Name: lumican extraction from amniotic membrane and determination of its storage temperature
Uploaded: 2022-10-14T14:30:00
Description: Watch this Scientific Journal Video about Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature at JoVE.com

The authors have no competing financial interests.

All the experimental procedures were approved by the Institutional Review Board (Project No. CEI-2020/06/04). The AM was obtained from the Instituto de Oftalmologia Conde de Valenciana amnion bank (from deidentified human subjects), which is prepared as described by Ch&#225;vez-Garc&#237;a et al.17.
1. Preparation of the amniotic membrane extract
<ol>
	<li>Obtain 100 mg of AM from the amnion bank. 
	NOTE: According to a previous report, 50 mg of ...

<ol>
	<li>Jirsova, K., Jones, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+in+ophthalmology:+properties,+preparation,+storage+and+indications+for+grafting-a+review.">Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting-a review.</a> Cell and Tissue Banking. 18 (2), 193-204 (2017).</li><li>Witherel, C., Yu, T., Concannon, M., Dampier, W., Spiller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunomodulatory+effects+of+human+cryopreserved+viable+amniotic+membrane+in+a+pro-inflammatory+environment+in+vitro.">Immunomodulatory effects of human cryopreserved viable amniotic membrane in a pro-inflammatory environment in vitro.</a> Cellular and Molecular Bioengineering. 10 (5), 451-462 (2017).</li><li>Ruiz-Ca&#241;ada, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+stimulates+cell+migration+by+modulating+transforming+growth+factor-&#946;+signalling.">Amniotic membrane stimulates cell migration by modulating transforming growth factor-&#946; signalling.</a> Journal of Tissue Engineering and Regenerative Medicine. 12 (3), 808-820 (2017).</li><li>Yeh, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+lumican+glycoprotein+purified+from+human+amniotic+membrane+promotes+corneal+epithelial+wound+healing.">Soluble lumican glycoprotein purified from human amniotic membrane promotes corneal epithelial wound healing.</a> Investigative Opthalmology &amp; Visual Science. 46 (2), 479 (2005).</li><li>Navas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-Inflammatory+and+anti-fibrotic+effects+of+human+amniotic+membrane+mesenchymal+stem+cells+and+their+potential+in+corneal+repair.">Anti-Inflammatory and anti-fibrotic effects of human amniotic membrane mesenchymal stem cells and their potential in corneal repair.</a> Stem Cells Translational Medicine. 7 (12), 906-917 (2018).</li><li>Maga&#241;a-Guerrero, F., Dom&#237;nguez-L&#243;pez, A., Mart&#237;nez-Aboytes, P., Buentello-Volante, B., Garfias, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+amniotic+membrane+mesenchymal+stem+cells+inhibit+neutrophil+extracellular+traps+through+TSG-6.">Human amniotic membrane mesenchymal stem cells inhibit neutrophil extracellular traps through TSG-6.</a> Scientific Reports. 7, 12426 (2017).</li><li>Garfias, Y., Zaga-Clavellina, V., Vadillo-Ortega, F., Osorio, M., Jimenez-Martinez, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+is+an+immunosuppressor+of+peripheral+blood+mononuclear+cells.">Amniotic membrane is an immunosuppressor of peripheral blood mononuclear cells.</a> Immunological Investigations. 40 (2), 183-196 (2010).</li><li>Koob, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+properties+of+dehydrated+human+amnion/chorion+composite+graft:+implications+for+chronic+wound+healing.">Biological properties of dehydrated human amnion/chorion composite graft: implications for chronic wound healing.</a> International Wound Journal. 10 (5), 493-500 (2013).</li><li>Miyagi, H., Thomasy, S., Russell, P., Murphy, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+hepatocyte+growth+factor+in+corneal+wound+healing.">The role of hepatocyte growth factor in corneal wound healing.</a> Experimental Eye Research. 166, 49-55 (2018).</li><li>Chen, S., Mienaltowski, M., Birk, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+corneal+stroma+extracellular+matrix+assembly.">Regulation of corneal stroma extracellular matrix assembly.</a> Experimental Eye Research. 133, 69-80 (2015).</li><li>Karamanou, K., Perrot, G., Maquart, F., Br&#233;zillon, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumican+as+a+multivalent+effector+in+wound+healing.">Lumican as a multivalent effector in wound healing.</a> Advanced Drug Delivery Reviews. 129, 344-351 (2018).</li><li>Theocharis, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-matrix+interactions:+focus+on+proteoglycan-proteinase+interplay+and+pharmacological+targeting+in+cancer.">Cell-matrix interactions: focus on proteoglycan-proteinase interplay and pharmacological targeting in cancer.</a> FEBS Journal. 281 (22), 5023-5042 (2014).</li><li>Saika, 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=Role+of+lumican+in+the+corneal+epithelium+during+wound+healing.">Role of lumican in the corneal epithelium during wound healing.</a> Journal of Biological Chemistry. 275 (4), 2607-2612 (2000).</li><li>Dom&#237;nguez-L&#243;pez, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+conditioned+medium+(AMCM)+reduces+inflammatory+response+on+human+limbal+myofibroblast,+and+the+potential+role+of+lumican.">Amniotic membrane conditioned medium (AMCM) reduces inflammatory response on human limbal myofibroblast, and the potential role of lumican.</a> Molecular Vision. 27, 370-383 (2021).