Jou nal o Con olled Release 335 (2021) 619–636
A ailable online 9 June 2021
0168-3659/© 2021 The Au ho (s). Published by Else ie B.V. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
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Cell mic oencapsula ion echnologies o sus ained d ug deli e y: La es
ad ances in e icacy and biosa e y
Tania B. Lopez-Mendez
a
,
b
, Edo a San os-Vizcaino
a
,
b
,
c
, Jose Luis Ped az
a
,
b
,
c
,
Go ka O i e
a
,
b
,
c
,
d
,
e
,
*
, Rosa Ma ia He nandez
a
,
b
,
c
,
*
a
NanoBioCel Resea ch G oup, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la Uni e sidad, 7, 01006 Vi o ia-Gas eiz, Spain
b
Biomedical Resea ch Ne wo king Cen e in Bioenginee ing, Bioma e ials and Nanomedicine (CIBER-BBN), Ins i u o de Salud Ca los III, C/Mon o e de Lemos 3-5,
28029 Mad id, Spain
c
Bioa aba, NanoBioCel Resea ch G oup, Vi o ia-Gas eiz, Spain
d
Uni e si y Ins i u e o Regene a i e Medicine and O al Implan ology - UIRMI (UPV/EHU-Fundaci´
on Edua do Ani ua), BTI Bio echnology Ins i u e, Vi o ia-Gas eiz,
Spain
e
Singapo e Eye Resea ch Ins i u e, The Academia, 20 College Road, Disco e y Towe , Singapo e
ARTICLE INFO
Keywo ds:
Cell encapsula ion
E icacy
Biosa e y
Biocompa ibili y
Bioma e ial
Algina e
ABSTRACT
The de elopmen o cell mic oencapsula ion sys ems began se e al decades ago. Howe e , oday ew sys ems
ha e been es ed in clinical ials. Fo his eason, in he las yea s, esea che s ha e di ec ed e o s owa ds
ying o sol e some o he key aspec s ha s ill limi e icacy and biosa e y, he wo majo c i e ia ha mus be
sa is ied o each he clinical p ac ice. Rega ding he e icacy, which is closely ela ed o biocompa ibili y,
subs an ial imp o emen s ha e been made, such as he pu i ica ion o chemical modi ica ion o he algina es ha
no mally o m he mic osphe es. Each o he componen s ha make up he mic ocapsules has been ca e ully
selec ed o a oid oxici ies ha can damage he encapsula ed cells o gene a e an immune esponse leading o
pe icapsula ib osis. As o he biosa e y, esea che s ha e de eloped biological ci cui s capable o ac i ely
esponding o he needs o he pa ien s o p ecisely and accu a ely elease he demanded d ug dose. Fu he mo e,
he s uc u e o he de ices has been subjec o s udy o adequa ely p o ec he encapsula ed cells and p e en
hei sp ead in he body. The objec i e o his e iew is o desc ibe he la es ad ances made by scien is o
imp o e he e icacy and biosa e y o cell mic oencapsula ion sys ems o sus ained d ug deli e y, also high-
ligh ing hose poin s ha s ill need o be op imized.
1. In oduc ion
Fo mo e han ou decades, di e en ma e ials, bo h o na u al and
syn he ic o igin, ha e been used o manu ac u e sus ained d ug deli e y
sys ems. Among hem, we ind hose ha allow he sus ained elease o
encapsula ed g ow h ac o s, p o eins o d ugs; bu also o he s, o
g ea e complexi y, ha a e capable o immobilizing and p o ec ing
li ing cells, selec i ely isola ing hem om hei en i onmen while hey
sec e e he he apeu ic molecules o in e es . Cell encapsula ion sys ems
ha e shown wide applicabili y in pa hologies wi h e y di e se cha -
ac e is ics, such as diabe es melli us (DM), anemia, hemophilia B o
pa hologies o he cen al ne ous sys em (CNS), among o he s [1]. They
a e especially con enien o pa hologies in which main aining a s ic
con ol o e he elease o he he apeu ic molecule is essen ial.
Cell encapsula ion can be classi ied based on he size o he sys em.
On he one hand, we ind cell mac oencapsula ion sys ems, in which he
cells a e immobilized in ela i ely la ge di usion chambe s, wi h
semipe meable p ope ies. They can ha e di e en shapes, such as discs,
la shee s o hollow ibe s. The applica ion o cell mac oencapsula ion
de ices ha e shown e y good esul s in i o demons a ing hei un-
deniable he apeu ic po en ial. Howe e , mac ocapsules a e cha ac e -
ized by a ela i ely small su ace/ olume a io, which is p obably hei
wo s disad an age, since his implies he need o la ge amoun s o
nu ien s and oxygen o achie e an adequa e di usion in o he chambe
and limi s he amoun o cells ha can be encapsula ed wi hou c ea ing
nec o ic nuclei in he inne mos and inaccessible a eas [2].
* Co esponding au ho s a : NanoBioCel Resea ch G oup, School o Pha macy, Uni e si y o he Basque Coun y (UPV/EHU), Paseo de la Uni e sidad, 7, 01006
Vi o ia-Gas eiz, Spain.
E-mail add esses: [email p o ec ed] (G. O i e), [email p o ec ed] (R.M. He nandez).
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Jou nal o Con olled Release 335 (2021) 619–636
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Cell mic oencapsula ion ep esen s a e y in e es ing al e na i e,
g ea ly imp o ing he su ace/ olume a io and inc easing he di usion
o nu ien s and oxygen inside he capsules. Cell mic oencapsula ion
s a egy is based on he immobiliza ion o cells ha p oduce he apeu-
ically ele an molecules in sphe ical pa icles be ween 100 and 1500
μ
m in diame e , app oxima ely. The pa icles a e elabo a ed wi h
biocompa ible ma e ials and usually su ounded by a semi-pe meable
polyme ic memb ane ha p e en s he passage o high molecula
weigh molecules — an ibodies and o he componen s o he immune
sys em —, p o ec ing hese cells om he hos 's immune esponse and
om he mechanical s ess ha may occu when he implan is placed in
he selec ed issue [3]. In addi ion, he mic ocapsule mus exe a igh
con ol o e he bidi ec ional di usion o molecules — en ance o nu-
ien s and oxygen; and elease o de no o syn hesized he apeu ic ac-
o s and me abolic subp oduc s —, and p o ide cells wi h a sui able
en i onmen o enhance and modula e hei unc ion. This echnology
also supp esses, o a leas educes, he ch onic adminis a ion o
immunosupp essi e agen s, hus a oiding some o he ad e se e en s
associa ed wi h o gan and issue ansplan a ion. On he o he hand, he
cons an imp o emen s in imaging echniques and obo ic su ge y
p ocedu es allow he access o di icul o each a eas o implan a ion
[4].
