Cell Transplantation, Vol. 9, pp. 2 5 - 3 1 , 2000 0963-6897/00 $20.00 + .

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Chitosan-Polyvinyl Pyrrolidone Hydrogels as Candidate for Islet

Immunoisolation: In Vitro Biocompatibility Evaluation
Makarand Risbud, Anandwardhan Hardikar, and Ramesh Bhonde

Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune 411 007, India

The success of immunoisolation devices for islet transplantation depends on the nature of semipermeable
membranes, which permit the crossover of micronutrients, glucose, and insulin and prevent the entry of
immunocytes and other transplant rejection mechanisms. In the present study we examined the properties
of chitosan-polyvinyl pyrrolidone (PVP) hydrogels for possible application as an immunoisolation device.
Hydrogels with two different proportions of chitosan-PVP (Ml 1:1 and M2 2:1, v/v) were synthesized by
cross-linking with glutaraldehyde. Hydrogels were characterized for their hydrophilic nature, protein adsorp­
tion, diffusion properties, cytotoxicity, and islet compatibility. Hydrogel membranes were found to be hydro­
philic as determined by high octane contact angle value ( M l : 142.9 ± 0 . 4 6 ; M2: 143.6 ±0.49). Protein ad­
sorption on the hydrogels was found to be low (0.0143 ± 0.0027 mg for M l and 0.0136 ± 0.0049 mg for
M2) compared to tissue culture polystyrene (TCPS) (0.0434 ± 0.001 mg) and pure chitosan (0.0214 ± 0.0025
mg) control. Hydrogel Ml was tested as a representative for diffusion studies. M l allowed regulated trans­
port of insulin and did not allow anti-insulin antibodies to pass through. In vitro biocompatibility of M l and
M2 was found to be excellent with no cytotoxic effects on the HeLa cells as determined by MTT and NR
assay. Mouse islets cultured on the hydrogel membranes retained their integrity and intact morphology as
assessed by image analysis study. Viability of islets cultured on hydrogels was comparable to that of controls
( M l : 97%; M2: 90.4%) as assessed by trypan blue dye exclusion test. Islets retained their functionality when
cultured on hydrogels, as judged by insulin secretion in response to glucose challenge (16.0 mM). Although
in vivo experiments are awaited, the present study provides sufficient documentation to consider chitosan-
PVP membranes as potential candidates for immunoisolation of islets.

Key words: Chitosan; Hydrogels; Biocompatibility; Islet; Immunoisolation

INTRODUCTION cells b y artificial barriers that p e r m i t c r o s s o v e r of low

T y p e I diabetes mellitus essentially involves a total/
partial destruction of islet P-cells leading to an insulin
deficiency a n d related diabetic c o m p l i c a t i o n s . T h e p r e s ­
ent line of therapy for such subjects involves administra­
tion of insulin with daily m o n i t o r i n g of pjasrna g l u c o s e
levels. Pancreas/islet transplantation w a s looked u p o n as
a s e e m i n g l y efficient and less traumatizing alternative.
However, pancreatic transplantation has limitations,
such as the shortage of H L A - m a t c h e d d o n o r p a n c r e a s ,
l o n g - t e r m i m m u n o s u p p r e s s i o n , and other c o m p l i c a t i o n s
(10). Studies h a v e also s h o w n destruction of p-cells with
p o o r (31) and perfect (29) H L A - m a t c h i n g p a n c r e a t i c
transplants d e s p i t e standard i m m u n o s u p p r e s s i v e therapy.
T h e r e f o r e , i m m u n o i s o l a t i o n d e v i c e s h a v e progressively
g a i n e d i m p o r t a n c e o v e r the traditional pancreatic/islet
transplant p r o c e d u r e s . I m m u n o i s o l a t i o n of transplanted
m o l e c u l a r w e i g h t s u b s t a n c e s , such as nutrients, electro­
lytes, o x y g e n , and b i o s e c r e t o r y p r o d u c t s , but not of im­
m u n o c y t e s and other transplant rejection effector m e c h ­
a n i s m s , p r o v i d e s great p r o m i s e for d e v e l o p i n g new
t e c h n o l o g i e s to o v e r c o m e these p r o b l e m s in a r e a s o n a b l e
time frame. V a r i o u s a p p r o a c h e s viz. A V shunt (6), h o l ­
low fibers ( 4 , 2 6 ) , a n d m i c r o c a p s u l e s (16,21) e m p l o y i n g
semipermeable membranes have been proposed. Such
s e m i p e r m e a b l e m e m b r a n e s allow regulated transport of
g l u c o s e and insulin (15) to and from islets b u t p r e v e n t
i m m u n o c y t e s from reaching islets. In this context s o m e
h y d r o g e l s h a v e o c c u p i e d the k e y role as i m m u n o b a r r i e r s
(14). W e h a v e u n d e r t a k e n the present study to d e m o n ­
strate t h e biocompatibility of c h i t o s a n - p o l y v i n y l p y r r o l ­
i d o n e ( P V P ) h y d r o g e l s in vitro and their possible c a n d i ­
d a c y as an i m m u n o b a r r i e r m e m b r a n e in the design of
i m m u n o i s o l a t i o n d e v i c e s for islets of L a n g e r h a n s .

