Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization

Summary

This manuscript outlines a detailed video protocol for culturing primary lens epithelial cells (LECs), aiming to improve reproducibility and aid research in cataracts and posterior capsule opacification (PCO). It offers step-by-step instructions on lens dissection, LECs isolation, and validation, serving as a valuable guide, especially for newcomers in the field.

Abstract

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, development, size, and transparency. Conversely, dysfunctional LECs can lead to cataract formation and posterior capsule opacification (PCO). Consequently, establishing a robust primary LEC culture system is important to researchers engaged in lens development, biochemistry, cataract therapeutics, and PCO prevention. However, cultivating primary LECs has long presented challenges due to their limited availability, slow proliferation rate, and delicate nature.

This study addresses these hurdles by presenting a comprehensive protocol for primary LEC culture. The protocol encompasses essential steps such as the formulation of an optimized culture medium, precise isolation of lens capsules, trypsinization techniques, subculture procedures, harvest protocols, and guidelines for storage and shipment. Throughout the culture process, cell morphology was monitored using phase-contrast microscopy.

To confirm the authenticity of the cultured LECs, immunofluorescence assays were conducted to detect the presence and subcellular distribution of critical lens proteins, namely αA- and γ-crystallins. This detailed protocol equips researchers with a valuable resource for cultivating and characterizing primary LECs, enabling advancements in our comprehension of lens biology and the development of therapeutic strategies for lens-related disorders.

Introduction

The lens of the eye plays a crucial role in vision by focusing incoming light onto the retina. It consists of a transparent, avascular structure composed of specialized cells, among which lens epithelial cells (LECs) are key players. LECs are located at the anterior surface of the lens and are responsible for maintaining its transparency, regulating water balance, and participating in lens growth and development^1,2. LECs are a unique type of cells located at the anterior part of the lens, playing a critical role in maintaining lens clarity and function by continuously producing lens fibers throughout life.

Cataracts are characterized by the progressive clouding of the lens, resulting in the distortion and scattering of light, leading to compromised vision^3,4. The precise mechanisms underlying cataract formation are complex and multifactorial, involving various cellular and molecular processes such as UV radiation, oxidative damage, and glycation^5,6. LECs have been found to contribute significantly to the development of cataracts, making them a vital focus of research^1,2,7,8,9.

Furthermore, one of the most pressing issues in ophthalmology today is the relatively high incidence of posterior capsule opacification (PCO), also known as secondary cataract. PCO remains the most common complication after cataract surgery, affecting up to 20-40% of adult patients and 100% of children within 5 years post surgery¹⁰. PCO is primarily caused by the residual LECs that remain in the capsular bag following cataract extraction. These cells undergo a multifaceted pathophysiological transformation involving not only epithelial-to-mesenchymal transition (EMT) but also the differentiation of LECs to lens fibers, resulting in a cell population that is a mixture of LECs, fibers, and myofibroblasts^11,12,13. The transformed cells proliferate and migrate across the posterior lens capsule, leading to visual impairment. Understanding the behavior and control mechanisms of LECs in culture models can provide valuable insights into the prevention and management of PCO. Therefore, this protocol of culturing LECs presents a vital tool for ophthalmic researchers aiming to study, understand, and ultimately combat this prevalent postoperative complication.

To unravel the intricacies of LEC biology and its role in cataract formation and PCO, it is essential to establish robust and reproducible in vitro primary cell culture systems. Primary LEC culture provides researchers with a controlled environment to study the functions, signaling, and molecular characteristics of LECs. Furthermore, it allows for the investigation of cellular processes and the effects of different experimental conditions, providing valuable insights into lens physiology and pathology.

Prior research has enriched our understanding of LEC culture techniques^{14,15,16,17,18,19,20}. Although these studies have employed various methodologies and yielded significant findings on LEC behavior and characteristics, a comprehensive and accessible video recording protocol for culturing LECs is absent in the current literature. This limitation can hinder novice researchers' ability to accurately reproduce the techniques and can lead to inconsistencies and variations in experimental results. By providing a video recording protocol, this research paper aims to bridge this gap and provide a standardized resource that can enhance reproducibility and facilitate knowledge transfer in the field of LEC culture.

Protocol

All animal experiments were performed in accordance with the Association for Research in Vision and Ophthalmology guidelines for the Use of Animals in Ophthalmic and Vision Research. Procedural approval was granted by the University of North Texas Health Science Center Animal Care and Use Committee (protocol number: IACUC-2022-0008). Young C57BL/6J mice, typically under 2 weeks of age, were used in these studies.

