This work was funded by an NIH grant Ey10540 to PAT.

1. Iris Cell Culture
<ol>
	<li>Collect eyeballs from 7 newts (anesthetized in 0.1% ethyl 3-aminobenzoate methane sulfonic acid prepared in PBS) and place in calcium magnesium free Hanks solution (CMF).</li>
	<li>Change gloves and work in the tissue culture hood.</li>
	<li>Sterilize eye balls in Lugol's-EtOH for 3 seconds.</li>
	<li>Wash 2 times in CMF.</li>
	<li>Transfer eyes to Hanks and begin dissection.</li>
	<li>Dissect iris-corneal complex and remove neural retina.</li>
	<li>Transfer complexes into a new dish of Hanks.</li>
	<li>Using #15 scalpel, separate complexes into dorsal and ventral halves.</li>
	<li>Remove iris fragments (ventral first) and place in a dish containing 1 ml L-15.</li>
	<li>Add 15% (150 ul) volume of dispase (7.5 units/ml concentration).</li>
	<li>Incubate at 27&deg;C for 2 hours (wrap dish with parafilm).</li>
	<li>Isolate IPE cells from stroma (Place trypsin solution at 27&deg;C).</li>
	<li>Collect IPE cells into a 1.5 ml tube.</li>
	<li>Centrifuge at 1000 rpm at RT (room temperature) for 2 minutes, remove supernatant.</li>
	<li>Wash with 1 ml Hanks and centrifuge at 1000 rpm at RT for 2 minutes, remove supernatant.</li>
	<li>Add 1 ml trypsin solution; incubate for 2 hours at 27&deg;C.</li>
	<li>Centrifuge at 1000 rpm at RT for 2 minutes, remove trypsin.</li>
	<li>Add 1 ml L-15 to wash, centrifuge at 1000 rpm for 2 minutes.</li>
	<li>Add 800 ul L-15, pipet slowly up and down ~ 10 times (more than 12 times reduces adherence).</li>
	<li>Plate the IPE cells on a 24 well collagen coated plate and incubate at 27&deg;C for 2 weeks (Usually, 4 dorsal and 4 ventral wells are obtained from 7 newts).</li>
	<li>At this stage cells are suitable for gene transfection or treatment with factors to determine their role in inducing or interfering with lens regeneration.</li>
</ol>
2. Aggregation of Iris Cells
<ol>
	<li>To the plates containing iris cells add 20&mu;l of dispase solution to 400 &mu;l medium, swirl gently.</li>
	<li>Incubate plates at 27&deg;C for overnight.</li>
	<li>Pipet gently up and down to dislodge cells</li>
	<li>Place cells into Eppendorf tube and spin for 2 minutes at 1000 rpm at RT.</li>
	<li>Remove medium and wash by adding 1 ml complete L-15, spin 2 minutes at 1000 rpm at RT, remove medium.</li>
	<li>Use 2000-7000 cells per aggregate. Add 200 ul L-15 per aggregate into each tube.</li>
	<li>Split cells (200 &mu;l) into new eppendorf tubes.</li>
	<li>Spin at 1000 rpm for 2 min at RT.</li>
	<li>Incubate for 48 hrs at 27&deg;C.</li>
</ol>
3. Implantation of Aggregrated Cells
<ol>
	<li>Make a slit in the cornea of newt eye and remove the lens.</li>
	<li>After lens removal, place the IPE cell aggregate just below the corneal tissue on the ventral side of the eye.</li>
	<li>The newts are kept for a month to let them regenerate the lens.</li>
</ol>
4. Embedding of Newt Eye
<ol>
	<li>Remove the newt eyes implanted with the aggregate and place it in phosphate buffered saline (PBS).</li>
	<li>Fix in 4% paraformaldehyde at 4&deg;C for at least 4 hrs or overnight.</li>
	<li>Wash in PBS, 4&deg;C for 30 minutes.</li>
	<li>Treat it with 0.85% saline: 4&deg;C, 30 minutes.</li>
	<li>Wash in Saline/Ethanol (1:1): RT for 15 minutes.</li>
	<li>Wash in 70% Ethanol: RT, 15 minutes. Repeat it once.</li>
	<li>Wash in 85% Ethanol and 95% Ethanol at RT, 30 minutes each</li>
	<li>Wash in 100% Ethanol: RT, 30 minutes. Repeat it once.</li>
	<li>Store at 4&deg;C or continue.</li>
	<li>Wash in 100% xylene: RT, 30 minutes. Repeat it once.</li>
	<li>Treat with xylene/paraffin (1:1): 60&deg;C, 45 minutes.</li>
	<li>Treat with 100% Paraffin: 60&deg;C, 20 minutes. Repeat it two more times.</li>
	<li>Embed in embedding molds.</li>
</ol>
5. Sectioning
<ol>
	<li>Prepare 15 &mu;m sections of the embedded eye using a microtome.</li>
	<li>Place the eye sections on a gelatin coated slide and leave it on a slide warmer.</li>
</ol>
6. Staining
<ol>
	<li>Place slides in xylene for 10 minutes. Repeat it once more.</li>
	<li>Hydrate in: 100%, 95%, 80%, 70%, 30% ethanol for 1 minute each</li>
	<li>Rinse in DI water for a minute.</li>
	<li>Wash in PBS for 15 minutes.</li>
	<li>Wash in PBST (0.2% Triton-X 100 in PBS) for 15 minutes.</li>
	<li>Wash in PBS for 15 minutes.</li>
	<li>Place in blocking solution (10% goat secondary antibody) for an hour.</li>
	<li>Place in primary antibody for overnight.</li>
	<li>Wash primary antibody in PBS for 15 minutes.</li>
	<li>Wash in PBST for 15 minutes.</li>
	<li>Wash in PBS for 15 minutes.</li>
	<li>Place in secondary antibody for 2 hours in dark (1:100 dilution in PBS).</li>
	<li>Wash in PBS, PBST, and PBS for 15 minutes each and then mount.</li>
	<li>Observe the slides under a fluorescence microscope.</li>
</ol>
7. Representative Results:
This procedure of culturing newt iris cells has been utilized to study the regeneration potential of the dorsal and the ventral IPE cells. Moreover, it is also possible to study specific genes that contribute towards the lens regeneration mechanism in newt eye. When the cells have been cultured for 2 weeks (Figure 1) they can be transfected by genes to examine their function in lens regeneration. Of particular interest are genes that might induce the ventral iris. Since the ventral iris cells cannot transdifferentiate to lens (Figure 2) the inductive function of a candidate gene can be studied. In the past, using this technique we have shown that when six-3was over expressed in the presence of retinoic acid lens induction was observed from the ventral iris 6. In Figure 3we can see that the ventral iris aggregate gave rise to a fully grown and differentiated lens (arrowhead), not different than the host lens from the dorsal iris (arrow).
<img src="/files/ftp_upload/2713/2713fig1.jpg" alt="Figure 1" /> 
 Figure 1. a) Dorsal iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. b) Ventral iris pigmented epithelial cells cultured in vitro for a period of 2 weeks. Note that pigmentation persists to this stage in both dorsal and ventral iris cells.
<img src="/files/ftp_upload/2713/2713fig2.jpg" alt="Figure 2" /> 
 Figure 2. Regeneration ability of cultured IPE cells. a) Lens induction from dorsal IPE cell aggregate (arrowhead). Host lens induction from dorsal iris (arrow), di: dorsal iris, vi: ventral iris, le: lens epithelium, lf: lens fibers. b) Absence of lens induction from ventral IPE cell aggregate (arrowhead). Host lens induction from dorsal iris (arrow).
<img src="/files/ftp_upload/2713/2713fig3.jpg" alt="Figure 3" /> 
 Figure 3. Lens induction from ventral aggregate transfected with six-3 and treated with retinoic acid. Host lens induction from dorsal iris (arrow). Lens induction from ventral aggregate (arrowhead).