Today, he esul s ob ained in he a ious clinical ials ca ied ou o
da e, make clea he ad an ages and po en ial applica ions o his
p omising echnology. Howe e , he e a e s ill aspec s ha need o be
imp o ed so ha cell mic oencapsula ion sys ems can be applied
ou inely in clinical p ac ice. Fo his eason, in he las yea s, e-
sea che s ha e di ec ed e o s owa ds ying o sol e some o he key
aspec s ha s ill limi e icacy and biosa e y, he wo majo c i e ia ha
mus be sa is ied o each he clinical p ac ice. Those wo concep s a e
closely ela ed o each o he and mus be ca e ully de ined and egula ed
due o hei implica ions ega ding pa ien well-being. The objec i e o
his e iew is, he e o e, o g oup and desc ibe he ex ensi e wo k ca -
ied ou wi h he aim o imp o e hese c i e ia, emphasizing he poin s
ha s ill need o be op imized.
2. E icacy - biocompa ibili y
When alking abou e icacy o cell mic oencapsula ion, biocom-
pa ibili y is one o he mos impo an aspec s o be conside ed. I will
de e mine implan ’s iabili y, unc ionali y and du abili y, becoming in
many cases a limi ing ac o o succeed. The biocompa ibili y o he
implan mus be gi en in 2 di ec ions (Fig. 1). F om ou side o inside, he
ma e ials used mus p o ec he immobilized cells, a oiding di ec
oxici y o he blockage o nu ien s and oxygen di usion. F om inside o
ou side, none o he sys em componen s — cells, bioma e ials, c oss-
linking agen s, e c. — o p ocedu es used mus be oxic o he pa ien o
elici an immune esponse in he hos . When his occu s, he o eign
body eac ion (Box 1) can e en ually isola e he implan wi hin a
ib o ic capsule, he eby p e en ing he access o essen ial molecules
and leading o g a ailu e. In addi ion, he biocompa ibili y mus las
o e ime, since li e cell he apies a e no mally used o long- e m
ea men s. In his sense, se e al expe s in he ield o cell encapsula-
ion ha e decided o de ine he e m "bio ole abili y", conside ing i
mo e app op ia e han "biocompa ibili y" [5].
Despi e he undeniable imp o emen occu ed in ecen decades, he
bioma e ials and cells ha a e used oday con inue o p oduce, o a
g ea e o lesse ex en , an in lamma o y esponse by he hos , so
sea ching o sui able componen s emains a p io i y. The inal pe o -
mance o he de ice will depend no only on he bioma e ials and cells
used, bu also on he si e o implan , he local applica ion o immuno-
supp essi e d ugs, o e en he size and shape o he implan .
2.1. Bioma e ials, c osslinke s and coa ings
2.1.1. Bioma e ials and c oss-linke s
On he one hand, he elabo a ed de ices mus p esen a sui able
s uc u e, esis an o unwan ed deg ada ion ha a oids con ac be-
ween he encapsula ed cells and he hos immune sys em. Fu he mo e,
he bioma e ials mus guide he p ocesses o p oli e a ion and di e -
en ia ion o encapsula ed cells, enhancing hei iabili y and unc ion-
ali y. On he o he hand, he choice o all he ma e ials mus be made
aking in o accoun possible oxici ies. The la e includes, in addi ion o
he main ma e ials, c oss-linke s, physicochemical modi ica ions and
possible deg ada ion byp oduc s.
Today, he ma e ials used include ce amics, plas ics and a ious
polyme s, among o he s. The la e can be classi ied as na u al (poly-
saccha ides, polypep ides and polynucleo ides) o syn he ic. Among
na u al polyme s, polysaccha ides a e he mos used because hey allow
ela i ely smoo h encapsula ion p ocesses ha a e compa ible wi h cell
iabili y. Examples o na u al polyme s a e algina e, aga ose, collagen,
o cellulose. On he o he hand, polye hylene glycol (PEG) con inues o
be he mos widely used op ion among syn he ic polyme s, along wi h
poly(lac ic-co-glycolic acid) (PLGA) and poly inyl alcohol (PVA)
[11,12].
Among all he a ailable polyme s, algina e is by a he mos widely
Fig. 1. Fac o s ha may comp omise implan
biocompa ibili y/bio ole abili y. Rega ding he
bioma e ials and coa ings used, ac o s such as he
pe meabili y o he mic ocapsule o p o-
in lamma o y molecules, he cha ac e is ics o he
implan su ace, he size and shape o he sphe e o
s uc u al de iciencies can igge an immune
esponse agains he capsules. The encapsula ed cells
can also elease p o-in lamma o y molecules o p o-
ude ou side he implan . Fu he mo e, he cha ac-
e is ics o he implan si e should also be conside ed
in de ail when ying o imp o e he biocompa i-
bili y/bio ole abili y o he sys em.
T.B. Lopez-Mendez e al.
Jou nal o Con olled Release 335 (2021) 619–636
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used bioma e ial in cell mic oencapsula ion sys ems, due o i s excellen
biocompa ibili y and easy handling [13]. Algina e is a na u al anionic
polysaccha ide ha c ea es h ee-dimensional s uc u es, going om sol
o gel, when i eac s wi h di alen ions. I is made up o di e en p o-
po ions o esidues o β-D-manu onic acid (M) and
α
-L-gulu onic acid
(G) ha c ea e di e en s uc u es acco ding o he a io o G and M.
De e mining and s anda dizing his p opo ion is essen ial since i has a
g ea in luence on some o algina e hyd ogels p ope ies, such as hei
biocompa ibili y, s abili y, mechanical esis ance and pe meabili y,
among o he s [13]. In gene al, algina es wi h a highe p opo ion o G
blocks a e s i e , compa ed o hose wi h a highe p opo ion o M
blocks ha ha e be e elas ic p ope ies, due o he g ea e a ini y o
gulu onic acid o di alen ions, and hese physical-mechanical di e -
ences a ec he way he immune sys em eac s agains he implan [14].
On he o he hand, he pu i y deg ee o he algina e is di ec ly ela ed
o i s biocompa ibili y. Low pu i y algina es con ain endo oxins, p o-
eins and polyphenols ha educe he biocompa ibili y o he implan s
and can damage he encapsula ed cells [15]. Se e al comme cial algi-
na es ha e been desc ibed o con ain pa hogen-associa ed molecula
pa e ns (PAMPs). These a e po en ini ia o s o in lamma o y esponses
[16] and p oduce he elease o small p oin lamma o y cy okines —
such as in e leukin 1β (IL-1β), he umo nec osis ac o
α
(TNF-
α
) o
in e leukin 6 (IL-6) —, which can come in o con ac wi h he encapsu-
la ed cells and cause damage. The mos common endo oxin ha can be
ound in algina e a e lipopolysaccha ides (LPS), which can bind o oll-
like ecep o s 4 (TLR-4) [17], p oducing an in lamma o y esponse
media ed by a a ie y o cells o he immune sys em [18]. The e o e, in
ecen yea s, di e en pu i ica ion me hods ha e been de eloped in
o de o ob ain ul a-pu e algina es wi h less immunogenici y in i o
[19–22]. Howe e , he e is g ea a iabili y be ween he p ocedu es
used in he di e en esea ch g oups and i is s ill necessa y o imp o e
he ools o he sc eening and elimina ion o hese and o he impu i ies,
such as pep idoglycans and lipo eicoic acid [23–27].
