Name: Yazar Spotlight: Retina Pigment Epitel Hücre Dondurma için Verimli ve Uygulanabilir Bir Yöntemin Geliştirilmesi
Uploaded: 2023-11-03T13:20:00
Description: Watch this Scientific Journal Video about Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage at JoVE.com

Bu çalışma, Çin Ulusal Doğa Bilimleri Vakfı (81970816) tarafından Mei Jiang'a finanse edildi; Çin Ulusal Doğa Bilimleri Vakfı (82201223) Xinyue Zhu'ya; ve Şanghay Bilim ve Teknoloji Komisyonu'nun (2014090067000) Bilim ve Teknoloji İnovasyon Eylem Planı'nı Haiyun Liu'ya teslim etti.

1. Hücre ayrışması
<ol><li>RPE hücrelerini daha önce tarif edildiği gibi koruyun17,22. NOT: Tüm hücreler, protokollerin süresi boyunca% 5 CO2 atmosferinde 37 ° C'de büyütülür.</li>
	<li>Gerekli miktarda PBS ve kültür ortamını 37 ° C'lik bir su banyosunda hazırlayın ve hücre ayrışma reaktifini oda sıcaklığına yerleştirin.</li>
	<li>Kültür çözeltisini atın ve plakaları oyuk başına 1 mL önceden ...

İnsan Kök Hücre Kaynaklı Retina Pigment Epitel Hücrelerinin Kriyo Depolamasının İlerletilmesi

