74
METABOLIC AND HORMONAL DISORDERS
Zdrav Vestn | January – February 2021 | Volume 90 | https://doi.org/10.6016/ZdravVestn.3001
1 Institute of Biomedical 
Sciences, Faculty of 
Medicine, University of 
Maribor, Maribor, Slovenia
2 Department of 
Pharmacology, Faculty 
of Medicine, University of 
Maribor, Maribor, Slovenia
3 Institute for physiology, 
Faculty of Medicine, 
University of Maribor, 
Maribor, Slovenia
Correspondence/
Korespondenca:
Marko Milojević, e: marko.
milojevic1@um.si
Key words:
in vitro models; pancreas; 
bioscaffolds; 3D printing; 
tissue slice method
Ključne besede:
modeli in vitro; trebušna 
slinavka; bionosilci; 3D tisk; 
metoda tkivne rezine
Received: 18. 10. 2019
Accepted: 12. 5. 2020
eng slo element
en article-lang
10.6016/ZdravVestn.3001 doi
18.10.2019 date-received
12.5.2020 date-accepted
Metabolic and hormonal disorders Metabolne in hormonske motnje discipline
Review article Pregledni znanstveni članek article-type
In vitro models of the endocrine pancreas Modeli in vitro endokrine trebušne slinavke article-title
In vitro models of the endocrine pancreas Modeli in vitro endokrine trebušne slinavke alt-title
in vitro models, pancreas, bioscaffolds, 3D 
printing, tissue slice method
modeli in vitro, trebušna slinavka, bionosilci, 3D 
tisk, metoda tkivne rezine
kwd-group
The authors declare that there are no conflicts 
of interest present.
Avtorji so izjavili, da ne obstajajo nobeni 
konkurenčni interesi. conflict
year volume first month last month first page last page
2021 90 1 2 74 90
name surname aff email
Marko Milojević 1 marko.milojevic1@um.si
name surname aff
Andraž Stožer 3
Uroš Maver 1,2
eng slo aff-id
Institute of Biomedical Sciences, 
Faculty of Medicine, University 
of Maribor, Maribor, Slovenia
Inštitut za biomedicinske vede, 
Medicinska fakulteta, Univerza v 
Mariboru, Maribor, Slovenija
1
Department of Pharmacology, 
Faculty of Medicine, University 
of Maribor, Maribor, Slovenia
Katedra za farmakologijo, 
Medicinska fakulteta, Univerza v 
Mariboru, Maribor, Slovenija
2
Institute for physiology, Faculty 
of Medicine, University of 
Maribor, Maribor, Slovenia
Inštitut za fiziologijo, Medicinska 
fakulteta, Univerza v Mariboru, 
Maribor, Slovenija
3
In vitro models of the endocrine pancreas
Modeli in vitro endokrine trebušne slinavke
Marko Milojević,1,2 Andraž Stožer,3 Uroš Maver1,2
Abstract
Ambitions to develop artificial tissue substitutes, combined with the need to study underlying 
mechanisms of disease under controlled conditions, shortcomings of animal models, as well 
as ethical constraints, were the main driving forces behind the development of advanced in vi-
tro models. These are defined as alternative experimental systems made by leveraging recent 
advances in tissue engineering and additive manufacturing that mimic tissue or organ level 
physiology in vitro. Simple in vitro models are already being used in many applications, how-
ever, due to their many drawbacks, they incompletely mimic dynamic responses of native tis-
sues. In order to construct functionally more relevant in vitro models, cells need to be grown in 
three-dimensional (3D) environments or bioscaffolds. Generally, bioscaffolds must recapitulate 
the microarchitecture, hierarchical structure, physical properties, and composition of native 
tissues. Nonetheless, progress towards building more complex models is hindered primarily by 
the diffusion of gases and nutrients into the constructs´ interior. Currently, 3D printing pres-
ents the most promising solution for the production of advanced bioscaffolds, which resolve 
the above-mentioned limitations. In addition to the technique´s ability to simultaneously use 
multiple biocompatible materials, 3D printing enables material deposition with micrometer spa-
tial resolution under cell-friendly conditions. The development of a functional in vitro pancreas 
model is governed by the desire to study diabetes aetiology and is one of the main goals of the in 
vitro modelling domain, which to date remains unfulfilled. Despite having some drawbacks, the 
tissue slice method presents the gold standard for basic and translational studies of the pancre-
as, while the currently most advanced 3D fabricated in vitro pancreas models mimic only basic 
functions of the organ. The purpose of this review is to provide an overview of in vitro models 
with a focus on in vitro models of the endocrine pancreas. We will highlight different model types 
and fundamental elements which need to be considered when constructing a model. Emphasis 
will be placed on more complex 3D fabricated in vitro models, tissue slices, bioscaffold material 
properties, and the use of 3D printing for the fabrication of advanced bioscaffolds. We believe 
that the simultaneous development of advanced materials, micro-manufacturing technologies, 
and advanced cell culture methods presents a very promising approach towards the construc-
tion of a functional in vitro pancreas model.
