.

[0113] The first lid 19 shown in Fig. 8b comprises ten apertures 28 and ten first protrusions 17, each comprising three tapered protrusion sections 18. The second lid 22 comprises ten second protrusions 23 extending through the apertures 28 of the first lid 19. The tapered protrusion sections 18 are arranged in an array 11 " with five tapered protrusion sections 18 being aligned in a first array dimension and six tapered protrusion sections 18 being aligned in a second array dimension.

[0114] The first lid 19 shown in Fig. 8c comprises twenty apertures 28 and twenty first protrusions 17, each comprising one tapered protrusion section 18. The second lid 22 comprises twenty second protrusions 23 extending through the apertures 28 of the first lid 19. The tapered protrusion sections 18 are arranged in an array 11 " with five tapered protrusion sections 18 being aligned in a first array dimension and four tapered protrusion sections 18 being aligned in a second array dimension. In Fig. 8d, a first lid 19 with forty apertures 28 and forty first protrusions 17 is shown, wherein each first protrusion 17 comprises one tapered protrusion section 18. The second lid 22 comprises forty second protrusions 23 extending through the apertures 28 of the first lid 19. Furthermore, the tapered protrusion sections 18 are arranged in an array 11 " with five tapered protrusion sections 18 being aligned in a first array dimension and eight tapered protrusion sections 18 being aligned in a second array dimension.

[0115] Fig. 9 shows a visualization of steps of demoulding a sample holder from the mould according the embodiment of Fig. 5.

[0116] After solidification of a casting material 34 forming a sample holder 1 , the second lid 22 is removed. This step involves pulling 42a the second protrusions 23 out of the recesses 4 of the sample holder 1 and the apertures 28 of the first lid 19. In a next step, the first lid 19 is moved in a direction 43 opposite to the protrusion direction 30 of the tapered protrusion sections 18 and into a space 44 exposed after removing the second lid 22. Subsequently, the first lid 19 is removed, comprising pulling 42b the first protrusions 17 out of the recesses 4 of the sample holder 1. In a last step, the sample holder 1 can be demoulded from the base mould 21 by gravity, inverting it, and, if necessary, additionally using a spatula and / or by bending and pressing the cured hydrogel out of the mould 16 by hand.

[0117] Fig. 10a-b show two different embodiments of a first protrusion of a first lid of the mould according to a further embodiment of the invention in a bottom view (left) and in a 3D-view from the bottom (right),

[0118] The first embodiment 17a (shown right in Fig. 10a-b) and the second embodiment 17b (shown left in Fig. 10a-b) of the first protrusion 17 each comprise a tapered protrusion section 18a, 18b with a rounded tip 53. A radius of the rounded tip 53 substantially corresponds to a radius of the tissue samples 2, which can be arranged in the tapered receiving section 5 formed by the tapered protrusion sections 18a, 18b. The first and second lids 19, 22 are arranged in an assembled state.

[0119] Furthermore, each of the embodiments 17a, 17b of the first protrusion 17 comprises a longitudinal section 54a, 54b (see Fig. 10b). Therein, the tapered protrusion section 18a of the first embodiment 17a is arranged on a lateral side 55 of the respective longitudinal section 54a and the tapered protrusion section 18b of the second embodiment 17b is arranged on a front end 56 of the respective longitudinal section 54b, in other words underneath the respective longitudinal section 54b. It is noted, that each tapered protrusion section 18a, 18b extends laterally with regard to the extension direction of the associated longitudinal section 54a, 54b. Consequently, each tapered receiving section 5 formed by the tapered protrusion sections 18a, 18b extends laterally with regard to the extension direction 4' and longitudinal axis 13 of the associated recess 4.

[0120] Fig. 11 a shows steps of a method for preparing tissue sample for histological analysis according to an embodiment of the present invention.

[0121] Fig. 11 b shows a visualization of steps of the method according to the embodiment of Fig. 11 a.

[0122] Fig. 11 a and 11 b refer to a method for preparing tissue samples for histological analysis. The tissue samples preferably comprise 3D model systems.