In his sense, he e a e di ided opinions on whe he o no i will be
possible o achie e an adequa e and su icien le el o pu i ica ion o he
algina e — so ha i becomes nea ly ine o he immune sys em — o
whe he i will also be necessa y o chemically modi y i s s uc u e.
Indeed, pe icapsula ib osis has been one o he majo d awbacks in
clinical s udies ca ied ou o da e wi h algina e as he main ma e ial.
Howe e , he composi ion o he algina e and he a iabili y be ween
adminis a ion p o ocols, cell ypes o he concomi an use o di e en
coa ing ma e ials, among o he s, make he compa ison complica ed.
In ecen yea s, algina e pu i ica ion p o ocols ha e been e ined
[27], while some g oups ha e begun o include chemical modi ica ions
in he algina es [28,29]. A ew yea s ago, Pa edes-Jua ez e al. c ea ed a
pla o m ha allows he iden i ica ion o pa e n ecogni ion ecep o
(PRR) ac i a ing polyme s, in o de o iden i y con aminan s in he
bioma e ials [16]. On he o he hand, in a s udy ca ied ou by Vegas e
al., a combina o ial app oach was used o gene a e a wide ange o
algina e a ian s wi h he aim o inding hose ha we e able o dec ease
he o eign body esponse [28]. A e a i s selec ion, he mos p om-
ising hyd ogel sphe es we e e alua ed in i o, in oden s and non-human
p ima es (cynomolgus macaques). Th ee iazole-con aining analogues
(Z2-Y12, Z1-Y15 and Z1-Y19) we e iden i ied, which signi ican ly
educed he o eign body esponse, when compa ed o con en ional
SLG20 algina e sphe es inhibi ing mac ophage ecogni ion and ib osis
o ma ion (Fig. 2). When implan ed in ape i oneally in o non-human
p ima es (n =3 each algina e a ian ), Z2-Y12, Z1-Y15 and Z1-Y19
sphe es wi h 1.5-mm diame e s displayed subs an ially educed
ib o ic esponses a e 4 weeks compa ed o 1.5-mm SLG20 sphe es.
SLG20 sphe es had mo e ex ensi e immune mac ophage and ib osis-
associa ed ac i a ed myo ib oblas co e age, consis en wi h he
isible ib o ic o e g ow h seen in he phase con as imaging. Z1-Y19
sphe es displayed mo e co e age by mac ophage and myo ib oblas s
han ei he Z2-Y12 o Z1-Y15, bu less compa ed o SLG20. Z2-Y12 and
Z1-Y15 sphe es had ma kedly lowe le els o smoo h muscle ac in
(SMA) p o ein, he majo mo phological cha ac e is ic o myo ib o-
blas s, as compa ed o SLG20 sphe es, bu Z1-Y19 sphe es did no ,
despi e he lowe a e age SMA measu ed. Hyd oxyp oline quan i ica-
ion e ealed lowe collagen le els o all h ee lead o mula ions
compa ed o SLG20.
In an in e es ing and mo e ecen s udy, Liu e al. p oposed a g oup o
zwi e ionic sul obe aine (SB) and ca boxybe aine (CB) modi ica ions o
algina e (SB-SLG20 and CB-SLG20) o educe cell accumula ion and
Box 1
Fo eign body eac ion agains bioma e ials.
Al hough he ma e ials and he apeu ic applica ions di e , he p ocess by which he body p oduces ejec ion agains implan s has many poin s
in common. This is known as " o eign body eac ion" and consis s o he ollowing phases:
Immedia ely a e he implan a ion and depending on he cha ac e is ics o i s su ace —ma e ial, shape, oughness, elec os a ic cha ge, e c. —
and he inju y caused du ing he su gical p ocess, a ious hos p o eins — such as albumin, ib onec in o complemen molecules — will s a o
adhe e o he su ace o he implan . This c ea es a chemoa ac i e g adien o he inna e immune esponse [6]. Neu ophils a e he i s cell
ype p esen a he implan si e and hei unc ion is o engul he mic oo ganism emains and dead cells. Neu ophils also sec e e p o eases,
lysozymes, eac i e species and o he enzymes o elimina e any ype o biodeg adable ma e ial. A he same ime, hey sec e e cy okines and
o he ac o s ha cause he ac i a ion o mac ophages (di e en ia ed om he ec ui ed monocy es), which will be he p edominan cell ype in
he ollowing phases o he o eign body esponse. This acu e phase o he in lamma o y eac ion would end wi h a e u n o homeos asis i he
ma e ial ecognized as o eign disappea s comple ely.
On he con a y, i he hos canno des oy he implan , i s con inued p esence can lead o a second phase o ch onic in lamma ion. The
“ us a ed” mac ophages s a o use in o mul inuclea ed cells a ound he implan , gi ing ise o o eign-body gian cells [7]. A he molecula
le el, p o-in lamma o y cy okines, such as IL-6, TNF-
α
, in e leukin 4 (IL-4), and in e leukin 13 (IL-13), ha e been epo ed o be o e exp essed
[8]. In his phase, he e is a con inuous p esence o monocy es and lymphocy es and a cons an ac i a ion o mac ophages and neu ophils,
which sec e e enzymes and eac i e species. A he same ime, neo ascula iza ion phenomena a e obse ed, wi h he appea ance o unc ional
capilla ies.
In he inal phase, ib oblas s, ac i a ed by mac ophages, deposi collagen ibe s o o m a dense and ib ous acellula capsule ha isola es he
implan om he su ounding issue [9]. This p e en s he passage o nu ien s and oxygen, and e en ually leads o comp omising he iabili y
o he encapsula ed cells.
In i o he ch onology a ies depending on he o ganism, anging om he appea ance o ib osis in jus 7 days, in he case o mini-pigs, o 14
days in a s [10].
T.B. Lopez-Mendez e al.
