Name: corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea
Uploaded: 2018-01-24T13:30:00.000+00:00
Duration: 7 min 35 s
Description: 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

Dr. Ben Fowler sayesinde bizim samimi-TEM deneyleri ile onun teknik yardım için genişletmek istiyorum.

Bu iletişim kuralı Üniversitesi Oklahoma Sağlık Bilimleri Merkezi/Kurumsal inceleme Kurulu (IRB #4509) kuralları izler. İletişim kuralının tüm parçaları Helsinki Bildirgesi ilkelerinin bir araya geldi. Kornea örnekleri ulusal kalkınma ve Araştırma Enstitüsü (NDRI) ve Oklahoma Lions göz Bankası elde edilmiştir.
1. Primer hücre izolasyon
<ol><li>İnsan kornea doku örneği alınması üzerine, dokularda bir 21,5 cm2 Petri dish 2 mL steril Dulbecco'nın fosfat tamponlu tuz (DPBS) içeren naklet (1 x).</li>
	<li>Scrape ve kornea epitel kornea kubbe yukarı doğru konumlandırma tarafından kaldırın. Tümüyle kaldırılmasını sağlamak için iyice bir 45 ° açıyla bir jilet kullanarak epitel tabaka kapalı kazımak. Ardından, kornea kubbe aşağı doğru çevirmek ve iyice endotel stroma tümüyle kaldırılmasını sağlamak için bir 45 ° açıyla bir tıraş bıçağı ile gelen kapalı kazımak. Stromal doku DPBS ile yıkayın.</li>
	<li>Stromal dokuları at DPBS kullanarak sürekli ıslak stromal parçaları tutmak bir 10 numara cerrahi bıçak kullanarak küçük parçalar halinde kesin. Stromal adet steril forseps ve yer ile bir T25 kültür şişesi ezelî kitle iletişim araçları (4 veya 5 adet şişe başına 2 x 2 mm dokusunun) içine al.</li>
	<li>Explants şişesi yaklaşık 30-40 dk 37 ° C'de altına bağlı kalmak izin verir. Bir kez yapıştırılır, yavaş yavaş % 10 fetal sığır serum (FBS) ve % 1'i içeren kartal'ın en az gerekli orta (EMEM) eklemek antibiyotik/Antimycotic explants bozmadan şişesi için.</li>
	<li>Yavaşça kuluçka 37 ° c, % 5 CO2şişeye koyun. Hücreleri izole başlayana explants rahatsız bekletin ve şişeye geçiş yapma. Yaklaşık 2-4 hafta sonra hücrelerin % 100 izdiham ulaştırılması gerekmektedir. % 100 izdiham, üzerine hücreleri doku kültürü medya kaldırarak geçiş ve DPBS hücrelerle yıkayın.</li>
	<li>Tripsin hücrelere ekleme ve sonra onları 37 ° C'de kuluçkaya Yaklaşık 5 dk sonra onlar ayırdıktan onaylamak için ışık mikroskobu altında hücreler görüntüleyin ve sonra taze medya ekleyerek proteolizis durdurmak.</li>
	<li>Santrifüjü 1000 x g. adlı için çıkarmak süpernatant dikkatle Pelet bozmadan hücre süspansiyon 15 mL konik tüp içine aktarmak ve taze EMEM hücrelerde % 10 askıya FBS % 1 antibiyotik. Hücreleri saymak ve T75 kültür şişesi için daha fazla genişleme ile taze EMEM hücrelerde yaklaşık 1 x 106 %1 tohum FBS % 1 antibiyotik.</li>
</ol>2. birincil HCFs kültür ve 3D birleştirme oluşturur
<ol><li>3D bir araya gelen yapıları explants daha önce yetişen EMEM % 10 FBS %1 antibiyotik/Antimycotic, geçit ve sayısı HCFs.</li>
	<li>Yaklaşık 1 x 106 hücreler/kuyu polikarbonat membran ekler ile birlikte 1.5 mL taze EMEM 3D yapılar üzerinden 0,4-µm gözenekleri üzerinde birincil HCFs % 10 tohum FBS % 1 antibiyotik/Antimycotic yapı dön. Aynı medya başka bir 1,5 mL her yapı altýna ekleyin.</li>
	<li>Hücre tohum 24 saat sonra % 10 EMEM hücrelerle kültür FBS % 1 antibiyotik/Antimycotic, ekleyerek 1,5 mL üstünde belgili tanımlık tepe ve her yapı altındaki 1,5 mL 0.5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini) ile uyarılmış.</li>
	<li>37 ° c, % 5 CO2 3 hafta için bir kuluçka kültürlerde korumak ve taze medya tüm çalışma süresi boyunca her gün verin. Daha fazla geçiş yok gereklidir.</li>
</ol>3. hücre farklılaşma ve ortak kültürler
<ol><li>3 hafta sonra 8 x 103 hücreleri/iyi SH-SY5Y insan Nöroblastom hücre önceden birleştirilmiş 3D üst yapıları içeren 1,5 mL % 10 içeren EMEM eklemek FBS, % 1 antibiyotik/Antimycotic ve 0,5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini).<ol>
<li>SH-SY5Y Nöroblastom hücreleri hücre farklılaşması işlemini başlatmadan önce en az 24 saat için kornea stromal hücreler üzerine büyümeye izin.</li>
	</ol>
</li>
	<li>SH-SY5Y hücre farklılaşması ile EMEM %1 FBS ve 10 µM 1,5 mL içeren hücreleri uyararak başlatmak retinoik asit üzerine inşa. EMEM % 10 FBS % 1 antibiyotik/Antimycotic 0,5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini) 1,5 mL yapı altýna ekleyin. Retinoik asit adım karanlıkta içermesi gerektiğini unutmayın.<ol>
<li>Bu farklılaşma medya hücrelerde 5 gün boyunca kültür devam ediyor.</li>
	</ol>
</li>
	<li>Hücrelere farklılaşma aşama ulaşmak, hücreleri için 1,5 mL serum-Ücretsiz medya içeren 2 geçiş beyin kaynaklı Nörotrofik faktör (BDNF) ve % 1 nM antibiyotik/Antimycotic eklendi EMEM için inşa en üstüne ekleyerek. EMEM % 10 FBS % 1 antibiyotik/Antimycotic 0,5 mM 2-O-α-D-glucopyranosyl-L-ascorbic asit (C vitamini) 1,5 mL yapı altýna ekleyin. BDNF adım karanlıkta içermesi gerektiğini unutmayın.</li>
	<li>Tablo 1' de gösterildiği gibi başka herhangi bir analiz için 2 ek gün önce işleme için hücreleri kültür.</li>
</ol>