Accepted August 16, 1999.

Address correspondence to Ramesh Bhonde, Tissue Engineering and Banking Laboratory, National Centre for Cell Science, Ganeshkhind, Pune
411 007, India. Tel: 91 20 5670922/31/41; Fax: 91 20 5672259; E-mail: mrisbud@hotmail.com

25
26
MATERIALS AND METHODS
Membrane Synthesis
Chitosan (Vishu A q u a t e c h , M a d r a s , India, deacytyla-
tion d e g r e e > 8 0 % ) solution 2 % (w/v) w a s p r e p a r e d b y
dissolving chitosan in acetic acid (0.1 N ) . P V P ( S R L ,
B o m b a y , India) solution 4 . 0 % (w/v) w a s p r e p a r e d in
d o u b l e distilled water, and t w o different c o m p o s i t i o n s
(Ml 1:1, M 2 2 : 1 , v/v) of chitosan and P V P w e r e p r e ­
p a r e d . T h e a q u e o u s solution of g l u t a r a l d e h y d e
B i o m e d i c a l s , A u r o r a , O H ) at 0 . 3 % of final v o l u m e w a s
then a d d e d with c o n t i n u o u s stirring. M e m b r a n e s w e r e
cast in tissue culture plates ( N u n c l o n , D e n m a r k ) and
kept for drying at 3 0 + 2 ° C for 4 8 h in sterile a t m o ­
sphere. Before u s i n g t h e m for islet seeding, m e m b r a n e s
w e r e w a s h e d extensively with P B S ( p H 7.4) to r e m o v e
any unreacted g l u t a r a l d e h y d e , followed b y t w o w a s h e s
with tissue culture m e d i u m .
Octane Contact Angle Determination
O c t a n e contact a n g l e m e t h o d w a s e m p l o y e d to deter­
m i n e the polar interactions across the p o l y m e r - w a t e r in­
terface. M e m b r a n e s w e r e m o u n t e d on the m i c r o s c o p e
slides and supported in an inverted fashion in a c o n ­
tainer. T h e c o n t a i n e r w a s filled with d o u b l e distilled w a ­
ter to i m m e r s e the slide. G o n i o m e t e r ( K e r n c o Instru­
m e n t s Co., Inc., T X ) w a s aligned and focused on the
polymer-water interface. A microsyringe containing
9 9 . 9 9 % « - o c t a n e w a s l o w e r e d in the w a t e r and a d r o p
of a r o u n d 0.1 p i w a s introduced on the p o l y m e r - w a t e r
interface. C o n t a c t a n g l e on b o t h sides of the d r o p w a s
immediately measured, assuming symmetry. Data repre­
sent m e a n ± S E M of at least 3 0 such angles on e a c h
membrane.
Protein Adsorption on Membrane Surface
Membrane pieces and tissue culture polystyrene
( T C P S , N u n c l o n e , D e n m a r k ) as control of 3 6 0 m m 2
face area w e r e i n c u b a t e d in D u l b e c c o ' s modified E a ­
g l e ' s m e d i u m ( D M E M ; G i b c o B R L , G r a n d Island, N Y )
with 1 0 % fetal calf s e r u m ( F C S ) for 2 4 h at 3 7 ° C . P i e c e s
w e r e r e m o v e d and w a s h e d t w i c e for 15 m i n in 5 0 0 p i
of 1 M saline. D e s o r b e d protein concentration w a s esti­
m a t e d using protein estimation kit (Pierce, Rockford,
IL). Results represent total a m o u n t of protein a d s o r b e d
on the m e m b r a n e s .
Cytotoxicity Testing by MTT and Neutral Red (NR)
Assay
Sterile m e m b r a n e p i e c e s of uniform w e i g h t (50 m g
each) w e r e incubated in 10 ml of D M E M at 3 7 ° C for
15 d a y s . T h i s extract, c o n t a i n i n g the m e m b r a n e leach-
o u t p r o d u c t s , w a s filter sterilized and used for testing
the effect of leaching p r o d u c t s on the cells. H e L a ( C C L -
