A fish tissue single cell suspension and preparation method thereof

Abstract

The present disclosure provides a fish tissue single cell suspension and a preparation method thereof, the method comprising: pre-treating the target tissue of the fish body into tissue fragments and placing it in a centrifuge tube equipped with a buffer solution; centrifuging the centrifuge tube, removing the supernatant, adding a BSA-DPBS solution, and blowing to resuspend the tissue fragments; removing the supernatant in the centrifuge tube, adding an enzyme dissociation reagent to digest the tissue fragments; removing the supernatant in the centrifuge tube, adding a BSA-DPBS solution, blowing to resuspend the digested tissue fragments to obtain a cell mixture; filtering the cell mixture through a cell filter screen, collecting the filtrate; adding DNase I to the filtrate, incubating at room temperature; centrifuging the mixture after incubation, removing the supernatant, resuspending in DPBS, blowing and dispersing cells, and obtaining a target tissue single cell suspension. Through the preparation method of the present invention, it is possible to achieve fast and efficient acquisition of fish tissue single cell suspensions, and ensure the viability of dissociated cells, and the quality of the single cell suspension meets the requirements of single cell sequencing.

Description

Technical Field

[0001] The present disclosure relates to the technical field of single cell suspension, and in particular to a fish tissue single cell suspension and a preparation method thereof. Background Art

[0002] Cells are the basic structural and functional units of organisms. Research at the single-cell level can reflect the heterogeneity of cells. In recent years, single-cell sequencing technology has been widely used in transcriptomics, genome, epigenomics and multi-omics research of model organisms such as mammals and zebrafish, and has made major breakthroughs, but there are few reports on economic fish.

[0003] The cell suspension for single-cell sequencing requires a cell survival rate of >80%, a concentration of no less than 700 cells / μL, and a clear cell suspension background without a large number of clumps, debris, and impurities. Currently, the preparation of aquatic fish cell suspensions is mainly for tissue cell culture, and most of them are mechanically broken and trypsin dissociated. There are problems such as insufficient dissociation, large cell damage, cell aggregation, and survival rate and concentration that cannot meet sequencing requirements, which limits the application of single-cell sequencing in aquaculture research. Summary of the invention

[0004] The present disclosure provides a fish tissue single cell suspension and a preparation method thereof, so as to at least solve the technical problems existing in the prior art of cell suspension, such as insufficient dissociation, significant cell damage, cell aggregation, and survival rate and concentration failing to meet sequencing requirements.

[0005] According to a first aspect of the present disclosure, a method for preparing a single cell suspension of fish tissue is provided, the method comprising:

[0006] Pre-process the target tissue of the fish body into tissue fragments, and place them in a centrifuge tube filled with a buffer solution;

[0007] Centrifuge the centrifuge tube, remove the supernatant, add BSA (bovine serum albumin)-DPBS (Dulbeccophosphate-buffered saline) solution, and pipette to resuspend the tissue fragments;

[0008] Remove the supernatant in the centrifuge tube and add enzyme dissociation reagent to digest the tissue fragments;

[0009] The supernatant in the centrifuge tube was removed, and BSA-DPBS solution was added, and the digested tissue fragments were resuspended by pipetting to obtain a cell mixture;

[0010] Filter the cell mixture through a cell strainer and collect the filtrate;

[0011] Add DNase I (deoxyribonuclease I) to the filtrate and incubate at room temperature;

[0012] The incubated mixture was centrifuged, the supernatant was removed, and the mixture was resuspended in DPBS. The cells were dispersed by pipetting to obtain a single cell suspension of the target tissue.

[0013] In one embodiment, the preprocessing of the target tissue of the fish body into tissue fragments comprises:

[0014] Take fresh target tissue from the fish body, remove fat and white membrane, clean the target tissue, place it in a sterile culture dish and cut it, and then place the tissue fragments in a centrifuge tube filled with buffer.

