PROGRESS REPORT
3D Skin Models
Toward Immunocompetent 3D Skin Models
Aleta Pupovac, Berna Senturk, Chiara Griffoni, Katharina Maniura-Weber,
Markus Rottmar,* and Sally L. McArthur*
3D human skin models provide a platform for toxicity testing, biomaterials
evaluation, and investigation of fundamental biological processes. However,
the majority of current in vitro models lack an inflammatory system,
vasculature, and other characteristics of native skin, indicating scope for
more physiologically complex models. Looking at the immune system, there
are a variety of cells that could be integrated to create novel skin models, but
to do this effectively it is also necessary to understand the interface between
skin biology and tissue engineering as well as the different roles the immune
system plays in specific health and disease states. Here, a progress report
on skin immunity and current immunocompetent skin models with a focus
on construction methods is presented; scaffold and cell choice as well as the
requirements of physiologically relevant models are elaborated. The wide
range of technological and fundamental challenges that need to be addressed
to successfully generate immunocompetent skin models and the steps
currently being made globally by researchers as they develop new models are
explored. Induced pluripotent stem cells, microfluidic platforms to control
the model environment, and new real-time monitoring techniques capable of
probing biochemical processes within the models are discussed.
1. Introduction
The in vitro development of skin models is an interdiscipli-
nary effort with a wide range of applications. Skin models pro-
vide 3D platforms to study various aspects of skin biology and
enable the testing of substances for cosmetic and pharmaceu-
tical industries (reviewed by Groeber et al.).[1] Engineering skin
Dr. A. Pupovac, Prof. S. L. McArthur
Faculty of Science
Engineering and Technology
Swinburne University of Technology
Hawthorn, Victoria 3122, Australia
E-mail: smcarthur@swin.edu.au
Dr. A. Pupovac, Prof. S. L. McArthur
Commonwealth Scientific and Industrial Research Organization (CSIRO)
Probing Biosystems Future Science Platform and Manufacturing
Clayton, Victoria 3168, Australia
Dr. B. Senturk, C. Griffoni, Dr. K. Maniura-Weber, Dr. M. Rottmar
Laboratory for Biointerfaces
Empa
Swiss Federal Laboratories for Materials Science and Technology
9014 St. Gallen, Switzerland
E-mail: markus.rottmar@empa.ch
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/adhm.201701405.
DOI: 10.1002/adhm.201701405
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www.advhealthmat.de
models remains challenging due to its
structural and functional complexity as it
encompasses a variety of specialized cells
and physiological subsystems, including
those responsible for controlling immune
responses.[2]
Different animal models have been
used for skin research, but mouse models
are most widely used because of easy han-
dling and lower cost. Mouse models are
regularly employed to study skin physi-
ology, biochemistry, and immunology,
and are often mandatory for in vivo trans-
lational research. While many facets of
anatomical, physiological, and immuno-
logical features are similar between mice
and humans, many comparative studies
show large differences between mouse
and human skin.[3] Human skin is struc-
turally and functionally different to mouse
skin; it is thicker and contains fewer hair
follicles. Further, the mouse epidermis
is composed of less keratinocyte layers,
which may contribute to decreased barrier
function and greater absorption observed in mouse skin com-
pared to human skin.[3] The skin immune system is composed
of a complex network of cells and extracellular matrix (ECM)
molecules. Immune responses maintain homeostasis and pro-
vide the host with defense against pathogenic microorganisms
in the tissue.[4] There are several differences between mouse
and human skin immune systems including the presence
and location of differing dominant T cells, a differing array of
chemokines expressed, and the presence or absence of antimi-
crobial peptides. These differences may contribute to the failure
of translating mouse in vivo skin model outcomes to human
skin disease.[3] 3D in vitro models that reconstitute the human
immune system would allow the development of new thera-
peutic materials for treatment of infectious diseases, delivery
agents, and also give insights into inflammatory pathologies.
However, the majority of current 3D skin models do not take
immune components into account, a factor that has been high-
lighted in recent reviews on skin disease models.[2,5]
Recent advances in tissue engineering and our under-
standing of immune cell biology have seen a number of
researchers start to include immune cells within 3D skin
models, making important steps toward recapitulating the func-
tions of native human skin. The main challenge of developing
an immunocompetent 3D skin model remains the effective
inclusion of multiple immune components and the creation
of an environment that allows and regulates the cross talk
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between those components.[6] It is only once these challenges

are addressed that it will be possible to properly mimic the Aleta Pupovac is currently a
pathogenesis of skin diseases in vitro. In this progress report, postdoctoral researcher at
the major components, challenges, and limitations of current the Commonwealth Scientific
skin models are discussed, with a focus on the immune com- and Industrial Research
ponent. Additionally, we discuss recent studies on applicability Organization (CSIRO) and
and limitations of endpoint and real-time monitoring tech- Swinburne University of
niques for inflammatory responses in skin models. Technology, Australia. She
received her Ph.D. in cell
biology and immunology at
the University of Wollongong,
2. Skin Immunity and Skin Models
Australia, and completed
Skin is a functionally heterogeneous and structurally complex 1.5 years of postdoctoral
organ. It is composed of three structural layers: the epidermis, research at Monash University, at the Biomedicine Discovery
dermis, and hypodermis. The epidermis is the avascular, out- Institute, Australia. Her current research interests include
ermost layer that consists of stratified epithelia exhibiting cru- infection and inflammatory pathways in 3D cell culture
cial regulatory and defensive functions including the regulation systems, tissue engineering, biomaterials, and biosensing.
