Method, sample holder and mould for preparing tissue samples for histological analysis

Abstract

A method for preparing tissue samples (1) for histological analysis, preferably wherein said tissue samples (1) comprise a two-dimensional form, comprises the steps of: - Preparing (S1) a sample holder (2), wherein said sample holder (2) comprises at least one slit-shaped recess (3), preferably a plurality of slit-shaped recesses (3), for receiving at least one tissue sample (1), - placing (S2) said at least one tissue sample (1) at least partially inside said at least one slit-shaped recess (3), - embedding (S3) said at least one tissue sample (1) to form a sample block (8) comprising said sample holder (2), such that said at least one tissue sample (1) is fixed in said sample block (8), and - sectioning (S5) said sample block (8) to form at least one sample block slice (14) to be analysed. Furthermore, the invention relates to a sample holder (2) and to a mould (5) for a sample holder (2).

Description

[0001] Method, sample holder and mould for preparing tissue samples for histological analysis

[0002] The present invention relates to a method for preparing tissue samples for histological analysis.

[0003] Further, the present invention relates to a sample holder to be used in a method for preparing tissue samples for histological analysis as well as to a mould for preparing such a sample holder.

[0004] The histological analysis of fixed tissues typically involves embedding the tissues 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). However, 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 an array (see, for example, Scientific Reports, 2022, 12:9991 , and Scientific Reports, 2019, 9:16287).

[0006] From US 11 ,320,349 B2 a spheroid tissue microarray comprising an array of tissue spheroids embedded within a porous mould is known. The product may be impregnated with a wax or resin and sectioned, and contains spheroids which are precisely located in a regular geometric grid. Furthermore, a method of manufacturing a spheroid tissue microarray is disclosed.

[0007] Similar methods for embedding tissue samples are described in

[0008] - Journal of Mammary Gland Biology and Neoplasia, 2022, 25:417-432;

[0009] - STAR Protocols 2023, 4: 102521 ;

[0010] - Scientific Data, 2017, 4:170170;

[0011] - Journal of Clinical and Translational Pathology, 2021 , 1 (1 ):28-31 ; and

[0012] - International Journal of Molecular Science, 2023, 24(18): 13903.

[0013] The known methods however, are not suitable for embedding planar tissue samples, for example a membrane from a cell culture insert (e.g. transwell) containing a cell monolayer or tissue extracted from a thin biomaterial using a puncher. When embedding such planar tissue samples in cylindrical mini wells, each tissue sample might be arranged and / or oriented differently therein and / or possibly even folded. Therefore, a slice of a sectioned sample block of such planar tissue samples might not comprise a cross-section of each tissue sample and / or comprise cross-sections of tissue sample, which are not suitable for histological analyses.

[0014] For histologically analysing such planar tissue samples, currently each sample has to be individually embedded, wherein a user holds the tissue sample vertically inside a metallic cassette using tweezers and fills its surrounding with paraffin while waiting for it to solidify. This procedure is challenging and can therefore only be performed by specially trained and well-experienced staff. Furthermore, this procedure is very time intensive and therefore very expensive, especially as downstream processing (e.g. sectioning, imaging and analysis) must be performed per sample. One of the objectives of the present invention is to provide a method, a mould and a sample holder for preparing tissue samples for histological analysis in a labourefficient manner and with easy means.

[0015] In an embodiment, the present invention may solve the objective by a method for preparing tissue samples for histological analysis, preferably wherein said tissue samples comprise a two-dimensional form, comprising the steps of:

[0016] Preparing a sample holder, wherein said sample holder comprises at least one slit-shaped recess, preferably a plurality of slit-shaped recesses, for receiving at least one tissue sample, placing said at least one tissue sample at least partially inside said at least one slit-shaped recess, 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 fixed in said sample block, and sectioning said sample block to form at least one sample block slice to be analysed.

[0017] In an embodiment, the present invention may solve the objective by a mould for preparing a sample holder to be used in a method for preparing tissue samples for histological analysis, preferably in a method according to any one of claims 1 to 8, wherein said sample holder comprises at least one recess, preferably a plurality of recesses, for receiving at least one tissue sample, wherein said mould comprises a base section, a side wall section and an opening opposite to said base section, wherein said base section and said side wall section delimit an inner space for receiving a casting material, wherein said base section and / or said side wall section comprise at least one protrusion, preferably a plurality of protrusions, corresponding to said at least one recess.

[0018] In an embodiment, the present invention may solve the objective by a sample holder to be used in a method for preparing tissue samples for histological analysis, preferably in a method according to any one of claims 1 to 8, wherein said sample holder comprises at least one recess, preferably a plurality of recesses, for receiving at least one tissue sample, wherein said sample holder is prepared with a mould according to any one of claims 9 to 14.

