A Protocol for Harvesting Single-cell Suspension from Mouse Corneas
April 3rd, 2026
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.
This protocol primarily introduces a method for obtaining high quality single cells from intact mouse corneas. Our method is viable single cells with high RNA integrity, making them compatible with downstream applications such as single cell on a thick thin and flow diametry. To begin, position a six to eight-week-old euthanized female C57BL6 mouse on a sterile dissection table.
After rinsing the closed eyelids with PBS, use ophthalmic scissors to make an incision along the posterior pole of the eyeball, and carefully place the isolated eye into a Petri dish containing ice cold supplemental hormonal epithelial medium. After rinsing the eyeball three times with 1XPBS supplemented with 5%penicillin streptomycin, use ophthalmic scissors and forceps to make an incision from the posterior pole toward the corneal limbus. Gently detach the cornea from the sclera and conjunctiva along the limbal border, then using two pairs of forceps, completely remove the lens and iris.
After washing the corneal tissue three times in PBS, transfer the clean tissue to a new tube. Add one milliliter of five milligrams per milliliter collagenase A solution, and mince the tissue into fragments of approximately 0.5 by 0.5 millimeters using ophthalmic scissors. Incubate the tube containing the corneal fragments with collagenase A solution in a cell culture incubator at 37 degrees Celsius for one hour.
Inspect the tissue every 10 minutes during the digestion. When no large tissue fragments remain, centrifuge the sample at 300G for five minutes at room temperature, then carefully aspirate the supernatant. Next resuspend the pellet in 200 microliters of 0.25%trypsin EDTA solution.
Place the tube in a cell culture incubator set to 37 degrees Celsius for 10 minutes to digest remaining tissue fragments. Finally, use a P1000 pipette fitted with a wide bore tip to gently triturate the mixture to obtain a single cell suspension. Centrifuge the cell suspension as demonstrated earlier, and resuspend the cell pellet in PBS containing 0.04%BSA.
Upon counting the isolated corneal cells using a hemocytometer, it was found that a total of 1.05 times 10 to the power of five cells were obtained from eight corneas with an average cell viability of 94.6%and a single cell ratio exceeding 96.3%confirming the suspension suitability for downstream applications. Immunofluorescence analysis showed that the cell suspension comprised 83.5%pan-CK-positive epithelial cells, 13.5%keratocan positive stromal cells, and approximately 3%cells negative for both markers. The RNA extracted from the single cell suspension exhibited a high RNA integrity number of around 8.8 and a 28S by 18S ribosomal RNA ratio of about 2.15, confirming excellent RNA quality.
After obtaining single cells, we can characterize corneal cell function and surface mark exploration. This vicinity the study of cell-to-cell interactions. A challenge in this method is virtually separating the cornea from other tissues.
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Overview
This article presents a robust protocol for isolating high-quality single-cell suspensions from whole mouse corneas. The method combines precise tissue dissection with sequential enzymatic digestion, enabling the preparation of viable single cells suitable for downstream applications such as single-cell RNA sequencing (scRNA-seq) and flow cytometry. The protocol yields high cell numbers, excellent viability, and preserves RNA integrity, facilitating detailed analysis of corneal cellular heterogeneity.
Key Study Components
Area of Science
- Ophthalmology
- Cell Biology
- Single-cell Analysis
Background
- The cornea is the outermost layer of the eye, essential for vision and ocular surface homeostasis.
- Single-cell RNA sequencing (scRNA-seq) requires high-quality single-cell suspensions.
- Corneal tissue is dense and complex, making cell dissociation challenging.
- Existing protocols often yield low cell viability or poor RNA quality.
Purpose of Study
- To establish a reliable protocol for generating single-cell suspensions from intact mouse corneas.
- To ensure high cell viability and RNA integrity for downstream applications.
- To enable accurate identification and quantification of major corneal cell types.
Methods Used
- Dissection of corneas from euthanized C57BL6 mice.
- Sequential enzymatic digestion using collagenase A and trypsin-EDTA.
- Mechanical mincing and gentle trituration to achieve single-cell suspension.
- Cell viability assessment with trypan blue staining and hemocytometer counting.
- Immunofluorescence staining for Pancytokeratin (epithelial cells) and Keratocan (stromal cells).
- RNA integrity analysis using a bioanalyzer.
Main Results
- Obtained approximately 1.05 × 105 cells from eight corneas.
- High cell viability (94.6%) and single-cell ratio (96.3%).
- Cell composition: 83.5% pan-CK-positive epithelial cells, 13.5% keratocan-positive stromal cells, ~3% negative for both markers.
- RNA integrity number (RIN) of ~8.8 and 28S/18S rRNA ratio of ~2.15, indicating excellent RNA quality.
Conclusions
- The protocol reliably produces high-quality single-cell suspensions from mouse corneas.
- Cell preparations are suitable for demanding downstream applications, including scRNA-seq.
- This method facilitates detailed studies of corneal cell function and cell-to-cell interactions.
