We are grateful for the financial support of the National Natural Science Funds of China (31770968) and the Tianjin Research Program of Application Foundation and Advanced Technology (17JCZDJC33400).

1. Fabrication of Heparin-incorporated Starpeg Nanocoating
<ol>
	<li>Synthesis of heparin succinimidyl succinate (Heparin-NHS)
	<ol>
		<li>Weigh out 0.69 g of heparin and dissolve in 2.0 mL of ice-cold deionized water.</li>
		<li>Weigh out 0.23 g of NHS and dissolve in 0.5 mL of ice-cold deionized water in a round-bottom flask.</li>
		<li>Weigh out 0.77 g of EDC and dissolve in 0.5 mL of ice-cold deionized water in a round-bottom flask.</li>
		<li>Mix the NHS and EDC solutions in a round-bottom flask. Leave the mixed solution on the bench at room temperature for 30 min at room temperature.</li>
		<li>Centrifuge the EDC NHS mixture at 5,031 x g for 10 min to remove excess EDC and NHS.</li>
		<li>Add 30 mL of cold ethanol to the mixture solution and centrifuge at 5,031 x g for 10 min.
		<ol>
			<li>Repeat twice.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of starPEG-heparin nanocoating
	<ol>
		<li>Add 1.0 mL of 10% (w/v) starPEG-(NH2)8 to a round-bottom flask.</li>
		<li>Add 0.67 mL of 10% (w/v) heparin-NHS to the same round-bottom flask.</li>
		<li>Place the mixed solution of starPEG-(NH2)8 and heparin-NHS in an incubator at 25 &#176;C for 20 min to obtain a clear solution with viscosity. 
		&#8203;NOTE: For experiments in which fluorescently labeled heparin (FAM-heparin) was required, the fabrication procedure is the same except that the star-PEG-(NH2)8 was dissolved in deionized water supplemented with 5(6)-carboxyfluorescein N-succinimidyl ester 0.1% (molar ratio) of starPEG-(NH2)8. )</li>
	</ol>
	</li>
	<li>Characterization of heparin-PEG nanocoating by Fourier Transform Infrared Spectroscopy (FT-IR)
	<ol>
		<li>Before starting, rinse the agate mortar, pestle and pelleting device (small disks with diameter of 13 mm) with acetone and deionized water. Dry the glassware in an oven before use.</li>
		<li>Mix approximately 0.1&#8211;1% heparin-PEG sample with 200&#8211;250 mg of fine KBr powder.</li>
		<li>Place the heparin-PEG and KBr mixture in the agate mortar and finely pulverize into small pellets with diameters of 2 &#181;m.</li>
		<li>Transfer the mixture into a pelleting device. Apply high compression force (8 T/cm2) in a vacuum for 1&#8211;2 min to form transparent pellets.</li>
		<li>Gently pull the sample pellets away from the pelleting device. Be careful not to pull too hard so not to incur cracks.</li>
		<li>Transfer the sample pellets very carefully to a Fourier Transform Infrared Spectroscope to determine sample chemical structure.</li>
	</ol>
	</li>
	<li>Examination of the internal porous structures of heparin-PEG nanocoating by scanned electron microscopy (SEM)
	<ol>
		<li>For the following procedures, prepare standard 48-well cell culture plate and pipettes.</li>
		<li>Pipette the heparin-PEG copolymer solution into the wells of a 48-well culture plate.</li>
		<li>Freeze the plate at -80 &#176;C for 24 h.</li>
		<li>Lypophilize the samples at -50 &#176;C in vacuum environment (0.1 Pa) for 24 h.</li>
		<li>Spread samples across the sticky surface of an aluminum stub.</li>
		<li>Coat the samples with gold and observe under the scanned electron microscope to observe internal porous structures.</li>
	</ol>
	</li>
	<li>Examination of the heparin-PEG nanocoating surface by atomic force microscopy (AFM)
	<ol>
		<li>Clean silica glass slides with piranha solution (70% H2SO4, 30% H2O2) at 80 &#176;C for 40 min.</li>
		<li>Sonicate the slides in methanol for 10 min and then in toluene for 10 min.</li>
		<li>Dry the slides by vacuum drying.</li>
		<li>Wash the slides with plenty 3-aminopropyl-triethoxysilane solution (2% in toluene), shake gently.</li>
		<li>Sonicate the slides in methanol for 10 min then in toluene for 10 min.</li>
		<li>Place 100 &#181;L of 3% heparin-PEG solution across the surface of the slides using a standard pipette.</li>
		<li>Examine the surface of heparin-PEG under an atomic force microscope (a resonant frequency of 50&#8211;80 kHz, force constant of 0.350 N m 21, tip radius of 600 nm).</li>
	</ol>
	</li>
</ol>
2. Mouse Islet Surface Engineering with Heparin-PEG Nanocoating
<ol>
	<li>Mouse islet surface coating with heparin-PEG nanocoating
	<ol>
		<li>Extract mouse islets by collagenase digestion and histopaque gradient purification as previously reported in JoVE19.</li>
		<li>Handpick 200 islets under a dissection microscope to a 1.5 mL tube.</li>
		<li>Add 500 &#181;L of PBS to the islets and centrifuge at 503 x g for 1 min.</li>
		<li>Take off the supernatant and add 250 &#181;L of the heparin-PEG solution and keep the mixture on ice for 10 min. Pipette up and down to allow the islets to mix well with the heparin-PEG solution.</li>
		<li>Centrifuge at 503 x g for 1 min.</li>
		<li>Remove the supernatant and add 500 &#181;L of PBS. Pipette up and down to mix well.</li>
		<li>Centrifuge at 503 x g for 1 min and remove the supernatant and keep the islets for further use. For examination of mouse islets coated with the heparin-PEG, FAM-heparin-PEG was used for islet coating following these steps.</li>
	</ol>
	</li>
	<li>Examination of mouse islets coated with the FAM-heparin-PEG nanocoating
	<ol>
		<li>Add 1&#8211;2 mL of RPMI 1640 supplemented with 10% fetal bovine serum to the coated islets and transfer these islets into a non-charged sterile bacterial culture dish.</li>
		<li>Observe the morphology and fluorescence signals of FAM-heparin-PEG nanocoating-coated islets under an inverted fluorescence microscope.</li>
	</ol>
	</li>
</ol>
3. Function of Heparin-PEG Coated Mouse Islets Compared to Non-coated Islets
<ol>
	<li>Viability of heparin-PEG coated mouse islets
	<ol>
