Block for preparing observation section, and use thereof

Abstract

The block for preparing an observation section according to the present disclosure is equipped with a cell assembly, the cell assembly being in a three-dimensional shape and embedded, and a continuous or intermittent marker extending in a preset direction.

Description

Blocks for preparing sections for observation and their use

[0001] The present invention relates to a block for preparing observation sections, a method for preparing observation sections, observation sections, and a method for observing observation sections.

[0002] Research has been conducted into the formation of vascular tissue, bronchial tissue, and the like by three-dimensionally culturing cell tissue in a culture gel (see, for example, Patent Documents 1 to 3). In these studies, a thick culture gel layer is typically formed in a dish or well, and cells or cell tissues are embedded in this culture gel layer to culture the cell tissue. Observation of the cultured cell tissue involves observing the cell tissue from above or below the culture gel layer using an inverted or upright microscope. In Patent Document 3, cells stained with a dye are observed using a confocal laser microscope.

[0003] In a confocal laser microscope, a laser beam is focused on a cell using an objective lens, and the autofluorescence of the fluorescent substance labeled on the cell at the focus can be detected. Therefore, a three-dimensional image of the cell can be created by superimposing two-dimensional images of multiple focal planes (xy planes) in the z direction.

[0004] In these conventional studies on 3D cell culture, only bright-field images of the cell tissue can be obtained from the top or bottom, making it difficult to recognize the 3D structure of the cell tissue in bright-field images. Furthermore, in the case of cell tissue with a large thickness in the vertical direction, light does not penetrate through areas with high cell density, making it difficult to observe the cell tissue.

[0005] In order to solve these problems, the inventors of the present application have proposed a cell culture vessel / observation sample cell that can culture cells or cell tissues three-dimensionally and that makes it easy to grasp the three-dimensional structure of the cultured cells or cell tissues through multifaceted observation (Patent Document 4, etc.).

[0006] Japanese Patent Publication No. 2009-213716 International Publication No. WO2004 / 084967 International Publication No. WO2012 / 147878 International Publication No. WO2017 / 094451

[0007] The cell culture vessel described in Patent Document 4 has the advantage that it allows for multi-faceted observation, and therefore the three-dimensional structure of cultured cells or cell tissues can be easily observed while they are stored in the vessel. However, for example, when imaging a weak signal derived from cells, it may be necessary to prepare a block containing the sample (cultured cells or cell tissue) for producing slices for observation, and then slice the sample for observation.

[0008] In this case, the block for preparing the observation slices used for sectioning is removed from the container. When using this block, it is difficult to determine whether the slices are being sectioned from the appropriate direction. Furthermore, even when observing the slices, it is difficult to grasp the positional relationship between the sample in the process prior to observation (e.g., cell culture process) and the sample on the slice. In particular, in the cell culture process, a concentration gradient is often applied to supply a substance to induce directional cell aggregates (e.g., organoids), making it increasingly important to understand the positional relationship between the sample in the process prior to observation and the sample on the slice.

[0009] An object of the present invention is to provide a block or the like for preparing sections for observation, which allows easy determination of whether the sectioning is being performed from an appropriate direction and retains positional information regarding the embedded sample.

[0010] In order to solve the above problems, the present invention includes the following aspects: <1> A block for preparing sections for observation, comprising an embedded three-dimensional cell aggregate and continuous or intermittent markers extending in a predetermined direction. <2> A jig for forming voids for seeding cells in a gel filled in a polyhedral cell culture vessel having at least one open surface, the jig comprising a frame body shaped to fit around the outer periphery of the open surface of the cell culture vessel, and a surface having protrusions located inside the frame body.

[0011] According to one aspect of the present invention, it is possible to provide a block or the like for preparing a section for observation, which holds positional information regarding an embedded sample.

