Preparation method of single-cell suspension from mineralized shell suitable for crossing animals

Abstract

The invention discloses a preparation method of a mineralized shell-derived single-cell suspension applicable across animals, and belongs to the technical field of cell suspension preparation. Aiming at the condition that a mineralized shell structure is relatively hard, decalcification pretreatment, decalcification liquid, a decalcification process, a digestion method and the like are optimized, and the high-quality single-cell suspension is obtained. The preparation method of the single-cell suspension is suitable for obtaining living cells from mineralized shells crossing animals, the mineralized shells comprise exoskeletons, shells and thorns, crushing treatment and non-enzymatic pure physical grinding digestion are adopted in the method, the experiment time consumption is shorter, the operation steps are simple and convenient, biological damage is greatly reduced, and the method is suitable for large-scale industrial production. The integrity and activity of the cells are reserved to the maximum extent, and the requirements of construction of various single cell sequencing platform libraries can be fully met.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell suspension preparation, and particularly relates to a method for preparing single-cell suspensions from mineralized shells (such as exoskeletons, shells, and spines) with strong universality, and is particularly suitable for preparing single-cell suspensions from mineralized shell samples of different invertebrate phyla (such as mollusks, echinoderms, and arthropods). Background Art

[0002] For a long time, the mineralized shells of invertebrates (such as mollusk shells, arthropod exoskeletons, etc.) have been regarded as inanimate inert mineralizations - "inanimate stones" in the biological field; in contrast, vertebrate bones are considered to be living "organs" with living cells in their internal bone marrow. Based on this cognitive difference, almost all existing single-cell suspension preparation technologies focus on the dissociation of cells from soft tissues such as vertebrate bone marrow, and the related research on invertebrate mineralized shells has been in a long-term blank state. The previous research of this research team has first subverted this traditional cognition: through live cell tracing and transcriptome evidence, it has been confirmed that there are living cells in the mineralized shells of invertebrates represented by shellfish. This has laid a biological foundation for single-cell research from invertebrate mineralized shells. Based on this, we have first achieved an attempt to prepare single-cell suspensions from invertebrate mineralized shells.

[0003] However, there are significant differences between the mineralized exoskeletons of vertebrates and invertebrates: the skeletons of vertebrates have hydroxyapatite (calcium phosphate) as the main mineral phase (accounting for 60-70%), and the mineral crystals have a porous nanostructure, which is beneficial for cell infiltration and material exchange; while the mineralized exoskeletons of invertebrates are mainly composed of aragonite / calcite (calcium carbonate) (mineralization degree > 95%), and the crystal arrangement is highly dense, forming an almost continuous mineralization barrier; the bone tissue of vertebrates presents a hierarchical porous structure of "mineral - collagen fiber", and cells are easy to release; while the mineralized exoskeleton of invertebrates is a sandwich structure of "mineralized matrix embedding organic matter layer", and living cells are deeply buried in micron-scale calcification units and are difficult to release. Therefore, the existing methods for preparing single-cell suspensions from vertebrate bones are not applicable to the mineralized exoskeletons of invertebrates, and cells cannot be released by simple pipetting or enzymatic digestion, etc. This leads to four major technical bottlenecks for traditional vertebrate bone treatment methods in invertebrate samples: 1) Decalcification kinetic barrier: The dissolution kinetics of calcium carbonate is significantly slower than that of calcium phosphate. The traditional EDTA decalcifying solution (0.5M) requires more than 72 hours for the exoskeletons of crustaceans, resulting in cells being exposed to an osmotic stress environment for a long time; 2) Aggravated chemical damage: Strong acid decalcifying agents (such as hydrochloric acid) cause a sudden drop in local pH (pH < 2.0) when dissolving calcium carbonate, directly dissolving cell membrane lipids and activating nucleases, resulting in RNA degradation and cell death; 3) Failure of enzymatic digestion strategy: The dense mineralized layer hinders the penetration of enzyme molecules, and the enzyme molecules cannot penetrate the mineral layer to reach the organic matter layer. Increasing the enzyme concentration / prolonging the digestion time will also damage cell surface receptors and interfere with the molecular fidelity of single-cell sequencing; 4) Mechanical dissociation damage: The remaining organic matter layer after decalcification has extremely high toughness, and mechanical external forces are likely to cause the plasma membrane of shell-forming cells without cell wall protection to rupture. The existing methods rely on high-speed centrifugation or violent grinding to break tissues after enzymatic digestion, and this step is likely to cause mechanical damage to cells, resulting in a decrease in the viable cell rate. In addition, the existing technologies generally have problems of complex operation procedures and long time consumption (usually several days to several weeks), which further limits their application in multi-species comparative studies.