[0123] In a first step S1 , a sample holder 1 is provided. The sample holder 1 comprises a plurality of recesses 4, wherein each recess 4 comprises three tapered receiving sections 5 for receiving tissue samples 2 (see Fig. 11 b). The tapered receiving sections 5 each comprise a conical shape and are arranged in an array 11 ' with five tapered receiving sections 5 being aligned in a first array dimension and six tapered receiving sections 5 being aligned in a second array dimension. The first and second array dimensions are perpendicular to each other. Furthermore, the tapered receiving sections 5 are equally oriented. The sample holder 1 is prepared by means of casting using a mould 16, wherein the mould 16 corresponds to the mould 16 shown in Fig. 3a-3c. Therefore, the mould 16 comprises a plurality of tapered protrusion sections 18 corresponding to the tapered receiving sections 5 of the sample holder 1 to be prepared.

[0124] In step S11 the mould is provided, wherein the mould 16 is preferably fabricated by means of 3D-printing with the use of a thermoplastic polymer and is provided in an assembled state. Subsequently, the mould 16 is, in step S12, filled with a casting material 34, preferably by means of gravitational force, wherein the casting material 34 is injected through the injection port 40 of the mould 16. The casting material 34 is a hydrogel, which comprises a polymer network with water as the dispersion medium and solidifies by curing. When filling the hydrogel 34 in the mould 16, the hydrogel 34 comprises a liquid form. For providing this liquid form, the hydrogel 34 may be warmed to a temperature of about 65°C. It is also conceivable, that the hydrogel 34 comprises a liquid form at room temperature and does not have to be warmed beforehand. In step S13, the filled hydrogel 34 is cured, preferably by cooling at a temperature between 4 and 5 °C, in particular for a duration of at least 20 min. After the solidification of the hydrogel 34, the sample holder 1 is partially demoulded in step S14 comprising removing the first and second lids 19, 22, preferably in accordance with the process described with regard to Fig. 9. The sample holder 1 is not demoulded from the base mould 21 .

[0125] In step S2, the tissue samples 2 are placed inside the recesses 4. The tissue samples 2 may be placed inside the recesses 4 manually with the help of a spatula and / or a pipetter or automatically with the help of a pipetting robot. For this step, the sample holder 1 is tilted in a way that the tapered receiving sections 5 are inclined to the vertical at an angle of 135°. This allows the tissue samples 2 to arrange themselves under the influence of gravitational force and the self-centering effect in a way that the centre 15 of each tissue sample 2 substantially lies on the central taper axis 7' of the respective tapered receiving section 5.

[0126] Subsequently, the tissue samples 2 are embedded to form a sample block 45 comprising the sample holder 1 , such that the tissue samples 2 are positioned in the sample block 45 (step S3). This embedding is also achieved by means of casting using a sample block mould 46, wherein the sample holder 1 forms the sample block mould 46. The casting procedure substantially coincides with the procedure, which is used for the preparation of the sample holder 1 described above. The sample block mould 46 is in step S31 filled with a casting material 34, preferably by means of gravitational force, up to a top side 10 of the sample holder 1 , so that the tissue samples 2 are surrounded by the casting material 34 and therefore fully embedded (see Fig. 11 b). The casting material 34 is the same hydrogel, which is used for the preparation of the sample holder 1 . When filling the hydrogel 34 in the sample block mould 46, the hydrogel 34 comprises a liquid form. In step S32, the filled hydrogel 34 is cured by cooling, preferably at a temperature between 4 and 5 °C, in particular for a duration of at least 20 min. After the solidification of the hydrogel 34, the sample block 45 is demoulded in step S33 and dehydrated in step S34.

[0127] In the next step S4, the sample block 45 is attached to a histology cassette 47, wherein the sample block 45 is dehydrated (step S41 ) followed by fixating the dehydrated sample block 45 in the fixing matrix in the form of paraffin (step S42). The sample block 45 may be placed into the histology cassette 47 and dehydrated prior to embedding. The step comprises embedding the sample block 45 in paraffin, wherein standard methods known in the art may be used.

