Download - Improving cellular function and immune protection via layer-by-layer nanocoating of pancreatic islet β-cell spheroids cocultured with mesenchymal stem cells

Improving cellular function and immune protection via layer-by-layernanocoating of pancreatic islet b-cell spheroids cocultured withmesenchymal stem cells

Tasneem Bhaiji, Zheng-Liang Zhi, John C. Pickup

Diabetes Research Group, King’s College London School of Medicine, Guy’s Hospital, London SE1 1UL, United Kingdom

Received 26 November 2011; accepted 26 January 2012

Published online 23 March 2012 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.34111

Abstract: Islet transplantation as a therapy for type 1 diabe-

tes is currently limited by lack of primary transplant material

from human donors and post-transplantation loss of islets

caused by adverse immune and nonimmune reactions. This

study aimed to develop a novel strategy to create microenvir-

onment for islets via integration of nanoencapsulation with

cell cocultures, thereby enhancing their survival and function.

The nanoencapsulation was achieved via layer-by-layer

deposition of phosphorycholine-modified poly-L-lysine/hepa-

rin leading to the formation of nanometer-thick multilayer

coating on islets. Spheroids formed by coculturing MIN6

b-cells with mesenchymal stem cells in suspension were

used as the tool for testing encapsulation. Coculturing MSCs

with MIN6 cells allowed the cell constructs to enhance structural

and morphologic stability with improved insulin secretory func-

tion and render them less susceptible to inflammatory cytokine-

induced apoptosis. Combining nanoencapsulation with cocul-

ture of MSCs/MIN6 resulted in higher glucose responsiveness,

and lower antibody binding and apoptosis-inducing effects of

cytokines. This strategy of nanoencapsulating islet cocultures

appears promising to improve cellular delivery of insulin for

treating type 1 diabetes. VC 2012 Wiley Periodicals, Inc. J Biomed

Mater Res Part A: 100A: 1628–1636, 2012.

Key Words: nanoencapsulation, immunoprotection, MIN6 b-cells, mesenchymal stem cells, insulin, diabetes mellitus

How to cite this article: Bhaiji T, Zhi Z-L, Pickup JC. 2012. Improving cellular function and immune protection via layer-by-layernanocoating of pancreatic islet b-cell spheroids cocultured with mesenchymal stem cells. J Biomed Mater Res Part A2012:100A:1628–1636.

INTRODUCTION

Despite the potential of pancreatic islet cell transplantationto offer a cure for type 1 diabetes, its widespread adoptionas a front-line therapy is currently limited by donor isletavailability and, in particular, by the post-transplantationloss of functional islets through deleterious immune andnonimmune reactions.1 Immunoisolation by encapsulation isbeing explored as a way of maintaining or extending long-term function of transplanted islets and offers severalpotential benefits.2,3 Thus, immunoisolation of islet graftmaterial may obviate the necessity for immune-suppressionand is of obvious clinical benefit to the graft recipient. Inaddition, colocalization of islets with other supporting cellsor factors within the encapsulation matrix could provide achronic delivery system for growth/proangiogenic factorswhich might further enhance survival and function of thetransplanted islets. Moreover, the encapsulation matrixcould also provide a physical support to maintain isletarchitecture post-transplantation.

Islet encapsulation strategies to date have mainly focusedon macrocapsules (encapsulation of the whole islet graft)4,5

and microcapsules (encapsulation of individual islets).5,6

These strategies have however experienced major drawbacksincluding hypoxic death due to poor diffusion of vital mole-cules such as oxygen and nutrients into the central cell mass,and unfavorable transplant volumes for the clinically pre-ferred intraportal route of transplantation.3–6 An attractive al-ternative strategy is to generate around the islets complexnanocoatings (nanoencapsulation) via a layer-by-layer deposi-tion of multilayers. Previous studies have shown the potentialof nanoencapsulation in terms of material biocompatibility,optimum molecular permeability of the capsule, and immuno-protection.5–9 Meanwhile, nanoencapsulation is a potentiallyclinically translational technique since there is a little or noincrease in size and volume of the islets postencapsulation,making nanoencapsulated islets suitable for implantation byclinically preferred intrahepatic infusion. Although nanoen-capsulation is a promising technology, islets might face detri-mental shear stresses post-transplantation, which a nanome-ter-thick shell would be unlikely to tolerate without a stromalenvironment. Also, nanoencapsulation layers may not directlyconnect to the islets with multiple biochemical signals.