of water loss,[7] defense against UV radiation[8] and infection.[9]
This layer predominantly consists of keratinocytes, which are Markus Rottmar is a group
organized into sublayers and exist at different stages of dif- leader in the Biointerfaces Lab
ferentiation. The dermis is the intermediate layer and is con- at Empa, the Swiss Federal
nected to the epidermis via the basement membrane. Blood Laboratories for Materials
and lymphatic vessels, nerves, sweat glands, and hair follicles Science and Technology,
reside in this extracellular-matrix-rich compartment which is Switzerland. He received his
secreted by resident fibroblasts. The deepest layer is the hypo- Ph.D. in the field of bioma-
dermis, which is mainly composed of loose connective tissue terials from ETH Zurich and
and adipose tissue that consists of fat-storing cells called adipo- with his team, he is interested
cytes. These inner layers provide nourishment, insulation, and in developing advanced,
energy reserves for the body.[10] A variety of cell types contribute predictive in vitro models
to immune regulation in human skin. The functions and ori- to understand and steer the
gins of these cells are listed in Table S1 and Figure S1 (Sup- interaction of human cells/tissues with materials relevant
porting Information). for skin wound healing and osseointegration.
Animal skin has a different structure and behavior compared
to human skin, making it difficult to unravel complex wound Sally L. McArthur is a
healing and disease processes in animal models. The develop- professor of biomedical
ment of functional in vitro skin models has also broadened the engineering at Swinburne
scope for the investigation of fundamental biological processes University of Technology
in human systems, which can in turn contribute to the devel- and a research science
opment of novel human therapies. Nonetheless, current 2D leader in biomaterials at the
and 3D in vitro models of healthy skin and skin disease do not Commonwealth Scientific
exhibit the complexity and heterogeneity of human skin. Sev- and Industrial Research
eral 2D models of skin disease have been described, including Organization (CSIRO) in
models of fibrosis, wounding, autoimmune disorders, cancer, Melbourne Australia. She
and infection.[2,11] However, it is well established that models has held academic roles in
utilizing 2D culture are not representative of in vivo conditions the United Kingdom and
and do not recapitulate the 3D environment of cells.[12] Australia for 15 years and leads research teams at both
More recent tissue engineered 3D skin models represent the CSIRO and Swinburne focusing on the development of 3D
most advanced constructs, and mimic the nature of skin by con- Cell Culture Systems and the development of coatings for
taining both epidermal and dermal layers. Current skin models, biointerface engineering, respectively.
with their respective components, applications, and limitations
are listed in Table 1. Applications of these models include basic,
pharmacological, and cosmetics research. The in vitro testing of
finished cosmetic products and cosmetic ingredients in Europe Epidermal 3D skin models[13,14] are actively used for in vitro
has been mandatory since 2013, since the European Commis- evaluation of ultra violet (UV) exposure,[15] bacterial adhesion,[16]
sion released a ban on animal research. The methods to eval- and penetration/permeation studies (Table 1).[17] However, these
uate irritation, corrosion, and sensitization of chemicals on skin models do not have a dermal layer or immune cells, which will
have been validated by the Organization for Economic Co-oper- likely affect data obtained in these studies. For example, the
ation and Development (OECD), who are responsible for the native epidermis contains melanocytes which are involved in
regulatory acceptance status of alternative methods. UV protection,[18] thus the inclusion of these cells are required

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Table 1.  Currently available skin equivalents and skin models, listed with their respective applications and limitations.

Model Components Application/study Limitations References

Reconstructed epidermis
models
Full-thickness skin models
Full-thickness skin model
containing macrophages
Full-thickness skin models
containing LCb) and/or DCc)
Full-thickness skin models
containing melanocytes and/
or endothelial cells
Full-thickness skin model
containing T cells
Organotypic tumor coculture
– Collagen, keratinocytes (EpiSkin)
–  Keratinocytes, melanocytes
– Normal or disease patient derived
differentiated keratinocytes on
fibroblast-populated dermis
– Fibroblasts, keratinocytes, RAW
264.7 cell
– Collagen, fibroblasts, keratinocytes,
mo-LCs and mo-DCsd)
–  MUTZ-3 derived LC
– mo-DC
– Normal or iPSCe)-derived fibroblasts,
keratinocytes, melanocytes, and
endothelial cells
– Collagen, fibroblasts, and
keratinocytes, CD4+ T cells
– Collagen, fibroblasts, macrophages,
squamous cell carcinoma cells
Skin irradiation, corrosion, penetration, No dermal layer, no immune [13,14,16]
UVa) damage, bacterial adhesion, cells
epidermal permeability
Dermal penetration, absorption No immune cells [17,19–25]
testing, fibrosis, autoimmune disease,
wound healing, skin infection
Test model for skin inflammation RAW 264.7 is a mouse cell [30]
line
Solar UV radiation testing, drug Other immune cells missing [32,35,36]
and chemical testing
High potential for drug discovery, Poor melanocyte viability, [26,27,29]
comparison of normal versus cancerous no immune cells
melanocytes, wound healing
Fungal infections Other immune cells missing [34]
Tumor progression No healthy keratinocytes; only [31]
represents cancerous skin

a)UV: ultraviolet; b)LC: Langerhans cell; c)DC: dendritic cell; d)mo-DC: monocyte derived DC; e)iPSC: induced pluripotent stem cell.

in the evaluation of UV exposure. 3D full thickness skin models
which contain both epidermal and dermal layers have also been
developed for a variety of applications and studies including
dermal penetration, absorption testing, fibrosis, autoimmune
disease, wound healing, and skin infection[19–22] (Table 1).
