Name: Autor em destaque: Desvendando os segredos da catarata - investigando enzimas de reparo redox em células epiteliais do cristalino
Uploaded: 2024-06-21T12:30:00
Description: Lens epithelial cells (LECs) play multiple important roles in maintaining the homeostasis and normal function of the lens. LECs determine lens growth, ...

Este trabalho foi apoiado pelo NEI R21EY033941 (para Hongli Wu); Departamento de Defesa W81XWH2010896 (para Hongli Wu); R15GM123463-02 (para Kayla Green e Hongli Wu)

Todos os experimentos com animais foram realizados de acordo com as diretrizes da Association for Research in Vision and Ophthalmology for the Use of Animals in Ophthalmic and Vision Research. A aprovação do procedimento foi concedida pelo Comitê de Cuidados e Uso de Animais do Centro de Ciências da Saúde da Universidade do Norte do Texas (número de protocolo: IACUC-2022-0008). Camundongos C57BL/6J jovens, tipicamente com menos de 2 semanas de idade, foram usados nesses estudos.
1....

Cultivo e caracterização de células epiteliais do cristalino

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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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+damage+and+the+prevention+of+age-related+cataracts.">Oxidative damage and the prevention of age-related cataracts.</a> Ophthalmic Research. 44 (3), 155-165 (2010).</li><li>Lou, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+regulation+in+the+lens.">Redox regulation in the lens.</a> Progress in Retinal and Eye Research. 22 (5), 657-682 (2003).</li><li>Charakidas, A., 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=Lens+epithelial+apoptosis+and+cell+proliferation+in+human+age-related+cortical+cataract.">Lens epithelial apoptosis and cell proliferation in human age-related cortical cataract.</a> European Journal of Ophthalmology. 15 (2), 213-220 (2005).</li><li>Lou, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutathione+and+glutaredoxin+in+redox+regulation+and+cell+signaling+of+the+lens.">Glutathione and glutaredoxin in redox regulation and cell signaling of the lens.</a> Antioxidants (Basel). 11 (10), 1973 (2022).</li><li>Wilhelm, J., Smistik, Z., Mahelkova, G., Vytasek, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Redox+regulation+of+proliferation+of+lens+epithelial+cells+in+culture.">Redox regulation of proliferation of lens epithelial cells in culture.</a> Cell Biochemistry &amp; Function. 25 (3), 317-321 (2007).</li><li>Zhang, Y., 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=Research+progress+concerning+a+novel+intraocular+lens+for+the+prevention+of+posterior+capsular+opacification.">Research progress concerning a novel intraocular lens for the prevention of posterior capsular opacification.</a> Pharmaceutics. 14 (7), 1343 (2022).</li><li>Konopinska, J., Mlynarczyk, M., Dmuchowska, D. A., Obuchowska, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+capsule+opacification:+A+review+of+experimental+studies.">Posterior capsule opacification: A review of experimental studies.</a> Journal of Clinical Medicine. 10 (13), 2847 (2021).</li><li>Pandey, S. K., Apple, D. J., Werner, L., Maloof, A. J., Milverton, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posterior+capsule+opacification:+A+review+of+the+aetiopathogenesis,+experimental+and+clinical+studies+and+factors+for+prevention.">Posterior capsule opacification: A review of the aetiopathogenesis, experimental and clinical studies and factors for prevention.</a> Indian Journal of Ophthalmology. 52 (2), 99-112 (2004).</li><li>Wei, Z., 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=Aged+lens+epithelial+cells+suppress+proliferation+and+epithelial-mesenchymal+transition-relevance+for+posterior+capsule+opacification.">Aged lens epithelial cells suppress proliferation and epithelial-mesenchymal transition-relevance for posterior capsule opacification.</a> Cells. 11 (13), 2001 (2022).</li><li>Creighton, M. O., Mousa, G. Y., Miller, G. G., Blair, D. G., Trevithick, 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=Differentiation+of+rat+lens+epithelial+cells+in+tissue+culture--iv.+Some+characteristics+of+the+process,+including+possible+in+vitro+models+for+pathogenic+processes+in+cataractogenesis.">Differentiation of rat lens epithelial cells in tissue culture--iv. Some characteristics of the process, including possible in vitro models for pathogenic processes in cataractogenesis.</a> Vision Research. 21 (1), 25-35 (1981).</li><li>Creighton, M. O., Mousa, G. Y., Trevithick, 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=Differentiation+of+rat+lens+epithelial+cells+in+tissue+culture.+(i)+effects+of+cell+density,+medium+and+embryonic+age+of+initial+culture.">Differentiation of rat lens epithelial cells in tissue culture. (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. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chicken+embryo+lens+cultures+mimic+differentiation+in+the+lens.">Chicken embryo lens cultures mimic differentiation in the lens.</a> Developmental Biology. 103 (1), 129-141 (1984).</li><li>Zelenka, P. S., Gao, C. Y., Saravanamuthu, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+culture+of+rat+lens+epithelial+explants+for+studying+terminal+differentiation.">Preparation and culture of rat lens epithelial explants for studying terminal differentiation.</a> Journal of Visualized Experiments. (31), 1591 (2009).</li></ol>

