心から感謝博士ベン ファウラー TEM 実験で彼の技術的な助けを拡張したいと思います。

このプロトコル オクラホマ州健康科学センター大学/機関審査委員会 (IRB #4509) のガイドラインに従います。プロトコルのすべての部分は、ヘルシンキ宣言の原則を会った。国立開発研究所 (NDRI) とオクラホマ州のライオンズ ・ アイバンクから角膜のサンプルを得た。
1. 初代培養細胞の分離
<ol><li>人間の角膜組織サンプルの受領、21.5 cm2シャーレ ダルベッコ滅菌リン酸緩衝生理食塩水 (DPBS) 2 mL を含む組織に転送 (1 x)。</li>
	<li>こすり、角膜ドームの顔アップを配置することで角膜の上皮を取り外します。完全な除去を確実に、45 ° の角度でかみそりの刃を使用して徹底的に上皮層をこすり落とします。次に、角膜ドーム下向きに反転し、徹底的に完全な除去を確実に 45 ° の角度でかみそりの刃と間質から血管内皮細胞をこすり落とします。DPBS と間質組織を洗浄します。</li>
	<li>間質組織を DPBS を使用してすべての時間で濡れている間質の部分を維持 10 番外科ブレードを使用して小さな断片にカットします。メディア (フラスコあたり 2 × 2 mm の組織の 4 または 5 の部分) を使用しないコート t25 フラスコ培養用フラスコに滅菌鉗子と場所を間質部分をピックアップします。</li>
	<li>約 30-40 分の 37 ° C でフラスコの底に付着する外植体を許可します。イーグル最小必須培地 (実装された EMEM) 10% 牛胎児血清 (FBS) および 1% を含む追加ゆっくりと付着、一度抗生物質/Antimycotic 外植片を妨げないでフラスコに。</li>
	<li>37 ° c、5% CO2インキュベーターでフラスコをそっと置きます。セルを分離する開始されるまで外植片を行わないでとフラスコ内で移行します。約 2-4 週間後電池が 100% の合流点に達する必要があります。100% 合流時に培養メディアを削除することによって、セルを通過し、DPBS とセルを洗ってください。</li>
	<li>セルにトリプシンを追加し、37 ° C でそれらを孵化させなさい約 5 分後に彼らが切り離されることを確認を光学顕微鏡下で細胞を表示し、新鮮なメディアを追加することによって、タンパク質の分解を停止します。</li>
	<li>1,000 x g. での遠心分離は、ペレットを乱すことがなく上澄みを慎重に削除の 15 mL コニカル チューブに細胞懸濁液を転送し、中断の新鮮な実装された EMEM のセル 10% FBS 1% 抗生物質。セルをカウントし、種子の新鮮な実装された EMEM のさらなる拡大のためコート t75 フラスコ培養用フラスコに約 1 × 106セル 1% FBS 1% 抗生物質。</li>
</ol>2. プライマリ HCFs 文化と 3 D 構造アセンブリ
<ol><li>3 D をアセンブルするから構造外植片の栽培実装された EMEM 10% FBS 1% 抗生物質/Antimycotic、通過カウント HCFs。</li>
	<li>種子の約 1 × 106細胞/ウェルと共に新鮮な EMEM の 1.5 mL の 3 D 構造から 0.4 μ m の細孔を有するポリカーボネート膜挿入の主な HCFs の 10% FBS 1% 抗生物質/Antimycotic コンス トラクターの先頭に。同じメディア別 1.5 mL を各構成要素の下に追加します。</li>
	<li>細胞播種の 24 h 後に実装された EMEM 細胞の培養 10% FBS 1% 上追加 1.5 mL と 1.5 mL の各構成要素の下に 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic 酸 (ビタミン C) で刺激した抗生物質/Antimycotic。</li>
	<li>37 ° c、3 週間 5% CO2インキュベーターの文化を維持し、全体の研究期間中に毎日新鮮なメディアを供給します。さらに通路は不要です。</li>
</ol>3. 細胞分化と共培養
<ol><li>8 x 103細胞/ウェル以前組み立てられた 3 D の上に SH SY5Y ひと神経芽腫細胞の 10% を含む実装された EMEM の含む 1.5 mL をコンス トラクターに追加 3 週間後 FBS、1% 抗生物質/Antimycotic、および 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic酸 (ビタミン C) です。<ol>
<li>SH SY5Y 神経芽細胞腫細胞分化を開始する前に、少なくとも 24 時間、角膜細胞の上に成長するを許可します。</li>
	</ol>
</li>
	<li>1 %fbs と 10 μ M の 1.5 mL を含む実装された EMEM の細胞を刺激することによって SH SY5Y 細胞分化の開始構造上にレチノイン酸。構成要素の下に実装された EMEM 10% FBS 1% 抗生物質/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic 酸 (ビタミン C) の 1.5 mL を追加します。暗闇の中でレチノイン酸のステップが実行されることに注意してください。<ol>
<li>5 日間この分化メディアで細胞の培養を続けます。</li>
	</ol>
</li>
	<li>細胞が分化段階に達したら、1.5 mL の 2 を含む無血清培にセルを切り替える脳由来神経栄養因子 (BDNF) と 1% の nM 抗生物質/Antimycotic は、コンス トラクターの先頭に追加することによって、実装された EMEM に追加。構成要素の下に実装された EMEM 10% FBS 1% 抗生物質/Antimycotic 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic 酸 (ビタミン C) の 1.5 mL を追加します。暗闇の中で BDNF ステップが実行されることに注意してください。</li>
	<li>表 1に示されるようにそれ以上の分析の処理の前に追加 2 日間培養します。</li>
</ol>