[0012] Figure 6 illustrates the procedure according to one embodiment of the present invention. Figure 7 illustrates the procedure according to one embodiment of the present invention. Figure 8 illustrates the procedure according to one embodiment of the present invention. Figure 9 illustrates images of a cube (container) and a concentration gradient chip according to one embodiment of the present invention. Figure 10 illustrates images of an observation section according to one embodiment of the present invention. Figure 11 illustrates the creation of a concentration gradient and localized differentiation using the Gradient-in-CUBE chip. Figure 12 (a) illustrates images of FITC-dextran and TRITC-dextran concentration gradients formed over a 5-day period. Imaging was performed every 24 hours after removing the used dextran, rinsing the medium chamber with DPBS, and adding fresh dextran to the chamber. The dimensions of the CUBE window were w = 2.75 mm; h = 3.5 mm, and the imaging field of view at 4x magnification was w = 3.6 mm; h = 2.7 mm. The scale bar is 1 mm. In (b), the concentration C was determined by linear correlation of the average intensity along the y-axis of each pixel in the central region of the fluorescence image from x to x' (w = 2.2 mm; h = 2.2 mm) obtained from standard curves of FITC-dextran and TRITC-dextran concentrations. The scale bar is 1 mm. Markers represent selected data points every 50 pixels (~0.3 mm), and the lines indicate the linear least-squares best fit. n = 5. The slopes of the individual lines for FITC are -0.0044 (Day 0), -0.5586 (Day 1), -0.5607 (Day 2), -0.5549 (Day 3), -0.5487 (Day 4), and -0.5391 (Day 5). The gradients for TRITC were 0.0095 (Day 0), 0.7644 (Day 1), 0.7062 (Day 2), 0.6504 (Day 3), 0.5897 (Day 4), and 0.6039 (Day 5). Day 0 showed a slight concentration gradient, while on day 1, opposing gradients of FITC and TRITC were formed from one end of the cube to the other. (c) in the figure shows the ratio of the concentration at the source end to the concentration at the sink end as an index of the steepness of the concentration gradient. The analyzed region was from x = 0.2 mm to x = 2.0 mm, roughly corresponding to the region where the spheroids were located.The concentration gradient was steepest on day 1, but decreased from day 2 onward, remaining constant over the five days. (d) shows the differentiation of hiPSC spheroids by applying mesoderm induction medium (M) to one side and neuroectoderm induction medium (NE) to the other side. Unlike the control, which used only M or NE, which had a nearly uniform morphology, spheroids with distinct morphologies were formed. The scale bar is 500 μm. Immunostaining imaging after cryosectioning and paraffin sectioning. (a) Cryosectioning and imaging. (i) The process of embedding and sectioning a cube-shaped frozen sample is shown. A cube frame was used as a reference marker for sample orientation. (ii) Immunostaining of hiPSC spheroids differentiated using an M-NE gradient showed localized expression patterns of mesodermal (Brachyury) and neuroectodermal (Sox2) markers, while the NE-only and M-only controls showed uniform distribution of the markers. (b) Paraffin sectioning and imaging. (i) To prevent sample loss, the process of embedding and sectioning the paraffin samples using a cube holder is shown. Next, the sample was removed from the cube and one end of the sample was cut to mark an orientation landmark. (ii) Immunostaining of hiPSC spheroids differentiated using an endoderm (END)-NE gradient showed localized expression patterns of endoderm (FoxA2) and neuroectodermal (Nestin) markers, while the NE-only and END-only controls showed uniform distribution of the markers. Detailed process showing sample preparation for paraffin sectioning. The cube holder was designed to prevent the risk of losing samples during the paraffinization process. After paraffinization, the samples were released into cubes and marked for orientation by cutting one end of the sample. Immunofluorescence of paraffin-sectioned samples of END-NE differentiated spheroids. Immunostaining showed localized expression patterns of endoderm (Sox17) and neuroectoderm (Sox2) markers in END-NE spheroids, while controls (NE only) and controls (END only) showed uniform distribution of the markers.

[0013] 1. Block for preparing sections for observation Blocks 100 and 101 for preparing sections for observation according to one embodiment of the present invention will be described in detail with reference to the drawings. The blocks include 1) an embedded three-dimensional cell aggregate and 2) continuous or discontinuous markers extending in a predetermined direction.

[0014] (Cell aggregate) The cell aggregate 12b contained in the blocks 100 and 101 is the object of observation. The cell aggregate broadly refers to a collection of multiple cells forming a three-dimensional shape, but is preferably a biological tissue or part of a tissue, or a cell aggregate produced in vitro by a method such as culture, and more preferably an organoid. Organoids are also called mini-organs formed three-dimensionally in vitro. Organoids are not particularly limited, but include tongue organoids, lung organoids, stomach organoids, intestinal organoids, prostate organoids, liver organoids, pancreatic organoids, kidney organoids, mammary organoids, thyroid organoids, thymus organoids, brain organoids, inner ear organoids, epithelial organoids, etc.

[0015] The size of the cell aggregate is not particularly limited, but from the viewpoint that its directionality is difficult to visually recognize and that it is more suitable for application of the present invention, it may be, for example, within a range of 10 mm or less, and may be preferably within a range of 5 mm or less. The lower limit of the size of the cell aggregate is not particularly limited, but may be, for example, preferably within a range of 1 mm or more.

[0016] In one example, the cell aggregates contained in the block are labeled for observation. Examples of the label for observation include 1) labeling by expression of a fluorescent protein or a luminescent protein, or 2) labeling by a fluorescent dye. Note that the cell aggregates may be labeled by immunostaining or other techniques after sectioning.

[0017] The cell aggregates contained in the block are embedded. Embedding is a term used in microscopy to mean solidifying an object, such as a cell aggregate that contains a lot of water, so that it can be sectioned. Techniques include, but are not limited to, paraffin embedding, resin embedding, and cryo-embedding.

[0018] (Markers) The markers included in blocks 100 and 101 are continuous or discontinuous markers extending in a predetermined direction. When the block is sectioned along a plane intersecting the predetermined direction, the markers appear on each section. When the block is sectioned in an appropriate direction, the markers appear on each section together with cell aggregates.

[0019] This marker facilitates determining whether the sectioning is performed from the appropriate direction and can also be used as positional information for the sample (cell aggregate) embedded in the block. That is, this marker can be used to determine the correspondence between the position of the sample in a process prior to sectioning or observation (such as a cell culture process) and the position of the sample on the section. In other words, the marker can be said to provide information regarding the direction of processing that the cell aggregate or its precursor has undergone.

