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 AM secretes a total of 10 ng/mL of lumican14. To obtain a higher concentration of lumican, use 100 mg of AM, equivalent to a total area of 32 cm2.</li>
	<li>If the AM is frozen, thaw it at room temperature. 
	NOTE: Perform the following procedures under a laminar flow hood class II B.</li>
	<li>Wash the AM in a Petri dish with 10 mL of sterile balanced salt solution (BSS, see Table of Materials) for 2 min.
	<ol>
		<li>Pour the BSS into a beaker.</li>
		<li>Repeat step 3 and visually confirm that glycerol medium is not present in Petri dish BSS. 
		NOTE: Repeat step 3. as needed until glycerol medium is not present in Petri dish BSS.</li>
	</ol>
	</li>
	<li>Incubate the AM with 10 mL of dispase II (1.7 IU/mL, see Table of Materials) at 37 &#176;C, 5% CO2 for 30 min. 
	NOTE: Dispase II is a neutral protease with gentle activity over epithelial cells. This enzyme effectively separates the intact epidermis from the dermis and isolates intact epithelial sheets18.</li>
	<li>After dispase incubation, perform a mechanical de-epithelialization14 with a rubber policeman (see Table of Materials). Confirm de-epithelialization by microscopy visualization. 
	NOTE: The process of de-epithelization is corroborated in an inverted microscope using 4x and 20x objectives. Visualize the tissue to exclude the presence of any cellular layer.</li>
	<li>Wash the AM in a Petri dish with 10 mL of BSS for 2 min. Pour the BSS into a beaker.</li>
	<li>Place the de-epithelialized AM (dAM) in a 2 mL microcentrifuge tube. Immerse the dAM in liquid nitrogen for 40 min.</li>
	<li>Manually ground the frozen dAM for 2-3 min in a precooled mortar at -85 &#176;C until a fine powder is obtained.</li>
	<li>In the mortar, solubilize the dAM powder with 2.5 mL of protease inhibitor solution (BSS with protease inhibitors). 
	&#8203;NOTE: Every tablet of protease inhibitor consists of the following mixture of enzymes: pancreas extract (0.02 mg/mL), thermolysin (metalloprotease) (0.0005 mg/mL), chymotrypsin (0.002 mg/mL), trypsin (0.02 mg/mL), and papain (0.33 mg/mL) (see Table of Materials).</li>
	<li>Collect the mixture with a micropipette, and clean the mortar walls with the help of a scalpel knife. Place the mixture in a 5 mL tube.</li>
	<li>Mix well with the vortex for 30 s.</li>
	<li>Homogenize the tissue mixture by centrifuging at 34 x g for 20 min at 4 &#176;C and immediately centrifuge at 3360 x g for 20 min at 4 &#176;C.</li>
	<li>The supernatant collected is the AME (Figure 1). Store 0.7 mL of each AME in different 2 mL microcentrifuge tubes for 6, 12, 20, and 33 days at the different temperature conditions of -20 &#176;C, 4 &#176;C, and room temperature (RT).</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64460/64460fig01.jpg" /> 
Figure 1: Process of AME preparation and lumican concentration measurement. 100 mg of AM were incubated with dispase II at 37 &#176;C for 30 min and mechanically de-epithelialized. The de-epithelialized AM was washed and immersed in liquid nitrogen for 40 min, and then crushed until a fine powder was obtained, which was solubilized with 2.5 mL of saline buffer with protease inhibitors and centrifuged. The supernatant was collected and stored at -20 &#176;C, 4 &#176;C and RT for 6, 12, 20, and 32 days until total protein and lumican quantitation. <a href="https://www.jove.com/files/ftp_upload/64460/64460fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. AME protein quantification
