We gratefully acknowledge funding from the Wellcome Trust Affordable Healthcare for India and an EPSRC Landscape Fellowship for Ilida Ortega as well as contributions from The Electrospinning Company Ltd.

Ethics Statement: The eyes used in this study were used according to the Declaration on Openness on Animal Research:
1. Fabrication of PLGA Biodegradable Membranes Equipped with Micropockets
<ol>
	<li>The ring scaffolds were created by a combination of microstereolithography and electrospinning techniques31. In essence, the process can be summarized in 2&#160;parts (i) creation of PEGDA templates by microstereolithography and (ii) electrospinning onto the templates for reproduction of the underlying PEGDA structure (in this case a microfabricated ring). These two steps are described in detail below (Figures 1 and 2).
	<ol>
		<li>Fabrication of PEGDA templates by microstereolithography (microSL)
		<ol>
			<li>Fabricate the rings using a 2&#160;layer model, with the first layer (L1) being the base of the structure and the second layer (L2) presenting 6 micropockets with horseshoe morphologies in a range of sizes from 300-500 &#181;m. The fabrication of PEGDA rings was recently described by this group22.
			<ol>
				<li>Create L1 by drawing a 1.2 cm black circle using any suitable drawing program.</li>
				<li>Create L2 in the same way but include 8 white 0.5 x 0.35 mm horseshoe-shaped structures. Distribute the small white horseshoe shapes within the black circle structure.</li>
				<li>Save L1 and L2 in JPEG format.</li>
			</ol>
			</li>
			<li>In a dark glass vial, mix polyethylene glycol diacrylate (PEGDA, Mn = 250 g/mol) with 1% w/w Camphorquinone, a photoinitiator, on a magnetic stirrer for 20 min.</li>
			<li>Expand the laser beam of the microstereolithography set-up using a telescopic lens arrangement and then project it onto a computer programmable digital multimirror device (UV-enabled DMD starter kit). NOTE: The DMD reflects the image (in this case a ring) onto a mirror via a 10 cm focal length tube lens. The image is then directed by the silver-coated mirror into a vial containing the photocurable polymer (PEGDA).</li>
			<li>Adjust and carefully clean the optics of the microstereolithography set up.</li>
			<li>Put 300 &#181;l of the PEGDA mixture into a well of a 12-well tissue culture plate. Make sure the wells are pre-coated with Teflon or other non-stick material for easy removal of the structure after curing.</li>
			<li>Switch on the blue laser (MBL-III 473 nm; 150 mW) and upload L1 into the ALP3-basic software previously installed on the PC. Irradiate the first layer for 60 sec. NOTE: ALP3-basic is a USB interface that provides the link between the PC and the Digital micromirror device.</li>
			<li>Add to the well 250 &#181;l more of the PEGDA, upload L2 as described above and irradiate L2 for 60 sec.</li>
			<li>Remove the uncured polymer and wash the ring with isopropanol O/N.</li>
		</ol>
		</li>
		<li>Fabrication of Biodegradable PLGA membranes using electrospinning 
		NOTE: The PEGDA rings were used as templates over which PLGA was electrospun with the PLGA reproducing the underlying topography as it is spun over these templates. After spinning, the sheet of PLGA polymer was peeled from the collector. The final PLGA membrane did not contain the PEGDA rings which were left attached to the metallic collector.
		<ol>
			<li>Distribute the PEGDA rings on an electroplated aluminum sheet (12 cm x 20 cm) for creating a static electrospinning collector.</li>
			<li>Attach the rings using conductive carbon tape. NOTE: These rings once formed can be re-used as templates for electrospinning.</li>
			<li>Prepare the polymer solution for spinning. Dissolve PLGA (50/50 DL-lactide (52 mol%): glycolide (48 mol%), 44 kg/mol) in dichloromethane (DCM) at 20% w/w concentration.</li>
			<li>Stir O/N before use.</li>
			<li>Place 4&#160;insulin syringes (blunt ended, 0.8 cm inner diameter needles) on a syringe pump. NOTE: Four syringes ensured more rapid electrospinning than working with a single syringe.</li>
			<li>Load 2.5 ml of PLGA solution in each syringe.</li>
			<li>Electrospin using a 30 &#181;l/min flow rate and voltages ranging from 12 to 15 kV. Leave a distance between the needles and the collector of 15 cm.</li>
			<li>Electrospin for 1 hr and 30 min and finally carefully peel the PLGA electrospun sheet from the collector supporting the PEGDA rings.</li>
			<li>Cut the electrospun scaffolds into 22 mm diameter circles using a circular hole punch and leaving the ring structure positioned in the center.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Long-term Storage of PLGA Microfabricated Membranes
Note: The PLGA rings were fabricated and sterilized by accredited external companies; samples were irradiated at an external dose range of 25-40 KGy.
<ol>
	<li>Mount the membrane in a small container (Plastic petri dish) and place it inside a medical grade bag.</li>
	<li>Use filter paper to create small filter bags for the desiccants. To create the bags, fold the filter paper (125 mm diameter) and cut the resulting semicircle in half; then seal the ends together using tape.
	<ol>
		<li>Fill three filter paper bags with 1 g&#160;of silica orange, cobalt (II) chloride, and copper (II) sulfate, respectively.</li>
		<li>Put the three bags of desiccant inside the medical grade bag along with the electrospun membrane.</li>
	</ol>
	</li>
	<li>Add to the bag a commercially available six spot humidity indicator card to detect any moisture accumulation during the storage period.</li>
	<li>Use a vacuum heat seal machine to vacuum and seal the bag.</li>