[0015] In one embodiment, the mass volume ratio of BSA to DPBS in the BSA-DPBS solution is 0.04-0.1 g:100 mL.

[0016] In one embodiment, the mass volume ratio of BSA to DPBS in the BSA-DPBS solution is 0.08 g:100 mL.

[0017] In one embodiment, the enzymatic dissociation reagent includes 90-95% by volume of collagenase and dispase solution, and 5%-10% of FBS (fetal bovine serum).

[0018] In one embodiment, the concentration of the collagenase is 0.1 U / mL, and the concentration of the dispase is 0.8 U / mL.

[0019] In one embodiment, filtering the cell mixture through a cell filter and collecting the filtrate comprises:

[0020] Rinse the cell filter with BSA-DPBS solution and place the rinsed cell filter in a centrifuge tube;

[0021] The cell mixture was poured into a cell filter, and the cell filter was rinsed with BSA-DPBS solution, and the filtrate at the bottom of the centrifuge tube was collected.

[0022] In one embodiment, the centrifugation condition is 4°C, 300g for 5 min.

[0023] In one embodiment, the blowing is blowing using a wide-mouth glass tube or a wide-mouth low-adsorption gun tip.

[0024] According to a second aspect of the present disclosure, a single-cell suspension of fish tissue is provided, and the single-cell suspension is prepared according to the above method.

[0025] The present invention discloses a single-cell suspension of fish tissue and a preparation method thereof, wherein the target tissue of the fish is pretreated, then a BSA-DPBS solution is added for re-suspending, an enzyme dissociation reagent is added for digesting tissue fragments, the suspension is re-suspended, the cells in the collected filtrate are incubated, and finally the mixture after incubation is centrifuged, the supernatant is removed, the suspension is re-suspended in DPBS, and the cells are dispersed by blowing to obtain a single-cell suspension of the target tissue. The present invention resuspends the cells by blowing through a wide-mouth glass, which can reduce damage to the cells during the blowing process; bovine serum albumin BSA and DNA enzyme (DNase I) are further used to reduce cell aggregation after digestion; through the preparation method of the present invention, a single-cell suspension of fish tissue can be obtained quickly and efficiently, and the viability of the dissociated cells is ensured, and the quality of the single-cell suspension meets the requirements of single-cell sequencing.

[0026] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:

[0028] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.

[0029] Figure 1 A schematic diagram showing a process of preparing a single-cell suspension of fish tissue according to an embodiment of the present disclosure is shown;

[0030] Figure 2 A trypan blue-stained microscope photograph of a single-cell suspension of tilapia intestinal tissue in an embodiment of the present disclosure is shown (scale 60 μm);

[0031] Figure 3 A trypan blue-stained microscope photograph of a single-cell suspension of tilapia intestinal tissue according to an embodiment of the present disclosure is shown (scale bar 50 μm). DETAILED DESCRIPTION

[0032] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.

[0033] At present, the preparation of existing aquatic fish cell suspension is mainly aimed at tissue cell culture, mostly mechanical crushing and trypsin dissociation. Mechanical crushing causes great damage to cells. Trypsin alone does not dissociate many tissues well, and it can cause DNA chain breakage and RNA degradation in cells. There are problems such as insufficient dissociation, cell aggregation, and viability and concentration that cannot meet sequencing requirements. Therefore, it is necessary to determine the most suitable enzyme or enzyme combination, optimize the specific tissue dissociation operation to reduce cell aggregation and cell damage, and obtain enough living cells. It is crucial, and an effective tissue dissociation scheme to obtain a high-quality single-cell suspension with high cell viability and complete nucleic acid information is an important basis for the successful development of single-cell sequencing. Based on this, the present invention proposes a method for preparing a single-cell suspension of fish tissue to solve the above-mentioned technical defects in the prior art.

[0034] like Figure 1 A schematic flow chart of a method for preparing a single cell suspension of fish tissue is shown, the method comprising the following steps:

[0035] S1. Pretreatment of target tissue: Pretreatment the target tissue of the fish body into tissue fragments, and place them in a centrifuge tube filled with buffer.