NOTE: The quantification of total protein in the AME must be carried out immediately after obtention. Quantify proteins using Lowry protein assay and follow the manufacturer&#39;s instructions (see Table of Materials). It is recommended that all standards and samples be assayed in triplicate.
<ol>
	<li>Pipette 40 &#181;L of each sample of AME into a 96-well microplate.
	<ol>
		<li>Prepare a standard curve into the same microplate using bovine serum albumin (BSA) standard for a final BSA concentration of 0-1,500 &#181;g/mL (0, 1, 5, 25, 125, 250, 500, 750, 1,000 and 1,500 &#181;g/mL).</li>
	</ol>
	</li>
	<li>Pipette 200 &#181;L of the modified Lowry reagent to each well. Immediately mix it on a plate mixer for 30 s.</li>
	<li>Cover the microplate with aluminum foil and incubate it at RT for 10 min.</li>
	<li>Pipette 20 &#181;L of 1x Folin-Ciocalteu reagent to each well. Immediately mix it on a plate mixer for 30 s. 
	NOTE: To prepare 1x Folin-Ciocalteu reagent, dilute 2x (2N) reagent 1:1 with ultrapure water. Prepare 1x Folin-Ciocalteu reagent on the same day of use as the diluted reagent is unstable.</li>
	<li>Cover the microplate from light with aluminum foil and incubate it at RT for 30 min.</li>
	<li>Measure the absorbance of samples at 660 nm in an ELISA plate spectrometer (see Table of Materials). 
	NOTE: Color can be measured at wavelengths between 650 nm and 750 nm.</li>
	<li>Average the 660 nm absorbance value of the standard blank samples and subtract it from other 660 nm values of standard and unknown samples.
	<ol>
		<li>Measure the absorbance with an ELISA plate spectrometer in an endpoint mode with low shaking for 10 s.</li>
	</ol>
	</li>
	<li>Use the standard curve to determine the protein concentration of each unknown sample.</li>
	<li>For the calculation of protein, determine the concentration from a lineal regression graph using the values of absorbance on the Y axis against the concentrations in mg/mL on the X axis of each standard BSA curve.
	<ol>
		<li>Obtain the equation of linear regression and r value to calculate the protein concentration. 
		&#8203;NOTE: Results are expressed as normalized relative concentration values of total protein relative to mg of AM (&#181;g/mL protein/mg AM tissue).</li>
	</ol>
	</li>
</ol>
3. Quantification of Lumican in AME
NOTE: The concentration of lumican must be measured in the AME stored at different storage conditions and time periods. Quantify lumican using sandwich ELISA and follow the manufacturer&#39;s instructions. It is recommended that all standards and samples be assayed in duplicate.
<ol>
	<li>Dilute the human lumican capture antibody (see Table of Materials) to the employed concentration in phosphate-buffered saline (PBS). 
	NOTE: The capture antibody vial contains 120 &#181;g of antibody. After reconstitution with 0.5 mL of PBS, dilute the capture antibody at a working solution of 2 &#181;g/mL.
	<ol>
		<li>Instantly pipette 100 &#181;L per well of the diluted capture antibody to a 96-well microplate. Enclose the plate and incubate it overnight at RT.</li>
	</ol>
	</li>
	<li>Aspirate each well and wash it by pipetting with 300 &#181;L of wash buffer: 0.05% polyoxyethylene sorbitan monolaurate 20 in PBS, pH 7.2-7.4 (see Table of Materials) using a multi-channel pipettor. Repeat three times. 