Izvleček
Želja po razvoju tkivnih nadomestkov, potrebe po preučevanju mehanizmov bolezni v kontroli-
ranih pogojih, pomanjkljivosti živalskih modelov in etične omejitve so bile povod za razvoj pod-
ročja naprednih modelov in vitro. Te definiramo kot alternativne eksperimentalne sisteme, ki 
z uporabo modernih tehnik tkivnega inženirstva in aditivne proizvodnje posnemajo strukturo 
in funkcionalnost tkiv ali organov in vitro. Enostavni modeli in vitro se že uporabljajo v številne 
namene, vendar zaradi mnogih pomanjkljivosti ne posnemajo dovolj dinamičnih odzivov izvor-
nih tkiv. Za izgradnjo funkcionalno kompleksnejših modelov in vitro je celice potrebno gojiti v 
tridimenzionalnem (3D) okolju ali bioloških nosilcih. Splošno morajo biološki nosilci posnemati 
mikroarhitekturo, hierarhično strukturo, fizikalne lastnosti in sestavo izvornega tkiva. Toda na-
predek k izgradnji kompleksnejših modelov omejuje predvsem difuzija plinov in hranil v notra-
Slovenian
Medical
Journal
75
REVIEW ARTICLE
In vitro models of the endocrine pancreas
1 Introduction
The main purpose of the majority of 
biomedical research is to decipher the 
origin and molecular mechanisms of hu-
man diseases with the goal of developing 
new or better preventive, diagnostic, and 
therapeutic approaches. For obvious rea-
sons, basic research alone cannot be per-
formed on humans, and animal models 
differ too much anatomically, physiolog-
ically, and genetically from humans, so 
they often do not mimic critical aspects of 
human healthy or pathologically altered 
tissues and organs, especially not with 
the desired molecular resolution (1). The 
development of alternative models that 
mimic human anatomy and physiology in 
vitro is therefore urgently needed. In par-
allel, the ultimate goal of the field of tis-
sue engineering (TE) is the production of 
functional tissues and organs in vitro that 
could be used as substitutes to repair or 
replace the damaged and diseased human 
body parts (2-4). Individual TE research 
njost konstrukta. Tehnika 3D tiska je trenutno najobetavnejša rešitev za proizvodnjo naprednih 
bioloških nosilcev, ki odpravljajo te pomanjkljivosti. 3D tisk poleg zmožnosti sočasne uporabe 
več biološko kompatibilnih materialov omogoča nalaganje materiala z mikrometrsko prostorsko 
ločljivostjo pri pogojih, primernih celicam. Vse od zasnove področja modelov in vitro je zaradi že-
lje po preučevanju nastanka sladkorne bolezni razvoj funkcionalnega modela trebušne slinavke 
eden od osrednjih, a še nedoseženih ciljev. Zlati standard za bazične in translacijske raziskave 
trebušne slinavke pa je kljub nekaterim pomanjkljivostim metoda tkivne rezine, medtem ko tre-
nutni najnaprednejši 3D zgrajeni modeli trebušne slinavke in vitro posnemajo le osnovne funkci-
je organa. Namen tega članka je predstavitev modelov in vitro s poudarkom na modelih trebušne 
slinavke. Predstavil bo tipe modelov in ključne elemente, ki jih je treba pri izgradnji upoštevati. 
Poudarek bo na kompleksnejših 3D zgrajenih modelih in vitro in tkivnih rezinah, materialnih la-
stnostih bioloških nosilcev ter tehniki 3D tiska za izgradnjo naprednih bioloških nosilcev. Meni-
mo, da je sočasni razvoj znanosti o materialih, mikroproizvodni tehnologiji in celičnih kulturah 
izjemno obetavna pot k izgradnji funkcionalnega modela trebušne slinavke in vitro.
Cite as/Citirajte kot: Milojević M, Stožer A, Maver U. In vitro models of the endocrine pancreas. Zdrav Vestn. 
2021;90(1–2):74–90.
DOI: https://doi.org/10.6016/ZdravVestn.3001
Copyright (c) 2021 Slovenian Medical Journal. This work is licensed under a
Creative Commons Attribution-NonCommercial 4.0 International License.
studies focus mainly on the development 
of individual milestones of this process 
in order to achieve the ultimate goal (e.g. 
development of advanced materials, pro-
duction methods and cellular resources, 
etc.) (5,6) and on the construction of sim-
ple in vitro models that mimic the basic 
functions of tissues and organs in vivo. 
In order to achieve the principles of 3Rs 
– replacement, reduction and refinement 
in the face of ethical limitations and short-
comings of animal models and the goals 
of tissue engineering to study the mecha-
nisms of disease in controlled conditions, 
they led to the development of complex in 
vitro models. The field of advanced in vi-
tro models lies at the intersection of TE, 
regenerative medicine, pathophysiology, 
advanced materials science, and additive 
manufacturing and focuses on mimicking 
the three-dimensional (3D) structure and 
functionality of tissues or organs in vitro.