[0020] Although not particularly limited, examples of markers include the following: 1) A block stored in a container, with the configuration of the container serving as the marker. The cryo-embedded block 101 in the examples described below corresponds to this type, with the cube frame 10a or PDMS side wall 10b constituting the container serving as the marker. 2) A block having a notch along the above-mentioned predetermined direction, with the notch serving as the marker. The paraffin-embedded block 100 in the examples described below, which is removed from the container (cube) and has one end (edge) cut off, corresponds to this type, with the cut-off portion 110 serving as the marker. 3) A structure embedded in the block along a predetermined direction, or a fine hole provided in the block along a predetermined direction, etc. Note that the container in 1) or the structure in 3) must be made of a material that can be sectioned. Examples of the container in 1) include a polycarbonate container (cube) or a Teflon (trademark, the same applies below) container (cube). On the other hand, the container (cube) of 2) is desired to be highly resistant to the organic solvent used in the paraffin embedding process, and examples thereof include an aluminum container (cube) or a Teflon container (cube).

[0021] (Shape of Block, etc.) The shape of the block is not particularly limited, but for example, it is a polyhedron, and preferably a rectangle such as a substantially rectangular parallelepiped or a substantially cubic shape. In the case of a rectangle, the length of one side is not particularly limited, but for example, it is about 1 mm to 10 mm, and preferably about 3 mm to 5 mm. The block is for preparing slices for observation, and refers to a block that can be set directly in a microtome or the like to be sliced.

[0022] 2. Method for preparing blocks for preparing sections for observation Blocks for preparing sections for observation are prepared, for example, through the following steps: 1) step A, in which a predetermined treatment is performed on cell aggregates or precursors thereof stored in a container; 2) step B, in which the cell aggregates stored in the container after step A are embedded; and 3) step C, which is performed as needed, in which the block containing the cell aggregates embedded in step B is removed from the container and a notch (marker) is formed in the removed block.

[0023] (Regarding Step A) The container 10 for storing the cell aggregate or its precursor 12a is not particularly limited, but examples thereof include those shown in Figures 1 and 2. The container comprises a polyhedral frame and a light-transmitting window covering the surface surrounded by the frame. The container 10 shown in Figures 1 and 2 comprises a substantially cubic frame (frame 10a) and windows (side walls 10b) provided on four of the six surfaces surrounded by the frame. Note that the window may be made of PDMS as shown in the examples, or may be made of a hydrogel such as agarose gel or polyacrylamide gel. Note that containers described in WO2017 / 094451, WO2018 / 150689, WO2022 / 080162, etc., can also be used.

[0024] The inside of the container is filled with a culture gel for culturing cell aggregates or their precursors. After filling the container with the unhardened culture gel, a mold cap 11 (described in the Examples) is placed over the container and the culture gel is hardened, thereby reproducibly forming a void for disposing cell aggregates or their precursors. In other words, the mold cap is a jig for forming a void for seeding cells in a gel filled in a polyhedral cell culture container having at least one open surface, the jig comprising a frame shaped to fit around the outer periphery of the open surface of the cell culture container and a surface having a protrusion located inside the frame. The frame accurately defines the positional relationship between the cell culture container and the mold cap, and the protrusion allows for reproducible formation of a void of a predetermined depth in the culture gel.

[0025] The precursor 12a of the cell aggregate refers to pluripotent stem cells such as ES cells and iPS cells, or other stem cells, which become cell aggregates when cultured in a culture gel.

[0026] The type of predetermined treatment performed on the cell aggregates or their precursors in step A is not particularly limited, but examples include culturing with the supply of a substance or drug treatment. Culturing with the supply of a substance includes cell aggregate induction using a morphogen concentration gradient, as shown in the examples. Drug treatment includes drug metabolism experiments or drug (candidate) screening experiments. One example of a predetermined treatment is a directional treatment with respect to the cell aggregates or their precursors (and relatively with respect to the container), such as the treatment using the concentration gradient of a substance described above. Note that a treatment with the supply of a substance falls under the category of directional treatment unless the substance is supplied uniformly from all directions to the cell aggregates or their precursors. An example of an apparatus for performing step A is the cell culture fluidic chip described in WO 2018 / 147032. This cell culture fluidic chip allows a container containing the cell aggregates or their precursors to be set in place and the treatment to be performed, and the container can be removed after treatment.

[0027] (Regarding Step B) Step B is a step of embedding the cell aggregate stored in the container after Step A. The embedding technique, such as paraffin embedding, resin embedding, or cryo-embedding, may be a method known in the field of preparing sections for observation. By undergoing this step, a cryo-embedded block for preparing sections for observation, as shown in FIG. 2, is produced.