<ol>
	<li>Pascolini, D., Mariotti, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+estimates+of+visual+impairment:+2010.">Global estimates of visual impairment: 2010.</a> Br J Ophthalmol. 96 (5), 614-618 (2012).</li><li>R&#246;nkk&#246;, S., Vellonen, K. -. S., J&#228;rvinen, K., Toropainen, E., 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=Human+corneal+cell+culture+models+for+drug+toxicity+studies.">Human corneal cell culture models for drug toxicity studies.</a> Drug Delivery and Translational Research. 6 (6), 660-675 (2016).</li><li>Ghezzi, C. E., Rnjak-Kovacina, J., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+tissue+engineering:+recent+advances+and+future+perspectives.">Corneal tissue engineering: recent advances and future perspectives.</a> Tissue Eng Part B Rev. 21 (3), 278-287 (2015).</li><li>Shafaie, S., Hutter, V., Cook, M. T., Brown, M. B., Chau, D. Y. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Cell+Models+for+Ophthalmic+Drug+Development+Applications.">In Vitro Cell Models for Ophthalmic Drug Development Applications.</a> BioResearch Open Access. 5 (1), 94-108 (2016).</li><li>Shaheen, B. S., Bakir, M., Jain, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Corneal+nerves+in+health+and+disease.">Corneal nerves in health and disease.</a> Surv Ophthalmol. 59 (3), 263-285 (2014).</li><li>Beuerman, R. W., Schimmelpfennig, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensory+denervation+of+the+rabbit+cornea+affects+epithelial+properties.">Sensory denervation of the rabbit cornea affects epithelial properties.</a> Exp Neurol. 69 (1), 196-201 (1980).</li><li>Heigle, T. J., Pflugfelder, 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=Aqueous+tear+production+in+patients+with+neurotrophic+keratitis.">Aqueous tear production in patients with neurotrophic keratitis.</a> Cornea. 15 (2), 135-138 (1996).</li><li>Wang, 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=In+vitro+3D+corneal+tissue+model+with+epithelium,+stroma,+and+innervation.">In vitro 3D corneal tissue model with epithelium, stroma, and innervation.</a> Biomaterials. 112, 1-9 (2017).</li><li>Ross, R. A., Spengler, B. A., Biedler, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinate+morphological+and+biochemical+interconversion+of+human+neuroblastoma+cells.">Coordinate morphological and biochemical interconversion of human neuroblastoma cells.</a> J Natl Cancer Inst. 71 (4), 741-747 (1983).</li><li>Griffith, L. G., Naughton, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+engineering+-+Current+challenges+and+expanding+opportunities.">Tissue engineering - Current challenges and expanding opportunities.</a> Science. 295 (5557), (2002).</li><li>Guo, X., 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=Morphologic+characterization+of+organized+extracellular+matrix+deposition+by+ascorbic+acid-stimulated+human+corneal+fibroblasts.">Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts.</a> Invest Ophthalmol Vis Sci. 48 (9), 4050-4060 (2007).</li><li>Karamichos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+tissue+engineering:+current+and+future+directions.">Ocular tissue engineering: current and future directions.</a> J Funct Biomater. 6 (1), 77-80 (2015).</li><li>Karamichos, D., Brown, R. A., Mudera, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+stiffness+regulates+cellular+contraction+and+matrix+remodeling+gene+expression.">Collagen stiffness regulates cellular contraction and matrix remodeling gene expression.</a> J Biomed Mater Res A. 83 (3), 887-894 (2007).</li><li>Ruberti, J. W., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prelude+to+corneal+tissue+engineering+-+gaining+control+of+collagen+organization.">Prelude to corneal tissue engineering - gaining control of collagen organization.</a> Prog Retin Eye Res. 27 (5), 549-577 (2008).</li><li>Karamichos, D., Guo, X. Q., Hutcheon, A. E., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+corneal+fibrosis:+an+in+vitro+model.">Human corneal fibrosis: an in vitro model.</a> Invest Ophthalmol Vis Sci. 51 (3), 1382-1388 (2010).</li><li>Chen, F. M., Liu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advancing+biomaterials+of+human+origin+for+tissue+engineering.">Advancing biomaterials of human origin for tissue engineering.</a> Prog Polym Sci. 53, 86-168 (2016).