A method for preparing a single cell suspension of mineralized shell sources applicable across phyla of animals

Abstract

The application discloses a preparation method of a single-cell suspension from a mineralized shell source applicable to animals of different phyla, and belongs to the technical field of cell suspension preparation. In view of the fact that the mineralized shell structure is relatively hard, the present application optimizes the pretreatment before decalcification, the decalcification solution, the decalcification process and the digestion method, and obtains a high-quality single-cell suspension. The single-cell suspension preparation method provided by the present application can be used for obtaining living cells from the mineralized shell source applicable to animals of different phyla, wherein the mineralized shell includes an exoskeleton, a shell and a spine. The method adopts crushing treatment and non-enzymatic pure physical grinding digestion, is shorter in experimental time, simple in operation steps, greatly reduces biological damage, maximally retains the integrity and activity of cells, and can fully meet the needs of library construction of various single-cell sequencing platforms.

Description

TECHNICAL FIELD

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

[0002] For a long time, the mineralized shells (such as mollusk shells and arthropod exoskeletons) of invertebrates have been regarded as inanimate and inert mineralized substances, i.e. "inanimate stones", in the field of biology; in contrast, the skeletons of vertebrates are considered to be living "organs", and the bone marrow inside the skeletons contains living cells. Based on this difference in cognition, existing single-cell suspension preparation techniques almost all focus on the dissociation of cells from soft tissues such as bone marrow of vertebrates, and the related research on mineralized shells of invertebrates has been in a blank state for a long time. The previous research of the research team overturned this traditional cognition for the first time: through evidence of living cell tracing and transcriptome, it was confirmed that there are living cells in the mineralized shells of invertebrates represented by mollusks. This laid a biological foundation for the single-cell research of mineralized shells of invertebrates, and based on this, we first realized the attempt of preparing a single-cell suspension from mineralized shells of invertebrates.

[0003] However, there are significant differences between vertebrate and invertebrate mineralized shells: the vertebrate bone is mainly composed of hydroxyapatite (calcium phosphate) with a porosity of 60-70%, and the mineral crystals have a porous nanostructure, which is conducive to cell infiltration and material exchange; while the invertebrate mineralized shell is mainly composed of aragonite / calcite (calcium carbonate) with a mineralization degree of >95%, and the crystals are highly dense and form a nearly continuous mineralization barrier; the vertebrate bone tissue has a hierarchical porous structure of "mineral-collagen fibers", and cells are easily released; while the invertebrate mineralized shell has a sandwich structure of "mineral matrix embedding organic layer", and living cells are deeply embedded in micron-sized calcified units, which are difficult to release. Therefore, the existing single-cell suspension preparation method for vertebrate bone is not suitable for invertebrate mineralized shells, and cells cannot be released by simple blowing or enzymatic digestion, which leads to four technical bottlenecks in traditional vertebrate bone processing methods in invertebrate samples: 1) decalcification kinetics barrier: the dissolution kinetics of calcium carbonate is significantly slower than that of calcium phosphate, and the traditional EDTA decalcification solution (0.5M) requires more than 72 hours for decalcification of crustacean exoskeleton, resulting in long-term exposure of cells to osmotic stress environment; 2) chemical damage intensifies: 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, causing RNA degradation and cell death; 3) enzymatic digestion strategy fails: the dense mineral layer hinders the penetration of enzyme molecules, and enzyme molecules cannot penetrate the mineral layer to reach the organic layer, while increasing enzyme concentration / lengthening digestion time will damage cell surface receptors and interfere with the molecular fidelity of single-cell sequencing; 4) mechanical dissociation damage: the residual organic layer after decalcification is extremely tough, and mechanical force easily causes the rupture of cell membranes without cell wall protection. The existing method relies on high-speed centrifugation or vigorous grinding to break down the tissue after enzymatic digestion, which easily causes mechanical damage to cells and leads to a decrease in the number of living cells. In addition, the existing technology generally has a complex operation process and a long time-consuming (usually several days to several weeks), which further limits its application in multi-species comparative studies.

