Name: isolation of retinal pigment epithelial cells from guinea pig eyes
Uploaded: 2023-05-09T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes at JoVE.com

This study is supported by the Japan Society for the Promotion of Science Overseas Research Fellowships (S.G.), a Loris and David Rich Postdoctoral Scholar (S.G.), and a grant from the National Eye Institute of the National Institutes of Health (R01EY012392; C.F.W.).

All animal care and treatments used in this study conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The experimental protocols were approved by the Animal Care and Use Committee of the University of California, Berkeley.
1. Enucleation of the guinea pig eye
<ol>
	<li>Euthanize a guinea pig&#160;with an intracardiac injection of sodium pentobarbital delivered under anesthesia (5% isoflurane in oxygen).</li>
	<li>Enucleate the eyes with ...

<ol>
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R., Siegwart, J. T., Norton, T. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+gene+expression+in+tree+shrew+retina+and+retinal+pigment+epithelium+produced+by+short+periods+of+minus-lens+wear.">Altered gene expression in tree shrew retina and retinal pigment epithelium produced by short periods of minus-lens wear.</a> Experimental Eye Research. 168 (3), 77-88 (2018).</li><li>Nickla, D. L., Wallman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+multifunctional+choroid.">The multifunctional choroid.</a> Progress in Retinal and Eye Research. 29 (2), 144-168 (2010).</li><li>Zhang, Y., Liu, Y., Wildsoet, C. 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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+vascular+patterns+in+domestic+animals.">Retinal vascular patterns in domestic animals.</a> Research in Veterinary Science. 47 (1), 34-42 (1989).</li><li>Araki, H., 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=Base-resolution+methylome+of+retinal+pigment+epithelial+cells+used+in+the+first+trial+of+human+induced+pluripotent+stem+cell-based+autologous+transplantation.">Base-resolution methylome of retinal pigment epithelial cells used in the first trial of human induced pluripotent stem cell-based autologous transplantation.</a> Stem Cell Reports. 13 (4), 761-774 (2019).</li><li>Sonoda, 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+protocol+for+the+culture+and+differentiation+of+highly+polarized+human+retinal+pigment+epithelial+cells.">A protocol for the culture and differentiation of highly polarized human retinal pigment epithelial cells.</a> Nature Protocols. 4 (5), 662-673 (2009).</li><li>Fernandez-Godino, R., Garland, D. 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In this article, we describe a method for isolating RPE, appropriate for RPE gene expression analyses, from the eyes of young, pigmented guinea pigs. The merits of this protocol are that it yields high-quality RPE samples that are relatively free from contamination, with&#160;RNA suitably preserved for RNA-specific analyses and, yet, is relatively simple and efficient. While in the example provided here, the RPE samples were collected from the eyes of a young (2 weeks old)&#160;guinea pig, the protocol has also been used...

