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>

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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><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Hyclone</td><td>AAJ207798</td><td></td></tr><tr><td>Streptozototin</td><td>Sigma</td><td>S0130</td><td></td></tr><tr><td>Histopaque</td><td>Sigma</td><td>10831</td><td></td></tr><tr><td>RPMI 1640</td><td>GIBCO, by Life Technologies</td><td>31800022</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>GIBCO, by Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>GIBCO, by Life Technologies</td><td>15140</td><td></td></tr><tr><td>Cell Dissociation Solution</td><td>GIBCO, by Life Technologies</td><td>13150-016</td><td></td></tr><tr><td>DMEM</td><td>GIBCO, by Life Technologies</td><td>12800017</td><td></td></tr><tr><td>D-(+)-Glucose solution</td><td>Sigma</td><td>G8644</td><td></td></tr><tr><td>488 phalloidin</td><td>Sigma</td><td>A12379</td><td></td></tr><tr><td>CFSE</td><td>Sigma</td><td>21888-25mg-F</td><td></td></tr><tr><td>Annexin V/PI apoptosis kit</td><td>Dojindo</td><td>AD10</td><td></td></tr><tr><td>DAPI Fluoromount-G</td><td>SouthernBiotech</td><td>0100-20</td><td></td></tr><tr><td>Collagenase from Clostridium, Type XI</td><td>Sigma</td><td>C7657</td><td></td></tr><tr><td>Heparin</td><td>Sigma-Aldrich</td><td>H3149</td><td></td></tr><tr><td>NHS</td><td>Sigma-Aldrich</td><td>56480</td><td></td></tr><tr><td>EDC</td><td>Sigma-Aldrich</td><td>3449</td><td></td></tr><tr><td>8-armed PEG</td><td>J&amp;amp;K Scientific Ltd</td><td>1685176</td><td></td></tr><tr><td>FAM</td><td>Sigma-Aldrich</td><td>M041100</td><td></td></tr><tr><td>5(6)-carboxyfluorescein N-succinimidyl ester</td><td>Sigma-Aldrich</td><td>21888</td><td></td></tr><tr><td>KBr</td><td>J&amp;amp;K Scientific Ltd</td><td>32036</td><td></td></tr><tr><td>3-aminopropyl-triethoxysilane&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A3648</td><td></td></tr><tr><td>toluene</td><td>J&amp;amp;K Scientific Ltd</td><td>S-15497-20X</td><td></td></tr><tr><td>Live/dead staining kit</td><td>Biovision, US</td><td>K501</td><td></td></tr><tr><td>BD Matrigel&lt;sup&gt;TM&lt;/sup&gt;, basement membrane matrix, growth factor reduced</td><td>BD Bioscience</td><td>354230</td><td></td></tr><tr><td>Sodium chloride, 99.5%</td><td>J&amp;amp;K Scientific Ltd</td><td>105864</td><td></td></tr><tr><td>Potassium chloride, 99%, extra pure</td><td>J&amp;amp;K Scientific Ltd</td><td>991468</td><td></td></tr><tr><td>Sodium bicarbonate, 99.7%, ACS reagent</td><td>J&amp;amp;K Scientific Ltd</td><td>988639</td><td></td></tr><tr><td>Magnesium chloride hexahydrate, 99%, ACS reagent</td><td>J&amp;amp;K Scientific Ltd</td><td>182158</td><td></td></tr><tr><td>Potassium dihydrogen phosphate, 99%, extra pure</td><td>J&amp;amp;K Scientific Ltd</td><td>128839</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate, 99%, for analysis</td><td>J&amp;amp;K Scientific Ltd</td><td>119370</td><td></td></tr><tr><td>Calcium chloride solution volumetric, 1.0 M CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>J&amp;amp;K Scientific Ltd</td><td>21114</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>V900933</td><td></td></tr><tr><td>Rat/Mouse Insulin ELISA kit</td><td>Millipore-linco</td><td>EZRMI-13K</td><td></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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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