Additional Supporting Information may be found in the online version of this article.

Correspondence to: Z.-L. Zhi; e-mail: [email protected]

Contract grant sponsor: EPSRC Science & Innovation Award; contract grant number: EP/D062861/1

1628 VC 2012 WILEY PERIODICALS, INC.

Several recent studies indicated that colocalization ofislets with mesenchymal stem/stromal cells (MSCs) couldprovide a delivery system for angiogenic growth factorswhich can promote angiogenesis, resulting in improvedpost-transplant vascularization and subsequent islet func-tion and engraftment.10–12 MSCs are also known to secretecytokines having anti-inflammatory and anti-apoptotic activ-ities.13,14 Colocalization of MSCs with islets thus has thepotential to provide modulation of the immune response,leading to prolonged graft survival and enhanced islet func-tion. These features make the MSCs an attractive cell sourceof coculturing for improving properties of islets.

The current shortage of donor islets is a major issue forallo-transplantation, therefore using insulin-producing cellsgenerated in vitro could be a solution for islet b-cell replace-ment in the treatment of diabetes. Studies are now neededto establish the methods for engineering the environmentand structure of the islets made from these cells. The mouseinsulinoma cell line, MIN6 is a b-cell line known to possessthe same vital characteristics as normal pancreatic b-cellsand is a cost-effective replacement for primary islets in b-cell research.15,16 MIN6 b-cells are able to self organize intoislet-like clusters called pseudoislets via reaggregation ofsingle cells. These organized three-dimensional structuresare important for the maintenance of normal insulin secre-tory responses as they closely mimic the native islet archi-tecture, thus maintaining better cell–cell interactions.17

These configured pseudoislets could benefit from coculturewith MSCs, which are able to secret the extracellular matrixand form a supportive stromal environment for the b-cells.In this study, we aimed to engineer pseudoislets by cocul-turing MSCs and MIN6 b-cells, and then to use the cocultureconstructs to assess whether nanoencapsulation could fur-ther enhance cellular function and immunoprotection capa-bility of the pseudoislets.

EXPERIMENTAL METHODS

Materials and reagentsTissue culture reagents, including Dulbecco’s ModifiedEagles Medium (DMEM), L-glutamine, penicillin, streptomy-cin, and trypsin-ethylenediaminetetraacetic acid (EDTA)were obtained from Sigma-Aldrich. Hanks buffered salt solu-tion (HBSS) was provided by PAA Laboratories, GmbH,(C€olbe, Germany). Fetal bovine serum (FBS) was purchasedfrom GIBCO. The CellTiter-BlueV

R

cell viability assay kit andthe Apo-ONEVR homogeneous caspase-3/7 assay kit werepurchased from Promega. Poly-L-lysine (PLL) (40–60 kDa),heparin (8–20 kDa), alginate (75–100 kDa), and chondroi-tin-4-sulphate (20 kDa) were purchased from Sigma Aldrich.Anti-mouse major histocompatibility complex (MHC) Class IIantibody was obtained from eBioscience (Hatfield, UK).Recombinant mouse IL-1b, TNF-a, and IFN-c were pur-chased from BioLegend (bottled at 200 lg/mL, carrier-free)(San Diego, USA). Liquid DAB þ substrate chromogen sys-tem (K347611) were purchased from Dako (Ely, UK). Rabbitanti-mouse insulin was purchased from GeneTex (Irvine,USA), and peroxidase-conjugated goat anti-rabbit IgG wasavailable from AnaSpec (Fremont, USA).