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2 In vitro models
2.1 Simple in vitro models
Simple two-dimensional (2D) in vi-
tro models are already being used for a 
number of purposes (Figure 1), such as 
the development and testing of new ther-
apeutical substances and for pharma-
cological and toxicological studies. For 
this purpose, transformed or immortal 
commercially available cell lines are most 
often used, but during prolonged cultiva-
tion (increase in the number of cell divi-
sions), they usually begin to differ genet-
ically, epigenetically and physiologically 
from primary cells. For the construction 
of in vitro models, commercial cell lines 
are, therefore, the worst choice (7,8). In 
contrast, isolated primary cell cultures 
are the best candidates for use in in vitro 
models, as they best represent functional 
units of the native tissue. The construction 
of in vitro models based on primary cell 
cultures is limited mainly due to the diffi-
cult availability and isolation of cells that, 
in addition, have difficulty proliferating in 
standard 2D cell cultures and have a short 
lifespan (i.e., Hayflick limit) (9). Recently, 
these problems have been overcome by us-
ing induced pluripotent stem cells (iPSCs), 
which can be obtained from somatic cells 
by a dedifferentiation process (10). iPSCs 
have the ability to self-regenerate and dif-
ferentiate into many mature cell types, 
making them extremely interesting for use 
in in vitro models where we study the on-
set and the course of disease development. 
The reason for the rare use of iPSCs is that 
the control of differentiation into mature 
cell types is extremely complex, and in 
addition, immature cell phenotypes are al-
ways present in cell culture (11,12).
Regardless of the source and type of 
cells, we regard as simple in vitro mod-
els the standard 2D single cell cultures 
(consisting of one type of cells grown in 
cell vessels) and their upgraded variants, 
such as 2D cocultures, 2D cultures grown 
in containers coated with extracellular 
matrix (ECM) components, and cultures 
grown on Transwell® plates. These are sim-
ple 2D cell cocultures grown on plates that 
mimic more complex three-dimensional 
(3D) intercellular signalling (13,14). In 
fact, such 2D in vitro models have many 
drawbacks. In addition to the fact that 
cell vessels differ statistically significantly 
from the native tissue in structure, me-
chanical properties, topography and com-
position, these cells cannot communicate 
with each other auto- and paracrineally 
in all spatial dimensions. Also, cells are 
unevenly exposed to concentration gra-
dients of oxygen, nutrients, and biologi-
cally active molecules, leading to the fact 
that cells grown in 2D cultures do not 
show the correct morphology and often 
do not express the appropriate pheno-
type long enough. Therefore, they do not Figure 1: Areas, where in vitro models are used.
In vitro models 
Regenerative 
medicine
Disease 
modelling
Pharmacological 
and toxicological 
studies
Personalized 
therapy
Detection 
and testing 
of active 
substances
Food testing 
and nutriceutics
Tissue 
engineering
Basic 
research
77
REVIEW ARTICLE
In vitro models of the endocrine pancreas
allow the performance of more complex 
and time-consuming experiments. As a 
result, simple in vitro models do not mim-
ic the key characteristics and functions of 
the native tissue (15,16). In contrast to 2D 
cultures, cells grown in 3D cultures estab-
lish complex interactions with the ECM 
and neighbouring cells in three spatial di-
mensions, thus better mimicking the bio-
chemistry and mechanics of the original 
microenvironment. Therefore, in order 
to build functionally more complex mod-
els, in vitro cells must be grown in a 3D 
environment or on 3D cell scaffolds, i.e., 
bioscaffolds that mimic the original ECM 
in which cells grow, differentiate, prolifer-
ate, and communicate in all spatial dimen-
sions (17).
2.2 Advanced in vitro models
Advanced in vitro models are defined 
as synthetic alternative experimental sys-
tems based on living human cells that 
mimic the physiology of a tissue or organ 
in vitro using modern TE approaches and 
micro-additive manufacturing. Within 
the framework of this definition, the aim is 
not to perfectly mimic the complex archi-
tecture and function of a tissue or organ, 
but rather the model reproduces, at least 
representatively, only the key functions 
that we want to mimic in vitro. Advanced 
3D in vitro models are particularly useful 
when conventional 2D cell cultures do not 
replicate the dynamic responses of native 
tissues well enough (16,18). The basic ele-
ments (Figure 2) that must be considered 
in the construction of the in vitro model 
are the source and type of cells (9), phys-
icochemical stimuli (19) and biologically 
active molecules (biochemical stimuli) 
(20,21), which promote the desired cellu-
lar phenotype.
The simplest 3D in vitro models include 
spheroids and organoids grown in special 
cell vessels to which cells do not adhere. 
With appropriate biochemical stimuli, 
cells organize themselves into a simple 
spherical 3D shape. iPSCs are the most 
commonly used. As the name suggests, 
ex vivo organoids mimic the basic hierar-
chical structure and physiology of organs, 
while spheroids do not have a clearly de-
fined internal structure and cellular orga-
nization (22). Organoids are mainly used 
for basic in vitro research dealing with or-
ganogenesis and the course of the disease 
(23). In addition to the fact that organoids 
differ significantly in size and shape, the 
main limitation of both models is size, as 
cells within the 3D construct rapidly nec-
rotize due to limited diffusion (24). To 
construct larger and more complex 3D 
constructs, it is extremely important to 
build cellular bioscaffolds with adequate 
porosity, facilitating the access of oxygen 
and nutrients to the cells (17,25). 
When constructing advanced 3D in 
vitro models, it is, therefore, necessary 
to additionally select appropriate build-
ing blocks for the production of cellular 
scaffolds with appropriate structural and 
mechanical properties. The nano-, micro- 
and macro-properties of in vitro models 
should be adapted to mimic the charac-
teristics of the native or diseased tissue. 