[0028] (Regarding Step C) Step C is performed as needed. For example, in the production of a paraffin-embedded block for preparing observation sections shown in FIG. 2, a block containing a paraffin-embedded cell aggregate is removed from a container in Step B, and a notch (marker) is formed in the removed block. In the block for preparing observation sections prepared in Step B, the configuration of the container serves as the marker. In addition, in the block for preparing observation sections prepared in Step C, the notch formed in the block serves as the marker. By performing Steps A to B or Steps A to C while understanding the correspondence between the direction of the treatment in Step A and each marker, the marker will indicate information regarding the direction of the treatment that the cell aggregate or its precursor has undergone.

[0029] [3. Method for preparing sections from a block for preparing sections for observation, method for observing a section, etc.] This method includes a step of slicing the block described in the above section [1. Block for preparing sections for observation] in a direction intersecting the predetermined direction (i.e., the direction in which the marker extends) to prepare sections for observation 100a / 101a. This method makes it possible to prepare sections for observation carrying a marker and a cell aggregate. The method for preparing sections from a block can be the same as that for preparing sections for microscopic observation; for example, the block can be set in a microtome and sections can be prepared.

[0030] The step of observing the prepared observation section with a microscope may be carried out according to a known observation method. The type of microscope is not particularly limited as long as it is an optical microscope. For example, either an upright or inverted microscope may be used, and the observation method may be bright-field observation, dark-field observation, phase-contrast observation, differential interference observation, HMC observation, polarized light observation, or the like. Fluorescence microscope observation may also be performed using an epifluorescence microscope, a total internal reflection fluorescence microscope, a confocal laser fluorescence microscope, a multiphoton excitation fluorescence microscope, or the like.

[0031] The sections prepared for observation are marked with markers. These markers provide information about the orientation of the processing of the cell aggregates or their precursors and can be referenced during the observation of each section. Observing the cutting of the markers also helps to ensure that the block is sectioned in the correct direction. Furthermore, by aligning the markers, images of multiple sections can be used to reconstruct a single stacked image.

[0032] (Summary) The present invention includes, for example, the following inventions. <1> A block for preparing observation sections, comprising an embedded three-dimensional cell aggregate and a continuous or intermittent marker extending in a predetermined direction. <2> The block according to <1>, which is frozen-embedded or paraffin-embedded. <3> The block according to <1>, which is stored in a container, and the configuration of the container serves as the marker. <4> The block according to <1>, which has a notch along the predetermined direction, and the notch serves as the marker. <5> The block according to <1>, in which the cell aggregate is an organoid. <6> The block according to <1>, in which the marker indicates information regarding the direction of a treatment that the cell aggregate or its precursor has undergone. <7> A method for preparing a section, comprising a step of slicing the block according to any one of <1> to <6> above in a direction intersecting the predetermined direction to prepare an observation section. <8> An observation section prepared by the method according to <7> above. <9> An observation method comprising the step of observing the observation section described in <8> above with an optical microscope. <10> A method for producing the block described in any of <1> to <6> above, comprising: step A: performing a predetermined treatment on a cell aggregate or a precursor thereof stored in a container; and step B: embedding the cell aggregate stored in the container after step A. <11> A jig for forming a void for seeding cells in a gel filled in a polyhedral cell culture container having at least one open surface, the jig comprising a surface having a frame body shaped to fit around the outer periphery of the open surface of the cell culture container and a protrusion located inside the frame. <12> A method for evaluating organoids, comprising the steps of: generating directional organoids by culturing cells in a culture container and providing a concentration gradient; and embedding and slicing the directional organoids generated in the step while maintaining positional information. <13> In the above <12>, in the sectioning step, when the organoid is embedded and sectioned, the culture vessel is also sectioned together, so that the section retains its positional information.<14> In the above <12>, in the sectioning step, the embedded block containing the organoids removed from the culture vessel is processed to remove a portion thereof before sectioning, so that the section retains its positional information.

[0033] An embodiment of the present invention will now be described.

[0034] <hiPSC culture> 0.5 μg / cm ２ Human iPSCs (1231A3; RIKEN BRC Cell Bank, HPS0381) were cultured on dishes coated with Laminin-511 E8 (iMatrix-511 Silk; Nippi, 892021) using StemFit AK02N (Ajinomoto Co., Inc., RC AK02N) and passaged using ReLeSR (STEMCELL Technologies, 05872).

[0035] <Cube (Container) Fabrication> Using Rhinoceros 3D software, we designed a 5 mm wide aluminum cube frame and ordered it from a machining company (Proto Labs, Japan). The upper frame of the cube was designed to be thicker so that an O-ring could be attached to the cube (Figure 1Ai). The O-ring was used to ensure a tighter seal between the cube and the PDMS chip to reduce medium leakage around the cube during concentration gradient culture. To form the cube's multiple sidewalls (windows), we first mixed an elastomer base and a curing agent in a 10:1 ratio to prepare uncured polydimethylsiloxane (PDMS; Silpot 184, Dow Toray, 04133124). Next, a thin layer of the mixture (uncured PDMS) was spread on a Petri dish and degassed to remove air bubbles. The cube frame was placed on this uncured PDMS, which was then degassed again and baked at 85°C for 15 minutes to cure the PDMS. Once cured, the PDMS was trimmed from the frame using a scalpel, and this process was repeated to cover the other three lateral openings of the cube with cured PDMS sidewalls, creating a cube with an open top and bottom (Fig. 1Aii). The cube with four PDMS sidewalls was washed twice by sonication, once with MilliQ water, and once with isopropanol, and then dried in an oven. For sterilization, the cube was autoclaved and then oven dried before use.

