Frozen blocks of islet cell-like cell clusters for spatiotemporalomics serial section analysis, methods and applications

Abstract

The application discloses a frozen embedding block of islet cell-like cell clusters meeting spatiotemporal omics continuous section analysis, a method and application thereof, and belongs to the field of frozen embedding block preparation. In order to solve the problem of RNA / DNA integrity and visualization of islet cell clusters, the method comprises the following steps: adding a buffer solution containing bromophenol blue drop by drop to the aggregated cell clusters on the inner surface of the bottom of a silk cloth to dye the cell clusters; keeping the selected area of the silk cloth in contact with the frozen embedding reagent surface of the lower half groove in the loading groove of a first mold, placing the first mold on dry ice, and completely freezing the frozen embedding reagent of the lower half groove and the cell clusters on the surface thereof; placing the first mold with the upper half groove frozen embedding reagent added in the loading groove on dry ice, completely freezing the frozen embedding reagent of the upper half groove and the cell clusters on the surface thereof, and making the frozen embedding reagent of the upper half groove and the frozen embedding reagent of the lower half groove be integrated into the frozen embedding block, and taking out the frozen embedding block, wherein the cell clusters are distributed at the middle position on the height of the frozen embedding reagent block.

Description

Technical Field

[0001] This invention belongs to the field of frozen embedding block preparation, and relates to frozen embedding blocks, methods and applications of pancreatic islet cell-like cell masses that meet the requirements of spatiotemporal omics serial section analysis. Background Technology

[0002] DNA sequencing is used to capture DNA expression, such as in single-cell sequencing (e.g., cancer cell sequencing). Since it operates on a single cell basis, it typically involves encapsulating a single cell in a droplet and sequencing it using a microfluidic chip. Other examples include commonly used methods such as cryopreservation, slicing, and DNA sequencing of tissues.

[0003] Pancreatic islets are clusters of cells of varying sizes and shapes scattered throughout the pancreas. Hormones produced by these islets (insulin, glucagon, etc.) effectively control glucose utilization in the body. They are generally 50-300 μm in diameter. The islet cell clusters are extremely small, making embedding them in embedding reagents extremely difficult. Direct cryopreservation, as with tissues, cannot guarantee a sufficient number of cell clusters embedded and their visualization within the frozen embedding block, hindering subsequent slicing and sequencing.

[0004] Because pancreatic islets function as cell clusters, meaning they can only secrete insulin and glucagon in the form of cell clusters, it is difficult to capture the expression of intercellular connections and interactions through sequencing if the cell clusters are broken down into single cells, encapsulated in droplets, and sequenced using microfluidic chips.

[0005] This invention utilizes cryopreservation of islet cell-like cell clusters for the cryopreservation of human islet cell clusters. The cryopreserved blocks are then subjected to spatiotemporal omics serial slicing for DNA sequencing of the human islet cell clusters. The challenges of this invention lie in: how to cryopreserve viable human islet cell clusters while maintaining cell viability throughout the process, ensuring visualization of the cell clusters during slicing, and guaranteeing sequencing accuracy; and how to effectively cryopreserve a sufficient number of cells while maintaining cellular RNA integrity to ensure sequencing is feasible, while also avoiding the waste of expensive viable cells.

[0006] CN116106108A discloses a method for embedding cell blocks and a small amount of frozen cell sections in paraffin, used for immunofluorescence staining of pancreatic islets or islet-like cells. Specifically, it discloses the following steps: (S.2) cell dehydration; (S.3) cell pre-embedding: (S.3.1) centrifuge at 300g for 3 minutes to completely remove the sucrose solution. After the liquid is removed, add a small amount of colored embedding agent (5 cell clusters per microliter of embedding agent, adjusting the volume of embedding agent according to the number of islet or islet-like cell clusters) to the bottom of the centrifuge column (Lecia, 3801480), and mix the cell clusters and embedding agent thoroughly; (S.3.2) place the centrifuge column above liquid nitrogen but not in contact with the liquid nitrogen until the embedding agent is completely solidified, and remove the embedded block from the bottom of the centrifuge column with tweezers. (S.4) Cell embedding: (S.4.1) Transfer the solidified pre-embedded block to a colorless embedding agent (SAKURA, 4583), ensuring the pre-embedded block is completely submerged in the colorless embedding agent and placed in the center. After the pre-embedded block has completely melted, place the embedding box on top of liquid nitrogen without contacting the liquid nitrogen for solidification; (S.4.2) After complete solidification, slice the cell wax block or store it at -80℃.

[0007] This method is used for cryo-embedding islets or islet-like cells, followed by sectioning of the embedded blocks for immunofluorescence staining. Cell viability is not a concern during staining; the method describes steps such as cell dehydration and centrifugation for 3 minutes, indicating that cell viability is not considered during embedding and it lacks steps to ensure cell viability during embedding. This is based on CN116106108A. Figure 2 The embedded block is cylindrical in shape and, based on the aforementioned dehydration and centrifugation equipment, occupies a significant portion of the operable space. The document states, "Add a small amount of colored embedding agent (5 cell clusters per microliter of embedding agent) to the bottom of the centrifuge column." If a single embedding volume of 2500 IEQ / block is to be achieved, the amount of colored embedding agent used would be approximately 0.3 ml or more. This results in an excessively large embedded block volume, dispersed cell clusters, and low concentration within the embedded block. The document also states, "After the embedded block has completely melted, place the embedding box on liquid nitrogen without contacting it for solidification." This indicates that the embedded block will further disperse during complete melting.