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Yazarlar hiçbir rakip mali çıkarlarının bildirin.

Çeşitli çalışmalarda de kornea hastalıkları daha iyi bir anlayış geliştirmelerine yardımcı olarak tedavileri keşfetmek çeşitli hayvan modelleri geliştirilmesi üzerinde duruldu. Ancak, bu çalışmalar insanlar için önemli bir değer doğrulanmadı. Bugüne kadar çeşitli vitro modelleri geliştirilmiş ve onların olağanüstü klinik önemi nedeniyle yaygın olarak araştırıldı. Bizim daha önce kurulan 3D vitro modeli önemli ölçüde vizyon araştırma gelişmeler tıp bilimin...

İnsan vücudunda kornea en yoğun innervated dokudur. Sinirleri dokunma, ağrı, sıcaklık gibi çeşitli duyumlar sorumludur ve iyileşmesi, refleksleri yanıp, gözyaşı üretim ve salgı5,6,7önemli bir rol olarak da mevcuttur. Kornea, stromal sinir mayo limbal pleksus ortaya ve periferik Korneal stroma Radyal girin. Stromal sinir organizasyonu için kollajen lamellae paralel ve daha fazla içine doğru yüzeysel stroma5,8devam ederken daha küçük fascicles şube. Sinir lifleri daha da epitel tabakası nüfuz ve böylece, innervasyon yaygın olarak kornea epitel ve stroma üzerinden dışarı yayılır. Bu nedenle, innervasyon kornea sağlıklı ve hastalıklı durumda önemli bir rol vardır. Bu protokol için in vivo sinir stromal etkileşimleri taklit etmek için kendi türünde ilk roman 3D vitro manken ilerlemesi ortaya koyuyor. Nöronal büyüme çalışırdım en köklü, iyi karakterize satırlardan birini olduğu gibi SH-SY5Y hücre kültürünü bu çalışma için kullanıldı. SH-SY5Y hücre kültürünü her iki substrat yapışık (S-type) üretmek için tanımlanmıştır ve neuroblastic (N-tipi) hücreleri transdifferentiation9tabi olabilir. Sonuç olarak, bu hücre kültürünü N-tipi hücre Çift Kişilik birbirini izleyen subclone seçimden türetilmiştir olsa bile, ayrıca S-type hücrelerin nöronlar aracılığıyla retinoik asit ve beyin kaynaklı içine farklılaşma geçiren küçük bir dizi içerir Nörotrofik faktör9. Bu kornea komplikasyon diabetik retinopati (DM) ve diğer göz hastalıkları ile ilgili daha iyi bir anlayış neden olabilir bir araç sağlar. Alma ve oküler hastalığı olan hastalarda neurons kültür ile ilgili nedenlerden dolayı nöronal etkileşimleri eğitim ve Korneal stroma ile sinyal önemli etkileri bu 3D vitro modeli sağlar.
Hastalıklı koşulları kez güvenliği aşılan bir yaşam kalitesi için önde gelen bir çok büyük ölçekli, vücudun çeşitli dokuları etkiler. Oküler dystrophies ortak komplikasyonlar genellikle sistemik hastalıklar ve kurşun görme keskinliği kaybı veya hatta kalıcı görme kaybı ile ilişkili vardır. Kapsamlı çalışmalar çoğu kez daha iyi hastalık durumu gibi Bazal hücresel düzeyde etkilerini anlamak için gerekli. Gibi hastalıklar etkileri çalışmaya, doku mühendisliği uygulamaları yardımıyla çeşitli in vivo ve in vitro modelleri geliştirilmiştir. Kornea doku mühendisliği uygulamaları bilim10,11,12,13,14çeşitli alanlarda büyük ilgi topladı var ama sırasında hala önemli sınırlamaları vardır fiili uygulama, kornea dahil greft reddi, enfeksiyonlar ve skarlasma10,11,12,13,14. Başarılı bir şekilde geliştirilen ve çeşitli vitro modelleri3,15,16,17,18, kurulan birçok çalışma vardır 19,20,21,22,23,24,25,26. 