[0036] The target tissue may be various organ tissues on the fish body, gill and spleen tissue, etc. In this embodiment, the target tissue takes fish intestinal tissue as an example.

[0037] In one example, pre-processing the target tissue of a fish body into tissue fragments includes:

[0038] Take fresh target tissue from the fish body, remove fat and white membrane, clean the target tissue, place it in a sterile culture dish and cut it, then place the tissue fragments in a centrifuge tube filled with buffer solution, the buffer solution is 1xPBS.

[0039] S2. Resuspend tissue fragments: centrifuge the centrifuge tube, remove the supernatant, add BSA-DPBS solution, and pipette to resuspend tissue fragments;

[0040] Place the centrifuge tube in a low-temperature centrifuge and centrifuge at 4°C, 300g for 5 minutes; remove the supernatant in the centrifuge tube and add ice-cold BSA-DPBS solution, in which the mass volume ratio of BSA to DPBS is 0.04-0.1g:100mL, that is, 0.04-0.1g of BSA is dissolved in 100mL of DPBS, and the mass concentration of BSA is 0.04-0.1%, and the mass concentration of BSA is preferably 0.08%; gently blow with a wide-mouth glass tube to resuspend the tissue fragments, and this step can be repeated 2 or more times.

[0041] S3, digestion of tissue fragments: remove the supernatant in the centrifuge tube, add enzyme dissociation reagent to digest the tissue fragments;

[0042] The centrifuge tube is placed in a low-temperature centrifuge, centrifuged at 4°C, 300g for 5 minutes; the supernatant in the centrifuge tube is removed, and an enzyme dissociation reagent is added, wherein the enzyme dissociation reagent includes a volume fraction of 90-95% collagenase and dispase solution, and 5%-10% FBS, preferably 95% collagenase and dispase solution, and 5% FBS, the concentration of collagenase is 0.1U / mL, and the concentration of dispase is 0.8U / mL. Digestion is carried out in a 37°C water bath, and the digestion time is adjusted according to the size of the tissue fragments, the amount of the tissue fragments, and the specific tissue site. During the digestion process, the centrifuge tube is turned upside down at intervals of a few minutes to allow the tissue fragments in the centrifuge tube to be fully digested.

[0043] It should be noted that if the single-cell suspension is used for single-cell RNA-seq (transcriptome sequencing), RNase inhibitor must also be added.

[0044] S4. Resuspend the digested tissue fragments: remove the supernatant in the centrifuge tube, add BSA-DPBS solution, and pipette to resuspend the digested tissue fragments to obtain a cell mixture;

[0045] Place the centrifuge tube in a low-temperature centrifuge, centrifuge at 4°C, 300g for 5 minutes, remove the supernatant in the centrifuge tube, add pre-cooled BSA-DPBS solution, and gently blow the cells with a wide-mouth glass tube for 1 minute; after centrifugation, add pre-cooled 0.08% BSA-DPBS and resuspend.

[0046] S5, filtration: filtering the cell mixture through a cell filter and collecting the filtrate;

[0047] In one example, the cell mixture is filtered through a cell strainer and the filtrate is collected, comprising:

[0048] Rinse the cell filter with BSA-DPBS solution and place the rinsed cell filter in a centrifuge tube;

[0049] The cell mixture was poured into a cell filter, and the cell filter was rinsed with BSA-DPBS solution, and the filtrate at the bottom of the centrifuge tube was collected.

[0050] S6, incubation: add DNase I to the filtrate and incubate at room temperature;

[0051] S7. Single-cell suspension of target tissue: centrifuge the incubated mixture, remove the supernatant, resuspend in DPBS, and pipette to disperse the cells to obtain a single-cell suspension of target tissue.