MIN6 cell culturingMIN6 cells were cultured in 75 mm flasks (passage 32–37)at 37�C (95% O2 and 5% CO2) in a humidified incubator in13 mL of medium: DMEM (with the glucose concentrationof 25 mM, supplemented with 10% FBS, 1% L-glutamine,2% penicillin/streptomycin) in accordance with that origi-nally reported for the cell line.18 Once monolayers reachedconfluence, the cells were trypsinized (0.1% trypsin, 0.02%EDTA) and used to make pseudoislets or seeded again tocontinue growth.

MSC culturingMesenchymal stem cells were available as a gift from MissRackham CL (King’s College London), and were originallyisolated from C57BL/6 mice via a standard procedure.12

MSC cells (passage 8–12) were cultured in flasks and keptin the same conditions as that of MIN6 cells.

Making pseudoisletsAfter trypsinization, the cell number and density were deter-mined by counting on a haemocytometer slide for both MIN6cells and MSCs. The cells were then adjusted to a density of4.9 � 104 cells/mL. The following ratios of MIN6 to MSCswere then made: 1:1, 1:3, and 1:5 into uncoated 90 mm Petridishes, with the population of MSCs kept constant. Pseudois-let constructs seeded with MIN6 cells (MIN6 alone) werealso prepared with a similar procedure. Pseudoislets wereused for experiments 6–8 days after static subculturing.

Deposition of nanoencapusulating layers onpseudoisletsPhosphorycholine-modified poly-L-lysine (PLL-PC) was syn-thesized according to the previously reported procedure.7 Tominimize the potential toxicity of the highly charged PLL, wegrafted PC to about 40% (graft ratio) of lysine residues inPLL. The working solution of PLL-PC (2 mg/mL) was pre-pared by dissolving the solids in HBSS (supplemented with2 mM Ca2þ). Working solutions of the heparin and chondroi-tin-4-sulfate (1 mg/mL) were prepared in the same way.Alginate was first made into a 1% stock solution with waterand further diluted to 1 mg/mL in HBSS. The pseudoisletswere nanoencapusulated with eight layers of PLL-PC/heparinunless specified. To start the coating, medium was removedfrom the pseudoislets, the cells were washed twice withHBSS in a 1.5-mL Eppendorf tube by gravity sedimentation.Since the overall surface charge of the pseudoislets is nega-tive, the polycation (PLL-PC) was added first (0.2 mL) to thecells for 5 min and mixed gently with a pipette. The remain-ing polycation was removed and the cells were washed twicewith (0.6 mL) HBSS before adding the next coating solution(polyanion). This method was repeated until the desirednumber of layers was reached. Note: an even number oflayers (negative outer charge) was always made.

Fluorescence microscopy studies of thenanoencapsulation layersTo label PLL, fluorescent dye Alexa Fluor 647 carboxylicacid succinimidyl ester (Invitrogen) (Paisley, UK) (0.1 mg)

ORIGINAL ARTICLE

JOURNAL OF BIOMEDICAL MATERIALS RESEARCH A | JUN 2012 VOL 100A, ISSUE 6 1629

was dissolved in dimethylsulfoxide (Sigma) (1 mg/mL). PLL(2 mg) was dissolved in 1 mL 10 mM phosphate buffer, pH7.2; 0.1 mL of the dye was then added to the PLL andmixed overnight at 4�C. To remove excess dye, the solutionwas dialysed against water for 16 h, and then HBSS for 4 h.This solution was then used for encapsulating the islets asthe fluorescence labeled polycation layer. The deposition ofthe eight-layer-coating was assessed by fluorescence micros-copy (Nikon Eclipse Ti-U, Japan) following the incorporationof a layer of the fluorescence-labeled PLL into the 7th layeras the fluorescent marker.

Transmission electron microscopy (TEM) analysis of thenanoencapsulated pseudoisletsSections were examined using a Hitachi H7600 transmissionelectron microscope (Hitachi, Tokyo, Japan) at 75 kV.