		<li>Handpick 20 of the heparin-PEG coated mouse islets and place them into one well of a 96-well cell culture plate.</li>
		<li>Fill a total of 10 wells with 20 heparin-PEG coated mouse islets for each well.</li>
		<li>Handpick 20 of noncoated control islets and place them into one well of the same 96-well cell culture plate.</li>
		<li>Fill a total of 10 wells with 20 noncoated control islets for each well.</li>
		<li>Put 200 &#181;L of RPMI 1640 supplemented with 10% fetal bovine serum into each well of the 96-well plate that contains islets and maintain in culture for 14 days.</li>
		<li>Replace islet culture media every 2 days.</li>
		<li>Treat the islets with a live/dead staining kit at the end of 14 days in culture and observed under an inverted fluorescence microscope.</li>
		<li>Count PI+ (red) cells within each islet under an inverted fluorescence microscope.</li>
	</ol>
	</li>
	<li>Revascularization of the heparin-PEG coated mouse islets in vitro
	<ol>
		<li>Pre-cool all experimental plastics including culture plate and pipette tips at least 12 h before use.</li>
		<li>Place a 24-well place on a cooling pad. Add 250 &#181;L of ice cold growth factor-reduced Matrigel into each well of a 24-well cell culture plate. Place the coated 24-well plate at 4 &#176;C for at least 30 min.</li>
		<li>While the matrix solution is setting in the fridge, trypsinize the mouse pancreatic islet endothelial Mile Sven 1 (MS1) cells.
		<ol>
			<li>Remove all culture media from the tissue culture flask. Rinse the flask with 5 mL of PBS.</li>
			<li>Remove the PBS and add 3 mL of trypsin-EDTA. Incubate at 37 &#176;C for 3 min in a standard incubator.</li>
			<li>Lightly tap the bottom of the flask to detach the cells and add 5 mL of serum-supplemented media.</li>
		</ol>
		</li>
		<li>Collect the cells into a 15 mL centrifuge tube and centrifuge at 1,000 x g for 5 min.</li>
		<li>Take off the supernatant and add 5 mL of PBS. Pipette up and down to mix well.</li>
		<li>Centrifuge at 1,000 x g for 5 min and take off the supernatant.</li>
		<li>Add serum-supplemented DMEM media into the tube and seed 50,000 cells into each well of the matrix-solution-coated 24-well plate.</li>
		<li>Add 5 heparin-PEG coated islets or noncoated control islets into each well.</li>
		<li>Add serum-supplemented DMEM media into each well to a total of 500 &#181;L per well.</li>
		<li>Observe tube formation after 4h and 24 h incubation under a light microscope.</li>
	</ol>
	</li>
	<li>Glucose-stimulated insulin secretion of heparin-PEG coated islets 
	&#8203;NOTE: To assess whether nanocoating would affect the function of mouse islets, glucose-stimulated insulin secretion of heparin-PEG coated and noncoated islets was assessed.
	<ol>
		<li>Handpick 30 heparin-PEG coated islets or noncoated control islets into one 1.5 mL tube. Prepare a total of 10 tubes for coated islets and noncoated islets each.</li>
		<li>Add 1 mL of physiological salt solution supplemented with 2 mmol/L glucose20 and incubate at 37 &#176;C for 2 h. 
		NOTE: For the recipe of the physiological salt solution, please refer to Table 1. Keep the cap of tubes loose during incubation in the incubator.</li>
		<li>Remove as much solution as possible.</li>
		<li>Stimulate islets by adding 600 &#181;L of physiological salt solution supplemented with 20 mmol/L glucose at 37 &#176;C for 30 min.</li>
		<li>Collect 200 &#181;L of supernatant from each tube for insulin ELISA quantification using a commercial insulin ELISA kit.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Ke, 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=Netrin-1+ameliorates+myocardial+infarction-induced+myocardial+injury:+mechanisms+of+action+in+rats+and+diabetic+mice.">Netrin-1 ameliorates myocardial infarction-induced myocardial injury: mechanisms of action in rats and diabetic mice.</a> Hum Gene Ther. 25 (9), 787-797 (2014).</li><li>Ke, 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=Co-transplantation+of+skin-derived+precursors+and+collagen+sponge+facilitates+diabetic+wound+healing+by+promoting+local+vascular+regeneration.">Co-transplantation of skin-derived precursors and collagen sponge facilitates diabetic wound healing by promoting local vascular regeneration.</a> Cell Physiol Biochem. 37 (5), 1725-1737 (2015).</li><li>Saxon, E., Bertozzi, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+engineering+by+a+modified+Staudinger+reaction.">Cell surface engineering by a modified Staudinger reaction.</a> Science. 287 (5460), 2007-2010 (2000).</li><li>Sarkar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+cell+homing.">Engineered cell homing.</a> Blood. 118 (25), 184-191 (2011).</li><li>Kato, M., Mrksich, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rewiring+cell+adhesion.">Rewiring cell adhesion.</a> J Am Chem Soc. 126 (21), 6504-6505 (2004).</li><li>Cheng, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cell+membrane+engineering+for+cell+rolling+using+peptide+conjugation+and+tuning+of+cell-selectin+interaction+kinetics.">Stem cell membrane engineering for cell rolling using peptide conjugation and tuning of cell-selectin interaction kinetics.</a> Biomaterials. 33 (20), 5004-5012 (2012).</li><li>Merzaban, J. 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=Cell+surface+glycan+engineering+of+neural+stem+cells+augments+neurotropism+and+improves+recovery+in+a+murine+model+of+multiple+sclerosis.">Cell surface glycan engineering of neural stem cells augments neurotropism and improves recovery in a murine model of multiple sclerosis.</a> Glycobiology. 25 (12), 1392-1409 (2015).</li><li>Silvescu, C. I., Sackstein, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-CSF+induces+membrane+expression+of+a+myeloperoxidase+glycvariant+that+operates+as+an+E-selectin+ligand+on+human+myeloid+cells.">G-CSF induces membrane expression of a myeloperoxidase glycvariant that operates as an E-selectin ligand on human myeloid cells.