2, h u m a n , epithelioid cervical c a r c i n o m a ) cells w e r e o b ­
RISBUD, HARDIKAR, AND BHONDE
tained from A T C C (Rockville, M D ) , seeded on 96-well
plates, and incubated at 3 7 ° C in 5 % C 0 , 9 5 % air. Cells
2
w e r e then e x p o s e d to varying c o n c e n t r a t i o n s ( 5 - 2 0 % )
of h y d r o g e l extracts for 2 4 h with cells u n e x p o s e d to
h y d r o g e l extracts serving as control. Plates w e r e then
incubated in the dark at 3 7 ° C for 3 h with M T T (0.5
m g / m l ) and N R (40 p g / m l ) d y e (5). A t the e n d of incu­
bation M T T - and N R - c o n t a i n i n g m e d i u m w a s aspirated
and color w a s d e v e l o p e d b y a d d i n g dimethyl s u l p h o x i d e
(ICN
( D M S O ) and acetic acid ( l % ) / e t h a n o l ( 5 0 % ) m i x t u r e ,
respectively. A b s o r b a n c e w a s read at 5 7 0 a n d 5 4 0 n m
on an E L I S A reader ( M R 7 0 0 , D y n a t e c h L a b o r a t o r i e s ,
U K ) for M T T and N R , respectively.
Islet Isolation and Biocompatibility Testing
Islets w e r e isolated following the protocol of G o t o h
et al. (7). Briefly, 8-week-old B A L B / c m i c e w e r e sacri­
ficed by cervical dislocation. T h e p a n c r e a s w a s d i s ­
sected, m i n c e d , and incubated in 1 m g / m l c o l l a g e n a s e P
( B o e h r i n g e r M a n n h e i m , G e r m a n y ) on a s h a k e r platform
for 2 0 m i n . Freshly isolated islets w e r e then cultured in
R P M I - 1 6 4 0 m e d i u m ( G i b c o B R L ) with 1 0 % F C S for 4 8
h. Islets (« = 30) w e r e h a n d p i c k e d and c u l t u r e d on the
h y d r o g e l - c o a t e d plates ( 3 5 - m m petri dishes, N u n c l o n e )
and their m o r p h o l o g y o b s e r v e d u n d e r p h a s e contrast m i ­
c r o s c o p e for 10 d a y s . Viability of islets cultured on h y ­
drogel m e m b r a n e s w a s d e t e r m i n e d by trypan b l u e d y e
exclusion test, using 0 . 0 4 % (w/v) trypan blue. Islets
staining b l u e w e r e scored dead while viable islets did
not take u p the d y e .
Insulin Diffusion
Only membrane M l w a s e m p l o y e d for diffusion
a s s a y s . A diffusion c h a m b e r w a s utilized to a s s e s s insu­
lin diffusion across the c h i t o s a n - P V P m e m b r a n e . T h e
membrane was clamped between the t w o compart­
sur­ m e n t s — l o a d i n g c o m p a r t m e n t ( L C ) and collecting c o m ­
p a r t m e n t ( C C ) — o f the diffusion c h a m b e r using multiple
supporting and sealing d e v i c e s . Both c o m p a r t m e n t s of
the c h a m b e r w e r e s i m u l t a n e o u s l y filled with equal vol­
u m e s of distilled water. A t time zero h u m a n insulin ( 4 0
U / m l , B o o t s P h a r m a c e u t i c a l s , India) w a s a d d e d to the
L C . A l i q u o t s ( 2 0 0 p i ) w e r e collected at 0, 5, 15, 30,
4 5 , and 6 0 m i n from the C C . I m m u n o r e a c t i v e insulin
concentration in the aliquots w a s d e t e r m i n e d using an
R I A kit ( D i a g n o s t i c P r o d u c t s Corporation, L A ) .
Antibody Diffusion Assay
M e m b r a n e permeability to anti-insulin antibody w a s
assessed using the diffusion c h a m b e r m e n t i o n e d p r e ­
viously. Anti-insulin IgG (purified from hybridoma
c l o n e C C 9 C 1 0 ) w a s loaded in L C and diffusion appara­