Dermal penetration studies and absorption tests are more rele-
vant in these models compared to 3D epidermal models, as they
better recapitulate native skin thickness and cell–cell interac-
tions. Skin infection and disease models have also been created,
including a model of percutaneous worm invasion,[23] bacterial
infection,[24] and psoriasis.[25] In vitro studies comparing normal
skin cells to psoriatic biopsy-derived cells in 3D constructs have
led to better characterization of the disease at a cellular level.[25]
However, these models also lack other cell types including
immune cells.
Other 3D full thickness models have incorporated addi-
tional cell types to understand the role of these cells in skin
health and disease, and to increase the complexity of cur-
rent skin models. Deeper understanding of the interaction
between melanocytes and the resident skin cell populations,
fibroblasts and keratinocytes, has been achieved using 3D
in vitro models[26,27] and currently, a standardized protocol
even enables the reproducible creation of a skin melanoma
model.[28] Melanocytes and endothelial cells have also been
used to create a more complex wound model to study cell
mobility in wound healing.[29] More comprehensive lists of
2D and 3D skin models and their applications and limita-
tions have been reviewed by Bergers et al.[2] and Mohammadi
et al.[11] It is apparent that in all of these models, including
those that have been used to study immune-based disease,
have largely neglected the role of immune cells and inflam-
matory processes.
Only a handful of full-thickness skin equivalent (FTSE)
models that contain immune cells have been created (Table 1,
Section 3). While these models also lack the structural and
immune complexity of native skin, they retain the foundations
required for the eventual realization of physiologically relevant
and immune competent skin.
3. Current Immune Competent 3D Skin Models
3.1. Construction Methods
3D culture provides the opportunity to coculture multiple cell
types leading to more complex cell systems. In 2D culture, cells
are usually grown in monolayers on polystyrene or glass, while
3D cultures grow in scaffold-free aggregates and spheroids,
or scaffold-based matrices.[12] In the context of infection and
inflammation, most current 3D models of immune competent
skin are reduced systems that are generated in Transwell,[30–34]
Snapwell,[35] or CellCrown[36] tissue culture inserts and plates.
The majority of these models comprise a fibroblast populated,
biologically derived collagen scaffold (dermal layer), seeded
with keratinocytes (epidermal layer) that are eventually cultured
at an air–liquid interface to generate a FTSE (Figure 1a). Others
have placed epidermal sheets from skin biopsies onto acellular
dermis (stratum corneum side up) and cultured them exposed
to air. Subsequently, these are placed onto fibroblast cultures
to allow for repopulation of the dermis and the generation of
a FTSE (Figure 1b).[37] Generally, these models are validated
against native skin by hematoxylin and eosin staining, and
immunostaining for markers expressed in the different skin
layers (Figure 1c).

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Figure 1.  Full thickness skin equivalent generation (FTSE). Fibroblast populated a) collagen or b) decellularized dermis are seeded with keratinocytes in
culture. After culture at air–liquid interface the differentiated keratinocytes represent the epidermis and the fibroblast-populated scaffolds represent the
dermis. c) Native skin and FTSEs generated with primary cells or cell lines after hematoxylin and eosin (H&E) staining and immunohistochemical staining
for specific markers vimentin, keratin 5, and keratin 10. Adapted with permission.[60] Copyright 2015, the author; published by Mary Ann Liebert, Inc.

To create immunocompetent models, up to two immune cell
types have been incorporated during or after the generation of
the FTSE. These are usually sourced from blood or are incor-
porated as cell lines (Section 3.3) and include langerhans cells
(LC),[32,35,37–39] dendritic cells (DC),[35,36] macrophages,[30,31]
and T cells.[34] Whole blood and peripheral blood mononu-
clear cells (PMBCs) (which contain different immune cell
types) have also been incorporated.[34] LCs and DCs have been
co-seeded with keratinocytes on the dermal layer during epi-
dermal equivalent generation (Figure 2a).[32,35,38] LCs have also
been placed beneath FTSEs and allowed to migrate to the epi-
dermis (Figure 2b).[37] These cells are usually visualized in the
models using immunostaining against cell-specific markers
including langerin, human leukocyte antigen DR-1, and den-
dritic-cell-specific intercellular adhesion molecule-3 grabbing
non-integrin (Figure 2a,b). Dendritic cells have also been
incorporated into an agarose-fibronectin layer that was placed
between a fibroblast-seeded synthetic microfiber scaffold and
a keratinocyte-populated microfiber scaffold, forming a sand-
wich style structure (Figure 2c). These cells were tracked in the
model using a fluorescent dye (Figure 2c).[36]
Spatial organization and the physical constraints provided by
3D culture influence gene expression and cellular behavior.[12]
In this regard, keratinocytes and fibroblasts require cross talk
in a bi-layered cellular construct for production of several rel-
evant growth factors and cytokines.[40] Thus, the presence of an
agarose-fibronectin immune cell layer in the FTSE (Figure 2)
may impede on fibroblast-keratinocyte cross talk. Furthermore,
the presence of high water content gel layers within the struc-
ture may complicate skin impedance readings.[36] While “sand-
wich” style construction may be advantageous for separation
of cells for analysis post use, ultimately the separation and/or
location of the inflammatory cells could be expected to influ-
ence cell–cell signaling. This, along with other processes needs
to be evaluated and validated for the specific application of the
skin models.