	<li>Send the ring membranes to an external company for &#947;-irradiation.</li>
	<li>Store &#947;-irradiated PLGA membranes at a wide range of temperatures from -20 &#176;C to +37 &#176;C in a moist environment within an incubator containing 5% CO2.</li>
	<li>Post storage, examine the humidity indicator to confirm that the level of humidity is beneath 30%.</li>
	<li>Use Scanning Electron Microscopy (SEM) to assess fiber integrity.</li>
</ol>
3. Isolation of Limbal Explants
Rabbit limbal explants were isolated from rabbit eyes (obtained from a farm where rabbits are bred for consumption).
<ol>
	<li>Disinfect the rabbit eyes using antiseptic solution (3%).</li>
	<li>Clean the eyes by removing any excess tissue surrounding the cornea.</li>
	<li>Separate the limbal region (identified as a thin circular area between the cornea (transparent) and the sclera (white)) from the rest of the cornea using a dissection microscope.</li>
	<li>Cut into segments (around 1.5 cm long) under the dissection microscope.</li>
	<li>Disinfect the limbal segments in 1.5% antiseptic solution for 1 min.</li>
	<li>Cut the limbal segments into small pieces (100-500 &#181;m) with a scalpel blade.</li>
	<li>Store the small pieces of tissue in culture media (DMEM+Glutamax: Ham&#180;s F12 (1:1), 10% fetal bovine serum, 1 U/ml penicillin, 100 mg/ml streptomycin, 2.5 &#181;g/ml amphotericin, 10 ng/ml of EGF, and 5 &#181;g/ml of insulin) at 37 &#176;C and 5% CO2 until use (no more than 60 min).</li>
</ol>
4. Outgrowth of Cells from Limbal Explants
Rabbit limbal explants were placed on both freshly spun microfabricated membranes and membranes which were vacuum-packed and stored for 6 months at -20&#160;&#176;C.
<ol>
	<li>Coat the ring scaffolds with 15 &#181;l of fibrin glue (1:1 mixture of fibrinogen from human plasma at a concentration of 18.75 mg/ml and thrombin from human plasma at a concentration of 2.5 U/ml) using a cell scraper for distributing the fibrin evenly.</li>
	<li>Place the tissue explants directly on the PLGA micropockets using a 25 G&#160;needle and a dissecting microscope.</li>
	<li>Add cell culture media very gently to avoid detaching the explants.
	<ol>
		<li>Change media every 3 days (media recipe described in section 3.7) and keep in culture for 2 weeks at in a humidified incubator at 37 &#176;C and 5% CO2.</li>
	</ol>
	</li>
	<li>Fix the samples with 3.7% buffered formaldehyde for 10 min&#160;followed by 3 washes with PBS.</li>
	<li>Counterstain by incubation in 1 &#181;g/ml of 4&#39;,6-diamidino-2-phenylindole (DAPI) or propidium iodide (PI) for 10 min&#160;at RT.</li>
	<li>Wash 3 times in PBS and store the samples covered with foil.</li>
	<li>Image the samples using a fluorescence microscope at wavelengths of 543 nm and 800 nm (two-photon).</li>
</ol>
5. Setting-up Rabbit Wounded Cornea 3D Models
<ol>
	<li>Clean and disinfect the rabbit eyes as described above.</li>
	<li>Wound the rabbit eyes by immersing them in 0.14% ammonium hydroxide for 5 min.</li>
	<li>Rinse the eyes in PBS and scrape away the epithelium with a sclerotome knife.</li>
	<li>Cut and isolate the cornea-scleral button removing any remaining tissue.</li>
	<li>Place the corneas epithelial side down onto a sterile cup and fill with 0.5% agar made in DMEM.</li>
	<li>Once set, put the corneas epithelial side up, in small petri dishes and add culture media (recipe described above) up to the limbal area. NOTE: Do not cover the whole cornea; keep the organ culture at the air-liquid interface.</li>
</ol>
6. Isolation of Limbal Explants and Inclusion of Ring Scaffolds in Rabbit Cornea 3D Models
<ol>
	<li>Coat the ring membranes with 15 &#181;l of fibrin glue (1:1 mixture of fibrinogen at a concentration of 18.75 mg/ml and thrombin at a concentration of 2.5 U/ml).Use a cell scraper as described above.</li>
	<li>Place the tissue explants directly on the PLGA micropockets using a 25 G needle and a dissecting microscope.</li>
	<li>Place the rings with the tissue explants on the denuded corneas with the explants facing up and at air-liquid interface conditions. (These conditions have been previously described22).</li>
	<li>Maintain the organ culture models for 4 weeks in a humidified incubator at 37 &#176;C and 5% CO2 changing the media every 2 days.</li>
</ol>
7. Assessment of Corneal Regeneration and Stem Cell Maintenance
<ol>
	<li>After 4&#160;weeks, fix the corneas using 3.7% formaldehyde.</li>
	<li>Process the corneas for conventional histology to produce 6 &#181;m paraffin sections.</li>
	<li>Stain with hematoxylin and eosin (H&#38;E).</li>
	<li>For immunocytochemistry, dewax the sections in xylene (3min) and rehydrate in 100% ethanol (1 min), 70% ethanol (1 min), and distilled water (2 min).</li>
	<li>Delineate the sections using a Dako pen to delimit small areas and avoid excessive use of antibody.
	<ol>
		<li>Treat the delineated areas with 0.05% trypsin (100 &#181;l) for 20 min&#160;(37 &#176;C).</li>
	</ol>
	</li>
	<li>Wash thoroughly with PBS and add 100 &#181;l of 10% goat&#8217;s serum (blocking) for 1 hr.</li>
	<li>Incubate the samples with 100 &#181;l of mouse monoclonal antibody cytokeratin 3 (CK3) and 100 &#181;l of p63 in 1% goat&#8217;s serum O/N at 4 &#176;C.</li>
	<li>Wash with PBS and treat with 100 &#181;l of biotinylated secondary anti-mouse antibody (1:1,000 in 1% goat serum) for 1 hr at RT.</li>
	<li>Add 100 &#181;l of FITC-streptavidin (1:100 in 1% goat serum) for 30 min at RT.</li>
	<li>Treat the samples with DAPI as described above.</li>
	<li>Image the samples using a fluorescence microscope at wavelengths of 800 nm (two-photon) and 488 nm.</li>
</ol>