tus w a s k e p t at 3 7 ° C for 2 4 h. A l i q u o t s of 2 0 0 p i w e r e
collected from the C C and tested by E L I S A to c h e c k for
ISLET COMPATIBILITY OF CHITOSAN-PVP HYDROGELS
the p a s s a g e of antibody across the m e m b r a n e . E L I S A
w a s p e r f o r m e d on insulin-coated plates using a H R P -
labeled secondary antibody.
Image Analysis
Islet m o r p h o m e t r y studies w e r e carried out on a so­
phisticated i m a g e analysis s y s t e m ( K o n t r o n Elektronik
G m b H , M u n c h e n , G e r m a n y ) c o n n e c t e d to a Z i e s s (Axi-
oplan 2) m i c r o s c o p e . Islets w e r e o b s e r v e d and i m a g e s
c a p t u r e d with a V a r i o C a m P C O C C D i m a g i n g c a m e r a
a n d p r o c e s s e d t o obtain binary i m a g e s . Binary i m a g e s
w e r e taken in further c o m p u t a t i o n s , using i m a g e analy­
sis software (KS400 ver. 2.0, Kontron Elektronik
G m b H ) . A r e a and d i a m e t e r of islets on tissue culture
p o l y s t y r e n e ( T C P S ) control and o n M l and M 2 h y d r o ­
gels w e r e m e a s u r e d .
Islet Insulin Release Determination
Islets w e r e cultured o n the h y d r o g e l m e m b r a n e s in
R P M I - 1 6 4 0 with 1 0 % F C S for 5 d a y s . Islets w e r e then
h a n d p i c k e d and p l a c e d in 1 ml of K r e b s R i n g e r bicar­
b o n a t e buffer ( p H 7.4) with 1 m g / m l b o v i n e s e r u m albu­
m i n ( S i g m a C h e m i c a l C o . , St. L o u i s , M O ) a n d 10 m M
H E P E S ( n o w referred to as K R B H ) s u p p l e m e n t e d with
5.5 m M g l u c o s e . T h e plates w e r e incubated at 3 7 ° C in
5% C 0 2 a t m o s p h e r e for 1 h. Islets w e r e also c h a l l e n g e d
with K R B H c o n t a i n i n g 16.0 m M g l u c o s e and incubated
for 1 h. A t the e n d of incubation s u p e r n a t a n t w a s col­
lected and i m m u n o r e a c t i v e insulin w a s a s s a y e d by an
R I A kit ( D i a g n o s t i c P r o d u c t s C o r p o r a t i o n ) .
Statistical Analysis
R e s u l t s are e x p r e s s e d as m e a n ± S E M for n o r m a l l y
distributed data or as m e d i a n and interquartile r a n g e
w h e n d a t a w e r e n o t n o r m a l l y distributed. Difference b e ­
t w e e n g r o u p s w a s tested b y /-test or M a n n - W h i t n e y test
as a p p r o p r i a t e . C o m p u t a t i o n s were performed using
S i g m a - S t a t statistical p a c k a g e (Jandel Scientific, version
2.0 for W i n d o w s 9 5 , S P S S Inc., C h i c a g o , I L ) .
RESULTS
Membrane Hydrophilicity and Protein Adsorption
H y d r o g e l s s h o w e d very high o c t a n e c o n t a c t angle
( 1 4 2 . 9 1 0 . 4 6 ° for M l and 143.6 ± 0 . 4 9 ° for M 2 ) , indi­
cating a highly h y d r o p h i l i c nature. Protein adsorption on
the h y d r o g e l s w a s found to be l o w ( 0 . 0 1 4 3 ± 0.0027 m g
for M l and 0 . 0 1 3 6 ± 0 . 0 0 4 9 m g for M 2 ) c o m p a r e d to
T C P S control ( 0 . 0 4 3 4 ± 0.001 m g ) and p u r e chitosan
m e m b r a n e s ( 0 . 0 2 1 4 1 0.0025 m g ) .
Hydrogel Biocompatibility
Biocompatibility testing of the h y d r o g e l s w a s carried
out using islet cell p r i m a r y cultures as well as an e s t a b ­
lished cell line ( H e L a ) . M T T and N R assay revealed n o
27
cytotoxic effects of h y d r o g e l ( M l and M 2 ) l e a c h - o u t
