A method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection

Abstract

The present invention discloses a method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection, comprising: extracting gill filament tissue from living shellfish; washing the gill filaments and drying the water; cutting the gill filament tissue into pieces; using an enzyme mixture composed of collagenase II, hyaluronidase and deoxyribonuclease I to enzymatically digest the gill filament tissue fragments; centrifuging and resuspending the cells; filtering to obtain a single-cell suspension. Aiming at the situation where the shellfish tissue structure is relatively loose, the present invention optimizes the cleaning solution, enzyme solution components, enzymatic hydrolysis time and centrifugal speed, etc., to obtain a high-quality single-cell suspension. The single-cell suspension preparation method provided by the present invention can be applied to the industry's demand for dynamic detection of shellfish traits through non-lethal sampling of marine shellfish. The method has more complete enzymatic hydrolysis, shorter experimental time, simple operation steps, more complete cell morphology, extremely low agglomeration rate and higher live cell rate, which can fully meet the needs of library construction of various single-cell sequencing platforms.

Description

Technical Field

[0001] The invention belongs to the technical field of cell suspension preparation, and in particular relates to a method for preparing a seawater shellfish single cell suspension suitable for dynamic detection. Background Art

[0002] Shellfish farming is an important part of my country's aquaculture industry, and its farming output accounts for about 70% of the total marine aquaculture output. According to the data in the "2024 China Scallop Industry In-depth Research Report", China's scallop farming output reached 1.792 million tons. In the process of shellfish farming and production, due to environmental stress (high temperature, low oxygen and lack of bait, etc.), pathogen infection (pathogens such as bacteria, viruses and parasites) or human factors, outbreaks of marine aquaculture shellfish diseases occur from time to time, causing huge economic losses, which seriously hinders the green and high-quality development of the marine aquaculture industry. Therefore, the selection and breeding of new stress-resistant varieties is urgently needed.

[0003] Yesso scallop ( Mizuhopecten yessoensis ) belongs to the bivalve marine organisms. Due to its huge commercial value, it has been rapidly developed in the northern coastal areas of my country since it was introduced from Japan in 1982. It has now become one of the most important marine aquaculture shellfish in northern China. Affected by global climate change, the large-scale death of the scallop in summer is characterized by concentrated outbreaks and high degree of harm. Accurately evaluating the heat resistance of individuals and analyzing the regulatory mechanism of their temperature response are necessary prerequisites for breeding new heat-resistant varieties. Previous studies have shown that under hypoxia and high temperature stress, the scallops show different changes in physiological metabolism and immune enzyme activity. However, most of the current evaluation indicators of shellfish stress resistance traits require individual dissection to obtain tissue samples, which is not only time-consuming and laborious, but also difficult to obtain shellfish individuals with excellent traits while obtaining stress resistance evaluation, which greatly reduces the efficiency of shellfish stress resistance material accumulation and new varieties of parent shellfish breeding. In addition, the evaluation of stress resistance traits requires tracking of dynamic processes, so it is necessary to explore the feasibility of using non-lethal gill filament live sampling methods for dynamic research on multiple stress resistance indicators.

[0004] The gills of the Yesso scallop not only play an important role in breathing, filtering food and toxin metabolism, but are also important temperature response organs. At the same time, the gill filaments have a loose structure and are easy to sample, making them a good object for live sampling. However, current research only stays at the level of gill tissue organs, and its results mainly reflect the sum of information of multiple cell types, ignoring the heterogeneity between cells, resulting in the loss of specific information in cell subpopulations. Therefore, in-depth exploration of the biological functions of shellfish gill tissues and the regulatory mechanisms of temperature response requires breaking through previous technical bottlenecks and conducting more detailed studies on molecular characteristics at the single-cell level.

[0005] Single-cell sequencing (SCS) is a high-throughput sequencing technology that can reveal changes in the genome, transcriptome or epigenome of a single cell. It aims to analyze genetic variation and gene expression differences between cells. It can be used to distinguish rare cells and generate cell maps, thereby revealing the heterogeneity characteristics between cells. The main processes include single-cell isolation, sequencing and data analysis, among which single-cell isolation and the preparation of high-quality single-cell suspensions are the most critical. However, the currently reported preparation conditions for single-cell suspensions of marine shellfish tissues cannot meet the needs of gill tissues with filamentous structures and relatively loose structures. For example, improper selection of enzymatic hydrolysis solutions and excessively long enzymatic hydrolysis times will make it impossible to obtain gill cell suspensions with high cell activity, which is not conducive to the normal development of subsequent single-cell experimental work. Therefore, it is very necessary to develop a preparation method for single-cell suspensions of gill filament tissues of shellfish.