<ol>
	<li>Ambati, B. K., 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=Corneal+avascularity+is+due+to+soluble+VEGF+receptor-1.">Corneal avascularity is due to soluble VEGF receptor-1.</a> Nature. 443, 993-997 (2006).</li><li>Reinach, P., Cap&#243;-Aponte, J., Mergler, S., Pokorny, K., Tombran-Tin, J., Barnstable, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ophthalmic+Diseases+And+Drug+DeliveryOphthalmology+Research.">Ophthalmic Diseases And Drug DeliveryOphthalmology Research.</a> Ch 2, Ocular Transporters. 2, 17-46 (2008).</li><li>Kinoshita, 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=Characteristics+of+the+Human+Ocular+Surface+Epithelium.">Characteristics of the Human Ocular Surface Epithelium.</a> Progress in Retinal and Eye Research. 20, 639-673 (2001).</li><li>Fatt, I., Bieber, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+steady-state+distribution+of+oxygen+and+carbon+dioxide+in+the+in+vivo+cornea:+I.+The+open+eye+in+air+and+the+closed+eye.">The steady-state distribution of oxygen and carbon dioxide in the in vivo cornea: I. The open eye in air and the closed eye.</a> Experimental Eye Research. 7, 103-112 (1968).</li><li>Shortt, A. J., 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=Characterization+of+the+Limbal+Epithelial+Stem+Cell+Niche:+Novel+Imaging+Techniques+Permit+In+Vivo+Observation+and+Targeted+Biopsy+of+Limbal+Epithelial+Stem+Cells.">Characterization of the Limbal Epithelial Stem Cell Niche: Novel Imaging Techniques Permit In Vivo Observation and Targeted Biopsy of Limbal Epithelial Stem Cells.</a> STEM CELLS. 25, 1402-1409 (2007).</li><li>Dua, H. S., Shanmuganathan, V. A., Powell-Richards, A. O., Tighe, P. J., Joseph, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+epithelial+crypts:+a+novel+anatomical+structure+and+a+putative+limbal+stem+cell+niche.">Limbal epithelial crypts: a novel anatomical structure and a putative limbal stem cell niche.</a> British Journal of Ophthalmology. 89, 529-532 (2005).</li><li>Thoft, R. A., Friend, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+X,+Y,+Z+hypothesis+of+corneal+epithelial+maintenance.">The X, Y, Z hypothesis of corneal epithelial maintenance.</a> Investigative Ophthalmolog., &amp; Visual Science. 24, 1442-1443 (1983).</li><li>Lehrer, M. S., Sun, T. T., Lavker, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+of+epithelial+repair:+modulation+of+stem+cell+and+transit+amplifying+cell+proliferation.">Strategies of epithelial repair: modulation of stem cell and transit amplifying cell proliferation.</a> Journal of Cell Science. 111, 2867-2875 (1998).</li><li>Djalilian, 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=Down-regulation+of+Notch+signaling+during+corneal+epithelial+proliferation.">Down-regulation of Notch signaling during corneal epithelial proliferation.</a> Molecular Vision. 14, 1041-1049 (2008).</li><li>Dua, H. S., Azuara-Blanco, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limbal+Stem+Cells+of+the+Corneal+Epithelium.+Survey+of+Ophthalmology.">Limbal Stem Cells of the Corneal Epithelium. Survey of Ophthalmology.</a> Survey. 44, 415-425 (2000).</li><li>Dravida, 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=A+biomimetic+scaffold+for+culturing+limbal+stem+cells:+a+promising+alternative+for+clinical+transplantation.">A biomimetic scaffold for culturing limbal stem cells: a promising alternative for clinical transplantation.</a> Journal of tissue engineering and regenerative medicine. 2, 263-271 (2008).</li><li>Rama, P., 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=Limbal+Stem-Cell+Therapy+and+Long-Term+Corneal+Regeneration.">Limbal Stem-Cell Therapy and Long-Term Corneal Regeneration.</a> New England Journal of Medicine. 363, 147-155 (2010).</li><li>Koizumi, N., Inatomi, T., Suzuki, T., Sotozono, C., Kinoshita, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivated+corneal+epithelial+stem+cell+transplantation+in+ocular+surface+disorders.">Cultivated corneal epithelial stem cell transplantation in ocular surface disorders.</a> Ophthalmology. 108, 1569-1574 (2001).</li><li>Fatima, 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=Technique+of+cultivating+limbal+derived+corneal+epithelium+on+human+amniotic+membrane+for+clinical+transplantation.">Technique of cultivating limbal derived corneal epithelium on human amniotic membrane for clinical transplantation.</a> Journal of Postgraduate Medicine. 52, 257-261 (2006).</li><li>Nubile, 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+transplantation+for+the+management+of+corneal+epithelial+defects:+an+in+vivo+confocal+microscopic+study.">Amniotic membrane transplantation for the management of corneal epithelial defects: an in vivo confocal microscopic study.</a> British Journal of Ophthalmology. 92, 54-60 (2008).</li><li>Nubile, 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=In+Vivo+Analysis+of+Stromal+Integration+of+Multilayer+Amniotic+Membrane+Transplantation+in+Corneal+Ulcers.">In Vivo Analysis of Stromal Integration of Multilayer Amniotic Membrane Transplantation in Corneal Ulcers.</a> American Journal of Ophthalmology. 151, 809-822 (2011).</li><li>Dua, H. S. <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.">Amniotic membrane transplantation.</a> British Journal of Ophthalmology. 83, 748-752 (1999).</li><li>Gicquel, J., 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=Epidermal+Growth+Factor+Variations+in+Amniotic+Membrane+Used+for+Ex+Vivo+Tissue+Constructs.">Epidermal Growth Factor Variations in Amniotic Membrane Used for Ex Vivo Tissue Constructs.</a> Tissue Engineering Part A. 15, 1919-1927 (2009).</li><li>Connon, C. J., 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+variation+in+transparency+of+amniotic+membrane+used+in+ocular+surface+regeneration.">The variation in transparency of amniotic membrane used in ocular surface regeneration.</a> British Journal of Ophthalmology. 94, 1057-1061 (2010).</li><li>Hopkinson, A., McIntosh, R. S., Tighe, P. J., James, D. K., Dua, H. S. <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+for+Ocular+Surface+Reconstruction:+Donor+Variations+and+the+Effect+of+Handling+on+TGF-&#946;+Content.">Amniotic Membrane for Ocular Surface Reconstruction: Donor Variations and the Effect of Handling on TGF-&#946; Content.</a> Investigative Ophthalmolog., &amp; Visual Science. 47, 4316-4322 (2006).</li><li>Sitalakshmi, G., 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=Ex+Vivo+Cultivation+of+Corneal+Limbal+Epithelial+Cells+in+a+Thermoreversible+Polymer+(Mebiol+Gel)+and+Their+Transplantation+in+Rabbits:+An+Animal+Model.">Ex Vivo Cultivation of Corneal Limbal Epithelial Cells in a Thermoreversible Polymer (Mebiol Gel) and Their Transplantation in Rabbits: An Animal Model.</a> Tissue Engineering Part A. 15, 407-415 (2008).</li><li>Ortega, I., Deshpande, P., Gill, A. A., MacNeil, S., Claeyssens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+microfabricated+artificial+limbus+with+micropockets+for+cell+delivery+to+the+cornea.">Development of a microfabricated artificial limbus with micropockets for cell delivery to the cornea.</a> Biofabrication. 5, (2013).</li><li>Deshpande, P., 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=Using+poly(lactide-co-glycolide)+electrospun+scaffolds+to+deliver+cultured+epithelial+cells+to+the+cornea.">Using poly(lactide-co-glycolide) electrospun scaffolds to deliver cultured epithelial cells to the cornea.</a> Regenerative Medicine. 5, 395-401 (2010).</li><li>Deshpande, P., 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=Simplifying+corneal+surface+regeneration+using+a+biodegradable+synthetic+membrane+and+limbal+tissue+explants.">Simplifying corneal surface regeneration using a biodegradable synthetic membrane and limbal tissue explants.</a> Biomaterials. 34, 5088-5106 (2013).</li><li>Wheeldon, I., Ahari, A. F., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microengineering+Hydrogels+for+Stem+Cell+Bioengineering+and+Tissue+Regeneration.">Microengineering Hydrogels for Stem Cell Bioengineering and Tissue Regeneration.</a> Charlottesv Va. 15, 440-448 (2010).</li><li>Moeller, H. C., Mian, M. K., Shrivastava, S., Chung, B. G., Khademhosseini, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microwell+array+system+for+stem+cell+culture.">A microwell array system for stem cell culture.</a> Biomaterials. 29, 752-763 (2008).</li><li>Fisher, O. Z., Khademhosseini, A., Langer, R., Peppas, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioinspired+Materials+for+Controlling+Stem+Cell+Fate.">Bioinspired Materials for Controlling Stem Cell Fate.</a> Accounts Chem. Res. 43, 419-428 (2010).</li><li>Raimondi, M. 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=Three-dimensional+structural+niches+engineered+via+two-photon+laser+polymerization+promote+stem+cell+homing.">Three-dimensional structural niches engineered via two-photon laser polymerization promote stem cell homing.</a> Acta Biomaterialia. 9, 4579-4584 (2013).</li><li>Gobaa, 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=Artificial+niche+microarrays+for+probing+single+stem+cell+fate+in+high+throughput.">Artificial niche microarrays for probing single stem cell fate in high throughput.</a> Nat Meth. 8, 949-955 (2011).</li><li>Truckenm&#252;ller, 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=Fabrication+of+cell+container+arrays+with+overlaid+surface+topographies.">Fabrication of cell container arrays with overlaid surface topographies.</a> Biomedical Microdevices. 14, 95-107 (2012).</li><li>Ortega, &. #. 2. 0. 5. ;., Ryan, A. J., Deshpande, P., MacNeil, S., Claeyssens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+microfabrication+and+electrospinning+to+produce+3-D+architectures+for+corneal+repair.">Combined microfabrication and electrospinning to produce 3-D architectures for corneal repair.</a> Acta Biomaterialia. 9, 5511-5520 (2013).</li><li>Blackwood, K. 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=Development+of+biodegradable+electrospun+scaffolds+for+dermal+replacement.">Development of biodegradable electrospun scaffolds for dermal replacement.</a> Biomaterials. 29, 3091-3104 (2008).</li></ol>