</a> Proc Natl Acad Sci U S A. 111 (29), 10696-10701 (2014).</li><li>Zhi, Z. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+bioactive+multilayered+nanocoatings+on+pancreatic+islet+cells:+incorporation+of+alpha1-antitrypsin+into+the+coatings.">Assembly of bioactive multilayered nanocoatings on pancreatic islet cells: incorporation of alpha1-antitrypsin into the coatings.</a> Chem Commun (Camb). 51 (53), 10652-10655 (2015).</li><li>Lou, 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=Pancreatic+islet+surface+bioengineering+with+a+heparin-incorporated+starPEG+nanofilm.">Pancreatic islet surface bioengineering with a heparin-incorporated starPEG nanofilm.</a> Mater Sci Eng C Mater Biol Appl. 78, 24-31 (2017).</li><li>Orlando, 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=Cell+replacement+strategies+aimed+at+reconstitution+of+the+beta-cell+compartment+in+type+1+diabetes.">Cell replacement strategies aimed at reconstitution of the beta-cell compartment in type 1 diabetes.</a> Diabetes. 63 (5), 1433-1444 (2014).</li><li>Kollmer, 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=Long-term+function+of+alginate-encapsulated+islets.">Long-term function of alginate-encapsulated islets.</a> Tissue Eng Part B Rev. 22 (1), 34-36 (2015).</li><li>Yang, H. K., Yoon, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+encapsulated+islet+transplantation.">Current status of encapsulated islet transplantation.</a> J Diabetes Complications. 29 (5), 737-743 (2015).</li><li>Kizilel, 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=Encapsulation+of+pancreatic+islets+with+nano-thin+functional+polyethylene+glycol+coatings+for+enhanced+insulin+secretion.">Encapsulation of pancreatic islets with nano-thin functional polyethylene glycol coatings for enhanced insulin secretion.</a> Tissue Eng. Part A. 16, 2217-2228 (2010).</li><li>Zhi, Z. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-scale+encapsulation+enhances+allograft+survival+and+function+of+islets+transplanted+in+a+mouse+model+of+diabetes.">Nano-scale encapsulation enhances allograft survival and function of islets transplanted in a mouse model of diabetes.</a> Diabetologia. 55, 1081-1090 (2012).</li><li>Gattas-Asfura, K. M., Stabler, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Layer-by-layer+deposition+of+antifouling+coatings+on+stainless+steel+via+catechol-amine+reaction.">Layer-by-layer deposition of antifouling coatings on stainless steel via catechol-amine reaction.</a> ACS Appl. Mater. Interfaces. 5, 9964-9974 (2013).</li><li>Kollmer, 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=Long-term+function+of+alginate-encapsulated+islets.">Long-term function of alginate-encapsulated islets.</a> Tissue Eng. Part B Rev. 22 (1), 34-46 (2015).</li><li>Yang, H. K., Yoon, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+status+of+encapsulated+islet+transplantation.">Current status of encapsulated islet transplantation.</a> J. Diabetes Complicat. 29, 737-747 (2015).</li><li>Zmuda, E. 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=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> J. Vis. Exp. (50), e2096 (2011).</li><li>Gey, G. O., Gey, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+maintenance+of+human+normal+cells+and+tumor+cells+in+continuous+culture.+I.+Preliminary+report:+Cultivation+of+mesoblastic+tumors+and+normal+tissue+and+notes+on+methods+of+cultivation.">The maintenance of human normal cells and tumor cells in continuous culture. I. Preliminary report: Cultivation of mesoblastic tumors and normal tissue and notes on methods of cultivation.</a> Am. J. Cancer. 27, 45-76 (1936).</li><li>Chen, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-selective+azide+incorporation+into+endogenous+RNase+A+via+a+"chemistry"+approach.">Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach.</a> Org. Biomol. Chem. 11, 353-361 (2013).</li><li>Cabric, 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=Anchoring+of+vascular+endothelial+growth+factor+to+surface-immobilized+heparin+on+pancreatic+islets:+implications+for+stimulating+islet+angiogenesis.">Anchoring of vascular endothelial growth factor to surface-immobilized heparin on pancreatic islets: implications for stimulating islet angiogenesis.</a> Tissue Eng. Part A. 16, 961-970 (2010).</li><li>Cabric, 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=Islet+surface+heparinization+prevents+the+instant+blood-mediated+inflammatory+reaction+in+islet+transplantation.">Islet surface heparinization prevents the instant blood-mediated inflammatory reaction in islet transplantation.</a> Diabetes. 56, 2008-2015 (2007).</li><li>Asif, 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=Heparinization+of+cell+surfaces+with+short+peptide-conjugated+PEG-lipid+regulates+thromboinflammation+in+transplantation+of+human+MSCs+and+hepatocytes.">Heparinization of cell surfaces with short peptide-conjugated PEG-lipid regulates thromboinflammation in transplantation of human MSCs and hepatocytes.</a> Acta Biomater. 35, 194-205 (2016).</li><li>Teramura, Y., 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=Microencapsulation+of+cells,+including+islets,+within+stable+ultra-thin+membrane+of+maleimide-conjugated+PEG-lipid+with+multifunctional+crosslinkers.">Microencapsulation of cells, including islets, within stable ultra-thin membrane of maleimide-conjugated PEG-lipid with multifunctional crosslinkers.</a> Biomaterials. 34 (11), 2683-2893 (2013).</li><li>Zhi, Z., 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=Multilayer+nanoencapsulation:+A+nanomedicine+technology+for+diabetes+research+and+management.">Multilayer nanoencapsulation: A nanomedicine technology for diabetes research and management.</a> Diabetes Res. Clin. Pract. 100, 162-169 (2013).</li></ol>