Live/dead cell staining of the encapsulated pseudoisletsin cultureThe live/dead cell test of the pseudoislets was done using atwo-color fluorescence method involving propidium iodide(PI) (marker for nonviable cells) and fluorescein diacetate(FDA) (marker for live cells), as reported previously.7

Antibody exclusion assayAn antibody exclusion assay with FITC-labeled anti-mousemajor histocompatibility antigens (MHC) Class II antibody(eBioscience) was used to examine the permeability of thenanocoating to large immunologically relevant molecules.Groups of 20 naked and nanoencapsulated pseudoisletswere each added to 100 lL of the blocking buffer (2% BSAand HBSS buffer), and then 1 lL of antibody-FITC solutionwas added. The solution was then incubated at 37�C for 30min. The islets were then rinsed three times with HBSS-0.1% Tween (0.5 mL) to remove any unbound antibody. Theislets were then transferred to a cover-slip for observationwith a fluorescence microscope.

Insulin secretion: Static incubationsThe formed pseudoislets were washed and incubated forovernight with 0.5 mL of 2 mM glucose (0.5% w/v BSA, 2mM Ca2þ) in a bicarbonate-buffered physiological salt solu-tion, the Gey & Gey buffer. Groups of five pseudoislets (bothencapsulated and nonencapsulated) were placed into 1.5-mL Eppendorf tubes in triplicate. The solution was thenremoved and the cells kept on ice to slow down any meta-bolic activity; 0.5 mL of 2 mM glucose was then added toeach tube. The tubes were then placed in a 37�C-incubatorfor 30 min. The pseudoislets were then placed on ice and0.1 mL of the medium was collected from each tube, induplicate. The rest of the solution in the tubes was removedand 0.5 mL of 20 mM glucose (0.5% BSA, 2 mM Ca2þ) inGey & Gey buffer added to the pseudoislets and againplaced in a 37�C incubator for 30 min. The tubes were thenplaced on ice and 0.1 mL of the solution was taken in dupli-cates. The insulin content in the samples was determinedby standard radioimmunoassay.17

Treatment of pseudoislets with cytokinesGroups of 50 pseudoislets (nanoencapsulated and control)were placed in 50-mm Petri dishes and the cytokinemixture was added to give a final concentration of IL-1b(5 ng/mL), TNF-a (10 ng/mL), and IFN-c (100 ng/mL)(ratio cytokine volume: medium 1:100). Both the control(no cytokine added) and experimental cells were placed ina 37�C (95% O2 and 5% CO2) humidified incubator for24 h. Cell viability and apoptosis were then examined bytaking five islets and 50 lL medium in each tube. The cellviability and apoptosis were measured using the Promega(Southampton, UK) kits: CellTiter-BlueV

R

cell viability assaykit and Apo-ONEV

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Homogeneous Caspase-3/7 Assay kit,according to the manufacturer’s instructions. A Spectra MaxGemini EM microwell plate-reader (Molecular Devices, UK)was used to measure the fluorescence intensity.

Immunohistological studies of insulin expression inpseudoisletsThe pseudoislets were fixed in 4% (vol/vol) neutral-buf-fered formalin and later paraffin-embedded, the specimenwas sectioned at a thickness of 5 lm using standard histo-logical procedures. Sections were stained with the primaryantibody polyclonal rabbit anti-insulin (used at 1:200 dilu-tions) in a humidifying chamber for 1 h at room tempera-ture. This was followed by a further incubation of 1 h withperoxidase-conjugated goat anti-rabbit IgG (diluted in block-ing buffer at a 1:200). DAB (obtained from Dako) solutionwas used to visualize the antibody binding. The brownstained regions identified areas of insulin which could beseen under a light microscope. For histological study of fluo-rescence-labeled islets, the histological procedure was thesame. However, the sectioned specimen was observed witha fluorescence microscope without any further treatment.

Statistical analysisIndependent t-tests were used to identify any significant dif-ference between individual groups of the insulin secretionand cytokine assay results and p values of less than 0.05were considered significant.