[0008] Existing technologies require cell fixation in solutions such as paraformaldehyde and dehydration in organic solvents before cryopreservation. Pre-embedding with colored frozen blocks makes it difficult to distinguish cell positions within the frozen block, hindering effective visualization and localization. This method can only achieve cryopreservation of a small number of cells, resulting in a limited cell count in the embedding field, which cannot meet the needs of spatiotemporal omics and immunofluorescence staining. This method can only ensure protein stability, not DNA / RNA integrity. Furthermore, this method involves two cumbersome embedding steps and requires complex equipment. Summary of the Invention

[0009] To address the issues of viable cryo-embedding of pancreatic islet cell clusters and improving the concentration of cell clusters, and to enable their use in human pancreatic islet cell cluster DNA sequencing, in a first aspect, a cryo-embedding method for islet cell clusters according to some embodiments of this application includes the following steps:

[0010] S10. Tilt the folded silk cloth into a funnel shape and aim it at the tip of the pipette containing the culture medium liquid containing cell clusters. Push the culture medium liquid containing cell clusters from the tip of the pipette toward the inner surface of the silk cloth tip, transferring and gathering the cell clusters on the inner surface of the silk cloth tip.

[0011] S20. Make regional contact between the outer surface of the bottom of the silk cloth and the absorbent paper laid on the plane below the silk cloth to separate the cell clusters from the culture medium liquid on the silk cloth.

[0012] S30. Add buffer solution containing bromophenol blue dropwise to the aggregated cell clusters on the inner surface of the bottom of the silk fabric to stain the cell clusters;

[0013] S40. Make regional contact between the outer surface of the bottom of the silk cloth and the absorbent paper laid on the plane below the silk cloth to separate the cell clusters from the buffer solution on the silk cloth.

[0014] S50. Unfold the silk cloth into a plane, and the area where the cell clusters on it are stained is the selected area of ​​the silk cloth;

[0015] S60. The lower half of the freezing embedding reagent is added to the receiving groove of the first mold placed horizontally, and placed on dry ice for incomplete freezing. The surface of the incompletely frozen embedding reagent is brought into contact with the surface of a selected area of ​​the silk cloth and kept there.

[0016] S70. Maintain contact between the selected area of ​​the silk fabric and the surface of the frozen embedding reagent in the lower half of the container of the first mold, place the first mold on dry ice, and completely freeze the frozen embedding reagent in the lower half of the container and the cell clusters on its surface.

[0017] S80. The selected area of ​​the separating fabric comes into contact with the surface of the frozen embedding reagent in the lower half of the container of the first mold;

[0018] S90. Place the first mold containing the upper half of the frozen embedding reagent in the container on dry ice to completely freeze the upper half of the frozen embedding reagent and the cell clusters on its surface, and freeze the upper half of the frozen embedding reagent and the lower half of the frozen embedding reagent into a single frozen embedding block and remove it, wherein the cell clusters are distributed in the middle position of the height of the frozen embedding reagent block.

[0019] The method for cryo-embedding pancreatic islet cell clumps according to some embodiments of this application further includes S100. Wrapping the cryo-embedding block in tin foil.

[0020] According to some embodiments of the present application, the method for cryo-embedding pancreatic islet cell clusters includes, in step S10, obtaining the pipette tip containing the culture medium liquid containing the cell clusters, which includes: shaking the culture device to gather the pancreatic islet cell clusters in the center of the culture device, the pipette drawing up the culture medium liquid containing the cell clusters, and the pipette being suspended to allow the cell clusters to settle to the bottom of the pipette tip.

[0021] According to some embodiments of the present application, a method for cryo-embedding pancreatic islet cell clusters is provided, wherein in step S10, culture medium liquid containing cell clusters is dispensed, and dispensing is stopped once the visible aggregated human islet cell clusters in the pipette tip have been transferred.

[0022] According to some embodiments of this application, the method for cryo-embedding pancreatic islet cell clusters includes step S40 further comprising: subsequently, adding buffer solution dropwise to the aggregated cell clusters on the inner surface of the bottom of a silk cloth, and then making regional contact between the outer surface of the bottom of the silk cloth and absorbent paper laid on the lower plane of the silk cloth, thereby separating the cell clusters from the buffer solution on the silk cloth.

[0023] According to some embodiments of this application, a method for cryo-embedding pancreatic islet cell clusters is used, wherein in step S50, a silk cloth is unfolded into a plane, and then the unfolded silk cloth is laid on absorbent paper placed on the plane. Buffer solution is added dropwise to the silk cloth along the sparse area of ​​the cell cluster toward the aggregation area, so that the cell clusters in the sparse area move and aggregate toward the cell clusters in the aggregation area.

[0024] A method for cryopreservation of pancreatic islet cell clusters according to some embodiments of this application, wherein the mold includes

[0025] The first mold includes a receiving groove, the edge of the upper opening of the receiving groove extending upward to form an opening groove;

[0026] The second mold includes a receiving groove, the edge of the upper opening of the receiving groove extending upward to form an opening groove;

[0027] In step S70, the selected area of ​​the silk fabric is kept in contact with the surface of the frozen embedding reagent in the lower half of the receiving groove of the first mold, and the opening groove of the second mold is limited in the opening groove of the first mold, so that the bottom surface of the receiving groove of the second mold is inserted into the receiving groove of the first mold and abuts against the selected area of ​​the silk fabric, so as to abut and keep the selected area of ​​the silk fabric in contact with the surface of the frozen embedding reagent in the lower half of the receiving groove of the first mold.