The authors have nothing to disclose.

In this article, we demonstrate an &#34;easy-to-adopt&#34; approach for living cell surface engineering a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of pancreatic islet surface membrane. Indeed, the amino groups within cell membranes are highly reactive, and as a result, earlier studies have reported interactions between primary amino groups with activated N-hydroxysuccinimidyl (NHS) ester under physiological conditions14,16,21. Furthermore, extensive research has reported that incorporation of heparin, a highly sulphated glycosaminoglycan and important component of the extracellular matrix, during islet encapsulation, could lead to enhanced post-transplantation revascularization and reduced IBMIR22,23. Considering the biocompatibility of PEG and multivalent properties of heparin, we used 8-armed PEG for maximal heparin loading during fabrication of the nanocoating. Heparin was modified with -NHS, which would subsequently react with the -NH2 groups on islet cell membrane. By enabling the covalent bond formation between -NH2 (of cell membrane) and -NHS of the Hep-PEG, the islets would be readily &#34;coated&#34; by the heparin-incorporated PEG, thus forming a nano-thin layer (nanocoating) on the outer surface of the pancreatic islets.
The present approach is different from previously published methods that also selected PEG as the major polymer for islet microencapsulation in that pseudo-bioorthogonal chemistry between the -NHS (of the nanocoating) and -NH2 of the islet cell membrane was used. Considering that stability of islet/cell coating, especially in a complex environment such as the plasma, is crucial to post-transplantation revascularization and survival, the formation between -NHS and -NH2 would be more stable compared to hydrophobic interaction between PEG and cell membrane24, electrostatic interactions9,15,24,25,26&#160;or biological linkage between biotin streptavidin14.
In addition, in contrast to islet coating approach that relies on the LBL approach with extended islet handling period for multi-layer deposition14,16,25, the present technique also requires minimal processing and very short coating period of the isolated islets. Both of these factors are essential for post-transplantation islet survival since islets viability is often already compromised following islet isolation due to damaged ECM during enzymatic digestion. However, one limitation of the present approach is that, unlike LBL, via which thickness of the outer coating could be controlled by increasing or reducing the number of layer deposition, thickness of the Hep-PEG nanocoating cannot be tailored for the time being.
Furthermore, due to the mild condition where chemical reaction between -NHS and -NH2 takes place, the present approach is applicable for living cell surface engineering not limited to pancreatic islets, but most cell therapy. Additionally, considering that heparin is known to interact with a range of cytokines and biologically active molecules, the Hep-PEG nanocoating also presents an open platform that has the potential for incorporation of unlimited biological mediators as well as interfaces for more complex cell surface engineering.