[0019] Said at least one slit-shaped recess substantially corresponds at least partially to said two-dimensional form of said at least one tissue sample. Therefore, said at least one two-dimensional tissue sample can be effectively immobilized by positioning said sample inside said at least one slit-shaped recess. Each tissue sample, placed in such a slit-shaped recess, is arranged in said sample holder with a position and an orientation corresponding to the respective slit-shaped recess. Therefore, the user can configure said method, in particular said at least one slit-shaped recess of said sample holder and / or said at least one protrusion of said mould, in a way that said sample block slice comprises the desired cross-section of said at least one tissue sample to be analysed. Said at least one tissue sample may be placed inside said at least one slit-shaped recess manually, for example with the help of tweezers, or automatically, for example with the help of a robotic device. 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. Therefore, comparability between the analysed samples may be ensured.

[0020] In addition, a high-throughput embedding with a significant reduction of labour- intensive and tedious manual procedures may be achieved. This may facilitate a high tissue sample density per sample block, decreasing reagent expenditure.

[0021] The tissue samples prepared according to the method of the present invention may subsequently be analysed comprising imaging of said 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. With regard to the mould, it has been recognized that the preparation of said 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 said mould. Said mould may also be reusable.

[0022] 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 in particular comprise organoids, being in vitro derived 3D cell aggregates derived from primary tissue or stem cells, and / or organoid based 2D cell cultures. Conceivable tissue samples might comprise 2D monolayer cultures, in particular 2D epithelial monolayer cultures, for example a membrane from a cell culture insert containing a cell monolayer, and / or 3D monolayer cultures, in particular 3D epithelial monolayer cultures, extracted from a thin hydrogel I 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 I matrix (e.g. with imprinted topography).

[0023] The term "two-dimensional form" is to be understood in the broadest sense and refers, in particular in the claims, preferably in the description, to a form, in particular of a tissue sample, with extensions in a first and a second dimension as well as a thickness extension, wherein a ratio of a thickness value, associated with the thickness extension, to values of the extensions in the first and the second dimension is less than or equal to 0.5, preferably less than or equal to 0.35. The two-dimensional form may be flat or comprise a curvature. Conceivable tissue samples with said two- dimensional form might be organoid based 2D cell cultures and / or 2D monolayer cultures, in particular 2D epithelial monolayer cultures, for example a membrane from a cell culture insert containing a cell monolayer. Further features, advantages and preferred embodiments are disclosed or may become apparent in the following.

[0024] According to a preferred embodiment of the invention, said at least one slit-shaped recess comprises a rectangular disc shape and / or, preferably, a curved disc shape. This may have the advantage that tissue samples with a form corresponding to said rectangular disc shape or said curved disc shape, are particularly well immobilized when positioned in said sample holder. Especially, during embedding a high resistance against loosening of said at least one tissue sample may be ensured. Forms corresponding to said rectangular disc shape or said curved disc shape are particularly common for tissue samples extracted from cell culture inserts or from biomaterial using a puncher. Said recess with said curved disc shape is particularly well suited for embedding tissue sample with a round disc shape, for example tissue samples extracted with a puncher. Said recess with said rectangular disc shape is particularly well suited for embedding tissue samples with a round disc shape that have been cut in half. Said curved disc shape may comprise a half round disc shape and / or a shape of a segment of a round disc.

[0025] According to a further preferred embodiment of the invention, said at least one slitshaped recess comprises a cross-section, being perpendicular to an axis of a depth extension of said at least one slit-shaped recess, with a width and a length, wherein a ratio of said width to said length is less than or equal to 0.5, preferably less than or equal to 0.35. Said at least one slit-shaped recess, which is dimensioned in this way, may effectively prevent said at least one tissue sample from folding and / or minimize tilting of said at least one tissue sample.

[0026] According to a further preferred embodiment of the invention, said at least one slitshaped recess comprises a cross-section, being perpendicular to an axis of a depth extension of said at least one slit-shaped recess, with a width of at least 20 pm and a maximum of 3 mm and / or a length of at least 200 pm and a maximum of 15 mm. Such dimensions are suitable for accommodating a wide range of tissue samples with said two-dimensional form that are commonly histologically analysed for diagnostic and research purposes.

[0027] According to a further preferred embodiment of the invention, said at least one slitshaped recess comprises a depth of at least a third, preferably half, of said length of said cross-section. This way, said at least one tissue sample may be effectively immobilized in said at least one slit-shaped recess.

[0028] According to a further preferred embodiment of the invention, said at least one slitshaped recess extends along at least one recess plane and said sample holder comprises a substantially planar bottom surface, wherein said bottom surface and said at least one recess plane are inclined to each other at an angle of 90° and / or of less than 90°, preferably 60°. At an angle of less than 90°, the cross-section of said at least one tissue sample in said at least one sample block slide may be larger than at an angle above 90° and therefore improve the quality of the histological analysis. Furthermore, in case of tissue samples, which comprise a stamped surface to make the surface more geometrically complex, e.g. with elevations and depressions, an angle of less than 90°, preferably 60°, may be advantageous with regard to the informative content of the results of the histological analysis.