[0006] In conclusion, the selection of parents with excellent traits is a key factor in determining the quality of scallop seedlings. It is particularly urgent to establish a method for the dynamic and continuous acquisition of single cells from the non-lethal gill filaments of scallops and apply it to the evaluation of individual stress resistance traits. In this process, individuals or populations with excellent performance can be preserved preferentially for the selection and breeding of new varieties. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection. The present invention achieves the dissociation of gill filaments of the scallop in a relatively short period of time while taking into account the cell survival rate. The single-cell suspension prepared using the method was successfully subjected to single-cell transcriptome sequencing analysis.

[0008] The technical solution of the present invention mainly includes the following contents:

[0009] A method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection comprises the following steps:

[0010] (1) Non-lethal sampling of gill filaments: extracting gill filament tissue from living shellfish;

[0011] (2) Rinse the gill filaments with pre-cooled phosphate buffer and dry them with absorbent paper; cut the gill filament tissue into small pieces;

[0012] (3) using an enzyme mixture to enzymatically digest the gill filament tissue fragments; the enzyme mixture comprises collagenase II, hyaluronidase and deoxyribonuclease I; and the enzymatic digestion time is 15 to 20 min;

[0013] (4) Centrifuge the enzymatic hydrolysate to obtain a cell precipitate; the centrifugation speed is 300-650 g, and the centrifugation time is 4-5 min;

[0014] (5) Resuspending the cells in phosphate buffer containing bovine serum albumin to obtain a cell suspension;

[0015] (6) Repeat steps (4) and (5) twice; the centrifugation speed is 650 g and the centrifugation time is 5 min;

[0016] (7) Filter the obtained cell suspension to obtain a single cell suspension.

[0017] Preferably, the non-lethal live sampling of gill filaments is as follows: select shellfish with good activity, wait for the shellfish to open its shell naturally, gently fix it with fingers, leaving a gap of 5 to 10 mm in width, use pointed tweezers to reach into the gap, and extract 2 gill filament tissues.

[0018] Preferably, the enzyme mixture comprises: 2 mg / mL collagenase II, 2 mg / mL hyaluronidase and 40 U / mL deoxyribonuclease I.

[0019] Preferably, the centrifugation temperature is 4°C.

[0020] Preferably, the filtration in step (7) is performed using a cell sieve with a pore size of 40 μm.

[0021] Preferably, the phosphate buffer is 3.5× phosphate buffer; and the enzyme mixture is prepared using 3.5× phosphate buffer.

[0022] Preferably, the enzymatic digestion time is 15 min.

[0023] Preferably, the phosphate buffer containing bovine serum albumin is a phosphate buffer containing 0.05% by volume of bovine serum albumin.

[0024] Preferably, the marine shellfish includes Yesso scallop.

[0025] Beneficial effects of the present invention:

[0026] 1. Taking non-lethal live gill filament sampling will not significantly affect the survival and growth of individuals, and the experimental shellfish can be used continuously for multiple times. The present invention proposes to carry out research on the preparation of single-cell suspension of Yesso scallops by a dynamic continuous detection sampling method, and evaluate the effect by detecting indicators such as the viable cell rate. It not only achieves the research purpose but also retains parent shellfish with excellent germplasm, and can be tracked for a long time. This method can also provide reference for the breeding of endangered shellfish (such as giant clams of the bivalve class) or other shellfish trait analysis that requires dynamic detection.

[0027] 2. The preparation method provided by the present invention has the shortest time consumption, higher viable cell rate and simple operation. Through the processes of live sampling, mixed enzymatic digestion, shortening the dissociation time, reducing the centrifugal speed, etc., a single cell suspension of gill tissue of the scallop can be quickly prepared. The method described in the present invention has higher enzymatic hydrolysis efficiency and better effect, and the experimental time consumption can be greatly shortened to about 1 h, the cell concentration can reach up to 1500 / μL or more, the cell activity is above 95%, and the agglomeration rate is less than 1%.