[0028] A method for cryo-embedding pancreatic islet cell clusters according to some embodiments of this application, wherein:

[0029] Preferably, the pipette is held vertically for 15-20 seconds;

[0030] Preferably, in step S10, the ejection is performed at a constant speed;

[0031] Preferably, in step S20, the silk cloth folded into a funnel shape is tilted at 60° and aligned with the tip of the pipette;

[0032] Preferably, in step S20, the silk fabric folded into a funnel shape is 400 mesh silk fabric;

[0033] Preferably, in step S30, the staining agent is bromophenol blue; more preferably, the mass-to-volume ratio of bromophenol blue to buffer is 1 mg: 10 mL.

[0034] Preferably, in step S50, the selected area is a region with an area of ​​0.5cm × 0.5cm;

[0035] Preferably, in step S90, the frozen state is 70%~100%, preferably 70%.

[0036] In a second aspect, frozen embedding blocks of pancreatic islet cell clumps prepared according to any one of the methods described in some embodiments of this application.

[0037] On a third-party level, the application of frozen embedded blocks according to some embodiments of this application in the spatiotemporal omics serial sections of pancreatic islet cell clusters for DNA sequencing.

[0038] Beneficial effects:

[0039] The first aspect of this invention ensures cell viability and integrity by employing silk cloth transfer, bromophenol blue staining, gradient liquid adsorption, and the proper combination of silk cloth and mold to prevent cell cluster dispersion. Figure 2-6 (Verification), RNA integrity (RIN value > 4, measured to be 6.42~6.69, Table 3), meeting the requirements for spatiotemporal transcriptome detection.

[0040] The second aspect of this invention uses a stepwise filling process with a cryo-embedding reagent (steps S8 and S11 in the examples) combined with dry ice freezing (steps S9 and S12 in the examples) to prevent cell damage and maintain DNA / RNA stability. Figure 3-4 (Permeability test verification).

[0041] The third aspect of this invention improves high-precision aggregation and positioning by using pipette suspension (step S3 in the example) in conjunction with a silk cloth Buchner funnel structure (step S4 in the example), especially by utilizing the identification function of staining during operation and the step of centralizing cell clusters, so that the single embedding amount meets the requirements and the cell recovery rate is high.

[0042] In a fourth aspect, the invention provides staining visualization (step S5 in the embodiment) and mold mounting groove matching design (step S9 in the embodiment) to ensure precise centered positioning of cell clusters. Figure 1 Embedded block section), quickly identify the target area during slicing ( Figure 2 ).

[0043] In a fifth aspect, the present invention optimizes operational efficiency and cost. The hydrophobic properties of the silk fabric (400 mesh) enable the near-non-destructive separation of cell clusters (step S10 in the example), avoiding contamination by expensive reagents. The regional adsorption of absorbent paper (steps S5 and S6 in the example) shortens the liquid processing time.

[0044] The sixth aspect of this invention uses a modular mold design (steps S8-S12 of the embodiment) to support standardized production, which is suitable for clinical-grade pancreatic islet cell embedding (sample verification in Table 1-2).

[0045] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0046] Figure 1 The frozen embedded block is shown in the example.

[0047] Figure 2 The slice shown in the example has a cluster of pancreatic islet cells in the center, and the purple color around it is due to the refraction of air bubbles.

[0048] Figure 3 This is a fluorescence image of insulin.

[0049] Figure 4 Images of HE-stained sections and fluorescence images from permeation tests.

[0050] Figure 5 This is the pre-slicing result of sample D0-E2.

[0051] Figure 6 The results are the pre-slice results for samples E3-E1.

[0052] Figure 7 This is the LabChip test result for sample D0.

[0053] Figure 8 This is the LabChip test result for sample E1.

[0054] Figure 9 This is a diagram illustrating the operation. Detailed Implementation

[0055] The embodiments of this application are described in detail below with reference to the accompanying drawings, examples of which are shown in the drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout.

[0056] Example 1: A method for cryo-embedding pancreatic islet cell-like cell clusters according to the present invention is used for cryo-embedding human islet cell clusters. The cryo-embedded blocks are sliced, and the DNA of the human islet cell clusters is sequenced. Based on this objective, the method should aim to solve the problem of cryo-embedding viable human islet cell clusters while striving to maintain cell viability during the process. A sufficient number of cells should be effectively cryo-embedded, such as a single embedding volume of not less than 2500 IEQ / block, and the integrity of cellular RNA should be ensured.

[0057] This objective also includes ensuring the aggregation of cell clusters during the process and the effective number of embedded cell clusters, avoiding cell cluster dispersion or splashing, which would result in the waste of expensive cells.

[0058] This also includes the ability to effectively identify extremely small cell clusters throughout the embedding process, especially whether the cells are aggregated before transfer, whether the cell clusters are aggregated and transferred to the surface of the frozen embedding reagent, and the ability to accurately center the embedding position at the height of the embedding reagent block.

[0059] This objective may also include the ability to accurately identify areas of the slice containing cell clusters.

[0060] The method in this embodiment includes the following steps:

[0061] S1. Gently shake the culture apparatus containing human islet cell clusters to gather them in the center. The apparatus is a six-well plate; the shaking is to encourage the cell clusters to concentrate in the culture medium. A six-well plate is a commonly used multi-well culture plate in cell culture experiments, typically made of transparent, sterile polystyrene (PS) with a hydrophilic surface suitable for adherent cell growth. Each six-well plate has six independent wells arranged in 2 rows × 3 columns, with each well having a volume of approximately 3-5 mL. The wells are usually circular with flat bottoms to ensure consistent cell adhesion. Each well has a diameter of approximately 35 mm, providing sufficient depth to hold the culture medium and cell suspension. Additional components for the six-well plate may include a cover plate, vents, edge protrusions, and a marking area. The cover plate is transparent to allow microscopic observation. Vents are used to balance internal and external gases. Edge protrusions prevent the well covers from directly contacting the culture medium when stacked, avoiding cross-contamination. The marked area is a number / letter label (such as A1, B2) on the edge of the hole or the cover plate to facilitate the differentiation of different experimental groups.

