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.bdigitaluss.remotexs.co/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>

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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 affect protein concentration on storage samples due to the crystallization of the liquid phase in the sample. The results suggest that this could have occurred as a process of freezing the concentration, affected by the storage time in the freezer. A higher concentration of proteins was observed at longer times of storage (32 and 20 days) and at the coldest temperatures (4 &#176;C and -20 &#176;C). However, the period with the highest concentration of protein did not have the highest concentration of lumican; this suggests that lumican could be affected by frozen temperatures and time conditions.
According to the results, lumican concentration was higher and more stable at 12 days of storage at 4 &#176;C and -20 &#176;C. Nonetheless, a lesser concentration of lumican was found at 6 days compared to 12 days. Some reports suggest that protein concentration could change after frozen storage conditions20. The results could be caused by a thermodynamic mechanism named ice-induced partial unfolding of protein structure, which happens during the freezing of samples. The interaction of proteins with water in the aqueous solutions reduces their interactions with other molecules. The crystallization process of water under frozen conditions allows proteins and some functional regions to interact with other molecules19. By the aforementioned, after 12 days of freezing lumican in an aqueous solution, it could be able to interact with the antibodies present in the ELISA quantification kit to result in a higher concentration. On the other hand, probably on the sixth day, the lumican could have been secluded in the aqueous solution.
In advancing innovations, the use of AM is the state-of-the-art treatment for corneal re-epithelialization regardless of etiology. As aforementioned, the benefits of AM are vast21,22,23,24,25,26. Many authors have demonstrated the benefits of lumican in AME, making it an affordable alternative specifically for developing countries1,2,3,4,5,6,7,8,9. Currently, there are many benefits of lumican in AME; its use as a treatment favors corneal re-epithelialization and improves the prognosis of corneal ulcers4,5,6,7,8,9,10,11,12,13,14,15. AM transplantation (AMT) has become a treatment with great benefits for improving various corneal disorders24,25. However, there are some chronic affections of the corneal tissue, such as persistent epithelial defects (PED) and limbal stem cell deficiencies (LESCD), that require constant treatment and maintenance of the presence of biological factors that help corneal repair21,24. Currently, there are no adjuvant treatments that allow long-term maintenance of the factors released by AMT on the corneal surface. However, a constant replacement of the AMT could not be recommended for patient safety26. For this reason, it is necessary to develop alternatives that allow the functions of AMT to assist and helps maintain the presence of factors released by AM in the corneal tissue such as lumican for a longer time, intending to favor the treatment of persistent problems of the cornea27.
Lumican is one of the factors present in AM with anti-inflammatory and antifibrotic functions, which has been reported to have functions in the corneal repair process6,7,8,9,10,11,12,13,14,15. That is why lumican suggests being a good candidate to aid in treating corneal affections; however, additional research is required to determine the efficacy of lumican in AME to achieve corneal re-epithelialization.
Lumican is a proteoglycan that has been shown to regulate the secretion of extracellular matrix compounds as collagen; also, it is involved in fibroblast activation and modulation of inflammatory cells and the angiogenesis process, having an important role in wound healing. According to the results, lumican can be extracted from AM tissue. Therapeutic applications of lumican are numerous; the use of lumican in the AME allows an attainable therapeutic option for ocular disorders4,13. The primary advantage of using AME is that it provides an easy application as a topical treatment for the ocular surface, given its aqueous composition. Likewise, other proteins with anti-inflammatory and immunoregulatory characteristics can be found within the components extracted from the AM tissue, which could present a greater benefit in treating de-epithelializing problems in the eye. For example, other anti-inflammatory factors such as TSG-6 present in the AM and cellular components have previously been reported to have immunoregulatory properties8. Hereby, a combined therapy of lumican and other extracellular matrix compounds and immunomodulatory molecules present in the AME could be useful in the re-epithelialization and wound healing process.
This method aims to demonstrate a straightforward technique for protein extraction, useful for the obtention of bountiful proteins and factors. One of the critical considerations for this method is the use of protease inhibitors, as it is fundamental for successful protein extraction as the AM is a tissue with loads of enzymatic compounds to prevent protein degradation27. Evidence has reported that using protein inhibitors together with an aqueous solution increases the extraction of other factors, such as HGF, in AM tissue26. The obtention of a fine powder after freezing and grounding the AM is necessary for the optimal obtention of AME, as this process is suitable for tissue and cellular disruption of structures needed for the extraction of cytosolic and other nuclear compounds28,29.
A few limitations of this extraction method are that the AM quantity used could not allow a high-scale extraction. This protocol allows protein extraction from a limited amount of tissue; since there is no evidence that this method allows obtaining a greater amount of protein from a larger tissue area. Troubleshooting to be considered in comparison with other extraction methods is to use a suitable concentration of protease inhibitor and incubation time, as the excess enzymes could affect proteins30 and reduce the technique&#39;s efficacy.
Part of this technique was modified from that reported by Mahbod et al.16, which describes that repeating the centrifugation and extraction process increases protein extraction. On contrary to Mahbod, the results did not report protein concentration after three cycles of centrifugation. When the total protein concentration was determined, it decreased by 80% in a second extraction and up to 97% in a third extraction. Performing only one extraction reduces the processing time and does not decrease the amount of total protein. With the preceding, the extraction method reported here requires only one centrifugation step with favorable results.
This technique could be used to extract other factors and proteins present in the AM and further, to obtain factors from other sources such as animals or vegetables. It could also have an application for obtaining proteins to carry out basic research or even the development of formulations and treatments.
These results suggest that lumican can be extracted from AM and stored for 12 days as AME under both -20 &#176;C and 4 &#176;C temperature conditions. It is important to consider its half-life to achieve the therapeutic effects of lumican as AME. Further studies are needed to determine the role of AME lumican in corneal epithelial cells and to determine the ideal dose of lumican for corneal re-epithelialization.
In conclusion, the results suggest obtaining factors such as lumican in the AME is possible. Likewise, temperature and storage time conditions influence the concentration of lumican present in the AME.