</li><li>Priyadarsini, S., Sarker-Nag, A., Rowsey, T. G., Ma, J. X., Karamichos, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+3D+In+Vitro+Model+to+Accelerate+the+Development+of+Human+Therapies+against+Corneal+Diabetes.">Establishment of a 3D In Vitro Model to Accelerate the Development of Human Therapies against Corneal Diabetes.</a> PLoS One. 11 (12), e0168845 (2016).</li><li>Karamichos, D., Hjortdal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratoconus:+tissue+engineering+and+biomaterials.">Keratoconus: tissue engineering and biomaterials.</a> J Funct Biomater. 5 (3), 111-134 (2014).</li><li>Wilson, S. L., Yang, Y., El Haj, 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=Corneal+Stromal+Cell+Plasticity:+In+Vitro+Regulation+of+Cell+Phenotype+Through+Cell-Cell+Interactions+in+a+Three-Dimensional+Model.">Corneal Stromal Cell Plasticity: In Vitro Regulation of Cell Phenotype Through Cell-Cell Interactions in a Three-Dimensional Model.</a> Tissue Engineering Part A. 20 (1-2), 225-238 (2014).</li><li>Proulx, 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=Reconstruction+of+a+human+cornea+by+the+self-assembly+approach+of+tissue+engineering+using+the+three+native+cell+types.">Reconstruction of a human cornea by the self-assembly approach of tissue engineering using the three native cell types.</a> Molecular Vision. 16 (234-236), 2192-2201 (2010).</li><li>Gonzalez-Andrades, 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=Establishment+of+a+novel+in+vitro+model+of+stratified+epithelial+wound+healing+with+barrier+function.">Establishment of a novel in vitro model of stratified epithelial wound healing with barrier function.</a> Sci Rep. 6, 19395 (2016).</li><li>Hopkins, A. M., DeSimone, E., Chwalek, K., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+in+vitro+modeling+of+the+central+nervous+system.">3D in vitro modeling of the central nervous system.</a> Prog Neurobiol. 125, 1-25 (2015).</li><li>Schulz, 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=Natural+Corneal+Cell-Based+Microenvironment+as+Prerequisite+for+Balanced+3D+Corneal+Epithelial+Morphogenesis:+A+Promising+Animal+Experiment-Abandoning+Tool+in+Ophthalmology.">Natural Corneal Cell-Based Microenvironment as Prerequisite for Balanced 3D Corneal Epithelial Morphogenesis: A Promising Animal Experiment-Abandoning Tool in Ophthalmology.</a> Tissue Engineering Part C-Methods. 20 (4), 297-307 (2014).</li><li>Gao, J., Wang, Y., Zhao, X., Chen, P., Xie, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-204-5p-Mediated+Regulation+of+SIRT1+Contributes+to+the+Delay+of+Epithelial+Cell+Cycle+Traversal+in+Diabetic+Corneas.">MicroRNA-204-5p-Mediated Regulation of SIRT1 Contributes to the Delay of Epithelial Cell Cycle Traversal in Diabetic Corneas.</a> Invest Ophthalmol Vis Sci. 56 (3), 1493-1504 (2015).</li><li>Koulikovska, 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=Enhanced+regeneration+of+corneal+tissue+via+a+bioengineered+collagen+construct+implanted+by+a+nondisruptive+surgical+technique.">Enhanced regeneration of corneal tissue via a bioengineered collagen construct implanted by a nondisruptive surgical technique.</a> Tissue Eng Part A. 21 (5-6), 1116-1130 (2015).</li><li>Ljubimov, A. V., Saghizadeh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+in+corneal+wound+healing.">Progress in corneal wound healing.</a> Progress in retinal and eye research. 49, 17-45 (2015).</li><li>Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+matrix+and+wound+healing.">Extracellular matrix and wound healing.</a> Curr Opin Ophthalmol. 12 (4), 237-241 (2001).</li><li>Karamichos, D., Hutcheon, A. E., Zieske, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor-beta3+regulates+assembly+of+a+non-fibrotic+matrix+in+a+3D+corneal+model.">Transforming growth factor-beta3 regulates assembly of a non-fibrotic matrix in a 3D corneal model.</a> J Tissue Eng Regen Med. 5 (8), e228-e238 (2011).</li><li>Karamichos, D., Lakshman, N., Petroll, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+experimental+model+for+assessing+fibroblast+migration+in+3-D+collagen+matrices.">An experimental model for assessing fibroblast migration in 3-D collagen matrices.</a> Cell Motil Cytoskeleton. 66 (1), 1-9 (2009).</li><li>Karamichos, 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=Novel+in+Vitro+Model+for+Keratoconus+Disease.">Novel in Vitro Model for Keratoconus Disease.</a> J Funct Biomater. 3 (4), 760-775 (2012).</li></ol>