RESULTS

Formation of MSCs/MIN6 pseudoisletsFigure 1 shows the pseudoislets formed after 3 and 7 days ofstatic coculturing with MSCs and MIN6 cells at varying ratios.Most of the spheroids formed at day 3 were very small andirregularly shaped, but we found that a 7-day culture periodwas the optimum time for growing pseudoislets, with all ratiosof MSCs/MIN6 cells forming round-shaped (ca. 150–200 lm) is-let-like morphology after this time. There was generally a higherfrequency of larger pseudoislets being formed in MIN6 cellsalone and in the ratio of 1:1 culture. Coculturing with a higherratio (e.g., 1:3 MSCs/MIN6) of the cells appeared to give a lowerfrequency of large pseudoislets (Fig. 1). Moreover, pseudoisletsformed from MSCs/MIN6 coculture appeared more robust andwere less easily breakable than that from MIN6 cells alone.

Live/dead staining with PI/FDA [Fig. 1(B)] showed thatvery large pseudoislets exhibited high levels of apoptosis or

1630 BHAIJI, ZHI, AND PICKUP NANOCOATING OF b-CELL SPHEROIDS COCULTURED WITH MSCs

necrosis (�37%, as analyzed by Image J analysis, see Sup-porting Information), especially at the centre of the struc-tures in comparison to the smaller pseudoislets (�0.4%).This is most likely due to hypoxia and poor nutrient trans-port resulting from the large diffusion distance imposed bythe size. Smaller pseudoislets were therefore consideredmore favorable for use in further study.

Time-dependent cell viability in nanoencapsulatedpseudoisletsFigure 2(A) shows the live/dead staining of the nanoencap-sulated (eight layers of PC-PLL/heparin) 1:3 MSCs/MIN6

pseudoislets at the beginning of the culturing (day 1) andafter 16 days postencapsulation in culture. Image J analysisshowed 0.6% of nonviable cells at day 1, and at day 16,there were �5% cells dead. These data were comparable tothat of the nonencapsulated pseudoislets in culture (datanot shown), suggesting that the islets tolerated the multi-step nanoencapsulation process and the deposited coatinglayers.

Confirmation of layer deposition and long-term stabilityof the nanolayersOur nanoencapsulation approach uses spontaneous deposi-tion of alternative layers of charged linear biopolymers(PC-PLL/heparin) as the coating materials. The deposition

FIGURE 1. A: Light microscopy image of MIN6 cells alone, MSCs/MIN6

pseudoislets for ratios 1:1 and 1:3 after 3 days and 7 days of culturing.

B: Fluorescence microscopy of live/dead MSCs/MIN6 pseudoislet after

9 days (viable cells: green and nonviable cells: red). (i) Large pseudois-

let (�400 lm): �37% of dead cells were detected (ii) small pseudoislet

(�165 lm): �0.4% of dead cells were detected. Nonviable cells were cal-

culated via Image J software. [Color figure can be viewed in the online

issue, which is available at wileyonlinelibrary.com.]

FIGURE 2. A: Fluorescence microscopic images of live/dead cells in

MSCs/MIN6 (1:3) pseudoislets encapsulated with alternate layers of

PLL-PC and heparin (eight layers). Nonviable cells at day 1 were 0.6%

and at day 16, 5%. B: Monitoring stability of the nanocoating formed

on islet surfaces. MSCs/MIN6 pseudoislets (1:3) were encapsulated

with alternate layers of PLL-PC and heparin (eight layers). The 7th

layer consisting of PLL tagged with Alexa Fluor 647 was incorporated

in the coating. [Color figure can be viewed in the online issue, which

is available at wileyonlinelibrary.com.]

ORIGINAL ARTICLE


of eight layers was assessed by fluorescence microscopy fol-lowing the incorporation of a layer of PLL tagged with AlexaFluor 647 into the 7th layer as a fluorescent marker. Asdepicted in Figure 2(B), the addition of the fluorescencedye-tagged layer onto the islets resulted in strong fluores-cence localized on the face of islet cells, which is consistentwith the extracellular architecture of the islets, indicating auniform and physically intact coating. Some bright spotsmay due to the density difference of either cells or extracel-lular matrix.

Figure 2(B) shows also the long-term coating stability inculture. Significant amounts of layer material were detecta-ble after at least 11 days in culture. The fluorescent signaldecreased considerably at day 20 either because the layersgenerated were unstable or degenerated or because ofintrinsic photoinstablity of Alexa Fluor 647.