[0029] According to a further preferred embodiment of the invention, said sample holder comprises a plurality of slit-shaped recesses, arranged in an array with at least two slit-shaped recesses being aligned in a first array dimension and / or at least two slitshaped recesses being aligned in a second array dimension. This way, multiple tissue samples can be analysed at the same time, improving the efficiency of the method. When arranged in said array, said slit-shaped recesses may be equidistantly spaced apart from each other and preferably equally oriented. Additionally or alternatively, said first and second array dimension may be perpendicular to each other.

[0030] According to a further preferred embodiment of the invention, said sample holder is prepared by means of casting using a sample holder mould. Casting simplifies the preparation of said sample holder, 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 casting.

[0031] According to a further preferred embodiment of the invention, said sample holder mould is fabricated by means of additive manufacturing, preferably by means of 3D- printing. Said sample holder mould may thus be easily manufactured with a high design complexity. Furthermore, additive manufacturing enables the production of custom-made sample holders in a short period of time.

[0032] According to a further preferred 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 said sample holder, at least partially, as a mould said at least one tissue sample may be easily and time-effectively embedded without having to install a complex embedding arrangement.

[0033] According to a further preferred 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 said at least one tissue sample may be achieved when filling said sample block mould with casting material until it reaches said upper edge of said surrounding enclosure. This may result in a sample block with a flat top surface, facilitating the handling and further processing of said sample block.

[0034] According to a further preferred 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 said surrounding enclosure said sample block mould may be easily customized depending on various boundary conditions, like special geometric dimensions of said at least one tissue sample. According to a further preferred embodiment of the invention, said casting by means of said sample holder mould and / or said sample block mould comprises the steps of: Filling a casting material inside the respective mould, preferably by means of gravitational force, solidifying said casting material, and demoulding said sample holder and / or said sample block.

[0035] These simple steps may be performed without the need of special equipment and provide a low risk for an incorrect execution by the user. Preferably, for casting said sample holder and / or said sample block the same casting material is used for ensuring sufficient bonding and therefore a high strength of said sample block. 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.

[0036] According to a further preferred embodiment of the invention, said casting material is a curable gel, preferably a hydrogel, wherein said gel solidifies by curing. Said curing may comprise gelation and / or polymerisation of said gel. Before curing, said 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 said gel in a liquid form into said sample holder mould and / or said sample block mould, it may be ensured that all cavities are filled and a forming of air pockets may be prevented. After solidification of said gel, said at least one tissue sample is fixed in said sample block. Said curable gel may comprise a polymer network with a dispersion medium. Said curable hydrogel may comprise a polymer network with water as the dispersion medium. Additionally or alternatively, said curable gel may comprise agarose. The cured gel may be flexible and therefore facilitate demoulding of said cured gel.

[0037] According to a further preferred embodiment of the invention, said curing is achieved by cooling said gel at a temperature between 0 °C and 10 °C, preferably between 4 and 5 °C, in particular for a duration of at least 20 min. It is pointed out that the skilled person can easily define the curing duration for a particular material.

[0038] According to a further preferred embodiment of the invention, said sample block is dehydrated, preferably wherein prior to dehydrating said sample block is transferred into a formalin bath, preferably for 4 hours. Dehydrating facilitates the subsequent embedding of said sample block in paraffin and the sectioning of said sample block. Dehydrating may be performed using a vacuum filter processor.

[0039] According to a further preferred embodiment of the invention, 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. Said histology cassette may facilitate the handling of said sample block for further steps of the histological analysis. Attaching said sample block may comprise the following steps. Said sample block may be distributed into said histology cassette and dehydrated. Subsequently, said sample block may be embedded in liquid paraffin in a metallic mould and capped with the said histology cassette. After polymerization of the paraffin, preferably at -20 °C, the metallic mould may be removed and said sample block with the attached histology cassette may be stored at -20 °C until sectioning.

[0040] According to a further preferred embodiment of the invention, said sectioning is performed by means of a microtome. With said microtome extremely thin slices of said sample block may be produced for observation under transmitted light or laser light. In particular, brightfield and / or confocal imaging may be used for analysing the slices of said sample block. A thickness of said slices may range from 3 pm to 15 pm. According to a further preferred embodiment of the invention, 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.

[0041] According to a further preferred embodiment of the invention, said mould comprises a polymer, preferably a thermoplastic polymer, and / or a metal, preferably aluminium. A mould comprising a polymer is easy to manufacture. In particular, a thermoplastic polymer may be used for additive manufacturing of said mould. A mould comprising a metal shows high strength, which may be beneficial when said at least one protrusion comprises a particularly filigree form.

[0042] According to a further preferred embodiment of the invention, an upper edge of said side wall section extends beyond said at least one protrusion. Hence, a bottom section of said sample holder with a flat bottom surface may be formed, when filling said sample holder mould with casting material up to said upper edge of said side wall section. This may facilitate the handling and further processing of said sample holder prepared with said sample holder mould.

