Name: corneal tissue engineering: an in vitro model of the stromal-nerve interactions of the human cornea
Uploaded: 2018-01-24T13:30:00
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

Vi vil gerne udvide vores oprigtige tak Dr. Ben Fowler hans teknisk hjælp til TEM eksperimenter.

Denne protokol følger retningslinjerne i University af Oklahoma Health Sciences Center/institutionelle review board (IRB #4509). Alle dele af protokollen mødte grundsætningerne af Helsinki-erklæringen. Hornhinde prøver blev indhentet fra de nationale udvikling og Research Institute (NDRI) og Oklahoma løver Eye Bank.
1. isolering af primærelementer
<ol><li>Ved modtagelsen af menneskelige hornhinden vævsprøver, overføre væv ind i en 21,5 cm2 petriskål indeholdende 2 mL s...

<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>

Flere undersøgelser har været fokuseret på at udvikle forskellige dyremodeller, der kan hjælpe med at udvikle en bedre forståelse af cornea sygdomme samt finde behandlinger. En betydelig værdi for mennesker fra disse undersøgelser er imidlertid ikke blevet bekræftet. Til dato, er forskellige in vitro- modeller blevet udviklet og udbredt undersøgt på grund af deres bemærkelsesværdige klinisk betydning. Vores tidligere etablerede 3D in vitro- model er en roman system, som væsentligt bidrager ...

I den menneskelige krop er hornhinden det tættest innerverede væv. Nerverne er ansvarlige for forskellige fornemmelser som berøring, smerte, temperatur, og har også en væsentlig rolle i sårheling, blinke reflekser, rive produktion og sekretion5,6,7. I hornhinden, stromale nerve kufferter opstår fra den limbal plexus og indtaste den perifere hornhindens stroma radialt. Stromale nerve organisation er parallel for kollagen lamellae og de yderligere forgrening ind i mindre fascicles når de går videre mod overfladiske stroma5,8. Nervefibre yderligere trænge epitel lag og således innervation er bredt fordelt i hele hornhindens epitel og stroma. Derfor har innervation en væsentlig rolle i den raske og syge tilstand af hornhinden. I denne protokol afslører vi fremme af roman 3D in vitro- model, som er først af sin art til at efterligne i vivo stromale-nerve interaktioner. SH-SY5Y cellelinje blev brugt til denne undersøgelse, da det er en af de mest etablerede, godt karakteriseret linjer bruges til at studere neuronal vækst. SH-SY5Y cellelinje er blevet beskrevet til at producere både substrat tilhænger (S-type) og neuroblastic (N-type) celler der kan gennemgå transdifferentiation9. Som et resultat, selv om denne cellelinie er afledt fra en tredobbelt successive subclone udvalg af N-type celler, indeholder det også et lille antal S-type celler kan undergå en differentiering af neuroner ved hjælp af retinoid syre og hjerne-afledte neurotrope faktor9. Dette giver et værktøj, der kan føre til en bedre forståelse af cornea komplikationer forbundet med diabetisk retinopati (DM) og andre okulær sygdomme. På grund af vanskelighederne med at opnå og dyrkning neuroner fra patienter med okulær sygdomme giver denne 3D i vitro model store konsekvenser i at studere de neuronale interaktioner og signalering med hornhindens stroma.
Syge betingelser påvirker ofte forskellige væv i kroppen på en meget stor skala, fører til en kompromitteret livskvalitet. Okulær dystrophies er fælles komplikationer ofte forbundet med systemiske sygdomme og føre til tab af synsstyrken eller endda permanent synstab. Omfattende undersøgelser er ofte afgørende for en bedre forståelse af sygdom tilstand samt effekter på basal cellulært niveau. For at studere virkningerne af sådanne sygdomme, er forskellige i vivo og in vitro- modeller blevet udviklet ved hjælp af tissue engineering applikationer. Hornhindevæv ingeniørmæssige anvendelser har vundet stor interesse på tværs af forskellige områder af videnskab10,11,12,13,14, men der er stadig store begrænsninger i løbet faktiske anvendelse, herunder hornhinde pode afvisninger, infektioner og ardannelse10,11,12,13,14. Der er flere undersøgelser, der har med held udviklet og etableret forskellige in vitro- modeller3,15,16,17,18, 19,20,21,22,23,24,25,26. 3D in vitro- modeller er den mest lovende og af stor videnskabelig interesse. 3D-modeller er kendt for at bedre spejl i vivo cellulære og fysiologiske begivenhederne, der kritisk under fibrose og sår healing15,27,28,29. Modellerne i vitro spille en integrerende rolle ved at finde nye terapeutiske tilgange til behandling af forskellige sygdomstilstande, herunder hornhinde komplikationer. Trods den kritiske rolle af innervation i cornea funktioner, er der sket lidt indsats at fremme perifere nerve spredning i hornhinden væv-manipuleret konstruktioner2,3. Dog efterligne cellekonstruktioner foreslåede 3D in vitro- målvæv for at nå den ønskede væv funktionalitet.
Mens diabetisk keratopati er en indlysende ansøgning om modellen beskrevet her på grund af de neuronale defekter, er der flere andre corneal sygdomme, der kan drage fordel af en menneskelig i vitro model herunder Keratoconus og Fuchs dystrophies. Vores 3D model fremgår af dette perspektiv og foreslår udviklingen af en in vitro- repræsentation af hornhindevæv at vurdere medicinafgivelse og sikkerheden af nye okulær lægemidler.