[0036] <Seeding hiPSCs into cubes> To control cell position during initial seeding, a mold cap was designed with 1) a cylindrical structure with a conical base that reaches the desired seeding position in the cube, and 2) a groove that fits into the top surface of the cube to ensure the cylindrical structure is correctly positioned within the cube (Fig. 1B). Prior to use, the mold cap was coated with 2-methacryloyloxyethyl phosphorylcholine (MPC; Lipidure, NOF Corporation, CM5206E) diluted to 5% in isopropanol and allowed to dry for 1 hour at room temperature (RT). A nitrile O-ring (AS ONE, 62-3049-63) was attached to the thick part of the aluminum frame of the cube, and the cube was placed on a dish with the O-ring side facing down. Matrigel® (Corning, 356231) was added to the cube, and then the mold cap was placed on top of the cube and incubated in an incubator (37°C, 5% CO). ２ The Matrigel was hardened for 25 minutes at 4°C (Invitrogen, 00455556). During this time, hiPSC cells were detached using Accutase® (Invitrogen, 00455556) and pelleted by centrifugation at 200 x g for 3 minutes at room temperature, and the supernatant was discarded. Immediately after the Matrigel hardened, the mold cap was removed and cells were seeded into the depressions created by the mold. 1 μL of cells pelleted from two 35 mm dishes or one 60 mm dish was seeded into one cube. The cube was returned to the incubator for 5 minutes to allow the cells to settle in the depression. Additional Matrigel® was then added to the top of the cube and allowed to harden for 25 minutes, after which the depression was closed (Figure 1Bii). After hardening, the cubes were transferred to a 48-well plate with 1 mL of StemFit medium plus 10 μM Rock inhibitor (Y-27632; Nacalai Tesque, 08945-84). For the next 2 days, the medium was switched to StemFit medium without Y-27632.

[0037] <HiPSC Differentiation Using a Concentration Gradient Formed on a Chip> After seeding hiPSCs into cubes and confirming their aggregation to form spheroids, the samples were transferred to the gradient-formed chip to initiate localized differentiation. The mold for creating the lid side of the PDMS gradient chip was designed with a groove to fit an O-ring and multiple ports for adding medium, while the mold for creating the base side of the PDMS gradient chip was designed to fit the cube, O-ring, and two separate medium chambers (Figure 2Ci). To create the PDMS chips, uncured PDMS was poured into the two molds, which were then degassed and baked at 85°C for 30 minutes to harden the PDMS. Immediately after hardening, the two formed chips were removed from the molds, washed, and sterilized in the same manner as the cubes. To assemble the concentration gradient chip, a sample cube with an O-ring attached was placed on the base of the chip. The lid and base of the chip were then sealed together using a PDMS adhesive film (NSD-100, NIPPA) (Figure 2Cii). After assembly, the two medium chambers were filled with STEMdiff Trilineage Ectoderm medium (STEMCELL Technologies, 05231) on one side and STEMdiff Trilineage Endoderm medium (STEMCELL Technologies, 05233) on the other side (Figure 2Ciii). The medium was replaced by discarding the spent medium and rinsing it once with DPBS before replacing it with fresh medium. Thus, a morphogen concentration gradient was formed between the two sides of the cube where the PDMS sidewalls were not present.

[0038] <Differentiation induction> Neuroectodermal differentiation medium (NE medium) includes KnockOut DMEM / F12 (Gibco, 12660-012), 10% KnockOut serum replacement (Gibco, 10828010), 1% MEM non-essential amino acid (Nacalai Tesque, 06344-56), 1% GlutaMAX (Gibco, 35050-061), 1μM LDN1913189 (Sigma, SML0559), 2μM SB431542 (Nacalai Tesque, 18176-54), 3μM CHIR99021 (Nacalai Tesque, 18764-44), 0.1 mM 2-mercaptoethanol (Nacalai Tesque, 21438-82) and 0.5 μM ascorbic acid The mesoderm basal medium contained a 1:1 mixture of Neurobasal (trademark) medium (Gibco, 21103-049) supplemented with RPMI medium 1640 (Gibco, 11875-093), 1% GlutaMAX, and 1% penicillin-streptomycin (Gibco, 15140122). For mesoderm differentiation, 5 μM CHIR99021 was added to the mesoderm basal medium on days 0 and 1. On days 2–4, CHIR99021 was removed and replaced with 100 ng / mL bFGF (Nacalai Tesque, 19155-36) and 1 μM all-trans retinoic acid (Stemgent, 04-0021). For endoderm differentiation, 5 μM CHIR99021 was added to mesoderm basal medium on day 0. On days 2 to 5, CHIR99021 was removed and replaced with 100 ng / mL Activin A (R&D Systems, 338-AC-050 / CF). Medium changes were performed daily by discarding the used medium, washing once with DPBS, and replacing with fresh medium.To observe the morphological changes of the spheroids in the cubes, phase contrast images of the spheroids were obtained using an Olympus CKX41 microscope.

