A Protocol for Harvesting Single-cell Suspension from Mouse Corneas

Summary

Here, we present a protocol to obtain high-quality single-cell suspensions from whole mouse corneas through sequential digestion with collagenase A and trypsin. This method yields viable single cells with high RNA integrity, making them compatible with downstream applications such as single-cell RNA sequencing and flow cytometry.

Abstract

As the outermost layer of the eyeball, the cornea plays a critical role in maintaining ocular surface homeostasis and vision. Recent advances in single-cell RNA sequencing (scRNA-seq) have enabled high-resolution analysis of corneal cellular heterogeneity, yet the preparation of high-quality single-cell suspensions from corneal tissue remains challenging due to its dense and complex structure. In this study, we establish a robust protocol for generating single-cell suspensions from whole mouse corneas. The procedure involves mincing the corneas into fine fragments followed by sequential enzymatic digestion using collagenase A and trypsin. The resulting cell suspension was assessed for quality through multiple metrics: trypan blue staining was used to evaluate cell viability and count; immunofluorescence staining for Pancytokeratin and Keratocan enabled identification and quantification of major corneal cell types; and RNA integrity was verified using a bioanalyzer. This protocol consistently yields single-cell preparations with high cell numbers, excellent viability (≥ 94.6%), and a high proportion of single cells (96.3%), fulfilling the requirements compatible with demanding downstream applications, including scRNA-seq.

Introduction

The cornea, situated at the outermost layer of the eyeball, is essential for its barrier and refractive functions, owing to a highly organized architecture comprising five distinct layers: the epithelium, Bowman's layer, stroma, Descemet's membrane, and the endothelium¹. The corneal epithelium is a non-keratinized stratified squamous structure that provides a physical barrier against external pathogens and exhibits robust antimicrobial and regenerative capacities. The stroma, composed of precisely aligned collagen fibrils, maintains corneal transparency and a uniform refractive index². The endothelium, a monocellular layer, regulates fluid transfer between the stroma and aqueous humor, thereby preserving corneal hydration and nutrient supply³. Nonetheless, this intricate structural and cellular complexity has historically posed significant challenges for investigating corneal biological functions and underlying mechanisms⁴.

To study intercellular interactions and identify cell-type-specific markers, techniques such as flow cytometry sorting⁵ and single-cell RNA sequencing (scRNA-seq)⁶ are widely employed. A major technical hurdle common to both approaches, however, is the preparation of high-quality single-cell suspensions. Parameters including total cell yield, proportion of single cells, and cell viability are critical for the reliability of downstream applications aimed at characterizing corneal cell function and surface marker expression⁷. Despite its importance, there remains a lack of standardized and efficient protocols for generating single-cell suspensions from whole mouse corneas.

Several methods have been established for obtaining single-cell suspensions from corneal tissue. Sequential enzymatic digestion using collagenase A, trypsin, or TrypLE has been employed to isolate single cells from both human and murine corneas⁸. Repeated trypsinization has also been utilized for corneal dissociation⁷. Additionally, commercial multi-tissue dissociation kits, such as those from Miltenyi Biotec, have been successfully applied to generate corneal single-cell suspensions⁹. In our previous comparative study of different corneal digestion methods, we demonstrated that sequential treatment with collagenase and trypsin constitutes the optimal approach. This method consistently yields high cell numbers, excellent viability (≥94.6%), a high percentage of single cells (96.3%), and well-preserved RNA integrity¹⁰.

In this protocol, we provide a detailed, step-by-step procedure for generating high-quality single-cell suspensions from whole mouse corneas using this optimized enzymatic digestion strategy, based on our established experience and prior findings.

Protocol

Female C57BL/6 (B6) mice, 6-8 weeks old, were supplied by the Experimental Animal Center of Guizhou Medical University. The animals were housed under a 12 h light/12 h dark cycle with ad libitum access to food and water. All procedures were approved by the Experimental Animal Ethics Committee of Guizhou Medical University (Approval No. 2305185) and conducted in compliance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