Emphasis should also be placed on mim-
icking the mechanical conditions in which 
cells grow. Therefore, suitable biocompati-
ble materials with appropriate mechanical 
properties must be selected. An important 
part of modelling also involves mimicking 
concentration gradients of nutrients, gas-
es, pH, and metabolites (9).
3 Pancreas
Ever since the development of the in 
vitro model, one of the primary goals has 
been to develop a functional in vitro mod-
el of pancreas, and in particular the islets 
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METABOLIC AND HORMONAL DISORDERS
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of Langerhans, due to the desire to study 
the course of type 1 and 2 diabetes (lat. 
Diabetes mellitus, DM). The normal func-
tioning of the human body depends on the 
precise regulation of blood glucose levels. 
The pancreas, more precisely the beta cells 
located in the islets of Langerhans, play a 
central role in maintaining the dynamic 
balance of glucose. These secrete insulin 
when the concentration of energy-rich 
molecules in the blood (especially glu-
cose) and certain hormones (e.g. GIP and 
GLP-1 incretins) increases and under the 
influence of neurotransmitters (e.g. CCK 
and acetylcholine) (26,27). The pancreas is 
a retroperitoneal organ about 14-18 cm in 
size, consisting of three main anatomical 
parts: the head, body and tail. It is a gland 
that is structurally and functionally divid-
ed into a larger (~ 95%), exocrine portion, 
consisting mainly of ductal and acinar 
cells, and a smaller (~ 5%), endocrine por-
tion, consisting of islets of Langerhans. 
Acinar cells secrete digestive enzymes 
indirectly into the duodenum, which are 
responsible for breaking down proteins, 
lipids and carbohydrates. Ductal cells, on 
the other hand, secrete fluid rich in bicar-
bonate anions (28). Endocrine tissues in 
humans are represented by about a million 
endocrine microorganisms, 50-500 µm in 
size, called islets of Langerhans. Each islet 
(even in different species) contains about 
1,000 cells. The most numerous are beta 
cells (50-60%), which secrete insulin, fol-
lowed by alpha cells (35-40%), which se-
crete glucagon, and delta cells (10-15%), 
which secrete somatostatin. Less repre-
sented are PP or gamma cells that secrete 
pancreatic polypeptide and epsilon cells 
that secrete ghrelin (29). DM types 1 and 
2 are considered to be among the most 
important disorders in the functioning 
of the endocrine portion of the pancreas. 
Diabetes is a metabolic disease character-
ized by the inability to regulate blood glu-
cose homeostasis. In particular, (auto) im-
mune-mediated type 1 diabetes is caused 
by an absolute lack of insulin due to the 
destruction of beta cells (30). DM type 2 
is a complex polygenic disease that is also 
influenced by a number of environmen-
tal and epigenetic factors. It is caused by 
a reduced sensitivity of the target tissues 
to insulin with initial compensatory in-
crease but relative insufficiency of insulin 
secretion. This ultimately leads to secreto-
ry dysfunction, beta cell failure, and abso-
lute insulin deficiency (31-33). Both types 
of disease can lead to acute and chronic 
complications, including cardiovascular 
problems, renal failure, neurological dam-
age, vision loss, and generally increased 
patient mortality (34,35). The most basic 
research on the origin and development of 
diabetes uses a number of different animal 
models (predominantly rodents, especial-
ly mice) (36). Despite many similarities, 
there are many structural and physiolog-
ical differences in the islets of Langerhans 
between humans and rodents, leading to 
differences in functional linkage between 
cells and finally to differences in the com-
plex dynamics of insulin secretion (26,28). 
This means that the results obtained in 
animal models cannot always be reliably 
transferred to humans in all respects.
4 In vitro models of the 
pancreas
The development of a functional hu-
man model of an in vitro pancreas would 
mark a breakthrough in basic diabetes re-
search and lead to the development of new 
therapeutical substances for the treatment 
of diabetes. At the same time, it would 
greatly reduce the need to use animal 
Figure 2: Key elements to consider when building advanced 3D in vitro models. After selecting the appropriate 
source and cell type, important biochemical and physicochemical stimuli must be mimicked in vivo which ensure the 
maintenance of the desired cellular phenotype. It is also essential that 3D bioscaffolds mimic the key physical and 
structural properties of the original ECM.
Advanced in 3D in vitro models
Commercially available cell lines
Primary cell cultures
Induced pluripotent stem cells
Source and type of cells
Biochemical stimuli
Physical and structural 
properties of bioscaolds
Physicochemical stimuli
Growth factors
Cytokines
Hormones
Steroids
Peptides
Geometry
Porosity
Topography
Hardness
High elastic properties
Concentration gradients
pH
Temperature
Charge
79
REVIEW ARTICLE
In vitro models of the endocrine pancreas
important disorders in the functioning 
of the endocrine portion of the pancreas. 