<ol>
	<li>Lakkaraju, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+the+retinal+pigment+epithelium.">The cell biology of the retinal pigment epithelium.</a> Progress in Retinal and Eye Research. 78, 100846 (2020).</li><li>Mcbain, V. A., Townend, J., Lois, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progression+of+retinal+pigment+epithelial+atrophy+in+stargardt+disease.">Progression of retinal pigment epithelial atrophy in stargardt disease.</a> American Journal of Ophthamology. 154 (1), 146-154 (2012).</li><li>George, S. M., Lu, F., Rao, M., Leach, L. L., Gross, J. 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+retinal+pigment+epithelium:+Development,+injury+responses,+and+regenerative+potential+in+mammalian+and+non-mammalian+systems.">The retinal pigment epithelium: Development, injury responses, and regenerative potential in mammalian and non-mammalian systems.</a> Progress in Retinal and Eye Research. 85, 100969 (2021).</li><li>Rizzolo, L. J., Nasonkin, I. O., Adelman, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinal+cell+transplantation,+biomaterials,+and+in+vitro+models+for+developing+next-generation+therapies+of+age-related+macular+degeneration.">Retinal cell transplantation, biomaterials, and in vitro models for developing next-generation therapies of age-related macular degeneration.</a> Stem Cells Translational Medicine. 11 (3), 269-281 (2022).</li><li>Bertolotti, E., Neri, A., Camparini, M., Macaluso, C., Marigo, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+as+source+for+retinal+pigment+epithelium+transplantation.">Stem cells as source for retinal pigment epithelium transplantation.</a> Progress in Retinal and Eye Research. 42, 130-144 (2014).</li><li>Da Cruz, ., L, , 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=Phase+1+clinical+study+of+an+embryonic+stem+cell-derived+retinal+pigment+epithelium+patch+in+age-related+macular+degeneration.">Phase 1 clinical study of an embryonic stem cell-derived retinal pigment epithelium patch in age-related macular degeneration.</a> Nature Biotechnology. 36 (4), 328-337 (2018).</li><li>Mehat, M. 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=Transplantation+of+human+embryonic+stem+cell-derived+retinal+pigment+epithelial+cells+in+macular+degeneration.">Transplantation of human embryonic stem cell-derived retinal pigment epithelial cells in macular degeneration.</a> Ophthalmology. 125 (11), 1765-1775 (2018).</li><li>Buchholz, D. E., 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=Rapid+and+efficient+directed+differentiation+of+human+pluripotent+stem+cells+into+retinal+pigmented+epithelium.">Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium.</a> Stem Cells Translational Medicine. 2 (5), 384-393 (2013).</li><li>Li, Q. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+assessment+of+cryopreserved+clinical+grade+hESC-RPE+cells+as+a+qualified+cell+source+for+stem+cell+therapy+of+retinal+degenerative+diseases.">Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.</a> Experimental Eye Research. 202, 108305 (2021).</li><li>Francois, 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=Cryopreserved+mesenchymal+stromal+cells+display+impaired+immunosuppressive+properties+as+a+result+of+heat-shock+response+and+impaired+interferon-gamma+licensing.">Cryopreserved mesenchymal stromal cells display impaired immunosuppressive properties as a result of heat-shock response and impaired interferon-gamma licensing.</a> Cytotherapy. 14 (2), 147-152 (2012).</li><li>Moll, G., 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=Do+cryopreserved+mesenchymal+stromal+cells+display+impaired+immunomodulatory+and+therapeutic+properties.">Do cryopreserved mesenchymal stromal cells display impaired immunomodulatory and therapeutic properties.</a> Stem Cells. 32 (9), 2430-2442 (2014).</li><li>Ekpo, M. D., 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=Incorporating+cryopreservation+evaluations+into+the+design+of+cell-based+drug+delivery+systems:+an+opinion+paper.">Incorporating cryopreservation evaluations into the design of cell-based drug delivery systems: an opinion paper.