Nanoencapsulating layer coverageWe examined histological appearances of the encapsulatedpseudoislets to investigate whether the nanolayers coveredthe entire surface of the islets, which is vital for completeimmune protection. Figure 3(A) shows the paraffin-embed-ded cross section of a coated islet (with the incorporation

of a layer of Alexa Fluor 647-labeled PLL), indicating that acomplete coverage of the exterior islet surfaces wasachieved and the coating was localized on the surface of pe-ripheral cells. The TEM ultrastructural image [Fig. 3(B)]showed an intact coating consisting of nanolayers of �60nm thickness that covered the outer surface of cells on thepseudoislet periphery.

Insulin staining of the pseudoislets: ImmunohistologicalanalysisImmunohistochemistry was used to confirm the functionality(insulin expression) of the pseudoislets prior and after nano-encapsulation. As can be seen in Figure 3(C,D), the intense in-sulin immune staining on the islet sections indicates that thepseudoislets were functional prior- and postencapsulation.Moreover, depending on the position of the section taken, itwas possible to distinguish and locate MSCs in the insulin-stained pseudoislet section. As shown in Figure 3(C,D), thebrown regions, likely to be insulin-producing MIN6 cells,were located mainly at the peripheral region of pseudoislets,whereas the white regions (MSCs, as they do not produceinsulin) were spread out randomly toward the islet centreand were well-incorporated within the coculture construct.

FIGURE 3. A: Cross section of the fluorescence-labeled MSCs/MIN6 (1:3) pseudoislet. B: TEM micrograph of a pseudoislet encapsulated by an

eight-layer of PLL-PC and heparin coating. C: immunohistological insulin staining of a nonencapsulated 1:3 MSCsMIN6 pseudoislet. D: immunohis-

tological insulin staining of a nanoencapsulated 1:3 MSCs/MIN6 pseudoislet. [Color figure can be viewed in the online issue, which is available at

wileyonlinelibrary.com.]


Insulin secretion from MSCs/MIN6 pseudoisletsStatic incubation was used to examine the insulin secretionand release from pseudoislets of varying MSCs/MIN6 ratios,including MIN6 alone, 1:3 and 1:5 ratio, in response toextracellular glucose change (2 vs. 20 mM). As shown in Fig-ure 4(A), the basal rate of insulin release into the mediumat a substimulatory glucose concentration (2 mM) wasincreased significantly when MSCs were introduced in theculture. Increasing the glucose concentration to a stimula-tory concentration (20 mM) significantly enhanced insulinsecretion from both the MIN6 cells alone and from 1:3 and1:5 MSCs/MIN6 cocultured pseudoislets. This glucose-induced insulin secretory response was further enhanced bythe nanoencapsulation (eight layers of PLL-PC/heparin) asshown in Figure 4(A).

When the data for insulin release were expressed rela-tive to the appropriate basal rate of secretion (stimulation

index) [Fig. 4(B)], the secretory responses induced by glu-cose were similar between the MIN6 cells alone group andthe MSCs/MIN6 coculture groups (p > 0.05). For the nano-encapsulated pseudoislets (eight layers of PLL-PC/heparin),the insulin secretion was little affected at 2 mM glucoseconcentration. However, at high glucose concentration (20mM), insulin secretion was significantly increased (p <

0.01) compared with the nonencapsulated islets. Thisresulted in higher ratios of insulin secretion, indicating thatthe nanocoating enhanced responsiveness to glucose of theb-cells.

We also found that a coating of 12 layers of PLL-PC/heparin exhibited a great decrease in insulin release andglucose responsiveness (data not shown), possibly due tothe nanolayer impairing the diffusion of the secreted insulinfrom the pseudoislets.

Immunoisolation of pseudoislets: Antibody bindingassayFigure 5 shows a fluorescence image of the naked andencapsulated (PLL-PC/heparin, eight layers) 1:3 pseudois-lets after being exposed to an FITC-antibody (�150 kDa)which targets MHC Class II antigens on the surface of themouse islets. Encapsulated pseudoislets were significantlymore effective at excluding the antibodies than the nonen-capsulated ones, where almost every islet had some anti-body binding to its MHC class II antigens. These resultsindicate that eight layers of PLL-PC/heparin coating is suffi-cient enough for the exclusion of biomacromolecules.