The authors have nothing to disclose.

This study describes (a) a technique for the fabrication of electrospun membranes containing microfeatures within them and (b) how to prepare such membranes for clinical use by vacuum packing, gamma irradiation and then storage prior to use. In this particular application we have developed PLGA membranes containing micropockets which mimic the physical features of the limbal stem cell niches. The aims of this study are (i) to describe methods to provide readers with the knowledge needed to design and fabricate scaffolds containing microfeatures for research into the contribution of stem cell niches to tissue regeneration and (ii) to provide the reader with a better understanding of how to store electrospun scaffolds for long periods of time.
In terms of clinical application, the storage of the ring membranes is of paramount importance. In this work, the degradation of the ring was studied over a period of 6 months. Degradation of the membranes is driven by hydrolysis so by simply keeping the membranes moisture-free the process is halted. Blackwood et al.&#160;reported that by varying the ratio of PLA to PGA, the degradation of the membrane changes32. This study also showed that by increasing the amount of PGA, the degradation rate of electrospun membranes increased in vivo22. Here it has been shown that with vacuum packing the membranes along with some desiccant and irradiating them and storing them at low temperatures for 6 months, there is no change in the fiber integrity and degradation. At present, 6 months is as far as we have studied with these membranes containing micropockets but storage data for 1 year has been reported on plain electrospun membranes at -20 &#176;C22 and we now have unpublished data for their storage at -20 &#176;C for 2 years without any signs of degradation. Thus for long term storage it would be recommended to store them dry at -20 &#176;C but it is possible to store them at RT even in India for at least 6 months (possibly much longer). The inclusion of a humidity indicator gives an easy means of checking that the packaging has kept membranes dry in which case they will be fit for the purpose.
Transfer of cells from these rings to 3D cornea models was shown when placing limbal explants within the micropockets. This group recently reported transfer of cells onto an in vitro rabbit cornea model by placing explants on plain PLGA membranes (membranes without ring structures)24. Using the present microfabricated scaffolds cell transfer has been taken one step further as we can now specifically locate tissue explants within the microfeatures. The ability to place the explants directly within the niches also allows the surgeon to use the membranes directly in the surgical theatre avoiding the need of a cleanroom to first expand the limbal stem cells. Although this piece of work has been focused on the development of devices for corneal disease, this microfabrication technology can be also applied for developing devices for many other applications. Future work will explore the fabrication of constructs for the regeneration of other tissues such as skin and bone.
Whilst the design and initial fabrication of the PEGDA microstructures can be time consuming, once fabricated the structures can be reused many times without degradation. Therefore, the subsequent fabrication of PLGA microstructured biodegradable membranes by electrospinning can be performed at a comparable rate to the production of plain (&#39;unstructured&#39;) membranes following the assembly of the collector. Although in this work we have used microstereolithography for fabricating the molds, other fabrication methods such as 3D-printing or injection-molding could be also used. Accordingly, the underlying mold could be made out of other polymers or metals instead of PEGDA. As such this technique is very versatile and researchers can easily adapt the method to match their own needs and facilities.
The in-house microstereolithography set-up used in this study will not allow the preparation of constructs with features under 30&#181;m; this is not a limitation for the corneal application described here but it could be crucial in the design of other models. In that case other techniques such as 2 photon polymerization (2PP) could be of interest however the electrospinning technique might not allow the reproduction of structures on the sub-micron scale (this is currently being studied by our group).
Critical steps within the fabrication process are (i) Avoiding the overcuring of the PEGDA templates which can be controlled by adjusting time and quantities of photoinitiator. (ii) Controlling electrospinning conditions such as temperature and humidity. (iii) Storing appropriately the electrospun ring membranes using vacuum-packing and desiccants.
In summary, by placing limbal tissue explants within the microfeatures of the membrane we have shown cell outgrowth from the explants on the niche areas, cell transfer onto a rabbit wounded cornea and subsequent re-epithelization of the cornea. The degradation of the membranes stored at different temperatures has also been studied and a packaging protocol which allows long term storage of membranes has been developed, the latter being essential in developing membranes for clinical use.

