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)

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
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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&#39;s phosphate buffered saline (DPBS) solution containing 10 &#181;g/mL gentamicin.</li>
	<li>Gently rinse the lenses with DPBS solution containing 10 &#181;g/mL gentamicin to remove any potential debris or contaminants, prepare the lenses for further processing, and maintain a sterile culture environment.</li>
	<li>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.</li>
</ol>
2. LECs isolation
<ol>
	<li>After completing the rinsing process, place the lens on a piece of filter paper, allowing it to dry.</li>
	<li>Once the lens is adequately dry, transfer it carefully to the cover of a Petri dish in preparation for the lens capsule removal.</li>
	<li>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 &#176;C, ensuring synchronized and uniform exposure.</li>
	<li>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.</li>
	<li>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 &#176;C. 
	NOTE: This step facilitates the breakdown of the lens capsule tissue and the subsequent release of individual epithelial cells.</li>
	<li>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.</li>
	<li>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 &#215; g for 5 min.</li>
	<li>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.</li>
	<li>Change the culture medium every 2-3 days.</li>
</ol>
3. LECs subculture
<ol>
	<li>Once the cells achieve confluence, remove the medium from the culture dish. Proceed to wash the cells 2x with 1 mL of DPBS.</li>
	<li>Add 200 &#181;L of trypsin-EDTA solution and place the cells in the incubator for 5 min.</li>
	<li>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.</li>
	<li>Add 1 mL of culture medium and gently pipette the cells 3-5x to detach all the cells.</li>
	<li>Transfer the cells to centrifuge tubes and centrifuge at 1,000 &#215; g for 5 min.</li>
	<li>Carefully remove the supernatant and resuspend the cells in the complete growth medium.</li>
	<li>If needed, count the cell number using a hemocytometer.</li>
	<li>Subdivide the cell suspension at a 1:2 or 1:3 ratio for subculturing purposes.</li>
	<li>When the culture becomes confluent again, repeat the aforementioned procedures. 
	&#8203;NOTE: LECs flourish in high-density culture conditions. Avoid excessively diluting the cells, as this may hinder their growth.</li>
</ol>
4. Storage and shipment
NOTE: The ideal cell number for storage is ~1 &#215; 106.
<ol>
	<li>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.</li>
	<li>Add 2 mL of complete culture medium and transfer the cell suspension to the centrifuge tube and centrifuge at 1,000 &#215; g for 5 min.</li>
	<li>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 &#215; 106 cells/mL. Transfer the cell suspension to the cryovial.</li>
	<li>Immediately move the cells to a -20 &#176;C environment for 1 h, followed by -80 &#176;C overnight, prior to permanent storage in liquid nitrogen. 
	NOTE: If liquid nitrogen is unavailable, the cells can be stored at -80 &#176;C after an initial hour at -20 &#176;C.</li>
	<li>If a shipment is required, ship the cells in the cryovial in a package with dry ice for overnight delivery.</li>
	<li>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. 
	&#8203;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.</li>
</ol>
5. LECs validation
<ol>
	<li>Plate LECs into 35 mm culture dishes with cover glasses and culture them for approximately 48 h.</li>
	<li>Wash the cells 2x with PBS and fix the cells with cold methanol for 10 min at -20 &#176;C.</li>
	<li>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.</li>
	<li>After blocking, incubate the cells overnight at 4 &#176;C with the primary antibodies (&#945;A-crystallin, &#947;-crystallin, and PROX1 antibodies) individually diluted at a 1:50 ratio in diluent buffer.</li>
	<li>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.</li>
	<li>Wash the cells for 3 x 5 min with PBS and stain the cells with 5 &#181;g/mL Hoechst 33342 in PBS for 10 min at room temperature to visualize the nuclei.</li>
	<li>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 &#945;A-, &#947;-crystallins, and PROX1.</li>
</ol>