Effect of cytokine exposure on pseudoisletsA combination of three cytokines (IL-1-b, TNF-a, and IFN-c)were employed to assess whether nanoencapsulation andcoculturing with MSCs could provide a cytoprotective effecton the MIN6 beta-cells. Previous studies have revealed thatthese cytokines exerted significant detrimental effects onthe function and viability of islet cells in vitro.19,20 As shownin Figure 6(A), cell viability decreased significantly (p <

0.05) for the pseudoislets seeded with MIN6 cells alonewhen exposed to the cytokines (2-day accumulation). How-ever, the viability was only slightly (but not significantly)affected for those pseudoislets from MSCs/MIN6 cocultures(p > 0.05). This suggests that addition of MSCs helped inhi-bition of the cytokine-mediated destruction to the b-cells.Figure 6(B) shows the level of apoptosis induced by cyto-kines in different groups of pseudoislets. Compared withthe apoptotic activity measured in MIN6 cells alone (cyto-kine-treated), the 1:3 MSCsMIN6 pseudoislets (naked)showed a significantly lower level of apoptosis (p < 0.01).

In search for the optimal coating layers to circumventcytokine-induced damage to the b-cells, three combinationsof coating layers were examined in this study: PLL-PC/hepa-rin, PLL-PC/chondroitin-4-sulfate, and PLL-PC/alginate. Allthese polyelectrolyte pairs showed negligible effect on apo-ptosis and viability of the pseudoislet cells when cytokineswere not applied in the culture, indicating that these coat-ings are cytocompatible. When exposed to the cytokines,they showed some but not complete protection for the

FIGURE 4. Insulin release of naked (control) and nanoencapsulated

MSCsMIN6 pseudoislets in response to glucose concentration change.

All encapsulation layers represent eight alternate layers of PLL-PC/

heparin. A: Insulin release was normalized against MIN6 pseudoislets

(2 mM glucose concentration). Error bars show 6 SEM (n ¼ 3). B: Ra-

tio of insulin release between high glucose (20 mM) and basal glu-

cose (2 mM) of each category (* p < 0.01). Error bars show relative

errors.

ORIGINAL ARTICLE


pseudoislets, as shown in Figure 6(B). Alginate and heparinwere found to be the most efficient in inhibiting cytokine-mediated damage to the pseudoislet cells, with a two thirddecrease in the apoptotic activity.

DISCUSSION

Although coculture of pancreatic islets with MSCs forenhancing b-cell function and islet graft survival has beenreported previously,13,21 there are no prior reports of engi-neering pseudoislets by coculturing single b-cells withMSCs. The unique advantage of the strategy developed inthis study is that it allows potential use of in vitro gener-ated insulin-producing cells for islet tissue engineering,therefore expanding the pool of available islet sources forbeta-cell replacement therapy of diabetes. Meanwhile, cocul-turing single beta-cells with MSCs offers additional benefitof maintaining better cell–cell contact between the two dif-ferent types of cells.

In this study, we demonstrated that addition of a smallfraction of MSCs into MIN6 b-cell culture resulted in the for-mation of pseudoislets with controllable size, similar to thatof primary islets. Small-sized pseudoislets are favorable forthe interest for minimizing the hypoxia and poor nutrienttransport that result from the diffusion distance imposed bythe large size. Immunohistological analysis with the formedcomposite pseudoislets revealed that MIN6 b-cells werelocated mainly at the peripheral regions of the pseudoislets,whereas the MSCs were spread out randomly toward thecentre of the structures. This indicates that MSCs which arewell-known for their adherent properties may have a roleas seeding cells, regulating the pseudoislet formation pro-cess and promoting the formation of cell clusters.