The RPE comprises a unique monolayer of pigmented cells located between the neural retina and the vascular choroid, and the RPE has well-recognized roles in the development and maintenance of normal retinal function, including phototransduction1,2. More recently, the RPE has been assigned an additional key role in eye growth regulation3 and, thus, the development of myopia4. This assignment is based on the RPE&#39;s critical location,&#160;interposed between the retina and choroid and the now broad acceptance that eye growth and, thus, refractive errors are regulated locally5. The RPE is believed to play a key role as a signal relay, linking the retina, the assumed source of growth modulatory signals, to the choroid and sclera, the two targets of the relayed signals6,7,8.
The increase in axial length that characterizes most myopia cannot be considered benign, with pathophysiological changes involving the retina, choroid, and/or sclera representing unavoidable and now well-recognized consequences of excessive ocular elongation7,9. In this context, the RPE is perhaps the most vulnerable, since, being a nonmitotic tissue, it is only able to accommodate the expanding vitreous chamber by the stretching and thinning of individual cells. While its role in myopia-related pathologies, such as myopic macular degeneration, is yet to be fully understood, the RPE has been implicated in the pathogenesis of a number of other retinal diseases, including geographic atrophy, one of the leading causes of blindness, which is associated with documented abnormalities in the retina, RPE, and choroid10,11,12.
The successful isolation of RPE cells, free from contamination from adjacent ocular tissues, potentially opens up many research opportunities to gain new insights into the mechanisms underlying a variety of eye/retinal diseases. However, the isolation of the RPE has proven challenging, with many published studies making use of combined retina/RPE or RPE/choroid samples for this reason13,14,15. Studies involving the successful isolation of the RPE of a quality suitable for molecular biology studies has been limited to chick and mouse eyes16,17. For example, the simultaneous RPE isolation and RNA stabilization (SRIRS) method described by Wang et al.18. To isolate RPE cells in mice does not appear to work well in guinea pig eyes. The protocol described here represents a refinement of an approach that was initially prototyped with tree shrew eyes by one of the authors (M.F.) and has proven to yield high-quality RPE samples, appropriate for RNA and other molecular biology analyses, from the eyes of young pigmented guinea pigs19.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Syringe with Slip Tip</td>
			<td>Bd Vacutainer Labware Medical</td>
			<td>22-253-260</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Invitrogen</td>
			<td>21985-023</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Tissue Culture Plate with Lid, Flat Bottom, Sterile</td>
			<td>pectrum Chemical Mfg. Corp</td>
			<td>970-95008</td>
			<td></td>
		</tr>
		<tr>
			<td>12 Well Tissue Culture Plate with Lid, Individual, Sterile</td>
			<td>Thomas Scientific LLC</td>
			<td>1198D72</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2100 Bioanalyzer</td>
			<td></td>
			<td></td>
			<td>automated electrophoresis to check RNA quality</td>
		</tr>
		<tr>
			<td>Balanced Salt Solutions</td>
			<td>Gibco</td>
			<td>10010031</td>
			<td></td>
		</tr>
		<tr>
			<td>Bonn Micro Forceps, Straight Smooth, 0.3 mm Tip, 7 cm</td>
			<td>Fine Science Tools, Inc.</td>
			<td>11083-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont forceps no. 5</td>
			<td>ROBOZ</td>
			<td>RS-5045</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic disposable needles</td>
			<td>Exelint International, Co.</td>
			<td>26419</td>
			<td></td>
		</tr>
		<tr>
			<td>Hypodermic disposable needles</td>
			<td>Exelint International, Co.</td>
			<td>26437</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniSpin Microcentrifuges</td>
			<td>Eppendorf</td>
			<td>540108</td>
			<td>Max. Speed: 8,000 g</td>
		</tr>
		<tr>
			<td>RNAlater Stabilization Solution</td>
			<td>Invitrogen</td>
			<td>AM7020</td>
			<td>tissue storage reagent</td>
		</tr>
		<tr>
			<td>RNeasy Mini kits</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td>RNA isolation kit</td>
		</tr>
		<tr>
			<td>Student Vannas Spring Scissors</td>
			<td>Fine Science Tools, Inc.</td>
			<td>91500-09</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of retinal pigment epithelial cells from guinea pig eyes

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential application in molecular biology studies, including gene expression analyses. In the context of eye growth regulation and myopia, the RPE likely plays a role as a cellular relay for growth modulatory signals, as it is located between the retina and the two walls of the eye, such as the choroid and sclera. While protocols for isolating the RPE have been developed for both chicks and mice, these protocols have proven not to be directly translatable to the guinea pig, which has become an important and widely used mammalian myopia model. In this study, molecular biology tools were used to examine the expression of specific genes to confirm that the samples were free of contamination from the adjacent tissues. The value of this protocol has already been demonstrated in an RNA-Seq study of RPE from young pigmented guinea pigs exposed to myopia-inducing optical defocus. Beyond eye growth regulation, this protocol has other potential applications in studies of retinal diseases, including myopic maculopathy, one of the leading causes of blindness in myopes, in which the RPE has been implicated. The main advantage of this technique is that it is relatively simple and&#160;once perfected, yields high-quality RPE samples suitable for molecular biology studies, including RNA analysis.

The analysis of the RPE samples collected using the above protocol showed well-preserved RNA (RIN &#62;8.0, Figure 2B), with 240.2 ng &#177; 35.1 ng per eye (n = 8, NanoDrop, Figure 2B). To further evaluate the quality of the isolated RPE samples, particularly the absence of choroidal and scleral contaminants, we examined the expression of representative genes for each of the latter tissues in the RPE samples19. The RPE samples demonstrat...

Watch this Scientific Journal Video about Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes at JoVE.com

Isolation of Retinal Pigment Epithelial Cells from Guinea Pig Eyes

We describe a simple and efficient method for isolating cells of the retinal pigment epithelium (RPE) cells from the eyes of young pigmented guinea pigs. This procedure allows for follow-up molecular biology studies on the isolated RPE, including gene expression analyses.

This protocol describes the isolation of cells of the retinal pigment epithelium (RPE) from the eyes of young pigmented guinea pigs for potential ...

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