</a> Frontiers in Immunology. 13, 967731 (2022).</li><li>Bailey, T. L., 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=Protective+effects+of+osmolytes+in+cryopreserving+adherent+neuroblastoma+(Neuro-2a)+cells.">Protective effects of osmolytes in cryopreserving adherent neuroblastoma (Neuro-2a) cells.</a> Cryobiology. 71 (3), 472-480 (2015).</li><li>Okumura, N., 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=Feasibility+of+a+cryopreservation+of+cultured+human+corneal+endothelial+cells.">Feasibility of a cryopreservation of cultured human corneal endothelial cells.</a> PLoS One. 14 (6), e0218431 (2019).</li><li>Pennington, B. O., 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=Xeno-free+cryopreservation+of+adherent+retinal+pigmented+epithelium+yields+viable+and+functional+cells+in+vitro+and+in+vivo.">Xeno-free cryopreservation of adherent retinal pigmented epithelium yields viable and functional cells in vitro and in vivo.</a> Scientific Reports. 11 (1), 6286 (2021).</li><li>Woods, E. J., Thirumala, S., Badhe-Buchanan, S. S., Clarke, D., Mathew, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Off+the+shelf+cellular+therapeutics:+Factors+to+consider+during+cryopreservation+and+storage+of+human+cells+for+clinical+use.">Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use.</a> Cytotherapy. 18 (6), 697-711 (2016).</li><li>Zhang, T., 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=Determining+the+optimal+stage+for+cryopreservation+of+human+embryonic+stem+cell-derived+retinal+pigment+epithelial+cells.">Determining the optimal stage for cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells.</a> Stem Cell Research &amp; Therapy. 13 (1), 454 (2022).</li><li>Leach, L. L., 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=Induced+pluripotent+stem+cell-derived+retinal+pigmented+epithelium:+a+comparative+study+between+cell+lines+and+differentiation+Methods.">Induced pluripotent stem cell-derived retinal pigmented epithelium: a comparative study between cell lines and differentiation Methods.</a> Journal of Ocular Pharmacology and Therapeutics. 32 (5), 317-330 (2016).</li><li>Reichman, 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=Generation+of+storable+retinal+organoids+and+retinal+pigmented+epithelium+from+adherent+human+iPS+cells+in+Xeno-free+and+feeder-free+conditions.">Generation of storable retinal organoids and retinal pigmented epithelium from adherent human iPS cells in Xeno-free and feeder-free conditions.</a> Stem Cells. 35 (5), 1176-1188 (2017).</li><li>Brandl, C., 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=In-depth+characterisation+of+retinal+pigment+epithelium+(RPE)+cells+derived+from+human+induced+pluripotent+stem+cells+(hiPSC).">In-depth characterisation of retinal pigment epithelium (RPE) cells derived from human induced pluripotent stem cells (hiPSC).</a> Neuromolecular Medicine. 16 (3), 551-564 (2014).</li><li>Hongisto, H., Ilmarinen, T., Vattulainen, M., Mikhailova, A., Skottman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xeno-+and+feeder-free+differentiation+of+human+pluripotent+stem+cells+to+two+distinct+ocular+epithelial+cell+types+using+simple+modifications+of+one+method.">Xeno- and feeder-free differentiation of human pluripotent stem cells to two distinct ocular epithelial cell types using simple modifications of one method.</a> Stem Cell Research &amp; Therapy. 8 (1), 291 (2017).</li><li>Foltz, L. P., Clegg, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+directed+differentiation+of+retinal+pigment+epithelial+cells+from+human+embryonic+or+induced+pluripotent+stem+cells.">Rapid, directed differentiation of retinal pigment epithelial cells from human embryonic or induced pluripotent stem cells.</a> Journal of Visualized Experiments: JoVE. (128), 10.3791/56274 (2017).</li><li>Marcantonini, G., 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=Natural+cryoprotective+and+cytoprotective+agents+in+cryopreservation:+a+focus+on+melatonin.">Natural cryoprotective and cytoprotective agents in cryopreservation: a focus on melatonin.</a> Molecules. 27 (10), 3254 (2022).</li></ol>