<ol>
	<li>Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+in+vertebrates.">Regeneration in vertebrates.</a> Dev Biol. 221, 273-284 (2000).</li><li>Tsonis, P. A., Madhavan, M., Tancous, E. E., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+newt's+eye+view+of+lens+regeneration.">A newt's eye view of lens regeneration.</a> Int. J. Dev. Biol. 48, 975-980 (2004).</li><li>Del Rio-Tsonis, K., Tsonis, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eye+regeneration+at+the+molecular+age.">Eye regeneration at the molecular age.</a> Dev. Dyn. , 226-2211 (2003).</li><li>Okamoto, M., Ito, M., Owaribe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+between+dorsal+and+ventral+iris+in+lens+producing+potency+in+normal+lens+regeneration+is+maintained+after+dissociation+and+reaggregation+of+cells+from+the+adult+newt,+Cynops+pyrrhogaster.">Difference between dorsal and ventral iris in lens producing potency in normal lens regeneration is maintained after dissociation and reaggregation of cells from the adult newt, Cynops pyrrhogaster.</a> Develop Growth Differ. 40, 11-18 (1998).</li><li>Hayashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+system+for+functional+gene+analysis+in+adult+amphibian+lens+regeneration.">Highly efficient transfection system for functional gene analysis in adult amphibian lens regeneration.</a> Develop Growth Differ. 43, 361-370 (2001).</li><li>Grogg, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+inhibition-driven+regulation+of+six-3+underlies+induction+of+newt+lens+regeneration.">BMP inhibition-driven regulation of six-3 underlies induction of newt lens regeneration.</a> Nature. 438, 858-862 (2005).</li><li>Nakamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+Newt+Lens+Regeneration:+Specific+Control+of+Proliferation+and+Evidence+for+miRNA+Networking.">miRNAs in Newt Lens Regeneration: Specific Control of Proliferation and Evidence for miRNA Networking.</a> PLoS One. 5, e12058-e12058 (2010).</li><li>Madhavan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+Pax-6+in+lens+regeneration.">The role of Pax-6 in lens regeneration.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 14848-14853 (2006).</li><li>Maki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oocyte-type+linker+histone+B4+is+required+for+transdifferentiation+of+somatic+cells+in+vivo.">Oocyte-type linker histone B4 is required for transdifferentiation of somatic cells in vivo.</a> FASEB J. 24, 3462-3467 (2010).</li></ol>