[0128] The sample block 45 is subsequently sectioned (step S5) by means of a microtome 48 to form at least one sample block slice 49 to be processed and analysed. Finally, the at least one sample block slice 49 is stained, preferably by means of haematoxylin and eosin staining and / or multiplex staining (step S6).

[0129] Fig. 12a shows steps of a method for preparing tissue samples for histological analysis according to a further embodiment of the present invention.

[0130] Fig. 12b shows a visualization of steps of the method according to the embodiment of Fig. 12a.

[0131] The steps of the method shown in Fig. 12a and 12b refer to a method, wherein the step of placing the tissue samples 2 inside the recesses 4 forms part of an automated process using a pipetting robot 50.

[0132] In a first step S101 , a pipette tip 51 of the pipetting robot 50 is coated with a coating agent by contacting the pipette tip 51 with a buffer. The buffer comprises the coating agent, wherein the coating agent is a protein, for example 10 % BSA (Bovine Serum Albumin) in a PBS (Phosphate Buffered Saline) solution.

[0133] For aspirating a tissue sample 2 in step S102, which is to be placed inside a recess 4, the pipette tip 51 is positioned at a distance of 0.6 mm to the tissue sample 2. Preferably, a pipette tip 51 with a wide bore may be used. Subsequently, at least 10 pL and a maximum of 15 pL of a solution, in which the tissue sample 2 is provided, is aspirated. The chosen amount of solution aspirated can depend on the size of the tissue sample 2. Next, the aspirated solution comprising the tissue sample 2 is dispensed into a recess 4 of the sample holder 1 (step S103). For this step, the sample holder 1 is tilted in a way that the central taper axes 7' of the tapered receiving sections 5 are inclined to the vertical at an angle 52 of 135°. This process is repeated for all tissue samples 2 to be transferred into the sample holder 1 .

[0134] The sample holder 1 is then tilted in a way that the central taper axes 7' of the tapered receiving sections 5 are inclined to the vertical at an angle 52 of 180° to accelerate the positioning of the tissue samples 2 inside the tapered receiving sections 5 (step S104).

[0135] Subsequently, the tissue samples 2 are embedded to form a sample block 45 comprising the sample holder 1 , such that the tissue samples 2 are positioned in the sample block 45. This embedding is achieved by means of casting, wherein the casting procedure substantially coincides with the procedures described above. The wide bore tip may be exchanged with a narrow bore tip. At the lowest possible speed (1 / 10), 5 pL of solution are aspirated from each recess 4, ensuring the pipette tip 51 goes as low as possible to the bottom of the recess 4 (step S105). In a next step S106, at a lower speed (3 / 10), 2.5 pL of the hydrogel 34 are dispensed to the bottom of each recess 4. After 3 minutes, 10 to 15 pL of hydrogel 34 are dispensed to the bottom of each recess 4 at a lower speed (3 / 10), preferably wherein a wide bore tip is used for this step S107. Subsequently, the sample holder 1 is in step S108 tilted in a way that the central taper axes 7' of the tapered receiving sections 5 are inclined to the vertical at an angle 52 of 135° and placed in the refrigerator at 4-5°C for 10 minutes to allow the hydrogel 34 to fully polymerize.

[0136] Many modifications and other embodiments of the invention set forth herein will come to mind to the one skilled in the art to which the invention pertains having the benefit of the teachings presented in the foregoing description and the associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation. Example

[0137] 1 ) Aim of the Experiment

[0138] The aim of this experiment is to compare the amount of each tissue sample in a single sample block slice that was prepared with a sample holder according to the invention to the amount of each tissue sample in a single sample block slice that was prepared with a sample holder comprising cylindrical recesses.

