Name: primary cell cultures from the mouse retinal pigment epithelium
Uploaded: 2018-03-16T13:30:00
Description: Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

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הליכים הכוללים נושאים בעלי חיים אושרו על ידי טיפול בעלי חיים מוסדיים ועל שימוש הוועדה (IACUC) ב אוניברסיטת פיטסבורג
1. מכינים פתרונות
<ol><li>להכין את מדיום הגידול על ידי וחדשניות Dulbecco ששינה נשר בינוני (DMEM), גלוקוז גבוהות עם 10% סרום שור עוברית (FBS), 1% פניצילין/סטרפטומיצין, 2.5 מ מגלוטמ...

<ol>
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H., Vargis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+culturing+retinal+pigment+epithelial+cells:+a+review+of+current+protocols+and+future+recommendations.">Methods for culturing retinal pigment epithelial cells: a review of current protocols and future recommendations.</a> J Tissue Eng. 7, (2016).</li><li>Rizzolo, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Barrier+properties+of+cultured+retinal+pigment+epithelium.">Barrier properties of cultured retinal pigment epithelium.</a> Exp Eye Res. 126, 16-26 (2014).</li><li>Lu, F., Yan, D., Zhou, X., Hu, D. N., Qu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+melanin-related+genes+in+cultured+adult+human+retinal+pigment+epithelium+and+uveal+melanoma+cells.">Expression of melanin-related genes in cultured adult human retinal pigment epithelium and uveal melanoma cells.</a> Mol Vis. 13, 2066-2072 (2007).</li><li>Boulton, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+melanin+and+lipofuscin+in+RPE+cell+culture+models.">Studying melanin and lipofuscin in RPE cell culture models.</a> Exp Eye Res. 126, 61-67 (2014).</li><li>Saini, J. S., Temple, S., Stern, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Retinal+Pigment+Epithelium+Stem+Cell+(RPESC).">Human Retinal Pigment Epithelium Stem Cell (RPESC).</a> Adv Exp Med Biol. 854, 557-562 (2016).</li><li>Akrami, 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=Retinal+pigment+epithelium+culture;a+potential+source+of+retinal+stem+cells.">Retinal pigment epithelium culture;a potential source of retinal stem cells.</a> J Ophthalmic Vis Res. 4, 134-141 (2009).</li><li>Hu, J., Bok, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+cultured+human+fetal+retinal+pigment+epithelium+in+studies+of+the+classical+retinoid+visual+cycle+and+retinoid-based+disease+processes.">The use of cultured human fetal retinal pigment epithelium in studies of the classical retinoid visual cycle and retinoid-based disease processes.</a> Exp Eye Res. , 46-50 (2014).</li><li>Mazzoni, F., Safa, H., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+photoreceptor+outer+segment+phagocytosis:+use+and+utility+of+RPE+cells+in+culture.">Understanding photoreceptor outer segment phagocytosis: use and utility of RPE cells in culture.</a> Exp Eye Res. 126, 51-60 (2014).</li><li>Reichhart, N., Strauss, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+and+transporters+of+the+retinal+pigment+epithelium.">Ion channels and transporters of the retinal pigment epithelium.</a> Exp Eye Res. 126, 27-37 (2014).</li><li>Valapala, 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=Lysosomal-mediated+waste+clearance+in+retinal+pigment+epithelial+cells+is+regulated+by+CRYBA1/&#946;A3/A1-crystallin+via+V-ATPase-MTORC1+signaling.">Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/&#946;A3/A1-crystallin via V-ATPase-MTORC1 signaling.