[0062] S2. Use a pipette tip to aspirate the human islet cell clusters and culture medium liquid aggregated in the center of the culture device. In this embodiment, a 50 μL pipette and a 200 μL tip are used. Shaking the aggregated cell clusters in the previous step is to ensure that as many dispersed cell clusters as possible can be aspirated in this step. It is understood that the human islet cells / islet cell clusters involved in this invention are viable.

[0063] S3. Hold the pipette tip vertically for a certain period of time to allow the human pancreatic islet cell clusters to gather at the bottom of the tip. The holding time in this step is 15-20 seconds. This holding allows the cell clusters to gather towards the bottom of the tip due to gravity, achieving cell cluster aggregation and ensuring that the upper part of the pipette tip is primarily liquid, with a significantly larger volume than the liquid at the bottom, creating a clear area distinction.

[0064] S4. Place the bottom of the folded Buchner funnel-shaped fabric near the tip of the tilted pipette at a 60° angle, aligning the inner surface of the bottom of the fabric with the pipette tip. Preferably, the fabric is 400-mesh fabric sterilized by autoclaving. Fabrics (such as silk or untreated synthetic fibers) are hydrophobic and have better toughness than filter paper. This invention utilizes these properties to provide physical support for cell clusters, facilitating more thorough separation of the buffer solution from the cell clusters. The utilization of these properties will be further explained below.

[0065] The purpose of tilting is to reduce the gravitational force exerted entirely on the cells due to their own weight, thereby reducing the disruption of cell aggregation during the dispensing of cell-containing fluid by the pipette.

[0066] In this step, the silk fabric is folded into a Buchner funnel shape, forming a pointed space at the bottom of the fabric. Cells are transferred to the fabric within this space and continue to maintain their aggregated state through the pointed space. The purpose of this maintenance is that the cell groups are very small and easily dispersed; once dispersed, they are difficult to re-aggregate.

[0067] S5. Push the pipette piston with a continuous and uniform force to allow the culture medium liquid of the aggregated human islet cell clusters to saturate the fabric substrate from the pipette tip until the visible aggregated human islet cell clusters in the pipette tip have been transferred. Stop pushing the pipette piston to prevent the liquid in the upper part of the pipette tip from transferring to the fabric.

[0068] After transfer, absorbent paper is laid flat and applied to the underside and outer surface of the silk fabric to regionally attach the cell clusters. This allows for timely absorption of the culture medium liquid after cell cluster transfer, separating the cell clusters from the liquid on the silk fabric and maintaining their aggregation. The contact is a gentle, area-specific contact to further maintain cell cluster aggregation. The reason for absorbing liquid with absorbent paper after transfer is that the increased frequency of absorption during transfer makes the silk fabric more prone to wrinkling and folding, which could cause other areas of the silk fabric—areas not currently occupied by the cell clusters—to come into contact with the cell clusters and disperse them.

[0069] The previous step achieved the aggregation of cell clusters in the liquid at the bottom of the pipette tip. During the transfer of cell clusters using the pipette, the cell clusters at the bottom of the tip are initially transferred to the fabric with a small amount of liquid. However, pancreatic islet cell clusters are extremely small. After the visible cell clusters at the bottom of the tip have been transferred, the pipette tip is mostly filled with culture medium liquid, which is no longer significant for cell cluster transfer. If this portion of liquid continues to be transferred from the pipette to the fabric, for a pneumatically driven pipette, it may cause the aggregated cell clusters at the bottom of the funnel-shaped fabric to be dispersed by the impact, causing the cell clusters to scatter on the fabric. To maintain cell cluster aggregation, once the visible aggregated pancreatic islet cell clusters in the pipette tip have been transferred (i.e., there are no more clearly visible cell clusters in the tip), the pipette piston should be stopped immediately to prevent the cell clusters transferred to the fabric from being dispersed by subsequent liquid impacts, thus affecting cell cluster aggregation. Furthermore, during and / or after the transfer, the absorbent paper is used to make regional contact with the outer surface of the bottom of the silk cloth, which allows for liquid absorption and separation of the human pancreatic islet cell clusters from the liquid. This achieves the aggregation and transfer of cell clusters to the silk cloth and their retention, while also achieving separation from the liquid.

[0070] S5. Using a disposable Pasteur pipette, slowly add a buffer solution containing bromophenol blue dropwise to the clusters of cells adhering to the inner surface of the bottom of the silk fabric, ensuring the buffer solution completely covers the islet cell clusters, thus staining the cell clusters. The dropwise addition of the bromophenol blue buffer solution using the Pasteur pipette utilizes gravity to draw the adsorbed cell clusters from the inner surface of the silk fabric towards the inner surface of the bottom of the Buchner funnel-shaped fabric. This process can be repeated until the islet cells show a distinct blue tint. In this example, 0.005g of bromophenol blue is dissolved in 50ml of PBS buffer solution and shaken thoroughly. If any bromophenol blue remains undissolved, small amounts of PBS solution can be added to adjust the concentration until the bromophenol blue solid is fully dissolved. The mass-to-volume ratio of bromophenol blue to buffer solution is 1mg:10mL.

[0071] S6. After staining, use absorbent paper laid flat to regionally attach the cell clusters to the underside and outer surface of the silk fabric. This allows for timely absorption of the buffer solution after staining, enabling the cell clusters to separate from the buffer solution on the silk fabric and remain aggregated. The attachment contact is a gentle contact over a defined area to further maintain cell cluster aggregation. Absorption after staining is necessary because aspiration during staining can easily cause wrinkles and folds in the silk fabric, allowing other areas of the fabric, i.e., areas outside the current cell cluster location, to come into contact with the cell clusters and disperse them.