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Fig. 2. Modi ied hyd ogels mi iga e o eign body esponse in non-human p ima es. Z2-Y12, Z1-Y15 and Z1-Y19 algina e sphe es signi ican ly educe ib osis in
cynomolgus macaques, while con en ional SLG20 algina e sphe es become ib o ic. a Phase con as imaging o sphe es e ie ed a e 4 weeks in he in ape i oneal
space show less ib osis on Z2-Y12, Z1-Y15 and Z1-Y19 sphe es han on SLG20. Scale ba s, 2,000
μ
m; n =3. b Con ocal imaging o e ie ed sphe es om a a e 4
weeks in he in ape i oneal space show signi ican ly less mac ophage (CD68, CD11b), myo ib oblas (SMA) and gene al cellula deposi ion (DAPI) on Z2-Y12
sphe es. Scale ba s, 200
μ
m; n =3. B igh ield images o he s ained sphe es a e inse ; scale ba s, 100
μ
m. c Wes e n-blo analysis o p o ein ex ac ed om he
op h ee algina e analog sphe es and con ol sphe es in a; n =3. Blo s we e s ained o SMA and loading was no malized o β-ac in. Do s ep esen measu emen s
om indi idual biological eplica es, and lines show he a e age o he h ee eplica es. One-way ANOVA wi h Bon e oni co ec ion was used o allow o s a is ical
compa ison o mul iple means. #P <0.05; **P <0.001; ns, no signi ican . d Collagen con en using a hyd oxyp oline quan i ica ion assay o p o ein ex ac ed om
he op h ee algina e analog sphe es and con ol sphe es in a. n =3. Do s ep esen measu emen s om indi idual biological eplica es and lines show he a e age o
he h ee eplica es. One-way ANOVA wi h Bon e oni co ec ion was used o allow o s a is ical compa ison o mul iple means. #P <0.05; **P <0.001; ns, no
signi ican . Rep in ed om e . [28], wi h pe mission om Sp inge Na u e.
T.B. Lopez-Mendez e al.
Jou nal o Con olled Release 335 (2021) 619–636
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ib o ic p ocesses a ound he capsules [30]. S udies in mice, dogs, and
pigs showed a signi ican educ ion in hese p ocesses. Finally, a
panc ea ic isle s immobilized in SB-SLG20 mic obeads (con ol g oup
wi h SLG20), we e ansplan ed, o 200 days, in o he pe i oneal ca i y
o s ep ozo ocin (STZ)-induced C57BL/6J diabe ic mice and ou ou o
six mice main ained no moglycemia by he end o he s udy ( he sho es
du a ion o glycemic con ol was ~135 days) (Fig. 3). An in ape i oneal
glucose ole ance es (IPGTT) 200 days a e ansplan a ion, showed
ha he mice (cu ed ones, n=3) in he SB-SLG20 g oup clea ed blood
glucose (BG) and es o ed no moglycemia a a a e compa able o ha o
non-diabe ic mice. An ex i o glucose-s imula ed insulin sec e ion (GSIS)
o isle s e ie ed om cu ed mice (n=3) indica ed again he no mal
unc ion o isle s. Da k- ield mic oscopic images and hema oxylin-eosin
(H&E) his ological analysis o e ie ed SB-SLG20 mic ocapsules om
no moglycemic mice a e 200 days e ealed no o minimal cellula
deposi ion on he mic ocapsules and he p esence o nume ous unc-
ional isle s inside.
Mic oencapsula ion sys ems based on he gela ion o algina e mainly
use ba ium and calcium as c osslinking agen s and, in some cases,
s on ium [31]. Today he e is no clea p e e ence be ween calcium o
ba ium and i s use depends mainly on he p o ocol adop ed in he
di e en esea ch g oups. The a gumen s in a o o using calcium as a
c osslinking ion e ol e a ound he lowe oxici y [32]. Howe e , he
esis ance o he sphe es c oss-linked wi h calcium is lowe han ha
achie ed wi h ba ium, and ha is why o he g oups op o his op ion
when de eloping hei sys ems [28,29]. Some au ho s a gue ha ,
ba ium-c oss-linked beads could be signi ican ly less immunogenic as
hey do no need subsequen coa ings o inc ease he immunoisola ion,
which a e usually necessa y when calcium is selec ed as he c osslinke
[33]. This las s a emen is s ill in doub , since some s udies seem o
indica e ha he le el o immunoisola ion would no be su icien in he
ba ium beads i hey do no ha e pos e io coa ings. In addi ion, he in
i o implan a ion o algina e and ba ium beads ha e o igina ed a
ib o ic esponse o he implan in di e en adminis a ion ou es [26].
On he o he hand, he elease o c oss-linking ions mus also be aken
in o accoun , especially when ba ium is selec ed, due o i s oxici y [34].
Howe e , despi e he ad an ages o algina e, he e a e s ill aspec s
ha need o be op imized. Among hem i s mechanical p ope ies, since
he sys ems made wi h algina e and di e en ions end o unde go
changes in size due o he osmo ic p ocesses ha occu in he physio-
logical en i onmen , inc easing he pe meabili y o he capsule, weak-
ening i s s uc u e and inally causing up u e o he sys em [31].
Ion concen a ion, he selec ed c osslinking agen o he algina e
composi ion a e de e mining ac o s in ob aining adequa e and homo-
geneous gela ion. Simply a ying he gelling condi ions, he spa ial
dis ibu ion o he algina e chains in he mic osphe e can a y om
homogeneous o e y he e ogeneous, wi h up o 10 imes mo e con-
cen a ion on he su ace han in he nucleus [31]. Some s udies ha e
sugges ed ha a uly homogeneous dis ibu ion o algina e chains can
only be achie ed by in e nal and ex e nal gela ion applied simul a-
neously [35].
Release o componen s om he mic ocapsules can also s imula e an
in lamma o y esponse. This includes deg ada ion p oduc s ha may
a ise om eac ions occu ing unde physiological condi ions, de-
achmen s o pa s o he sys em o ion exchange, among o he s. Algi-
na e is subjec ed o hyd oly ic and enzyma ic deg ada ion. I has been
desc ibed o ha e a e y low a e o hyd olysis a physiological pH and
he low molecula weigh chains eleased a e exc e ed ia he u ina y
ac . These deg ada ion p ocesses ha e been ex ensi ely s udied, in i o
and in i o [36,37], in he subcu aneous space, he pe i oneum and in
Fig. 3. Sul obe aine-algina e (SB-SLG20) mic ocapsules imp o e diabe es co ec ion in mice in a 200-day s udy. a Blood glucose concen a ions o mice (n=6
mice pe ea men g oup). b In ape i oneal glucose ole ance es (IPGTT) be o e e ie al (n=3). c Ex i o glucose-s imula ed insulin sec e ion es (GSIS) o he
e ie ed a isle s om SB-SLG20 mic ocapsules, n=3, Mean±SEM, *P<0.05. d A da k- ield phase con as image o e ie ed isle -con aining SB-SLG20 mic o-
capsules. (n=6; scale ba , 2mm). e An hema oxylin-eosin (H&E) s ained c oss-sec ional image o e ie ed isle -con aining SB-SLG20 mic ocapsules. Scale ba ,
500
μ
m. Immunohis ochemical s aining o a isle s in e ie ed SB-SLG20 mic ocapsules. Insulin is s ained ed and nuclei a e s ained blue (Scale ba : 50
μ
m). g A
da k- ield phase con as image o e ie ed isle -con aining SLG20 mic ocapsules. (n=6; scale ba , 2mm). h An H&E s ained c oss-sec ional image o e ie ed isle -
con aining SLG20 mic ocapsules. Scale ba , 500
μ
m. i Immunohis ochemical s aining o a isle s in e ie ed SLG20 mic ocapsules. Insulin s aining is nega i e and
nuclei a e s ained blue. Scale ba , 500
μ
m. Rep in ed om e . [30] h p://c ea i ecommons.o g/licenses/by/4.0/.
