Single-Cell Suspension Preparation from Nile Tilapia Intestine for Single-Cell Sequencing

Summary

Here, we demonstrate the preparation of a high-quality single-cell suspension of tilapia intestine for single-cell sequencing.

Abstract

Nile tilapia is one of the most commonly cultured freshwater fish species worldwide and is a widely used research model for aquaculture fish studies. The preparation of high-quality single-cell suspensions is essential for single-cell level studies such as single-cell RNA or genome sequencing. However, there is no ready-to-use protocol for aquaculture fish species, particularly for the intestine of tilapia. The effective dissociation enzymes vary depending on the tissue type. Therefore, optimizing the tissue dissociation protocol by selecting the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. This study illustrates an optimized protocol to prepare a high-quality single-cell suspension from Nile tilapia intestine with an enzyme combination of collagenase/dispase. This combination is highly effective for dissociation with the utilization of bovine serum albumin and DNase to reduce cell aggregation after digestion. The cell output satisfies the requirements for single-cell sequencing, with 90% cell viability and a high cell concentration. This protocol can also be modified to prepare a single-cell suspension from the intestines of other fish species. This research provides an efficient reference protocol and reduces the need for additional trials in the preparation of single-cell suspensions for aquaculture fish species.

Introduction

Cells are the fundamental units of organisms. Compared to bulk-tissue studies, single-cell level studies can reflect cell heterogeneity and provide higher-resolution information¹. In recent years, researchers have applied single-cell sequencing technologies for genome, transcriptome, epigenome, or multi-omic studies at the single-cell level in mammals, zebrafish, and other model organisms and reported great breakthroughs^2,3,4,5,6,7. While most studies have focused on model organisms, there are few reference protocols or commercial dissociation kits for single-cell sequencing in economic fish species, which limits the application of single-cell sequencing in aquaculture research. Therefore, developing tissue dissociation protocols that produce high-quality single-cell suspensions with high cell viability and nucleic acid integrity is crucial.

Optimizing the tissue dissociation protocol with the appropriate enzyme or enzyme combination to obtain enough viable cells with minimum damage is essential. The most effective enzyme for tissue dissociation varies depending on the tissue type. In mammals, several enzymes have been used to prepare single-cell suspensions for mammalian solid tissues, including collagenase, dispase, trypsin, papain, elastase, hyaluronidase, liberase, accutase, and trypLE^8,9. Trypsin digestion combined with mechanical disruption has commonly been used to dissociate tissues for cell culture in fish^{10,11,12,13,14}. Trypsin has also been used or added into the digestion cocktail for tissue dissociation in the rat intestine¹⁵ and zebrafish gill tissue¹⁶. However, for several reasons, trypsin is not the best option for single-cell sequencing. Trypsin alone is usually ineffective for tissue dissociation. Additionally, trypsin induces DNA strand breaks^17,18 and RNA degradation¹⁹.

Papain degrades the proteins that make up the tight junctions between cells. In mammalian nerve and smooth muscle cells, papain is more efficient and less destructive than other proteases^20,21. However, like trypsin, papain results in the free DNA-induced aggregation of cells due to the cell lysis that occurs during enzymatic digestion⁹. Elastase breaks down elastin, which is typically found in the skin, lungs, ligaments, tendons, and vascular tissues²². It is often used in combination with collagenase, dispase, or trypsin for dissociating lung tissue⁸. Hyaluronidase cleaves the glycosidic bonds of hyaluronan, contributing to the digestion of the extracellular matrix in various connective tissues and skin^9,23.

In general, collagenase and dispase are good options for extracellular matrix breakdown. They have been used in the dissociation of human, mouse, and zebrafish intestines^24,25,26,27. Collagenase destroys the peptide bond in collagen, promotes the digestion of the extracellular matrix, and releases the cells into suspension, and, thus, collagenase is often used for human and mouse solid tissue dissociation, including for the liver^28,29, spleen³⁰, pancreas³¹, and intestine²⁵. Dispase is a protease that hydrolyzes the N-terminal peptide bonds of nonpolar amino acid residues and is milder than collagenase. It cleaves the extracellular matrix components, such as fibronectin, type IV collagen, and to a lesser extent, type I collagen, without affecting cell-cell junctions. Dispase is used separately or with other enzymes for tissue dissociation, such as for intestine^25,32, brain³³, liver³⁴, etc. In addition, commercially available digestion cocktails, including liberase, accutase, and trypLE, are also good alternatives for solid tissue dissociation, especially for the skin, liver, and kidney^8,9.