[0072] As one implementation method, a disposable Pasteur pipette can be used to slowly drop PBS buffer onto the aggregated cell clusters attached to the inner surface of the bottom of the silk fabric. After rinsing off excess PBS buffer containing bromophenol blue, absorbent paper is laid flat and regionally attached to the bottom and outer surface of the silk fabric. Utilizing the hydrophobic properties of the silk fabric and the absorbent properties of the absorbent paper, the absorbent paper absorbs the buffer buffer in a timely manner, leaving the silk fabric with virtually no PBS buffer. This achieves complete separation of the cell clusters and the buffer buffer, aiming to avoid liquid adhesion to the surface of the cell clusters, which would prevent the subsequent DNA transcription chip from directly contacting the cells themselves. Furthermore, it avoids dilution of the DNA concentration by the liquid, which would reduce the detection sensitivity.

[0073] S7. Unfold the silk fabric into a flat surface. After staining, the cell clusters turn blue, forming blue aggregation areas of varying degrees on the silk surface. This allows for clear differentiation between the silk fabric and the cell clusters, while unstained cell clusters show less differentiation on the silk. This clear distinction makes it easy to differentiate the distribution of cell clusters on the silk. In one embodiment, the silk fabric is laid on absorbent paper placed flat. Using a disposable Pasteur pipette, buffer solution is slowly added dropwise to the silk fabric along the sparse areas of the cell clusters towards the aggregation areas. This causes the cell clusters in the sparse areas to aggregate towards the aggregation areas, maximizing the aggregation of cell clusters within a specific area. During this process, the absorbent paper absorbs the buffer solution, separating the cell clusters from the buffer solution. In this embodiment, an area of ​​0.5cm × 0.5cm is selected. This method relies on the clear partitioning of cell clusters formed by staining in the previous steps. In another embodiment, the silk fabric is unfolded into a plane, and a certain area with as many clustered cells as possible is selected. In this embodiment, the area is 0.5cm × 0.5cm. This method relies on the clear partitioning of the cell clusters formed by staining in the steps. This distinction not only distinguishes the cell clusters from the silk fabric, but also clearly distinguishes the degree of aggregation of the cell clusters.

[0074] S8. Add cryoembedding reagent, which is a gel-like liquid, to the receiving groove of the first mold. The mold includes a receiving groove, which is rectangular, preferably square, with the edge of the upper opening extending upwards to form an opening recess. The cryoembedding reagent is added until it reaches half the volume of the receiving groove of the first mold, i.e., the depth of the cryoembedding reagent in the receiving groove reaches half the height of the groove. Thus, the first mold has a first groove extending upwards from the opening edge of the receiving groove, forming an upper opening, and a second groove opening at the bottom. The first mold is placed on dry ice for incomplete freezing, such as 70% frozen into a solid state and 30% still in a gel-like liquid state. Preferably, the bottom surface of the receiving groove is placed on dry ice so that freezing occurs from the bottom of the cryoembedding reagent upwards.

[0075] S9. The shape and area of ​​the receiving opening of the first mold receiving groove are basically consistent with the shape and area of ​​the selected area of ​​the silk fabric. The unfolded flat silk fabric is placed on one hand (such as the left hand) with the cell cluster facing upwards, and the first mold is placed on the other hand (such as the right hand) with the receiving opening of the first mold facing upwards. Move it horizontally so that the selected area of ​​the silk fabric is basically flush with the receiving opening of the first mold (positional correspondence). One hand (e.g., left hand) inverts an unfolded flat piece of silk fabric. The inverted fabric is moved, aligning a selected area with the receiving opening of the first mold held by the other hand (e.g., right hand). This alignment process also facilitates the staining of the cell clusters. The selected area (cell aggregation area) of the silk fabric adheres to the surface of the cryopreservation reagent inside the receiving groove of the first mold. The bottom surface of the receiving groove of the second mold extends into the receiving groove of the first mold, covering the selected area of ​​the silk fabric. The opening groove of the second mold is confined within the opening groove of the first mold, and the bottom surface of the receiving groove of the second mold extends into the receiving groove of the first mold, so that the bottom surface of the receiving groove of the second mold lightly touches the selected area of ​​the silk fabric. The silk fabric outside the selected area is scattered on the inclined inner wall of the first mold. Because the cell clusters are extremely small and light, the silk fabric's adsorption capacity means that the cell clusters will not detach from the silk fabric for a certain period after inversion. Therefore, this invention selects an easily manipulated area of ​​the silk fabric for inversion and flipping.

[0076] Understandably, incomplete freezing involves maintaining a certain surface area of ​​the cryo-embedding reagent in the first mold's container as a gel-like liquid, allowing cell clusters to adhere to and embed in the gel-like liquid, thus preserving them within the cryo-embedding reagent. Incomplete freezing of the cryo-embedding reagent in the first mold before contact with the selected area of ​​the fabric is necessary because the second mold applies pressure to the first mold. Incomplete freezing enhances the support of the cryo-embedding reagent in the first mold, while maintaining its gel-like liquid state and binding with the cell clusters. Furthermore, incomplete freezing before contact reduces the freezing time required for cell embedding after contact (i.e., the time required for complete freezing), saving experimental time and reducing the time spent manipulating the cell clusters.

[0077] Place the first mold and the second mold on dry ice, preferably with the bottom surface of the first mold in contact with the dry ice, or with a larger contact area, so that the cryo-embedding reagent is completely frozen.