Bu çalışmada, araştırma ve klinik ihtiyaçlar için hESC türevi RPE hücreleri için başarılı bir donma-çözülme protokolü tanımlanmıştır. Ölümsüzleştirilmiş RPE hücre hattı ARPE-19'un aksine, kök hücre türevli RPE hücreleri gibi uygun karakteristik epitelyal fenotip ve fonksiyona sahip RPE hücreleri, kriyoprezervasyona daha duyarlıdır. Hücrelerin% 32'sinden daha azı, uygun şekilde korunmadığı takdirde, çözülmeden 24 saat sonra kaldı17. Kriyoprezervasyon zam...

Retina pigment epiteli (RPE), retinanın düzgün işlevini sürdürmek için gerekli olan pigmentli bir tek hücre tabakasıdır1. RPE disfonksiyonu ve ölümü, yaşa bağlı makula dejenerasyonu, retinitis pigmentosa ve Stargardt hastalığı 2,3 dahil olmak üzere birçok retinal dejeneratif hastalıkla yakından ilişkilidir. RPE replasman tedavisi, bu hastalıklar için en umut verici tedavi rejimlerinden biridir 4,5,6,7. Donör RPE hücrelerinin stabil bir şekilde tedarik edilmesi, hücre tedavisi için hayati önem taşır. İnsan embriyonik kök hücresinden (hESC) türetilen RPE hücreleri, birincil RPE hücrelerinin işlevini taklit ettikleri ve teorik olarak sınırsız bir tedarik üretebildikleri için hücre tedavisi için ideal bir hücre kaynağıdır8. Bununla birlikte, farklılaşma süreci zahmetlidir ve elde edilen RPE hücrelerinin raf ömrü, sonraki epitelyal-mezenkimal geçiş (EMT) nedeniyle nispeten kısadır. Bu nedenle, hESC'den türetilen RPE hücrelerinin dondurularak saklanması, uzun süreli depolama ve isteğe bağlı dağıtım için gerekli olan vazgeçilmez bir adımdır9.
Kriyoprezervasyonun neden olduğu hücresel hasar, yanlışlıkla terapötik etkinliği tehlikeye atabilir10,11. Bu nedenle, kriyoprezervasyon ile ilgili son çalışmalar, hücre tedavileri tasarlanırken optimal kriyojenik depolama koşullarının belirlenmesi gerektiğini önermiştir12. Başarılı kriyoprezervasyon, donma-çözülme döngüsünden sonra verimli hücre geri kazanımı, yüksek canlılık ve hücre fonksiyonunun restorasyonunu garanti eder. Bununla birlikte, memeli hücrelerinin yapışık tek tabakalarının dondurularak saklanması üzerine yapılan önceki çalışmalar, çözülmeden sonra oldukça değişken (%35-%95) sağkalım oranları bildirmiştir 13,14,15. Birçok faktör, özellikle dondurma aşamasında kriyoprezervasyonun sonuçlarını önemli ölçüde etkiler16,17. Son araştırmalar, farklı zaman noktalarında donmuş RPE hücrelerinin çözüldükten sonra çeşitli iyileşme gösterdiğini gösterdi17. Bildiğimiz kadarıyla, kök hücre kaynaklı RPE hücreleri için en uygun dondurma süresi penceresinin belirlenmesine yönelik çalışmalar eksiktir. Farklı çalışmalarda, hücreler çeşitli aşamalarda donduruldu: bazı hücreler geçişten kısa bir süre sonra veya birleşme veya pigmentasyondanönce donduruldu 8,15,18, diğerleri ise diğer zaman noktalarında 9,19,20,21 donduruldu. Ayrıca, kriyoprezervasyon için kullanılan RPE hücrelerinin fazının veya aşamasının çözüldükten sonra RPE fonksiyonunu etkileyip etkilemediğine dair net bir kanıt yoktur. Önceki çalışmamızda, hücre büyümesinin üstel fazının (P2D5), hücre canlılığı ve hücresel özelliklerin ve işlevlerin geri kazanılması açısından hESC'den türetilmiş RPE hücrelerinin dondurularak saklanması için en iyi aşama olduğunu ilk kez gösterdik17.
Burada oluşturulan yöntem, çözüldükten sonra hücre canlılığı ve işlevi açısından en iyi korumayı elde etmek için hESC'den türetilen RPE'yi optimal bir aşamada kriyoprezervasyon yapmayı amaçlamaktadır. Kriyoprezervasyondan önce DNA sentezinin üstel fazını tespit etmek için 5-etinil-2'-deoksiüridin (EdU) etiketleme testini kullanarak, çözülmüş RPE hücreleri %>80 canlılık ve bağlanma oranı, RPE'ye özgü gen ekspresyonu, polarize hücre morfolojisi, pigment epitel kaynaklı faktör sekresyonyonu, uygun transepitelyal direnç ve fagositik yetenek sergiledi 8,17,22. Bu protokol hESC'den türetilmiş RPE hücrelerine odaklanmasına ve tüm terapötik hücrelerin eşit şekilde dondurularak saklanmamasına rağmen, üstel fazda dondurma stratejisi diğer birçok terapötik hücreye uygulanabilir.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40 &#956;m Cell strainer</td>
			<td>Corning</td>
			<td>431750</td>
			<td></td>
		</tr>
		<tr>
			<td>Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 488 Dye</td>
			<td>Thermo Fisher Scientific</td>
			<td>C10337</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo freezing container</td>
			<td>Nalgene</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>CryoStor CS10</td>
			<td>Biolife Solutions</td>
			<td>07930</td>
			<td>cryopreservation medium #1</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190144</td>
			<td></td>
		</tr>
		<tr>
			<td>Genxin</td>
			<td>Selcell</td>
			<td>YB050050</td>
			<td>cryopreservation medium #2</td>
		</tr>
		<tr>
			<td>Human embryonic stem cells</td>
			<td>provided by Wicell, USA</td>
			<td>H9 cell line</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel, hESC-Qualified Matrix</td>
			<td>Corning</td>
			<td>354277</td>
			<td>basement membrane matrix</td>
		</tr>
		<tr>
			<td>ThawSTAR CFT2 Automated Cell Thawing System</td>
			<td>BioLife Solutions</td>
			<td>AST-601</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue solution 0.4%</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>TryPLE Select</td>
			<td>Thermo Fisher Scientific</td>
			<td>12563029</td>
			<td>cell dissociation reagent</td>
		</tr>
		<tr>
			<td>XVIVO-10 medium</td>
			<td>Lonza</td>
			<td>BEBP04-743Q</td>
			<td>RPE culture medium</td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Selleck</td>
			<td>S1049</td>
			<td></td>
		</tr>
	</tbody>
</table>

cryopreservation of human embryonic stem cell-derived retinal pigment epithelial cells at the optimal stage