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 2.7 &#177; 0.3 &#181;g/mL without a significant difference between the samples evaluated. However, when the samples were stored for 12, 20, and 32 days, variability in protein concentration with respect to basal concentration was observed. Interestingly, protein concentration increased in the AME at 4 &#176;C and -20 &#176;C with respect to the RT at all times of storage.
Similarly, when protein concentration was compared between storage times, it changed after 12, 20, and 32 days. A significant difference (p &#60; 0.05) was found in the AME of 32 and 20 days at 4 &#176;C and -20 &#176;C in comparison to the RT condition (Figure 2), suggesting that temperature is important for protein conservation in the different AME obtained.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64460/64460fig02.jpg" /> 
Figure 2: Total protein concentration in AME affected by time and storage temperature. The concentration of protein extraction on AME was quantified before and after temperature and time storage conditions. Storage time evaluated was 6 days (black triangles), 12 days (pink triangles), 20 days (purple square), and 32 days (brown circles) in comparison with three different temperature conditions (RT &#176;C, 4 &#176;C, and -20 &#176;C). Basal protein concentration was similar among all AME. There was a significant difference in protein concentration in the AME of 20 and 32 days with respect to basal protein concentration at different temperature conditions. In each condition n = 3. Data is expressed as median of &#181;g/mL of protein &#177; SE *p &#60; 0.05 (S1 32 days vs. S1 20, 12, and 6 days at 4 &#176;C); (S1 32 days vs. S1 20, 12, and 6 days at -20 &#176;C). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64460/64460fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Lumican concentration was affected by storage time and temperature conditions. Fewer concentration of lumican was found in the AME stored for 6, 20, and 32 days, compared with 12 days of storage. Significantly, the AME of 12 days had a higher concentration of lumican than 20 and 32 days of storage (p &#60; 0.05).
When lumican concentration in the AME was compared between storage temperatures, a higher concentration of lumican was found if stored at -20 &#176;C and 4 &#176;C for 12 days (Figure 3). Interestingly, even a higher (p &#60; 0.05) concentration of lumican was found in the 12 days AME if stored at -20 &#176;C in comparison with 4 &#176;C.
This suggests that the concentration of lumican is affected by temperature conditions and storage time, suggesting that the appropriate storage time and temperature to achieve the highest concentration of lumican is 12 days at -20 &#176;C.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64460/64460fig03.jpg" /> 
Figure 3: Total lumican concentration in AME affected by time and storage temperature. Lumican concentration was affected by storage time and temperature conditions. The concentration of lumican in AME was quantified before and after temperature and time storage conditions. Storage time evaluated was 6 days (black triangles), 12 days (pink triangles), 20 days (purple square), and 32 days (brown circles) in comparison with three different temperature conditions (RT &#176;C, 4 &#176;C, and -20 &#176;C). Lumican in the AME of 12 days was significantly higher in comparison with the AME of 32, 20, and 6 days, at temperature conditions of -20 &#176;C and 4 &#176;C. *p &#60; 0.05 (S1 12 days vs. S1 32, 20, and 6 days at 4 &#176;C); ***p &#60; 0.001 (S1 12 days vs. S1 32, 20 and 6 days at -20&#176;C). The highest concentration of lumican in AME was at 12 days stored at -20 &#176;C. ++p &#60; 0.01 (S1 12 days 4 &#176;C vs. S1 12 days -20 &#176;C). There was no significant difference between lumican in the AME of 32 and 20 days compared with storage temperature conditions (n.s). In each condition (n = 3), data are expressed as the median of ng/mL of protein. Data were normalized with respect to mg of tissue &#177; SE. (n.s.) not statistical significance. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/64460/64460fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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