</li><li>Vij, N., Roberts, L., Joyce, S., Chakravarti, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lumican+regulates+corneal+inflammatory+responses+by+modulating+Fas-Fas+Ligand+signaling.">Lumican regulates corneal inflammatory responses by modulating Fas-Fas Ligand signaling.</a> Investigative Opthalmology &amp; Visual Science. 46 (1), 88 (2005).</li><li>Mahbod, 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=Amniotic+membrane+extract+preparation:+What+is+the+best+method.">Amniotic membrane extract preparation: What is the best method.</a> Journal of Ophthalmic and Vision Research. 9 (3), 314-319 (2014).</li><li>Ch&#225;vez-Garc&#237;a, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ophthalmic+indications+of+amniotic+membrane+transplantation+in+Mexico:+an+eight+years+Amniotic+Membrane+Bank+experience.">Ophthalmic indications of amniotic membrane transplantation in Mexico: an eight years Amniotic Membrane Bank experience.</a> Cell and Tissue Banking. 17 (2), 261-268 (2015).</li><li>Stenn, K. S., Link, R., Moellmann, G., Madri, J., Kuklinska, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dispase,+a+neutral+protease+from+Bacillus+polymyxa,+is+a+powerful+fibronectinase+and+type+IV+collagenase.">Dispase, a neutral protease from Bacillus polymyxa, is a powerful fibronectinase and type IV collagenase.</a> Journal of Investigative Dermatology. 93 (2), 287-290 (1989).</li><li>Bhatnagar, B. S., Bogner, R. H., Pikal, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+stability+during+freezing:+separation+of+stresses+and+mechanisms+of+protein+stabilization.">Protein stability during freezing: separation of stresses and mechanisms of protein stabilization.</a> Pharmaceutical Development and Technology. 12 (5), 505-523 (2007).</li><li>McClain, A. K., McCarrel, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+four+different+freezing+conditions+and+time+in+frozen+storage+on+the+concentration+of+commonly+measured+growth+factors+and+enzymes+in+equine+platelet-rich+plasma+over+six+months.">The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months.</a> BMC Veterinary Research. 15 (1), 292 (2019).</li><li>Tamhane, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+amniotic+membrane+transplantation+as+an+adjunct+to+medical+therapy+as+compared+with+medical+therapy+alone+in+acute+ocular+burns.">Evaluation of amniotic membrane transplantation as an adjunct to medical therapy as compared with medical therapy alone in acute ocular burns.</a> Ophthalmology. 112 (11), 1963-1969 (2005).</li><li>Shtein, R., 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=Autologous+serum-based+eye+drops+for+treatment+of+ocular+surface+disease.">Autologous serum-based eye drops for treatment of ocular surface disease.</a> Ophthalmology. 127 (1), 128-133 (2020).</li><li>Shahriari, H., Tokhmehchi, F., Reza, M., Hashemi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+the+effect+of+amniotic+membrane+suspension+and+autologous+serum+on+alkaline+corneal+epithelial+wound+healing+in+the+rabbit+model.">Comparison of the effect of amniotic membrane suspension and autologous serum on alkaline corneal epithelial wound healing in the rabbit model.</a> Cornea. 27 (10), 1148-1150 (2008).</li><li>Schuerch, K., Baeriswyl, A., Frueh, B., Tappeiner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficacy+of+amniotic+membrane+transplantation+for+the+treatment+of+corneal+ulcers.">Efficacy of amniotic membrane transplantation for the treatment of corneal ulcers.</a> Cornea. 39 (4), 479-483 (2019).</li><li>Chen, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+transplantation+for+persistent+corneal+ulcers+and+perforations+in+acute+fungal+keratitis.">Amniotic membrane transplantation for persistent corneal ulcers and perforations in acute fungal keratitis.</a> Cornea. 25 (5), 564-572 (2006).</li><li>Guo, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+effectiveness+between+amniotic+membrane+homogenate+and+transplanted+amniotic+membrane+in+healing+corneal+damage+in+a+rabbit+model.">A comparison of the effectiveness between amniotic membrane homogenate and transplanted amniotic membrane in healing corneal damage in a rabbit model.</a> Acta Ophthalmologica. 89 (4), 315-319 (2011).</li><li>Sabater, A., Perez, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amniotic+membrane+use+for+management+of+corneal+limbal+stem+cell+deficiency.">Amniotic membrane use for management of corneal limbal stem cell deficiency.</a> Current Opinion in Ophthalmology. 28 (4), 363-369 (2017).</li><li>Ahmad, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autolysis+of+bovine+skin,+its+endogenous+proteases,+protease+inhibitors+and+their+effects+on+quality+characteristics+of+extracted+gelatin.">Autolysis of bovine skin, its endogenous proteases, protease inhibitors and their effects on quality characteristics of extracted gelatin.</a> Food Chemistry. 265, 1-8 (2018).</li><li>Mullegama, S. 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In this study, the presence of lumican was analyzed in the AME and its direct correlation with its stability under different storage conditions. Interestingly, when the total protein concentration in AME was quantified, protein concentration increased after storage. Evidence suggests three mechanisms that could change protein concentration in frozen storage: cold denaturation, the frozen concentration of solutes, and ice-induced partial unfolding of protein structure19. The freezing process could ...