著者は競合する金銭的な利益を宣言しません。

いくつかの研究ができる角膜疾患のよりよい理解の開発を助けると同様の治療法を発見する様々 な動物モデルの開発で注目されています。しかし、これらの研究から人間に重要な価値は確認されていません。日には、様々 な生体外モデルを開発し、広くその顕著な臨床的意義のため調査が。私たち以前に確立された 3 Dの in vitroモデルです新規のシステムで大幅ビジョン研究の?...

人間の体内では、角膜は、難解な神経組織です。神経は接触、苦痛、温度のような様々 な感覚を担当し、また創傷治癒、反射を点滅、涙の生産と分泌5,6,7で重要な役割を持っています。角膜間質の神経幹は輪部の神経叢から発生し、放射状周辺の角膜実質を入力します。間質の神経組織はコラーゲン ラメラに平行、彼らはさらに小さい線維束彼らは実質5,8に向かって進むように分岐します。神経線維はさらに上皮層に浸透し、したがって、神経支配は広く広がって角膜上皮と実質の間で。したがって、神経支配は、角膜の健康と病気の状態の重要な役割を持ちます。このプロトコルでは、体内の神経間質の相互作用を模倣する、その種の最初である新しい 3 Dの in vitroモデルの進歩を明らかにします。SH SY5Y 細胞株は、神経の成長を観察するために使用も特徴と、最も確立されたラインの 1 つであるこの研究に使われました。SH SY5Y 細胞ラインは両方の基板の付着 (S 型) を生成する記載されているし、neuroblastic (N 型) 細胞を分化転換9を受けることができます。結果として、この細胞ラインは N 型セルのトリプルの連続 subclone 選択から派生するにもかかわらずそれも含まれていますレチノイン酸と脳由来を使用してニューロンへの分化過程を受けることができる S 型セルの数が少ない神経栄養因子の9。これは角膜合併症糖尿病網膜症 (DM) やその他の眼の疾患に関連付けられているのより良い理解につながる可能性がありますツールを提供します。ために取得し、眼疾患の患者から神経細胞の培養に伴う困難、この 3 Dの in vitroモデルはニューロン間相互作用の勉強と角膜実質とシグナル伝達における実質的な意味を提供します。
病的状況しばしば妥協の生活の質につながる、非常に大規模で体のさまざまな組織に影響を与えます。眼ジストロフィーは、全身疾患と視力の損失や永久的な視力低下につながる多くの場合関連付けられている一般的な合併症です。包括的な研究は、基底の細胞レベルでの効果と同様に、病気の状態の理解を深めるために不可欠。このような病気の影響を研究するには、様々 な生体内と体外のモデルはティッシュ エンジニア リング アプリケーションの助けを借りて開発されています。Corneal ティッシュ エンジニア リング アプリケーションは、科学10、11,12,13,14では、様々 な分野で大きな関心を集めているが、時に重大な制限があります。実際のアプリケーションは、拒絶反応、感染症、瘢痕10,11,12,13,14を移植角膜を含みます。正常に開発され、様々 な生体外モデル3,15,16,17,18,を設置するいくつかの研究がある19,,2021,22,23,24,25,26。3 Dの in vitroモデルは、最も有望な偉大な科学的な興味の。3 D モデルより体内の細胞および生理学的イベント線維化に重要であり、創傷治癒15,27,28,29をミラーする知られています。これらの in vitroモデルは、角膜合併症を含む異なる病状を治療するための新しい治療上のアプローチを見つけることに不可欠な役割を果たします。角膜の機能に神経支配の重要な役割にもかかわらず角膜組織設計構造2,3で末梢神経の増殖を促進するために少しの努力をしました。ただし、目的の組織機能を達成するために標的組織を模倣する提案 3 D培養細胞構造。
糖尿病角膜症神経欠陥のためここで説明するモデルのための明白なアプリケーションですが、円錐角膜、Fuchs のジストロフィーを含む人間の in vitroモデルの恩恵を受けることができますいくつかの他の角膜疾患があります。当社の 3 D モデルは、この見通しから出現し、ドラッグデリバリー システムを評価するために角膜組織の体外表現と新しい眼薬の安全性の開発を提案します。