</a> Autophagy. 10, 480-496 (2014).</li><li>Shang, P., 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=The+amino+acid+transporter+SLC36A4+regulates+the+amino+acid+pool+in+retinal+pigmented+epithelial+cells+and+mediates+the+mechanistic+target+of+rapamycin,+complex+1+signaling.">The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling.</a> Aging Cell. , (2017).</li><li>Fernandez-Godino, R., Garland, D. L., Pierce, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation,+culture+and+characterization+of+primary+mouse+RPE+cells.">Isolation, culture and characterization of primary mouse RPE cells.</a> Nat Protoc. 11, 1206-1218 (2016).</li><li>Pfeffer, B. A., Philp, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+culture+of+retinal+pigment+epithelium:+Special+Issue.">Cell culture of retinal pigment epithelium: Special Issue.</a> Exp Eye Res. 126, 1-4 (2014).</li><li>Singh, S., Woerly, S., McLaughlin, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+and+artificial+substrates+for+retinal+pigment+epithelial+monolayer+transplantation.">Natural and artificial substrates for retinal pigment epithelial monolayer transplantation.</a> Biomaterials. 22, 3337-3343 (2001).</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> Nat Protoc. 4, 662-673 (2009).</li><li>Ho, T. C., Del Priore, L. V., Kaplan, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+culture+of+retinal+pigment+epithelium+following+isolation+with+a+gelatin+matrix+technique.">Tissue culture of retinal pigment epithelium following isolation with a gelatin matrix technique.</a> Experimental eye research. 64, 133-139 (1997).</li><li>Toops, K. A., Tan, L. X., Lakkaraju, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detailed+three-step+protocol+for+live+imaging+of+intracellular+traffic+in+polarized+primary+porcine+RPE+monolayers.">A detailed three-step protocol for live imaging of intracellular traffic in polarized primary porcine RPE monolayers.</a> Exp Eye Res. , 74-85 (2014).</li><li>Gibbs, D., Kitamoto, J., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+phagocytosis+by+retinal+pigmented+epithelium+that+lacks+myosin+VIIa,+the+Usher+syndrome+1B+protein.">Abnormal phagocytosis by retinal pigmented epithelium that lacks myosin VIIa, the Usher syndrome 1B protein.</a> Proc Natl Acad Sci USA. 100, 6481-6486 (2003).</li><li>Gibbs, D., Williams, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+primary+mouse+retinal+pigmented+epithelial+cells.">Isolation and culture of primary mouse retinal pigmented epithelial cells.</a> Adv Exp Med Biol. 533, 347-352 (2003).</li><li>Pitk&#228;nen, L., Ranta, V. P., Moilanen, H., Urtti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+of+retinal+pigment+epithelium:+effects+of+permeant+molecular+weight+and+lipophilicity.">Permeability of retinal pigment epithelium: effects of permeant molecular weight and lipophilicity.</a> Invest Ophthalmol Vis Sci. 46, 641-646 (2005).</li><li>Mao, Y., Finnemann, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+photoreceptor+outer+segment+phagocytosis+by+RPE+cells+in+culture.">Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture.</a> Methods Mol Biol. 935, 285-295 (2013).</li></ol>