Diabetes is a metabolic disease character-
ized by the inability to regulate blood glu-
cose homeostasis. In particular, (auto) im-
mune-mediated type 1 diabetes is caused 
by an absolute lack of insulin due to the 
destruction of beta cells (30). DM type 2 
is a complex polygenic disease that is also 
influenced by a number of environmen-
tal and epigenetic factors. It is caused by 
a reduced sensitivity of the target tissues 
to insulin with initial compensatory in-
crease but relative insufficiency of insulin 
secretion. This ultimately leads to secreto-
ry dysfunction, beta cell failure, and abso-
lute insulin deficiency (31-33). Both types 
of disease can lead to acute and chronic 
complications, including cardiovascular 
problems, renal failure, neurological dam-
age, vision loss, and generally increased 
patient mortality (34,35). The most basic 
research on the origin and development of 
diabetes uses a number of different animal 
models (predominantly rodents, especial-
ly mice) (36). Despite many similarities, 
there are many structural and physiolog-
ical differences in the islets of Langerhans 
between humans and rodents, leading to 
differences in functional linkage between 
cells and finally to differences in the com-
plex dynamics of insulin secretion (26,28). 
This means that the results obtained in 
animal models cannot always be reliably 
transferred to humans in all respects.
4 In vitro models of the 
pancreas
The development of a functional hu-
man model of an in vitro pancreas would 
mark a breakthrough in basic diabetes re-
search and lead to the development of new 
therapeutical substances for the treatment 
of diabetes. At the same time, it would 
greatly reduce the need to use animal 
Figure 2: Key elements to consider when building advanced 3D in vitro models. After selecting the appropriate 
source and cell type, important biochemical and physicochemical stimuli must be mimicked in vivo which ensure the 
maintenance of the desired cellular phenotype. It is also essential that 3D bioscaffolds mimic the key physical and 
structural properties of the original ECM.
Advanced in 3D in vitro models
Commercially available cell lines
Primary cell cultures
Induced pluripotent stem cells
Source and type of cells
Biochemical stimuli
Physical and structural 
properties of bioscaolds
Physicochemical stimuli
Growth factors
Cytokines
Hormones
Steroids
Peptides
Geometry
Porosity
Topography
Hardness
High elastic properties
Concentration gradients
pH
Temperature
Charge
models. Despite many years of developing 
in vitro models, there are many problems 
remaining in the area of the pancreas. The 
first problem in the cultivation of in vitro 
pancreatic cells arises with the demanding 
process of isolation and purification of vi-
able pancreatic islets and cells within the 
islet. Islet of Langerhans cells are no longer 
capable of auto- and paracrine communi-
cation soon after isolation, when they lose 
homo- and heterotypic intercellular con-
tacts. In addition, they lose critical ECM 
contacts and basement membrane con-
tacts, which ultimately leads to reduced 
viability and loss of cell function. The 
additionally high metabolic requirements 
and size of the islets limit the availability 
and access of oxygen and nutrients to the 
cells inside the islet. Due to limited dif-
fusion, necrotic cell death begins rapidly. 
All this contributes to the fact that current 
2D in vitro pancreatic cell models do not 
mimic the critical dynamics of insulin se-
cretion when stimulated with glucose (37). 
2D substrates thus restrict the growth of 
pancreatic cells, prevent the formation of 
complex 3D in vivo morphology, and do 
not mimic ECM cell-type contacts that are 
critical for normal endocrine cell function. 
Even more in favour of the importance of 
the 3D environment for the differentia-
tion, growth and functionality of the islets 
of Langerhans is the fact that recently a di-
rect link between the morphology of the 
pancreatic islet and endocrine differentia-
tion was confirmed. In the differentiation 
of endocrine progenitor cells, the alpha 
cells that develop first migrate to the out-
side of the islet and form a mantle, while 
the beta cells that form later remain in the 
nucleus of the islet. Such temporal and 
spatial proportionality leads to the typical 
spherical 3D architecture of the human 
pancreatic islet (alpha cell mantle and be-
ta cell nucleus), which is essential for the 
normal functioning of the islet (38).
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METABOLIC AND HORMONAL DISORDERS
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4.1 3D Biomimetic 
scaffolds mimic the ECM 
of the native tissue
In the most elementary sense, the ECM 
is a natural biologically active cellular 
scaffold composed of structural (colla-
gen, laminins, fibronectins) and signalling 
proteins, polysaccharides, glycoproteins, 
proteoglycans, biologically active mole-
cules, electrolytes, and water (39). In in vi-
vo conditions, the ECM creates a complex 
3D framework by providing mechanical 
support to cells and, in addition to inter-
cellular communication in all spatial di-
mensions, enables key cellular processes 
such as adhesion, migration, proliferation, 
and differentiation (40). ECM plays a key 
role in the microenvironment of pancre-
atic cells (41), so it is extremely important 
to mimic the basic properties of the origi-
nal ECM using 3D cellular scaffolds in the 
construction of an advanced in vitro mod-
el of the pancreas (42,43). 
In general, cellular scaffolds must rep-
licate the complex 3D microarchitecture, 
hierarchical structure, and ECM compo-
sition of the native tissue to be mimicked 
in vitro. The basic building blocks, 3D 
structure, and composition of the pancre-
atic ECM are best mimicked by scaffolds 
constructed of natural polymers (primari-
ly polysaccharides). These form hydrogels 
and have a positive effect on the growth 
and viability of pancreatic cells (44). 