[0039] Fixation and Sectioning for Imaging: When the samples were ready for analysis, the sample cubes were removed from the gradient chip and transferred to a 48-well plate for fixation. Each sample cube was washed twice with DPBS for 5 minutes each, fixed in 4% paraformaldehyde for 20 minutes, and then washed twice with DPBS for 10 minutes each. For frozen sections, a PTFE cube was used instead of an aluminum cube. After fixation, the samples were embedded in a frozen section embedding solution (White Tissue-Coat, Yuaikasei). After freezing, the samples were sectioned together with the cube (Fig. 2, Di). In frozen sections, the direction connecting the two sides of the cube frame without the PDMS sidewalls corresponds to the direction of the morphogen concentration gradient during hiPSC differentiation induction. For paraffin sections, the cubed samples were embedded in paraffin using the following procedure: 70% ethanol twice for 30 minutes, 80% ethanol for 1 hour, 95% ethanol for 1 hour, 95% ethanol for 1 hour, 99% ethanol for 1 hour, 100% isopropanol for 1 hour twice, isopropanol:xylene (7:3 by volume) for 1 hour, isopropanol:xylene (1:1 by volume) for 1 hour, isopropanol:xylene (3:7 by volume) for 1 hour, 100% xylene for 1 hour, and paraffin for 1 hour twice.

[0040] The cube was then excised from the paraffin, and the sample (paraffin block) was removed from the cube by cutting the paraffin along the inside of the cube frame using a scalpel and pressing the cube onto an extrusion jig (Fig. 2Dii). One edge of the sample (paraffin block) was cut off to mark the direction of the concentration gradient, and the sample was then cut into multiple slices.

[0041] The resulting slices were deparaffinized using the following method: 100% xylene twice for 10 minutes, 99% ethanol twice for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, 70% ethanol for 5 minutes, and MilliQ water for 5 minutes. Heat-induced antigen retrieval was then performed by placing the samples in boiling 10 mM citrate buffer (pH 6) for 10 seconds, followed by 10 seconds of cooling. This boiling and cooling cycle was repeated six times. Upon cooling, the samples were washed with MilliQ water for 5 minutes, followed by three 5-minute washes with DPBS. When sections of the paraffin-embedded blocks were sliced, the cut-off portion was positioned at the bottom right, and the vertical direction of the section corresponded to the direction of the morphogen concentration gradient during hiPSC differentiation induction.

[0042] Immunostaining: Cryosection samples were permeabilized with 0.5% Triton X-100 for 10 minutes and then washed three times with 100 mM glycine for 10 minutes each. Immunofluorescence buffer (IF buffer) was prepared by adding 0.5% Tween 20, 2% Triton X-100, and 10% bovine serum albumin (BSA; Sigma, 126615) to DPBS. Samples were incubated with IF+G containing 10% goat serum (Gibco, 16210064) for 30 minutes, followed by blocking with IF+G containing 1% goat anti-mouse IgG (Bethyl Laboratories, A90-116A) for 20 minutes. Antibodies were diluted (1:200 or 1 μg / mL) according to Table 1 below. The primary antibody was incubated for 90 minutes, and the secondary antibody (Alexa fluor, 1:200, Thermo Fisher Scientific) was incubated for 50 minutes. After each antibody incubation, the samples were washed three times for 15 minutes with IF buffer. Nuclei were stained with DAPI for 20 minutes and then washed three times for 5 minutes with DPBS.

[0043]

[0044] Example: Cubes capable of controlling cell position were prepared using the method described above, hiPSCs were seeded, and the cubes were transferred to a 48-well plate (Fig. 3 i, Fig. 4 i). The sample was then transferred to a chip on which a concentration gradient was formed using Mesoderm differentiation medium and Neuroectoderm differentiation medium (NE medium) and cultured for 5 days (Fig. 3 ii, Fig. 4 ii). When the sample was ready for analysis, the sample cube was removed from the concentration gradient chip and frozen sections were prepared using the method described above (Fig. 3 iii). Images of the frozen sections are shown in Fig. 5. In Fig. 5, the three leftmost images are labeled with Dapi, βIII-tublin, and Brachyury, respectively, and the rightmost image is an overlay of the respective images. Fig. 5 also shows an image of a gradient formed with Mesoderm differentiation medium from the top and Neuroectoderm differentiation medium from the bottom.

[0045] Concentration gradient formation and localized differentiation using the Gradient-in-CUBE chip. Daily imaging was performed over a 5-day period to monitor the progress of the formation of two concentration gradients within the cube, using FITC-dextran and TRITC-dextran to simulate the addition of two different types of media to the cube (Figure 6a).

[0046] The average concentrations (C) of FITC and TRITC across the central region (xx') of the cube window were measured. FITC and TRITC exhibited opposing concentration gradients, with higher concentrations at the source and lower concentrations at the sink, due to the migration of dextran molecules from the higher concentration side to the lower concentration side (Fig. 6b).