The therapeutic efficacy of cell-based therapies is limited by low cell retention and poor survival1,2. In order to improve the outcome of cell therapies, cell surface engineering via enzymatic manipulation, peptide conjugation, bioorthogonal chemistry and physical encapsulation with biomaterials has been exploited3,4,5,6,7,8,9,10. The current protocol aims to achieve surface engineering of living cells using an &#34;easy-to-adopt&#34; method by applying an ultrathin heparin-incorporated starPEG (heparin-PEG) nanocoating to the cell surface. Surface engineering of pancreatic islets was presented here as an example due to the heterogeneous nature of islets of Langerhans and the disparaging outcomes of current clinical islet transplantation.
Indeed, clinical islet transplantation is currently performed by direct injection of isolated islets into the hepatic portal vein and this procedure is only available for selective patients because of the scarcity of donor materials and low therapeutic efficacy11. Conventionally, alginate has been the most commonly used biomaterial for islet encapsulation and surface modification, although it is less than ideal due to the chemical instability of alginate and inflammatory-related fibrosis12,13. Furthermore, compared to the natural size of islets that ranges between 100 to 200 &#181;m, the alginate-islet microcapsules are larger, ranging between 400 and 800 &#181;m, which exceed the physiological diffusing distance of oxygen. Conformal islet encapsulation, i.e., encapsulating islets without significant alteration of islet volume, was then developed. Thus, deposition of nanomembranes composed of PEG, tetrafluoroethylene, silicon membrane or multi-layered nanocoating (also known as the &#34;layer-by-layer&#34; [LBL] technique) has been reported, resulting in improved in vitro islet survival14,15,16,17,18, although the LBL approach often requires extensive islets handing period for deposition of multiple layers, which may compromise islet viability. Moreover, instability of nanomembranes that relies on electrostatic or covalent interactions between biomembrane layers or hydrophobic interactions between nanomembranes and the islet surface also raises concerns9,14,15,16,22,23,24,25,26.
Another limiting factor that encumbers the therapeutic outcome of intraportal islet transplantation is the instant blood-mediated inflammatory reaction (IBMIR) caused by direct contact of implanted islets with blood, resulting in platelet aggregation, coagulation and adverse immune effect or undesired cellular activation9. To address these problems, an ultra-thin nanocoating composed of star-shaped polyethylene glycol (starPEG) was prepared for its established biocompatibility and versatility as islet housing material. Heparin, a highly sulphated glycosaminoglycan, was also incorporated in the starPEG nanocoating for its anti-inflammatory, anti-coagulant properties and ability to facilitate vascularization by recruiting pro-angiogenic growth factors22,23.