O protocolo apresentado neste artigo fornece um guia passo a passo abrangente para o isolamento, a cultura e a subcultura bem-sucedidos de LECs primários, completo com a documentação em vídeo que o acompanha. O guia visual detalhado, juntamente com as instruções escritas, aumenta a clareza e a acessibilidade do protocolo, promovendo seu uso e reprodutibilidade entre os pesquisadores da área. O objetivo final é contribuir para a expansão do corpo de conhecimento em torno do papel das CELs na formação da catarat...

A lente do olho desempenha um papel crucial na visão, concentrando a luz recebida na retina. Consiste em uma estrutura avascular transparente composta por células especializadas, entre as quais as células epiteliais do cristalino (LECs) são peças-chave. As CLEs estão localizadas na superfície anterior do cristalino e são responsáveis por manter sua transparência, regular o balanço hídrico e participar do crescimento e desenvolvimento do cristalino 1,2. LECs são um tipo único de células localizadas na parte anterior da lente, desempenhando um papel crítico na manutenção da clareza e função da lente, produzindo continuamente fibras da lente ao longo da vida.
A catarata caracteriza-se pela turvação progressiva do cristalino, resultando em distorção e espalhamento da luz, levando ao comprometimento da visão 3,4. Os mecanismos precisos subjacentes à formação da catarata são complexos e multifatoriais, envolvendo vários processos celulares e moleculares, como radiação UV, dano oxidativo e glicação 5,6. Descobriu-se que as LECs contribuem significativamente para o desenvolvimento da catarata, tornando-as um foco vital de pesquisa 1,2,7,8,9.
Além disso, uma das questões mais prementes em oftalmologia atualmente é a incidência relativamente alta de opacificação da cápsula posterior (PCO), também conhecida como catarata secundária. A PCO continua sendo a complicação mais comum após a cirurgia de catarata, afetando até 20-40% dos pacientes adultos e 100% das crianças dentro de 5 anos após a cirurgia10. A PCO é causada principalmente pelas LECs residuais que permanecem na bolsa capsular após a extração da catarata. Essas células sofrem uma transformação fisiopatológica multifacetada, envolvendo não apenas a transição epitélio-mesenquimal (EMT), mas também a diferenciação das LECs em fibras do cristalino, resultando em uma população celular que é uma mistura de LECs, fibras e miofibroblastos11,12,13. As células transformadas proliferam e migram através da cápsula posterior do cristalino, levando à deficiência visual. A compreensão do comportamento e dos mecanismos de controle das LECs em modelos de cultura pode fornecer informações valiosas sobre a prevenção e o manejo da PCO. Portanto, este protocolo de cultivo de LECs apresenta-se como uma ferramenta vital para os pesquisadores oftalmológicos com o objetivo de estudar, compreender e, finalmente, combater essa complicação pós-operatória prevalente.
Para desvendar os meandros da biologia LEC e seu papel na formação de catarata e PCO, é essencial estabelecer sistemas de cultura de células primárias robustos e reprodutíveis. A cultura primária de LEC fornece aos pesquisadores um ambiente controlado para estudar as funções, sinalização e características moleculares de LECs. Além disso, permite a investigação de processos celulares e dos efeitos de diferentes condições experimentais, fornecendo informações valiosas sobre a fisiologia e patologia do cristalino.
Pesquisas anteriores enriqueceram nosso entendimento sobre as técnicas de cultura LEC 14,15,16,17,18,19,20. Embora esses estudos tenham empregado várias metodologias e produzido achados significativos sobre o comportamento e as características do LEC, um protocolo de videogravação abrangente e acessível para a cultura de LECs está ausente na literatura atual. Essa limitação pode dificultar a capacidade de pesquisadores iniciantes em reproduzir com precisão as técnicas e pode levar a inconsistências e variações nos resultados experimentais. Ao fornecer um protocolo de gravação de vídeo, este artigo de pesquisa visa preencher essa lacuna e fornecer um recurso padronizado que possa aumentar a reprodutibilidade e facilitar a transferência de conhecimento no campo da cultura LEC.