The cornea, the avascular central outer most tissue of the eye, is one of the most important tissues involved in vision1. There are several types of cells that maintain the function of the cornea. The top outermost layer of the cornea comprises of epithelial cells which can be about 5-7 layers in thickness2. This layer prevents bacterial invasion into the cornea3 and allows entry of oxygen4. It has been reported that the stem cells of the corneal epithelium lie in niches or crypts (with sizes of 120-150 &#181;m) at the peripheral region of the cornea known as the limbus5,6. As the stem cells divide, the daughter cells also known as transient amplifying cells travel out of the niches and as division continues the cells move centripetally inwards and upwards resulting in terminally differentiated cells at the central corneal region7,8. These cells are routinely wiped away with the blink of the eye exposing newer cells underneath9.
In addition to being the location of the epithelial stem cells, the limbus also plays a role in keeping the vascularized conjunctiva away from the cornea region10. Damage to the limbus could be caused by thermal/chemical burns, radiation and also genetic diseases10. When this happens, the limbus barrier is broken down allowing the conjunctival cells to move onto the cornea, vascularizing the region, causing pain and blindness in some cases. The condition is known as limbal stem cell deficiency (LSCD)10.
Different natural substrates have been reported as possible stem cell carriers for aiding in corneal regeneration. For example, collagen-based membranes were used by Dravida et al.11 and Rama and coworkers12 reported the use of fibrin in a study with 112 patients. At present however the most commonly used method of treatment is to use human amniotic membrane from a tissue bank and culture limbal epithelial cells on its surface13,14. Once a monolayer has formed, the amniotic membrane is glued cell-side up onto the damaged cornea which has all the conjunctival cells and scar tissue surgically removed from it prior to this cell transplantation14. The amniotic membrane degrades within weeks to months leaving the epithelial cells attached to the denuded area to regenerate the epithelium15,16. This technique has been successful in restoring vision however there are still a few practical issues which restrict its widespread uptake clinically. As the amniotic membrane is human tissue it needs to undergo screening using good tissue banking procedures before being used for cell transplantation on patients. This screening only lowers the risk of transmission of diseases but cannot completely eliminate it17. In addition to this there have been reports of variability in the performance of the amniotic membrane due to inter donor variation18,19 and different processing methods19,20. Alongside the small risk of disease transmission there is the requirement for surgical centers to have access to well-run tissue banks, not available to all.
Although the amniotic membrane is relatively successful, there is a need for the development of new synthetic biodegradable cell carrier alternatives for the treatment of corneal disease. Synthetic carriers would overcome the need for banking procedures as well as eliminating the small risk of disease transmission and inter-donor variability. In this sense, materials such as polyethylene glycol21,22 and PLGA23,24 have been studied.
In developing a synthetic alternative to the human amniotic membrane there is also the possibility to design into it desirable features to hopefully help the survival of the cultured cells. The inclusion of microfeatures within biomaterial devices for the specific control of cell behavior is an emerging area of interest. Many authors have reported work towards the development of artificial stem cells niches25-30. This group has recently reported the creation of a microfabricated PEGDA fibronectin-biofunctionalized artificial limbus for the delivery of limbal epithelial cells22 and a methodology for the fabrication of electrospun biodegradable membranes containing microfabricated pockets for the support of limbal epithelial cells31.
The aim of this work is to develop a new manufacturing technology for the development of biomaterial devices containing microfeatures which mimic to an extent the microenvironments in which stem cells reside in the body. We have developed a technique which combines microstereolithography and electrospinning that allows the fabrication of biodegradable microstructured membranes that show great potential for tissue regeneration applications.
It is important to notice that although in this work this technique has been applied to the fabrication of rings for corneal regeneration, the technology can be applied to the fabrication of devices for the regeneration of a broad range of epithelial tissues, e.g., skin, oral mucosa, intestine, respiratory, and bladder epithelia. Specifically, in this study we have developed a synthetic biodegradable membrane which functions in a similar manner to the amniotic membrane to deliver cells to the cornea. This membrane contains micropockets of around 300 &#181;m (larger than the limbal crypts of the Pallisades of Vogt (around 150 &#181;m)). Finally, we have established a packaging protocol which allows these membranes to be stored at -20 &#176;C for more than 6 months without showing any signs of breakdown.