Staining Protocols for Human Pancreatic Islets

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and beta mass ranges from 500 to 1500 mg 1-3. With this known heterogeneity, a standard processing and staining procedure was developed so that pancreatic regions were clearly defined and islets characterized using rigorous histopathology and immunolocalization examinations. 
Standardized procedures for processing human pancreas recovered from organ donors are described in part 1 of this series. The pancreas is processed into 3 main regions (head, body, tail) followed by transverse sections. Transverse sections from the pancreas head are further divided, as indicated based on size, and numbered alphabetically to denote subsections. This standardization allows for a complete cross sectional analysis of the head region including the uncinate region which contains islets composed primarily of pancreatic polypeptide cells to the tail region.
The current report comprises part 2 of this series and describes the procedures used for serial sectioning and histopathological characterization of the pancreatic paraffin sections with an emphasis on islet endocrine cells, replication, and T-cell infiltrates. Pathology of pancreatic sections is intended to characterize both exocrine, ductular, and endocrine components. The exocrine compartment is evaluated for the presence of pancreatitis (active or chronic), atrophy, fibrosis, and fat, as well as the duct system, particularly in relationship to the presence of pancreatic intraductal neoplasia4. Islets are evaluated for morphology, size, and density, endocrine cells, inflammation, fibrosis, amyloid, and the presence of replicating or apoptotic cells using H&amp;E and IHC stains. 
The final component described in part 2 is the provision of the stained slides as digitized whole slide images. The digitized slides are organized by case and pancreas region in an online pathology database creating a virtual biobank. Access to this online collection is currently provided to over 200 clinicians and scientists involved in type 1 diabetes research. The online database provides a means for rapid and complete data sharing and for investigators to select blocks for paraffin or frozen serial sections.

This video demonstrates procedures for characterization of human pancreatic islets using hematoxylin and eosin (H&E) and immunohistochemistry (IHC). Pancreatic sections from head, body, and tail regions are stained by both H&amp;E and IHC to determine islet endocrine composition (insulin, glucagon, and pancreatic polypeptide), cell replication (Ki67), and inflammatory infiltrates (H&E, CD3). The uncinate region is localized using IHC for pancreatic polypeptide.

Estimates of islet area and numbers and endocrine cell composition in the adult human pancreas vary from several hundred thousand to several million and ...

Medicine

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 ...

A Murine Pancreatic Islet Cell-based Screening for Diabetogenic Environmental Chemicals

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to the development of type 2 diabetes mellitus (T2DM). Although the mechanisms for the chemical-induced &#946; cell damage were unclear and likely to be complex, one recurring finding is that these chemicals induce oxidative stress leading to the generation of excessive reactive oxygen species (ROS) which induce damage to the &#946; cell. To identify potential diabetogenic environmental chemicals, we isolated pancreatic islet cells from C57BL/6 mice and cultured islet cells in 96-well cell culture plates; then, the islet cells were dosed with chemicals and the ROS generation was detected by 2',7'-dichlorofluorescein (DCFH-DA) fluorescent dye. Using this method, we found that bisphenol A (BPA), Benzo[a]pyrene (BaP), and polychlorinated biphenyls (PCBs), could induce high levels of ROS, suggesting that they may potentially induce damage in islet cells. This method should be useful for screening diabetogenic xenobiotics. In addition, the cultured islet cells may also be adapted for in vitro analysis of chemical-induced toxicity in pancreatic cells.

Here we present a protocol to isolate mouse pancreatic islet cells for screening the ROS inductions by the xenobiotics in order to identify the potential diabetogenic xenobiotic chemicals.

Exposure to certain environmental chemicals in human and animals has been found to cause cellular damage of the pancreatic &#946; cells which will lead to ...

Immunology and Infection

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 ...