[0052] Place the centrifuge tube in a low-temperature centrifuge and centrifuge at 4°C, 300g for 5 minutes. Remove the supernatant in the centrifuge tube and resuspend the cells in 400 μL pre-cooled DPBS. Use a wide-mouth low-adsorption pipette tip to gently blow the cells to disperse them. Repeat this step 2 or more times to obtain a single-cell suspension of the target tissue.

[0053] The present invention discloses a single-cell suspension of fish tissue and a preparation method thereof, wherein the target tissue of the fish is pretreated, then a BSA-DPBS solution is added for re-suspending, an enzyme dissociation reagent is added for digesting tissue fragments, the suspension is re-suspended, the cells in the collected filtrate are incubated, and finally the mixture after incubation is centrifuged, the supernatant is removed, the suspension is re-suspended in DPBS, and the cells are dispersed by blowing to obtain a single-cell suspension of the target tissue. The present invention resuspends the cells by blowing through a wide-mouth glass, which can reduce damage to the cells during the blowing process; bovine serum albumin BSA and DNA enzyme (DNase I) are further used to reduce cell aggregation after digestion; through the preparation method of the present invention, a single-cell suspension of fish tissue can be obtained quickly and efficiently, and the viability of the dissociated cells is ensured, and the quality of the single-cell suspension meets the requirements of single-cell sequencing.

[0054] Example 1

[0055] This example takes tilapia as an example, and the target tissue takes the intestine of tilapia as an example, because the intestine of fish is not only an organ for fish to digest and absorb nutrients, but also a habitat for microbial populations and an important immune tissue. It is an important component of the mucosal immune system and has important research significance in terms of fish metabolism, nutrition, intestinal flora and immunity.

[0056] A method for preparing a fish intestinal single cell suspension comprises the following steps:

[0057] 1. Take 3-5cm of fresh intestinal tissue of tilapia, remove the fat and mesentery on the surface of the intestine, and use a syringe to draw pre-cooled 1xPBS to flush the intestinal contents, avoiding scraping;

[0058] 2. Place the cleaned intestinal tissue in a clean sterile culture dish and cut the tissue into 1-2 mm 3 The tissue pieces were removed and transferred into a 1.5 mL centrifuge tube containing 1 mL of pre-cooled 1x PBS;

[0059] 3. Place the centrifuge tube in a low-temperature centrifuge and centrifuge (300g) at 4°C for 5 minutes. Gently remove the supernatant and add 1 mL of ice-cold 0.08% BSA-DPBS. Gently pipette with a wide-mouth glass tube to resuspend the tissue fragments. Repeat this step twice.

[0060] 4. Place the centrifuge tube in a low-temperature centrifuge, centrifuge (300g) at 4°C for 5 minutes, gently remove the supernatant, add 1mL of enzyme dissociation reagent, and digest the tissue fragments in a 37°C water bath; the enzyme dissociation reagent consists of 95% by volume of collagenase and dispase (collagenase: 0.1U / mL, dispase: 0.8U / mL) solution and 5% by volume of FBS; dissociate in a water bath for 30min-1h, during which the centrifuge tube is turned upside down twice every 5min (the incubation time varies depending on the tissue, amount and tissue shear size. The number of free cells is observed under a microscope and the digestion of the tissue block is observed with the naked eye to determine the dissociation effect).

[0061] 5. After digestion, place the centrifuge tube in a low-temperature centrifuge, centrifuge (300g) at 4°C for 5 minutes, and gently remove the supernatant;

[0062] 6. Add 1 mL of pre-cooled 0.08% BSA-DPBS, gently blow the cells with a wide-mouth glass tube for 1 min, centrifuge, add pre-cooled 0.08% BSA-DPBS again and resuspend;

[0063] 7. Rinse the 40 μm cell filter with 0.08% BSA-DPBS and place the cell filter in a 50 mL centrifuge tube. Pass the cell mixture (the product obtained in step 6) through the cell filter and rinse the filter with 0.08% BSA-DPBS. Collect the filtrate at the bottom of the centrifuge tube.