İnsan embriyonik kök hücrelerinden (hESC'ler) türetilen retinal pigment epitel (RPE) hücreleri, retinal dejeneratif hastalıkları olan bireylerde hücre replasman tedavisi için üstün hücre kaynaklarıdır; Bununla birlikte, bu terapötik hücrelerin istikrarlı ve güvenli bankacılığı ile ilgili çalışmalar azdır. Kriyoprezervasyon sonrası RPE hücrelerinin oldukça değişken hücre canlılığı ve fonksiyonel geri kazanımı en sık karşılaşılan sorunlardır. Bu protokolde, orijinal deney koşullarına dayalı olarak dondurma için en uygun hücre fazını seçerek çözdürme işleminden sonra en iyi hücre geri kazanım oranını elde etmeyi amaçladık. Hücreler, çözüldükten sonra hücre canlılığını ve geri kazanım oranını artıran 5-etinil-2'-deoksiüridin etiketleme testi kullanılarak belirlenen üstel fazda donduruldu. Stabil ve fonksiyonel hücreler, uzun bir farklılaşma sürecinden bağımsız olarak, çözülmeden kısa bir süre sonra elde edildi. Burada açıklanan yöntemler, hESC'den türetilen RPE hücrelerinin basit, verimli ve ucuz bir şekilde korunmasına ve çözülmesine izin verir. Bu protokol RPE hücrelerine odaklansa da, bu dondurma stratejisi diğer birçok farklılaşmış hücre tipine uygulanabilir.

The retinal pigment epithelium (RPE) is a pigmented monolayer of cells required for maintaining the proper function of the retina1. RPE dysfunction and death are closely associated with many retinal degenerative diseases, including age-related macular degeneration, retinitis pigmentosa, and Stargardt disease2,3. RPE replacement therapy is one of the most promising treatment regimens for these diseases4,5,6,7. A stable supply of donor RPE cells is vital for cell therapy. Human embryonic stem cell (hESC)-derived RPE cells are an ideal cell source for cell therapy because they mimic the function of primary RPE cells and can produce a theoretically unlimited supply8. However, the differentiation process is laborious and the shelf-life of the obtained RPE cells is relatively short because of subsequent epithelial-mesenchymal transition (EMT). Therefore, the cryopreservation of hESC-derived RPE cells is an indispensable step required for long-term storage and on-demand distribution9.
Cryopreservation-induced cellular damage can inadvertently compromise therapeutic efficacy10,11. Therefore, recent studies on cryopreservation have proposed that optimal cryogenic storage conditions should be determined when designing cell therapies12. Successful cryopreservation guarantees efficient cell recovery, high viability, and cell function restoration after the freeze-thaw cycle. However, previous studies on the cryopreservation of the adherent monolayers of mammalian cells have reported highly variable (35%-95%) survival rates after thawing13,14,15. Many factors considerably affect the outcomes of cryopreservation, particularly during the freezing stage16,17. Recent research showed that RPE cells frozen at different time points exhibited varied recovery after thawing17. To the best of our knowledge, studies on the determination of the optimal freezing time window for stem cell-derived RPE cells are lacking. In different studies, the cells were frozen at various stages: some cells were frozen shortly after passaging or before confluency or pigmentation8,15,18, whereas others were frozen at other time points9,19,20,21. Furthermore, there is no clear evidence of whether the phase or stage of RPE cells used for cryopreservation affects RPE function after thawing. In our previous study, we demonstrated for the first time that the exponential phase of cell growth (P2D5) is the best stage for the cryopreservation of hESC-derived RPE cells in terms of cell viability and the recovery of cellular properties and functions17.
The method established here aims to cryopreserve hESC-derived RPE at an optimal stage to achieve the best preservation in terms of cell viability and function after thawing. Using the 5-ethynyl-2&#39;-deoxyuridine (EdU) labeling assay to detect the exponential phase of DNA synthesis before cryopreservation, thawed RPE cells exhibited &#62;80% viability and attachment rate, RPE-specific gene expression, polarized cell morphology, pigment epithelium-derived factor secretion, appropriate transepithelial resistance, and phagocytic ability8,17,22. Although this protocol focuses&#160;on hESC-derived RPE cells and not all therapeutic cells are equally cryopreserved, the strategy of freezing in the exponential phase may be applied to many other therapeutic cells.