<table border="0" cellpadding="0" cellspacing="0" width="692">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:183px;">Name of&#160; reagents/material/equipment</td>
			<td style="width:173px;">Company</td>
			<td style="width:157px;">Catalogue number</td>
			<td style="width:180px;">Comments (optional)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:183px;">Poly lactic-co-glycolic acid</td>
			<td style="width:173px;">Purac</td>
			<td style="width:157px;">PDLG5004</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:183px;">Dichloromethane</td>
			<td style="width:173px;">Sigma Aldrich Or Fisher</td>
			<td style="width:157px;">270997 Or D/1850/17</td>
			<td style="width:180px;">&#62;99.8% contains 50-150 ppm amylene stabiliser</td>
		</tr>
		<tr height="113">
			<td height="113" style="height:113px;width:183px;">Digital Micromirror Device (DMD) Discovery 1100 Controller Board &#38; Starter Kit</td>
			<td style="width:173px;">Texas Instruments</td>
			<td style="width:157px;">1076N732 (UV)</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">473 nm Laser</td>
			<td style="width:173px;">Laser 2000</td>
			<td style="width:157px;">MBL-III</td>
			<td style="width:180px;">150 mW</td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:183px;">Poly (ethylene glycol) diacrylate</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">475629</td>
			<td style="width:180px;">Mn = 250g/mol&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 500 ml</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">DEMEM + Glutamax</td>
			<td style="width:173px;">Fisher</td>
			<td style="width:157px;">12077549</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:183px;">Ham&#8217; s F12</td>
			<td style="width:173px;">Labtech biosera</td>
			<td style="width:157px;">LMH1236/500</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Fetal Bovine Serum</td>
			<td style="width:173px;">Labtech biosera</td>
			<td style="width:157px;">FB-1090/50</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">EGF</td>
			<td style="width:173px;">R&#38;D</td>
			<td style="width:157px;">236-EG-200</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Insulin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">91077C-1G</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Amphotericin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">A2942-100ml</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Penicillin/Streptomycin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">P0781-100ml</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="22">
			<td height="39" rowspan="2" style="height:39px;width:183px;">DAPI</td>
			<td rowspan="2" style="width:173px;">Sigma Aldrich</td>
			<td rowspan="2" style="width:157px;">32670</td>
			<td rowspan="2" style="width:180px;"></td>
		</tr>
		<tr height="17">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Propidium Iodide</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">P4864</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Thrombin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">T9326</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Fibrinogen</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">F3879</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">p63</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">P3737</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">CK3</td>
			<td style="width:173px;">Merck Millipore</td>
			<td style="width:157px;">CLB218</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Hematoxylin</td>
			<td style="width:173px;">SLS</td>
			<td style="width:157px;">HHS16-500ML</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:183px;">Eosin</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">HT110232-1L</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:183px;">Medical grade bag (PET/Foil/LDPE) Peelable pouch</td>
			<td style="width:173px;">Riverside Medical Ltd. Derby, UK</td>
			<td style="width:157px;">Foil laminate PET/Foil/LDPE, (12,7,50)</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="140">
			<td height="140" style="height:140px;width:183px;">gamma- irradiation (Sterilisation)</td>
			<td style="width:173px;">Applied Sterilisation Technologies (Synergy Health Laboratory Services (SHLS), Abergavenny UK) - external dose range of 25-40KGy</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:183px;">Silica gel orange</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">10087</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:183px;">Cobalt (II) chloride</td>
			<td style="width:173px;">Sigma Aldrich</td>
			<td style="width:157px;">232696</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:183px;">Copper (II) sulphate</td>
			<td style="width:173px;">Sigma-Aldrich</td>
			<td style="width:157px;">C-1297</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:183px;">Six spot humidity indicator card</td>
			<td style="width:173px;">SCC, USA</td>
			<td style="width:157px;">6HIC200</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:183px;">Vacuum heat seal machine</td>
			<td style="width:173px;">Andrew James UK Ltd, Bowburn, UK</td>
			<td style="width:157px;">VS518</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="71">
			<td height="71" style="height:71px;width:183px;">Andrew James Vacuum Sealer rolls 28cm X 40 metre rolls</td>
			<td style="width:173px;">Andrew James UK Ltd, Bowburn, UK</td>
			<td style="width:157px;">BR2805</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:183px;">Scanning Electron Microscopy (SEM)</td>
			<td style="width:173px;">Philips/FEI XL-20 SEM</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:183px;">Confocal Microscope</td>
			<td style="width:173px;">Zeiss LSM 510 META</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="32">
			<td height="32" style="height:32px;width:183px;">Videne Antiseptic Solution</td>
			<td>Ecolab, Swindon, UK</td>
			<td style="width:157px;">N/A</td>
			<td style="width:180px;">3%</td>
		</tr>
	</tbody>
</table>

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.

