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 monocell....

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, ....

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