[0004] To solve the above problems, there is an urgent need in the art to develop a mineralized shell-derived single-cell suspension preparation method that is widely applicable, has excellent cell activity, and is simple to operate across animal phyla, especially to solve the key technical bottlenecks of invertebrate sample decalcification kinetics, non-enzymatic mild digestion, and short-time high-efficiency processing, to meet the stringent requirements of single-cell multi-omics research on cell integrity, activity, and preparation throughput. SUMMARY

[0005] In light of the shortcomings of existing technologies, the present invention provides a method for preparing single-cell suspensions derived from mineralized shells that is applicable across animal phyla. Through an innovative ECTP composite decalcification solution and a non-enzymatic physical digestion system, the method achieves: 1) a balance between decalcification efficiency and cell activity: the ECTP decalcification solution (pH 7.0-7.4) accelerates the dissolution of calcium carbonate by synergistically chelating calcium ions with a polycarboxyl integrator, while trehalose maintains cell membrane stability, shortening decalcification time and achieving cell viability exceeding 90%. 2) non-enzymatic physical digestion: eliminating exogenous enzymes and employing gradient centrifugation (400-600g) combined with gentle pipetting / trituration to maximize the integrity of cell surface proteins. 3) cross-species compatibility: by adjusting the ECTP buffer osmotic pressure and trehalose concentration, the method is tailored to the physiological needs of different phyla, such as mollusks and crustaceans.

[0006] The technical solution of the present invention mainly includes the following contents:

[0007] A method for preparing a single cell suspension derived from mineralized shells applicable across animal phyla comprises the following steps:

[0008] (1) Mineralized shell crushing: Mineralized shells are obtained from living species; they are cleaned, polished, and broken into small pieces;

[0009] (2) placing the small fragments in an ECTP composite decalcification solution containing EDTA decalcification solution, 50-200 mM sodium citrate, 100-500 mM trehalose, and phosphate buffer;

[0010] (3) Physical digestion: The decalcified organic layer is washed with a cleaning solution, and the washed organic matter is collected, ground, and blown;

[0011] (4) Centrifugation: Filter the digestion solution obtained after the treatment in step (3) through a cell sieve, collect the filtered cell suspension, centrifuge at low temperature, and collect the cell precipitate A;

[0012] (5) Resuspending cells: Remove the supernatant and resuspend the cells in washing solution to obtain cell suspension A;

[0013] (6) Second centrifugation: centrifuge the cell suspension A at low temperature to collect the cell pellet B;

[0014] (7) Secondary cell resuspension: Remove the supernatant and resuspend the cells with washing solution to obtain a single cell suspension with mineralized shell.

[0015] Preferably, the mineralized shell is derived from an invertebrate, and the mineralized shell comprises an exoskeleton, a shell or spines.

[0016] Preferably, the invertebrate comprises a mollusk, an echinoderm or an arthropod.

[0017] Preferably, the invertebrate comprises a scallop, a sea urchin or a crab.

[0018] Preferably, the cleaning solution is phosphate buffer.

[0019] Preferably, the cell sieve is a cell sieve with a pore size of 40 μm.

[0020] Preferably, the centrifugation temperature is 4°C.

[0021] Preferably, the centrifugal speed is 400-600 g and the centrifugal time is 4-5 min.

[0022] Preferably, the crushing is as follows: selecting species with good activity, brushing the surface clean with a brush in sea water or clean water, grinding the surface into a thin layer with a grinding gun, and breaking it into small pieces of about 0.5 cm with tweezers or pliers.

[0023] The present invention further relates to a single-cell suspension derived from the mineralized shell of an invertebrate, produced using the single-cell suspension preparation method described above. The single-cell suspension retains the original biological properties and activity of the cells, exhibits good cell morphology, and exhibits no clumping, meeting the requirements of various single-cell sequencing platforms.

[0024] Beneficial effects of the present invention:

[0025] 1. The mineralized shell is cleaned and polished, and then broken into small pieces for crushing, which accelerates the mineral removal process, significantly improves decalcification efficiency and shortens decalcification time, while maximizing the integrity and activity of cells.