3D vitro en umut verici ve büyük bilimsel ilgi modeller. 3D modelleri daha iyi fibrozis sırasında kritik olan ve şifa15,27,28,29yara vivo içinde hücresel ve fizyolojik olaylar ayna olduğu bilinmektedir. Bu vitro modelleri kornea komplikasyonlar da dahil olmak üzere farklı hastalık tedavi için yeni tedavi yaklaşımları bulma konusunda önemli bir rol oynamak. İnnervasyon kornea işlevlerinde önemli rol rağmen küçük bir çaba periferik sinirleri yayılması kornea dokusu mühendislik yapıları2,3içinde tanıtmak için yapılmıştır. Ancak, önerilen 3D vitro hücre yapıları istenen doku işlevselliği elde etmek için hedef doku taklit.
Diyabetik keratopathy nöronal kusurları nedeniyle burada açıklanan model için bariz bir uygulama olmakla birlikte, Keratokonus ve Fuchs dystrophies de dahil olmak üzere bir insan vitro modelinden yararlanabilir birçok kornea hastalıkları vardır. Bizim 3D modeli bu umudu ortaya çıkar ve vitro gösterilişinin ilaç dağıtım değerlendirmek için kornea dokusunun ve Emanet yeni oküler ilaçların geliştirilmesi öneriyor.

<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

Doku Mühendisliği ile yaklaşık 10 milyon kişi kornea vizyon kaybı1ile acı dünya çapında insan kornea replasmanları için yüksek talep nedeniyle önemli tanıma kazanmıştır. Uygun insan kornealar, üç boyutlu (3D) doku içinde önemli bir ilerleme için talep adrese Mühendisliği2,3,4yapılmıştır. Bu kornea modelleri arasında değişir basit monolayer sistemleri çok katmanlı modelleri, 3D tam-kalınlık kornea eşdeğerleri2' ye lider.
Ancak, çok katmanlı 3D kornea dokusu yapı, fonksiyon ve farklı hücre türleri (örneğin, sinir, ağ için tarihi yoksun benzerlik için bir 3D doku Mühendisliği kornea vitro hastalık modelleri bağlamında kullanımı eğitimi epitel, stroma ve endotel)2,3. Buna ek olarak, talep vitro kornea dokusu modelleri için bir girişim için eczacılık ürünleri hayvan test azaltmak için artmıştır. Böylece, daha gelişmiş modeller daha iyi sistemleri insan fizyolojik gereksinimleri için maç için gerekli olan ve daha çok hasta popülasyona ilgili bir model geliştirilmesi kesinlikle gereklidir. Birden fazla hücre türü kornea hastalıkları ve dystrophies, Keratokonus, diyabetik Keratopathy ve Fuchs, gibi etkilenir verilen bu birincil insan kornea fibroblastlar (HCFs) sağlıklı bağış ve neurons 3D ortak kültür modeli bu modeli içerir SH-SY5Y hücre satırı. Bu bizim için ilk kez insan kornea dokusu içinde iki hücre tipleri arasındaki etkileşimler araştırmak için izin verir. Biz bu model potansiyel sinir hasar oluşturan kornea hastalıkları sinir stromal etkileşimleri ile ilgili temel mekanizmaları incelemek inanıyoruz. Bu 3D model kornea dokusu içinde vivo temel anatomik ve fizyolojik doğasını yansıtır ve gelecekte kornea kusurları araştıran yanı sıra çeşitli ajanların etkinliği hayvan test etmeden önce eleme için bir araç olarak kullanılabilir.