[0004] To address the above problems, there is an urgent need in the field to develop a method for preparing single-cell suspensions from mineralized exoskeletons that is highly universal, can maintain excellent cell viability, and has a simple operation and is applicable across animal phyla. In particular, it is necessary to solve the key technical bottlenecks such as the decalcification kinetic differences of invertebrate samples, non-enzymatic mild digestion, and short-time and high-efficiency treatment, so as to meet the stringent requirements of single-cell multi-omics research for cell integrity, viability, and preparation throughput. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides a method for preparing a single-cell suspension derived from a mineralized shell applicable across different animal phyla. Through an innovative ECTP composite decalcifying solution and a non-enzymatic physical digestion system, the following are achieved: 1) Balance between decalcification efficiency and cell viability: The ECTP decalcifying solution (pH 7.0 - 7.4) strongly chelates calcium ions through the synergistic effect of multi-carboxyl chelating agents to accelerate the dissolution of calcium carbonate, while trehalose maintains the stability of the cell membrane, shortening the decalcification time and achieving a cell survival rate > 90%; 2) Non-enzymatic physical digestion: Excluding exogenous enzymes, gradient centrifugation (400 - 600g) combined with gentle pipetting / grinding is adopted to maximize the preservation of the integrity of cell surface proteins; 3) Cross-species adaptability: By adjusting the osmotic pressure of the buffer solution and the concentration of trehalose in ECTP, the physiological requirements of different phyla such as mollusks and crustaceans are adapted.

[0006] The technical solution of the present invention mainly includes the following content: A method for preparing a single-cell suspension derived from a mineralized shell applicable across different animal phyla, comprising the following steps: (1) Crushing treatment of the mineralized shell: Obtain the mineralized shell from a living species; after cleaning, polish and break it into small pieces; (2) Place the small pieces in the ECTP composite decalcifying solution, which contains EDTA decalcifying solution, 50 - 200 mM sodium citrate, 100 - 500 mM trehalose and phosphate buffer solution; (3) Physical digestion: Wash the decalcified organic matter layer with a washing solution, collect the washed organic matter, and perform grinding and pipetting; (4) Centrifugation: Filter the digestion solution obtained in step (3) through a cell sieve, collect the filtered cell suspension, centrifuge at low temperature, and collect cell pellet A; (5) Resuspend the cells: Remove the supernatant, and resuspend the cells with a washing solution to obtain cell suspension A; (6) Secondary centrifugation: Centrifuge cell suspension A at low temperature and collect cell pellet B; (7) Secondary resuspension of the cells: Remove the supernatant, and resuspend the cells with a washing solution to obtain a single-cell suspension of the mineralized shell.

[0007] Preferably, the mineralized shell is derived from invertebrates, and the mineralized shell includes exoskeletons, shells or spines.

[0008] Preferably, the invertebrates include mollusks, echinoderms or arthropods.

[0009] Preferably, the invertebrates include scallops, sea urchins or crabs.

[0010] Preferably, the washing solution is phosphate buffer solution.

[0011] Preferably, the cell sieve is a cell sieve with a pore size of 40 μm.

[0012] Preferably, the centrifugation temperature is 4°C.

[0013] Preferably, the centrifugation speed is 400 - 600 g, and the centrifugation time is 4 - 5 min.

[0014] Preferably, the crushing is as follows: Select species with good activity, brush the surface clean with a brush in seawater or clear water, grind its surface layer into a thin layer with a grinding gun, and break it into small pieces of about 0.5 cm with tweezers or pliers.

[0015] The present invention further relates to a single - cell suspension derived from the mineralized shell of invertebrates, and the single - cell suspension is prepared by the above - described single - cell suspension preparation method. The single - cell suspension maintains the original biological characteristics and activity of the cells, has good cell morphology, no agglomeration, and meets the requirements for loading onto various single - cell sequencing platforms.

[0016] The beneficial effects of the present invention are as follows: 1. By adopting crushing treatment, after cleaning and grinding the mineralized shell and further breaking it into small pieces for pulverization, the process of removing minerals is accelerated, the decalcification efficiency is significantly improved, the decalcification time is shortened, and at the same time, the integrity and activity of the cells are maximally retained.