[0026] 2. The core advantage of the ECTP composite system proposed in the present invention as a decalcification solution lies in the synergistic effect of the various components on decalcification efficiency and cell activity: EDTA, as a classic strong chelating agent, can widely and efficiently complex metal ions such as calcium and iron by virtue of its stable hexadentate coordination structure formed by four carboxyl groups and two amino groups, and is suitable for deep decalcification; however, its large molecular weight and rigid structure lead to a relatively slow chelation rate. Sodium citrate, as a tridentate organic acid salt, has a limited chelation range, but with the advantages of small molecular weight and flexible structure, it can quickly bind free Ca 2+, significantly improving the initial decalcification rate. The combination of EDTA and sodium citrate not only achieves the rapid removal of initial calcium ions, but also ensures long-term complex stability, thereby significantly improving the overall decalcification efficiency. At the same time, trehalose, as an osmotic protectant, can stabilize the lipid bilayer structure of the cell membrane and protect intracellular proteins when the ion concentration of the decalcification solution changes and causes osmotic pressure fluctuations, 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 the decalcification process and prevent strong acid from damaging the cell membrane. The synergistic effect of trehalose and sodium citrate jointly improves cell survival rate. In addition, by adjusting the concentration of phosphate buffer, the stability of the ion environment during the decalcification process is further optimized, reducing cell rupture caused by osmotic pressure fluctuations. The present invention also proposes to adopt a phased decalcification strategy and monitor the decalcification saturation in real time, and replace fresh decalcification solution in time to avoid damage to the soft tissue matrix caused by excessive decalcification.

[0027] 3. The non-enzymatic, purely physical grinding method proposed in this invention processes the decalcified organic layer. Repeated crushing with a grinding rod or repeated pipetting with a pipette effectively releases single cells, significantly minimizing biological damage and preserving the cells' original biological properties and activity. This method is time-efficient, simple to operate, and produces a high yield of viable cells.

[0028] 4. The single-cell suspension prepared using this method has been successfully applied to the preparation of mineralized shell cell suspensions from three different species. Data output was successfully obtained using the 10× Genomics platform, with results meeting or even exceeding expectations. This demonstrates the method's high cross-species applicability and reliable experimental results, providing effective technical support for the study of mineralized shell cells from diverse species. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 : Mineralized shell fragmentation diagram.

[0030] Figure 2 The broken mineralized shell in Example 1 of the present invention is placed in an ECTP decalcification solution for decalcification to finally obtain an organic layer.

[0031] Figure 3 : Microscopic photograph of the mineralized shell organic layer after physical grinding, digestion, filtration, centrifugation at 400-600 g and staining in Example 1 of the present invention.

[0032] Figure 4 Single-cell map of the mineralized shell single-cell suspension of the Japanese scallop (Scallop ocellatus) in Example 1 of the present invention. 0: long-term hematopoietic stem cell; 1: short-term hematopoietic stem cell; 2: blood cell; 3: mesenchymal stem cell; 4: macrophage. "Cell ratio" in the figure refers to the ratio of each cell population.

[0033] Figure 5 : In Example 4 of the present invention, the mineralized shell of the sea urchin is crushed and finally decalcified to obtain the organic layer.

[0034] Figure 6 : Microscopic photograph of the organic layer of the mineralized shell of the sea urchin in Example 4 of the present invention after physical grinding, digestion, filtration, centrifugation at 400-600g and staining.

[0035] Figure 7 : Single-cell UMAP images obtained from scRNA-seq analysis of the mineralized shell of sea urchins in Example 4 of the present invention. Wherein, SnC0: hematopoietic stem cell-like cells; SnC1: blood cells; SnC2: mesenchymal stem cells; SnC3: macrophages.

[0036] Figure 8 : The mineralized shell of the swimming crab is obtained and the organic layer map is finally obtained in Example 5 of the present invention.

[0037] Figure 9 Microscopic photograph of the organic layer of the mineralized shell of a swimming crab after physical grinding, digestion, filtration, centrifugation at 400-600 g, and staining in Example 5 of the present invention. Red circles mark dead cells.