One of the most used treatments for corneal affections is amniotic membrane transplantation; however, in recent years, new proposals have emerged for using various components of amniotic tissue as alternative and adjuvant treatments. Among the most studied components of AM are those obtained from the AM extract (AME)1,2,3,4,5,6,7. AM contains multiple soluble factors such as antiangiogenic proteins, interleukins (IL), tissue inhibitors of metalloproteinases (TIMPs), anti-inflammatory proteins mediated by TSG-6 that inhibit neutrophil extracellular traps, growth factors: epidermal growth factor (EGF), transforming growth factor (TGF) (alpha and beta), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and lumican, which maintains corneal transparency by regulating collagen fibrillogenesis1,2,3,4,5,6,7,8,9.
Lumican is a small leucine-rich proteoglycan (SLRP), one of the main extracellular components of interstitial collagenase in the corneal stroma matrix, responsible for organizing collagen fibers and maintaining corneal transparency4,10,11. Proteoglycans are molecules in the extracellular matrix (ECM), which are the main ones in carrying out cell signaling and maintaining intracellular homeostasis12. ECM proteins have been reported to drive the cellular processes of proliferation, differentiation, and migration during wound healing11.
Evidence indicates the possible participation of lumican in the process of corneal re-epithelialization. Saika et al., in a study, showed that after a corneal injury, lumican could be detected in corneal keratocytes between the first 8 h and up to 3 days after injury. Presenting the highest concentration of lumican on the second and third day, this proteoglycan is subsequently undetectable on the seventh day13. These data suggest the participation of lumican in the activation of the corneal re-epithelialization process. On the other hand, in another study, it was reported that the absence of lumican delays re-epithelialization; interestingly, adding lumican could accelerate the re-epithelialization process4,11,13. Likewise, a recent study has reported that lumican can modulate the inflammatory functions of corneal limbus fibroblasts14, which suggests a role for lumican as a modulator of the inflammatory, antifibrotic and re-epithelializing response. Similarly, lumican can modulate the corneal response by interacting with signaling molecules such as Fas-FasL. Also, the absence of lumican in a knockout Lum-/- mouse model demonstrated that the lack of lumican signaling prevents adequate corneal repair15.
Primarily, this method aims to demonstrate a feasible and approachable way to extract lumican from AM. With this advantageous method of lumican extraction, it is possible to obtain similar concentrations of proteins, decreasing the processing time and making it more convenient for investigators compared to the previous studies16. Furthermore, this AME lumican could be used as an adjuvant for corneal repair and re-epithelialization processes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 N H2SO4 stop solution</td>
			<td>R&#38;D Systems</td>
			<td>DY994</td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1000 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm Petri dish</td>
			<td>Symlaboratorios</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>18 G Needle (1.2 mm x 40 mm)</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>305211</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>Z606340</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL plastic syringe&#160;</td>
			<td>BD Becton Dickinson&#160;</td>
			<td>302562</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>20-200 &#956;L micropipette</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL microcentrifuge tube</td>