[0028] 3. Aiming at the situation that the shellfish tissue structure is relatively loose, the present invention optimizes the cleaning solution, enzyme solution components, enzymatic hydrolysis time and centrifugal speed, etc., which can not only obtain a large number of living cells from a small amount of tissue (2 gill filaments tissue), but also the cell fluid has high quality (high activity, complete cell morphology, and no agglomeration).

[0029] 4. The single-cell suspension prepared by the combined enzymatic hydrolysis method (collagenase II, hyaluronidase and DNase I) of the present invention can fully meet the requirements of library construction of various single-cell sequencing platforms, and has successfully carried out single-cell transcriptome sequencing, which has important application value for the genetic breeding research of marine shellfish. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 : Non-lethal gill filament sampling diagram.

[0031] Figure 2 : Microscopic photographs of gill filaments of the scallop in the present invention after being dissociated with an enzyme mixed solution for 15 minutes and centrifuged at a speed of 300-650 g; wherein, A: gill filament cells; B: gill filament cells stained with trypan blue. Red circles mark dead cells, and the scale bar shown in the figure is 100 μm.

[0032] Figure 3 : Single-cell sequencing diagram of the single-cell suspension of gill tissue of the Yesso scallop in an embodiment of the present invention. Figure 3 The Chinese description of each field is shown in Table 1.

[0033] Figure 4 : t-SNE projection diagram of cells in an embodiment of the present invention. Figure 4 t-SNE: t-Distributed Stochastic Neighbor Embedding, is a technique specialized for dimensionality reduction and visualization.

[0034] Figure 5 : Microscopic photographs of gill filaments of the scallop in Comparative Example 1 of the present invention after being dissociated with 0.25% trypsin solution for 15 min and centrifuged at 300-650 g; wherein, A: gill filament cells; B: gill filament cells stained with trypan blue. Arrows indicate incompletely dissociated tissue blocks, and red circles mark dead cells.

[0035] Figure 6: Microscopic photographs of gill filaments of the scallop in Comparative Example 2 of the present invention after being dissociated with an enzyme mixed solution for 40 minutes and centrifuged at a speed of 300-650 g; wherein, A: gill filament cells; B: gill filament cells stained with trypan blue. Red circles mark dead cells.

[0036] Figure 7 : Microscopic photographs of gill filaments of the scallop in Comparative Example 3 of the present invention after being dissociated with an enzyme mixed solution for 15 minutes and centrifuged at 650 g; wherein, A: gill filament cells; B: gill filament cells stained with trypan blue. Arrows indicate debris and impurities. DETAILED DESCRIPTION

[0037] 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.

[0038] In the following examples and comparative examples, unless otherwise specified, PBS (phosphate buffered saline) was purchased from Shanghai Bioengineering, China (Cat. No.: SB0627); collagenase II was purchased from Solarbio, China (Cat. No.: C8150); 0.25% trypsin was purchased from HyClone, USA (Cat. No.: SH30042.02); DNase I was purchased from Vazyme, China (Cat. No.: EN401); hyaluronidase was purchased from Solarbio, China (Cat. No.: H8030); bovine serum albumin (BSA) was purchased from Solarbio, China (Cat. No.: A8020-5g); 0.4% trypan blue dye was purchased from Solarbio, China (Cat. No.: C0040).

[0039] Example 1: Preparation method of marine shellfish single cell suspension suitable for dynamic detection

[0040] (1) Non-lethal sampling of gill filaments: Select active scallops. After the scallops open their shells naturally, gently fix them with your fingers, leaving a gap of 5 to 10 mm in width. Use pointed tweezers to reach into the gap and extract two gill filaments. Figure 1 As shown, place in a culture dish.

[0041] (2) Rinse the gill filaments 2-3 times with pre-cooled 3.5× PBS in a culture dish and dry with absorbent paper. Use sterile surgical scissors to cut the gill filament tissue into 1 mm 3 The cleaned tissue fragments were removed and transferred to a sterile 1.5 mL centrifuge tube.

[0042] (3) Enzymatic digestion: Add 1 mL of enzyme mixture solution (2 mg / mL collagenase II, 2 mg / mL hyaluronidase and 40 U / mL DNase I) to the centrifuge tube, invert it several times, and place it at room temperature (25-27°C) for 15 min. During this time, place the EP tube on a rotator and digest until the tissue blocks disappear. After digestion, gently blow with a sterile wide-mouth resin-coated pipette tip for 2 min. Observe the dissociation state of the gill filaments under a microscope. The results are as follows: Figure 2 shown.