	NOTE: After the last wash, remove any remaining wash buffer by everting the plate and gently tap it against paper towels.</li>
	<li>Block plates by adding 300 &#181;L of reagent diluent: 1% BSA in PBS, pH 7.2-7.4, 0.2 &#181;m filtered (see Table of Materials) to each well. Incubate at RT for 1 h.</li>
	<li>Repeat step 2.</li>
	<li>Prepare a standard curve into a 96-well microplate using two-fold serial dilutions from 0-8,000 pg/mL for final concentrations of 125, 250, 500, 1,000, 2,000, 4,000 and 8,000 pg/mL. The ELISA lumican kit contains a recombinant lumican standard of 75 ng (see Table of Materials).</li>
	<li>Add 100 &#181;L of samples and the standard curve in the capture antibody-coated 96-well microplate.</li>
	<li>Cover the microplate and incubate for 2 h at RT with low agitation in a compact rocker maintaining the velocity between 2-3 rpm.</li>
	<li>Repeat step 2.</li>
	<li>Add 100 &#181;L of the biotinylated-detection antibody (see Table of Materials) to each well. Cover from light and incubate 2 h at RT with low agitation in a compact rocker maintaining the velocity between 2-3 rpm. 
	NOTE: The biotinylated-detection antibody vial contains 24 &#181;g of antibody. After reconstitution with 1.0 mL of reagent diluent, dilute the biotinylated-detection antibody at a working solution of 400 ng/mL.</li>
	<li>Repeat step 2.</li>
	<li>Add 100 &#181;L of the working dilution of streptavidin-horseradish peroxidase (HRP, see Table of Materials) to each well. Cover the microplate from light and incubate for 20 min at RT. 
	NOTE: The reactive streptavidin-HRP was 40-fold concentrated. The work solution 1x of streptavidin- HRP was made with reagent diluent. 
	NOTE: Avoid placing the plate in direct light.</li>
	<li>Repeat step 2.</li>
	<li>Finally, add 100 &#181;L of substrate tetramethylbenzidine (TMB, see Table of Materials) solution to each well. 
	NOTE: Prepare TMB solution with an equal volume of stabilized hydrogen peroxide 30% solution provided in the kit. 
	NOTE: Prepare the solution immediately before use and maintain it at room temperature.</li>
	<li>Incubate for 30 min at RT in a dark place. 
	NOTE: Avoid placing the plate in direct light. Do not aspirate the TMB solution as no further washing is needed.</li>
	<li>Add 50 &#181;L of 1N H2SO4 stop solution to stop the colorimetric reaction. Gently tap the plate to ensure thorough mixing.</li>
	<li>Immediately determine the absorbance of each well using a microplate reader set to 450 nm in an ELISA plate spectrometer.</li>
	<li>Measure the absorbance with an ELISA plate spectrometer in an endpoint mode with low shaking for 10 s.</li>
	<li>Average the 450 nm absorbance value of the standard blank samples and subtract it from other 450 nm values of standard and unknown samples.</li>
	<li>Use the standard curve to determine the lumican concentration of each unknown sample.</li>
	<li>For the calculation of lumican concentration, make a lineal regression graph using the values of absorbance on the Y axis against the concentrations in pg/mL on the X axis of each standard lumican curve.
	<ol>
		<li>Obtain the equation of linear regression and r value to calculate the lumican concentration. 
		NOTE: The concentration of lumican was normalized with respect to the mg of tissue extracted. Results are expressed as normalized relative concentration values of lumican to mg AM (ng/mL lumican/mg AM tissue).</li>
	</ol>
	</li>
</ol>