<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&#8217;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&#8217;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-&#956;m pores</td>
			<td>Corning Costar</td>
			<td>3412</td>
			<td></td>
		</tr>
		<tr>
			<td>2-O-&#945;-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

Vævsmanipulering har fået betydelig anerkendelse på grund af den store efterspørgsel for menneskelige hornhinden udskiftninger med omkring 10 millioner mennesker verden over lider af cornea vision tab1. Til at håndtere efterspørgslen efter bæredygtige menneskelige hornhinder, store fremskridt i tre-dimensionelle (3D) væv er manipulation gjort2,3,4. Disse hornhinden modeller spænder fra simple éncellelag systemer til flerlaget modeller, fører til 3D full-tykkelse hornhinde ækvivalenter2.
Men brugen af en 3D væv-manipuleret hornhinden i forbindelse med in vitro- sygdomsmodeller studerede til dato mangler lighed med flerlaget 3D hornhindevæv struktur, funktion og netværk af forskellige celletyper (dvs., nerve, epitel, stroma og endotelet)2,3. Derudover steget efterspørgslen efter in vitro- hornhinden væv modeller i et forsøg på at reducere dyreforsøg for farmaceutiske produkter. Således mere avancerede modeller er forpligtet til at bedre at matche systemer til menneskets fysiologiske behov, og udviklingen af en model, der er mere relevant for den patientgruppe er absolut nødvendigt. Eftersom at flere celletyper i hornhinden påvirkes af sygdomme og dystrophies, Keratoconus, diabetisk keratopati og Fuchs, omfatter denne model en 3D Co kultur model af primære menneskelige hornhinde fibroblaster (HCFs) fra raske donorer og neuroner fra SH-SY5Y cellelinje. Dette giver os for første gang at undersøge interaktioner mellem to celletyper i den menneskelige hornhindevæv. Vi mener, at denne model potentielt kunne dissekere de underliggende mekanismer forbundet med stromale-nerve interaktioner af cornea sygdomme, der udviser nerve skader. Denne 3D model afspejler den grundlæggende anatomiske og fysiologiske karakter af hornhindevæv i vivo og kan bruges i fremtiden som et redskab til undersøge hornhinde defekter samt screening effekten af forskellige agenter før dyreforsøg.

Figur 1 er en trinvis repræsentativt billede af 3D i vitro arbejdsmodel. På det første trin er celler isolerede fra menneskets hornhinder. Derefter, de er dyrket på en polycarbonat membran og stimuleret med C-Vitamin til at samle en selvstændig udskilles 3D matrix. Denne 3D konstruktion system inducerer syntesen af et flerlaget cellulære i vivo-ligesom stromale matrix. Derefter, er neuroblastoma celler seedet på toppen af de stromale ...

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

Denne protokol beskriver en ny tre-dimensionelle in vitro- model, hvor hornhinde stromale celler og differentieret neuronale celler dyrkes sammen til at bistå med gennemgang og forståelse af samspillet mellem to celletyper.

Hornhindevæv Engineering: En In Vitro Model af den menneskelige hornhinden stromale-nerve interaktioner

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

Research

JoVE Journal

Developmental Biology

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

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Analyse af zebrafisk Larver Skeletmuskel Integritet med Evans blå farvestof

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Differentiation of the SH-SY5Y Human Neuroblastoma Cell Line

Differentiering af SH-SY5Y human neuroblastomcellelinje

Having appropriate in vivo and in vitro systems that provide translational models for human disease is an integral aspect of research in neurobiology and ...

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

En Metode til Lineage Sporing af hornhindeceller Brug Multi-color Fluorescent Reporter Mus

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Udvikling af et<em&gt; In vitro</em&gt; Assay til Evaluer kontraktionsfunktion af mesenchvmal-celler der undergik Epithelial-Mesenchymal Overgangseffekter

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Conditional Reprogramming of Pediatric Human Esophageal Epithelial Cells for Use in Tissue Engineering and Disease Investigation

Betinget Omprogrammering af Pediatric Menneskelig Esophageal epitelceller til brug i Tissue Engineering og Sygdom Investigation

Identifying and expanding patient-specific cells in culture for use in tissue engineering and disease investigation can be very challenging. Utilizing ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

En 5-mC prikkeblot-analyse, der kvantificerer DNA-methyleringsniveauet for chondrocyt-differentiering<em&gt; In Vitro</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

In Vitro Growth of Mouse Preantral Follicles Under Simulated Microgravity

In Vitro Vækst af mus Preantral follikler Under simulerede vægtløshed

14 day-old mouse ovarian tissue and preantral follicles isolated from same-aged mice were incubated in a simulated microgravity culture system. We ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Dissektion og immunfluorescent farvning af champignon krop og fotoreceptor neuroner i voksen Drosophila melanogaster hjerner

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Celle sammenlægning Assays til at evaluere bindingen af Drosophila Notch med Trans-ligander og dens hæmning af Cis-ligander

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...