Macrophages have been placed into models either via
seeding at the bottom of transwell plates,[30] or seeding into col-
lagen scaffolds together with fibroblasts (Figure 3a,b).[31] They
have been visualized using cell-specific markers including the
macrophage non-inflammation marker, CD209, and pan mac-
rophage marker CD68 (Figure 3b).[31] Whole blood, PBMCs, or
T cells have been incorporated into collagen and placed under-
neath FTSEs in transwell inserts (Figure 3c).[34] During model
generation, the addition of these cells into the scaffold represents
a more 3D-like approach that mimics natural skin structure
compared to cells seeded at the bottom of transwell plates,
which does not allow migration and cell–cell contact. Never-
theless, while these immune competent models may resemble
some components of native skin, in terms of immune cell inte-
gration their entirety remains more reminiscent of “sandwich”
style cocultures rather than true 3D representations. Others
have used human skin explants to generate full-thickness
models that recapitulate complexity of human skin, including
the presence of innate LCs and T cells, however culture times
of these models are limited to 7 d before cell viability becomes
an issue.[33] Future developments should examine alternative
methods of construction that represent more complex and rel-
evant skin. This may be achieved by integrating immune cells
that can form interactions with adjacent cells types with min-
imal support and interference (see Section 3.4).[41]
3.2. Scaffold Choice
The majority of immune competent FTSEs contain a scaffold
generated from rat tail collagen I gels.[30–32,34,39] Collagen I
hydrogels are a popular extracellular matrix choice for 3D fibro-
blast culture, as collagen I is the most abundant ECM compo-
nent found in native skin tissue and because these hydrogels
can be formed with appropriate matrix densities, stabilize cells,
and contain cell adhesion sites.[42] Collagen gels also undergo

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Figure 2.  Dendritic and Langerhans cell locations in full thickness skin equivalent (FTSE) models. a) The schematic (left panel) shows that dendritic
cells and Langerhans cells are coseeded with keratinocytes during FTSE generation. After culture at air–liquid interface they migrate into the dermis
and epidermis, respectively. Experimental results (right panel) show that monocyte-derived Langerhans cells (mo-LC) are visualized in the epidermis
after immunostaining of langerin and human leukocyte antigen DR-1 (HLA-DR). Dermal dendritic cells (mo-DDC) are visualized in the dermis after
staining of HLA-DR, and dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) markers. The dotted line represents
the dermal–epidermal junction. Scale bar: 20 µm (Adapted with permission.[35] Copyright 2007, Mary Ann Liebert, Inc.). b) The schematic (left panel)
shows that FTSEs cultured with decellularized dermis are placed onto cultured Langerhans cells, which migrate to the epidermis during culture at
air–liquid interface. Experimental results (right panel) show immunostaining of markers for Langerhans cells including langerin and HLA-DR. Scale
bar: 20 µm (Reproduced with permission.[37] Copyright 2012, Elsevier). c) The schematic (left panel) shows that microfiber scaffolds are seeded with
keratinocytes or fibroblasts to form separate layers, while dendritic cells are incorporated into an agarose-fibronectin layer. These layers are assembled
in a “sandwich” style so that the dendritic cells sit between the dermis and epidermis. Experimental results (right panel) show monocyte-derived
DCs (middle layer) visualized using Cell Tracker Red between microfiber scaffolds (top and bottom layers) that contain keratinocytes and fibroblasts,
respectively. Scale bar: 20 µm (Adapted under the terms of the CC BY 3.0 license.[36] Copyright 2013, IOP Publishing Ltd.).

cell-mediated contraction, which occurs as a result of mechan-
ical interaction of keratinocytes and fibroblasts with the col-
lagen hydrogel.[43,44] Cell-mediated contraction is an important
process in wound healing[45] and has been used as a measure of
cell activation in many models.[46–48] While this feature is neces-
sary in wound healing processes, it can be a source of variation
that impedes on the generation of standardized skin models.
Moreover, the variability associated with the use of different
batches of collagen makes it difficult to achieve the same struc-
tural and mechanical properties. Hence, collagen alone may
not be useful for models with applications that require testing
skin responses to drugs. Additionally, fibroblasts, keratinocytes,
and macrophages secrete collagenases such as matrix metal-
loproteinases that degrade collagen gels,[49–51] therefore this
scaffold may also not be useful for models requiring long-term
skin culture or skin survival. Other ECM components such as
collagen type-III, elastin, laminin, or fibronectin play important
roles in native skin but are largely neglected in current FTSEs.
Yet, these components are likely important for such models,
as different ECM fragments are known to be involved in skin
inflammatory responses.[52] For example, Almine et al. showed
that the interaction of elastin with human dermal fibroblasts
induces the expression of matrix metalloproteinase-12 and
chemokines that trigger a pro-inflammatory response.[53]
To address some of the issues associated with the use of
collagen-only scaffolds, immune competent FTSEs have been
generated using modified collagens. Bechetoille et al. have
generated a dermal scaffold in the form of a porous sponge,
composed of types I and II bovine collagen containing chi-
tosan and the glycosaminoglycan, chondroitin-4 sulfate.[35]
While Kuhbacher et al. have combined the glycosaminoglycans,
chondroitin-4 sulfate, and chondroitin-6-sulfate with rat tail

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Figure 3.  Macrophage and T cell locations in full thickness skin equivalents (FTSEs). The schematics show that macrophages are a) seeded at the bottom of
transwell plates or b) (left panel) coseeded with keratinocytes, and migrate to the dermis after culture at air–liquid interface. b) (right panel) Experimental results
show macrophage migration to the tumor epithelium (TE) and the pan macrophage marker CD68 and macrophage M2 polarization marker, CD209 were
visualized in the dermal equivalent (DE) in the presence or absence of interleukin 4 (IL-4) treatment (Reproduced under the terms of the CC BY 3.0 license.[31]
Copyright 2012, the authors, published by PLOS). c) The schematic shows that T cells are incorporated into a collagen scaffold and placed underneath the FTSE.