[0043] (4) Centrifugation: Place the enzymatic hydrolysate in a 4°C centrifuge and centrifuge at 300 g for 4-5 min to collect the cell precipitate A.

[0044] (5) Resuspending cells: Remove the supernatant and add pre-cooled 3.5× PBS buffer containing 0.05% (volume percentage, the same below) bovine serum albumin (BSA) 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.

[0045] (6) Second centrifugation: Place the cell suspension A in a 4°C centrifuge and centrifuge at 650 g for 4-5 min to collect the cell pellet B.

[0046] (7) Secondary resuspension of cells: Remove the supernatant and add pre-cooled 3.5× PBS buffer containing 0.05% BSA to the cell pellet B. Use a sterile wide-mouth pipette tip to gently pipette 10 times to resuspend the cells to obtain cell suspension B.

[0047] (8) Three centrifugations: Place cell suspension B in a 4°C centrifuge and centrifuge at 650 g for 4-5 min to collect the cell pellet C.

[0048] (9) Resuspend cells three times: Remove the supernatant, add pre-cooled 3.5× PBS buffer containing 0.05% BSA to the cell pellet C, and gently pipette 10 times with a sterile wide-mouth pipette tip to resuspend the cells to obtain cell suspension C.

[0049] (10) Filtration: Filter the cell suspension C through a 40 μm cell sieve to obtain a single cell suspension of gill filaments of the Yesso scallop.

[0050] (11) Staining: Mix the single cell suspension with 0.4% trypan blue dye at a volume ratio of 9:1 and stain for 2 min. Take a small amount of the stained cells and count the cell survival rate using a hemocytometer. The results are as follows: Figure 2 shown.

[0051] Result description:

[0052] Figure 2The 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 gill filaments of the Yesso scallop could reach 1500 / μL, the cell survival rate was 96%, there were no cell clusters larger than 40 μm or other impurities, and the cell clumping rate was less than 1%, which could meet the requirements of various single-cell sequencing platforms. The single-cell suspension of gill tissue obtained was sequenced for single-cell transcriptome. The results are as follows Figure 3 and Figure 4 shown. Figure 3 The Chinese description of each field is shown in Table 1.

[0053] Table 1 Description of sequencing results

[0054]

[0055] like Figure 3 As shown, the number of cells after sequencing is 8,660, the median number of UMIs (unique molecular identifiers) per cell is 2,102, the median number of genes per cell is 781, and the average number of reads per cell is 22,333; the turning point of the cell curve is obvious, indicating that the sequencing quality is very good and the interference background is small; the total number of genes detected is 17,474; the total number of sequenced reads shown in Sequencing is 193,408,018, and the total number of reads containing valid barcodes is 94.6%; the proportion of reads mapped to the genome in Mapping is 88.2%, the proportion of reads mapped to intron regions is 9.0%, the proportion of reads mapped to exon regions is 43.3%, and the proportion of reads mapped to the antisense region of the gene is 0.5%.

[0056] Figure 4 In the figure, A is the t-SNE (t-distributed stochastic neighbor embedding) projection of cells colored by UMI counts. Each point represents a cell, and the color of each point is colored according to the number of UMIs. The redder the color, the higher the UMI, and the bluer the color, the lower the UMI. A high UMI may indicate a high RNA content in the cell. Figure 4 In the figure, B is the t-SNE projection of automatically clustered and colored cells. 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.

[0057] Example 2

[0058] Based on Example 1, the enzymatic hydrolysis time in step (3) was changed to 20 min. The results were the same as those in Example 1. Data omitted.

[0059] Comparative Example 1

[0060] Based on Example 1, the type of enzyme in step (3) was changed to 0.25% trypsin. The specific operation was as follows: 1 mL of 0.25% trypsin solution was added to the centrifuge tube, and the tube was placed at room temperature (25-27°C) for digestion for 15 min. During this period, the EP tube was placed on a rotator. After digestion, the tube was gently blown with a sterile wide-mouth resin-coated pipette tip for 2 min. The dissociation state of the gill filaments was observed under a microscope. The results were as follows: Figure 5 The other operations are the same as those in the embodiment.