[0038] Figure 10 : Single-cell UMAP images 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 cell; PtC1: blood cell; PtC2: mesenchymal stem cell; PtC3: macrophage.

[0039] Figure 11 Microscopic photograph of the mineralized shell of the Japanese scallop in Comparative Example 1 of the present invention after decalcification in EDTA decalcification solution, physical grinding, digestion, filtration, centrifugation at 400-600 g, and staining. Red circles mark dead cells

[0040] Figure 12 This study used the 10× Genomics platform to perform single-cell transcriptome sequencing on the ECTP-decalcified group (Example 1) and the EDTA-decalcified group (Comparative Example 1). Correlation analysis was performed with the bulk transcriptome data from the original mineralized shell of the Japanese scallop. Figures A and B show a correlation scatterplot of the bulk transcriptome between the EDTA-decalcified group and the original mineralized shell.

[0041] Figure 13Microscopic photograph of the mineralized shell of the Japanese scallop in Comparative Example 2 of the present invention after dissociation with an enzyme mixture (2 mg / mL collagenase II and 0.25% trypsin), filtration, centrifugation at 400-600 g, and staining. Red circles mark dead cells.

[0042] Figure 14 This study used the 10× Genomics platform to perform single-cell transcriptome sequencing on the non-enzymatic digestion group (Example 1) and the enzymatic digestion group (Comparative Example 2). Correlation analysis was performed with the bulk transcriptome data from the original mineralized shell of the scallop (Yoshino scallop). Figure A: Correlation scatter plot of the bulk transcriptome between the enzymatic digestion group and the original mineralized shell; Figure B: Correlation scatter plot of the bulk transcriptome between the non-enzymatic digestion group and the original mineralized shell. DETAILED DESCRIPTION

[0043] In order to better understand the technical content of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0044] In the following examples, unless otherwise specified, the materials used are conventional commercially available products.

[0045] PBS (phosphate-buffered saline) was purchased from Shanghai Bioengineering, China (catalog number: SB0627); EDTA decalcification solution (pH 7.2) was purchased from Solarbio, China (catalog number: E1171-500 ml); sodium citrate (pH 7.0) was purchased from Beyotime, China (catalog number: ST368); D-trehalose was purchased from Solarbio, China (catalog number: G8570); 0.4% trypan blue dye was purchased from Solarbio, China (catalog number: C0040); collagenase II was purchased from Solarbio, China (catalog number: C8150); and 0.25% trypsin was purchased from HyClone, USA (catalog number: SH30042.02).

[0046] The components of ECTP decalcification solution are shown in Table 1.

[0047] Table 1 ECTP composite decalcification solution component ratio

[0048]

[0049] Example 1: Method for preparing a single cell suspension derived from mineralized shells applicable across animal phyla

[0050] (1) Mineralized shell crushing: Mineralized shells were obtained from living species of the Japanese scallop; the surface was cleaned with a brush in water, and then polished and broken into small pieces with a diameter of about 0.5 cm, such as Figure 1 As shown;

[0051] (2) The broken pieces were placed in a pre-prepared ECTP composite decalcification solution. The composition ratio of the ECTP composite decalcification solution is shown in Table 2. The pieces were placed on a shaker. The decalcification saturation was observed in real time during the decalcification process, and fresh decalcification solution was replaced in time.

[0052] Table 2 ECTP composite decalcification solution component ratio

[0053]

[0054] (3) Physical digestion: The decalcified organic layer (such as Figure 2 After washing in phosphate buffer, transfer to a sterile 1.5 mL centrifuge tube and crush several times with a sterile grinding rod in phosphate buffer until the organic matter disappears. Then, use a sterile wide-mouth resin-coated pipette tip to gently pipette.

[0055] (4) Centrifugation: The digestion solution obtained after treatment in step (3) was filtered through a 40 μm cell sieve. The filtered cell suspension was collected and placed in a 4°C centrifuge. Centrifuged at 400 g for 4-5 min to collect the cell pellet A.