[0078] S10. After the cryo-embedding reagent has completely frozen and solidified, separate the second mold from the first mold by lifting the second mold upwards. Gently separate the fabric from the surface of the cryo-embedding reagent in the first mold's receiving groove. As mentioned above, the fabric (such as silk or untreated synthetic fibers) has good toughness. This invention utilizes this characteristic to provide physical support for the cell clusters and to completely separate them from the cryo-embedding reagent and cell clusters, ensuring that virtually no fabric fragments remain in the formed embedding block.

[0079] The aggregated cell clusters, by applying slight pressure between the fabric and the first mold, can fall onto the surface of the cryo-embedding reagent. Because the reagent is not completely frozen, a certain area of ​​the surface remains in a gel-like liquid state, allowing for good embedding. Once the reagent is completely frozen and solidified, the cell clusters can be stably fixed to the reagent surface. If, as in the previous step, the bottom surface of the first mold is in contact with dry ice, or has a larger contact area, then after the reagent is completely frozen and solidified, the resilient fabric can be completely separated from the cryo-embedding reagent, with minimal fabric fragmentation. In particular, if the area of ​​the fabric is much larger than the 0.5cm × 0.5cm area of ​​the container in which the fabric is in frozen contact, the fabric can be completely separated, achieving separation of the fabric from the cell clusters.

[0080] The fabric is separated only after the cryo-embedding reagent has completely frozen and solidified. This is because if the fabric is separated before complete freezing, the binding between the cryo-embedding reagent and the cell clusters is poor, causing the cell clusters to disperse or even detach from the reagent during separation. Furthermore, separating the fabric at this point, with the bottom surface of the second mold's container in contact with the reagent surface, also causes the cell clusters to disperse or detach, making it difficult to maintain their aggregation.

[0081] In particular, using an open freezing method can affect DNA sequencing of cell clusters. For example, enzyme-induced degradation of DNA and RNA within the cell clusters reduces their concentration, impacting cell viability and sequencing accuracy. However, common materials may easily adhere to gel-like liquid reagents during freezing, making them difficult to remove. For instance, filter paper, after freezing, tends to fragment and become embedded in the frozen block. This invention uses silk fabric with a contact surface much larger than its contact surface with the reagents, allowing the fabric to be removed intact after complete freezing, minimizing the impact of material removal on cell cluster aggregation.

[0082] In a preferred embodiment, before the frozen embedding reagent is extruded from the reagent tube, the reagent tube is inverted to remove air bubbles, and then extruded into the first mold receiving groove, and smoothed with a tool.

[0083] S11. Cell clusters are fixed and aggregated on the upper surface of the frozen embedding reagent in the container of the first mold. The frozen embedding reagent is added to the other half of the container volume of the first mold, that is, the depth of the frozen embedding reagent in the container reaches the height of the container. The first mold with the upper half of the container containing the frozen embedding reagent is placed on dry ice to freeze the upper half of the frozen embedding reagent and the cell clusters on its surface, so that the upper half of the frozen embedding reagent and the lower half of the frozen embedding reagent are formed into a single frozen embedding reagent block. Preferably, the bottom surface of the first mold is in contact with the dry ice, or has a larger contact area. At this time, the cell clusters are distributed in the container, or in other words, in the middle of the frozen embedding reagent with its shape, and this distribution is a precise positional distribution.

[0084] S12. After the cryo-embedding reagent has solidified, the frozen-formed cryo-embedding reagent block can be removed from the receiving slot of the first mold using a small spoon or similar tool. This yields a cryo-embedding block in which human pancreatic islet cells are precisely distributed in the middle of the height of the reagent block. The purpose of the cell cluster being in the middle of the height of the reagent block is to provide strong protection for the cells and to support the cell cluster during the slicing process, thereby ensuring the integrity of the slices during the cryo-slicing process.

[0085] S13. Wrap the frozen embedded block in aluminum foil, record the information, and freeze it for transport purposes.

[0086] This invention aims to preserve cell viability through cryopreservation. A sufficient number of cells are effectively cryopreserved to ensure cellular RNA integrity. The embedding process employs various methods to minimize cell dispersion and maintain cell aggregation, ensuring that pancreatic islet cell clusters aggregate and are embedded within a specific central region of the embedding reagent block. Cell cluster staining effectively visualizes cell clusters that are difficult to distinguish visually during embedding, ensuring cell aggregation and accurately identifying areas containing cell clusters on the slide. This invention ensures that almost all cells taken from the culture medium are cryopreserved, minimizing cell waste and maintaining cell viability.

[0087] In this invention, through two transfers of viable cell clusters, stable and aggregated manipulations, along with staining and recognition techniques, enable the formation of a planar layer of aggregated cell clusters at the mid-height of the frozen embedding block. The cell clusters aggregate in a very small area (0.5cm × 0.5cm) (the actual aggregated area is smaller than this minimum area), distributing the cell clusters within a very small space—a thin layer at the mid-height of the frozen embedding block—significantly increasing the number of cell clusters per section. Based on a section size, from the initial discovery of cell clusters to the point where no more cell clusters are found, the thickness of the planar layer of aggregated cell clusters is generally around 3-4mm.

[0088] Experimental example: such as Figure 1 The image shows a frozen embedding block of human insulin cell clusters; the blue areas represent stained cells. The example shown is... Figure 1 The frozen embedded blocks shown were subjected to cryosectioning, and the sections were as follows: Figure 2 As shown. The cell staining method described above in this invention can accurately indicate the presence of cells in the slide.