[0047] 10 μM dextran was added to the source reservoir, and the average maximum concentrations at the source end of the FOV were 2.618 μM for FITC and 3.255 μM for TRITC, and the average minimum concentrations at the sink end were 0.024 μM for FITC and 0.026 μM for TRITC. The concentration gradient was I for FITC. ｘ ０．２ / I ｘ ２．０For TRITC, it is calculated as the ratio of I ｘ ２．０ / I ｘ ０．２ The values ​​showing no gradient and a higher ratio represent a steeper gradient (Fig. 6c).

[0048] The concentration gradient increased in steepness and peaked over the first 24 hours as the molecules penetrated the gel. However, by day 2, the gradient decreased in steepness as the molecules accumulated in the gel within the cube as well as on the other side. Despite daily washing with DPBS and replacement with fresh dextran, the medium maintained a constant concentration gradient over 5 days without reaching equilibrium.

[0049] Here, we used only the diffusion of 40 kDa dextran through agarose gel as a model for the diffusion of various growth factors through ECM hydrogels. These were chosen because Wnt, which plays an important signaling role in development, has a molecular weight of approximately 40 kDa, and because dextran and agarose are widely used and readily available reagents in the field of mass transport and diffusion research. Based on the assumption that a concentration gradient spanning one or a few cell lengths is sufficient to provide positional information for cells (Vetter, R. & Iber, Nat. Commun. 13, 1145 (2022); Matos, I. et al. Elife 9, e54304 (2020)), we hypothesized that the concentration gradient formed in the Gradient-in-CUBE chip would be sufficient to induce localized differentiation at opposite ends of spheroids. Another approach to counteracting morphogen accumulation is to supply morphogen inhibitors from the opposite end of the gradient. Morphogens and their inhibitors are important for tissue patterning in vivo. For example, Wnt and Nodal agonists Dkk1, Lefty-1, and Cer-1 at the anterior end of the embryo restrict Wnt / Nodal to the posterior end of the embryo, forming the anterior-posterior axis (Carlson, B. Human Embryology and Developmental Biology (Elsevier, 2018)). To demonstrate the application of the Gradient-in-CUBE chip, we used the Gradient-in-CUBE chip to differentiate a single spheroid into two distinct regions by supplying neuroectodermal differentiation medium (NE) from one end of the CUBE and mesodermal differentiation medium (M) from the other end.The mesoderm differentiation medium (based on the protocol of Lam, AQ et al. J. Am. Soc. Nephrol. 25, 1211-1225 (2014)) contained a high concentration of the Wnt activator CHIR99021, while the neuroectoderm differentiation medium (based on the protocol of Bianchi, F. et al. Stem Cell Res. 32, 126-134 (2018)) contained a lower concentration of the Wnt activator along with the Nodal / Activin inhibitor SB431542, which prevented mesoderm differentiation on the neuroectoderm side. After 4 days of differentiation, morphological changes were observed at both ends of the spheroids. Compared to the smooth features of the neuroectoderm side, the mesoderm side became uneven and more protruding. In contrast, control spheroids exhibited a more uniform external appearance. That is, the mesodermal control had rough protrusions all over its surface, while the neuroectodermal control had a smoother surface (Fig. 6d).

[0050] Maintaining Orientation During Sectioning and Imaging Due to the limited penetration range of lasers, the low sensitivity of low-magnification objectives, and the short focal length of high-sensitivity objectives, it is often difficult to visualize the expression of cellular pluripotency or differentiation markers in large-scale 3D samples such as organoids. Therefore, organoids are sometimes embedded in freezing medium or paraffin and sliced ​​into thin sections for staining and imaging. However, after sample recovery from a typical gradient-forming device, the sample orientation is often lost. The advantage of the cube device is that cells can be contained within the cube and recovered without damaging the sample, and the gradient orientation can be easily marked for later recognition. The following experiment demonstrates that samples within the cube can be processed for freezing and paraffin sectioning.

[0051] For cryosectioning, one of the cube frames was made thicker, and an O-ring attached to it served as an orientation marker (Figure 7a-i). After sample recovery from the gradient-formed cube chip, the cube could be easily immersed in sucrose and cryoembedding medium before freezing. Once frozen, the cube was sliced ​​along with the sample, and the cube frame and PDMS outer wall served as references to maintain the orientation of the sample (Figure 2d-i, Figure 7a). Immunofluorescent staining of hiPSC spheroids differentiated in the NE-M gradient revealed localization of the neuroectodermal marker Sox2 and the mesodermal marker Brachyury on both sides of the spheroid, whereas in the control sample, both markers were uniformly expressed throughout the spheroid (Figure 7b). These results demonstrate a method for maintaining the orientation of morphogen gradient-differentiated spheroids by sectioning the sample directly within the cube. However, a drawback of this method is that repeatedly cutting through hard acrylic materials can damage the microtome blade, potentially requiring frequent replacement of the microtome. This problem can be solved by using an easy-to-cut material for the cube frame (e.g., polycarbonate or Teflon). Alternatively, microtome blades or diamond-coated blades commonly used for cutting hard materials such as bone can be used to cut the cube frame.