Nile tilapia (Oreochromis niloticus) belongs to the family Cichlidae of the order Perciformes. It is one of the most cultured freshwater fish species in tropical and subtropical areas, with an annual production of 4.5 million tons in 2022³⁵. It is one of the best-studied aquaculture fish species with a well-annotated genome. Nile tilapia is an ideal research model for aquaculture fish species due to its short generation time, ease of rearing, and adaptability to a wide range of rearing environments. The intestine is of great research interest as it is the organ of nutrition digestion and absorption, metabolism, and mucosal immunity. The intestine is the habitat of microbial populations and is an essential immune tissue³⁶. It is immunologically active due to the presence of numerous immune cell types, including macrophages, B cells, granulocytes, and T cells.

In the current study, we developed a protocol for preparing a high-quality single-cell suspension from the Nile tilapia intestine to facilitate single-cell level studies in aquaculture fish species. According to the characteristics of these tissue-specific enzymes and preliminary work, collagenase/dispase is appropriate for dissociating tilapia intestine tissue. The final enzyme type to consider in preparing single-cell suspensions is DNase-I, which prevents cell aggregation by degrading free DNA released through dead cell lysis during enzymatic digestion without initiating apoptotic pathways⁹ and increases the live cell yield³⁶. Additionally, bovine serum albumin (BSA) is added to the washing buffer to reduce cell clumping and improve cell viability. Several reagent companies describe BSA as an enzyme stabilizer. The addition of 0.04%-1% BSA to PBS (phosphate-buffered saline) has been used to develop a washing solution for preparing single-cell sequencing suspensions without adverse effects³⁸. The addition of a low ratio of BSA could help maintain cell viability and avoid the free DNA-induced aggregation of cells due to cell lysis. This protocol can also provide a valuable reference for developing cell dissociation protocols from the intestines of other aquaculture fish species.

Protocol

All animal protocols during this study were approved by the Hainan University Institutional Animal Care and Use Committee (Protocol number: HNUAUCC-2022-00063; Approval date: 2022-03-03). A list of the equipment and supplies used in this experiment can be found in the Table of Materials. A summary of the current protocol is illustrated in Figure 1.

1. Fish preparation

1. Obtain 6 month old Nile tilapia with a mean body weight of 100 g from a reliable source. Select fish that are free of any signs of disease.
2. Stop feeding the fish 24 h before proceeding to the next step.
   NOTE: Fish fasting reduces the volume of intestinal contents and facilitates the preparation of intestinal segments.
3. Rinse the fish in well-aerated sterile water for 15 min to wash off loosely bound bacteria on the body surface.
4. Euthanize the fish with 300 mg/L tricaine methanesulfonate (MS-222) for about 10 min until all movement stops.

2. Reagent preparation

1. Prepare 0.08% BSA-Dulbecco's phosphate-buffered saline (BSA-DPBS, for cell rinsing and resuspension). Dissolve 0.08 g of BSA (0.8% w/v) in 100 mL of 1x DPBS. Store at 4 °C, and pre-cool on ice when used.
   NOTE: The addition of BSA is beneficial for minimizing cell clumping. The BSA ratio can be increased from 0.04% to 1% according to the cell clumping conditions.
2. Prepare the enzymatic cell dissociation reagent.
   1. Dilute the collagenase/dispase with 1x PBS to a final concentration of 1 mg/mL (0.1 U/mL collagenase and 0.8 U/mL dispase). Ensure that PBS and not DPBS is used.
      NOTE: PBS provides the calcium ions needed for the enzyme to function properly. DPBS should not be used in this step because it is Ca²⁺/Mg²⁺ free. Additionally, collagenase type I is used in the current protocol.
   2. Filter the dilution through a sterile 0.22 µm membrane filter.
   3. Prepare a cell dissociation reagent that is composed of a 95% volume of collagenase/dispase solution and a 5% volume of fetal bovine serum (FBS).
      NOTE: The dissociation reagent should be made fresh every time it is used. Using 5% FBS helps maintain the cell viability during digestion.
3. Prepare trypan blue solution.
   1. Take the trypan blue solution from the 4 °C refrigerator, and keep it at 15-20 °C for a few minutes before use to prevent precipitation.
   2. Filter the solution through a sterile 0.22 µm membrane.