<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. V., 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=Nucleic+acid+extraction+from+human+biological+samples.">Nucleic acid extraction from human biological samples.</a> Methods in Molecular Biology. 1897, 359-383 (2019).</li><li>Skog, 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=The+effect+of+enzymatic+digestion+on+cultured+epithelial+autografts.">The effect of enzymatic digestion on cultured epithelial autografts.</a> Cell Transplantation. 28 (5), 638-644 (2019).</li></ol>

The study was funded by the Support Program for Research and Technological Innovation Projects of the Universidad Nacional Autonoma de Mexico (Grant No. PAPIIT IN203821), and Ministry of Education, Science, Technology and Innovation (Grant No. SECTEI 250/2019).

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><tbody><tr><td>1 N H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt; stop solution</td><td>R&amp;amp;D Systems</td><td>DY994</td><td></td></tr><tr><td>100 &amp;mu;L micropipette</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>1000 &amp;mu;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&amp;nbsp;</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&amp;nbsp;</td><td>BD Becton Dickinson&amp;nbsp;</td><td>302562</td><td></td></tr><tr><td>20 &amp;mu;L micropipette</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>20-200 &amp;mu;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&amp;nbsp;</td><td>BioRender</td><td></td><td>figures design&amp;nbsp;</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&lt;br/&gt; 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 &amp;amp; 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&amp;nbsp;</td><td>35401106</td><td>Multiscan</td></tr><tr><td>Freezer</td><td></td><td></td><td></td></tr><tr><td>GraphPad Prism&amp;nbsp;</td><td>GraphPad Software, Inc</td><td>version 9</td><td>statistical analysis and graphic program&amp;nbsp;</td></tr><tr><td>Human lumican DuoSet ELISA kit</td><td>R&amp;amp;D Systems</td><td>DY2846-05</td><td>includes human Lumican capture antibody</td></tr><tr><td>Incubator&amp;nbsp;</td><td>Forma Scientific&amp;nbsp;</td><td>3326 S/N 36481-7002</td><td></td></tr><tr><td>Inverted light Microscope</td><td>Olympus&amp;nbsp;</td><td>6A13921</td><td>to confirm de-epithelialization&amp;nbsp; Mod.CK2</td></tr><tr><td>Laminar flow hood</td><td>Forma Scientific&amp;nbsp;</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&amp;amp;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&amp;amp;D Systems</td><td>DY992</td><td></td></tr><tr><td>Reagent diluent</td><td>R&amp;amp;D Systems</td><td>DY995</td><td>1% BSA in PBS, pH 7.2-7.4, 0.2 &amp;mu;m filtered</td></tr><tr><td>Refrigerated centrifuge</td><td>centurion scientific Ltd&amp;nbsp;</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&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>part 893975</td><td></td></tr><tr><td>Substrate tetramethylbenzidine (TMB) solution</td><td>R&amp;amp;D Systems</td><td>DY999</td><td></td></tr><tr><td>Toothed tweezers</td><td>Invent Germany</td><td>6b</td><td>inox&amp;nbsp;</td></tr><tr><td>Ultrapure water</td><td>PISA</td><td></td><td></td></tr><tr><td>Wash buffer</td><td>R&amp;amp;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 ...

lumican-extraction-from-amniotic-membrane-determination-its-storage

Research

JoVE Journal

Medicine

1.3K Views.  Instituto de Oftalmologia Fundacion Conde de Valenciana IAP. The present protocol describes the extraction of lumican from the amniotic membrane (AM) and their storing conditions as AM extract (AME) at -20 °C, 4 °C, and room temperature (RT) for 6, 12, 20, and 32 days to quantify its proteins and lumican concentration.

Video: Lumican Extraction from Amniotic Membrane and Determination of its Storage Temperature

Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation -&#160;is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques.

In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins.

In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions.

The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps are suitable for subsequent proteomic and lipidomic analysis.

Two techniques for isolating cellular lipid droplets from 1) yeast cells and 2) human placentas are presented. The centerpiece of both procedures is density gradient centrifugation, where the resulting floating layer containing the droplets can be readily visualized by eye, extracted, and quantified by Western Blot analysis for purity.

Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of ...

Bioengineering

Reprogramming Primary Amniotic Fluid and Membrane Cells to Pluripotency in Xeno-free Conditions

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic fluid and membrane mesenchymal stem cells, represent a unique type of undifferentiated cells with promise in tissue engineering and for reprogramming into iPSC for future pediatric interventions and stem cell banking. The protocol presented here describes an optimized procedure for extracting and culturing primary amniotic fluid and membrane mesenchymal stem cells and generating episomal induced pluripotent stem cells from these cells in fully chemically defined culture conditions utilizing human recombinant vitronectin and the E8 medium. Characterization of the new lines by applying stringent methods &#8211; flow cytometry, confocal imaging, teratoma formation and transcriptional profiling &#8211; is also described. The newly generated lines express markers of embryonic stem cells &#8211; Oct3/4A, Nanog, Sox2, TRA-1-60, TRA-1-81, SSEA-4 &#8211; while being negative for the SSEA-1 marker. The stem cell lines form teratomas in scid-beige mice in 6-8 weeks and the teratomas contain tissues representative of all three germ layers. Transcriptional profiling of the lines by submitting global expression microarray data to a bioinformatic pluripotency assessment algorithm deemed all lines pluripotent and therefore, this approach is an attractive alternative to animal testing. The new iPSC lines can readily be used in downstream experiments involving the optimization of differentiation and tissue engineering.

This protocol describes the reprogramming of primary amniotic fluid and membrane mesenchymal stem cells into induced pluripotent stem cells using a non-integrating episomal approach in fully chemically defined conditions. Procedures of extraction, culture, reprogramming, and characterization of the resulting induced pluripotent stem cells by stringent methods are detailed.

Autologous cell-based therapies got a step closer to reality with the introduction of induced pluripotent stem cells. Fetal stem cells, such as amniotic ...

Developmental Biology

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.