פרוטוקול מפורט הציג מאפשרת הקמת אמין של העכבר הראשי תרבויות RPE להגיע confluency לאחר שבוע 1, להציג את מאפייני RPE העיקריים כגון צורת משושה פיגמנטציה לאחר 2 שבועות. תאי RPE שהושג יכול לשמש עבור מספר יישומים במורד הזרם כגון חילוף החומרים ויטמין A9, phagocytosis של מקטעי החיצוני קולט אור<sup class="xref...

אפיתל הפיגמנט ברשתית (RPE) היא שכבה אחת של תאי אפיתל מקוטב הממוקם בין עצבי הרשתית דמית העין. הפונקציונליות של תאי RPE ויושר טפט RPE הם קריטיים עבור חזון כי RPE ממלא תפקיד חשוב בתהליכים מרובים כגון שמירה על המחסום דם-רשתית החיצוני, הובלת מים, יונים בין הרשתית ו מקלעת דמית העין, אור הקליטה, הגנה מפני סטרס חמצוני, שליטה על חילוף החומרים retinoid, phagocytosis המקטעים החיצוני של1,photoreceptors2. המיקום של RPE מאחורי העין, וכן תפקידה מחסום מניעת סמים מנוהל מערכתית עובר מהדם אל נוזל, להקשות ללמוד את המורכבות של ה RPE פונקציה ויוו. לפיכך, יש צורך גדול להקמתה של תרביות תאים RPE ללמוד תאי RPE גמיש, בסביבה מבוקרת3,4.
מספר קווי תאי RPE הוקמה קיימות, מתן דרך קלה ונוחה של קבלת ואחסון של התאים; עם זאת, תאים passaged יש כמה חסרונות לעומת תאים ראשי-2,-3,-4. ראשית, הם לעיתים קרובות מאופיינים על ידי שינויים מורפולוגיה תאים. לדוגמה, אף אחד ממחקרי התא הקיימים נמצאו בהכשרת מחקר אמין של המאפיינים מכשול RPE בשל האובדן של פנוטיפ קוטביות תא והעלמות חלקית של צמתי צר4. בנוסף אובדן קוטביות והתקשרויות לתא המתאים, הקווים תאי RPE לאבד במהירות שלהם פיגמנטציה בשל היעדרם של אנזימים melanogenesis מפתח ב RPE למבוגרים5. ניתן לשחזר את הפיגמנטציה, אבל ניתוח מקיף של המנגנון של פיגמנטציה מחדש אשר כוללים שילוב של מיקרוסקופ אלקטרונים הילוכים, ג'ין ביטוי מבחני ניתוח וכימיקלים לאשר הנוכחות של מלנין מעולם כבר בביצוע6. מגבלה אחת יותר הוא הקווים תאי RPE חיי תאים המורחבת (לפעמים - אלמוות), בתנאים מסוימים יכול להפוך עצמי המתחדש תאי גזע multipotent כי ניתוק מן המצע טופס צף מושבות7, 8. מגבלה זו עושה את זה בלתי אפשרי להשתמש הקווים תא עבור השתלת ניסויים3.
בהתחשב החסרונות של הקווים תאי RPE הוקמה העיקרי תרביות תאים RPE המתקבל רקמות טרי עשוי לשמש מודל יותר ביולוגית רלוונטית ללמוד את RPE. ראשי תאי RPE שימשו לא רק ללמוד פונקציות ייחודיות RPE כגון ויטמין A חילוף החומרים9, phagocytosis של קולט אור מקטעי החיצוני10 ויון תחבורה11, אלא גם ללמוד ביולוגיה של התא בסיסיים כגון אפיתל התא קוטביות2 , הומאוסטזיס lysosomal autophagy12,13.
בחודשיים האחרונים של שנים היו מספר פרסומים על ביסוס ראשוני RPE תרבויות, המציין עניין הולך וגובר באזור זה של מחקר3,14,15. פרוטוקולים רבים עבור תאי RPE אדם ותאים RPE אנושיות כגון תאי RPE שור וחזירי היו שפורסמו16,17,18,19. עם זאת, קשה יותר להתמודד עם תאי RPE העכבר בשל גודלם קטן בהרבה. אף-על-פי מספר פרסומים תיארו פרוטוקולים בידוד תאי RPE העכבר14,20,21, עדיין ישנם חוקרים רבים נאבקים לבודד תאי RPE ללא זיהום של תאים דמית העין או תאים מפני פסולת עצבי הרשתית. כאן אנו מציגים את הפרוטוקול להקמת העכבר הראשי RPE תרבית תאים, לרבות קבלת העיניים של העכבר, דיסקציה של העיניים ובידוד של הסדינים RPE להניב את התאים עבור culturing. פרוטוקול וידאו זה יהיה שימושי במיוחד עבור חוקרים שמתחילים לעבוד עם העכבר הראשי RPE תרבויות, צריכים הדרכה על הטכניקות לנתיחה.