type I collagen.[34] The addition of these components endows
mechanical strength to the scaffold without compromising cell
attachment and growth.[54] During culture, the collagen-glycosa-
minoglycan-chitosan based dermal scaffold does not contract,
unlike its collagen-only counterpart.[35] This scaffold also allows
for long-term culture (up to 49 d) of the FTSE, which may be
valuable in infection and inflammation studies that incorporate
immune cells and living infectious agents.[34]
Others have facilitated a FTSE with similar characteristics
to native skin by cross-linking succinimidyl glutarate poly-
ethylene glycol with a collagen hydrogel to create mechanical
strength and less cell-mediated contraction.[55] Modified col-
lagens may improve model variability and the complexity of
3D immune competent skin by allowing the incorporation of
vasculature (see Section 3.4). This was exhibited with the use
of a cross-linked porous bovine I collagen gel that was seeded
with endothelial cells suspended in a fibrin gel.[56] Using col-
lagen in conjunction with fibrin provided the model with a
greater vascular capacity compared to fibrin alone. As com-
plexity increases, scaffold choice and how it is utilized becomes
increasingly important with respect to physiological relevance.
Considering this, Chau et al. have separately populated a syn-
thetic, porous microfiber-based scaffold with fibroblasts or
keratinocytes as shown in Figure 2. This scaffold is commer-
cially available and does not significantly affect cell viability.[36]
However, this scaffold allowed keratinocytes to permeate
through it, consequently disrupting the brick-and-mortar like
structure of the epidermis.[36] Even though the authors show
staining of some differentiation markers, this model is not
ideal when comparing the structure to native skin in terms of
promoting a more natural 3D structure.
Decellularized tissue scaffolds have been used both in vitro
and in vivo to investigate the cellular interactions, organ forma-
tion and transplantation in tissue engineering and regenerative
medicine.[57] While human decellularized dermis is currently
the best scaffold option for the creation of clinically utilized
bioengineered skin constructs,[58] whether it is the best scaffold
option for the creation of immune competent skin models that
are needed to ascertain immunological mechanisms in disease
remains to be determined. Human dermis is usually harvested
from cadavers, surgical skin donations, or animal organs and
thus may not be easily sourced or applicable for skin models
that require high throughput applications. Decellularized
dermis has been used as a scaffold to create an immune com-
petent skin model to study chemokine-driven migration of
LC-like cells.[37] The same group has also used a collagen-based
scaffold to study the migration of these cells.[32] While this indi-
cates that decellularized dermis may not be required for cer-
tain applications, a decellularized porcine jejunum scaffold has
been successfully used to create a perfusable vascularized skin
model and such vascularization may be necessary for the crea-
tion of immune cell flow (see Section 3.4).[59]
3.3. Cell Types and Sources
The cells used to generate immune competent FTSEs are usu-
ally partially or fully sourced from donor skin samples and
peripheral blood.[30–32,34,35] Primary cells may be harder to
source regularly and are endowed with donor variation.[5] To
overcome donor variation in LCs sourced from blood, Ouwe-
hand et al. incorporated LCs differentiated from the human

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acute myeloid leukemia cell line, MUTZ-3 (MUTZ-LC) into
their FTSE. However, the FTSE itself was generated using
primary skin samples.[32] Others have used cell lines to create
models in a disease context, where mouse or human FTSEs
were generated to resemble skin in a tumor-associated envi-
ronment.[31] These contained an epidermal equivalent gen-
erated from the mouse or human squamous cell carcinoma
cell lines PDVA and A-5RT3, respectively, but the monocytes/
macrophages in these models were sourced from peripheral
blood.[31]
Using the telomerase reverse transcriptase (TERT)-immortal-
ized, N/TERT-1 keratinocyte and BJ-5ta fibroblast cell lines, an
all cell line FTSE was developed that was comparable to native
skin (Figure 1c).[60] However, it did not contain any immune
cells. To the best of our knowledge, no one has created an
immune competent FTSE composed entirely out of cell lines.
It would be vital to determine whether incorporating immune
cell lines such as MUTZ-LC into an all cell line model, allevi-
ates the variability associated with donor samples, and whether
the immune response in such a model is comparable to that
of its primary counterparts. It is also important to consider
whether the cell types being used to create immune competent
skin are physiologically relevant. In this regard, Chung et al.
used a mouse macrophage cell line within a human FTSE to
study lipopolysaccharide (LPS)-induced immune response.[30]
Mouse and human macrophages have a different response to
LPS treatment,[61] so using human monocyte/macrophage cell
lines such as THP-1 or U937 would be more physiologically
relevant in such a model. Others have created FTSEs using
HaCaT keratinocytes, which display deficient epidermal differ-
entiation[62] and therefore do not appropriately mimic the native
epidermis.
Despite considerable efforts to create immunocompetent
skin models to date, a limited variety of immune cells (max-
imum of two) have been incorporated into current immune
competent skin models.[35] To increase the complexity of skin
models, several immune cell types should be incorporated. Con-
versely, increasing the number of cell types may also increase
variability, thus a trade-off exists between model complexity and
variability. Recently, Kuhbacher et al. incorporated whole blood
and PBMCs into their FTSEs to study skin infection by Candida
albicans (C. albicans).[34] In terms of immune cell variety, this
FTSE represents the most sophisticated immune model to date.