products on HeLa cells. Moreover, these extracts
s h o w e d g r o w t h - p r o m o t i n g effects on the cells u p to c o n ­
centrations of 2 0 % (Fig. 1).
H y d r o g e l m e m b r a n e s ( M l and M 2 ) w e r e found to b e
b i o c o m p a t i b l e to islets of L a n g e r h a n s as tested b y their
g r o w t h supportive ability. Islets cultured on h y d r o g e l s
M l and M 2 exhibited e x c e l l e n t m o r p h o l o g y t h r o u g h o u t
10 d a y s of study (Fig. 2). Islets cultured o n h y d r o g e l s
M l and M 2 revealed n o significant deviation (p > 0.05)
in d i a m e t e r a n d area from the control islets ( T a b l e 1).
Islets seeded o n M l and M 2 s h o w e d high viability ( 9 7 %
on M l and 9 0 . 4 % o n M 2 ) c o m p a r a b l e to the islets on
T C P S control ( 9 1 % ) as assessed b y trypan b l u e d y e e x ­
clusion test.
Insulin and Antibody Diffusion Studies
Hydrogel M l a l l o w e d diffusion of insulin (Fig. 3)
across the m e m b r a n e . Insulin w a s detected in the C C at
5 m i n after its introduction in the L C . A r o u n d 3 3 % of
the initial a m o u n t of insulin p a s s e d t h r o u g h the m e m ­
b r a n e M l b y 6 0 m i n . M e m b r a n e M l did not allow p a s ­
s a g e of anti-insulin antibody as c o n f i r m e d b y E L I S A .
Islet Functionality
Islets m a i n t a i n e d their functionality during the entire
t e n u r e of study. B a s a l insulin release c o n c e n t r a t i o n s
w e r e c o m p a r a b l e to t h o s e in the control g r o u p islets (Ta­
b l e 2). Islets cultured on h y d r o g e l s r e s p o n d e d to a 16.0
m M g l u c o s e c h a l l e n g e , with released insulin c o n c e n t r a ­
tions c o m p a r a b l e to t h o s e in t h e control g r o u p ( T a b l e 2 ) .
DISCUSSION
H y d r o g e l s are p o l y m e r i c material that are insoluble
in w a t e r at physiological t e m p e r a t u r e , p H , and ionic
strength (22). I m p o r t a n t criteria in selecting the m a t e r i ­
als for i m m u n o i s o l a t i o n is their b i o c o m p a t i b i l i t y and se­
lective p e r m e a b i l i t y . A l g i n a t e h a s b e e n w i d e l y used in
i m m u n o i s o l a t i o n b u t h a s also b e e n r e p o r t e d to elicit in­
f l a m m a t o r y reactions b y activating c y t o k i n e p r o d u c t i o n
(28). A n o t h e r major limitation p r e v e n t i n g s u c c e s s of
such p o l y m e r s in transplantation p r o c e d u r e s h a s b e e n
t h e issue of fibrous o v e r g r o w t h associated w i t h the
m e m b r a n e s . T o date, several reports h a v e b e e n p u b ­
lished u s i n g similar i m m u n o i s o l a t i o n d e v i c e s in r o d e n t
m o d e l s , s o m e d e m o n s t r a t i n g l o n g - t e r m success, w h i l e
o t h e r s reporting s e v e r e fibrous o u t g r o w t h and graft m a l ­
function within a short p e r i o d following i m p l a n t a t i o n
(27).
Studies (19,25) h a v e reported fibroblast g r o w t h - i n ­
hibitory p r o p e r t i e s of chitosan in vitro. C h i t o s a n , a cat-
ionic p o l y m e r , is a deacetylated p r o d u c t of chitin (1),
nontoxic and bioabsorbable (20). Nonthrombogenic
properties of chitosan are well d o c u m e n t e d (2) and h a v e
28 RISBUD, HARDIKAR, AND BHONDE