<table><tbody><tr><td>Healthy corneal tissue</td><td>NDRI</td><td></td><td>Samples from donors with no ocular trauma or systemic disease</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Solution (1X)</td><td>Gibco by Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>Sterile forceps</td><td>Fischer Scientific</td><td>13-812-42</td><td>Fisherbrand Dissecting Extra-Fine-Pointed Splinter Forceps</td></tr><tr><td>Single edge razor blades</td><td>Personna</td><td>270100</td><td></td></tr><tr><td>Sterile surgical scalpel blades No.10</td><td>Feather Surgical Blade</td><td>2976#10</td><td></td></tr><tr><td>Eagle&amp;rsquo;s Minimum Essential Medium</td><td>ATCC</td><td>30-2003</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Atlanta Biologicals</td><td>S11550</td><td>10% FBS is required for media preparation</td></tr><tr><td>Antibiotic-Antimycotic (100X)</td><td>Gibco by Life Technologies</td><td>15240-062</td><td>1% Antibiotic-Antimycotic is required for media preparation</td></tr><tr><td>0.05% Trypsin EDTA(1X)</td><td>Gibco by Life Technologies</td><td>25300062</td><td></td></tr><tr><td>Polycarbonate membrane inserts with 0.4-&amp;mu;m pores</td><td>Corning Costar</td><td>3412</td><td></td></tr><tr><td>2-O-&amp;alpha;-Dglucopyranosyl-L-ascorbic acid (Vitamin C)</td><td>Sigma-Aldrich</td><td>SMB00390-14</td><td>A concentration of 0.5 mM should be used for the study</td></tr><tr><td>Wax block</td><td>VWR</td><td>50-949-027</td><td></td></tr><tr><td>SH-SY5Y Neuroblastoma cells</td><td>ATCC</td><td>SHSY5YATCC CRL-2266</td><td></td></tr><tr><td>Retinoic Acid</td><td>Sigma-Aldrich</td><td>SRP3014-10UG</td><td>Final concentration of 10uM needs to be used</td></tr><tr><td>BDNF</td><td>Sigma-Aldrich</td><td>R2625-100MG</td><td>Final concentration of 2nM needs to be used</td></tr><tr><td>Dimethyl Sulfoxide(DMSO)</td><td>VWR-Alfa Aesar</td><td>67-68-5</td><td>Ultra Pure Grade-Sterile DMSO to be used</td></tr><tr><td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T25)</td><td>Fisher Scientific</td><td>12-565-351</td><td></td></tr><tr><td>Thermo Scientific Nunc Cell Culture Treated EasYFlasks (T75)</td><td>Fisher Scientific</td><td>12-565-349</td><td></td></tr></tbody></table>

corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea

ティッシュ ・ エンジニア リングは、世界角膜視力損失1に苦しんで推定 1000 万人ひと角膜交換のための需要が高いため実質的な認識を得ています。実行可能な人間の角膜、三次元 (3 D) 組織で重要な進歩のための要求に対処するため工学2,3、4を行われています。これら角膜モデル範囲簡単な単分子膜システムから多層モデルでは、3 D の完全厚さ角膜同等2に 。
ただし、3 D ティッシュ エンジニア リングの角膜疾患モデルの in vitroのコンテキストでの使用を検討多層 3 D 角膜組織構造、機能、および異なる種類の細胞 (すなわち、神経ネットワークに欠けている類似している日付上皮、実質、内皮細胞) の2,3。さらに、医薬品のための動物テストを削減する試みで角膜の in vitro組織モデルの需要が増加しています。したがってより洗練されたモデルは人間の生理的要求するシステムに合わせて必要なより患者の人口に関連するモデルの開発が絶対に必要です。主な角膜線維芽細胞 (HCFs) 健康なドナーからから神経細胞の三次元共培養モデルを搭載、角膜内の複数セル型ジストロフィー、フックス、糖尿病角膜症、円錐角膜などの病気やを受けますことを考えるSH SY5Y セルの行。これにより、初めて人間の角膜組織内の 2 つのセル型間の相互作用を調査します。このモデルが神経損傷を示す角膜疾患の神経間質相互作用に関連付けられている基になるメカニズムを解剖して可能性があると考えています。この 3 D モデルは生体内で角膜組織の基本的な解剖学的および生理学的な性質を反映し、角膜欠損を調査だけでなく、様々 なエージェントの有効性を動物実験の前にスクリーニング ツールとして将来的に使用できます。

図 1は、モデル 3 D培養作業の順を追って代表イメージです。最初のステップでは、細胞は人間の角膜から分離されます。その後、ポリカーボネート膜上に成長した、自己分泌の 3 D マトリックスを組み立てるためビタミン C と刺激します。この 3 D 構築システム、多層細胞の生体内での合成を誘導する-間質マトリックスのような。?...