[0017] 2. The ECTP composite system proposed in the present invention as a decalcifying solution has its core advantage in the synergistic effect of each component on the decalcification efficiency and cell activity: As a classic strong chelating agent, EDTA can widely and efficiently complex metal ions such as calcium and iron with its stable six - tooth coordination structure formed by four carboxyl groups and two amino groups, and is suitable for deep decalcification; however, due to its large molecular weight and rigid structure, the chelating rate is relatively slow. Sodium citrate, as a tridentate organic acid salt, although its chelating range is limited, can quickly bind free Ca 2+, significantly improving the initial decalcification rate. The combination of EDTA and sodium citrate not only enables the rapid removal of calcium ions in the initial stage but also ensures long-term complexation stability, thus significantly enhancing the overall decalcification efficiency. Meanwhile, as an osmoprotectant, trehalose can stabilize the lipid bilayer structure of the cell membrane and protect intracellular proteins when the osmotic pressure fluctuates due to changes in the ion concentration of the decalcifying solution, effectively reducing cell death caused by osmotic stress. Sodium citrate also has good buffering capacity, which can neutralize the local acidic microenvironment that may be generated during decalcification and avoid damage to the cell membrane by strong acids. The synergistic effect of trehalose and sodium citrate together improves the cell survival rate. In addition, by adjusting the concentration of phosphate buffer solution, the stability of the ion environment during decalcification is further optimized, reducing cell rupture caused by osmotic pressure fluctuations. The present invention also proposes to adopt a staged decalcification strategy and monitor the decalcification saturation in real time, and replace the fresh decalcifying solution in a timely manner, so as to avoid damage to the soft tissue matrix caused by over-decalcification.

[0018] 3. The non-enzymatic pure physical grinding method proposed by the present invention is used to treat the decalcified organic matter layer. By repeatedly rolling it with a grinding rod or repeatedly blowing and sucking it with a pipette, single cells can be effectively released, greatly reducing biological damage and maintaining the original biological characteristics and activity of the cells. This method is time-consuming, simple to operate, and has a high yield of viable cells.

[0019] 4. The single-cell suspension prepared by the present invention has been successfully applied to the preparation of single-cell suspensions of mineralized shells of three different species, and data output has been successfully obtained through the 10× Genomics platform. The results meet or even exceed expectations. It is proved that this method has high cross-species applicability and reliable experimental results, providing effective technical support for the research on mineralized shell cells of different species. Description of the Drawings

[0020] Figure 1 : Diagram of the fragmentation of the mineralized shell.

[0021] Figure 2 : In Example 1 of the present invention, the broken mineralized shell was placed in the ECTP decalcifying solution for decalcification, and finally the organic matter layer was obtained.

[0022] Figure 3 : Microscopic photograph of the organic matter layer of the mineralized shell in Example 1 of the present invention after physical grinding digestion, filtration, centrifugation at a speed of 400 - 600 g, and staining.

[0023] Figure 4 : Single-cell atlas of the single-cell suspension of the mineralized shell of Patinopecten yessoensis in Example 1 of the present invention. Among them, 0: long-term hematopoietic stem cells; 1: short-term hematopoietic stem cells; 2: blood cells; 3: mesenchymal stem cells; 4: macrophages. "cellratio" in the figure refers to the proportion of each cell population.

[0024] Figure 5 : In Example 4 of the present invention, the mineralized shell of the sea urchin was broken and finally decalcified to obtain the organic matter layer.

[0025] Figure 6 : Microscopic photograph of the organic matter layer of the mineralized shell of the sea urchin in Example 4 of the present invention after physical grinding digestion, filtration, centrifugation at 400 - 600 g rotation speed, and staining.

[0026] Figure 7 : Single - cell UMAP map obtained by scRNA - seq analysis of the mineralized shell of the sea urchin in Example 4 of the present invention. Among them, SnC0: hematopoietic stem cell - like cells; SnC1: blood cells; SnC2: mesenchymal stem cells; SnC3: macrophages.

[0027] Figure 8 : Diagram of obtaining the mineralized shell of the swimming crab and finally obtaining the organic matter layer in Example 5 of the present invention.

[0028] Figure 9 : Microscopic photograph of the organic matter layer of the mineralized shell of the swimming crab in Example 5 of the present invention after physical grinding digestion, filtration, centrifugation at 400 - 600 g rotation speed, and staining. Dead cells are marked with red circles.