Additionally, it is the only FTSE containing immune cells that
has incorporated a living infectious agent to study skin infec-
tion. It was demonstrated that whole blood or PBMCs (which
contain T cells), and CD4+ T cells alone inhibited C. albicans-
induced dermal invasion to similar degrees. As efficient protec-
tion was achieved with the incorporation of CD4+ T cells alone,
FTSEs containing these cells were used to determine mecha-
nisms of C. albicans invasion.[34] Although less physiologically
representative, one cell type achieved the same response and
allowed a simpler approach to relevant model generation.
Avoiding the inclusion of redundant cell types may be advanta-
geous in terms of reproducibility, while achieving similar out-
comes. Such an approach to cell choice may also be useful when
generating a model of skin infection where immune response
mechanisms are undefined. It is challenging to precisely regu-
late the microenvironment where cells are introduced, making
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it difficult to fine-tune and maintain cell differentiation toward
the desired phenotype. For example, macrophages are respon-
sible for both pro-inflammatory (M1) responses as well as the
switch from a pro-inflammatory to a reparative (M2) environ-
ment, and the resolution of inflammation in skin wounds.[63]
However, this dualistic role and functional heterogeneity also
represents a limitation when considering their implementation
into 3D in vitro skin models. Nonpolarized (M0) macrophages
that are derived from peripheral blood monocytes can easily
be isolated from blood and differentiated into M1 or M2-like
macrophages. While macrophage colony stimulating factor or
granulocyte-macrophage colony stimulating factor is required
for monocyte survival and differentiation into macrophages,[64]
both cytokines distinctly influence the cells’ expression profiles
and by that, prime them toward M1- or M2-like macrophage
populations.[65] The M1-like phenotype is normally induced
by supplementing the medium with interferon-γ [66] and LPS
or tumor necrosis factor-α,[67] while M2-like macrophages are
obtained when supplementing the medium with the cytokines
interleukin-4 and/or IL-13.[68] Cell survival also depends on
macrophage types, M2-like macrophages survive longer than
M0 and M1-like macrophages.[69] Thus, defining the purpose
and biological processes involved in skin infection, injury and
disease is critical before the introduction of relevant immune
cell types.
3.4. Requirements for Physiologically Relevant Skin Models
The ideal skin model would contain many different cell types,
appendages, adipose tissue, and vasculature that recapitulate
skin structure and function. This model should support long-
term culture and encompass devices that monitor real-time cel-
lular behavior in health and disease (Figure 4).[11] Yet, several
hurdles are faced when trying to construct the ideal or physio-
logically relevant skin model for research purposes. Skin models
tend to be designed in a highly context dependent manner and
thus require unique design concerns. First, the biological con-
text and complexity of the model must be established. Once
established, several considerations are apparent including the
effect of multiple cell types, that is, cell–cell ratios and cell–cell
interactions, which will affect the expression of physiological
surface markers and the migration of cells within the model.[70]
Concurrently, matrix considerations including scaffold choice
and spatial interactions of cells within the model (2D vs 3D),
as well as the effect of culture requirements including culture
times, media constituents, and media diffusion are relevant.[70]
These considerations are required to maintain a physiologically
relevant model while achieving research purposes.
To the best of our knowledge, a complex immunocom-
petent skin model, containing more than a few immune cell
types in long-term culture has not been generated. Incorpo-
rating a wide variety of cells to generate complexity may be
realized with the use of induced pluripotent stem cells. iPSCs
are derived from differentiated somatic cells that are repro-
grammed to a pluripotent state with the potential to become
many different cell types.[71] iPSCs have been used to gen-
erate keratinocytes and fibroblasts to create a whole iPSC-
derived FTSE,[27] iPSC-derived melanocytes,[27] and endothelial
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
Figure 4.  The ideal skin model. The ideal skin model should encompass native skin structure tant for the realization of more complex
and function by containing many different cell types, appendages, adipose tissue, and vasculature.
It should support immune cell flow, long-term culture, and encompass devices that monitor
real-time cellular behavior and biomarkers in health and disease.
cells [72] have also been integrated into FTSEs, exemplifying
the capability of iPSCs to produce different skin cell types and
increase the complexity of current skin models. In the near
future, iPSCs may be used to introduce immune cells into
the skin models. Furthermore, stem cells have been used to
generate 3D models that can be cultured for up to 45–50 d,
and therefore may be advantageous for developing models in
long-term culture.[73,74] Currently, it is known that iPSCs can be
differentiated into multiple functional lymphocyte lineages,[75]
however skin-specific immune cells such as LCs have not
been generated. Human iPSC-derived macrophages, which are
developmentally related to tissue resident macrophages such as
LCs[76] may warrant use in FTSEs. iPSCs may also facilitate the
development of personalized skin models, enabling drug and
other treatment methodologies to be evaluated and tailored to
the specific patient’s needs.[5] Specialized medium is required
to maintain different cell types, thus the combination of many
cell types in one model containing one type of medium may
result in aberrant marker expression that may not be physio-
logically relevant. This will subsequently affect cell differentia-
tion, viability, and migration within the model. As iPSCs allow
for directed differentiation,[71] they may be used as a future tool
to control marker expression of cells. Although for this to be
possible, the use of this tool must be accompanied with the
creation of a medium or a blood substitute that can successfully
maintain many cell types at a time.