[0043] According to a further preferred embodiment of the invention, said side wall section comprises an inwardly facing surface narrowing from said opening to said base section, preferably wherein said inwardly facing surface is inclined at an angle of less than or equal to 85°, preferably less than or equal to 80°, relative to said base section. One advantage of such an inwardly facing surface is that said sample holder may be easily demoulded. According to a further preferred embodiment of the invention, said base section comprises a surrounding recess surrounding said at least one protrusion, preferably wherein said surrounding recess and said side wall section merge into each other. Due to this surrounding recess, said sample holder casted with said mould comprises a surrounding wall portion forming the surrounding enclosure, which facilitates the embedding of said tissue samples. In other words, said surrounding recess of said sample holder mould corresponds to the form of said surrounding enclosure of said sample holder.

[0044] According to a further preferred embodiment of the invention, said at least one protrusion corresponds to a slit-shaped recess and / or comprises a rectangular disc shape and / or, preferably, a curved disc shape. With a slit-shaped recess, a tissue sample comprising a two-dimensional form may be effectively immobilized. Each tissue sample, placed in a slit-shaped recess corresponding to such a protrusion, is arranged in said sample holder with a position and an orientation corresponding to the respective slit-shaped recess. Said rectangular disc shape and / or a curved disc shape may have the advantage that tissue samples with a form corresponding to said rectangular disc shape or said curved disc shape, are particularly well immobilized when positioned in said sample holder. Especially, during embedding a high resistance against loosening of said at least one tissue sample may be ensured. Forms corresponding to said rectangular disc shape or said curved disc shape are particularly common for tissue samples extracted from cell culture inserts or from biomaterial using a puncher. Said curved disc shape may comprise a half round disc shape and / or a shape of a segment of a round disc.

[0045] According to a further preferred embodiment of the invention, said at least one protrusion comprises a cross-section, being perpendicular to a protrusion axis of said at least one protrusion, with a width and a length, wherein a ratio of said width to said length is less than or equal to 0.5, preferably less than or equal to 0.35. A slit-shaped recess corresponding to a protrusion dimensioned in such a way may effectively prevent said at least one tissue sample from folding and / or minimize tilting of said at least one tissue sample.

[0046] According to a further preferred embodiment of the invention, said at least one protrusion comprises a cross-section, being perpendicular to a protrusion axis of said at least one protrusion, with a width of at least 20 pm and a maximum of 3 mm and / or a length of at least 200 pm and a maximum of 15 mm. The dimensions of a slit-shaped recess corresponding to a protrusion dimensioned in such a way are suitable for accommodating a wide range of tissue samples with said two-dimensional form that are commonly histologically analysed for diagnostic and research purposes.

[0047] According to a further preferred embodiment of the invention, said at least one protrusion comprises a protrusion depth of at least a third, preferably half, of said length of said cross-section. This way, said at least one tissue sample may be effectively immobilized in said at least one slit-shaped recess corresponding to said at least one protrusion.

[0048] According to a further preferred embodiment of the invention, said at least one protrusion extends along a protrusion axis and said base section comprises a substantially planar bottom surface, wherein said bottom surface and said protrusion axis are inclined to each other at an angle of 90° and / or of less than 90°, preferably 60°. At an angle of less than 90°, the cross-section of a tissue sample in a sample block slide prepared by using said sample holder mould may be larger than at an angle of 90° and therefore improve the quality of the histological analysis. Furthermore, in case of tissue samples, which comprise a stamped surface to make the surface more geometrically complex, e.g. with elevations and depressions, an angle of less than 90°, preferably 60°, may be advantageous with regard to the informative content of the results of the histological analysis.

[0049] According to a further preferred embodiment of the invention, said mould comprises a plurality of protrusions, wherein said protrusions are arranged in an array with at least two protrusions aligned in a first array dimension and / or at least two protrusions aligned in a second array dimension. This way, multiple tissue samples can be analysed at the same time, improving the efficiency of the method. When arranged in said array, said protrusions may be equidistantly spaced apart from each other and preferably equally oriented. Additionally or alternatively, said first and second array dimension may be perpendicular to each other.

[0050] The following preferred embodiments in particular refer to the sample holder according to claim 15.

[0051] According to a preferred embodiment of the invention, said at least one recess comprises a rectangular disc shape and / or, preferably, a curved disc shape.

[0052] According to a further preferred embodiment of the invention, said at least one recess has the form of a slit-shaped recess, wherein said at least one slit-shaped recess comprises a cross-section, being perpendicular to an axis of a depth extension of said at least one slit-shaped recess, with a width and a length, wherein a ratio of said width to said length is less than or equal to 0.5, preferably less than or equal to 0.35.

[0053] According to a further preferred embodiment of the invention, said at least one recess has the form of a slit-shaped recess, wherein said at least one slit-shaped recess comprises a cross-section, being perpendicular to an axis of a depth extension of said at least one slit-shaped recess, with a width of at least 20 pm and a maximum of 3 mm and / or a length of at least 200 pm and a maximum of 15 mm.

[0054] According to a further preferred embodiment of the invention, said at least one slitshaped recess comprises a depth of at least a third, preferably half, of said length of said cross-section.