[0029] Figure 10 : Single - cell UMAP map obtained by scRNA - seq analysis of the mineralized shell of the swimming crab in Example 5 of the present invention. Among them, PtC0: hematopoietic stem cell - like cells; PtC1: blood cells; PtC2: mesenchymal stem cells; PtC3: macrophages.

[0030] Figure 11 : Microscopic photograph of the mineralized shell of the Patinopecten yessoensis in Comparative Example 1 of the present invention after decalcification treatment with EDTA decalcifying solution, physical grinding digestion, filtration, centrifugation at 400 - 600 g rotation speed, and staining. Dead cells are marked with red circles Figure 12 : The present invention uses the 10× Genomics platform to perform single - cell transcriptome sequencing on the ECTP decalcification group (Example 1) and the EDTA decalcification group (Comparative Example 1), and performs a correlation analysis diagram with the Bulk transcriptome data of the original mineralized shell of the Patinopecten yessoensis. Among them, A: Scatter diagram of the correlation between the EDTA decalcification group and the Bulk transcriptome of the original mineralized shell; B: Scatter diagram of the correlation between the ECTP decalcification group and the Bulk transcriptome of the original mineralized shell.

[0031] Figure 13 : Microscopic photograph of the mineralized shell of the Patinopecten yessoensis in Comparative Example 2 of the present invention after dissociation with an enzyme mixture solution (2 mg / mL collagenase II, 0.25% trypsin), filtration, centrifugation at 400 - 600 g rotation speed, and staining. Dead cells are marked with red circles.

[0032] Figure 14 : In this invention, the 10× Genomics platform was used to perform single-cell transcriptome sequencing on the non-enzymatic digestion group (Example 1) and the enzymatic digestion group (Comparative Example 2), and a correlation analysis graph was made with the Bulk transcriptome data of the original mineralized shell of the Yesso scallop. Among them, A: Scatter plot of the correlation between the enzymatic digestion group and the Bulk transcriptome of the original mineralized shell; B: Scatter plot of the correlation between the non-enzymatic digestion group and the Bulk transcriptome of the original mineralized shell. Detailed implementation manners

[0033] To better understand the technical content of this invention, the following will further illustrate this invention in combination with specific examples and drawings.

[0034] In the following examples, unless otherwise specified, the substances used are all conventional commercially available products.

[0035] PBS (phosphate buffer solution) was purchased from Sangon Biotech, China (product number: SB0627); EDTA decalcifying solution (pH 7.2) was purchased from Solarbio, China (product number: E1171-500ml); sodium citrate (pH 7.0) was purchased from Beyotime, China (product number: ST368); D-trehalose was purchased from Solarbio, China (product number: G8570); 0.4% trypan blue staining solution was purchased from Solarbio, China (product number: C0040); collagenase II was purchased from Solarbio, China (product number: C8150); 0.25% trypsin was purchased from HyClone, USA (product number: SH30042.02).

[0036] The components of the ECTP decalcifying solution are shown in Table 1.

[0037] Table 1 Composition ratio of the ECTP composite decalcifying solution

[0038] Example 1: Preparation method of single-cell suspension from mineralized shell applicable across animal phyla (1) Crushing treatment of the mineralized shell: Obtain the mineralized shell from the living Yesso scallop species; brush the surface clean with a brush in water, wash it, polish it, and further break it into small pieces with a diameter of about 0.5 cm, as Figure 1 shown; (2) Place the broken small pieces into the pre-prepared ECTP composite decalcifying solution. The composition ratio of the ECTP composite decalcifying solution is shown in Table 2. Place it on a shaker and observe the decalcification saturation in real time during the decalcification process, and replace the fresh decalcifying solution in a timely manner.

[0039] Table 2 Composition ratio of the ECTP composite decalcifying solution

[0040] (3) Physical digestion: Place the decalcified organic matter layer (as shown in Figure 2 ) in phosphate buffer, wash it, and transfer it to a sterile 1.5 mL centrifuge tube. In the phosphate buffer, use a sterile grinding rod to crush it several times until the organic matter disappears, and then gently pipette it with a sterile wide-mouth resin-coated pipette tip.

[0041] (4) Centrifugation: Filter the digested solution obtained in step (3) through a 40 μm cell strainer, collect the filtered cell suspension, place it in a 4°C centrifuge, and centrifuge it at 400 g for 4 - 5 min to collect cell pellet A.