Yazar Spotlight: Retina Pigment Epitel Hücre Dondurma için Verimli ve Uygulanabilir Bir Yöntemin Geliştirilmesi

İnsan Embriyonik Kök Hücre Kaynaklı Retina Pigment Epitel Hücrelerinin Optimal Aşamada Dondurularak Saklanması

Our lab aims to better understand the molecular mechanism of retinal diseases, with a focus on unmet medical needs of patients. We aim to develop effective treatments, such as stem-cell-based therapies, for them. Cryopreservation of stem-cell-derived RPE cells is an important step for RPE replacement therapy.Currently, the cells are frozen at various stages, resulting in highly variable survival rates for thawing. This is needed to determine the optimal cell stage for freezing these cells. Our protocol provides a simple, efficient, and inexpensive ways of freezing RPE cells by determining the exponential phase of RPE cells for freezing.It's less dependent of differentiated methods or cell lines.

Burada, P1D35'teki hESC'den türetilmiş RPE hücreleri pasajlandı ve 105 / cm2'lik bir yoğunlukta tohumlandı. Tohumlamadan sonraki bir hafta içinde, karakteristik altıgen morfoloji ve pigmentasyon, gecikme fazında (yaklaşık 2 gün) kayboldu. RPE hücreleri, üstel fazda (yaklaşık 5 gün, Şekil 1A) altıgen morfolojiyi kademeli olarak yeniden benimsedi ve daha poligonal bir morfoloji ile yavaşlama fazına (yaklaşık 6 gün) girdi. Hücre kültürü bir haft...

Watch this Scientific Journal Video about Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage at JoVE.com

Bu çalışma, insan kök hücresinden türetilmiş retina pigment epitel hücrelerinin etkili bir şekilde dondurularak saklanması için ayrıntılı bir protokol sunmaktadır.

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in ...

Laboratuvarımız, hastaların karşılanmamış tıbbi ihtiyaçlarına odaklanarak retina hastalıklarının moleküler mekanizmasını daha iyi anlamayı amaçlamaktadır. Onlar için kök hücre bazlı tedaviler gibi etkili tedaviler geliştirmeyi amaçlıyoruz. Kök hücre kaynaklı RPE hücrelerinin dondurularak saklanması, RPE replasman tedavisi için önemli bir adımdır.Şu anda, hücreler çeşitli aşamalarda dondurulmakta ve bu da çözülme için oldukça değişken hayatta kalma oranları ile sonuçlanmaktadır. Bu, bu hücrelerin dondurulması için en uygun hücre aşamasını belirlemek için gereklidir. Protokolümüz, dondurma için RPE hücrelerinin üstel fazını belirleyerek RPE hücrelerini dondurmanın basit, verimli ve ucuz bir yolunu sağlar.Farklılaştırılmış yöntemlere veya hücre hatlarına daha az bağımlıdır.

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Research

JoVE Journal

Medicine

Cryopreservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells at the Optimal Stage

Retinal pigment epithelial&#160;(RPE) cells derived from human embryonic stem cells (hESCs) are superior cell sources for cell replacement therapy in individuals with retinal degenerative diseases; however, studies on the stable and secure banking of these therapeutic cells are scarce. Highly variable cell viability and functional recovery of&#160;RPE cells after cryopreservation are the most commonly encountered issues. In the present protocol, we aimed to achieve the best cell recovery rate after thawing by selecting the optimal cell phase for freezing based on the original experimental conditions. Cells were frozen in the exponential phase determined by using the 5-ethynyl-2&#8242;-deoxyuridine labeling assay, which improved cell viability and recovery rate after thawing. Stable and functional cells were obtained shortly after thawing, independent of a long differentiation process. The methods described here allow the simple, efficient, and inexpensive preservation and thawing of hESC-derived RPE cells. Although this protocol focuses on RPE cells, this freezing strategy may be applied to many other types of differentiated cells.