1. Preparation

1. Preparation of surgical instruments: Clean two pairs of ophthalmic scissors, two pairs of ophthalmic forceps, one pair of tissue forceps, and one 2 mL centrifuge tube by rinsing twice with double-distilled water (DDW). Sterilize the instruments and tube using a steam autoclave. Dry all items overnight in a conventional oven at 65 °C.
2. Solution preparation
   1. Supplemental Hormonal Epithelial Medium (SHEM): Prepare SHEM by supplementing DMEM/F12 medium with the following components: 5% (v/v) fetal bovine serum (FBS), 0.5% (v/v) dimethyl sulfoxide (DMSO), 10 ng/mL mouse epidermal growth factor, 5.5 µg/mL transferrin, 0.5 µg/mL hydrocortisone, 1% (v/v) penicillin-streptomycin (100 U/mL penicillin, 100 µg/mL streptomycin).
      ​NOTE:SHEM is used to maintain cell viability during processing. It can be substituted with alternative media such as basal DMEM or phosphate-buffered saline (PBS) containing at least 10% (v/v) FBS.
   2. Phosphate-buffered saline (PBS)
      1. Dissolve 22.4 g of PBS powder in 2 L of double-distilled water (DDW) under stirring using a magnetic stirrer to obtain 1× PBS.
      2. Autoclave the PBS solution one day in advance and allow it to cool.
      3. Under sterile conditions, transfer 47.5 mL of sterile 1× PBS to a 50 mL centrifuge tube and add 2.5 mL of 1% (v/v) penicillin-streptomycin.
         NOTE: The 1× PBS formulation (2 L) contains 137 mM NaCl, 8 mM Na₂HPO₄, and 2 mM NaH₂PO₄, pH 7.2.
   3. Collagenase A solutions
      1. Stock solution: Dissolve 1 g of collagenase A powder in 40 mL of DDW. Filter sterilize through a 0.2 µm filter and store at -20 °C.
      2. Working solution: Dilute 1 mL of collagenase A stock solution in 4 mL of sterile DDW to obtain a final concentration of 5 mg/mL.
   4. Trypsin solution: Thaw commercially available 0.25% (w/v) trypsin/EDTA solution (containing 0.91 mM EDTA) on ice before use.
   5. Trypan Blue solution (0.4%): Dissolve 0.4 g of Trypan Blue in 100 mL of DDW. Filter using filter paper and store at 4 °C.
      NOTE: The 0.4% Trypan Blue solution is used for assessing total cell count, single-cell proportion, and viability. It may be substituted with commercial live/dead staining kits or automated cell-counting systems.

2. Preparation of mouse corneas

1. Sterilize the dissecting microscope and work area by wiping with 75% ethanol, then expose the area to UV light inside a biological safety cabinet for 30 min.
2. Prepare the following in sterile conditions: One 60 mm Petri dish with 5 mL of SHEM medium, three 60 mm Petri dishes each containing 5 mL of 1× PBS supplemented with 5% penicillin-streptomycin (P-S), three 60 mm Petri dishes each with 5 mL of plain 1× PBS, and one sterile 2 mL centrifuge tube
3. Euthanize the mouse using CO₂ asphyxiation, followed by cervical dislocation.
4. Rinse the mouse head and closed eyelids thoroughly by immersion three times in PBS containing 5% P-S before proceeding with enucleation.
5. Using ophthalmic scissors, make an incision along the posterior pole of each eyeball and carefully place the isolated eyes into the Petri dish containing ice-cold SHEM medium (Figure 1A).
   NOTE: To maximize cell viability, all solutions should be pre-chilled to 4 °C and all dissection steps must be performed on ice.
6. Immediately transfer the Petri dish with eyeballs to a biological safety cabinet. Wash the eyeballs three times in 1× PBS containing 5% P-S, incubating for 5 min per wash.
7. Under a dissecting microscope, carefully separate the corneal tissue: Using ophthalmic scissors and forceps, make an incision from the posterior pole toward the corneal limbus. Gently detach the cornea from the sclera and conjunctiva along the limbal border. Remove the lens and iris completely using two pairs of forceps (Figure 1B).
8. Using ophthalmic forceps, transfer the corneal tissue to a Petri dish containing 1× PBS and wash three times (5 min each) to remove any residual non-corneal tissue and antibiotics.
   NOTE: The iris often adheres tightly to the cornea. Ensure it is gently and completely removed using forceps to avoid contamination, leaving only clear corneal tissue.
9. Using ophthalmic forceps, transfer the cleaned corneal tissue to a 2 mL centrifuge tube and add 1 mL of collagenase A working solution (5 mg/mL).
10. Using ophthalmic scissors, mince the corneal tissue into approximately 0.5 mm × 0.5 mm fragments directly inside the tube.
11. Incubate the tube containing the corneal fragments and collagenase A solution in a cell culture incubator at 37 °C for 1 h (Figure 2A-C).
12. During collagenase A digestion, monitor the tissue every 10 min. Terminate digestion when no large tissue fragments remain by proceeding to centrifugation. Centrifuge the sample at 300 x g for 5 min at room temperature using a centrifuge. Carefully aspirate the supernatant.
13. Resuspend the pellet in 200 µL of 0.25% trypsin/EDTA solution.
14. Incubate the tube in a cell culture incubator at 37 °C for 10 min to digest remaining tissue fragments.