Watch this Scientific Journal Video about Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea at JoVE.com

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

このプロトコルでは、新規三次元の in vitroモデル、角膜間質細胞と分化した神経細胞が一緒に培養試験と 2 つの細胞のタイプの相互作用の理解を支援するについて説明します。

角膜組織工学: In Vitroのモデル人間の角膜の神経間質相互作用

Tissue engineering has gained substantial recognition due to the high demand for human cornea replacements with an estimated 10 million people worldwide ...

corneal-tissue-engineering-an-vitro-model-stromal-nerve-interactions

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

8.8K ビュー.  University of Oklahoma Health Sciences Center. このプロトコルでは、新規三次元の in vitroモデル、角膜間質細胞と分化した神経細胞が一緒に培養試験と 2 つの細胞のタイプの相互作用の理解を支援するについて説明します。

ビデオ: Corneal Tissue Engineering: An In Vitro Model of the Stromal-nerve Interactions of the Human Cornea

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema and diminished visual acuity. However, human CECs are prone to compromised proliferative potential. Furthermore, stimulation of cell growth is often complicated by gradual endothelial-mesenchymal transition (EnMT). Therefore, understanding the mechanism of EnMT is necessary for facilitating the regeneration of CECs with competent function. In this study, we prepared a primary culture of bovine CECs by peeling the CECs with Descemet's membrane from the corneal button and demonstrated that bovine CECs exhibited the EnMT process, including phenotypic change, nuclear translocation of &#946;-catenin, and EMT regulators snail and slug, in the in vitro culture. Furthermore, we used a rat corneal endothelium cryoinjury model to demonstrate the EnMT process in vivo. Collectively, the in vitro primary culture of bovine CECs and in vivo rat corneal endothelium cryoinjury models offers useful platforms for investigating the mechanism of EnMT.

In Vitro and In Vivo Models to Study Corneal Endothelial-mesenchymal Transition

A primary culture of bovine corneal endothelial cells was used to investigate the mechanism of corneal endothelial-mesenchymal transition. Furthermore, a rat corneal endothelium cryoinjury model was used to demonstrate corneal endothelial-mesenchymal transition in vivo.

in vitroおよびin vivoモデルで角膜内皮間葉転換を研究します

Corneal endothelial cells (CECs) play a crucial role in maintaining corneal clarity through active pumping. A reduced CEC count may lead to corneal edema ...

発生生物学

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as Aniridia or Steven Johnson Syndrome as well as by external factors such as chemical burns or radiation. Current treatments are (i) the use of corneal grafts and (ii) the use of stem cell expanded in the laboratory and delivered on carriers (e.g., amniotic membrane); these treatments are relatively successful but unfortunately they can fail after 3-5 years. There is a need to design and manufacture new corneal biomaterial devices able to mimic in detail the physiological environment where stem cells reside in the cornea. Limbal stem cells are located in the limbus (circular area between cornea and sclera) in specific niches known as the Palisades of Vogt. In this work we have developed a new platform technology which combines two cutting-edge manufacturing techniques (microstereolithography and electrospinning) for the fabrication of corneal membranes that mimic to a certain extent the limbus. Our membranes contain artificial micropockets which aim to provide cells with protection as the Palisades of Vogt do in the eye.

We report a technique for the fabrication of micropockets within electrospun membranes in which to study cell behavior. Specifically, we describe a combination of microstereolithography and electrospinning for the production of PLGA (Poly(lactide-co-glycolide)) corneal biomaterial devices equipped with microfeatures.

角膜再生のためのニッチを装備膜を製造するためのマイクロ光とエレクトロスピニングの組み合わせ

Corneal problems affect millions of people worldwide reducing their quality of life significantly. Corneal disease can be caused by illnesses such as ...

生物工学

Efficient and Scalable Directed Differentiation of Clinically Compatible Corneal Limbal Epithelial Stem Cells from Human Pluripotent Stem Cells

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis and visual clarity. Human pluripotent stem cell (hPSC)-derived LESCs provide a promising cell source for corneal cell replacement therapy. Undefined, xenogeneic culture and differentiation conditions cause variation in research results and impede the clinical translation of hPSC-derived therapeutics. This protocol provides a reproducible and efficient method for hPSC-LESC differentiation under xeno- and feeder cell-free conditions. Firstly, monolayer culture of undifferentiated hPSC on recombinant laminin-521 (LN-521) and defined hPSC medium serves as a foundation for robust production of high-quality starting material for differentiations. Secondly, a rapid and simple hPSC-LESC differentiation method yields LESC populations in only 24 days. This method includes a four-day surface ectodermal induction in suspension with small molecules, followed by adherent culture phase on LN-521/collagen IV combination matrix in defined corneal epithelial differentiation medium. Cryostoring and extended differentiation further purifies the cell population and enables banking of the cells in large quantities for cell therapy products. The resulting high-quality hPSC-LESCs provide a potential novel treatment strategy for corneal surface reconstruction to treat limbal stem cell deficiency (LSCD).