[0055] According to a further preferred embodiment of the invention, said at least one recess extends along at least one recess plane and said sample holder comprises a substantially planar bottom surface, wherein said bottom surface and said at least one recess plane are inclined to each other at an angle of 90° and / or of less than 90°, preferably 60°.

[0056] According to a further preferred embodiment of the invention, said sample holder comprises a plurality of recesses, arranged in an array with at least two recesses being aligned in a first array dimension and / or at least two recesses being aligned in a second array dimension. When arranged in said array, said recesses may be equidistantly spaced apart from each other and preferably equally oriented. Additionally or alternatively, said first and second array dimension may be perpendicular to each other.

[0057] 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 patent claims 1 and 9 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.

[0058] In the drawing

[0059] Fig. 1 a shows steps of a method according to an embodiment of the present invention,

[0060] Fig. 1 b shows a visualization of steps of the method according to the embodiment of Fig. 1 a,

[0061] Fig. 2a shows a mould according to an embodiment of the invention in a three- dimensional view, Fig. 2b shows the mould according to the embodiment of Fig. 2a in a cross- sectional view,

[0062] Fig. 2c shows the mould according to the embodiment of Fig. 2a in a further three-dimensional view,

[0063] Fig. 3a shows a mould according to a further embodiment of the invention in a three-dimensional view,

[0064] Fig. 3b shows the mould according to the embodiment of Fig. 3a in a cross- sectional view,

[0065] Fig. 3c shows the mould according to the embodiment of Fig. 3a in a further three-dimensional view,

[0066] Fig. 4a shows a mould according to a further embodiment of the invention in a three-dimensional view,

[0067] Fig. 4b shows the mould according to the embodiment of Fig. 4a in a cross- sectional view,

[0068] Fig. 4c shows the mould according to the embodiment of Fig. 4a in a further three-dimensional view, and

[0069] Fig. 5 shows a sample holder according to an embodiment of the present invention in a three-dimensional view.

[0070] Figure 1a shows steps of a method according to an embodiment of the present invention. Steps of the method according to the embodiment of Figure 1 a are visualized in Figure 1 b.

[0071] Figures 1 a and 1 b refer to a method for preparing tissue samples 1 for histological analysis. Said tissue samples 1 comprise a two-dimensional form with a round disc shape and, in this example, are membranes from cell culture inserts 1 a containing a cell monolayer.

[0072] In a first step S1 , a sample holder 2 is prepared. Said sample holder 2 comprises a plurality of slit-shaped recesses 3 for receiving tissue samples 1 (see Figure 1 b). Said slit-shaped recesses 3 each comprise a rectangular disc shape and are arranged in an array 4a with three slit-shaped recesses 3 being aligned in a first array dimension and five slit-shaped recesses 3 being aligned in a second array dimension. Said first and second array dimension are perpendicular to each other. Furthermore, said slitshaped recesses 3 are equidistantly spaced apart from each other and equally oriented. Said sample holder 2 is prepared by means of casting using a sample holder mould 5. Therefore, said sample holder mould 5 comprises a plurality of protrusions 6 corresponding to said slit-shaped recesses 3 of said sample holder 2 to be prepared.

[0073] Said sample holder mould 5 is preferably fabricated (step S11 ) by means of 3D- printing with the use of a thermoplastic polymer. Subsequently, said sample holder mould 5 is, in step S12, filled with a casting material 7, preferably by means of gravitational force. The casting material 7 is a hydrogel, which comprises a polymer network with water as the dispersion medium and solidifies by curing. When filling the hydrogel 7 in said sample holder mould 5, the hydrogel 7 comprises a liquid form. For providing this liquid form, the hydrogel 7 may be warmed to a temperature of about 65°C. It is also conceivable, that the hydrogel 7 comprises a liquid form at room temperature and does not have to be warmed beforehand. In step S13, the filled hydrogel 7 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 7, said sample holder 2 is demoulded in step S14 by gravity, inverting it, and, if necessary, additionally using a spatula and / or by bending and pressing the cured hydrogel 7 out of the sample holder mould 5 by hand.

[0074] In step S2, said tissue samples 1 are partially placed inside said slit-shaped recess 3, wherein about half of each tissue sample 1 extends beyond said slit-shaped recess 3. Said tissue samples 1 may be placed inside said slit-shaped recesses 3 manually with the help of tweezers or automatically with the help of a robotic device.