<table border="0" cellpadding="0" cellspacing="0" width="980">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">animals</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td style="width:388px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">2~3-week old wildtype mice</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Name</td>
			<td style="width:123px;">Company</td>
			<td style="width:123px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">reagents</td>
			<td style="width:123px;"></td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:347px;">Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM), high glucose</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">11965092</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dispase II</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:123px;">D4693</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:123px;">F4135</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">15140122</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">MEM Non-Essential Amino Acids Solution (100X)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">11140050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Phosphate-buffered saline (PBS), 1X, pH 7.4</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">HEPES</td>
			<td style="width:123px;">Cellgro</td>
			<td style="width:123px;">61-034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">KOH</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">1310-58-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">NaCl</td>
			<td style="width:123px;">Ambion</td>
			<td style="width:123px;">AM9759</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">L-Glutamine (200 mM)</td>
			<td style="width:123px;">GIBCO</td>
			<td style="width:123px;">25030-081</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Ethyl Alcohol</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:123px;">111000200</td>
			<td></td>
		</tr>
		<tr height="37">
			<td height="37" style="height:38px;width:347px;">RPE65 antibody</td>
			<td style="width:123px;">A gift from Dr. Michael Redmond</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Fluorescein Phalloidin&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">F432</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Goat anti-Rabbit IgG (H+L), Alexa Flour 568&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">A11011&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Goat serum</td>
			<td style="width:123px;">Sigma-Aldrich</td>
			<td style="width:123px;">G9023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">DAPI</td>
			<td style="width:123px;">Invitrogen</td>
			<td style="width:123px;">D1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Paraformaldehyde (PFA)</td>
			<td style="width:123px;">Polysciences, Inc</td>
			<td style="width:123px;">00380</td>
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			<td height="37" style="height:38px;width:347px;">ZO-1 Polyclonal Antibody</td>
			<td style="width:123px;">ThermoFisher Scientific</td>
			<td style="width:123px;">40-2200</td>
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			<td height="34" style="height:34px;width:347px;">Laminar flow cabinet</td>
			<td style="width:123px;">Baker</td>
			<td style="width:123px;">SterilGARD SG403A</td>
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			<td height="20" style="height:20px;width:347px;">Dissecting microscope (Zoom stereomicroscope)</td>
			<td style="width:123px;">Nikon</td>
			<td style="width:123px;">SMZ1500</td>
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			<td height="20" style="height:20px;width:347px;">CO2 incubator with hot air sterilization</td>
			<td style="width:123px;">Binder</td>
			<td style="width:123px;">C150</td>
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			<td height="20" style="height:20px;width:347px;">Centrifuge</td>
			<td style="width:123px;">Eppendorf</td>
			<td style="width:123px;">5702</td>
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			<td height="20" style="height:20px;width:347px;">Petri dishes</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:123px;">0875712</td>
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			<td height="35" style="height:35px;width:347px;">12 mm Polyester Membrane Inserts Pre-Loaded in 12-Well Culture Plates, Pore Size: 0.4 &#181;m, Sterile</td>
			<td style="width:123px;">Corning Incorporated</td>
			<td style="width:123px;">COR-3460</td>
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			<td height="35" style="height:35px;width:347px;">Westcott tenotomy scissors, std blades, sharp</td>
			<td style="width:123px;">STEPHENS instruments</td>
			<td style="width:123px;">S7-1320</td>
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			<td height="35" style="height:35px;width:347px;">Castroviejo suturing forceps 0.12mm</td>
			<td style="width:123px;">Stroz Ophthalmic Instruments</td>
			<td style="width:123px;">E1796</td>
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			<td height="35" style="height:35px;width:347px;">Crescent straight knife</td>
			<td style="width:123px;">Beaver-Visitec International</td>
			<td style="width:123px;">373808</td>
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			<td height="35" style="height:35px;width:347px;">Dumont Tweezers #5, 11 cm, Straight, 0.1x0.06 mm Tips, Dumostar</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">500233</td>
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			<td height="35" style="height:35px;width:347px;">Vannas Scissors, 8 cm, 45&#176; Angle, Standard</td>
			<td style="width:123px;">World Precision Instruments</td>
			<td style="width:123px;">500260</td>
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			<td height="20" style="height:20px;width:347px;">Millex-GS Syringe Filter Unit, 0.22 &#181;m</td>
			<td style="width:123px;">Millipore</td>
			<td style="width:123px;">SLGL0250S</td>
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			<td height="20" style="height:20px;width:347px;">Syringe, 5 mL</td>
			<td style="width:123px;">BD</td>
			<td style="width:123px;">309632</td>
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			<td height="20" style="height:20px;width:347px;">Inverted Laboratory Microscope&#160;Leica DM IL LED</td>
			<td style="width:123px;">&#160;Leica&#160;</td>
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			<td height="21" style="height:21px;width:347px;">Pipette</td>
			<td style="width:123px;">Gilson</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Barrier and non-filtered pipette tips</td>
			<td style="width:123px;">Thermo Scientific</td>
			<td style="width:123px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