[0042] (5) Resuspension of cells: Remove the supernatant, add pre-cooled PBS buffer to cell pellet A, and gently pipette it 10 times with a sterile wide-mouth pipette tip to resuspend the cells, obtaining cell suspension A.

[0043] (6) Secondary centrifugation: Place cell suspension A in a 4°C centrifuge and centrifuge it at 600 g for 4 - 5 min to collect cell pellet B.

[0044] (7) Secondary resuspension of cells: Remove the supernatant, add 50 μL of pre-cooled PBS buffer to cell pellet B, and gently pipette it 10 times with a sterile wide-mouth pipette tip to resuspend the cells, enriching to obtain a single-cell suspension of the mineralized shell.

[0045] (8) Staining: Mix the single-cell suspension with 0.4% trypan blue staining solution at a volume ratio of 9:1 for staining. Pipette a small amount of the stained cells, and use a hemocytometer to count the cell viability. The results are as shown in Figure 3 .

[0046] Results description: Figure 3 The results show that there are no obvious debris in the background, the cells are clearly visible, in good morphology, and there is almost no cell clumping; the cell concentration of the single-cell suspension of the mineralized shell of Patinopecten yessoensis is relatively high, there are no cell clusters or other impurities larger than 40 μm, and the cell clumping rate is less than 1%, all meeting the requirements for loading onto various single-cell sequencing platforms. Perform single-cell transcriptome sequencing on the obtained single-cell suspension of the mineralized shell. The results are as shown in Figure 4 .

[0047] As shown in Figure 4 , construct a single-cell atlas for 11,336 high-quality cells from the mineralized shell, where each point represents a cell, and each color represents a cell cluster (Cluster). Cells with similar gene expression patterns are clustered together, and there are obvious expression differences between the clusters.

[0048] Figure 4The UMAP is a visualization method based on non-linear dimensionality reduction that maps high-dimensional data into two or three dimensions while preserving the relative distances and structures between the data, making clustering, heterogeneity, and differences between samples more evident.

[0049] Example 2 Based on Example 1, adjust the dosages of sodium citrate and trehalose to 50 mM and 100 mM respectively.

[0050] Example 3 Based on Example 1, adjust the dosages of sodium citrate and trehalose to 200 mM and 500 mM respectively.

[0051] The experimental results of Example 2 and Example 3 are comparable to those of Example 1, so the experimental data are omitted in this application.

[0052] Example 4 Based on Example 1, change the species and conduct experiments on sea urchins. The acquisition and pretreatment of the mineralized shell are as Figure 5 shown, and the results are as Figures 6 - 7 shown. Other operations are the same as those in Example 1.

[0053] Result description: Figures 6 - 7 The results show that for the single-cell suspension of the sea urchin mineralized shell, the cells are clearly visible and in good morphology, meeting the requirements for loading onto the single-cell sequencing platform. Moreover, data output was successfully obtained for the single-cell suspension of the mineralized shell through the 10× Genomics platform, and the results met expectations, achieving unsupervised clustering of the cells in the sea urchin mineralized shell.

[0054] Example 5 Based on Example 1, change the species and conduct experiments on swimming crabs. The acquisition and pretreatment of the mineralized shell are as Figure 8 shown, and the results are as Figures 9 - 10 shown. Other operations are the same as those in Example 1.

[0055] Result description: Figures 9 - 10 The results show that for the single-cell suspension of the swimming crab mineralized shell, the cells are clearly visible and in good morphology, meeting the requirements for loading onto the single-cell sequencing platform. Moreover, data output was successfully obtained for the single-cell suspension of the mineralized shell through the 10× Genomics platform, and the results met expectations, achieving unsupervised clustering of the cells in the swimming crab mineralized shell.

[0056] Comparative Example 1 Based on Example 1, change the type of decalcifying solution in step (2) and use the EDTA decalcifying solution. The subsequent processes of single-cell suspension preparation, 10× Genomics library construction, and sequencing are exactly the same as those in Example 1, and the results are asFigures 11 - 12 as shown

[0057] Result description: Figure 11 The results show that, compared with Example 1 ( Figure 3 ), the cell survival rate in the single-cell suspension of the mineralized shell of Patinopecten yessoensis is lower, and the number of single cells in the prepared suspension is smaller.

[0058] Figure 12 The results show that single-cell transcriptome sequencing was performed on the ECTP decalcification group (Example 1) and the EDTA decalcification group (Comparative Example 1) using the 10× Genomics platform, and compared with the Bulk transcriptome data of the original mineralized shell, the correlation (B) between the ECTP decalcification group and the Bulk transcriptome was significantly higher than that of the EDTA decalcification group (A). It is proved that the ECTP decalcifying solution can better maintain molecular fidelity.