Figure 1. (a) Viability of HeLa cells after exposure to hydrogel extract by MTT assay, (b) Viabil­
ity of HeLa cells after exposure to hydrogel extract by NR assay. Percent hydrogel extract repre­
sents the percent of hydrogel leach-out products in DMEM. 100 ul of this medium containing
various percentages of hydrogel leach-out products was added in each experimental well of a
96-well plate while control wells received 100 ul of plain DMEM without hydrogel leach-out
products.

also b e e n e x p l o r e d in d e v e l o p i n g dialysis m e m b r a n e s there are n o reports on the suitability of c h i t o s a n - P V P

(3). Polyvinyl p y r r o l i d o n e ( P V P ) , a synthetic p o l y m e r ,
has b e e n s h o w n to be b i o c o m p a t i b l e and largely used as
vitreous h u m o r substitute (11,33) and also as a blood
p l a s m a e x p a n d e r d u e to its suitable o s m o t i c properties.
Chitosan has b e e n used previously to e n c a p s u l a t e m a m ­
m a l i a n cells (8,18). C h i t o s a n - a l g i n a t e c a p s u l e s
also b e e n reported for islet encapsulation (9,23). W e
h a v e already studied the physical properties and d e m o n ­
strated b l o o d biocompatibility of c h i t o s a n - P V P m e m ­
b r a n e s (24). H o w e v e r , to the best of our k n o w l e d g e ,
b l e n d s for i m m u n o i s o l a t i o n of the islets of L a n g e r h a n s .
High o c t a n e contact angle values of h y d r o g e l m e m ­
b r a n e s ( M l and M 2 ) are indicative of their highly h y d r o -
philic nature c o m p a r e d to the T C P S control, w h i c h is
reported to be relatively h y d r o p h o b i c (35). T h i s property
have of surface hydrophilicity has been negatively correlated
to protein adsorption by m a n y w o r k e r s (32). P V P is a
h y d r o p h i l i c p o l y m e r and has been used as a coating sur­
face with properties of low coefficient of friction, p r o ­
tein repulsion, and t h r o m b o r e s i s t a n c e (12,30). T h e in-
ISLET COMPATIBILITY OF CHITOSAN-PVP HYDROGELS 29

Figure 2. (a) Islet morphology on TCPS control after 2 days in culture (x300). Note that islets have normal rounded morphology,
(b) Islet morphology on TCPS control after 8 days in culture (x300). Note that islets have stuck to the bottom and formed a colony
of cells, (c) Islet morphology on membrane M l after 8 days in culture (x300). Note the rounded morphology, (d) Islet morphology
on membrane M2 after 8 days in culture (x300). Note the rounded morphology.

herent surface hydrophilicity of the c h i t o s a n - P V P blend the m e m b r a n e indicates that the m e m b r a n e a l l o w e d p a s ­

would thus induce low protein adsorption, as is seen in
the protein adsorption profiles. L o w adsorption of p r o ­
tein on the surface of these hydrogels is an important
property, especially upon transplantation in a hostile en­
vironment. M e m b r a n e s with such properties will thus
ensure increased biocompatibility, reduced cellular ad­
hesion, and m a i n t e n a n c e of the diffusion properties.
Chitosan, a cationic polysaccharide, has primary
a m i n o groups, which interact with water molecules. This
indicates better w a t e r diffusion/permeability properties
for water-soluble solutes. Insulin transfer (Fig. 3) across
sive diffusion of insulin. M o l e c u l e s such as g l u c o s e (be­
ing m u c h smaller than insulin) will also be transported
easily across hydrogel m e m b r a n e s . T h u s , in conditions
of glucose-stimulated insulin secretion, the hydrogel
m e m b r a n e will ensure faster availability of the released
insulin. Chitosan m e m b r a n e s are reported to be highly
p o r o u s and fragile. In o r d e r to control the diffusion and
m e m b r a n e porosity, P V P w a s added, which is k n o w n to
check the porosity and i m p r o v e m e c h a n i c a l p e r f o r m a n c e
of the p o l y m e r i c b l e n d s (13,15). Hydrogel ( M l ) did not
allow anti-insulin antibody to pass through, indicating

Table 1. Islet Morphometry Determined by Image Analysis System (n = 100)

Control (TCPS) Ml M2

Area (pm ) 2
11875 ± 1201.4 11750+ 1164.5 12754+ 1932.31
Diameter (pm) 104.7 + 5.81 102.4 + 5.93 103.6 + 7.2
30 RISBUD, HARDIKAR, A N D BHONDE