The cutaneous vascular system, which is missing in estab-
lished immunocompetent skin models, is critical for homeo-
stasis and inflammatory responses in native skin. Blood and
lymphatic vessels supply oxygen and nutrients, and allow
immune cell and cytokine infiltration to the skin during
pathogen invasion. Many inflammatory skin diseases are
associated with insufficient or overactive vasculature.[77] Due
to the absence of vasculature systems in current immuno-
competent skin models, understanding the mechanisms
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of inflammatory diseases with vascular
involvement remains a major challenge
in skin immunology. Additionally, the vas-
cularization of skin models is required to
address diffusion limitations of media in
models with matrix-rich environments and
remains a solid requirement for the suc-
cessful implementation of shared nutrients
or media, and immune cell flow.[70] There
are several vascularization strategies in skin
tissue engineering including those that are
scaffold-based, cell-based, and emerging.[78]
Modifying scaffolds can increase intrinsic
vascularization (see Section 3.2)[56] and cell-
based technologies such as iPSCs can be
used to create endothelial cells that generate
vascular structures.[72] Likewise, emerging
strategies such as 3D bioprinting, cell sheet
engineering, and microfluidics are impor-
immunocompetent skin.[78] The bioprinting
of skin models is gaining traction as a result
of the need to reduce the amount and cost
of animal experimentations.[79] Several skin
constructs have been bioprinted including models that mimic
wound healing, in vitro models for validation and testing, and
constructs that encompass appendages, pigmentation, and vas-
culature, all of which, to some extent recapitulate the native
skin. Nevertheless, bioprinting faces challenges in replicating
native skin, particularly the complex ECM structure.[79] To intro-
duce vasculature into skin models, the major approaches have
been printing endothelial and smooth muscle cell on hydrogel
layers[80] and printing channels that are subsequently seeded
with cells.[81,82]
The aforementioned strategies will provide additional user
control of construction factors and scaffold choice,[11] hence a
combination of vascularization strategies may generate physi-
ologically relevant models. This is becoming evident with the
construction of several user-controlled vascular models. A
human FTSE that contains spatially controlled perfusable vas-
culature was produced using microfabrication and micropat-
terning techniques and iPSC-derived endothelial cells.[72] Addi-
tionally, a novel FTSE containing blood and lymph-like capil-
laries was constructed using a cell accumulation technique
whereby cell surface coating was produced using layer-by-layer
assembled nanofilms of extracellular matrix and the coculture
of human umbilical vein endothelial cells and human dermal
lymphatic microvascular endothelial cells.[83] This process
eliminated the use of collagen hydrogels and provided more
reliability between models.[83] Moreover, this model contained
lymph-like capillaries that may subsequently form lymphatic
structures that allow immune cell travel. Spatial control exhib-
ited by these models will also be useful in mimicking skin het-
erogeneity in disease versus healthy states.
While these models are relevant for the analysis of static top-
ical delivery of drugs or infectious agents, systemic delivery of
drugs, infectious agents, or immune cells cannot be achieved
without blood and lymphatic flow. Recently, a model of vascu-
larized skin was generated that may allow flow of immune cells
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
through the construct. Groeber et al. described a FTSE based
on decellularized porcine jejunum with a perfused vascular
network generated using a bioreactor for dynamic culture.[59]
Using this system they were able to obtain similar physiolog-
ical perfusion of capillary blood flow and bidirectional mass
transport.[59] By increasing the complexity of models through
the introduction of flow, the stress applied from flow force on
cells may affect cell viability and activate cells through mecha-
notransduction.[70] The mechanical signal to cells is converted
to signaling pathways that regulate gene expression, cell prolif-
eration, and differentiation.[84] Understanding the mechanisms
involved in mechanotransduction signaling in skin models
and controlling the duration and frequency of the flow rate in
models may eventually help to control cell viability and main-
tain physiological levels of cell proliferation and differentiation.
Microfluidics platforms are being utilized to create skin-
on-chip models in pursuit of a controlled environment and an
exchange of immune cells (reviewed by van den Broek et al.).[5]
Noticeably, many of these are reduced models that lack a FTSE
and immune cells. One skin-on-chip model, dubbed as being
immunocompetent, includes the U937 monocyte/macrophage
cell line with the HaCaT keratinocyte cell line in a device with
a perfusion setup.[85] This device allowed the monitoring of
monocyte and keratinocyte interactions after UV irradiation or
LPS stimulation in dynamic culture. It also contained an in-
built barrier function readout using transepithelial electrical
resistance.[85] While this device did not utilize a FTSE, real-time
monitoring of skin-relevant processes is evident. Ultimately, a
combination of the aforementioned technologies and real-time
monitoring of skin processes can lead to the creation of the
ideal skin model (Figure 4).