<table border="0" cellpadding="0" cellspacing="0" style="width:531px;" width="531">
	<colgroup>
		<col />
		<col />
		<col span="2" />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Reagent</td>
			<td style="width:160px;"></td>
			<td style="width:120px;"></td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">PBS</td>
			<td style="width:160px;">Hyclone</td>
			<td style="width:120px;">AAJ207798</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Streptozototin</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:120px;">S0130</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Histopaque</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:120px;">10831</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:131px;">RPMI 1640</td>
			<td style="width:160px;">GIBCO, by Life Technologies</td>
			<td style="width:120px;">31800022</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:131px;">Fetal Bovine Serum</td>
			<td style="width:160px;">GIBCO, by Life Technologies</td>
			<td style="width:120px;">16000-044</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Penicillin Streptomycin</td>
			<td style="width:160px;">GIBCO, by Life Technologies</td>
			<td style="width:120px;">15140</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:131px;">Cell Dissociation Solution</td>
			<td style="width:160px;">GIBCO, by Life Technologies</td>
			<td style="width:120px;">13150-016</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:131px;">DMEM</td>
			<td style="width:160px;">GIBCO, by Life Technologies</td>
			<td style="width:120px;">12800017</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">D-(+)-Glucose solution</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:120px;">G8644</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">488 phalloidin</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:120px;">A12379</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">CFSE</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:120px;">21888-25mg-F</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:131px;">Annexin V/PI apoptosis kit</td>
			<td style="width:160px;">Dojindo</td>
			<td style="width:120px;">AD10</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">DAPI Fluoromount-G</td>
			<td style="width:160px;">SouthernBiotech</td>
			<td style="width:120px;">0100-20</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">Collagenase from Clostridium, Type XI</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:120px;">C7657</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Heparin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:120px;">H3149</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">NHS</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:120px;">56480</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">EDC</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:120px;">3449</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">8-armed PEG</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">1685176</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">FAM</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:120px;">M041100</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:131px;">5(6)-carboxyfluorescein N-succinimidyl ester</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:120px;">21888</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">KBr</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">32036</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">3-aminopropyl-triethoxysilane&#160;</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:120px;">A3648</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">toluene</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">S-15497-20X</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:131px;">Live/dead staining kit</td>
			<td style="width:160px;">Biovision, US</td>
			<td style="width:120px;">K501</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="79">
			<td height="79" style="height:79px;width:131px;">BD MatrigelTM, basement membrane matrix, growth factor reduced</td>
			<td style="width:160px;">BD Bioscience</td>
			<td style="width:120px;">354230</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">Sodium chloride, 99.5%</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">105864</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">Potassium chloride, 99%, extra pure</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">991468</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">Sodium bicarbonate, 99.7%, ACS reagent</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">988639</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:131px;">Magnesium chloride hexahydrate, 99%, ACS reagent</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">182158</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="76">
			<td height="76" style="height:76px;width:131px;">Potassium dihydrogen phosphate, 99%, extra pure</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">128839</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:131px;">Magnesium sulfate heptahydrate, 99%, for analysis</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">119370</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="57">
			<td height="57" style="height:57px;width:131px;">Calcium chloride solution volumetric, 1.0 M CaCl2</td>
			<td style="width:160px;">J&#38;K Scientific Ltd</td>
			<td style="width:120px;">21114</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">Bovine Serum Albumin</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:120px;">V900933</td>
			<td style="width:120px;"></td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:131px;">Rat/Mouse Insulin ELISA kit</td>
			<td style="width:160px;">Millipore-linco</td>
			<td style="width:120px;">EZRMI-13K</td>
			<td style="width:120px;"></td>
		</tr>
	</tbody>
</table>

surface engineering of pancreatic islets with a heparinized starpeg nanocoating

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and survival post-transplantation. This protocol aims to achieve surface engineering of pancreatic islets using an ultrathin heparin-incorporated starPEG (Hep-PEG) nanocoating. To generate the Hep-PEG nanocoating for pancreatic islet surface engineering, heparin succinimidyl succinate (Heparin-NHS) was first synthesized by modification of its carboxylate groups using N-(3-dimethylamino propyl)-N'-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The Hep-PEG mixture was then formed by crosslinking of the amino end-functionalized eight-armed starPEG (starPEG-(NH2)8) and Heparin-NHS. For islet surface coating, mouse islets were isolated via collagenase digestion and gradient purification using Histopaque. Isolated islets were then treated with ice cold Hep-PEG solution for 10 min to allow covalent binding between NHS and the amine groups of islet cell membrane. Nanocoating with the Hep-PEG incurs minimal alteration to islet size and volume and heparinization of the islets with Hep-PEG may also reduce instant blood-mediated inflammatory reaction during islet transplantation. This "easy-to-adopt" approach is mild enough for surface engineering of living cells without compromising cell viability. Considering that heparin has shown binding affinity to multiple cytokines, the Hep-PEG nanocoating also provides an open platform that enables incorporation of unlimited functional biological mediators and multi-layered surfaces for living cell surface bioengineering.