Electrospun microfabricated rings were manufactured using a combination of microstereolithography and electrospinning (Figures 1 and 2). PEGDA rings of different sizes were fabricated using microstereolithography (Figure 3); this technique allows the fabrication structures in the order of cm and the simultaneous incorporation of microfeatures. In this case, rings of diameters ranging from 1.2-1.6 cm containing micropockets of 350-500 &#181;m were fabricated (Figure 4).
In terms of producing, sterilizing and packaging of materials for future clinical use it was found that vacuum packing in medical grade bags significantly improved the ability to achieve long term storage of PLGA membranes (Figure 5); the use of a medical grade bag (PET/Foil/LDPE) with thickness of 0.12 mm allowed us to achieve a longer shelf life. This was investigated by sending membranes to our collaborators in India and membranes were stored for a period of months at -20 &#176;C, at RT and at 37 &#176;C in deliberately moist conditions (a moist incubator). Figure 5 shows that using the deliberately provocative conditions of storage at 37 &#176;C under moist conditions, membranes were only stable for approximately 1 month under non-vacuum packed conditions, but achieved 3 months storage under vacuum packed conditions (Figure 5 and Table 1).
Table 1 demonstrates the improvement in storage conditions that can be achieved even under conditions selected to be conducive to water uptake and fiber swelling if one pays attention to the choice of bag used.
The rings supported cell outgrowth from limbal explants in different conditions (i) rings freshly made and (ii) rings stored for 6 months (Figure 6). Cell transfer was achieved after 4 weeks when placing the PLGA membranes on 3D wounded models. Cells grew out from the tissue explants placed on the membranes creating a new epithelium on the previously denuded corneas (Figure 7). Positive (corneas without any treatment) and negative controls (wounded corneas) were also maintained in culture for the same periods of time. The negative controls confirmed the lack of formation of a new epithelium in the absence of any added cells. Immunocytochemistry demonstrated that the cells growing out from the explants were corneal epithelial cells since they were positive for the corneal differentiation marker CK3 (Figure 7E).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig1highres.jpg" width="500" /> 
Figure 1. Schematic of microstereolithography set up for the creation of PEGDA rings.
<img alt="Figure 2" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig2highres.jpg" width="500" /> 
Figure 2. Schematic of electrospinning process using PEGDA microfabricated rings as a templates.
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig3highres.jpg" width="500" /> 
Figure 3. (A)&#160;shows an example of a static collector (electroplated aluminum sheet with PEGDA rings) for the spinning of microfabricated PLGA membranes. (B) and (E) show different electrospun mats being peeled from static collectors. (C) shows PEGDA templates of different sizes highlighting the versatility of using microstereolithography for the fabrication of the underlying surface. (D) shows a PLGA microfabricated replica. <a href="https://www.jove.com/files/ftp_upload/51826/51826fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 4" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig4highres.jpg" width="500" /> 
Figure 4. SEM image of a PEGDA ring with a horseshoe microfeature (A); high magnification SEM image of a microfabricated pocket (B). Phase contrast image of a PLGA ring with a horseshoe microfeature (C); high magnification phase contrast image of a microfabricated pocket (D). <a href="https://www.jove.com/files/ftp_upload/51826/51826fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 5" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig5highres.jpg" width="500" /> 
Figure 5. Effect of temperature and time on storage of vacuum and non-vacuum packed PLGA (50/50) membranes (44 kg/mol) with micro-fabricated rings over 6 months. Membrane integrity was scored as fully intact fibers (+++), some fiber swelling (++), fiber merging (+) or no intact fibers (-). SEM images and three desiccants (silica orange, cobalt (II) chloride, and copper (II) sulfate) show no changes in fiber integrity or humidity. <a href="https://www.jove.com/files/ftp_upload/51826/51826fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 6" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig6highres.jpg" width="500" /> 
Figure 6. Fluorescence images showing outgrowth of LEC from limbal explants on freshly made biodegradable PLGA rings (A, B) and on rings after 6 months storage at -20 &#176;C (C, D). Images (A) and (B) correspond to cells stained with DAPI (blue) and propidium iodide (red) respectively. Image b is an orthogonal view from a confocal z-stack of an explant placed on a microfabricated pocket. Images (C) and (D) show positive staining for p63 (green). <a href="https://www.jove.com/files/ftp_upload/51826/51826fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 7" fo:content-width="5in" src="/files/ftp_upload/51826/51826fig7highres.jpg" width="500" /> 
Figure 7. (A)&#160;shows a rabbit wounded cornea model with a ring scaffold and tissue explants located on the scaffold which was previously coated with fibrin glue. (B) and (C) are positive and negative controls; the positive control is a fresh rabbit cornea and the negative control a cornea where the epithelium was deliberately removed (the negative control was also cultured for 4 weeks). (D) is a H&#38;E image of a tissue engineered cornea after 4 weeks in culture; the figure shows the new multi-layered epithelium formed by the cells coming out from the explants placed on the niches. (E) is an immunocytochemistry image showing cell outgrowth from a limbal explant; nuclei are stained with DAPI (blue) and the cells shows positive staining for cytokeratin 3, a corneal differentiation marker (green). <a href="https://www.jove.com/files/ftp_upload/51826/51826fig7highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always" width="611">
	<tbody>
		<tr height="22">
			<td height="43" rowspan="2" style="height:43px;width:163px;">PLGA (50/50)</td>
			<td colspan="7" style="width:448px;">Fiber integrity for membranes stored at 37 &#176;C moist</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:64px;">Day 0</td>
			<td style="width:64px;">Month 1</td>
			<td style="width:64px;">Month 2</td>
			<td style="width:64px;">Month 3</td>
			<td style="width:64px;">Month 4</td>
			<td style="width:64px;">Month 5</td>
			<td style="width:64px;">Month 6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Non-Vacuum Packed</td>
			<td style="width:64px;">+++</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:163px;">Vacuumed (Bag A) (PE, PA Composite) Thickness: 0.14 mm</td>
			<td style="width:64px;">+++</td>
			<td style="width:64px;">+</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:163px;">Vacuumed (Bag B) (PET / Foil / LDPE) Thickness: 0.75 mm</td>
			<td style="width:64px;">+++</td>
			<td style="width:64px;">+++</td>
			<td style="width:64px;">+++</td>
			<td style="width:64px;">+</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
			<td style="width:64px;">-</td>
		</tr>
	</tbody>
</table>
Table 1. Effect of vacuum and different storage bags on integrity of PLGA (50/50) membranes (44 kg/mol) examined over 6 months of storage. Membrane integrity was scored as fully intact fibers (+++), some fiber swelling (++), fiber merging (+) or no intact fibers (-).