3. Equipment preparation

1. Pre-cool the refrigerated centrifuge to 4 °C before use.
2. Obtain sterile RNase-free 1.5 mL centrifuge tubes, 50 mL conical tubes, wide-bore glass pipettes and tips, membrane filters, cell strainers, and all the low retention tubes and tips.
3. Autoclave glass pipettes and dissection tools, including forceps, scissors, and scalpels, and pre-treat them with the RNase removal solution to prevent the adverse effects of RNase for single-cell RNA-seq.

4. Tissue dissection and cell dissociation

1. Put the euthanized fish on ice for dissection. Aseptically, collect 3-4 cm of the mid-section of the intestine, and excise as much fat and mesentery as possible. Carefully remove the fat attached to the intestinal surface and the layer of elastic and malleable clear mucosa with forceps.
   NOTE: The fat and mesentery are attached to the external surface of the intestine.
2. Remove as much fat as possible to prevent its adverse effects on the dissociation protocol. Using a syringe, gently rinse the fragments with sterile ice-cold PBS several times to wash off the intestinal contents and mucus. Avoid rough handling.
3. Dissect the segment of intestine into small pieces, and transfer them to a 1.5 mL tube filled with 1 mL of ice-cold PBS.
4. Centrifuge at 300 × g for 5 min at 4 °C. Remove the supernatant gently, and replace with 1 mL of ice-cold 0.08% BSA-DPBS. Resuspend the tissue fragments by gentle aspiration using a glass pipette. Repeat this step twice.
5. Remove the supernatant, and digest the tissue fragments using 1 mL of the enzymatic dissociation reagent (950 µL of collagenase/dispase solution and 50 µL of FBS) in a 37 °C water bath for 30-60 min.
   1. Additionally, add 5 µL of RNase inhibitor (40 U/µL in the stock solution) to the dissociation reagent if the samples are prepared for single-cell RNA sequencing.
      NOTE: The final concentration of the collagenase/dispase mix is 1 mg/mL, including collagenase at 0.1 U/mL and dispase at 0.8 U/mL.
6. Manually invert the tube at 5 min intervals during the 30-60 min water bath.
   NOTE: The exact incubation time varies according to the tissues used. Check the progress of dissociation under a microscope until no bulk tissue is seen. Place 10 µL of the cell suspension on a glass slide with a pipette, and examine it under the microscope. Increase the digestion time if the cells are not completely dissociated.
7. Spin down the tubes at 300 × g for 5 min at 4 °C, and gently remove the enzymatic cell dissociation reagent using wide-bore tips. Add 1 mL of ice-cold 0.08% BSA-DPBS, and gently pipette the cells up and down using a wide-bore glass pipette for 1 min.
   1. Repeat this step twice.
      NOTE: Pipetting up and down should be performed with care using wide-bore and low-retention tips to prevent cell disruption.
8. Pre-wet a 40 µm cell strainer with 0.08% BSA-DPBS, and place it into a 50 mL conical tube. Pass the cell suspension through the strainer. Tap and rinse with an additional 200 µL DPBS, and collect the suspension at the bottom of the tube.
9. Transfer the suspension into a 1.5 mL tube. Add 5 µL of DNase I (1 U/µL), and incubate for 15 min at 15-20 °C. Centrifuge at 300 × g for 5 min at 4 °C.
10. Remove the supernatant, and resuspend the cell pellet in 400 µL of ice-cold DPBS (Ca²⁺/Mg²⁺ free). Gently pipette up and down with wide-bore tips to mix the cells. Repeat this step twice.