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

Engineering Transplantation-suitable Retinal Pigment Epithelium Tissue Derived from Human Embryonic Stem Cells

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms of retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Cell therapy is one of the most promising therapeutic strategies proposed to cure these diseases, with already encouraging preliminary results in humans. However, the method of preparation of the graft has a significant impact on its functional outcomes in vivo. Indeed, RPE cells grafted as a cell suspension are less functional than the same cells transplanted as a retinal tissue. Herein, we describe a simple and reproducible method to engineer RPE tissue and its preparation for an in vivo implantation. RPE cells derived from human pluripotent stem cells are seeded on a biological support, the human amniotic membrane (hAM). Compared to artificial scaffolds, this support has the advantage of having a basement membrane that is close to the Bruch&#39;s membrane where endogenous RPE cells are attached. However, its manipulation is not easy, and we developed several strategies for its proper culturing and preparation for grafting in vivo.

We describe a method to engineer a retinal tissue composed of retinal pigment epithelial cells derived from human pluripotent stem cells cultured on top of human amniotic membranes and its preparation for grafting in animal models.

Several pathological conditions of the eye affect the functionality and/or the survival of the retinal pigment epithelium (RPE). These include some forms ...

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

Biochemistry

In Vitro Culture of Epithelial Cells from Different Anatomical Regions of the Human Amniotic Membrane

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that the amniotic epithelium is a homogeneous layer. The human amnion can be divided into three anatomical regions: reflected, placental, and umbilical. Each region has different physiological roles, such as in pathological conditions. Here, we describe a protocol to dissect human amnion tissue in three sections and maintain it in vitro. In culture, cells derived from the reflected amnion displayed a cuboidal morphology, while cells from both placental and umbilical regions were squamous. Nonetheless, all the cells obtained have an epithelial phenotype, demonstrated by the immunodetection of E-cadherin. Thus, because the placental and reflected regions in situ differ in cellular components and molecular functions, it may be necessary for in vitro studies to consider these differences, because they could have physiological implications for the use of HAEC in biomedical research and the promising application of these cells in regenerative medicine.

This protocol describes the isolation of epithelial cells from different anatomical regions of the human amniotic membrane to determine their heterogeneity and functional properties for possible application in clinical and physiopathological models.

Several protocols have been reported in the literature for the isolation and culture of human amniotic epithelial cells (HAEC). However, these assume that ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Biology

A Protocol to Isolate, Culture, and Identify Human Limbal Niche Cells

Isolation and Identification of Limbal Niche Cells

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used for LNCs isolation. The tissue was divided into 12 pieces under aseptic conditions and digested for 18 h at 37 &#176;C in the cell culture incubator using collagenase A to obtain cell clusters with LNCs and limbal&#160;epithelial progenitor cells. The cell clusters were further digested for 15 min at 37 &#176;C using 0.25% trypsin-EDTA to obtain single cells and then cultured in modified embryonic stem cell medium (MESCM) on a plastic surface coated with 5% Matrigel. Cells were passaged upon 70% confluence, and LNCs were identified using immunofluorescence, real-time quantitative PCR (qPCR), and flow cytometry. Primary LNCs were isolated and passaged more than 12 times. The proliferation activity of LNCs from P4 to P6 was the highest. LNCs expressed higher stem cell markers than BMMSCs (SCF, Nestin, Rex1, SSEA4, CD73, CD90, MSX1, P75NTR, and PDGFR&#946;). Furthermore, results showed that P4 LNCs uniformly expressed VIM, CD90, CD105, and PDGFR&#946;, but not Pan-CK, which could be used as a marker for the identification of LNCs. Flow cytometric analysis showed that approximately 95%, 97%, 92%, and 11% of LNCs expressed CD73, CD90, CD105, and SCF respectively, while they were&#160;68%, 99%, 20%, and 3% in&#160;BMMSCs. The standard process for LNC&#160;isolation and identification could provide&#160;a reliable laboratory basis for the widespread use of LNCs.

Author Spotlight: Standardizing Limbal Niche Cell (LNC) Isolation and Characterization to Support Widespread LNC Research 

Here, we present a protocol to isolate and identify the human limbal niche cells.

Here we report a standard procedure for the isolation and identification of limbal niche cells (LNCs). Limbus tissue obtained from an eye bank was used ...