The heparin-PEG nanocoating was synthesized by conjugation of starPEG-(NH2)8 and heparin using EDC and NHS as coupling agents (Figure 1). Chemical structure of the heparin-PEG nanocoating was examined by FT-IR and as shown in Figure 2, characteristic peaks of heparin could be observed at 3,300&#8211;3,600 cm-1, corresponding to the hydroxyl groups of heparin (Figure 2 red). The decrease in amplitude of the peak at 3,300&#8211;3,600 cm-1 (Figure 2 blue) represents conjugation between the starPEG-(NH2)8 amide groups and heparin carbonyl group. Amplitude of peak 1,650 cm-1 corresponds to the amide carbonyl stretching vibration was also reduced, indicating sufficient reaction between the carboxylate groups of heparin with succinimidyl succinate and amine of starPEG-(NH2)8. Surface structure of the heparin-PEG nanocoating was examined by atomic force microscopy and it is shown from Lou et al., the nanocoating was approximately 30 nm in height and 2 &#181;m in width with small porous features (dark spots) ranging between 100 to 200 nm in diameter10. Data obtained from scanned electronic microscopy also confirm the highly interconnected porous structure of the heparin-PEG (Figure 3), suggesting that it could be suitable for cell survival during in vivo delivery.
Surface coating of isolated mouse islets was examined and as presented in Figure 4, thin layer of nanocoating, shown by green fluorescence was evenly deposited across the surface of coated islets without causing evident changes on islet volume/size. During coating, it is recommended to keep the islets on ice to maintain their viability. Similarly, the coating period, 10 min in this case, was also optimized to allow maintenance of islets viability. It is worth noting that for better observation of the nanocoating, electron microscopy that examines the cross-sections of coated islets as previously reported9,16 would be more appropriate, although the data would be generated from fixed and embedded islets instead of living islets in culture.
Regarding survival and function of coated islets, islet revascularization and functions in have been assessed in vitro. Considering the beneficial properties of heparin, heparin functionalization onto the islet surface could facilitate islet revascularization in culture and consequently its survival. We have observed that the heparin-PEG coated mouse islets exhibited robust islet viability in culture (Figure 5). Significantly more advanced vascular formation was also evident from islet endothelial cells (MS1) that were co-cultured with heparin-PEG coated islets, indicated by elongated microvessel-like structures and network-like vascular structures (Figure 6).
The nanocoating process incurred no effect glucose-stimulated insulin secretory ability of the heparin-PEG coated islets. Low level of insulin secretion was observed in all treatment groups when islets were perfused with physiological salt solution supplemented with sub-stimulatory level of glucose (2 mmol/L; Figure 7). When islets were stimulated with a supra-physiological level of glucose (20 mmol/L glucose), increase in insulin secretion was observed in all treatment groups.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56879/56879fig1v2.jpg" /> 
Figure 1: Chemical structure of Hep-PEG nanocoating for pancreatic islet surface engineering. Islet coating was achieved by covalent crosslinking between the heparin-NHS and primary amines within the cell membrane and star-PEG-(NH2)8. Each heparin molecule possesses multiple carboxyl groups, which were modified to have an NHS group each. The NHS-activated carboxyl groups will react with primary amines of protein to form amide linkages, via which nanocoating of Hep-PEG and islets is stabilized.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56879/56879fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56879/56879fig2v2.jpg" /> 
Figure 2: Infrared spectrum of star-PEG-(NH2)8, heparin and Hep-PEG nanocoating in dried state. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56879/56879fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56879/56879fig3.jpg" /> 
Figure 3: Scanned electronic microscopy images of Hep-PEG in dried state. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56879/56879fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56879/56879fig4.jpg" /> 
Figure 4: Representative images of heparin-PEG coated islets under fluorescence microscope.&#160; 
Heparin was pre-labelled with FAM (shown in green). Images are representative of 100 islets. Scale bar = 100 &#181;m.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56879/56879fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56879/56879fig5.jpg" /> 
Figure 5: Heparin-PEG coated islets exhibited robust islet viability. (A) Data presented as mean&#177; standard error of means, n = 50 islets per group. (B) Living cells were shown in green and dead cells in red. Scale bar = 100 &#181;m. Images are representative of 50 islets.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56879/56879fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56879/56879fig6.jpg" /> 
Figure 6: Heparin-PEG nanocoating facilitates intra-islet revascularisation. Matrigel tube formation of MS1 cells co-cultured with heparin-PEG coated islets and noncoated control islets. Images were taken at 4 and 24 h. Scale bar = 100 &#181;m.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56879/56879fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/56879/56879fig7.jpg" /> 
Figure 7: Heparin-PEG nanocoating incurs no change on islet insulin secretion function. Heparin-PEG coated islets and noncoated control islets (30 each) were exposed to 2 mmol/L (white bar) or 20 mmol/L (black bar) glucose for 30 min. Insulin secretion in response to 20 mmol/L glucose was comparable between the coated and control islets. Data are shown as mean &#177; standard error of means, n = 10.&#160;<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56879/56879fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating at JoVE.com

Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Cell surface engineering can protect implanted cells from host immune attack. It can also reshape cellular landscape to improve graft function and ...

surface-engineering-pancreatic-islets-with-heparinized-starpeg

Nanyang Technological University

Research

JoVE Journal

Bioengineering

7.3K Views.  Chinese Academy of Medical Science & Peking Union Medical College. This protocol aims to achieve surface engineering of pancreatic islets using a heparin-incorporated starPEG nanocoating via pseudo-bioorthogonal chemistry between the N-hydroxysuccinimide groups of the nanocoating and the amine groups of islet cell membrane.