			<td>Eppendorf</td>
			<td>30119401</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well microplate</td>
			<td>SARSTEDT</td>
			<td>821581</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Amniotic membrane</td>
			<td>Instituto de Oftalmologia Conde de Valenciana Amnion Bank</td>
			<td></td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>Balanced salt solution</td>
			<td>Bausch + Lomb</td>
			<td>BSS-403802</td>
			<td></td>
		</tr>
		<tr>
			<td>Beaker</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>BioRender&#160;</td>
			<td>BioRender</td>
			<td></td>
			<td>figures design&#160;</td>
		</tr>
		<tr>
			<td>Compact Rocker</td>
			<td>BioRad</td>
			<td>970822DD</td>
			<td>Mod. 5202SD-BIO</td>
		</tr>
		<tr>
			<td>complete, EDTA-free, Protease inhibitor 
			cocktail tablets</td>
			<td>Roche</td>
			<td>11 873 580 001</td>
			<td>Protease Inhibitor</td>
		</tr>
		<tr>
			<td>Daiggner vortex Genie 2</td>
			<td>A.Daigger &#38; Co. , INC</td>
			<td>22220A</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plate spectrometer</td>
			<td>Thermo Labsystems&#160;</td>
			<td>35401106</td>
			<td>Multiscan</td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism&#160;</td>
			<td>GraphPad Software, Inc</td>
			<td>version 9</td>
			<td>statistical analysis and graphic program&#160;</td>
		</tr>
		<tr>
			<td>Human lumican DuoSet ELISA kit</td>
			<td>R&#38;D Systems</td>
			<td>DY2846-05</td>
			<td>includes human Lumican capture antibody</td>
		</tr>
		<tr>
			<td>Incubator&#160;</td>
			<td>Forma Scientific&#160;</td>
			<td>3326 S/N 36481-7002</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted light Microscope</td>
			<td>Olympus&#160;</td>
			<td>6A13921</td>
			<td>to confirm de-epithelialization&#160; Mod.CK2</td>
		</tr>
		<tr>
			<td>Laminar flow hood</td>
			<td>Forma Scientific&#160;</td>
			<td>14753-567</td>
			<td>Mod.1184</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Mortar</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-channel pipettor</td>
			<td>Eppendorf</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrogen Tank</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td>Mod. Biocan 20</td>
		</tr>
		<tr>
			<td>Paper towels</td>
			<td>N/A</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline</td>
			<td>R&#38;D Systems</td>
			<td>DY006</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Modified Lowry Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23240</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate sealers</td>
			<td>R&#38;D Systems</td>
			<td>DY992</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent diluent</td>
			<td>R&#38;D Systems</td>
			<td>DY995</td>
			<td>1% BSA in PBS, pH 7.2-7.4, 0.2 &#956;m filtered</td>
		</tr>
		<tr>
			<td>Refrigerated centrifuge</td>
			<td>centurion scientific Ltd&#160;</td>
			<td>15877</td>
			<td>Mod. K2015R</td>
		</tr>
		<tr>
			<td>Rubber policeman cell scraper</td>
			<td>NEST</td>
			<td>710001</td>
			<td>for mechanical de-epithelialization</td>
		</tr>
		<tr>
			<td>Scalpel knife</td>
			<td>Braun</td>
			<td>BB521</td>
			<td>No. 10 or 21</td>
		</tr>
		<tr>
			<td>Streptavidin-HRP 40-fold concentrated&#160;</td>
			<td>R&#38;D Systems</td>
			<td>part 893975</td>
			<td></td>
		</tr>
		<tr>
			<td>Substrate tetramethylbenzidine (TMB) solution</td>
			<td>R&#38;D Systems</td>
			<td>DY999</td>
			<td></td>
		</tr>
		<tr>
			<td>Toothed tweezers</td>
			<td>Invent Germany</td>
			<td>6b</td>
			<td>inox&#160;</td>
		</tr>
		<tr>
			<td>Ultrapure water</td>
			<td>PISA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wash buffer</td>
			<td>R&#38;D Systems</td>
			<td>WA126</td>
			<td>0.05% Tween 20 in PBS, pH 7.2-7.4</td>
		</tr>
	</tbody>
</table>