No conflicts of interest declared.

This protocol has established an in vitro system to study lens regeneration mechanisms in newts. Since the aggregates (either dorsal or ventral faithfully follow their in vivo behavior during regeneration this technique can alleviate the tremendous effort required for transgenesis in newts and can be used for gain of function as well as loss of function experiments 7,8,9 . Also, the aggregates or the irises as a whole can easily be treated with growth factors and examine their effects as described in this protocol. For example the role of BMP pathway has been studied using this technique 6.

<table><tbody><tr><td>Lugol&amp;rsquo;s solution</td><td>Sigma-Aldrich</td><td>L-6146</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H-4034</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonic acid</td><td>Sigma-Aldrich</td><td>E-10521</td><td></td></tr><tr><td>Dispase</td><td>GIBCO, by Life Technologies</td><td>17105-041</td><td></td></tr><tr><td>Trypsin 1:250</td><td>GIBCO, by Life Technologies</td><td>27250018</td><td></td></tr><tr><td>L-15 ( Leibovitz)</td><td>Sigma-Aldrich</td><td>L-4386</td><td></td></tr><tr><td>DNase 1</td><td>Sigma-Aldrich</td><td>D5025-150KU</td><td></td></tr><tr><td>Kanamycin Sulfate</td><td>GIBCO, by Life Technologies</td><td>100x, 15160</td><td></td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F4135</td><td></td></tr><tr><td>Fungizone (amphotericin B solution)</td><td>Sigma-Aldrich</td><td>A2942</td><td></td></tr><tr><td>24 well collagen coated plate</td><td>BD Biosciences</td><td>354408</td><td></td></tr></tbody></table>

a system for culturing iris pigment epithelial cells to study lens regeneration in newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

Watch this Scientific Journal Video about A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt at JoVE.com

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

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Universidad de Cartagena UDEC

Research

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Biology

11.7K Views.  University of Dayton. In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Video: A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Neuroscience