[0075] Subsequently, said tissue samples 1 are embedded to form a sample block 8 comprising said sample holder 2, such that said tissue samples 1 are fixed in said sample block 8 (step S3). This embedding is also achieved by means of casting using a sample block mould 9, wherein said sample holder 2 forms said sample block mould 9. As illustrated in Figure 1 b, said sample block mould 9 comprises a surrounding enclosure 10, wherein an upper edge 11 of said surrounding enclosure 10 extends beyond said tissue samples 1 . Said surrounding enclosure 10 is integrally formed with said sample holder 2. The casting procedure substantially coincides with the procedure, which is used for the preparation of said sample holder 2 described above. Said sample block mould 9 is in step S31 filled with a casting material 7, preferably by means of gravitational force, up to said upper edge 11 of said surrounding enclosure 10, so that said tissue sample 1 are surrounded by the casting material 7 and therefore fully embedded (see Figure 1 b). The casting material 7 is the same hydrogel, which is used for the preparation of said sample holder 2. When filling the hydrogel 7 in said sample block mould 9, the hydrogel 7 comprises a liquid form. In step S32, the filled hydrogel 7 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 7, said sample block 8 is demoulded in step S33 and dehydrated in step S34.

[0076] In the next step S4, said sample block 8 is attached to a histology cassette 12, wherein said sample block 8 is dehydrated (step S41 ) followed by fixating the dehydrated sample block 8 in said fixing matrix in the form of paraffin. Said sample block 8 may be placed into said histology cassette and dehydrated prior to embedding. The step comprises embedding said sample block 8 in paraffin, wherein standard methods known in the art may be used.

[0077] Said sample block 8 is sectioned (step S5) by means of a microtome 13 to form at least one sample block slice 14 to be processed and analysed. Finally, said at least one sample block slice 14 is stained, preferably by means of haematoxylin and eosin staining and / or multiplex staining (step S6).

[0078] Figures 2a, 2b and 2c show a mould according to an embodiment of the invention in different views.

[0079] Figures 3a, 3b and 3c as well as 4a, 4b and 4c show moulds according to a further embodiments of the invention in different views.

[0080] The mould 5, illustrated in Figures 2a-2c, may be used for preparing a sample holder 2 to be used in a method for preparing tissue samples 1 for histological analysis, wherein said sample holder 2 comprises a plurality of recesses 3 for receiving said tissue samples 1 .

[0081] Said mould 5, being a sample holder mould, comprises a base section 15, a side wall section 16 and an opening 17 opposite to said base section 15, wherein said base section 15 and said side wall section 16 delimit an inner space 18 for receiving a casting material 7. Said base section 15 comprises a plurality of protrusions 6 corresponding to said plurality of recesses 3.

[0082] Said side wall section 16 comprises an upper edge 19 that extends beyond said protrusions 6. Said side wall section 16 further comprises an inwardly facing surface 20 narrowing from said opening 17 to said base section 15, wherein said inwardly facing surface 20 is inclined, preferably at an angle 21 a of 80° relative to said base section 15. Additionally, said base section 15 comprises a surrounding recess 22 surrounding said protrusions 6, wherein said surrounding recess 22 and said side wall section 16 merge into each other. In other words, said inwardly facing surface 20 forms part of said surrounding recess 22. Said surrounding recess 22 of said sample holder mould 5 corresponds to the form of said surrounding enclosure 10 of said sample holder 2.

[0083] The above description of the features of the mould 5 shown in Figures 2a-2c analogously apply to the moulds 5 shown in Figures 3a-3c and 4a-4c. However, values of the angles 21 a of inclination of said inwardly facing surface 20 relative to said base section 15 may vary.

[0084] The protrusions 6 of the mould 5 illustrated in Figures 2a-2c are arranged in an array 4b with three protrusions 6 aligned in a first array dimension and five protrusions 6 aligned in a second array dimension. Each protrusion 6 comprises a rectangular disc shape and a cross-section 23, being perpendicular to a protrusion axis 24 of the protrusion 6, for example with a width 25 of 1.2 mm and a length 26 of 7 mm. Therefore, a ratio of said width 25 to said length 26 is equal to 0.17. A protrusion depth 27 of said protrusion 6 is for example equal to 4 mm and therefore amounts to more than half of said length 26 of said cross-section 23. Each base section 15 comprises a substantially planar bottom surface 28, wherein said bottom surface 28 and said protrusion axis 24 are inclined to each other at an angle 21 b of 90°. A sample holder 2, prepared with the mould 5 illustrated in Figures 2a-2c, is, for example, well suited for holding membranes extracted from standard 96-well cell culture plates.

[0085] The protrusions 6 of the mould 5 illustrated in Figures 3a-3c are arranged in an array 4b with five protrusions 6 aligned in a first array dimension and ten protrusions 6 aligned in a second array dimension. Each protrusion 6 comprises a curved disc shape. A cross-section 23 of said protrusion 6, being perpendicular to a protrusion axis 24, for example comprises a width 25 of 1 mm and a length 26 of 3.5 mm, leading to a ratio of said width 25 to said length 26 of 0.29. A protrusion depth 27 of said protrusion 6 is for example equal to 1.75 mm and therefore amounts to half of said length 26 of said cross-section 23. Each base section 15 comprises a substantially planar bottom surface 28. Said bottom surface 28 and said protrusion axis 24 are inclined to each other at an angle 21 b of 90°. A sample holder 2, prepared with the mould 5 illustrated in Figures 3a-3c, is, for example, well suited for holding hydrogelbased cell layers, that are typically extracted with a puncher.