[0064] 8. Add 5 μL DNase I to the collected filtrate and incubate at room temperature for 15 min;

[0065] 9. Transfer the incubated mixture to a 1.5 mL centrifuge tube, place the centrifuge tube in a low-temperature centrifuge, centrifuge (300 g) at 4°C for 5 minutes, and gently remove the supernatant;

[0066] 10. Resuspend the cells in 400 μL pre-cooled DPBS, and gently blow the cells with a wide-mouth low-adsorption pipette tip to disperse them. Repeat this step twice to obtain a single-cell suspension of intestinal tissue.

[0067] Microscopic examination of live cell yield: aspirate the single cell suspension and dilute it to an appropriate concentration, add trypan blue dye and an equal volume of intestinal tissue single cell suspension, mix thoroughly, add to a blood cell counting plate, count under a microscope, and measure the live cell ratio. This method was used to dissociate the tilapia intestine. The intestinal tissue was about 4 cm, and a single cell suspension with up to 90% cell viability was obtained after dissociation. The cell concentration was about 1.4×10 7 / mL, the cell suspension background should be clean, without a large number of clumps, debris and impurities.

[0068] like Figure 2-3Shown is a microscopic photograph of a single-cell suspension of tilapia intestinal tissue stained with trypan blue (the image has been processed in grayscale), in which bright white represents living cells and dark gray represents dead cells.

[0069] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solutions disclosed in this disclosure can be achieved, and this document does not limit this.

[0070] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features. In the description of the present disclosure, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0071] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A method for preparing a single cell suspension of fish tissue, characterized in that: The method comprises: Pre-process the target tissue of the fish body into tissue fragments, and place them in a centrifuge tube filled with a buffer solution; Centrifuge the centrifuge tube, remove the supernatant, add BSA-DPBS solution, and blow to resuspend the tissue fragments; the blowing is done by blowing with a wide-mouth glass tube or a wide-mouth low-adsorption gun tip; The supernatant in the centrifuge tube is removed, and an enzyme dissociation reagent is added to digest the tissue fragments; the enzyme dissociation reagent includes 90-95% collagenase and dispase solution by volume, and 5%-10% FBS; The supernatant in the centrifuge tube was removed, and BSA-DPBS solution was added, and the digested tissue fragments were resuspended by pipetting to obtain a cell mixture; Filter the cell mixture through a cell strainer and collect the filtrate; DNase I was added to the filtrate and incubated at room temperature; The incubated mixture was centrifuged, the supernatant was removed, and the mixture was resuspended in DPBS. The cells were dispersed by pipetting to obtain a single cell suspension of the target tissue.

2. The method according to claim 1, characterized in that: The method of pre-processing the target tissue of the fish body into tissue fragments comprises: Take fresh target tissue from the fish body, remove fat and white membrane, clean the target tissue, place it in a sterile culture dish and cut it, and then place the tissue fragments in a centrifuge tube filled with buffer.

3. The method according to claim 1, characterized in that: The mass volume ratio of BSA to DPBS in the BSA-DPBS solution is 0.04-0.1 g:100 mL.

4. The method according to claim 3, characterized in that The mass volume ratio of BSA to DPBS in the BSA-DPBS solution is 0.08 g:100 mL.

5. The method according to claim 4, characterized in that The concentration of the collagenase was 0.1 U / mL, and the concentration of the dispase was 0.8 U / mL.

6. The method according to claim 1, characterized in that The step of filtering the cell mixture through a cell filter screen and collecting the filtrate comprises: Rinse the cell filter with BSA-DPBS solution and place the rinsed cell filter in a centrifuge tube; The cell mixture was poured into a cell filter, and the cell filter was rinsed with BSA-DPBS solution, and the filtrate at the bottom of the centrifuge tube was collected.

7. The method according to claim 1, characterized in that The centrifugation conditions are 4°C, 300g for 5 min.

8. A single cell suspension of fish tissue, characterized in that: The single cell suspension is prepared according to the method according to any one of claims 1-7.

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