T.B. Lopez-Mendez e al.
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some a eas o he b ain.
Some g oups ha e de ised s a egies o imp o e he mechanical
s abili y o algina e hyd ogels, co alen ly c osslinking i wi h di e en
polyme s, ia pho oc osslinking solu ions o enzyma ic eac ions, o
example [38–40]. In a ecen s udy, sodium algina e was unc ionalized,
wi h c oss- eac i e PEG de i a i es p esen ing a e minal hiol and
ca bon elec ophile unc ionali ies, and he sphe es o med by a com-
bina ion o Ca-algina e in e ac ions and sul u -ca bon co alen bonds.
The esul ing sphe es showed g ea e mechanical esis ance and be e
p ese ed shape, compa ed o he simple algina e and calcium beads.
When hese sphe es we e implan ed in he in ape i oneal space o
immunocompe en mice, issue adhe ence was no obse ed and in eg-
i y was no comp omised in he 30 days o he s udy [41]. In ano he
in e es ing s udy, i was shown ha modi ying he algina e wi h 2-ami-
nome hyl me hac yla e hyd ochlo ide can dec ease immune eac ions
agains he implan . The au ho s pe o med a i s ionic c osslinking,
ollowed by he applica ion o UV ligh o o m he co alen bonds. This
showed g ea e mechanical s abili y when i was e alua ed in i o o 3
weeks [42].
Ma e ial selec ion is especially demanding in his ype o sys em
since he esul ing pa icles mus no only ha e a high du abili y a e
implan a ion, hey mus also be capable o esponding o he biological
needs o he immobilized cells o long pe iods o ime. Howe e , many
ma e ials, such as algina e, do no ha e cellula signaling mo i s and
mus be bio unc ionalized o imp o e hei in e ac ion wi h encapsu-
la ed cells. In his ega d, in ecen yea s, di e en p o eins — such as
collagen, laminin o ib onec in — o small sho pep ides — such as
RGD (a ginine-glycine-aspa ic acid) — ha e been inco po a ed in o
mic ocapsules, ying o imi a e he physical and biomechanical cha -
ac e is ics o he na i e en i onmen o he encapsula ed cells o
imp o e and con ol cellula beha io [43–46]. In his ega d, he e a e
di ided opinions on whe he i is mo e app op ia e o use comple e
ex acellula ma ix p o eins, such as ib onec in o collagen, o small
syn he ic pep ides, such as RGD [47,48]. In e es ingly, he bes s a egy
in e e y case seem o be s ongly cell-dependen .
Fo example, Ga a e e al. e alua ed he in luence o RGD unc ion-
aliza ion o algina e encapsula ing C
2
C
12
myoblas s, baby hams e kid-
ney (BHK) ib oblas o s omal mesenchymal cells (MSCs) and he
esul s showed di e en op imal concen a ions o RGD in e e y case
[47,49,50]. In his sense, Gonzalez-Pujana e al. designed a sensi i e
analy ical ool ha pe mi s he e alua ion o di e en cell adhesion
kine ics, bu also he in eg in p o iling and hei con ibu ion o cell
a achmen and adhesion s eng hening ia clus e ing, which allows he
design o speci ic bio unc ionaliza ion s a egies depending on he cell
ype [51].
O he componen s o he ex acellula ma ix (ECM), such as hyal-
u onic acid (HA) ha e also been added o he algina e ma ix o mi-
c ocapsules. Recen ly, panc ea ic pseudo-isle s de i ed om MSCs we e
immobilized in algina e-HA mic ocapsules and he esul s showed be e
cell iabili y, wi h lowe le els o ini ial apop osis [52]. Mo eo e , he
inclusion o HA in he algina e ma ix, enhanced he di e en ia ion o
he MSCs owa ds panc ea ic p ogeni o s and inc eased he insulin
elease [53,54].
2.1.2. Coa ing ma e ials
In some cases, and depending on he applica ion, he mic obeads
composed o di e en bioma e ials and cells, a e he inal p oduc o be
adminis e ed. Howe e , ob ained po e size in mos cases is oo la ge and
does no p esen a eal ba ie agains he h ea s ha he implan will
ace once implan ed. The e o e, many g oups coa hese beads wi h
di e en polyme s o elabo a e mic ocapsules ha con ol he molecules
and cells ha can come in o con ac wi h he immobilized cells.
Cu en ly, his il e ing is ca ied ou by de ining a minimum molecula
weigh — molecula weigh cu -o (MWCO) — o solu e ha is o ally
excluded by he semipe meable memb ane [55]. This de ini ion can be
misleading since molecules o simila molecula weigh can ha e e y
di e en sizes, as is he case wi h p o eins and polysaccha ides.
Today, he e is s ill no consensus ega ding wha should be he mos
op imal c i e ion o he exclusion o molecules ha can access he
in e io o he mic ocapsule, despi e Chick e al. al eady named he
concep o he immuno-ba ie in 1977 [56]. Adequa ely de ining his
concep is essen ial o de elop biocompa ible and bio ole able sys ems
and i should be a p io i y issue.
To c ea e a biocompa ible and bio ole able en i onmen , he semi-
pe meable memb ane mus i s a oid con ac o he encapsula ed cells
wi h he cellula componen s o he immune sys em and he an ibodies.
A MWCO o a ound 70 kDa seem o be adequa e o many d ug deli e y
applica ions, bu i has been ound ha his is no enough i bi-
di ec ional low o an igenic, chemo ac ic and cy o oxic molecules —
such as eac i e oxygen species o p o-in lamma o y cy okines — is
allowed. Wi h he classic app oach o size-exclusion, low molecula
weigh molecules such as IL-1β (17.5 kDa) o TNF-
α
(51 kDa) will be able
o easily access he in e io o he capsule and cause damage o he
encapsula ed cells, as hey a e e en smalle han some o he he apeu ic
molecules ha a e usually eleased om hese sys ems. On he o he
hand, encapsula ed cells sec e e an igens — e.g. chemokines as low as 8-
13 kDa in molecula weigh — o he ex e io o he mic ocapsules ha
a e esponsible o ec ui ing cells om he hos immune sys em.
Fo he elabo a ion o he semi-pe meable memb ane, di e en
polyme s ha e been used, such as chi osan, oligo-chi osan o poly
(me hylene-co-guanidine) (PMCG), bu bo h in p eclinical s udies and
in human ials, he mos used molecules a e poly-L-lysine (PLL) and
poly-L-o ni hine (PLO) [57–59]. Howe e , bo h molecules a e known o
be immunogenic, so many g oups choose o add a las laye o algina e
on he pa icles o mask he posi i e cha ges ha would o he wise be
exposed o he componen s o he immune sys em. Resul ing mic o-
capsules a e known as APA (algina e-poly-L-lysine-algina e o algina e-
poly-L-o ni hine-algina e) [60,61]. This s a egy has been in ensely
deba ed since he e a e s udies showing ha his second laye o algina e
may no be su icien o inac i a e all he exposed posi i e cha ges
[62–64]. In ac , he s udies ca ied ou o analyze he su ace o he
mic ocapsules coa ed wi h hese polyca ions, showed ha hese mole-
cules a e exposed — and in g ea quan i y — in he ou e mos 1-2
monolaye s o he memb ane, hus he ou e algina e laye appea s o
o e lap wi h he PLL laye , a he han o m an addi ional ou e mem-
b ane [65,66]. In addi ion, bo h polyca ions show limi ed physico-
chemical p ope ies bu mos wo ks chose PLO o appa en ly ha ing
g ea e mechanical s abili y, biocompa ibili y and pe meabili y [63].