[0086] In Figures 4a-4c a mould 5 comprising a plurality of protrusions 6 with curved disc shapes is shown. The protrusions 6 are arranged in an array 4b with five protrusions 6 aligned in a first and in a second array dimension. A cross-section 23 of said protrusion 6, being perpendicular to a protrusion axis 24, for example comprises a width 25 of 1 mm and a length 26 of 3 mm, leading to a ratio of said width 25 to said length 26 of 0.33. A protrusion depth 27 of said protrusion 6 is for example equal to 1.5 mm. Each base section 15 comprises a substantially planar bottom surface 28. Said bottom surface 28 and said protrusion axis 24 are inclined to each other at an angle 21 b of 60°. A sample holder 2, prepared with this mould 5, is, for example, also well suited for holding hydrogel-based cell layers extracted with a puncher.

[0087] It is pointed out that the form of the sample holder moulds 5 shown in the Figures at least partially correspond to the forms of the sample holders 2 prepared with these sample holder moulds 5. In each case, the form of the recesses 3 corresponds to the form of the protrusions 6, in particular with regard to the angles 21 a, 21 b, the crosssection 23, the protrusion axis 24, the width 25 and length 26 of the cross-section 23 and the protrusion depth 27. Furthermore, in each case, the form of the surrounding recess 22 of the sample holder mould 5 corresponds to the form of the surrounding enclosure 10 of the sample holder 2.

[0088] In this regard, it is referred to the sample holder according to an embodiment of the present invention shown in a three-dimensional view of Fig. 5. This sample holder 2 corresponds to the sample holder 2 used in the method according to the embodiment of Fig. 1a and may be prepared using the mould according to the embodiment of Fig. 2a.

[0089] Said sample holder 2 comprises a plurality of slit-shaped recesses 3 for receiving a plurality of tissue samples 1. Each slit-shaped recess 3 comprises a rectangular disc shape. Furthermore, each slit-shaped recess 3 comprises a cross-section 23', being perpendicular to an axis of a depth extension 24' of said at least one slit-shaped recess 3, for example with a width 25' of 1 .2 mm and a length 26'of 7 mm. A ratio of said width 25' to said length 26' is equal to 0.17. A depth 27' of each slit-shaped recess 3 is for example equal to 4 mm and therefore amounts to more than half of said length 26' of said cross-section 23'. Said sample holder 2 comprises a substantially planar bottom surface 28'. Furthermore, each slit-shaped recess 3 extends along a recess plane, wherein said bottom surface 28' and said recess plane are inclined to each other at an angle of 90°.

[0090] 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.

Claims

- 23 -C l a i m s1. Method for preparing tissue samples (1 ) for histological analysis, preferably wherein said tissue samples (1 ) comprise a two-dimensional form, comprising the steps of:Preparing (S1 ) a sample holder (2), wherein said sample holder (2) comprises at least one slit-shaped recess (3), preferably a plurality of slit-shaped recesses (3), for receiving at least one tissue sample (1 ), placing (S2) said at least one tissue sample (1 ) at least partially inside said at least one slit-shaped recess (3), embedding (S3) said at least one tissue sample (1 ) to form a sample block (8) comprising said sample holder (2), such that said at least one tissue sample (1 ) is fixed in said sample block (8), and sectioning (S5) said sample block (8) to form at least one sample block slice (14) to be analysed.

2. Method according to claim 1 , characterized in that said at least one slit-shaped recess (3) comprises a rectangular disc shape and / or, preferably, a curved disc shape.

3. Method according to claim 1 or 2, characterized in that said at least one slitshaped recess (3) comprises a cross-section (23'), being perpendicular to an axis of a depth extension (24') of said at least one slit-shaped recess (3), with a width (25') and a length (26'), wherein a ratio of said width (25') to said length (26') is less than or equal to 0.5, preferably less than or equal to 0.35, and / or wherein said at least one slit-shaped recess (3) comprises a cross-section (23'), being perpendicular to an axis of a depth extension (24') of said at least one slit-shaped recess (3), with a width (25') of at least 20 pm and a maximum of 3 mm and / or a length (26') of at least 200 pm and a maximum of 15 mm,preferably wherein said at least one slit-shaped recess (3) comprises a depth (27') of at least a third, preferably half, of said length (26') of said cross-section (23').

4. Method according to any one of claims 1 to 3, characterized in that said at least one slit-shaped recess (3) extends along at least one recess plane and that said sample holder (2) comprises a substantially planar bottom surface (28'), wherein said bottom surface (28') and said at least one recess plane are inclined to each other at an angle of 90° and / or of less than 90°, preferably 60°, and / or wherein said sample holder (2) comprises a plurality of slit-shaped recesses (3), arranged in an array (4a) with at least two slit-shaped recesses (3) being aligned in a first array dimension and / or at least two slit-shaped recesses (3) being aligned in a second array dimension.

5. Method according to any one of claims 1 to 4, characterized in that said sample holder (2) is prepared (S2) by means of casting using a sample holder mould (5), preferably wherein said sample holder mould (5) is fabricated (S11 ) by means of additive manufacturing, preferably by means of 3D-printing.