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic age-related macular degeneration (AMD) which represents 90% of the cases. Therapies are needed to slow or retard the development of geographic atrophy (GA), and understanding Bruch&#39;s membrane pathology is part of this process. Alterations in human Bruch&#39;s membrane precede the progression of AMD by contributing to the damage of retinal pigment epithelial (RPE) cells. Given the lack of sufficient animal models to study AMD, ex vivo models of aged human Bruch&#39;s membrane serve as a useful tool to study the behavior of RPE cells from immortalized and primary cell lines as well as RPE lines derived from induced pluripotent stem cells (iPSCs). Here, we present a detailed method that allows one to determine the effects of RPE cell behavior seeded on harvested human Bruch&#39;s membrane explants from human donors, including attachment, apoptosis and proliferation, ability to phagocytize photoreceptor outer segments, establishment of polarity, and gene expression. This assay provides an ex vivo model of aged Bruch&#39;s membrane to assess the functional characteristics of RPE cells when seeded on aged/compromised extracellular matrix.

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

The goal of this protocol is to demonstrate the culturing of retinal pigment epithelial (RPE) cells on aged and/or diseased human Bruch&#39;s membrane. This method is suitable to study RPE cell behavior on a compromised extracellular matrix.

Aside from vitamins and antioxidants recommended by the Age-Related Eye Disease Study, there is no effective therapy for &#34;dry,&#34;&#160;or atrophic ...

Developmental Biology

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Primary Cell Cultures to Study the Regeneration Potential of Murine M&#252;ller Glia after MicroRNA Treatment

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates such as fish, MG-driven regeneration occurs naturally; in mammals, however, stimulation with certain factors or genetic/epigenetic manipulation is required. Since MG comprise only 5% of the retinal cell population, there is a need for model systems that allow the study of this cell population exclusively. One of these model systems is primary MG cultures that are reproducible and can be used for a variety of applications, including molecule/factor screening and identification, testing of compounds or factors, cell monitoring, and/or functional tests. This model is used to study the potential of murine MG to convert into retinal neurons after supplementation or inhibition of microRNAs (miRNAs) via transfection of artificial miRNAs or their inhibitors. The use of MG-specific reporter mice in combination with immunofluorescent labeling and single-cell RNA sequencing (scRNA-seq) confirmed that 80%-90% of the cells found in these cultures are MG. Using this model, it was discovered that miRNAs can reprogram MG into retinal progenitor cells (RPCs), which subsequently differentiate into neuronal-like cells. The advantages of this technique are that miRNA candidates can be tested for their efficiency and outcome before their usage in in vivo applications.

M&#252;ller glia primary cultures obtained from mouse retinas represent a very robust and reliable tool to study the glial conversion into retinal progenitor cells after microRNA treatment. Single molecules or combinations can be tested before their subsequent application of in vivo approaches.

M&#252;ller glia (MG) are the predominant glia in the neural retina and can function as a regenerative source for retinal neurons. In lower vertebrates ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Cultivation and Characterization of Lens Epithelial Cells

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.

Author Spotlight: Unlocking the Secrets of Cataracts &#8211; Investigating Redox Repair Enzymes in Lens Epithelial Cells

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, ...

Isolating IPE and RPE Cells: A Technique for Obtaining Ocular Pigment Epithelial Cells from Rabbit Eye

Source: Bascuas, T., et al. <a href="https://jove.unicartagenaproxy.elogim.com/v/62145">Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy.</a> J. Vis. Exp. (2021).
In this video, we show a procedure to obtain ocular pigment epithelial cells from a rabbit model. The cells are helpful in developing therapeutic strategies for retinal degenerative diseases.

Source: Bascuas, T., et al. Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy. J. Vis. ...

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Summary

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Chapters in this video

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

Organotypic Culture of Adult Rabbit Retina

Culturing of Retinal Pigment Epithelial Cells on an Ex Vivo Model of Aged Human Bruch's Membrane

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

Primary Cell Cultures to Study the Regeneration Potential of Murine Müller Glia after MicroRNA Treatment

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

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

Isolating IPE and RPE Cells: A Technique for Obtaining Ocular Pigment Epithelial Cells from Rabbit Eye