The inc eased immunogenici y o mic ocapsules coa ed wi h his
ype o polyca ions is mainly due o he physicochemical changes ha
a ec p o ein adhesion on he su ace o he mic ocapsules, such as ze a
po en ial, hyd ophobici y o oughness. On he one hand, he ze a po-
en ial o his ype o implan mus be nega i e and simila o ha o he
memb anes o adjacen cells. In a s udy ca ied ou by De Vos e al. [67],
i was ound ha he ze a po en ial o APA-PLL mic ocapsules showed a
mo e nega i e alue be o e hey we e implan ed. Al hough all he alues
we e less nega i e han hose desc ibed in o he s udies o he uncoa ed
algina e mic obeads [68]. On he o he hand, in a s udy ca ied ou by
Lekka e al. [69], a lowe su ace oughness, o less han 1 nm deep, was
associa ed wi h he uncoa ed algina e mic osphe es and wi h he PLL-
coa ed mic ocapsules, compa ed o much highe alues, o up o 14.4
nm, o PMCG-sul a e coa ed mic ocapsules. Finally, he addi ion o
coa ings o he algina e mic obeads inc eases he hyd ophobici y o he
sys em [63], al hough he ype o algina e used in each case also in-
luences he inal esul .
In a e y in e es ing s udy by Roks ad e al. [62] a lepi udine-based
human whole blood model was used as a ool o measu ing he
biocompa ibili y o di e en mic ocapsules. The esul s showed ha
algina e polyca ion (AP) o APA capsules igge he complemen ac i-
a ion, whe eas Ca/Ba algina e do no . Fig. 4 shows ha he deposi ion
o complemen componen 3 (C3) on he bead su ace is highe in AP o
APA beads han Ca/Ba beads.
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Taking in o accoun he ob ious need o imp o e he coa ings o
hese pa icles, in ecen yea s se e al g oups ha e analyzed o he
molecules ha may be app op ia e, bo h o subs i u e he PLL/PLO
molecules o in combina ion wi h hem [70–72]. In a ecen s udy,
genipin was used in associa ion wi h PLL [73]. Using o ce spec oscopy-
based simul aneous opog aphical and mechanical cha ac e iza ion o
s udy polyme o polyme in e ac ion, he s udy concluded ha genipin
c osslinking a oided memb ane de achmen in algina e mic osphe es
wi h double polyca ion coa ings.
A emp s ha e also been made o imp o e he biocompa ibili y o he
mic ocapsules by coa ing hem wi h polyme s capable o educing
p o ein adso p ion and he ib o ic esponse o he implan . By coa ing
he algina e mic ocapsules wi h hyd ophilic polyme s such as PEG
[74–76], he biocompa ibili y o he implan can be imp o ed, al hough
he le el o p o ein adso p ion will depend on he densi y, leng h and
con o ma ion o i s chains. In one s udy, algina e-PEG mic ocapsules
con aining allogenic isle s we e e alua ed and hei biocompa ibili y
was imp o ed when ansplan ed in o he in ape i oneal space, bu no
in o he epididymal a pad [77]. The s a egy o coa ing he algina e
mic ocapsules wi h PEG and apamycin, e alua ed by ano he g oup,
was also able o educe mac ophage p oli e a ion and ib o ic esponse
[78].
Modi ying he su ace o he mic ocapsules wi h a pa en ed mac o-
molecula hepa in conjuga e has also been shown o imp o e biocom-
pa ibili y and signi ican ly educe he ib o ic esponse agains he
implan , in syngeneic and allogeneic ansplan models [79]. Wi h a
simila s a egy, bu coa ing he algina e mic ocapsules wi h he C-X-C
mo i chemokine 12 (CXCL12) (also known as "s oma-de i ed ac o 1",
SDF-1), he biocompa ibili y was also imp o ed bu , in his case, a long-
e m imp o emen in xenogeneic panc ea ic isle su i al and
Fig. 4. Deposi ion o C3 on he mic osphe e su ace a e incuba ion in human lepi udin an i-coagula ed whole blood. A–L 3D p ojec ions made by
sec ioning en i e mic osphe es a e incuba ion o 30, 120 and 360 min. M–P P ojec ions h ough he equa o o e laid wi h ansmi ed ligh images a e 360 min.
Q–T Con ols a e gi en in he lowe panels as p ojec ions (black pic u es). The inse s show ansmi ed ligh equa o ial sec ions o isualiza ion. Ba s a e 100
μ
m.
Rep in ed om e . [62] wi h pe mission om Else ie .
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unc ionali y was also achie ed, due o he ec ui men o immuno-
supp essi e egula o y T cells o he implan si e [80]. Recen ly, in
ano he s udy, coa ing he algina e sphe es wi h chi osan also signi i-
can ly educed he ib o ic esponse agains he implan , imp o ing i s
biocompa ibili y, while main aining glucose le els o one yea , in a
canine allogeneic ansplan model and in a xeno ansplan in oden s
[81].
Ano he s a egy ha may be e y in e es ing is o inco po a e mo i s
ha ha e an i-in lamma o y p ope ies in o he design o he mic o-
capsule. Sul a ed algina es [82] o he in e leukin-1 ecep o (IL-1R)
[83] a e good examples o his, as hey dec ease he p oduc ion o some
cy okines and imp o e he iabili y o encapsula ed cells.
In some sys ems, such as hose made by he algina e-PLL combina-
ion, i is no possible o independen ly adjus he mechanical s abili y
and he pe meabili y o he mic ocapsules, which is a no able limi a ion
[84]. Howe e , he e a e s udies in which his pe meabili y-s i ness
ela ionship has been di ided using a ious polyme s [85]. The possi-
bili y o independen ly modi ying c i ical pa ame e s o cell encapsu-
la ion, such as capsule size, hickness, mechanical esis ance and
memb ane pe meabili y, o e s g ea ad an ages in he design o his
ype o sys em.
Poo mechanical esis ance can lead o p o usion o encapsula ed
cells, a phenomenon ha needs o be ixed when designing hese sys ems
[86]. In his sense, Johnson e al. ca ied ou an analysis quan i ying cell
p o usion in algina e mic ocapsules, coa ed wi h PLL and 50% hyd o-
lyzed poly(me hyl inyle he -al -maleic anhyd ide) (PMM). Acco ding
o he esul s ob ained, a ound 30% o he encapsula ed INS-1E β cells
we e loca ed in he las 20
μ
m o he algina e-PLL-PMM50 laye , wi h
7% o he cells p o uding [87]. Rein o cing he capsules wi h c oss-
linked shells may help p e en ing cell exposu e and scape.