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix (ECM) Production

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those obtained with whole pancreas transplantation. The protocols currently used for human islet isolation are under scrutiny because they are based on the enzymatic digestion of the organ, whereby the pancreas is demolished, its connections to the body are lost and islets are irreversibly damaged. Islet damage is characterized by critical factors such as the destruction of the extracellular matrix (ECM), which represents the 3D framework of the islet niche and whose loss is incompatible with islet euphysiology. Researchers are proposing the use of ECM-based scaffolds derived from the mammalian pancreas to address this problem and ultimately improve islet viability, function, and lifespan. Currently available methods to obtain such scaffolds are harsh because they are largely detergent based. Thus, we propose a new, detergent-free method that creates less ECM damage and can preserve critical components of pancreatic ECM. The results show that the newly developed decellularization protocol allowed the achievement of complete DNA clearance while the ECM components were retained. The ECM obtained was tested for cytotoxicity and encapsulated with human pancreatic islets which showed a positive cellular behavior with insulin secretion when stimulated with glucose challenge. Collectively, we propose a new method for the decellularization of the human pancreas without the use of conventional ionic and non-ionic chemical detergents. This protocol and the ECM obtained with it could be of use for both in vitro and in vivo applications.

Detergent-Free Decellularization of the Human Pancreas for Soluble Extracellular Matrix ECM Production

The protocol described in this manuscript explains the steps for the fabrication of a soluble extracellular matrix (ECM) from the human pancreas. The solubilized ECM powder obtained through this protocol may be used for the recapitulation of pancreatic islets&#8217; microenvironment in vitro and, potentially, in vivo settings.

Islet transplantation (ITx) has the potential to become the standard of care in beta cell replacement medicine but its results remain inferior to those ...

A Method for Mouse Pancreatic Islet Isolation and Intracellular cAMP Determination

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The &#946;-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the &#946;-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on &#946;-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased &#946;-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3H]-thymidine incorporation, protein abundance, and mRNA expression.

Assaying in vitro &#946;-cell function using isolated mouse islets of Langerhans is an important component in the study of diabetes pathophysiology and therapeutics. While many&#160;downstream applications are available, this protocol specifically describes the measurement of intracellular cyclic adenosine monophosphate (cAMP) as an essential parameter determining &#946;-cell function.

Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing ...

Biology

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

A Simple High Efficiency Protocol for Pancreatic Islet Isolation from Mice

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other important biological functions. The islets primarily consist of five types of hormone-secreting cells: &#945; cells secrete glucagon, &#946; cells secrete insulin, &#948; cells secrete somatostatin, &#949; cells secrete ghrelin, and PP cells secrete pancreatic polypeptide. Sixty to 80% of the cells in the islets are &#946; cells, which are the most important cell population to study insulin secretion. Pancreatic islets are a crucial model system to study ex vivo insulin secretion. Acquiring high quality islets is of great importance for diabetes research. Most islet isolation procedures require technically difficult to access site of collagenase injection, harsh and complex digestion procedures, and multiple density gradient purification steps. This paper features a simple high yield mouse islet isolation method with detailed descriptions and realistic demonstrations, showing the following specific steps: 1) injection of collagenase P at the ampulla of Vater, a small area joining the pancreatic duct and the common bile duct, 2) enzymatic digestion and mechanical separation of the exocrine pancreas, and 3) a single gradient purification step. The advantages of this method are the injection of digestive enzyme using the more accessible ampulla of Vater, more complete digestion using combination of enzymatic and mechanical approaches, and a simpler single gradient purification step. This protocol produces approximately 250&#8212;350 islets per mouse; and islets are suitable for various ex vivo studies. Possible caveats of this procedure are potentially damaged islets due to enzymatic digestion and/or prolonged gradient incubation, all of which can be largely avoided by careful ad justification of incubation time.