6. Method according to any one of claims 1 to 5, characterized in that said at least one tissue sample (1 ) is embedded (S3) by means of casting using a sample block mould (9), wherein said sample holder (2) at least partially forms said sample block mould (9), preferably wherein said sample block mould (9) comprises a surrounding enclosure (10), preferably wherein an upper edge (11 ) of said surrounding enclosure (10) extends beyond said at least one tissue sample (1 ), preferably wherein said surrounding enclosure (10) is integrally formed with said sample holder (2) or is formed by a separate part.

7. Method according to claim 5 or 6, characterized in that said casting by means of said sample holder mould (5) and / or said sample block mould (9) comprises the steps of:Filling (S12, S31 ) a casting material (7) inside the respective mould (5, 9), preferably by means of gravitational force, solidifying (S13, S32) said casting material (7), and demoulding (S14, S33) said sample holder (2) and / or said sample block (8), preferably wherein said casting material (7) is a curable gel, preferably a hydrogel (7), wherein said gel (7) solidifies by curing (S13, S32), preferably wherein said curing (S13, S32) is achieved by cooling said gel (7) at a temperature between 0 °C and 10 °C, preferably between 4 and 5 °C, in particular for a duration of at least 20 min.

8. Method according to any one of claims 1 to 7, characterized in that, prior to sectioning (S5), said sample block (8) is attached (S4) to a histology cassette (12) 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 (13) and / or wherein said at least one sample block slice (14) is stained (S6), preferably by means of haematoxylin and eosin staining and / or multiplex staining.

9. Mould (5) for preparing a sample holder (2) to be used in a method for preparing tissue samples (1 ) for histological analysis, preferably in a method according to any one of claims 1 to 8, wherein said sample holder (2) comprises at least one recess (3), preferably a plurality of recesses (3), for receiving at least one tissue sample (1 ), wherein said mould (5) comprises a base section (15), a side wall section (16) and an opening (17) opposite to said base section (15), wherein said base section (15) and said side wall section (16) delimit an inner space (18) for receiving a casting material (7), wherein said base section (15) and / or said side wall section (16) comprise at least one protrusion (6), preferably a plurality of protrusions (6), corresponding to said at least one recess (3).- 26 -10. Mould (5) according to claim 9, characterized in that said mould (5) comprises a polymer, preferably a thermoplastic polymer, and / or a metal, preferably aluminium, and / or wherein an upper edge (19) of said side wall section (16) extends beyond said at least one protrusion (6).11 . Mould (5) according to claim 9 or 10, characterized in that said side wall section (16) comprises an inwardly facing surface (20) narrowing from said opening (17) to said base section (15), preferably wherein said inwardly facing surface (20) is inclined at an angle (21a) of less than or equal to 85°, preferably less than or equal to 80°, relative to said base section (15), and / or wherein said base section (15) comprises a surrounding recess (22) surrounding said at least one protrusion (6), preferably wherein said surrounding recess (22) and said side wall section (16) merge into each other, and / or wherein said at least one protrusion (6) corresponds to a slit-shaped recess (3) and / or comprises a rectangular disc shape and / or, preferably, a curved disc shape.

12. Mould (5) according to any one of claims 9 to 11 , characterized in that said at least one protrusion (6) comprises a cross-section (23), being perpendicular to a protrusion axis (24) of said at least one protrusion (6), with a width (25) and a length (26), wherein a ratio of said width (25) to said length (26) is less than or equal to 0.5, preferably less than or equal to 0.35, and / or wherein said at least one protrusion (6) comprises a cross-section (23), being perpendicular to a protrusion axis (24) of said at least one protrusion (6), with a width (25) of at least 20 pm and a maximum of 3 mm and / or a length (26) of at least 200 pm and a maximum of 15 mm.

13. Mould (5) according to claim 12, characterized in that said at least one protrusion (6) comprises a protrusion depth (27) of at least a third, preferably half, of said length (26) of said cross-section (23).

14. Mould (5) according to any one of claims 9 to 13, characterized in that said at least one protrusion (6) extends along a protrusion axis (24) and that said base section (15) comprises a substantially planar bottom surface (28), wherein said bottom surface (28) and said protrusion axis (24) are inclined to each other at an angle (21 b) of 90° and / or of less than 90°, preferably 60°, and / or wherein said mould (5) comprises a plurality of protrusions (6), wherein said protrusions (6) are arranged in an array (4b) with at least two protrusions (6) aligned in a first array dimension and / or at least two protrusions (6) aligned in a second array dimension.

15. Sample holder (2) to be used in a method for preparing tissue samples (1 ) for histological analysis, preferably in a method according to any one of claims 1 to 8, wherein said sample holder (2) comprises at least one recess (3), preferably a plurality of recesses (3), for receiving at least one tissue sample (1 ), wherein said sample holder (2) is prepared with a mould (5) according to any one of claims 9 to 14.

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