Video: Surface Engineering of Pancreatic Islets with a Heparinized StarPEG Nanocoating

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Automated Robotic Dispensing Technique for Surface Guidance and Bioprinting of Cells

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using an automated robotic dispensing system. The particular bioink was selected as it allows cells to sediment towards and sense the features. The dispensing system bioprints viable cells in hydrogel bioinks using a backpressure assisted print head. However, by replacing the print head with a sharpened stylus or scalpel, the dispensing system can also be employed to create topographical cues through surface etching. The stylus movement can be programmed in steps of 10 &#181;m in the X, Y and Z directions. The patterned grooves were able to orientate mesenchymal stem cells, influencing them to adopt an elongated morphology in alignment with the grooves&#39; direction. The patterning could be designed using plotting software in straight lines, concentric circles, and sinusoidal waves. In a subsequent procedure, fibroblasts and mesenchymal stem cells were suspended in a 2% gelatin bioink, for bioprinting in a backpressure driven extrusion printhead. The cell bearing bioink was then printed using the same programmed coordinates used for the etching. The bioprinted cells were able to sense and react to the etched features as demonstrated by their elongated orientation along the direction of the etched grooves.

This protocol describes a bioprinting methodology using an automated robotic depositing system that incorporates etched topographical guidance cues with the precision deposition of a cell bearing hydrogel bioink. The printed cells are directly delivered to the etched features and are able to sense and orientate with them.

This manuscript describes the introduction of cell guidance features followed by the direct delivery of cells to these features in a hydrogel bioink using ...

Sustained Administration of &#946;-cell Mitogens to Intact Mouse Islets Ex Vivo Using Biodegradable Poly(lactic-co-glycolic acid) Microspheres

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the pancreatic &#946;-cells. In addition, biomaterial particles, hydrogels, and scaffolds also provide a unique opportunity to administer sustained, controllable drug delivery to &#946;-cells in culture and in transplanted tissue models. These technologies allow the study of candidate &#946;-cell proliferation factors using intact islets and a translationally relevant system. Moreover, determining the effectiveness and feasibility of candidate factors for stimulating &#946;-cell proliferation in a culture system is critical before moving forward to in vivo models. Herein, we describe a method to co-culture intact mouse islets with biodegradable compound of interest (COI)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres for the purpose of assessing the effects of sustained in situ release of mitogenic factors on &#946;-cell proliferation. This technique describes in detail how to generate PLGA microspheres containing a desired cargo using commercially available reagents. While the described technique uses recombinant human Connective tissue growth factor (rhCTGF) as an example, a wide variety of COI could readily be used. Additionally, this method utilizes 96-well plates to minimize the amount of reagents necessary to assess &#946;-cell proliferation. This protocol can be readily adapted to use alternative biomaterials and other endocrine cell characteristics such as cell survival and differentiation status.

Here, we present methodology to generate and administer compound of interest-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres to intact mouse islets in culture with subsequent immunofluorescence analysis of &#946;-cell proliferation. This method is suitable for determining the efficacy of candidate &#946;-cell mitogens.

The development of biomaterials has significantly increased the potential for targeted drug delivery to a variety of cell and tissue types, including the ...

Scaffold-supported Transplantation of Islets in the Epididymal Fat Pad of Diabetic Mice

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy may incur acute whole blood reactions and result in poor islet engraftment. Here, we report a robust protocol for the transplantation of islets at the extrahepatic transplantation site-the epididymal fat pad (EFP)-in a diabetic mouse model. A protocol to isolate and purify islets at high yields from C57BL/6J mice is described, as well as a transplantation method performed by seeding islets onto a decellularized scaffold (DCS) and implanting them at the EFP site in syngeneic C57BL/6J mice rendered diabetic by streptozotocin. The DCS graft containing 500 islets reversed the hyperglycemic condition within 10 days, while the free islets without DCS required at least 30 days. The normoglycemia was maintained for up to 3 months until the graft was explanted. In conclusion, DCS enhanced the engraftment of islets into the extrahepatic site of the EFP, which could easily be retrieved and might provide a reproducible and useful platform for investigating the scaffold materials, as well as other transplantation parameters required for a successful islet engraftment.

This protocol demonstrates murine islet isolation and seeding onto a decellularized scaffold. Scaffold-supported islets were transplanted into the epididymal fat pad of streptozotocin (STZ)-induced diabetic mice. Islets survived at the transplantation site and reversed the hyperglycemic condition.

Islet transplantation has been clinically proven to be effective at treating type 1 diabetes. However, the current intrahepatic transplantation strategy ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...