[0056] (5) Resuspending cells: Remove the supernatant and add pre-cooled PBS buffer to the cell pellet A. Use a sterile wide-mouth pipette tip to gently pipette 10 times to resuspend the cells to obtain cell suspension A.

[0057] (6) Second centrifugation: Place the cell suspension A in a 4°C centrifuge and centrifuge at 600 g for 4-5 min to collect the cell pellet B.

[0058] (7) Secondary resuspension of cells: Remove the supernatant and add 50 μL of pre-cooled PBS buffer to the cell pellet B. Use a sterile wide-mouth pipette tip to gently pipette 10 times to resuspend the cells and enrich the mineralized shell single cell suspension.

[0059] (8) Staining: Single cell suspension was mixed with 0.4% trypan blue dye at a volume ratio of 9:1. A small amount of stained cells was taken and the cell survival rate was counted on a hemocytometer. The results were as follows: Figure 3 shown.

[0060] Result description:

[0061] Figure 3 The results showed that there was no obvious debris in the background, the cells were clearly visible, the morphology was good, and there was almost no clumping; the cell concentration of the single-cell suspension of the mineralized shell of the scallop was high, there were no cell clusters larger than 40 μm or other impurities, and the cell clumping rate was less than 1%, which met the requirements of various single-cell sequencing platforms. The obtained mineralized shell single-cell suspension was subjected to single-cell transcriptome sequencing. The results are as follows Figure 4 shown.

[0062] like Figure 4 As shown, a single-cell atlas was constructed from 11,336 high-quality cells from the mineralized shell, where each point represents a cell and each color represents a cell cluster. Cells with similar gene expression patterns are clustered together, and cells between clusters show obvious expression differences.

[0063] Figure 4 UMAP is a visualization method based on nonlinear dimensionality reduction, which maps high-dimensional data into two-dimensional or three-dimensional space and maintains the relative distance and structure between data, making clustering, heterogeneity and differences between samples more obvious.

[0064] Example 2

[0065] Based on Example 1, the dosages of sodium citrate and trehalose were adjusted to 50 mM and 100 mM, respectively.

[0066] Example 3

[0067] Based on Example 1, the dosages of sodium citrate and trehalose were adjusted to 200 mM and 500 mM, respectively.

[0068] The experimental results of Example 2 and Example 3 are equivalent to those of Example 1, so their experimental data are omitted in this application.

[0069] Example 4

[0070] On the basis of Example 1, the species was changed and the experiment was conducted on sea urchins. The mineralized shells were obtained and pre-treated as follows. Figure 5 As shown, the results are Figures 6-7 Other operations are the same as those in Example 1.

[0071] Result description:

[0072] Figures 6-7 The results showed that the cells in the single-cell suspension of sea urchin mineralized shells were clearly visible and had good morphology, meeting the requirements of the single-cell sequencing platform. The single-cell suspension of mineralized shells successfully obtained data output through the 10× Genomics platform. The results were in line with expectations, realizing unsupervised clustering of sea urchin mineralized shell cells.

[0073] Example 5

[0074] On the basis of Example 1, the species was changed and the experiment was conducted on swimming crabs. The mineralized shell was obtained and pre-treated as follows. Figure 8 As shown, the results are Figures 9-10 Other operations are the same as those in Example 1.

[0075] Result description:

[0076] Figures 9-10The results showed that the cells in the single-cell suspension of the mineralized shell of the swimming crab were clearly visible and had good morphology, which met the requirements of the single-cell sequencing platform. The single-cell suspension of the mineralized shell successfully obtained data output through the 10× Genomics platform. The results were in line with expectations and achieved unsupervised clustering of the mineralized shell cells of the swimming crab.

[0077] Comparative Example 1

[0078] Based on Example 1, the type of decalcification solution in step (2) was changed to EDTA decalcification solution. The subsequent single cell suspension preparation, 10× Genomics library construction and sequencing process were exactly the same as in Example 1. The results are as follows: Figures 11-12 shown.