[0139] 1 .2 Experimental Protocol

[0140] The experimental procedure involved testing two different geometries of the sample holder to assess their performance in providing a high amount of each tissue sample in a single sample block slice. The following steps were likely involved:

[0141] Two sample holder geometries with differently shaped recesses were created, including recesses with a cylindrical shape and a flat bottom (“Control”) as well as recesses according to an embodiment of the invention with a tapered receiving section with a taper extending along a central taper axis, wherein the central taper axis extends in a common plane being parallel to a cutting plane (“3D histomold”). Retinal organoids were placed inside the recesses of the sample holders using a spatula or a pipetter. The sample holders were made of HistoGel®.

[0142] The organoids were embedded with hydrogel to form two sample blocks with one of the sample holders each, such that the organoids were positioned in the sample blocks.

[0143] Subsequently, the sample blocks were sectioned along several cutting planes to form several sample block slices. Each sample block slice was prepared by H&E staining and imaged.

[0144] The obtained images of the sample block slices were analysed to determine the area of each organoid. The values of the organoid areas were normalized using a maximum organoid area among all the sample block slices. This maximum organoid area corresponds approximately with the centre of the organoid.

[0145] The normalized values of the organoid areas of the different sample holders were subsequently compared with each other. 1 .3 Results

[0146] Fig. 13 shows the sample holder “Control” with retinal organoids of different sizes placed in the recesses, a sample block formed with this sample holder after embedding as well as four H&E stained sample block slices obtained from sectioning this sample block at different cutting planes.

[0147] Fig. 14 shows the sample block formed with a sample holder “3D histomold” with retinal organoids of different sizes placed in the recesses after embedding as well as three H&E stained sample block slices obtained from sectioning this sample block at different cutting planes.

[0148] Fig. 15 shows (a) thresholding of an original H&E stained sample block slice, wherein only the outer rim of the organoids is considered to calculate their area, irrespective of inner cavities, (b) values of the organoid areas per sample block slice obtained from the sample block of Fig. 13 (“Control”) and obtained from the sample block of Fig. 14 (“3D histomold”), normalized to the maximum organoid area within those sample block slices, wherein colour gradients between red and green indicate proximity to the respective maximum area, and (c) percentage of organoids in each sample block slice that reach at least 80% of their maximum area.

[0149] The results indicated that the sample holder geometry comprising recesses with a tapered receiving section (“3D histomold”) performed the best compared to the other geometry. Specifically:

[0150] For the sample holder geometry with cylindrically shaped recesses, the best sample block slice (section 1 ) contains a maximum of organoids but misses a significant portion of them. In contrast, for the sample holder geometry comprising recesses with a tapered receiving section, section 3 cuts through all the organoids with a maximized organoid area.

[0151] For the sample holder geometry with cylindrically shaped recesses, the center of the organoids varies between sections 0 and 1 for small organoids and sections 1 -3 for large organoids. In contrast, for the other sample holder geometry, most organoid centers are found in section 2.

[0152] More specifically, under the assumption that any normalized organoid area larger than 80% is valid, 93% of the organoids in the best section for the sample holder geometry comprising recesses with a tapered receiving section correspond to this condition (28 out of 30). For the sample holder geometry with cylindrically shaped recesses, the best section only reaches 67% (16 out of 24).

[0153] In conclusion, the sample holder geometry comprising recesses with a tapered receiving section was identified as the optimal design for maximizing the amount of each tissue sample in a single sample block slice.

[0154] References

[0155] Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P, Leighton S, Torhorst J, Mihatsch MJ, Sauter G and Kallioniemi OP. Tissue microarrays for high throughput molecular profiling of tumor specimens. Nature Medicine. 1998; 4:844-847.

[0156] Packeisen J, Korsching E, Herbst H, Boecker W, Buerger H. Demystified... tissue microarray technology. Mol Pathol. 2003 Aug;56(4): 198-204. doi:

[0157] 10.1136 / mp.56.4.198. PMID: 12890740; PMCID: PMC1187321.

[0158] De Hoogt, R., Estrada, M., Vidic, S. et al. Protocols and characterization data for 2D, 3D, and slice-based tumor models from the PREDECT project. Sci Data 4, 170170 (2017). https: / / doi.Org / 10.1038 / sdata.2017.170.