lumican extraction from amniotic membrane and determination of its storage temperature

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen fibers, maintaining corneal transparency. In the present work, a method for protein extraction from AM to obtain lumican is proposed. Additionally, the stability of lumican in the AM extract (AME) stored at different temperatures and time periods is evaluated. 100 mg of AM were thawed and mechanical de-epithelialized. The de-epithelialized AM was frozen and crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged for protein extraction. The supernatant was collected and stored at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days. Afterward, lumican was quantified in each AME. This technique allows an accessible and acquirable protocol for lumican extraction from AM. Lumican concentration was affected by storage time and temperature conditions. Lumican in the AME of 12 days stored at -20 &#176;C and 4 &#176;C was significantly higher than other AME. This lumican extraction could be useful for developing treatments and pharmaceutical solutions. Further studies are needed to determine the uses of AME lumican in re-epithelialization and wound healing process.

Results are reported as the mean value &#177; standard deviation (SD). Student&#39;s t-tests and analysis of variance (ANOVA) were performed. P-values &#60; 0.05 was considered statistically significant. Statistical analysis was performed using statistics software (see Table of Materials).
The total protein quantity in the AME was affected by time and storage conditions. The basal protein concentration was similar among all AME; the range of total protein was...

Watch this Scientific Journal Video about Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature at JoVE.com

Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 &#176;C, 4 &#176;C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.

Lumican is a small leucine-rich proteoglycan in the human amniotic membrane (AM) that promotes corneal epithelialization and the organization of collagen ...

This method aims to demonstrate a straightforward technique for protein extraction, useful for the obtaintion of bountiful proteins and other factors. This technique could be used to extract other factors and proteins present in the amniotic membrane and also, to obtain proteins from other sources such as animals and vegetal tissues. Begin by thawing 100 milligrams of the amniotic membrane obtained from the amnion bank.Once thawed, add 10 milliliters of the sterile balanced salt solution to a Petri dish and wash the membrane for two minutes. Then, remove the used balanced salt solution into a beaker and repeat the process until no trace of glycerol medium is visible in the Petri dish. Next, add 10 milliliters of dispase II to the membrane and incubate at 37 degrees Celsius and 5%carbon dioxide for 30 minutes.At the end of the incubation, perform a mechanical de-epithelialization with a rubber policeman cell scraper. Then, visualize the membrane under an inverted microscope to confirm de-epithelialization. Next, wash the de-epithelialized amniotic membrane with the sterile balanced salt solution as demonstrated earlier, and place it in a two-milliliter microcentrifuge tube.Then, immerse the tube containing the membrane in liquid nitrogen for 40 minutes. Post freezing with liquid nitrogen, manually grind the membrane for two to three minutes in a mortar, pre-chilled at minus 85 degrees Celsius, until a fine powder is obtained. Next, add 2.5 milliliters of protease inhibitor solution and solubilize the finely-powdered membrane.Clean the mortar walls with the help of a scalpel knife and transfer the mixture to a five-milliliter tube. Vortex the tube for 30 seconds. Then, homogenize the tissue mixture by first centrifuging at 34 G for 20 minutes at four degrees Celsius, and immediately centrifuging it again at 3, 360 G for 20 minutes at four degrees Celsius.At the end of centrifugation, collect the supernatant, which is the amniotic membrane extract, in a new tube. Then, aliquot 0.7 milliliters in a different two-milliliter microcentrifuge tube for various time points and temperatures. The total protein quantity and lumican concentration in the amniotic membrane extract were affected by time and storage conditions.Basal protein concentration was similar among all the amniotic membrane extracts. However, a significantly high protein concentration was noted in the extract stored for 32 days at four degrees Celsius and 20 days at minus 20 degrees Celsius. Lumican in the amniotic membrane extract was significantly higher in the sample stored for a period of 12 days compared with the samples stored for 30, 20, and six days at four and minus 20 degrees Celsius.These results suggested that the appropriate storage time and temperature to achieve the highest concentration of lumican is 12 days at minus 20 degrees Celsius. Exercise utmost care while grounding the frozen amniotic membrane. The grounding should be gentle as it could fall out from the mortar.Further studies are needed to determine the role of amniotic membrane extract lumican in corneal epithelial cells and to determine the ideal dose of lumican for corneal re-epithelialization.

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