Therefore, to build advanced in vitro mod-
els of the pancreas (islets of Langerhans), 
great effort is invested in the development 
of 3D biomimetic cellular scaffolds that 
mimic the basic building blocks of the 
original microenvironment (ECM) with 
which pancreatic cells are surrounded. A 
key criterion in the construction of the 
scaffold is the choice of the appropriate 
material. It must be based on the mechan-
ical properties of the tissue, as the surface 
of the material plays an important role in 
directing the growth and development of 
cells, and it is also the central interface for 
intercellular interactions (45). The pan-
creas is a nonlinear visco-elastic soft tissue 
with low shear modulus (46). Therefore, 
natural polymer hydrogels, which have 
a high water content and exhibit similar 
structural and mechanical properties as 
pancreatic ECM, are the best materials for 
building cellular scaffolds (47). In addition 
to biological compatibility, the basic prop-
erties that must be taken into account in 
the construction of the bioscaffold are the 
structure and elementary building blocks 
of the scaffold. Topographic, material, and 
physical characteristics of all size classes 
(e.g. macro-, nano-, and microtopography, 
macro-, and microporosity) are structur-
al stimuli that guide cell behaviour. The 
choice of biocompatible materials must 
be based on the fact that both the mi-
cro- and macro-properties of the scaffolds 
mimic the characteristics of the native or 
pathologically altered pancreatic tissue. 
The surface properties of scaffolds (e.g. 
roughness), their mechanical properties, 
microstructures (e.g. pore size and shape), 
and other characteristics (e.g. swelling and 
biodegradability of scaffolds) significantly 
affect the growth and phenotype of the cell 
(48). Pancreatic cells grown in vitro on 3D 
cellular scaffolds were able to differentiate 
into physiologically appropriate tissue, 
and at the same time, their morphology 
differed significantly from cells grown in 
2D cell cultures. Compared to 2D sub-
strates, 3D scaffolds made of polysaccha-
rides also promoted better adhesion, pro-
liferation, and survival of cells (49-51). 
Although materials of natural ori-
gin represent a good choice for the con-
struction of 3D biomimetic scaffolds, 
they cannot mimic the complexity of the 
ECM of the native tissue down to the last 
detail. Recently developed decellularized 
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In vitro models of the endocrine pancreas
extracellular matrix (dECM) techniques 
promise the possibility of building dECMs 
cellular scaffolds that almost completely 
mimic the complexity of the native tissue. 
DECM cell scaffolds can be obtained from 
a variety of tissues by a decellularization 
method, which typically involves the lysis 
and removal of cells from the tissue by per-
fusion with deionized water or detergents 
(52,53). Thus, only tissue-specific ECM 
remains after the process, which typical-
ly involves macromolecules such as col-
lagens, laminins, fibronectin, elastin, and 
other tissue-specific glycosaminoglycans, 
cytokines, and growth factors. Recently, a 
group of scientists demonstrated that such 
dECM-specific scaffolds also significant-
ly affect the function of pancreatic cells. 
Using the decellularization method, they 
successfully prepared cellular scaffolds 
from the pancreas with a unique composi-
tion, physical structure, and biological ac-
tivity. iPSC cells grown on specific pancre-
atic scaffolds spontaneously differentiated 
into cells, similar to pancreatic cells, which 
also began to express corresponding genes 
(54). DECM prepared from pancreatic tis-
sue has also been successfully used for 3D 
printing of biologically mimetic cellular 
scaffolds. Such 3D printed dECM scaffolds 
promoted the differentiation of iPSCs 
against the pancreatic phenotype while 
maintaining the long-term viability and 
function of isolated islets of Langerhans 
(55). The biggest problem with the use of 
dECM remains the fact that during the de-
cellularization process, the highly specific 
spatial arrangement of proteins and other 
molecules is disrupted. Therefore, the cur-
rent goal in development remains to find a 
balance between the complete removal of 
cellular components and the preservation 
of small vessels (capillaries) and other tis-
sue structures. Toxic effects were also ob-
served on cells grown on dECM scaffolds, 
most likely due to residual detergents used 
during the process (56). 
Currently, the biggest limitation in the 
construction of 3D scaffolds that support 
the growth of pancreatic cells in the long 
run is the inadequate mechanical prop-
erties of materials, especially hydrogels. 
Various crosslinking techniques (e.g. ionic 
crosslinking), inorganic/organic additives 
(cellulose fibres, various nanoparticles) 
(57,58), or other synthetic materials (poly-
caprolactone) are used to improve the 
mechanical stability of hydrogels. These 
improve the mechanical properties of the 
scaffold or provide a suitable basic frame-
work (57,59). An additional problem is the 
fact that it is extremely difficult to adjust 
the aforementioned key characteristics of 
scaffolds (porosity, topography, mechani-
cal properties, rate of decay and water up-
take, rheological properties of materials) 
to fully mimic the properties of the native 
tissue. The development of hydrogel for-
mulations, on the basis of which it would 
be possible to produce long-term stable 
cellular scaffolds and to which the desired 
properties could be additionally arbitrari-
ly adjusted (e.g. visco-elastic, mechanical, 
surface), would mean remarkable progress 
in the construction of advanced in vitro 
models (60).