Universidad San Sebastian

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

A Simple Dewar/Cryostat for Thermally Equilibrating Samples at Known Temperatures for Accurate Cryogenic Luminescence Measurements

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and a CCD spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, fast sample freezing, fast alignment of the sample, accurate and stable sample temperatures, and small size and portability of the Dewar/cryostat cryogenic unit. When coupled with the fast data acquisition rates of the CCD spectrograph, this Dewar/cryostat is capable of supporting cryogenic luminescence spectroscopic measurements on luminescent samples at a series of known, stable temperatures in the 77-300 K range. A temperature-dependent study of the oxygen quenching of luminescence in a rhodium(III) transition metal complex is presented as an example of the type of investigation possible with this Dewar/cryostat. In the context of this apparatus, a stable temperature for cryogenic spectroscopy means a luminescent sample that is thermally equilibrated with either liquid nitrogen or gaseous nitrogen at a known measureable temperature that does not vary (&#916;T &#60; 0.1 K) during the short time scale (~1-10 sec) of the spectroscopic measurement by the CCD. The Dewar/cryostat works by taking advantage of the positive thermal gradient dT/dh that develops above liquid nitrogen level in the Dewar where h is the height of the sample above the liquid nitrogen level. The slow evaporation of the liquid nitrogen results in a slow increase in h over several hours and a consequent slow increase in the sample temperature T over this time period. A quickly acquired luminescence spectrum effectively catches the sample at a constant, thermally equilibrated temperature.

A simple liquid nitrogen Dewar/cryostat apparatus comprised of a small fused silica optical Dewar, a thermocouple, and a charge-coupled device (CCD) spectrograph are described. The experiments for which this Dewar/cryostat is designed require fast sample loading, freezing, and alignment, accurate and stable sample temperatures, and small size/portability.

The design and operation of a simple liquid nitrogen Dewar/cryostat apparatus based upon a small fused silica optical Dewar, a thermocouple assembly, and ...

Engineering

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 ...

Bioengineering

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 ...

Membrane Transport Processes Analyzed by a Highly Parallel Nanopore Chip System at Single Protein Resolution

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.
The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.
High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.
The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.

The presented protocol describes the analysis of membrane protein mediated transport on the single transporter level using pore-spanning solvent-free lipid bilayers. This is achieved by the creation of bulk produced nanopore array chips, combined with highly parallel data acquisition and analysis, enabling the future establishment of membrane protein effector screenings.

Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable ...

Biology

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 ...