primary cell cultures from the mouse retinal pigment epithelium

אפיתל הפיגמנט ברשתית (RPE) הוא אפיתל רב תפקודי מקוטב מאוד הממוקם בין עצבי הרשתית דמית העין. . זה גיליון אחד של תאי פיגמנט hexagonally ארוז, המחוברים באמצעות צמתי צר... הפונקציות העיקריות של RPE כוללים ספיגת אור, phagocytosis מקטעי החיצוני של קולט אור המחסן, אגירה המרחבי של יונים, הובלה של חומרים מזינים, יונים, מים, כמו גם מעורבות פעילה במחזור חזותי. עם כזה פונקציות חשובות ומגוונות, חשוב באורח קשה ללמוד את הביולוגיה של תאי RPE. מספר קווי תאי RPE הוקמו; עם זאת, תאים passaged מונצחים ידועים במהירות לאבד חלק מאפיינים פיזיולוגיים מורפולוגיים של תאי RPE טבעי. לפיכך, ראשי תאים מתאימים יותר ללמוד היבטים שונים של ביולוגיה של התא RPE ותפקוד. תרבית תאים RPE העיקרי של העכבר הוא שימושי מאוד עבור חוקרים מאז מודלים העכבר נמצאים בשימוש נרחב במחקרים ביולוגיים, אולם איסוף RPE תאים מן העכבר גם מאתגר מאוד בשל גודלם הקטן. כאן, אנו מציגים פרוטוקול להקמת תרביות תאים RPE העכבר הראשי הכוללת העקירה, דיסקציה של העיניים ובידוד של הסדינים RPE להניב את התאים עבור culturing. שיטה זו מאפשרת שחזור היעילה תא. תאי RPE המתקבל שני עכברים יכולים להגיע confluency על אחד הוספה ממברנה פוליאסטר של 12 מ מ, טעון מראש בצלחת תרבות לאחר שבוע של תרבות להציג חלק מהמאפיינים המקוריים של RPE בתום תאים כגון צורת משושה, פיגמנטציה לאחר שני שבועות של תרבות.

העכבר הראשי RPE התרבות נוצרו מ- 2 עכברים ב 12 מ מ ממברנה פוליאסטר הוספה מגיע ל 90-95% confluency לאחר שבוע 1 בתרבות. לאחר 2 שבועות של תרבות, התאים הגיעו 100% confluency והתחלתי ליצור פסיפס של תאים משושה, פיגמנט ו- bi-nucleated. על ידי 3 שבועות של תרבות, התאים המשיך כדי ליצור את הצורה והסגנון של פיגמנטצ...

Watch this Scientific Journal Video about Primary Cell Cultures from the Mouse Retinal Pigment Epithelium at JoVE.com

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

אפיתל הפיגמנט ברשתית (RPE) הוא אפיתל רב תפקודי של העין. כאן אנו מציגים פרוטוקול ליצירת תרביות תאים ראשי נגזר RPE מאתר...