5 3D printing for building in 
vitro models
Despite all the technological advances 
in TE, the development of physiologically 
relevant tissues, especially the pancreas, 
remains difficult. Due to the limitation in 
the diffusion of oxygen and nutrients, the 
building of constructs larger than 200 µm 
is extremely problematic (25). Progress 
towards building larger tissue constructs 
is hampered by the fact that vascular 
functionality must be incorporated into 
the scaffold to ensure and improve the 
transfer of oxygen and nutrients to the 
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cells while promoting the removal of their 
waste products. Construction of 3D vas-
cular networks within tissue constructs 
plays a key role in the long-term survival 
and maintenance of cell viability in in vitro 
models (61,62). To date, researchers have 
not been able to develop cellular scaffolds 
to build in vitro soft tissue models that 
demonstrate adequate resolution, struc-
tural integrity, and required biocompat-
ibility at the same time (63). Several TE 
approaches (64-67) have been developed 
to address these problems, but most are 
limited to inducing in vitro angiogenesis. 
In addition to the fact that such approach-
es require long-term incubation for ves-
sel formation as well as the use of costly 
growth factors (e.g. vascular endothelial 
growth factor, VEGF), their main draw-
back is limited repeatability and the inabil-
ity to spatially control the distribution of 
blood vessels within the construct, which 
often does not allow perfusion at all. In re-
cent years, additive approaches such as 3D 
printing have been used more frequently 
to produce cellular scaffolds based on bio-
compatible materials, advanced in vitro 
models, and tissue constructs with blood 
vessels included (68). In doing so, various 
additive production techniques are devel-
oped and used simultaneously (e.g. litho-
graphic techniques, i.e., ink-jet printing, 
microextrusion methods) to build 3D cel-
lular scaffolds that better mimic the archi-
tecture, biochemistry, and functionality of 
native tissues. Individual techniques show 
certain advantages and disadvantages (52). 
Among them, 3D bioprinting represents 
a new and most promising method used, 
which is expected to revolutionize the 
field of building advanced in vitro models. 
In addition to the ability to simultaneously 
use a wide range of biocompatible materi-
als and exceptional application versatility 
(69,70), the 3D biological printer allows 
the loading of material with a micrometre 
spatial resolution (71) under cell-friendly 
conditions such as low shear forces (72,73). 
This gives the 3D printer a great advantage 
over other conventional techniques for the 
preparation of cellular scaffolds, which 
are often limited by the control of the 3D 
shape, the spatial arrangement of individ-
ual components of the material and thus 
the local distribution of the density of ma-
terial and cells (74).
5.1 Core/Shell 3D printing 
technique for building 
advanced in vitro models
As already mentioned, for the develop-
ment of larger and physiologically more 
complex models in vitro, it is extremely 
important to build cellular scaffolds that 
allow the smooth flow of cellular medium 
or even mimic the basic functionality of 
the vessel lumen. Probably the best and 
simplest approach to building a 3D print-
ed in vitro flow model is to include a con-
nected network of hollow channels inside 
the tissue scaffold. The construction of 
such a bioscaffold would reduce the like-
lihood of the formation of necrotic areas 
in the construct and also solve many oth-
er already mentioned shortcomings of the 
existing models (73). Endothelial cells can 
be further populated into the hollow ca-
nals of the scaffolds, thus gaining the abil-
ity to mimic synthetic vessels in 3D tissue 
models (75). In addition to the already 
mentioned facilitated diffusion of oxygen, 
nutrient inflow, and uninterrupted remov-
al of CO2 and metabolites, such lumens of 
the channels can also fulfil other import-
ant physiological tasks depending on the 
specifics of in vitro cultured tissue. In vitro 
endocrine tissue models allow secretion 
collection and assessment of the secreto-
ry function of the construct, and exocrine 
tissues allow secretion and its collection 
for quantification. In order to build an 
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In vitro models of the endocrine pancreas
advanced 3D in vitro model of the entire 
pancreas, which would include both endo-
crine and exocrine portions, in addition to 
the lumens where endocrine cells release 
their secretions, ducts for the release of 
enzymes must be additionally included in 
the biological scaffold, because their acti-
vation could lead to autodigestion or even 
trigger in vitro pancreatitis of cultured 
tissue. 
Recently, a new version of the method 
of 3D printing of hollow channels (i.e., 
core/shell printing) (76-79) has attracted 
a lot of attention, as it promises fast, sim-
ple, and repeatable construction of stable 
cellular scaffolds with built-in flow chan-
nels. Using a coaxial nozzle allows so-
called core/shell printing, 3D printing of 
two materials at the same time, one being 
extruded as a core filament (the core) and 
the other as a shell around it (the shell). 
The technique opens the possibility that 
by choosing biological materials with dif-
ferent mechanical properties, the harder 
material structurally supports the softer 
material during and after printing. If to 
construct the scaffold a material is used 
that can be chemically crosslinked (e.g. al-
ginate) and it is extruded as a shell while 
at the same time the crosslinking agent 
(e.g. CaCl2) is extruded into the core, then 
it is possible to construct a stable bios-
caffold with hollow filaments in a single 
process step (80). Coaxial printing has 
already been used to build scaffolds with 
solid (81), so-called core/shell filaments 
(82), and hollow filaments (83). However, 
the materials used have not yet been op-
timized for simultaneous cellular viabili-
ty and even mechanical robustness of the 
constructs. Despite all the advantages of 
both ordinary 3D printing and so-called 
core/shell printing, the development of ap-
propriate material formulations (ink) that 
simultaneously demonstrate all the nec-
essary properties suitable for 3D printing 
and meet all biological requirements still 
presents a great challenge (84).