This protocol introduces a simple two-step method for differentiating corneal limbal epithelial stem cells from human pluripotent stem cells under xeno- and feeder cell-free culture conditions. The cell culture methods presented here enable cost-efficient, large-scale production of clinical quality cells applicable to corneal cell therapy use.

効率的かつスケーラブルな臨床的に互換性のある角膜輪部上皮幹細胞ひと多能性幹細胞からの分化

Corneal limbal epithelial stem cells (LESCs) are responsible for continuously renewing the corneal epithelium, and thus maintaining corneal homeostasis ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

生体物質膜上におけるヒトおよび羊角膜内皮細胞層の増殖

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

Ex Vivo Corneal Organ Culture Model for Wound Healing Studies

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two dimensional (2D) and three dimensional (3D) culture has generated a wealth of information not only about corneal biology but also about wound healing, myofibroblast biology, and scarring in general. The goal of the protocol is an assay system for quantifying myofibroblast development, which characterizes scarring. We demonstrate a corneal organ culture ex vivo model using pig eyes. In this anterior keratectomy wound, corneas still in the globe are wounded with a circular blade called a trephine. A plug of approximately 1/3 of the anterior cornea is removed including the epithelium, the basement membrane, and the anterior part of the stroma. After wounding, corneas are cut from the globe, mounted on a collagen/agar base, and cultured for two weeks in supplemented-serum free medium with stabilized vitamin C to augment cell proliferation and extracellular matrix secretion by resident fibroblasts. Activation of myofibroblasts in the anterior stroma is evident in the healed cornea. This model can be used to assay wound closure, the development of myofibroblasts and fibrotic markers, and for toxicology studies. In addition, the effects of small molecule inhibitors as well as lipid-mediated siRNA transfection for gene knockdown can be tested in this system.

A protocol for an ex vivo corneal organ culture model useful for wound healing studies is described. This model system can be used to assess the effects of agents to promote regenerative healing or drug toxicity in an organized 3D multicellular environment.

前のヴィヴォ角膜器官培養モデルの創傷治癒の研究

The cornea has been used extensively as a model system to study wound healing. The ability to generate and utilize primary mammalian cells in two ...

医学

A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for experimental investigations as they are normally needed for transplantation. Endothelial cell cultures often do not translate well to in vivo situations. Due to the biostructural characteristics of non-human corneas, stromal swelling during cultivation induces substantial corneal endothelial cell loss, which makes it difficult to perform cultivation for an extended period of time. Deswelling agents such as dextran are used to counteract this response. However, they also cause significant endothelial cell loss. Therefore, an ex vivo organ culture model not requiring deswelling agents was established. Pig eyes from a local slaughterhouse were used to prepare split corneal buttons. After partial corneal trephination, the outer layers of the cornea (epithelium, bowman layer, parts of the stroma) were removed. This significantly reduces corneal endothelial cell loss induced by massive stromal swelling and Descemet&#39;s membrane folding throughout longer cultivation periods and improves general preservation of the endothelial cell layer. Subsequent complete corneal trephination was followed by the removal of the split corneal button from the remaining eye bulb and cultivation. Endothelial cell density was assessed at follow-up times of up to 15 days after preparation (i.e., days 1, 8, 15) using light microscopy. The preparation technique used allows a better preservation of the endothelial cell layer enabled by less stromal tissue swelling, which results in slow and linear decline rates in split corneal buttons comparable to human donor corneas. As this standardized organo-typically cultivated research model for the first time allows a stable cultivation for at least two weeks, it is a valuable alternative to human donor corneas for future investigations of various external factors with regards to their effects on the corneal endothelium.

Here, a step-by-step protocol for the preparation and cultivation of porcine split corneal buttons is presented. As this organo-typically cultivated organ culture model shows cell death rates within 15 days, comparable to human donor corneas, it represents the first model allowing long-term cultivation of non-human corneas without adding toxic dextran.

分割角膜ボタンを用いてのブタ角膜内皮器培養モデル

Experimental research on corneal endothelial cells is associated with several difficulties. Human donor corneas are scarce and rarely available for ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮細胞損失の非侵襲的レーザー支援実験モデルの開発

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, and result in a large, persistent corneal epithelial defect. Epithelial defect with the following corneal opacity and peripheral neovascularization result in irreversible visual impairment and hinder future management, especially keratoplasty. Since the animal model can be used as an effective drug development platform, models of corneal injury to the mouse and alkali burn to rabbit corneal epithelium are developed here. New Zealand white rabbit is used in the alkali burn model. Different concentrations of sodium hydroxide can be applied onto the central circular area of the cornea for 30 s under intramuscular and topical anesthesia. After copious isotonic normal saline irrigation, residual loose corneal epithelium was removed with corneal burr deep down to the Bowman's layer within this circular area. Wound healing was documented by fluorescein staining under Cobalt blue light. C57BL/6 mice were used in the traumatic model of murine corneal epithelium. The murine central cornea was marked using a skin punch, 2 mm in diameter, and then debrided by a corneal rust ring remover with a 0.5 mm burr under a stereomicroscope. These models can be prospectively used to validate the therapeutic effect of eye drops or mixed agents such as stem cells, which potentially facilitate corneal epithelial regeneration. By observing corneal opacity, peripheral neovascularization, and conjunctival congestion with stereomicroscope and imaging software, therapeutic effects in these animal models can be monitored.