התא העיקרי של אפיתל הפיגמנט ברשתית העכבר תרבויות

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

The overall goal of this procedure is to demonstrate a feasible way to obtain retinal pigment epithelium cells from mouse eyes, and to culture them in vitro while retaining their original properties. This method will help RPE researchers to obtain dissection skills that are required for establishing of mouse RPE cell cultures, and to demonstrate how to do that in an easy way. The main advantage of this procedure is that it is simple and feasible.The only thing required is practice, and the visual demonstration of RPE isolation and the microscopy literally demonstrates how to obtain the required skills. To isolate the mouse eyeballs, first euthanize two mice using any approved method, and wearing gloves place it on an absorbent pad. Position a thumb and index finger around the eye and gently push the skin to bulge out the eyeball.Once the eyeball is protruded, tuck the tip of a pair of angled scissors between the skin and the eyeball and carefully cut the tissue around the eye until it is released. Then, briefly dip the isolated eye in the 70%ethanol and submerge it in PBS in a Petri dish. After collecting both eyes from each of the mice, proceed with the dissection.Under the dissecting microscope, carefully remove the majority of connective tissue from around the eye. It is not necessary to keep the eye submerged during this step. To do so, use suturing forceps to lift the connective tissues from the eyeball, and cut them out using Vannas scissors.Do not cut the sclera because it is practically impossible to obtain the intact posterior eye cups if the sclera gets cut. After cleaning off the connective tissue, submerge the eyeballs in PBS, and switch to working in a laminar flow cabinet under sterile conditions. Now, use forceps to transfer the eyes into a dish of warm DMEM containing a high concentration of glucose.After 20 to 30 seconds, replace the wash solution using aspiration for a second wash. Transfer eyeballs to warmed 2%Dispase II working solution in 15 mL tubes and let the eyes incubate at 37 degrees celsius for 45 minutes to dissociate. After the incubation, the eyeballs should be soft.Next, wash the eyes two times in the same vessel with warmed growth medium. After the second wash, replace the growth medium with fresh medium, and transfer the eyeballs and the medium to a new dish to continue the dissection. Continue working in a clean bench with the help of a dissection microscope.Start the dissection by securing the eye with forceps and make an incision around the ora serrata using Vannas scissors. Keeping the eyeball in the solution, carefully remove the anterior cornea by extending the cut around the ora serrata circumference. After completing the cut, pull off the anterior cornea.Next, remove the lens capsule and associated iris pigmented epithelium by gently pulling it out of the eye cup using toothed forceps. Then, repeat the dissection on the remaining eyes and incubate the posterior eye cups in growth medium for 20 minutes at 37 degrees celsius to facilitate separation of the neural retina from the RPE. After 20 minutes, cut the posterior eye cups into four petals.Make the cuts long enough to flatten the eye cup, but short enough to keep the petals connected. Next, flatten the eye cup to remove the neural retina. Secure the remaining RPE choroid sclera complex with the non-dominant hand and very gently tug away the retina from the edges.Once the neural retina is removed, transfer the quartered RPE choroid sclera complex into a new sterile culture dish filled with warm growth medium. In the Petri dish, secure one petal of the quartered RPE choroid sclera complex with super fine forceps, and using a microsurgical crescent knife, gently peel off the intact sheets of RPE from the underlying basement membrane. The brownish RPE should be clearly distinguishable from the dark, sticky, fluffy choroid.Then, wet a p200 micropipette tip with growth medium and use the tip to transfer the RPE sheets into the new dish containing warmed growth medium. Now, wash the RPE sheets three times in warmed growth medium. After each wash step, carefully collect the RPE sheets while avoiding other tissues, such as the debris from the retina or choroid.After the last wash, transfer the RPE sheets to a 1.5 mL tube, and allow them to sediment by gravity. Then, in the laminar flow cabinet remove the extra growth medium and re-suspend the cells in freshly made warm growth medium. Now, gently triturate the tissue using a p200 micropipette without making bubbles.Triturate between 40 and 50 times, just enough to make a single cell suspension. Be careful as too much trituration can be lethal. Now, plate the RPE cells collected from both mice in one 12 mL polyester membrane insert in the well of a 12 well culture plate.A high cell density is desired so that the RPE cells retain their hexagonal shape and pigmentation. A mouse primary RPE culture established from two mice in a 12 mm polyester membrane insert reached 90 to 95%confluency after one week in culture. After two weeks in culture, the cells were 100%confluent and started to form a mosaic of hexagonal pigmented and bi-nucleated cells.By week three, the cells continued to change in shape and pigmentation. However, after four weeks a portion of the cells became hyperpigmented. The purity of the primary RPE cell culture was assessed using the RPE65 antibody, which labels retinoid isomerohydrolase, an enzyme critical to the vertebrate visual cycle.The integrity of the cell to cell junctions and hexagonal cell shape was demonstrated by staining with phalloidin. Tight junctions were visualized by analysis of ZO-1 protein expression, which could be visualized using an amino stain and validated by western blotting. Overall, the cultures are able to proliferate, retain pigmentation and their hexagonal shape while forming tight junctions and expressing functional markers, such as RPE65.After watching this video, you should have a good understanding of how to isolate RPE cells from the mouse eyes and culture them properly in vitro. Once mastered, this technique can be done in three hours if it is performed properly. While attempting this procedure, it is important to remember to always keep the eyes wet and try to operate as fast as you can.