5. Staining and microscopic examination

1. For staining, mix the cell suspension with 0.4% trypan blue solution at a 1:1 ratio. Incubate for 3 min at room temperature.
2. Add 5-10 µL of the mixture onto a hemocytometer, and examine under the microscope.
   NOTE: Viable cells will have unstained cytoplasm, while dead cells will have a blue cytoplasm (Figure 2).
3. Count the viable and dead cells in three different fields under the microscope. Calculate the cell viability percentage by dividing the number of viable cells by the total number of cells in a field.
   NOTE: The cell viability should be more than 85%, and cell concentration should not be less than 1 x 10⁵-1 x 10⁷/mL. The cell suspension background should be clean, without clumps, fragments, or impurities.

Results

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine for single-cell sequencing (Figure 1). This research shows that the collagenase/dispase mix has a good dissociation effect and is mild for intestine tissue. The selection of the optimal digestion enzyme is essential for preparing a high-quality single-cell suspension. In the preliminary work, the dissociation efficiencies of several commonly used enzymes were compared, and the results a...

Discussion

This protocol describes the preparation of a high-quality single-cell suspension of Nile tilapia intestine. Before dissociation, the removal of fat and mesentery from the intestine is necessary, particularly for carnivorous fish intestines with much fat. Using a syringe instead of hard scraping to wash off the intestinal contents reduces the mechanical damage to the cells. To ensure cell viability, it is also essential to maintain the temperature at 20 °C or below for the tissue dissection and rinsing steps. The was...

Materials

Name	Company	Catalog Number	Comments
0.22-μm Sterile Filter	Solarbio Life Sciences	SLGV033RB	It is used to filter and sterilize the enzyme solution.
40-μm Cell Strainer	Solarbio Life Sciences	F8200	Cell Strainer is applied to eliminate undigested tissue pieces.
Bovine serum albumin (BSA)	Sigma-Aldrich	SRE0098	Powder; dilute 0.04 g BSA with 100 mL 1× DPBS to prepare 0.04% BSA-DPBS washing bffer. Store at 2 - 8 °C.
Collagenase II	Sangon Biotech	A004202	Dilute with PBS to a final concentration of 1 mg/mL.
Collagenase/dispase	Roche	10269638-001	Dilute with PBS to a final concentration of 1 mg/mL.
Dispase	Sigma-Aldrich	D4818	Dilute with PBS to a final concentration of 1 mg/mL.
DNase I	Sigma-Aldrich	AMPD1	DNase I helps reduce cell clumping.
Dulbecco's phosphate-buffered saline (DPBS), Ca2+/Mg2+-free	Solarbio Life Sciences	E607009-0500	Store at room temperature.
Elastase	Sangon Biotech	A600438	Dilute with PBS to a final concentration of 0.5 mg/mL.
Fetal bovine serum (FBS)	Gibco	16000-044	Serum, used at volume of 5% in digetstion solution.
Inverted Microscope	Leica	qTOWER3G	It is used to examine cell viability.
Liberase	Roche	5401119001	Dilute with PBS to a final concentration of 0.25 mg/mL.
Nile tilpia (Oreochromis niloticus)	ProGift Aquaculture Technology Co. Ltd.	NA	Healthy fish with no disease signs (Mean body weight: 100 g).
Phosphate-buffered saline (PBS)	Solarbio Life Sciences	P1020	Store at room temperature.
Refrigerated Centrifuge	Eppendorf	5424	It is used to spin down the tissue and cell petet.
RNase inhibitor	NEB	M0314L	Inhibit RNase activity
Solid-phase RNase-Be-Gone Reagent	Sangon Biotech	B644201-0050	It is used to remove the RNase from tools such as dissecting scissors and glass pipettes. Store at room temperature.
Tricaine methanesulfonate (MS-222)	Sigma-Aldrich	E10521	For fish euthanasia.
Trypan Blue	Invitrogen	C0040	It is used for staining dead cells.
Trypsin	Sangon Biotech	E607001	Dilute with PBS to a final concentration of 1 mg/mL.