It is apparent from our data [Fig. 4(A)] that insulinsecretion from MIN6 pseudoislets alone is outperformed bypseudoislets that were cocultured with MSCs, which is con-sistent with results reported previously for primary islets.13

However, the presence of MSCs in MIN6 cell pseudoislets

did not influence the ability of the b-cells to detect andrespond appropriately to changes in extracellular glucose(responsiveness). This is most likely due to the fact that thehighest insulin expression MIN6 cells are mainly located onthe surfaces of a pseudoislet, as confirmed by the immuno-histochemistry staining, and MSCs are located in the interiorof the structure, therefore having little effect on those cellslocated outside.

Electrostatic interaction-based layers were found in pre-vious studies to sometimes exhibit decomposition due tounstable binding,22 especially for weakly ionic polymers. Inthis study, we chose heparin which carries strongly ionicsulfate groups as one of the complexation-pair components(anionic) in the coating. Heparin contributes greatly to thestability of the layered nanofilm, making it a durable coatingunder physiological conditions. Nanoencapsulation withalternate layers of PLL-PC/heparin nanocoating resulted innot only higher insulin release but also enhanced glucoseresponsiveness of the b-cells, as shown in Figure 4(B). Thisis possibly due to the activity of heparin that was depositedon the surface of the pseudoislets. Heparin is known tobind to a variety of soluble growth factors that can aid incell survival and function.23 Therefore, localization of hepa-rin at the islet may result in an accumulation of extracellu-lar matrix around the islet cells which promotes cellularfunction.

Cytokines such as IFN-c, TNF-a, and IL-1b are releasedby infiltrating mononuclear cells like activated macrophagesand effective T-cells and are damaging to pancreatic isletsvia apoptosis induction. In this study, we found that thepresence of MSCs in pseudoislets suppressed the inflamma-tory cytokine-mediated apoptosis. This finding is also con-sistent with that of Karaoz et al. who found low percentageof apoptotic cells in the coculture of MSCs and mouseislets.13 The elevated levels of immunomodulatory cytokineslike IL-6 and TGF-b1 were thought to aid the cytoprotectiveproperties of the MSCs.

FIGURE 5. Fluorescence images of MSCs/MIN6 (1:3) pseudoislets after the antibody (anti-MHC-FITC) binding assay. A: Naked pseudoislets; B:

Nanoencapsulated pseudoislets (layered with eight layers of PLL-PC/heparin). [Color figure can be viewed in the online issue, which is available

at wileyonlinelibrary.com.]


One of the major concerns of nanoencapsulation is thatit does not always offer complete coverage for the islets,making them still vulnerable to immune attacks. Any suc-cess of islet nanoencapsulation in immune-isolation is thusgoverned by the potential for the nanocoating to locallyblock a variety of specific protein–protein interactionsinvolved in islet rejection.24 The image of the fluorescence-labeled pseudoislet section in this study suggested the com-plete coverage of the nanolayer on the islet, and our nano-coating with eight layers of PLL-PC/heparin also displayed asuccessful exclusion of the antibody probe, confirming acomplete or near-complete coverage of the nanolayer coat-ing. The nanocoatings comprising of multilayers of PLL-PC/heparin, PLL-PC/alginate, and PLL-PC/chondroitin-4-sulfatewere all found to be cytoprotective, significantly suppress-ing apoptosis induced by the cytokines. Therefore, the con-

cept of integrating nanoencapsulation with coculture ofMSCs has significant potential for use in modulation of theimmune response and enhancing islet function in transplan-tation applications.

In conclusion, the nanocoating developed in this studymaintained insulin secretory function and enhanced glucoseresponsiveness of pseudoislets. The encapsulation materialsand the encapsulation procedure used in this study werenot damaging to pseudoislet cells. Addition of small frac-tions of MSCs significantly enhanced insulin production inMIN6 b-cell cocultures and conferred antiapoptotic andimmunosuppressive properties. Furthermore, the nanocoat-ing also effectively minimized antibody binding to the pseu-doislets and had the superior property of suppressing cyto-kine-mediated damage to the pseudoislets. Future studiesnow need to test the short- and long-term functioning andgraft survival of nanoencapulated composite MSCs/b-cellpseudoislets after transplantation.

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