[0089] like Figure 1 As shown, the experiment aimed to establish the final spatiotemporal transcriptome of pancreatic islet cells. To achieve this goal, preliminary quality assessment and tissue permeation fluorescence imaging were required. The quality assessment requirements were a RIN value >7 for the sample, satisfactory permeation, and a visible and clear fluorescence imaging result.

[0090] Table 1. Spatiotemporal transcriptome analysis of three groups of human islet samples.

[0091]

[0092] Figure 4 Table 1 shows HE staining images and fluorescence images from the permeation test of the samples. Figure 4 Based on the fluorescence imaging results from the permeation test, tissue samples A3 and B8 showed complete and clear tissue morphology after 18 minutes, with the high-resolution original image revealing tissue texture consistent with the H&E results. Tissue sample C6 showed complete and clear tissue morphology after 12 minutes, with the high-resolution original image revealing tissue texture consistent with the H&E results. Based on the H&E results and fluorescence signal intensity, recommended permeation times for the corresponding samples are proposed.

[0093] Table 2 Spatiotemporal transcriptome analysis samples from fourteen groups of human islet samples

[0094]

[0095] Figure 5 and Figure 6The results are as follows: 14 samples were sectioned in this experiment. During sectioning, samples D1, D7, and E3 detached completely and were subsequently collected after OCT adhesion. H&E staining was performed on the sections generated during the experiment. Some samples were not stained quickly, and no obvious abnormalities were observed on the sections of the stained tissues.

[0096] in:

[0097] The complete detachment of samples D1, D7, and E3 was due to temperature fluctuations during sample storage and transportation, which caused the upper and lower frozen embedded blocks to separate from each other.

[0098] The presence of cavitation in the frozen section of sample E1 is essentially due to the formation of ice crystals or the creation of physical gaps between water molecules within the tissue during freezing. Temperature fluctuations during sample storage and transportation cause recrystallization of remaining water molecules in certain areas. During slicing, temperature fluctuations within the microtome lead to recrystallization of water within the embedding block, preventing sufficient penetration of OCT into the tissue interstitial spaces.

[0099] Table 3. Results of RNA integrity testing for samples

[0100]

[0101] a. Take 10-20 10μm / 20μm slices from solid tissues such as heart, kidney, and brain. The amount of RNA extracted is sufficient for detection. If the tissue RNA is very little or difficult to extract, the number of genes captured by the chip may not meet the analysis requirements.

[0102] b. Normally embedded samples have a RIN value ≥4. If the quality control conditions are not met, it may affect library construction (such as the reverse transcription step) and data results, such as the number of MIDs or the number of genes identified.

[0103] Results: RNA was extracted from all samples in groups D and E in a single tube for integrity testing. The result was the RIN value of the combined samples. Both groups had RIN values ​​> 4, indicating that RNA integrity met the requirements.

[0104] This invention aims to visualize frozen-embedded and serially sectioned pancreatic islet cell-like cell clumps without affecting their function and DNA / RNA integrity. To this end, this invention stains the islet cell-like cell clumps without affecting the expression of macromolecules such as proteins, DNA, and RNA, as well as small molecules. Through standardized procedures, frozen-embedding of islet cell-like cell clumps is achieved, resulting in a sufficiently large sample volume within a specific embedding frame. This sample can then be analyzed via serial sections, and the RNA expression levels meet the requirements for spatiotemporal omics analysis, facilitating further omics analysis.

[0105] This invention addresses the lack of visibility of pancreatic islet cell-like cell clusters during cryo-embedding by using bromophenol blue staining to give the target cell clusters a blue hue, enabling visualization during embedding and sectioning. A 400-mesh silk cloth is used to filter moisture around the target cell clusters, ensuring their morphology and staining while removing surrounding moisture. The OCT (occlusive transdermal filler) at the bottom of the embedding cassette is cooled on dry ice to 100% or 70% freeze-thaw (adjusting from 100% freeze-thaw to 70% to embed more cell clusters). The stained cell clusters on the silk cloth are then inverted onto the cooled OCT, and another embedding cassette is used to press the target cell clusters down onto dry ice. After all the OCT has solidified, the silk cloth is removed, revealing the blue cell clusters in the target embedding area. The remaining space in the embedding cassette is then filled with OCT and placed back on dry ice to complete the cryo-embedding process. The single embedding volume shall not be less than 2500 IEQ / block (IEQ is the unit of counting pancreatic islet cell clusters, and a cell cluster with a particle size of 150μm is 1IEQ).

[0106] This invention offers the following advantages: it enables the visualization of cell clusters resembling pancreatic islet cells that are difficult to distinguish with the naked eye, without affecting their RNA, DNA, and protein expression. It allows for the cryopreservation of pancreatic islet cells aggregated within specific regions, ensuring that the RNA and DNA expression levels in the target region meet the requirements for spatiotemporal omics analysis and subsequent immunofluorescence experiments. Furthermore, it enables the generation of serial sections of islet cell-like cell clusters, allowing for the analysis and study of cells within cell clusters with different spatial distributions.

[0107] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," "counterclockwise," "axial," "radial," and "circumferential" indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing this invention and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0108] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0109] In this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," "linking," and "fixing," etc., should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection, an electrical connection, or a connection that allows communication between them; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components, unless otherwise explicitly limited. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0110] In this invention, unless otherwise explicitly specified and limited, "above" or "below" the second feature can mean that the first feature is in direct contact with the second feature, or that the first feature is in indirect contact with the second feature through an intermediate medium. Furthermore, "above," "over," and "on top" of the second feature can mean that the first feature is directly above or diagonally above the second feature, or simply that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature can mean that the first feature is directly below or diagonally below the second feature, or simply that the first feature is at a lower horizontal level than the second feature.