[0052] For paraffin sectioning, additional steps are required to maintain the integrity of the sample and the orientation of the gradient within the cube (Figure 2, Dii, Figure 7, Bi, Figure 8). While frozen cubes made by combining an acrylic frame and soft PDMS walls allow for cube sectioning, sectioning paraffin cubes is extremely difficult because the cube is entirely made of acrylic and PDMS requires modification due to its incompatibility with the organic solvents used in the paraffin embedding process. During the initial dehydration process before paraffin embedding, the hydrogel loses a significant amount of volume and shrinks within the cube. To avoid the risk of losing the sample, which could potentially detach from the cube, we designed a cube holder with a lid and base to cover most of the open surfaces on the top and bottom of the cube while allowing reagents to access the sample. The cube was connected to the holder with wire to maintain it in place. After paraffin processing, the sample was removed from the cube using an extrusion tool, and a marker for orientation was created by cutting off one corner of the sample. To demonstrate the applicability of the paraffin method, hiPSC spheroids from one cube were differentiated using NE and endoderm (END) differentiation media (see the protocol in Lam, AQ et al. J. Am. Soc. Nephrol. 25, 1211-1225 (2014)). Immunostaining for the neuroectoderm markers Nestin and Sox2, and the endoderm markers FoxA2 and Sox17, demonstrated localization of NE and END at both ends of the spheroid, respectively (Figure 7(b) and Figure 9). In contrast, control samples without gradient culture showed uniform expression of the markers throughout the spheroid.

[0053] Summary: The objective of this example was to develop a simple concentration gradient culture platform for controlling the differentiation of PSCs into organoids with desired patterns, while preserving the integrity of the sample and the directionality of the concentration gradient during culture for subsequent image analysis experiments. To achieve this, we used a previously developed cube culture device (Hagiwara et al., 2018a; Hagiwara et al., 2018b) to improve the ease of sample handling with ECM and the reproducibility of cell seeding and pattern formation. In this example, we first demonstrated a method for precisely positioning cells at the desired location within the cube culture device. Furthermore, we demonstrated that simply transferring the cube culture device to a two-compartment chip device can generate a concentration gradient within the cube that induces dorsal-ventral differentiation of iPSCs. Finally, we present the concentration gradient platform along with a different post-experimental processing method for imaging and analysis that preserves the directionality of the concentration gradient. Using this intracube gradient platform, organoids with controlled axial differentiation can be achieved, providing a more complex in vitro model in which the effects of morphogen gradients can be systematically applied and studied in directing cell differentiation and the development of tissues, organs, and ultimate body systems to further understand developmental processes in humans.

[0054] The main unique features demonstrated in this example can be summarized as follows: (1) a simple technique; (2) the placement or positioning of the sample in the device is tightly controlled; and (3) the sample can be retrieved from the device for further analysis after the experiment without significant damage to the sample or loss of information about the sample's orientation.

[0055] The present invention provides a block for preparing sections for observation that allows easy determination of whether the sectioning is performed from the appropriate direction and retains positional information regarding the embedded sample. This allows for the seamless generation, observation, sampling, and evaluation of organoids in the Organ-on-a-Chip field, which is attracting attention in the basic research and non-clinical trial stages of pharmaceutical development.

[0056] REFERENCE SIGNS LIST 10 Cube (container) 10a Frame (container configuration) 10b Side wall (container configuration) 11 Mold cap (jig) 12a Precursor of cell aggregate 12b Cell aggregate 100 Block (block for preparing slices for observation) 100a Slice 101 Block (block for preparing slices for observation) 101a Slice

Claims

1. A method for preparing a block for preparing a section for observation, comprising the steps of: The above block is an in vitro generated cell aggregate having an embedded three-dimensional shape; and a continuous or intermittent marker extending in a predetermined direction; A step A of treating a cell aggregate or a precursor thereof stored in a container using a concentration gradient of a substance; A step B of embedding the in vitro produced cell aggregate stored in the container after the step A; The method includes:

2. The method of claim 1, wherein the block is frozen-embedded or paraffin-embedded.

3. The method of claim 1 , wherein the block is in a form stored in a container, the configuration of the container being the marker.

4. The method of claim 1 , wherein the block is a block removed from a container and includes a notch along the predetermined direction, the notch being the marker.

5. The method of claim 1, wherein the in vitro generated cell aggregate is an organoid.

6. The method of claim 1 , wherein the marker provides information regarding the direction of a treatment that the cell aggregate or its precursor has undergone.

7. A method for preparing slices for observation, comprising the step of slicing a block for preparing slices for observation, prepared by the method according to any one of claims 1 to 6, in a direction intersecting the predetermined direction to prepare slices for observation.

8. A section for observation prepared by the method according to claim 6.

9. An observation method, comprising the step of observing the observation section according to claim 8 with an optical microscope.

10. An in vitro generated cell aggregate having an embedded three-dimensional shape; and a continuous or intermittent marker extending in a predetermined direction; The marker is a block for preparing slices for observation, which indicates information regarding the direction of treatment using a concentration gradient of a substance that a cell aggregate or its precursor has undergone.