40 analysis studies. Islets that w e r e cultured on hydrogel

0 10 20 30 40 50 60
Time (min)
Figure 3. Insulin diffusion kinetics through membrane M 1 .
that the m e m b r a n e p o r e size w a s small e n o u g h to p r e ­
vent p a s s a g e of anti-insulin antibodies. S u c h porosity
properties of the blend w o u l d b e m o s t suitable for the
intended applications of i m m u n o i s o l a t i o n . M e m b r a n e s
with such m o l e c u l a r w e i g h t cutoff will n o t allow the
i m m u n o c y t e s like m a c r o p h a g e s and m o n o c y t e s to reach
the islets, thereby e n s u r i n g their isolation from t h e host
i m m u n e system.
T h e hydrogel m e m b r a n e s M l and M 2 p r o v e d bio­
c o m p a t i b l e b e c a u s e there w a s n o cytotoxicity associated
with m e m b r a n e extracts as determined b y M T T and N R
assay. High viability of H e L a cells is suggestive of the
fact that hydrogel leach-out products had n o deleterious
effects on the mitochondrial and lysosomal functionality
of these cells. Increase in cell n u m b e r could b e due to
the stimulation of cells by leaching products, b e c a u s e
chitosan is k n o w n to stimulate cell proliferation (17).
Islets w e r e seen t o attach o n t o T C P S control, dissociate,
and form cell colonies b y day 3 of culture. H o w e v e r ,
islets cultured on the surface of the hydrogel m e m b r a n e s
m e m b r a n e s had perfect, round m o r p h o l o g y a n d n o cells
w e r e seen dissociating from the e d g e . T h e r e w e r e n o
significant c h a n g e s in the area of the islets cultured on
h y d r o g e l s from that of t h e control islets. T h i s is s u g g e s ­
tive of compatibility and the nontoxicity of the h y d r o ­
gels t o w a r d s islets. High viability ( 9 7 % and 9 0 . 4 % for
M l and M 2 , respectively) of islets cultured on h y d r o g e l s
also confirms the nontoxic and inert nature of the m e m ­
b r a n e s . H y d r o g e l nontoxicity t o w a r d s islets is also sup­
ported b y the m a i n t e n a n c e of pancreatic islet functional­
ity as assessed b y insulin release. Islets on h y d r o g e l s
m a i n t a i n e d their responsive nature t o the e n v i r o n m e n t a l
c h a n g e s in glucose concentration, and there w e r e n o sig­
nificant differences in insulin release in r e s p o n s e to glu­
cose challenge c o m p a r e d to the control islets (p > 0.05).
70 Graft malfunctioning d u e to fibrous o v e r g r o w t h r e ­
lated to the islet-encapsulating s e m i p e r m e a b l e m e m ­
branes has b e e n reported previously (27). Preliminary in
vitro studies at o u r laboratory (Risbud et al., u n p u b ­
lished data) also pointed out that c h i t o s a n - P V P h y d r o ­
gels arrest the fibroblast growth, thus p e r h a p s r e d u c i n g
the complications leading to graft rejection b y fibrosis.
In short, c h i t o s a n - P V P hydrogels maintained the
morphological and functional integrity of the pancreatic
islets. B o t h M 1 a n d M 2 w e r e seen to b e c o m p a t i b l e for
the m a i n t e n a n c e of islet population. Hydrogel M l al­
l o w e d passive diffusion of insulin and at the s a m e time
prevented transfer of antibodies across the m e m b r a n e .
T h u s , p r o p o s e d hydrogel blends of c h i t o s a n - P V P are
ideally suited for islet immunoisolation d u e to their d o c ­
u m e n t e d biocompatibility and selective diffusion of in­
sulin, prohibiting p a s s a g e of anti-insulin antibodies. T h e
p r o p o s e d hydrogel can b e used for islet i m m u n o i s o l a t i o n
in the form of microcapsules following the protocols d e ­
scribed earlier for chitosan microcapsule generation
(34). Although in v i v o studies are awaited, the present
study provides sufficient d o c u m e n t a t i o n to c o n s i d e r
these m e m b r a n e s as potential candidates for i m m u n o i s o ­
lation of islets.
ACKNOWLEDGMENTS: Authors wish to thank Dr. C. P.

did not attach or dissociate but maintained their m o r p h o ­ Sharma and Willi Paul, Shree Chitra Tirunal Institute for
Medical Sciences and Technology, Trivandrum, India for their
logical integrity, as evident from microscopic and i m a g e
help in octane contact angle measurements. We also thank Dr.
P. B. Parab, NCCS, Pune for the generous gift of anti-insulin
antibody.
Table 2. Insulin Release of Islets After 5 Days of Culture
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