<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

As células epiteliais do cristalino (LECs) desempenham múltiplos papéis importantes na manutenção da homeostase e da função normal do cristalino. Os LECs determinam o crescimento, o desenvolvimento, o tamanho e a transparência das lentes. Por outro lado, as CLEs disfuncionais podem levar à formação de catarata e opacificação da cápsula posterior (PCO). Consequentemente, o estabelecimento de um sistema de cultura LEC primário robusto é importante para pesquisadores envolvidos no desenvolvimento de lentes, bioquímica, terapêutica de catarata e prevenção de PCO. No entanto, o cultivo de LECs primárias há muito tempo apresenta desafios devido à sua disponibilidade limitada, taxa de proliferação lenta e natureza delicada. 
Este estudo aborda esses obstáculos apresentando um protocolo abrangente para cultura LEC primária. O protocolo engloba etapas essenciais, como a formulação de um meio de cultura otimizado, isolamento preciso das cápsulas do cristalino, técnicas de tripsinização, procedimentos de subcultivo, protocolos de colheita e diretrizes para armazenamento e embarque. Durante todo o processo de cultivo, a morfologia celular foi monitorada por meio de microscopia de contraste de fase. 
Para confirmar a autenticidade das LECs cultivadas, ensaios de imunofluorescência foram conduzidos para detectar a presença e a distribuição subcelular de proteínas críticas do cristalino, ou seja, αA- e γ-cristalinos. Este protocolo detalhado fornece aos pesquisadores um recurso valioso para cultivar e caracterizar LECs primárias, permitindo avanços em nossa compreensão da biologia do cristalino e o desenvolvimento de estratégias terapêuticas para distúrbios relacionados ao cristalino.

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.

Autor em destaque: Desvendando os segredos da catarata - investigando enzimas de reparo redox em células epiteliais do cristalino

Otimizando a cultura de células epiteliais primárias de lente de camundongo: um guia abrangente para tripsinização

Our research objective is to explore the underlying causes of cataracts with a particular emphasis on the function of redox repair enzymes. Our aim is to identify the critical role this enzyme play in lens protection, and to establish their potential as drug targets for cataract prevention and treatment. Among many exciting technologies, our research uses lens epithelial cells as a foundational platform.This approach is critical for unraveling the mechanism behind cataract formation, posterior capsule opacification, and the discovery of new anti-cataract drugs. Moving forward, we will focus on understanding the impact of antioxidant enzymes and aging on lens functionality. Our goal is to discover innovative antioxidant treatments that can delay lens aging, prevent cataracts, and provide effective intervention for PCO.

Como mostrado na Figura 2, seguindo esse protocolo, LECs primárias de camundongos C57BL/6J aderiram às placas em um período de 4 h. Notadamente, havia resquícios visíveis de outros tecidos, como cortes da cápsula posterior e células de fibras do cristalino. No entanto, esses elementos não intencionais não se prenderam ao prato e, portanto, poderiam ser removidos com a mudança do meio de cultura. Posteriormente, entre o terceiro e o quinto dia, as LECs iniciaram sua fase de prolifer...

Este manuscrito descreve um protocolo de vídeo detalhado para a cultura de células epiteliais primárias do cristalino (LECs), com o objetivo de melhorar a reprodutibilidade e auxiliar a pesquisa em catarata e opacificação da cápsula posterior (PCO). Ele oferece instruções passo a passo sobre dissecção de lentes, isolamento de LECs e validação, servindo como um guia valioso, especialmente para recém-chegados no campo.

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

Nosso objetivo de pesquisa é explorar as causas subjacentes da catarata com uma ênfase particular na função das enzimas de reparo redox. Nosso objetivo é identificar o papel crítico desta enzima na proteção do cristalino e estabelecer seu potencial como alvos de drogas para a prevenção e tratamento da catarata. Entre muitas tecnologias interessantes, nossa pesquisa usa células epiteliais de lente como uma plataforma fundamental.Essa abordagem é fundamental para desvendar o mecanismo por trás da formação da catarata, a opacificação da cápsula posterior e a descoberta de novas drogas anticatarata. No futuro, nos concentraremos em entender o impacto das enzimas antioxidantes e do envelhecimento na funcionalidade das lentes. Nosso objetivo é descobrir tratamentos antioxidantes inovadores que podem retardar o envelhecimento do cristalino, prevenir a catarata e fornecer uma intervenção eficaz para a PCO.

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

Research

JoVE Journal

Biology

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.

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.