[0061] Result description:

[0062] Figure 5 The results showed that the cell survival rate in the single-cell suspension of gill filaments of the Yesso scallop was relatively high, but there were still obvious differences compared with the examples, such as incomplete dissociation of the gill filament tissue, clearly visible tissue blocks, and a small number of single cells in the prepared suspension, which had a high risk of failure when running on various single-cell sequencing platforms.

[0063] Comparative Example 2

[0064] Based on Example 1, the enzymatic hydrolysis time in step (3) was changed to 40 min. The specific operation was as follows: 1 mL of the enzyme mixture solution was added to the centrifuge tube, and the tube was placed at room temperature (25-27°C) for digestion for 40 min. During this period, the EP tube was placed on a rotator. After digestion, the tube was gently blown with a sterile wide-mouth resin-coated pipette tip for 2 min. The dissociation state of the gill filaments was observed under a microscope. The results were as follows Figure 6 The other operations are the same as those in the embodiment.

[0065] Result description:

[0066] Figure 6 The results showed that there was a lot of cell death in the single-cell suspension of gill filaments of the Yesso scallop, which was significantly different from that in the example, and the cells were severely broken and the proportion of fragments was high, which had an extremely high risk of failure when running on various single-cell sequencing platforms.

[0067] Comparative Example 3

[0068] Based on Example 1, the speed and time of centrifugation in step (4) were changed. The specific operation was as follows: the enzymatic hydrolysate was placed in a centrifuge at 4°C, centrifuged at 650 g for 5 min, and the cell precipitate A was collected. The other operations were the same as in Example 1.

[0069] Result description:

[0070] Figure 7 The results showed that the cell survival rate of the single-cell suspension of gill filaments of the Yesso scallop was relatively high, with no significant difference compared with Example 1. However, the prepared single-cell suspension contained more impurities and a higher proportion of fragments, which posed a higher risk of failure for single-cell sequencing.

[0071] Table 2 Comparison of the preparation effects of single cell suspension of gill filaments of Yesso scallop

[0072]

[0073] In summary, the single-cell suspension of gill filaments of Yesso scallops digested with an enzyme mixture for 15 min and centrifuged at 300-650 g had more complete cell morphology, lower clumping rate and the highest viable cell rate, and the cell stability was significantly improved.

[0074] 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 protection scope of the present invention.

Claims

1. A method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection, characterized in that: The following steps are involved: (1) Non-lethal live sampling of gill filaments: extracting gill filament tissue from live shellfish; the marine shellfish include the Yesso scallop; (2) Rinse the gill filaments with pre-cooled phosphate buffer and dry them with absorbent paper; cut the gill filament tissue into small pieces; (3) using an enzyme mixture to enzymatically digest the gill filament tissue fragments; the enzyme mixture comprises: 2 mg / mL collagenase II, 2 mg / mL hyaluronidase and 40 U / mL deoxyribonuclease I; the enzymatic digestion time is 15-20 min; (4) Centrifuge the enzymatic hydrolysate to obtain a cell pellet at a speed of 300 g for 4-5 min. (5) resuspending the cells with a washing solution to obtain a cell suspension; the washing solution is a phosphate buffer containing 0.05% by volume of bovine serum albumin; (6) Repeat steps (4) and (5) twice; the centrifugation speed is 650 g and the centrifugation time is 4-5 min; (7) Filter the obtained cell suspension to obtain a single cell suspension.

2. The method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection according to claim 1, characterized in that: The non-lethal live sampling of gill filaments is as follows: select shellfish with good activity, wait for the shellfish to open its shell naturally, gently fix it with fingers, leaving a gap of 5 to 10 mm in width, use pointed tweezers to reach into the gap, and extract 2 gill filament tissues.

3. The method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection according to claim 1, characterized in that: The centrifugation temperature was 4°C.

4. The method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection according to claim 1, characterized in that: The phosphate buffer is 3.5× phosphate buffer.

5. The method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection according to claim 1, characterized in that: The filtration in step (7) is performed using a cell sieve with a pore size of 40 μm.

6. The method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection according to claim 1, characterized in that: The enzyme mixture is prepared using 3.5× phosphate buffer.

7. The method for preparing a single-cell suspension of marine shellfish suitable for dynamic detection according to claim 1, characterized in that: The enzymatic digestion time is 15 min.

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