[0059] Comparative Example 2 On the basis of Example 1, the enzymatic digestion was changed in step (3). The specific operation was as follows: 1 mL of enzyme mixed solution (2 mg / mL collagenase II, 0.25% trypsin) was added to the centrifuge tube, the mineralized shell membrane was cut into pieces with sterile scissors, and after inverting several times, it was placed on a rotator for digestion at room temperature for 15 - 20 min. After digestion, it was pipetted with a sterile wide-mouth pipette tip for 2 min. The subsequent preparation of single-cell suspension, 10× Genomics library construction and sequencing processes were exactly the same as those in Example 1, and the results are as Figures 12 - 13 shown

[0060] Result description: Figure 13 The results show that, compared with the Example ( Figure 3 ), the cell survival rate after enzymatic treatment is significantly worse than that of non-enzymatic digestion, and the proportion of cell debris is higher.

[0061] Figure 14 The results show that single-cell transcriptome sequencing was performed on the non-enzymatic digestion group (Example 1) and the enzymatic digestion group (Comparative Example 2) using the 10× Genomics platform, and compared with the Bulk transcriptome data of the original mineralized shell, the correlation (B) between the non-enzymatic digestion group and the Bulk transcriptome was significantly higher than that of the enzymatic digestion group (A). It shows that the non-enzymatic physical digestion strategy effectively avoids the destruction of the molecular integrity of cells by exogenous enzymes.

[0062] Table 3 Comparison of the preparation effects of single-cell suspensions of the mineralized shell of Patinopecten yessoensis

[0063] In summary, the single-cell suspension of the mineralized shell obtained by crushing the mineralized shell, improving the decalcification system, and non-enzymatic pure physical grinding digestion greatly improves cell viability, reduces biological damage, and has high cross-species applicability and reliable experimental results.

[0064] The above are only partial embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall fall within the protection scope of the present invention.

Claims

1. A method for preparing a single-cell suspension from a mineralized shell applicable across animal phyla, characterized in that, Comprising the following steps: (1) Mineralized shell fragmentation treatment: Obtain the mineralized shell from a living species; Wash it, polish it, and break it into small pieces; (2) Place the small pieces in an ECTP composite decalcifying solution, which contains an EDTA decalcifying solution, 50 - 200 mM sodium citrate, 100 - 500 mM trehalose, and a phosphate buffer solution; (3) Physical digestion: Wash the decalcified organic matter layer with a washing solution, collect the washed organic matter, and grind and pipette it; (4) Centrifugation: Filter the digestion solution obtained in step (3) through a cell sieve, collect the filtered cell suspension, centrifuge it at a low temperature, and collect cell pellet A; (5) Resuspend the cells: Remove the supernatant and resuspend the cells with a washing solution to obtain cell suspension A; (6) Secondary centrifugation: Centrifuge cell suspension A at a low temperature and collect cell pellet B; (7) Secondary resuspension of cells: Remove the supernatant and resuspend the cells with a washing solution to obtain a single - cell suspension of the mineralized shell.

2. The method for preparing a single cell suspension according to claim 1, wherein The mineralized shell is derived from invertebrates, and the mineralized shell includes an exoskeleton, a shell, or spines.

3. The method for preparing a single cell suspension according to claim 2, wherein The invertebrates include mollusks, echinoderms, or arthropods.

4. The method for preparing a single-cell suspension according to claim 2, wherein The invertebrates include scallops, sea urchins, or crabs.

5. The method for preparing a single-cell suspension according to claim 1, wherein, The washing solution is a phosphate buffer solution.

6. The method for preparing a single-cell suspension according to claim 1, wherein The cell sieve is a cell sieve with a pore size of 40 μm.

7. The method for preparing a single cell suspension according to claim 1, wherein, The centrifugation temperature is 4°C.

8. The method for preparing a single-cell suspension according to claim 1, wherein The centrifugation speed is 400 - 600 g, and the centrifugation time is 4 - 5 min.

9. The method for preparing a single cell suspension according to claim 1, wherein The diameter of the small pieces is 0.5 cm.

10. A single-cell suspension derived from the mineralized shell of an invertebrate, characterized in that, The single - cell suspension is prepared by the method for preparing a single - cell suspension according to any one of claims 1 - 9.

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