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Optimizing Mouse Primary Lens Epithelial Cell Culture: A Comprehensive Guide to Trypsinization
In This Article
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 development1,2. 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.
....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
- Pr.......
Representative 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.......
Acknowledgements
This work was supported by NEI R21EY033941 (to Hongli Wu); Department of Defense W81XWH2010896 (to Hongli Wu); R15GM123463-02 (to Kayla Green and Hongli Wu)
....Materials
| 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 |
References
- Bermbach, G., Mayer, U., Naumann, G. O. Human lens epithelial cells in tissue culture. Experimental Eye Research. 52 (2), 113-119 (1991).
- Reddy, V. N., Lin, L. R., Arita, T., Zigler, J. S., Huang, Q. L. Crystallins and their s....
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