Ex Vivo and In Vivo Animal Models for Mechanical and Chemical Injuries of Corneal Epithelium

Here, animal models based on mouse and rabbit are developed for mechanical and chemical injury of corneal epithelium to screen new therapeutics and the underlying mechanism.

角膜上皮の機械的および化学的損傷のためのEx Vivoおよびin vivo動物モデル

Corneal injury to the ocular surface, including chemical burn and trauma, may cause severe scarring, symblepharon, corneal limbal stem cells deficiency, ...

A Human Corneal Organ Culture Model of Descemet's Stripping Only with Accelerated Healing Stimulated by Engineered Fibroblast Growth Factor 1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the whole cornea or Descemet's membrane. Recent developments in ocular surgery have established Descemet's Stripping Only (DSO), a surgical technique in which a central circle of guttae-dense Descemet's membrane is removed to allow for the migration of CECs onto the smooth stroma, restoring function and vision to the cornea. While this potential treatment option is of high interest in the field of ophthalmic research, no successful ex vivo models of DSO have been established and clinical data is limited. This work presents a novel wound-healing model simulating DSO in human donor corneas. Using this approach to evaluate the efficacy of the human engineered FGF1 (NM141), we found that treatment accelerated healing via stimulation of migration and proliferation of CECs. This finding was confirmed in 11 pairs of human corneas with signs of dystrophy reported by the eye banks in order to verify that these results can be replicated in patients with Fuchs' Dystrophy, as the target population of the DSO procedure.

Descemet's Stripping Only is an experimental procedure wherein patients with central corneal guttae resulting from Fuchs Endothelial Corneal Dystrophy have Descemet's membrane stripped for peripheral cells to regenerate the endothelial layer. We present novel methodology simulating DSO in dystrophic human corneas ex vivo with accelerated healing stimulated by eFGF1 (NM141).

操作された線維芽細胞増殖因子によって刺激された加速治癒を伴うデスメのストリッピングのみのヒト角膜器官培養モデル1

Fuchs Endothelial Corneal Dystrophy (FECD) results from dysfunctional corneal endothelial cells (CECs) and is currently treated by transplantation of the ...

軸索再生研究のための角膜感覚線維損傷プロトコル

Three Strategies to Induce Neurotrophic Keratitis and Nerve Regeneration in Murine Cornea

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation provides sensation and trophic function to the eye and contributes to preserving corneal integrity. The pathological disruption of this innervation is termed neurotrophic keratitis. This can be triggered by injury to the eye, surgery, or disease. In this study, we propose three different protocols for inflicting damage on the innervation in ways that recapitulate the three types of cases generally encountered in the clinic.
The first method consists in making an abrasion of the epithelium with an ophthalmic burr. This involves the removal of the epithelial layer, the free nerve endings, and the subbasal plexus in a manner similar to the photorefractive keratectomy surgery performed in the clinic. The second method&#160;only targets the innervation by sectioning it at the periphery with a biopsy punch, maintaining the integrity of the epithelium. This method is similar to the first steps of lamellar keratoplasty and leads to a degeneration of the innervation followed by regrowth of the axons in the central cornea. The last method damages the innervation of a transgenic mouse model using a multiphoton microscope, which specifically localizes the site of cauterization of the fluorescent nerve fibers. This method inflicts the same damage as photokeratitis, an overexposure to UV light.
This study describes different options for investigating the physiopathology of corneal innervation, particularly the degeneration and regeneration of the axons. Promoting regeneration is crucial for avoiding such complications as epithelium defects or even perforation of the cornea. The proposed models can help test new pharmacological molecules or gene therapy that enhance nerve regeneration and limit disease progression.

著者スポットライト:革新的なモデルによる角膜神経支配研究の進歩 

Here, we propose three different methods of damaging the sensory fibres innervating the cornea. These methods facilitate the study of axon regeneration in mice. These three methods, which are adaptable to other animal models, are ideal for the study of corneal innervation physiology and regeneration.

マウス角膜の神経栄養性角膜炎と神経再生を誘導する3つの戦略

The cornea is a transparent tissue that covers the eye and is crucial for clear vision. It is the most innervated tissue in the body. This innervation ...