6 Tissue slice as a model of the 
pancreas in vitro
Due to the many limitations and the 
complexity of building good 3D models 
of the pancreas, because this organ has a 
complex structure and function (28,85), 
isolated endocrine cells, especially beta 
cells, isolated acinar cells, isolated islets 
of Langerhans, and isolated ducts and ac-
inuses are still mostly used for basic and 
translation studies of the pancreas (86,87). 
The results of these studies are often com-
plemented by various in vivo measure-
ments at the level of the whole organism 
(88). An important step towards the best 
possible 3D model for pancreatic tissue, 
which is alternative in many respects and 
partly complements the development of 
3D models with the help of various media 
and 3D printing, has recently been devel-
oped and the method of pancreatic tissue 
slices is increasingly being used (86,89).
In this method, the pancreatic tis-
sue of various types of model organisms 
(most often mice, possibly rats or pigs) 
or humans is cut into tissue slices about 
100 micrometres thick. In contrast to the 
isolation of cells and islets, no enzymes 
are used, but only minimal mechanical 
stress due to cutting. Tissue slices contain 
intact islets of Langerhans, cut islets of 
Langerhans, large areas of intact acinus-
es, and long sections of ducts of different 
orders of magnitude. One of the most im-
portant properties of the tissue slices thus 
obtained is that the cells within the islets 
of Langerhans and within the acinuses are 
interconnected by various intercellular 
contacts while preserving the paracrine 
interaction within the endocrine portion, 
within the exocrine portion and between 
the two portions. To a greater extent than 
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in the isolation of islets and acinuses, the 
vessels, basement membrane, connective 
envelopes, immune cells, and other ele-
ments of the mesenchyme are also pre-
served, and thus also the 3D structure of 
the tissue (86,87,90,91). Acute pancreatic 
slice is thus a special form of primary cell 
culture that can be used for at least 24–48 
hours without special additional scaffolds 
(87,92). 
In combination with electrophysio-
logical measurements, intracellular cal-
cium ion concentration measurements, 
secretion measurements, and various 
morphological measurements, the tissue 
slice method has been shown to be at least 
equivalent, in terms of results and repeat-
ability, to cell and islet or acinus isolation 
methods (93-99). In many respects, how-
ever, the tissue slice allows for more phys-
iological data, especially when it comes 
to assessing communication between 
different cells (37,100-102). An addition-
al important advantage of the tissue slice 
method is that it is well compatible with 
many different model organisms with flu-
orescently labelled cells that interest us, 
and at the same time also with diabetes 
models that lead to the decay of most be-
ta cells (e.g. streptozotocin model) and so 
they are not compatible with the isolation 
of cells or islets, as in these cases too little 
isolate is obtained (103,104). The results of 
morphological and functional measure-
ments in the slice are becoming the gold 
standard in this field, as well as an import-
ant reference for measurements in other 
3D models.
Finally, we should emphasize that tis-
sue slices have recently been used in com-
bination with special scaffolds that extend 
the lifespan of the ex vivo slice and its 
usefulness for studies by at least one week 
(87). When using human tissue, it is an-
ticipated that scaffolds will need to allow 
tissue transfer for a duration of several 
days so that it can be used in studies in 
those parts of the world where there is no 
local source of human tissue or for mea-
surements in specialized laboratories us-
ing methods that are not available at the 
location of the source of human tissue. We 
are assuming that a larger or smaller part 
of the tissue from the slice can in the fu-
ture also be used to build 3D models using 
the already mentioned advanced materials 
and techniques of combining cells with 
these materials (60).
7 A glance into the future 
Despite remarkable advances in both 
the broader interdisciplinary domain of 
tissue engineering and the narrower field 
of in vitro pancreas models, both fields still 
face many problems that need to be over-
come. Currently, the most advanced 3D in 
vitro models mimic only the basic func-
tions of individual tissues and organs, so 
despite the shortcomings, tissue slices are 
used as the gold standard for performing 
basic studies (especially with the pancre-
as). A step in the right direction means the 
development of two seemingly unrelated 
fields of microfluidics and the production 
of computer microchips. The so-called 
organs-on-a-chip are cell cultures grown 
on advanced microfluidic devices (105). 
Microchip manufacturing techniques 
(e.g. soft lithography) (106) can produce 
high-precision flow scaffolds that better 
mimic physicochemical (concentration 
gradients of gases and nutrients), struc-
tural (nano-topology), and biochemical 
stimuli (concentration gradients of bio-
logically active molecules) of the tissues 
that are being mimicked. In addition, the 
cells in such devices are not grown in a 
static environment of cell vessels or 3D 
bioscaffolds, but are continuously exposed 
to the flow of cellular medium. Medium 
perfusion mimics key physicochemical 
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