Watch this Scientific Journal Video about Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration at JoVE.com

Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

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 ...

combination-microstereolithography-electrospinning-to-produce

Research

JoVE Journal

Bioengineering

12.4K ビュー.  University of Sheffield. 私たちは、細胞の挙動を研究する中でエレクトロスピニング膜内micropocketsを製造するための技術を報告している。具体的には、マイクロ光の組み合わせを記述し、微小特徴を備えたPLGA（ポリ（ラクチド - コ - グリコリド））角膜の生体材料デバイスの製造のためのエレクトロスピニング。 

ビデオ: Combination of Microstereolithography and Electrospinning to Produce Membranes Equipped with Niches for Corneal Regeneration

Electrospinning Fundamentals: Optimizing Solution and Apparatus Parameters

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. Electrospinning is a process in which a charged polymer jet is collected on a grounded collector; a rapidly rotating collector results in aligned nanofibers while stationary collectors result in randomly oriented fiber mats. The polymer jet is formed when an applied electrostatic charge overcomes the surface tension of the solution. There is a minimum concentration for a given polymer, termed the critical entanglement concentration, below which a stable jet cannot be achieved and no nanofibers will form - although nanoparticles may be achieved (electrospray). A stable jet has two domains, a streaming segment and a whipping segment. While the whipping jet is usually invisible to the naked eye, the streaming segment is often visible under appropriate lighting conditions. Observing the length, thickness, consistency and movement of the stream is useful to predict the alignment and morphology of the nanofibers being formed. A short, non-uniform, inconsistent, and/or oscillating stream is indicative of a variety of problems, including poor fiber alignment, beading, splattering, and curlicue or wavy patterns. The stream can be optimized by adjusting the composition of the solution and the configuration of the electrospinning apparatus, thus optimizing the alignment and morphology of the fibers being produced. In this protocol, we present a procedure for setting up a basic electrospinning apparatus, empirically approximating the critical entanglement concentration of a polymer solution and optimizing the electrospinning process. In addition, we discuss some common problems and troubleshooting techniques.

Electrospinning techniques can create a variety of nanofibrous scaffolds for tissue engineering or other applications. We describe here a procedure to optimize the parameters of the electrospinning solution and apparatus to obtain fibers with the desired morphology and alignment. Common problems and troubleshooting techniques are also presented.

Electrospun nanofiber scaffolds have been shown to accelerate the maturation, improve the growth, and direct the migration of cells in vitro. ...

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Preparation of Light-responsive Membranes by a Combined Surface Grafting and Postmodification Process

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is presented. The polymerization from the membrane surface is induced by plasma treatment of the membrane, followed by reacting the membrane surface with a methanolic solution of 2-hydroxyethyl methacrylate (HEMA). Special attention is given to the process parameters for the plasma treatment prior to the polymerization on the surface. For example, the influence of the plasma-treatment on different types of membranes (e.g.&#160;polyester, polycarbonate, polyvinylidene fluoride) is studied. Furthermore, the time-dependent stability of the surface-grafted membranes is shown by contact angle measurements. When grafting poly(2-hydroxyethyl methacrylate) (PHEMA) in this way, the surface can be further modified by esterification of the alcohol moiety of the polymer with a carboxylic acid function of the desired substance. These reactions can therefore be used for the functionalization of the membrane surface. For example, the surface tension of the membrane can be changed or a desired functionality as the presented light-responsiveness can be inserted. This is demonstrated by reacting PHEMA with a carboxylic acid functionalized spirobenzopyran unit which leads to a light-responsive membrane. The choice of solvent plays a major role in the postmodification step and is discussed in more detail in this paper. The permeability measurements of such functionalized membranes are performed using a Franz cell with an external light source. By changing the wavelength of the light from the visible to the UV-range, a change of permeability of aqueous caffeine solutions is observed.

A plasma-induced polymerization procedure is described for the surface-initiated polymerization on polymer membranes. Further postmodification of the grafted polymer with photochromic substances is presented with a protocol of conducting permeability measurements of light-responsive membranes.

In order to modify the surface tension of commercial available track-edged polymer membranes, a procedure of surface-initiated polymerization is ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

Synthesis of Hydrogels with Antifouling Properties As Membranes for Water Purification

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and thus achieving stable water permeability through membranes over time. Here, we report a facile method to prepare hydrogels based on zwitterions for membrane applications. Freestanding films can be prepared from sulfobetaine methacrylate (SBMA) with a crosslinker of poly(ethylene glycol) diacrylate (PEGDA) via photopolymerization. The hydrogels can also be prepared by impregnation into hydrophobic porous supports to enhance the mechanical strength. These films can be characterized by attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) to determine the degree of conversion of the (meth)acrylate groups, using goniometers for hydrophilicity and differential scanning calorimetry (DSC) for polymer chain dynamics. We also report protocols to determine the water permeability in dead-end filtration systems and the effect of foulants (bovine serum albumin, BSA) on membrane performance.

This paper reports practical methods to prepare hydrogels in freestanding films and impregnated membranes and to characterize their physical properties, including water transport properties.

Hydrogels have been widely utilized to enhance the surface hydrophilicity of membranes for water purification, increasing the antifouling properties and ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Assembly and Characterization of Biomolecular Memristors Consisting of Ion Channel-doped Lipid Membranes

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the cognitive powers of the brain with comparable efficiency and density. To date, silicon-based three-terminal transistors and two-terminal memristors have been widely used in neuromorphic circuits, in large part due to their ability to co-locate information processing and memory. Yet these devices cannot achieve the interconnectivity and complexity of the brain because they are power-hungry, fail to mimic key synaptic functionalities, and suffer from high noise and high switching voltages. To overcome these limitations, we have developed and characterized a biomolecular memristor that mimics the composition, structure, and switching characteristics of biological synapses. Here, we describe the process of assembling and characterizing biomolecular memristors consisting of a 5 nm-thick lipid bilayer formed between lipid-functionalized water droplets in oil and doped with voltage-activated alamethicin peptides. While similar assembly protocols have been used to investigate biophysical properties of droplet-supported lipid membranes and membrane-bound ion channels, this article focuses on key modifications of the droplet interface bilayer method essential for achieving consistent memristor performance. Specifically, we describe the liposome preparation process and the incorporation of alamethicin peptides in lipid bilayer membranes, and the appropriate concentrations of each constituent as well as their impact on the overall response of the memristors. We also detail the characterization process of biomolecular memristors, including measurement and analysis of memristive current-voltage relationships obtained via cyclic voltammetry, as well as short-term plasticity and learning in response to step-wise voltage pulse trains.

Soft, low-power, biomolecular memristors leverage similar composition, structure, and switching mechanisms of bio-synapses. Presented here is a protocol to assemble and characterize biomolecular memristors obtained from insulating lipid bilayers formed between water droplets in oil. The incorporation of voltage-activated alamethicin peptides results in memristive ionic conductance across the membrane.

The ability to recreate synaptic functionalities in synthetic circuit elements is essential for neuromorphic computing systems that seek to emulate the ...