3. Isolation of corneal single cells

1. Gently triturate the mixture (tissue and trypsin/EDTA solution) repeatedly using a P1000 pipette with a wide-bore tip to dissociate the tissue into a single-cell suspension.
2. Centrifuge the cell suspension at 300 x g for 5 min and discard the supernatant. Resuspend the cell pellet in 1 mL of PBS containing 0.04% bovine serum albumin (BSA) to obtain a single-cell suspension.
   NOTE: For cells intended for sequencing or flow cytometry, resuspend in serum-free PBS. For cells that will be cultured, use 1x PBS supplemented with 0.04% BSA.
3. Filter the cell suspension through a pre-wetted 40 µm cell strainer into a new 50 mL centrifuge tube to remove cell clumps and debris.
   NOTE: Pre-wet the cell strainer with PBS to minimize cell loss. If necessary, the filtered cells may be centrifuged again and resuspended in a desired volume for concentration.

4. Cell counting

1. Combine 9 µL of the cell suspension with 1 µL of 0.4% Trypan Blue solution and mix gently.
2. Slowly load the mixture into a hemocytometer chamber via the designated loading channel. Count the cells using the four-quadrant method to determine total cell number, viability, and the proportion of single cells. Perform at least two technical replicates.

Results

The quality of the single-cell suspensions was assessed based on total cell yield, proportion of single cells, and cell viability. The suspension was stained with Trypan Blue and analyzed using phase-contrast microscopy across multiple fields (Figure 3). From four mouse eyes (eight corneas), a total of 1.05 × 10⁵ cells were obtained, with an average cell viability of 94.6% and a single-cell ratio exceeding 96.3%. These results confirm that the prepared cell suspension is suitable ...

Discussion

The introduction of single-cell RNA sequencing has markedly increased the use of genomic, transcriptomic, epigenetic, and proteomic technologies in ocular research^9,10,11,12. Consequently, the preparation of high-quality single-cell suspensions has become increasingly critical, as it allows the detection of signaling variations among individual cells and provides deeper insights into cellular h...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 594 donkey anti-rabbit	Abcam	ab150064
Antikeratin	Abcam	ab230175
Anti-pancytokeratin	Thermo Fisher Scientific	AE1/AE3, Alexa FluorTM 488
Autoclave sterilizer	An Shen	LDZH-100KBS
Bovine Serum Albumin V	Solarbio	A8020
Cell filter	NEST	258369	40 μm
Centrifugal machine	Eppendorf AG	22331 Hamburg
Collagenase A	Roche	10103578001
Confocal microscope	Oberkochen	Zeiss LSM 880
Counting plate	MARIENFELD	M407859
Centrifugal tube	NEST	620611	2 mL, 50mL
Coverslip	MARIENFELD	M407859
DAPI	Solarbio	C0065
Dissecting microscope	Carl ZEISS	VISU150
DMEM/F12  media	Gibco	11320033
DMSO	Solarbio	D8371
Fetal  bovine  serum	Gibco	16140071
Hydrocortisone	Solarbio	IH0100
ITS	Gibco	23337619
Mouse epidermal growth factor	Invitrogen	PMG8041
ophthalmic surgical  forceps	Dumot	0208-5-PO
ophthalmic surgical scissors	FST	15021-15
Oven	AISITE	DH6000 II
Penicillin &Streptomycin	Gibco	15070063	100mL
petri dish	NEST	705201	60mm
Phosphate buffered saline	Solarbio	P1010
Thermostatic incubator	Thermo Fisher Scientific	3111
Triton-X 100	Solarbio	T8200
Trypan Blue	Solarbio	T8070
Trypsin/EDTA	Gibco	R001100
Ultra-cleaning table	You Qi	VS-840-2
4% Paraformaldehyde	Solarbio	P1110