This study provides a detailed protocol for the efficient cryopreservation of human stem cell-derived retinal pigment epithelial cells.

Tıp

Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cell Dissociation and Optimal Cryopreservation Stage Determination

To begin, dilute a vial of thawed cold basement membrane matrix with 12 milliliters of DM EM F12. Mix the suspension well. Add one cover slip into each well of a 24-well plate.Then, pipette 250 microliters of the dilute matrix solution into each well. Discard the culture medium of the cultured hESC derived retinal pigment epithelial cell plates. Wash the plates two times with one milliliter of preheated PBS per well.Next, add 0.5 milliliters of the cell dissociation reagent to the wells of the culture plates, and incubate them at 37 degrees Celsius for 15 minutes to initiate digestion. After incubation, observe the cells under the microscope to confirm the end of digestion. Now with a one milliliter pipette, gently pipette the cell suspension up and down 10 times.Add preheated culture medium to dilute the suspension. Then, centrifuge it at 250 G for three minutes at room temperature. Pour out the supernatant from the centrifuge tube.Then, re-suspend the cell pellet in two milliliters of the culture medium. Use a pipette to mix the cells 10 to 15 times. Filter the cell suspension through a 40 micrometer cell strainer.Next, count the retinal pigment epithelial cells. Aspirate the coating solution before plating. Then, add one milliliter of the culture medium into each well and seed the cells on the cover slips.After EDU incorporation and staining, capture the images of five random fields with a fluorescence microscope. Calculate the percentage of EDU positive cells. Then, plot the corresponding proportion time curves to generate a growth curve.Lastly, determine the freezing window from the exponential phase for each cell line. The retinal pigment epithelial cells initially lost their characteristic hexagonal morphology, but later regained it in the exponential phase of growth. When the cells were cultured for an additional week, cell proliferation decreased.EDU proliferation assay confirmed that P2 day five cells exhibited a higher proliferation rate, whereas P2 day 11 cells had entered the deceleration phase.

İnsan Embriyonik Kök Hücre Kaynaklı Retina Pigment Epitel Hücre Dissosiyasyonu ve Optimal Kriyoprezervasyon Aşaması Tayini

Preservation of Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells with Optimized Cryopreservation

To begin, dissociate the hESC-derived retinal pigment epithelial cells, prepare the cell suspension, and then count the cells. Now, centrifuge the cells at 250g for three minutes at room temperature. Pour out the supernatant, then, resuspend the cell pellet in cryopreservation medium.Next, transfer one milliliter of the cell suspension to a 1.2-milliliter cryogenic vial. Place the cryogenic vials in a freezing container and freeze overnight at minus 80 degrees Celsius. Transfer the vials to liquid nitrogen for long-term storage.To thaw the frozen vials, first heat the culture medium in metallic beads heated to 37 degrees Celsius, pre-fill 10 milliliters of the warmed culture medium into a 15-milliliter tube. Now, remove the cryogenic vials from the liquid nitrogen and place them in an automated thawing system for rapid thawing. Drop 0.5 to one milliliter of the preheated culture medium into the cryogenic vial to ensure gradual cell adaptation.Then, pipet 1.5 to two milliliters of the cell suspension to the 15-milliliter tube with medium. Centrifuge the cells at 250g for three minutes at room temperature. Then, discard the supernatant.Resuspend the pellet in two milliliters of warm medium. Load the cells in a hemocytometer and count them to determine the recovery and survival rates. Culture the thawed cells on basement membrane coated plates in a culture medium with Y27632.The retinal pigment epithelial cells that were frozen on day five in their exponential phase displayed a higher attachment rate after thawing. Relative to their characteristic hexagonal morphology, the cells frozen at other time points had a fibroblastic phenotype. P2 day-five cells displayed higher expression and more properly localized cell markers 28 days after thawing.It was noted that different cryopreservation media performed equally well in achieving high cell viability and attachment post-thawing.