1. Culture medium preparation and lens dissection

1. Prepare culture medium by adding 50 mL of fetal bovine serum (FBS) and 0.1 mL of 50 mg/mL gentamicin to 450 mL of DMEM.
2. Humanely euthanize C57BL/6J mice younger than 2 weeks.
   NOTE: We euthanized using the CO₂ inhalation method. An optimal flow rate for CO₂ euthanasia systems should displace 30% to 70% of the chamber or cage volume/min.
3. Gently remove the eyelids using surgical scissors and apply delicate pressure with curved tweezers on opposite sides of the eye socket, causing the eye to protrude outward. Make a careful incision on the cornea using the cataract knife and carefully extract the lens using curved tweezers, ensuring no damage to the lens or its capsule.
   NOTE: Exercise caution while performing these steps to maintain the integrity of the lens capsule. Due to the delicate nature of the lens, it is important to use dissecting tools with curved and blunt tips to minimize the risk of lens damage.
4. Utilize curved tweezers with blunt tips to transfer the lenses into a 60 mm plastic tissue culture dish filled with 5 mL of prewarmed and sterile Dulbecco's phosphate buffered saline (DPBS) solution containing 10 µg/mL gentamicin.
5. Gently rinse the lenses with DPBS solution containing 10 µg/mL gentamicin to remove any potential debris or contaminants, prepare the lenses for further processing, and maintain a sterile culture environment.
6. To obtain an adequate number of LECs, pool four lenses for a 24-well culture plate and six lenses for a 6-well culture plate.

2. LECs isolation

1. After completing the rinsing process, place the lens on a piece of filter paper, allowing it to dry.
2. Once the lens is adequately dry, transfer it carefully to the cover of a Petri dish in preparation for the lens capsule removal.
3. Rotate the lens upward, ensuring that the anterior segment is facing upward. While using the tweezers to hold the anterior capsule, employ the capsulorhexis forceps in the dominant hand to create a small tear in the capsule. Gently pull the two tools in opposite directions to remove the capsule and put it in DPBS until all the lens dissections are completed.
   NOTE: To avoid any discrepancies, researchers should promptly dissect each lens epithelial capsule and temporarily store them in DPBS. Only after completing all dissections are the capsules collectively transferred to trypsin maintained at 37 °C, ensuring synchronized and uniform exposure.
4. Carefully transfer the lens capsule to a 6-well plate. Add 1 mL of 0.05% trypsin solution to each well to initiate the enzymatic digestion process.
5. Gently agitate the trypsin solution to ensure even permeation. Place the plate in a cell culture incubator and allow the capsule to be digested for 8-10 min at 37 °C.
   NOTE: This step facilitates the breakdown of the lens capsule tissue and the subsequent release of individual epithelial cells.
6. After the incubation, carefully mince the digested lens capsule using dissecting scissors to break down any remaining tissue clumps and promote cell separation.
   NOTE: Thoroughness in tissue mincing is emphasized to ensure efficient cell release from the digested lens capsules.
7. Add 0.5 mL of the culture medium containing 10% FBS to quench the trypsin. Transfer the tissue samples to a centrifugation tube and centrifuge at 1,000 × g for 5 min.
8. Carefully remove the supernatant without disturbing the cell pellet. Use 1 mL of culture medium to resuspend the cells and seed the cells in a 24-well plate.
9. Change the culture medium every 2-3 days.

3. LECs subculture

1. Once the cells achieve confluence, remove the medium from the culture dish. Proceed to wash the cells 2x with 1 mL of DPBS.
2. Add 200 µL of trypsin-EDTA solution and place the cells in the incubator for 5 min.
3. After incubation, remove the cells from the incubator and inspect them under a microscope to confirm that they have detached from the culture dish and begun to float.
4. Add 1 mL of culture medium and gently pipette the cells 3-5x to detach all the cells.
5. Transfer the cells to centrifuge tubes and centrifuge at 1,000 × g for 5 min.
6. Carefully remove the supernatant and resuspend the cells in the complete growth medium.
7. If needed, count the cell number using a hemocytometer.
8. Subdivide the cell suspension at a 1:2 or 1:3 ratio for subculturing purposes.
9. When the culture becomes confluent again, repeat the aforementioned procedures.
   ​NOTE: LECs flourish in high-density culture conditions. Avoid excessively diluting the cells, as this may hinder their growth.

4. Storage and shipment

NOTE: The ideal cell number for storage is ~1 × 10⁶.