Cultivation and Characterization of Lens Epithelial Cells

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+global+state+of+cataract+blindness.">The global state of cataract blindness.</a> Current Opinion in Ophthalmology. 28 (1), 98-103 (2017).</li><li>Khairallah, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Number+of+people+blind+or+visually+impaired+by+cataract+worldwide+and+in+world+regions,+1990+to+2010.">Number of people blind or visually impaired by cataract worldwide and in world regions, 1990 to 2010.</a> Investigative Ophthalmology &amp; Visual Science. 56 (11), 6762-6769 (2015).</li><li>Beebe, D. C., Holekamp, N. M., Shui, Y. 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(i) effects of cell density, medium and embryonic age of initial culture.</a> Differentiation. 6 (3), 155-167 (1976).</li><li>Ibaraki, N., Ohara, K., Shimizu, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Explant+culture+of+human+lens+epithelial+cells+from+senile+cataract+patients.">Explant culture of human lens epithelial cells from senile cataract patients.</a> Japanese Journal of Ophthalmology. 37 (3), 310-317 (1993).</li><li>Sundelin, K., Petersen, A., Soltanpour, Y., Zetterberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+growth+of+lens+epithelial+cells+from+cataract+patients+-+association+with+possible+risk+factors+for+posterior+capsule+opacification.">In vitro growth of lens epithelial cells from cataract patients - association with possible risk factors for posterior capsule opacification.</a> The Open Ophthalmology Journal. 8, 19-23 (2014).</li><li>Parreno, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodologies+to+unlock+the+molecular+expression+and+cellular+structure+of+ocular+lens+epithelial+cells.">Methodologies to unlock the molecular expression and cellular structure of ocular lens epithelial cells.</a> Frontiers in Cell and Developmental Biology. 10, 983178 (2022).</li><li>Andjelic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+and+proliferative+studies+on+ex+vivo+cultured+human+anterior+lens+epithelial+cells+-+relevance+to+capsular+opacification.">Morphological and proliferative studies on ex vivo cultured human anterior lens epithelial cells - relevance to capsular opacification.</a> Acta Ophthalmologica. 93 (6), e499-e506 (2015).</li><li>Andjelic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+for+establishing+adherent+ex+vivo+explant+cultures+from+human+eye+pathologies+for+use+in+subsequent+calcium+imaging+and+inflammatory+studies.">A simple method for establishing adherent ex vivo explant cultures from human eye pathologies for use in subsequent calcium imaging and inflammatory studies.</a> Journal of Immunology Research. 2014, 232659 (2014).</li><li>Andley, U. P., Rhim, J. S., Chylack, L. T., Fleming, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+and+immortalization+of+human+lens+epithelial+cells+in+culture.">Propagation and immortalization of human lens epithelial cells in culture.</a> Investigative Ophthalmology &amp; Visual Science. 35 (7), 3094-3102 (1994).</li><li>Zhou, M., Leiberman, J., Xu, J., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hierarchy+of+proliferative+cells+exists+in+mouse+lens+epithelium:+Implications+for+lens+maintenance.">A hierarchy of proliferative cells exists in mouse lens epithelium: Implications for lens maintenance.</a> Investigative Ophthalmology &amp; Visual Science. 47 (7), 2997-3003 (2006).</li><li>Wolffe, A. P., Glover, J. F., Tata, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+shock.+Synthesis+of+heat-shock-like+proteins+in+fresh+primary+cell+cultures.">Culture shock. Synthesis of heat-shock-like proteins in fresh primary cell cultures.</a> Experimental Cell Research. 154 (2), 581-590 (1984).</li><li>Haykin, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioimage+analysis+of+cell+physiology+of+primary+lens+epithelial+cells+from+diabetic+and+non-diabetic+cataract+patients.">Bioimage analysis of cell physiology of primary lens epithelial cells from diabetic and non-diabetic cataract patients.</a> Biotechnology &amp; Biotechnological Equipment. 35 (1), 170-178 (2021).</li><li>Menko, A. S., Klukas, K. A., Johnson, R. 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The authors declare that they have no conflicts of interest.

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...

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.
Cataracts are characterized by the progressive clouding of the lens, resulting in the distortion and scattering of light, leading to compromised vision3,4. The precise mechanisms underlying cataract formation are complex and multifactorial, involving various cellular and molecular processes such as UV radiation, oxidative damage, and glycation5,6. LECs have been found to contribute significantly to the development of cataracts, making them a vital focus of research1,2,7,8,9.
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 myofibroblasts11,12,13. 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 techniques14,15,16,17,18,19,20. 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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Thermo Fisher</td>
			<td>#25300054</td>
			<td>For LECs dissociation</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Secondary Antibody&#160;</td>
			<td>Jackson ImmunoResearch</td>
			<td>#715-545-150</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Alexa Fluor 647 AffiniPure Goat Anti-Rabbit IgG (H+L)</td>
			<td>Jackson ImmunoResearch</td>
			<td>111-605-003</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Antibody dilution buffer</td>
			<td>Licor</td>
			<td>#927-60001</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Beaver safety knife</td>
			<td>Beaver-Visitec International</td>
			<td>#3782235</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>Blocking buffer</td>
			<td>Licor</td>
			<td>#927-60001</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Capsulorhexis forceps</td>
			<td>Titan Medical Instruments</td>
			<td>TMF-124</td>
			<td>For lens capsule isolation</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6429</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>#D2650</td>
			<td>For making freezing medium&#160;</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline&#160;</td>
			<td>Thermo Fisher</td>
			<td>#J67802</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>Dumont tweezers</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-4976</td>
			<td>For lens capsule isolation</td>
		</tr>
		<tr>
			<td>EpiCGS-a (optional)</td>
			<td>ScienCell</td>
			<td>4182</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma Aldrich</td>
			<td>F2442</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>Gentamicin (50&#160;mg/mL)</td>
			<td>Sigma-Aldrich</td>
			<td>G1397</td>
			<td>For LECs culture medium</td>
		</tr>
		<tr>
			<td>Hoechst 33342 solution</td>
			<td>Thermo Fisher</td>
			<td>#62249</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Micro-dissecting scissors</td>
			<td>Roboz Surgical Instrument&#160;</td>
			<td>RS-5983</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>Micro-dissecting tweezers</td>
			<td>Roboz Surgical Instrument&#160;</td>
			<td>RS5137&#160;</td>
			<td>For lens dissection</td>
		</tr>
		<tr>
			<td>PROX1 antibody</td>
			<td>Thermo Fisher</td>
			<td>11067-2-AP</td>
			<td>For cell validation</td>
		</tr>
		<tr>
			<td>Vannas micro-dissecting spring scissors</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5608</td>
			<td>For lens capsule isolation</td>
		</tr>
		<tr>
			<td>&#945;A-crystallin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-28306</td>
			<td>For cell validation&#160;</td>
		</tr>
		<tr>
			<td>&#947;-crystallin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-365256</td>
			<td>For cell validation</td>
		</tr>
	</tbody>
</table>

optimizing mouse primary lens epithelial cell culture: a comprehensive guide to trypsinization

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 &#945;A- and &#947;-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.

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

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...

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.

Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...

optimizing-mouse-primary-lens-epithelial-cell-culture-comprehensive

Research

JoVE Journal

Biology

1.2K Views.  University of North Texas Health Science Center. 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.