[0079] Result description:

[0080] Figure 11 The results showed that compared with Example 1 ( Figure 3 ) Compared with the single-cell suspension of mineralized shell of the Yesso scallop, the cell survival rate is lower and the number of single cells in the prepared suspension is less.

[0081] Figure 12 The results showed that single-cell transcriptome sequencing of the ECTP-decalcified group (Example 1) and the EDTA-decalcified group (Comparative Example 1) using the 10× Genomics platform and comparison with the bulk transcriptome data of the original mineralized shell showed a significantly higher correlation between the ECTP-decalcified group and the bulk transcriptome (B) than the EDTA-decalcified group (A), demonstrating that the ECTP decalcified solution can better maintain molecular fidelity.

[0082] Comparative Example 2

[0083] Based on Example 1, step (3) was changed to enzymatic digestion. The specific operation was as follows: 1 mL of enzyme mixture solution (2 mg / mL collagenase II, 0.25% trypsin) was added to the centrifuge tube, and the mineralized shell membrane was cut into pieces with sterile scissors. After inverting several times, the cell was placed on a rotator and digested at room temperature for 15-20 minutes. After digestion, the cell suspension was pipetted with a sterile wide-mouth pipette tip for 2 minutes. The subsequent single-cell suspension preparation, 10× Genomics library construction and sequencing process were exactly the same as in Example 1. The results are shown in Figure 2. Figures 12-13 shown.

[0084] Result description:

[0085] Figure 13 The results show that, compared with the example ( Figure 3 ) Compared with non-enzymatic digestion, the cell survival rate after enzymatic digestion was significantly lower, and the proportion of cell fragments was higher.

[0086] Figure 14The results showed that single-cell transcriptome sequencing of the non-enzymatic digestion group (Example 1) and the enzymatic digestion group (Comparative Example 2) using the 10× Genomics platform, and comparison with the bulk transcriptome data of the original mineralized shell, showed that the correlation between the non-enzymatic digestion group and the bulk transcriptome (B) was significantly higher than that of the enzymatic digestion group (A). This indicates that the non-enzymatic physical digestion strategy effectively avoids the damage of exogenous enzymes to the integrity of cellular molecules.

[0087] Table 3 Comparison of the preparation effects of single cell suspensions of mineralized shells of the Yesso scallop

[0088]

[0089] In summary, the single-cell suspension of mineralized shells that was prepared by crushing the mineralized shells, improving the decalcification system, and non-enzymatically digesting them by pure physical grinding greatly improved cell survival rate, reduced biological damage, and had high cross-species applicability and reliable experimental results.

[0090] The above descriptions are only some embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for preparing a single cell suspension derived from mineralized shells applicable across animal phyla, characterized in that: The following steps are involved: (1) Mineralized shell crushing: obtaining mineralized shells from living species; After cleaning, grind it and break it into small pieces; (2) placing the small fragments in an ECTP composite decalcification solution containing EDTA decalcification solution, 50-200 mM sodium citrate, 100-500 mM trehalose, and phosphate buffer; (3) Physical digestion: The decalcified organic layer is washed with a cleaning solution, and the washed organic matter is collected, ground, and blown; (4) Centrifugation: Filter the digestion solution obtained after the treatment in step (3) through a cell sieve, collect the filtered cell suspension, centrifuge at low temperature, and collect the cell precipitate A; (5) Resuspending cells: Remove the supernatant and resuspend the cells in washing solution to obtain cell suspension A; (6) Second centrifugation: centrifuge the cell suspension A at low temperature to collect the cell pellet B; (7) Secondary cell resuspension: Remove the supernatant and resuspend the cells with washing solution to obtain a single cell suspension with mineralized shell.

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

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

4. The method for preparing a single cell suspension according to claim 2, wherein: The invertebrate includes a scallop, a sea urchin or a crab.

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

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

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

8. The method for preparing a single cell suspension according to claim 1, wherein: The centrifugal speed is 400~600g and the centrifugal 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 was 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 single cell suspension preparation method according to any one of claims 1 to 9.

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