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Research

JoVE Journal

Biology

Primary Neuronal Cultures from the Brains of Late Stage Drosophila Pupae

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the removal of brains from animals at 70-78 hrs after puparium formation. The isolated brains are shown after brief incubation in papain followed by several washes in serum-free growth medium. The process of mechanical dissociation of each brain in a 5 ul drop of media on a coverslip is illustrated. The axons and dendrites of the post-mitotic neurons are sheered off near the soma during dissociation but the neurons begin to regenerate processes within a few hours of plating. Images show live cultures at 2 days. Neurons continue to elaborate processes during the first week in culture. Specific neuronal populations can be identified in culture using GAL4 lines to drive tissue specific expression of fluorescent markers such as  GFP or RFP. Whole cell recordings have demonstrated the cultured neurons form functional, spontaneously active cholinergic and GABAergic synapses. A short video segment illustrates calcium dynamics in the cultured neurons using Fura-2 as a calcium indicator dye to monitor spontaneous calcium transients and nicotine evoked calcium responses in a dish of cultured neurons. These pupal brain cultures are a useful model system in which genetic and pharmacological tools can be used to identify intrinsic and extrinsic factors that influence formation and function of central synapses.

This video demonstrates the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. Views of live cultures show neurite outgrowth and imaging of calcium levels using Fura-2.

תרבויות עצבית ראשונית של המוח של Late Stage תסיסנית גלמים

In this video, we demonstrate the preparation of primary neuronal cultures from the brains of late stage Drosophila pupae. The procedure begins with the ...

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

ראשי ניתק דופמין במוח התיכון Cell תרבויות מן ילודים מכרסמים

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

הכנת ראשי בתרביות תאים hematopoietic ממח העצם Murine עבור electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

תא תרבויות ראשי מ תסיסנית Gastrula עוברים

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

פרופילי הביטוי microRNA של תאי iPS האנושי, רשתית פיגמנט האפיתל נגזר משב&quot;ס, ועוברי הרשתית פיגמנט האפיתל

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

ניתוח יעיל ותרבות של תאי אפיתל פיגמנט רשתית העכבר הראשי

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...

Primary Culture of Porcine Retinal Pigment Epithelial Cells

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. Multiple functions, including phagocytosis, nutrient/metabolite transportation, vitamin A metabolism, etc., are conducted by the RPE on a daily basis. RPE cells are terminally differentiated epithelial cells with little or no regenerative capacity. Loss of RPE cells results in multiple eye diseases leading to visual impairment, such as age-related macular degeneration. Therefore, the establishment of an in vitro culture model of primary RPE cells, which more closely resembles the RPE in vivo than cell lines, is critical for the characteristic and mechanistic studies of RPE cells. Considering the fact that the source of human eyeballs is limited, we create a protocol to culture primary porcine RPE cells. By using this protocol, RPE cells can be easily dissociated from adult porcine eyeballs. Subsequently, these dissociated cells attach to culture dishes/inserts, proliferate to form a confluent monolayer, and quickly re-establish key features of epithelial tissue in vivo within 2 wks. By qRT-PCR, it is demonstrated that primary porcine RPE cells express multiple signature genes at comparable levels with native RPE tissue, while the expressions of most of these genes are lost/highly reduced in human RPE-like cells, ARPE-19. Moreover, the immunofluorescence staining shows the distribution of tight junction, tissue polarity, and cytoskeleton proteins, as well as the presence of RPE65, an isomerase critical for vitamin A metabolism, in cultured primary cells. Altogether, we have developed an easy-to-follow approach to culture primary porcine RPE cells with high purity and native RPE features, which could serve as a good model to understand RPE physiology, study cell toxicities, and facilitate drug screenings.