Las ly, in some cases, o example i he sec e ed molecule is espe-
cially la ge, i will be necessa y o op imize he sys em so ha i allows
he passage o he he apeu ic molecule ou o he capsule, wi hou
comp omising he p o ec ion o he encapsula ed cells. In his sense,
ecen ly Mon anucci e al. modi ied he pe meabili y o algina e mi-
c ocapsules o allow he con inuous sec e ion o immunoglobulin M
(IgM), wi h no signs o in lamma ion [88].
2.2. Cell sou ce and a ge pa hology
Bo h allogenic and xenogeneic cells ha e been inco po a ed in o he
mic oencapsula ion sys ems. In case o human o igin cells, hei acqui-
si ion can be complica ed and expensi e. Besides, hey can be subjec o
biological, e hical and legal limi a ions. The e o e, he use o xenogeneic
cells has sp ead in he ield o cell mic oencapsula ion, hanks o he
immunoisola ion p oduced by he semipe meable memb ane [89].
Howe e , he sys ems used o da e o encapsula e bo h cell ypes ha e
been p ac ically iden ical, wi hou aking in o accoun ha he di e en
immunological esponses caused by allogenic o xenogenic cells equi e
capsula con igu a ions capable o p o ec ing he cellula con en
agains a iable immunological en i onmen s.
In he case o allogeneic ansplan s, i is p obably su icien o a oid
con ac be ween he encapsula ed cells and he cells o he hos 's im-
mune sys em [90]. The e o e, he simples mic ocapsules o ca ions and
algina e, wi hou g ea limi a ions in he di usion o molecules, may be
sui able. When a xeno ansplan a ion is pe o med, he scena io is mo e
complex and he simples sys ems may no be e ec i e in a oiding im-
mune ejec ion (Fig. 5). These cells p oduce xenogenic epi opes, such as
galac osyl (Gal) esidues, ha a e sec e ed ou side he capsule and a e
ecognized by he immune sys em o highe mammals, including
humans. In addi ion, in ecen yea s he ole o N-glycolyl neu aminic
acid (Neu5Gc), ano he pig xenoan igen, is being s udied as a possible
obs acle in xeno ansplan a ion [91].
The complexes o med by Gal esidues and he an ibodies linked o
hem, a e powe ul ac i a o s o he classical complemen pa hway. As
hese complexes begin o accumula e on he capsula su ace, chemo-
axis o di e en cell ypes, such as neu ophils [92] occu s, which
ini ia e powe ul in lamma o y eac ions. Du ing his i s phase, many
Fig. 5. P o-in lama o y molecule sec e ion om he mic ocapsules ac i a es he immune esponse in di e en scena ios. Xenogeneic ansplan . Some
an ibodies a e able o en e he capsule o con ac he encapsula ed cells loca ed in he mos supe icial laye s o he implan . These an ibodies ecognize sequences
ha a e no p esen in he hos species, such as he Gal ca bohyd a e in he case o p ima es, and his leads o he ac i a ion o complemen pa hways. This ac i a ion
p oduces di ec cell lysis and he elease o molecules ha p omo e in lamma ion and he ec ui men o immune cells, such as neu ophils, o he implan si e. The
ec ui ed cells elease small cy okines ha can c oss he mic ocapsule memb ane and damage he encapsula ed cells. In mo e ad anced phases, ib o ic p ocesses
appea , which could end up isola ing he implan and comp omising he supply o nu ien s and oxygen o he in e io o he capsule. In acapsula nec osis.
Nec o ic cells elease he so-called DAMPs o ala mins, such as high-mobili y g oup box-1 (HMGB1), hea shock p o eins (HSPs), S100 p o eins, DNA/RNA agmen s
e c., o he ex acellula luid. These small molecules can di use ou side he mic ocapsules and ac i a e cells o he immune sys em, such as mac ophages, dend i ic
cells, neu ophils o lymphocy es, binding PRRs, such as TLRs. These cells, in esponse, will sec e e p o-in lamma o y cy okines, such as IL-1β, IL-6, in e leukin-8 (IL-
8), o TNF-
α
, which will p oduce in lamma ion and ec ui mo e immune cells o he a ea. These molecules can en e he mic ocapsules and damage he encapsula ed
cells. I he si ua ion pe sis s o e ime, he adap i e immune esponse may be ac i a ed.
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small cy okines a e able o c oss he semipe meable memb ane o he
mic ocapsules, causing damage o he encapsula ed cells.
Following he i s inna e esponse, a second IgM-media ed [92]
delayed hype sensi i i y esponse o xenogeneic epi opes begins, p o-
mo ing he ec ui men o new cells o he immune sys em o he implan
si e and he sec e ion o mo e chemokines and cy okines. A e hese
e en s, he mic ocapsules a e usually su ounded by in lamma o y cells
and ib oblas s ha hinde he passage o nu ien s and oxygen,
comp omising he su i al o he encapsula ed cells. Finally, he
appea ance o ib osis can lead o o al isola ion o he implan .
Due o hese di e ences be ween allo and xenog a s, he la e
equi e sys ems ha p o ec encapsula ed cells agains mo e po en
h ea s. The memb anes mus be less pe meable and, ideally, p e en he
passage o molecules p oduced by he immune sys em, while p e en ing
he exi o hype in lamma o y xenogeneic epi opes, such as Gal esidues.
Ano he way o ac i a ing he immune esponse may occu when cell
nec osis appea s inside he mic ocapsules [93] (Fig. 5). Un o una ely,
his is s ill qui e common, when he e a e p oblems in he di usion o
nu ien s and oxygen, due o insu icien pe meabili y o he bio-
ma e ials, ib o ic p ocesses associa ed wi h o eign body eac ion o an
excess o encapsula ed cell mass [94]. Nec o ic phenomena a e di ec ly
ela ed o damage-associa ed molecula pa e ns (DAMPs). These mol-
ecules a e no mally ound inside he cells, bu a e eleased ou side when
cell damage occu s [95]. Some examples a e hea shock p o eins o
DNA/RNA agmen s. The mammalian immune sys em has speci ic e-
cep o s o his ype o signals, he PRRs, such as Toll-like ecep o s
(TLRs). The DAMPs eleased om he mic ocapsules a e powe ul ac i-
a o s o he immune sys em, ac i a ing in lamma o y and angiogenesis
p ocesses, which media e he elease o la ge amoun s o cy okines ha
jeopa dize he su i al o he encapsula ed cells [96].