Şekil 1 çalışma 3D vitro modeli hakkında adım adım bir temsilcisi görüntüdür. İlk adımda, insan kornealar izole hücrelerdir. O zaman, onlar bir polikarbonat membran üzerinde yetiştirilen ve kendini salgılanan 3D matris montajı için C vitamini ile uyarılmış. Bu 3D yapı sistemi çok katmanlı bir hücresel vivo içindesentezi indükler-stromal matris gibi. Bundan sonra Nöroblastom hücreleri nöronal hücre farklılaşm...

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

Bu iletişim kuralı muayene ve etkileşimleri iki hücre tiplerinin anlayış yardımcı olmak için nerede kornea stromal hücreler ve farklılaştırılmış nöronal hücre birlikte kültürlü bir roman üç boyutlu vitro modeli, açıklar.

Kornea doku Mühendisliği: İnsan kornea sinir Stromal etkileşimlerin bir Vitro modeli

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

Research

JoVE Journal

Developmental Biology

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

8.8K Görüntüleme.  University of Oklahoma Health Sciences Center. Bu iletişim kuralı muayene ve etkileşimleri iki hücre tiplerinin anlayış yardımcı olmak için nerede kornea stromal hücreler ve farklılaştırılmış nöronal hücre birlikte kültürlü bir roman üç boyutlu vitro modeli, açıklar.

Video: 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.

Vitro ve in vivo Modelleri Kornea Endotel-mezenkimal Geçiş Çalışması için

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

Gelişim Biyolojisi

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.

Microstereolithography ve ELEKTROÜRETİM kombinasyonu Kornea Yenilenme Nişli ile donatılmış Membranlarını Üretiliyor

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

Biyomühendislik

Investigating Scarless Tissue Regeneration in Embryonic Wounded Chick Corneas

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of transparency due to fibrotic scarring. The tissue integrity of injured embryonic corneas is intrinsically restored with no detectable scar formation. Given its accessibility and ease of manipulation, the chick embryo is an ideal model for studying scarless corneal wound repair. This protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. First, eggs are windowed at early embryonic ages to access the eye. Second, a series of in ovo physical manipulations to the extraembryonic membranes are conducted to ensure access to the eye is maintained through later stages of development, corresponding to when the three cellular layers of the cornea are formed. Third, linear cornea wounds that penetrate the outer epithelial layer and the anterior stroma are made using a microsurgical knife. The regeneration process or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure. Studies to date using this model have revealed that wounded embryonic corneas display activation of keratocyte differentiation, undergo coordinated remodeling of ECM proteins to their native three-dimensional macrostructure, and become adequately re-innervated by corneal sensory nerves. In the future, the potential impact of endogenous or exogenous factors on the regenerative process could be analyzed in healing corneas by using developmental biology techniques, such as tissue grafting, electroporation, retroviral infection, or bead implantation. The current strategy identifies the embryonic chick as a crucial experimental paradigm for elucidating the molecular and cellular factors coordinating scarless corneal wound healing.

The present protocol demonstrates the different steps involved in wounding the cornea of an embryonic chick in ovo. The regenerating or fully restored corneas can be analyzed for regenerative potential using various cellular and molecular techniques following the wounding procedure.&#160;

Embriyonik Yaralı Civciv Kornealarında İzsiz Doku Rejenerasyonunun İncelenmesi

Chick embryonic corneal wounds display a remarkable capacity to fully and rapidly regenerate, whereas adult wounded corneas experience a loss of ...

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.

Ex vivo kornea Organ kültür modeli için yaranın çalışmaları

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 ...

Tıp

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

Kornea Greftlerinde Stromal Özelliklerin Histoloji Benzeri Analizi için Tam Alan Optik Koherens Mikroskopisi

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

İmmünoloji ve Enfeksiyon

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.

Split Korneal Düğmeleri Kullanarak Bir Domuz Korneal Endotel Organ Kültür Modeli

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).

Kornea Endotel Hücre Kaybının Noninvaziv, Lazer Destekli Deneysel Modelinin Geliştirilmesi

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.

Kornea Epitelinin Mekanik ve Kimyasal Yaralanmaları için Ex Vivo ve In Vivo Hayvan Modelleri

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).

Descemet'in Sadece Mühendislik Fibroblast Büyüme Faktörü 1 Tarafından Uyarılan Hızlandırılmış İyileşme ile Sıyırılmasının İnsan Kornea Organ Kültürü Modeli

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

Akson Rejenerasyon Çalışmaları için Kornea Duyusal Fiber Hasar Protokolleri

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.

Yazar Spotlight: Yenilikçi Modellerle Kornea İnnervasyonu Araştırmalarını Geliştirme 

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.

Murin Korneasında Nörotrofik Keratit ve Sinir Rejenerasyonunu İndüklemek için Üç Strateji

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 ...