[0159] Olli-P. Kallioniemi, Urs Wagner, Juha Kononen, Guido Sauter, Tissue microarray technology for high-throughput molecular profiling of cancer, Human Molecular Genetics, Volume 10, Issue 7, 1 April 2001 , Pages 657-662, https: / / doi.Org / 10.1093 / hmg / 10.7.657.

[0160] Heub, S., Navaee, F., Migliozzi, D. et al. Coplanar embedding of multiple 3D cell models in hydrogel towards high-throughput micro-histology. Sci Rep 12, 9991 (2022). https: / / doi.Org / 10.1038 / s41598-022-13987-4.

[0161] Gabriel, J., Brennan, D., Elisseeff, J.H. et al. Microarray Embedding / Sectioning for Parallel Analysis of 3D Cell Spheroids. Sci Rep 9, 16287 (2019). https: / / doi.Org / 10.1038 / s41598-019-52007-w.

[0162] Kabadi, Pranita K., et al. "Into the depths: Techniques for in vitro three-dimensional microtissue visualization." Biotechniques 59.5 (2015): 279-286.

[0163] Weisler, W., Miller, S., Jernigan, S. et al. Design and testing of a centrifugal fluidic device for populating microarrays of spheroid cancer cell cultures. J Biol Eng 14, 7 (2020). https: / / doi.Org / 10.1186 / s13036-020-0228-6.

Claims

- 35 -C l a i m s1. Sample holder (1 ) for preparing tissue samples (2), preferably 3D model systems, for histological analysis, comprising a base body (3) with at least one recess(4), preferably a plurality of recesses (4), for receiving a tissue sample (2), characterized in that each of said at least one recess (4) comprises at least one tapered receiving section (5) with a taper (6') extending along a central taper axis (7'), wherein each of said at least one tapered receiving section (5) extends laterally, preferably from the associated recess (4), with regard to an extension direction (4') of the associated recess (4), wherein said central taper axis (7') extends in a plane (8), preferably a common plane (8) for a plurality of tapered receiving sections (5), preferably being at least essentially parallel to a cutting plane (9, 9a, 9b).

2. Sample holder (1 ) according to claim 1 , characterized in that said taper (6') comprises a conical, a pyramidal, a hemispherical or a hemiellipsoidal shape or a segmented shape thereof and / or wherein said taper (6') of each of said at least one tapered receiving section(5) extends along said respective central taper axis (7') with a common orientation and / or wherein each of said at least one recess (4) comprises a plurality of tapered receiving sections (5), preferably three tapered receiving sections (5) and / or wherein a plurality of tapered receiving sections (5) is provided, wherein said tapered receiving sections (5) are arranged in an array (11 ') with at least two tapered receiving sections (5) being aligned in a first array dimension and / or at least two tapered receiving sections (5) being aligned in a second array dimension.

3. Sample holder (1 ) according to claim 1 or 2, characterized in that each of said at least one tapered receiving section (5) comprises a cross-section, being perpendicular to its central taper axis (7'), with a maximum width and a depth extending along its central taper axis (7'), wherein a ratio of said maximum width to said depth is at least 0.1 and less than or equal to 10, preferably at least 0.5 and less than or equal to 3, more preferably equal to 1 .33, and / or wherein each of said at least one tapered receiving section (5) comprises a cross-section, being perpendicular to its central taper axis (7'), with a maximum width- 36 - of at least 0.5 mm and less than or equal to 5 mm, preferably at least 2 mm and less than or equal to 3 mm, more preferably of 1 .2 mm or 2.4 mm, and / or wherein each of said at least one tapered receiving section (5) comprises a depth extending along its central taper axis (7') of at least 0.5 mm and less than or equal to 5 mm, preferably at least 1 mm and less than or equal to 2.5 mm, more preferably of 0.9 mm or 1 .8 mm.

4. Sample holder (1 ) according to any one of claims 1 to 3, characterized in that each of said at least one recess (4) comprises a first section (12) extending along a longitudinal axis (13), preferably being aligned with said extension direction (4') of said at least one recess (4), wherein the associated tapered receiving section (5) extends laterally from said first section (12) with regard to said longitudinal axis (13), and / or wherein said longitudinal axis (13) and / or said extension direction (4') of each of said at least one recess (4) is inclined to said central taper axis (7') at an angle (14a) of at least 90° and less than or equal to 150°, preferably at an angle (14a) of 90°.