Here, an easy-to-follow method to culture primary porcine retinal pigment epithelial cells in vitro is presented.

תרבית ראשונית של תאי אפיתל פיגמנט רשתית חזירית

The retinal pigment epithelium (RPE) is a monolayer of polarized pigmented epithelial cells, located between the choroid and neuroretina in the retina. ...

Isolation of Primary Mouse Retinal Pigmented Epithelium Cells

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical roles in maintaining the photoreceptors' function. Thus, the RPE structure and function are essential to sustain normal vision. This manuscript presents an established protocol for primary mouse RPE cell isolation. RPE isolation is a great tool to investigate the molecular mechanisms underlying RPE pathology in the different mouse models of ocular disorders. Furthermore, RPE isolation can help in comparing primary mouse RPE cells isolated from wild-type and genetically modified mice, as well as testing drugs that can accelerate the development of therapy for visual disorders. The manuscript presents a step-by-step RPE isolation protocol; the entire procedure, from enucleation to seeding, takes approximately 4 hours. The media shouldn't be changed for 5-7 days after seeding, to allow the growth of the isolated cells without disturbance. This process is followed by the characterization of morphology, pigmentation, and specific markers in the cells via immunofluorescence. Cells can be passaged a maximum of three or four times.

This manuscript describes a simplified protocol for the isolation of retinal pigmented epithelium (RPE) cells from mouse eyes in a stepwise manner. The protocol includes the enucleation and dissection of mouse eyes, followed by the isolation, seeding, and culturing of RPE cells.

בידוד של תאי אפיתל פיגמנטים ברשתית של עכברים ראשוניים

The retinal pigmented epithelium (RPE) layer lies immediately behind the photoreceptors and harbors a complex metabolic system that plays several critical ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

ייצור יעיל ועקבי של אפיתל פיגמנט רשתית/כורואיד שטוח מעיני אדם לניתוח היסטולוגי

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

מידול וניתוח ניוון הרשתית

LipidUNet-Machine Learning-Based Method of Characterization and Quantification of Lipid Deposits Using iPSC-Derived Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a monolayer of hexagonal cells located at the back of the eye. It provides nourishment and support to photoreceptors and choroidal capillaries, performs phagocytosis of photoreceptor outer segments (POS), and secretes cytokines in a polarized manner for maintaining the homeostasis of the outer retina. Dysfunctional RPE, caused by mutations, aging, and environmental factors, results in the degeneration of other retinal layers and causes vision loss. A hallmark phenotypic feature of degenerating RPE is intra and sub-cellular lipid-rich deposits. These deposits are a common phenotype across different retinal degenerative diseases. To reproduce the lipid deposit phenotype of monogenic retinal degenerations in vitro, induced pluripotent stem cell-derived RPE (iRPE) was generated from patients&#39; fibroblasts. Cell lines generated from patients with Stargardt and Late-onset retinal degeneration (L-ORD) disease were fed with POS for 7 days to replicate RPE physiological function, which caused POS phagocytosis-induced pathology in these diseases. To generate a model for age-related macular degeneration (AMD), a polygenic disease associated with alternate complement activation, iRPE was challenged with alternate complement anaphylatoxins. The intra and sub-cellular lipid deposits were characterized using Nile Red, boron-dipyrromethene (BODIPY), and apolipoprotein E (APOE). To quantify the density of lipid deposits, a machine learning-based software, LipidUNet, was developed. The software was trained on maximum-intensity projection images of iRPE on culture surfaces. In the future, it will be trained to analyze three-dimensional (3D) images and quantify the volume of lipid droplets. The LipidUNet software will be a valuable resource for discovering drugs that decrease lipid accumulation in disease models.

מחבר זרקור: חשיפת הפתוגנזה של ניוון מקולרי הקשור לגיל וגילוי טיפולים פוטנציאליים 

Degenerative eye diseases that affect the retinal pigment epithelium layer of the eye have monogenic and polygenic origins. Several disease models and a software application, LipidUNet, have been developed to study mechanisms of disease, as well as potential therapeutic interventions.