1. Thoroughly wash the cells 3x with 1 mL of DPBS. After washing, add 1 mL of trypsin-EDTA solution and place the cells in the incubator for 5 min.
2. Add 2 mL of complete culture medium and transfer the cell suspension to the centrifuge tube and centrifuge at 1,000 × g for 5 min.
3. Discard the supernatant and resuspend the cells in a freezing medium composed of 90% FBS and 10% DMSO, aiming for a cell density of 1 × 10⁶ cells/mL. Transfer the cell suspension to the cryovial.
4. Immediately move the cells to a -20 °C environment for 1 h, followed by -80 °C overnight, prior to permanent storage in liquid nitrogen.
   NOTE: If liquid nitrogen is unavailable, the cells can be stored at -80 °C after an initial hour at -20 °C.
5. If a shipment is required, ship the cells in the cryovial in a package with dry ice for overnight delivery.
6. Upon receipt of the cells, ensure swift recovery and place the cells in the subculture. If immediate culture is not feasible, transfer the cells to liquid nitrogen for prolonged storage.
   ​NOTE: If the cells are to be shipped, ensure the samples are deeply buried in the dry ice to prevent potential damage from temperature fluctuations.

5. LECs validation

1. Plate LECs into 35 mm culture dishes with cover glasses and culture them for approximately 48 h.
2. Wash the cells 2x with PBS and fix the cells with cold methanol for 10 min at -20 °C.
3. Wash the fixed cells for 3 x 5 min with PBS and incubate the fixed cells with blocking buffer for 1 h at room temperature to prevent non-specific binding.
4. After blocking, incubate the cells overnight at 4 °C with the primary antibodies (αA-crystallin, γ-crystallin, and PROX1 antibodies) individually diluted at a 1:50 ratio in diluent buffer.
5. Wash the cells for 3 x 5 min with PBS and incubate the cells with the secondary antibody diluted 1:100 in diluent buffer for 1 h.
6. Wash the cells for 3 x 5 min with PBS and stain the cells with 5 µg/mL Hoechst 33342 in PBS for 10 min at room temperature to visualize the nuclei.
7. Wash the cells for 2 x 5 min with PBS to remove excess staining solution and capture fluorescent images of the cells using a fluorescence microscope using the DAPI channel for nuclei and FITC channel for αA-, γ-crystallins, and PROX1.

Results

As shown in Figure 2, by following this protocol, primary LECs from C57BL/6J mice adhered to the dishes within a period of 4 h. Notably, there were visible remnants of other tissues such as sections of the posterior capsule and lens fiber cells. However, these unintended elements did not attach to the dish and could, therefore, be removed by changing the culture medium. Subsequently, between the third and fifth day, the LECs initiated their proliferation phase. Rapid growth, characteristic o...

Discussion

The protocol presented in this paper provides a comprehensive, step-by-step guide to the successful isolation, culture, and subculture of primary LECs, complete with accompanying video documentation. The detailed visual guide alongside the written instructions enhances the clarity and accessibility of the protocol, promoting its use and reproducibility among researchers in the field. The ultimate aim is to contribute to the expanding body of knowledge surrounding the role of LECs in cataract formation and PCO, a prevalen...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.05% Trypsin-EDTA	Thermo Fisher	#25300054	For LECs dissociation
Alexa Fluor 488 Secondary Antibody	Jackson ImmunoResearch	#715-545-150	For cell validation
Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L)	Jackson ImmunoResearch	111-605-003	For cell validation
Antibody dilution buffer	Licor	#927-60001	For cell validation
Beaver safety knife	Beaver-Visitec International	#3782235	For lens dissection
Blocking buffer	Licor	#927-60001	For cell validation
Capsulorhexis forceps	Titan Medical Instruments	TMF-124	For lens capsule isolation
DMEM	Sigma Aldrich	D6429	For LECs culture medium
DMSO	Sigma Aldrich	#D2650	For making freezing medium
Dulbecco's Phosphate Buffered Saline	Thermo Fisher	#J67802	For lens dissection
Dumont tweezers	Roboz Surgical Instrument	RS-4976	For lens capsule isolation
EpiCGS-a (optional)	ScienCell	4182	For LECs culture medium
FBS	Sigma Aldrich	F2442	For LECs culture medium
Gentamicin (50 mg/mL)	Sigma-Aldrich	G1397	For LECs culture medium
Hoechst 33342 solution	Thermo Fisher	#62249	For cell validation
Micro-dissecting scissors	Roboz Surgical Instrument	RS-5983	For lens dissection
Micro-dissecting tweezers	Roboz Surgical Instrument	RS5137	For lens dissection
PROX1 antibody	Thermo Fisher	11067-2-AP	For cell validation
Vannas micro-dissecting spring scissors	Roboz Surgical Instrument	RS-5608	For lens capsule isolation
αA-crystallin antibody	Santa Cruz	sc-28306	For cell validation
γ-crystallin antibody	Santa Cruz	sc-365256	For cell validation