5. Sample holder (1 ) according to any one of claims 1 to 4, characterized in that said base body (3) comprises a top side (10), from which said at least one recess (4) extends, wherein said top side (10) is parallel to said at least one central taper axis (7'), preferably wherein said longitudinal axis (13) is inclined to said top side (10) at an angle (14b) of at least 30° and less than or equal to 90°, preferably at an angle (14b) of 90°, and / or wherein a material of said sample holder (1 ) comprises a curable gel (34), preferably a hydrogel, and / or a polymer.

6. Mould (16) for preparing a sample holder (1 ) according to any one of claims 1 to 5, comprising a base mould (21 ) with a bottom section (31 ), a side wall section (32) and an opening opposite to said bottom section (31 ), wherein said bottom section (31 ) and said side wall section (32) delimit an inner space (33) for receiving a casting material (34), and at least one first protrusion (17), preferably a plurality of first protrusions (17), each comprising at least one tapered protrusion section (18) corresponding to said at least one tapered receiving section (5) of said sample holder (1 ).

7. Mould (16) according to claim 6, characterized in that said at least one first protrusion (17) forms an integral part of said base mould (21 ) and / or forms part of a separate element of said mould (16), preferably wherein said separate element is a first lid (19) which can be arranged on said base mould (21 ) to form an assembled state of said mould (16), preferably wherein said mould (16) comprises a second lid (22) with at least one second protrusion (23), preferably a plurality of second protrusions (23), wherein said second lid (22) can be arranged on said first lid (19) and / or on said base mould (21 ) to form an assembled state of said mould (16), wherein, in said assembled state, each first and second protrusions (17, 23) form a protrusion pair associated with one of said at least one recess (4) of said sample holder (1 ), wherein for said protrusion pair said second protrusion (23) is arranged on a side of said first protrusion (17) opposite to a protrusion direction (30) of said at least one tapered protrusion section (18), preferably wherein said first lid (19) comprises at least one aperture (28), preferably a plurality of apertures (28), associated with said at least one first protrusion (17), wherein, in an assembled state, said at least one second protrusion (23) extends through said at least one aperture (28).

8. Mould (16) according to claim 6 or 7, characterized in that said base mould (21 ) and / or said first lid (19) and / or said second lid (22) form an injection port (40) and / or wherein said first lid (19) comprises at least one stiffening element, for example a wall, arranged between at least two of said tapered protrusion sections (18) and / or wherein said side wall section (32) forms at least one restraint element (35), for example at least one contact edge, for preventing a lateral displacement of said first and / or second lid (19, 22) in at least one lateral direction (36, 36a, 36b, 36c) in an assembled state, preferably wherein said at least one lateral direction (36, 36a, 36b, 36c) comprises a lateral direction (36a) corresponding to a protrusion direction (30) of said at least one tapered protrusion section (18) and / or one or both lateral directions (36b, 36c) being perpendicular to said protrusion direction (30) of said at least one tapered protrusion section (18).

9. Mould (16) according to any one of claims 6 to 8, characterized in that said bottom section (31 ) and / or said side wall section (32) comprises a plurality of grooves (37), preferably parallel grooves (37), on a side facing said inner space (33) and / or wherein said side wall section (32) comprises at least one undercut (41 ), preferably a circumferential undercut (41 ), preferably wherein said at least one undercut (41 ) extends laterally at a level of said bottom section (31 ), and / or wherein said base mould (21 ) and / or said first lid (19) and / or said second lid (22) is fabricated by means of additive manufacturing, preferably by means of 3D- printing, and / or wherein said base mould (21 ) and / or said first lid (19) and / or said second lid (22) comprises a hydrophobic material.