[0111] In this invention, the term "and / or" describes the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following associated objects are in an "or" relationship. "At least one" refers to one or more; "at least one of A and B," similar to "A and / or B," describes the relationship between associated objects, indicating that three relationships can exist. For example, at least one of A and B can represent: A alone, A and B simultaneously, or B alone.

[0112] In this invention, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0113] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for cryopreservation of pancreatic islet cell clusters, characterized in that, Includes the following steps: S10. Tilt the folded silk cloth into a funnel shape and aim it at the tip of the pipette containing the culture medium liquid containing cell clusters. Push the culture medium liquid containing cell clusters from the tip of the pipette toward the inner surface of the silk cloth tip, transferring and gathering the cell clusters on the inner surface of the silk cloth tip. S20. Make regional contact between the outer surface of the bottom of the silk cloth and the absorbent paper laid on the plane below the silk cloth to separate the cell clusters from the culture medium liquid on the silk cloth. S30. Add buffer solution containing staining agent dropwise to the aggregated cell clusters on the inner surface of the bottom of the silk fabric to stain the cell clusters; S40. Make regional contact between the outer surface of the bottom of the silk cloth and the absorbent paper laid on the plane below the silk cloth to separate the cell clusters from the buffer solution on the silk cloth. S50. Unfold the silk cloth into a plane, and the area where the cell clusters on it are stained is the selected area of ​​the silk cloth; S60. The lower half of the freezing embedding reagent is added to the receiving groove of the first mold placed horizontally, and placed on dry ice for incomplete freezing. The surface of the incompletely frozen embedding reagent is brought into contact with the surface of a selected area of ​​the silk cloth and kept there. S70. Maintain contact between the selected area of ​​the silk fabric and the surface of the frozen embedding reagent in the lower half of the container of the first mold, place the first mold on dry ice, and completely freeze the frozen embedding reagent in the lower half of the container and the cell clusters on its surface. S80. The selected area of ​​the separating fabric comes into contact with the surface of the frozen embedding reagent in the lower half of the container of the first mold; S90. Place the first mold containing the upper half of the frozen embedding reagent in the container on dry ice to completely freeze the upper half of the frozen embedding reagent and the cell clusters on its surface, and freeze the upper half of the frozen embedding reagent and the lower half of the frozen embedding reagent into a single frozen embedding block and remove it, wherein the cell clusters are distributed in the middle position of the height of the frozen embedding reagent block.

2. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, It also includes S100. Foil-wrapped frozen embedded block.

3. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, in, The pipette tip for obtaining culture medium liquid containing cell clusters in step S10 includes: shaking the culture device to gather the pancreatic islet cell clusters in the center of the culture device, the pipette drawing up the culture medium liquid containing the cell clusters, and the pipette being suspended to allow the cell clusters to settle to the bottom of the pipette tip.

4. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, in, In step S10, culture medium liquid containing cell clusters is dispensed, and dispensing stops once the visible aggregated human islet cell clusters in the pipette tip have been transferred.

5. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, in, Step S40 further includes: then, adding buffer solution dropwise to the aggregated cell clusters on the inner surface of the bottom of the silk cloth, and then making regional contact between the outer surface of the bottom of the silk cloth and the absorbent paper laid on the plane below the silk cloth, so as to separate the cell clusters from the buffer solution on the silk cloth.

6. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, in, In step S50, the silk cloth is unfolded into a flat surface. Then, the unfolded silk cloth is laid on the absorbent paper placed on the flat surface. Buffer solution is added drop by drop to the silk cloth along the sparse area of ​​the cell clusters toward the aggregated area, so that the cell clusters in the sparse area move and aggregate toward the cell clusters in the aggregated area.

7. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, in, Mold includes The first mold includes a receiving groove, the edge of the upper opening of the receiving groove extending upward to form an opening groove; The second mold includes a receiving groove, the edge of the upper opening of the receiving groove extending upward to form an opening groove; In step S70, the selected area of ​​the silk fabric is kept in contact with the surface of the frozen embedding reagent in the lower half of the receiving groove of the first mold, and the opening groove of the second mold is limited in the opening groove of the first mold, so that the bottom surface of the receiving groove of the second mold is inserted into the receiving groove of the first mold and abuts against the selected area of ​​the silk fabric, so as to abut and keep the selected area of ​​the silk fabric in contact with the surface of the frozen embedding reagent in the lower half of the receiving groove of the first mold.

8. The method for cryopreservation of pancreatic islet cell clusters according to claim 3, characterized in that, in: Hold the pipette vertically for 15-20 seconds.

9. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, In step S10, the ejection is performed at a constant speed.

10. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, In step S10, the silk cloth folded into a funnel shape is tilted at 60° and aligned with the tip of the pipette.

11. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, In step S10, the silk cloth folded into a funnel shape is 400 mesh silk cloth.

12. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, In step S30, the staining agent is bromophenol blue.

13. The method for cryopreservation of pancreatic islet cell clusters according to claim 12, characterized in that, The mass-to-volume ratio of bromophenol blue to buffer solution is 1 mg: 10 mL.

14. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, In step S50, the selected area is a region with an area of ​​0.5cm × 0.5cm.

15. The method for cryopreservation of pancreatic islet cell clusters according to claim 1, characterized in that, In step S90, the frozen state is 70%~100%.

16. The method for cryopreservation of pancreatic islet cell clusters according to claim 15, characterized in that, The frozen state is 70%.

17. A frozen embedding block of pancreatic islet cell clumps prepared by the method of any one of claims 1-16.

18. The use of the frozen embedded block of claim 17 in spatiotemporal omics serial sections of pancreatic islet cell clusters.

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