In his sense, he e a e di e en s udies ha ied o imp o e he
iabili y o mic oencapsula ed cells, inco po a ing chemical compounds
capable o gene a ing oxygen [97,98] o h ough s a egies ha p omo e
he ascula iza ion o he implan . Fo he la e , se e al s a egies ha e
been es ed. On he one hand, he abili y o di e en angiogenesis-
inducing g ow h ac o s, such as ib oblas g ow h ac o (FGF)
[99,100] o ascula endo helial g ow h ac o (VEGF) [101,102] has
been exploi ed o p omo e he neo ascula iza ion o he implan , hus
imp o ing he esul s o he he apy. On he o he hand, he implan a-
ion o he mic ocapsules in p e- ascula ized spaces is also conside ed as
a bene icial op ion, ei he gene a ed in he hos ’s o ganism o in mac-
ode ices [103,104].
E en i he isks associa ed wi h xeno ansplan a ion a e being
educed, he ad ances in he ield o s em cell use ha e unlocked an
un hinkable po en ial. The abili y o di e en ia e human s em cells,
om di e en sou ces, o ob ain he desi ed cell ype o he possibili y o
ep og amming adul cells o induced plu ipo en s em cells (iPSCs)
[105,106] ha e de ined he pa h o a su icien sou ce o human cells.
Fu he mo e, in he pa icula case o iPSCs, he e a e no e hical e-
s ic ions [107].
In his ein, he s udies ca ied ou o da e ha e shown ha i is
possible o ob ain ully unc ional be a cells o panc ea ic p ogeni o s,
s a ing om human plu ipo en s em cells (hPSCs) — ei he human
emb yonic s em cells (hESCs) [108–113] o iPSC [114]. In ecen yea s,
hese cells, known as human s em cell de i ed β cells (SC-β), ha e been
seen as an excellen sou ce o unlimi ed panc ea ic cells [30,111,115].
Fo example, Vegas e al. implan ed human SC-β, immobilized on algi-
na e beads, in he in ape i oneal space o immunocompe en C57BL/6J
mice p e iously ea ed wi h s ep ozocin. C-pep ide le els and blood
glucose concen a ion showed he apeu ically ele an esul s up o 174
days, wi hou he need o immunosupp essi e ea men [116]. In
ano he ecen s udy, he ma u a ion o SC-β was s imula ed by o ming
agg ega es, simila in size o panc ea ic isle s, which make hem espond
o glucose s imula ion in jus 3 days a e ansplan a ion [117].
Likewise, s em cells om o he o igins, such as amnio ic luid o
adipose issue, can also be ans o med in o insulin-p oducing cells,
which can be encapsula ed and ansplan ed in diabe ic animal models
o no malize blood glucose alues [118–120]. Fo example, Mon anucci
e al. managed o emi hype glycemia in diabe ic mice, implan ing
human umbilical co d Wha on jelly-de i ed mesenchymal s em cells
(hUCMS), immobilized in algina e and PLO mic ocapsules [121].
Mo eo e , in a mo e ecen s udy by he same au ho s [122], hUCMS
cells we e co-encapsula ed wi h human panc ea ic isle -de i ed p o-
geni o cells (hIDC) and implan ed in o non-obese diabe ic mice. The
objec i e o his syne gy was o main ain ace insulin ou pu by hIDC,
while exploi ing he immuno egula o y p ope ies o hUCMS. A decline
o blood glucose le els was obse ed in i o.
MSCs ha e demons a ed o be e y sui able o hei use in cell
encapsula ion sys ems, due o hei hypoimmunogenic and immuno-
modula o y cha ac e is ics [123–125]. These cells inhibi immune e-
sponses by sec e ing cy okines and soluble g ow h ac o s ha p oduce a
local immunosupp essi e e ec in he su ounding cells [126]. In ecen
s udies, e o s ha e ocused on analyzing he beha io o immo alized
MSCs, gene ically modi ied o sec e e e y h opoie in (EPO), o he
ea men o anemia [50,127–129]. In addi ion, hei bene i s ha e also
been e alua ed in hepa ic pa hologies [130,131], as an al e na i e o
po cine hepa ocy es [132,133]. Mo eo e , MSCs no only a e a e y
in e es ing op ion as a sec e o y cell [50,128], bu also as a co-
encapsula ed auxilia y cell [134–137]. In a ecen s udy, panc ea ic is-
le s and MSCs we e co-encapsula ed in algina e and PEG mic ocapsules
and implan ed in o he in ape i oneal space o a diabe ic mouse model
[138]. The esul s showed ha MSCs in e ac wi h N-cadhe in and in-
c ease insulin sec e ion, in addi ion o p o iding s uc u al suppo o
he isle s, imp o ing hei iabili y and unc ionali y.
I is also impo an o highligh ha he cus omizable en i onmen
gene a ed in hese 3D s uc u es can no ably imp o e cell iabili y and
cellula unc ion. In his ega d, cell mic oencapsula ion echnologies
a e used a beyond sus ained elease pu poses, o example, o
enhanced cell cul u e [139] o o ecapi ula ing umo mic oen i on-
men o in i o disease models [140], among o he s.
2.3. Mic ocapsule size and shape
The op imal size o cell mic oencapsula ion sys ems emains a
ma e o deba e. On he one hand, i is e iden ha a la ge capsule size
could be an obs acle o he di usion o nu ien s and oxygen o he
nucleus o he sys em. This would lead he encapsula ed cells o si ua-
ions o hypoxia and cell dea h, as well as o slowe esponses o he
s imuli om hei en i onmen [141]. In ac , in a ecen s udy, i was
sugges ed ha , in he case o panc ea ic isle s, he maximum dis ance
be ween hese and he ex acapsula luid should no be mo e han 100
μ
m, o allow adequa e exchange o nu ien s and oxygen [142].
The e o e, many ha e been he s udies aimed a ob aining smalle
capsules. Coaxial ai low and low ocusing echnologies we e p esen ed
as a ac i e al e na i es o he usual me hods o making mic ocapsules
by means o elec os a ic d ipping, making i possible o manu ac u e
capsules o 100-200
μ
m in diame e , ha allow o mo e complica ed
ou es o adminis a ion, such as in ac anial adminis a ion (in he case
o CNS pa hologies) o e en in a i eal [143,144]. Fu he mo e, ying
o educe he size o he capsules as much as possible, in ecen yea s
nanoencapsula ion s a egies ha e also been e alua ed — such as
con o mal coa ing o laye -by-laye coa ing —, especially o he
immunop o ec ion o he isle s o Lange hans [124].
Con o mal coa ing is a o m o non-sphe ical encapsula ion ha e-
duces he di usion dis ance and he olume o he implan [145].
Howe e , he p ocess o en in ol es mul iple s eps ha can cause
damage o he encapsula ed cells, and a conclusion has no ye been
eached ega ding whe he hese ype o coa ings a e hick enough o
hei use in clinical p ac ice [146,147]. Some s udies [148] ha e sug-
ges ed ha he con o mal coa ing has a lowe immunop o ec i e ca-
paci y, compa ed o hyd ogel mic ocapsules, bu in ecen yea s se e al
new s a egies ha e demons a ed he po en ial o his echnology
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