4. Monitoring Infection and Inflammation in
Immunocompetent Skin Models
The barrier function provided by skin is an important mech-
anism of protection against infection and disease.[86] Many
skin models have used transepithelial impedance measure-
ments as an indicator of barrier function in response to treat-
ment.[36,59,83,85] Skin impedance measurements are minimally
invasive and permit real-time measurement. However, when
used alone, this technique does not serve as a relevant bio-
marker of infection and inflammation in the skin. Many of the
methods used to study skin immune responses are endpoint
measurements that involve deconstructing the 3D structure
of models. Cell fluorescence imaging and immunostaining
are abundantly used to track the migration and phenotype of
DCs[32,35] and macrophages.[30,31] Furthermore, antibody array
assays and enzyme-linked immunosorbent assays are routinely
used to show that the microenvironment of the models con-
taining immune cells successfully maintain their phenotypes,
and that the model responds to treatment through the meas-
urement of cytokine expression and secretion.[30,35,36] Other
methods used to detect immune responses include zymog-
raphy, which has been used to observe collagenolytic activity
and expression of matrix metalloproteinases after macrophage
integration into FTSEs,[31] and dual RNA-sequencing of FTSEs
infected with C. albicans, which allowed the assessment of a
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www.advhealthmat.de
wide range of responses, including the expression of cytokines
such as IL-1β, IL-6, and IL-8.[34]
The pro-inflammatory cytokines TNF-α, IL-1α, IL-1β, IL-6,
and IL-8, as well as the immune suppressive IL-10 have been
measured in response to a variety of inflammation or infection
inducing agents including UV radiation,[35] LPS,[30] and sensi-
tizing agents.[36] Soluble cytokines, chemokines, and their recep-
tors can act in complex networks making it difficult to target a
particular cytokine or chemokine as a biomarker of inflamma-
tion and infection.[87] In future immunocompetent models, tar-
geting a variety of inflammatory biomarkers including cytokines
may be more effective. When the skin barrier is compromised
during inflammation and disease, this affects skin pH, protease
activity, and cytokine secretion.[87] Other biological indicators of
inflammation include the increased production and release of
antimicrobial peptides and damage associated molecular pat-
terns.[87] These features may be useful as potential novel bio-
markers of skin inflammation and disease in new generation
skin models. Further, the activation properties of immune cells
may also be explored as potential novel biomarkers. Incidentally,
several smart fluorescent probes have been developed for detec-
tion of macrophage activity including migration, reactive oxygen
and nitrogen species release, and matrix metalloproteinase and
cathepsin activation.[88] Others have created reporter cell lines
that have been integrated into FTSEs to elucidate mechanisms
of infection progression. Kuhbacher et al. used photometric
measurement of secreted alkaline phosphatase by S1F fibro-
blasts containing the nuclear factor kappa-light-chain-enhancer
of activated B cells (NF-κB) reporter gene, which allowed meas-
urement of toll-like receptor activity.[34] Methods such as these
may be useful for future immunocompetent skin designs that
take advantage of in situ measurements of activity.[2]
5. Conclusion
Much of research and development in skin health and disease has
traditionally been centered on the use of 2D cultures, cocultures,
and animal models. Since the European Union’s ban on animal
testing of cosmetics in 2013, there has been a rise in the construc-
tion and implementation of more physiologically relevant and 3D
human skin models. This has resulted in the development of a
variety of epidermal and FTSE models to study disease, as well as
skin substitutes for clinical applications. However, current models
do not fully recapitulate native skin. A handful of groups have
constructed skin models that have integrated several immune
cell types including LCs, DCs, macrophages, and T cells into 3D
FTSEs, paving the way for the development of more relevant and
immunocompetent skin models. Construction approaches of
these models focus heavily on the use of transwell systems, col-
lagen-based scaffolds, and human-sourced primary cells. Although
useful, coculture type systems are not truly representative of native
3D skin. Further, the need exists to design scaffolds that withstand
long-term culture and treatment with infectious agents, as well
as appropriate and easily sourced cells. Together, these alterations
may deliver more representative and reproducible results.
Successful future creation of relevant models lies in inte-
grating and implementing knowledge from materials science,
engineering, and cell biology. Designing and incorporating
© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advancedsciencenews.com
appropriate scaffolds and cells into models of disease is con-
text-dependent and requires a solid understanding of biological
processes involved in the disease, as well as materials engi-
neering and design needs. Several technologies are paramount
in designing physiologically relevant immunocompetent skin
models. The integration of iPSCs into models may allow for faster
sourcing of cells, as well as more relevant cell choice to model
disease. It may also provide a more personalized or patient-spe-
cific approach to study disease, which would offer industry and
clinicians alternative test models for drug development. Using
current and newly emerging bioengineering techniques such as
bioprinting, cell sheet engineering, and microfluidics to integrate
vasculature into immunocompetent models will eventually allow
immune cell flow and more relevant cell–cell interactions in skin
models. Further, in situ measurements of model activity at the cel-
lular and molecular level will provide important biomarker read-
outs in skin disease without the need to disturb or process the
model. Taking advantage of cellular processes and engineering
requirements during the construction of new and more relevant
models will allow smart ways of integrating in situ sensors into
future skin models. Together the aforementioned aspects may
lead to the creation of an ideal skin model that is physiologically
relevant and biologically informative in its contextual use.
Supporting Information
Supporting Information is available from the Wiley Online Library or
from the author.
Acknowledgements
A.P. and S.L.M. gratefully acknowledge funding from the Commonwealth
Scientific and Industrial Research Organization (CSIRO) Probing
Biosystems Future Science Platform, CSIRO OCE Science Leader
Program and Swinburne University of Technology. B.S. and M.R.
gratefully acknowledge funding from the Commission for Technology
and Innovation CTI (Grant No. 18524.2 PFLS-LS). S.L.M. and M.R.
gratefully acknowledge the Swiss National Science Foundation for the
Visiting Fellowship (Grant No. IZK0Z1_171124) with which support this
review was developed.
Conflict of Interest
The authors declare no conflict of interest.
Keywords
3D skin models, inflammation, inflammatory cells, skin immunity
Received: December 1, 2017
Revised: January 29, 2018
Published online: March 15, 2018
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