10. Method for preparing a sample holder (1 ) with a mould (16) according to any one of claims 6 to 9, comprising the steps of:Providing (S11 ) said mould (16), filling (S12) a casting material (34) inside said mould (16), preferably by means of gravitational force, solidifying (S13) said casting material (34), and at least partially demoulding (S14) said sample holder (1 ), preferably comprising removing a first and / or a second lid (19, 22) of said mould (16).

11. Method according to claim 10, characterized in that said at least partially demoulding (S14) of said sample holder (1 ) comprises the steps of:Removing said second lid (22), preferably comprising pulling (42a) said at least one second protrusion (23) out of said at least one recess (4) of said sample holder (1 ) and said at least one aperture (28), moving said first lid (19) in a direction (43) opposite to a protrusion direction (30) of said at least one tapered protrusion section (18), preferably into a space (44) exposed after removing said second lid (22), and removing said first lid (19), preferably comprising pulling (42b) said at least one first protrusion (17) out of said at least one recess (4) of said sample holder (1 ).

12. Method for preparing tissue samples (2), preferably 3D model systems, for histological analysis with a sample holder (1 ) according to any one of claims 1 to 5, comprising the steps of:- 39 -Providing (S1 ) said sample holder (1 ), placing (S2) at least one tissue sample (2) inside said at least one recess (4), preferably using a spatula and / or a pipetter, embedding (S3) said at least one tissue sample (2) to form a sample block (45) comprising said sample holder (1 ), such that said at least one tissue sample (2) is positioned in said sample block (45), and sectioning (S5) said sample block (45) along said cutting plane (9) to form at least one sample block slice (49) to be analysed, preferably wherein said sample holder (1 ) is prepared by means of casting (S11 , S12, S13, S14), preferably using a method according to any one of claims 24 to 26.

13. Method according to claim 12, characterized in that said at least one tissue sample (2) is embedded (S3) by means of casting using a sample block mould (46), wherein said sample holder (1 ) at least partially forms said sample block mould (46), preferably wherein said sample block mould (46) comprises a surrounding enclosure, preferably wherein an upper edge of said surrounding enclosure extends beyond said at least one tissue sample (2), preferably wherein said surrounding enclosure is integrally formed with said sample holder (1 ) or is formed by a separate part.

14. Method according to claim 12 or 13, characterized in that said casting using said sample block mould (46) comprises the steps of:Filling (S31 ) a casting material (34) inside said sample block mould (46), preferably by means of gravitational force, solidifying (S32) said casting material (34), and demoulding (S33) said sample block (45), and / or wherein said sample holder (1 ) is arranged in a way that said central taper axis (7') of said at least one tapered receiving section(5) is inclined to the vertical at an angle (52) of at least 90° and less than or equal to 180°, preferably at an angle (52) of at least 120° and less than or equal to 180°, more preferably at an angle (52) of 135°, in particular during placing (S2) said at least one tissue sample (2) inside said at least one recess (4) and / or during embedding (S3) said at least one tissue sample (2), and / or wherein said sample holder (1 ) is arranged in a way that said longitudinal axis (13) of said at least one recess (4) is inclined to the vertical at an angle of at least 0°and less than or equal to 45°, preferably at an angle of 30°, in particular during placing (S2) said at least one tissue sample (2) inside said at least one recess (4) and / or during embedding (S3) said at least one tissue sample (2).

15. Method according to any one of claims 12 to 14, characterized in that, prior to sectioning (S5), said sample block (45) is attached (S4) to a histology cassette (47) using a fixing matrix, preferably wherein said fixing matrix comprises a paraffin and / or a resin, and / or wherein said sectioning (S5) is performed by means of a microtome (48) and / or wherein said at least one sample block slice (49) is stained (S6), preferably by means of haematoxylin and eosin staining and / or multiplex staining, and / or wherein at least one of said steps (S1 , S11 , S12, S13, S14, S2, S3, S31 , S32, S33, S34, S4, S41 , S42, S5, S6) forms part of an automated process, preferably wherein said placing (S2) and / or embedding (S3) of said at least one tissue sample (2) is performed using a pipetting robot (50).

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