This islet isolation protocol described a novel route of collagenase injection to digest the exocrine tissue and a simplified gradient procedure to purify the islets from mice. It involves enzymatic digestion, gradient separation/purification, and islet hand-picking. Successful isolation can yield 250&#8211;350 high quality and fully functional islets per mouse.

Pancreatic islets, also called the Islets of Langerhans, are a cluster of endocrine cells which produces hormones for glucose regulation and other ...

Pancreatic Islet Embedding for Paraffin Sections

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a mixed cell population in a structured architecture that impacts function, and human islets are widely variable in cell type composition. Current frequently used methods to study cultured islets include molecular studies performed on whole islets, lumping disparate islet cell types together, or microscopy or molecular studies on dispersed islet cells, disrupting islet architecture. For in vivo islet studies, paraffin-embedded pancreas sectioning is a powerful technique to assess cell-specific outcomes in the native pancreatic environment. Studying post-culture islets by paraffin sectioning would offer several advantages: detection of multiple outcomes on the same islets (potentially even the exact-same islets, using serial sections), cell-type-specific measurements, and maintaining native islet cell-cell and cell-substratum interactions both during experimental exposure and for analysis. However, existing techniques for embedding isolated islets post-culture are inefficient, time consuming, prone to loss of material, and generally produce sections with inadequate islet numbers to be useful for quantifying outcomes. Clinical pathology laboratory cell block preparation facilities are inaccessible and impractical for basic research laboratories. We have developed an improved, simplified bench-top method that generates sections with robust yield and distribution of islets. Fixed islets are resuspended in warm histological agarose gel and pipetted into a flat disc on a standard glass slide, such that the islets are distributed in a plane. After standard dehydration and embedding, multiple (10+) 4 - 5 &#181;m sections can be cut from the same islet block. Using this method, histological&#160;and immunofluorescent analyses can be performed on mouse, rat, and human islets. This is an effective, inexpensive, time-saving approach to assess cell-type-specific, intact-architecture outcomes from cultured islets.

Ex vivo pancreatic islet studies are important for diabetes research. Existing techniques to study cultured islets in their native 3-dimensional architecture are time consuming, inefficient, and infrequently used. This work describes a new, simple, and efficient method for generating high-quality paraffin sections of whole cultured islets.

Experiments using isolated pancreatic islets are important for diabetes research, but islets are expensive and of limited abundance. Islets contain a ...

Isolated Pancreatic Islet Treatment and Apoptosis Measurement

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of fluorescence staining techniques and confocal microscopy to assess cell viability, apoptosis, and &#946;-cell-specific death. Isolated mouse islets were treated with varying concentrations of a cytokine cocktail, including TNF-&#945;, IL-1&#946;, and IFN-&#947;, to mimic immune-mediated apoptosis during the development of type 1 diabetes. The viability of islet cells was evaluated with FDA/PI dual staining, where FDA conversion to fluorescein indicated viable cells, and PI marked membrane-compromised cells. YOPRO-1 and nuclear staining provided additional data on apoptosis, with Annexin-V confirming early apoptotic cells. Quantitative analysis revealed significant increases in apoptosis and cell death rates in cytokine-treated islets. To specifically assess effects on &#946;-cells, Zn2+ selective indicator staining was used to label insulin-producing cells through the zinc association in insulin granules, revealing substantial &#946;-cell loss following treatment of islets with pro-inflammatory cytokines for 24 h. These multi-staining protocols effectively capture and quantify the extent of cytokine-induced damage in islets and can be used to evaluate therapeutics designed to prevent &#946;-cell apoptosis in early type 1 diabetes.

This study utilizes multi-staining fluorescence-based markers of cell death and apoptosis combined with confocal microscopy to assess cytokine-induced apoptosis and &#946;-cell-specific death in pancreatic islets. It reveals spatial and temporal changes in cell death and apoptosis in response to extracellular stimuli.

This study investigates the effect of pro-inflammatory cytokines on pancreatic islets, particularly insulin-producing &#946;-cells, using a combination of ...