Macromolecular

Chemistry and Physics Trend

Cell Microencapsulation by Droplet

Microfluidic Templating
Torsten Rossow, Philipp S. Lienemann, David J. Mooney*

Microgels are hydrogel particles with micro-scale dimensions in the range of 10–1000 µm. They
have gained particular importance for cell microencapsulation, because they allow for highly
specific creation of a myriad of discrete and miniaturized mimics of the three-dimensional
natural extracellular matrix of tissues. This trend article highlights and critically reviews cur-
rent droplet-based microfluidic approaches for the formation of such cell-laden microgels. It
addresses the technical issues of particle formation, the most
common polymers used for microgel preparation, and the dif-
ferent types of polymer crosslinking. Furthermore, remaining
technical challenges in the field are discussed and perspec-
tives for further development and potential future applica-
tions are provided.

1. Introduction
In vitro recapitulation of the three-
dimensional (3D) microenvironment
that cells face in the human body
has emerged as powerful approach
for addressing biomedical chal-
lenges such as control of stem cell-
fate or assessment of drug efficacy
within a close to physiological and
therefore biomedically insightful
environment.[1–3] The concept of cul-
turing cells in a milieu that resem-
bles their in vivo situation stands in
strong contrast to the typical in vitro
Dr. T. Rossow, Dr. P. S. Lienemann,
Prof. D. J. Mooney
John A. Paulson School of Engineering
and Applied Sciences
Harvard University
Cambridge, MA 02138, USA
E-mail: mooneyd@seas.harvard.edu
Dr. T. Rossow, Dr. P. S. Lienemann,
Prof. D. J. Mooney
Wyss Institute for Biologically Inspired
Engineering
Cambridge, MA 02138, USA
experiments where isolated cells
are cultured on stiff plastic in a set-
ting that typically does not resemble
the situation in the human body.
It has become clear in recent years
that standard 2D culture often does
not capture key features of in vivo
biology.[4,5] Moreover, compared
to actual in vivo testing in animal
models, advantages of engineered
3D microenvironments for cell cul-
ture include high reproducibility and
throughput, the ability to routinely
use human or even patient-spe-
cific cells, and diminished ethical
concerns.
In order to recapitulate the com-
plex in vivo microenvironment, bio-
chemical and biophysical aspects of
the 3D cellular microenvironment
are typically imitated. The indi-
vidual cellular microenvironment is
defined by the extracellular matrix
(ECM; polysaccharides and proteins),
soluble and immobilized agents such
as growth factors and cytokines, and
neighboring cells, which altogether
result in a tissue specific biochemical
and biophysical profile (Figure 1A).[6]
Droplet-based microfluidics has
emerged as a powerful tool to rec-
reate such microenvironments with
very high throughput and tight con-
trol over cells, biomolecules, and ECM
properties (Figure 1B).[7] Application
of droplet-based microfluidics can
result in the creation of hundreds of
such microenvironments per second
in the form of microgels, which are
hydrogel particles with micro-scale
dimensions in the range of 10–1000
µm. Usage of microgels, as opposed
to larger gels, to address biomedical
questions in 3D allows with high sta-
tistical power, to minimize material
consumption, and to largely bypass
issues related to impaired nutrient
and waste transfer during culture.[8]
Cells encapsulated in microgels
can be readily manipulated using
syringes and pipettes, and imaged
with optical microscopy. Micro-
gels are also potentially very useful
for in vivo applications, as their
small dimensions allow them to be
delivered to a target site by injection.

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 Macromolecular
Cell Microencapsulation by Droplet Microfluidic Templating Chemistry and Physics
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Overall, microgels allow for cells to
be encapsulated, stored, and mani­
pulated in a very controlled manner
ex-vivo, and subsequently injected
into the body.[9]
In order to recapitulate the in
vivo milieu as closely as possible
using droplet-based microfluidics,
selection of the appropriate input
materials and settings regarding cell
types, cell numbers, biomolecules,
and ECM properties, is essential. The
design and usage of suitable artifi-
cial ECM poses a significant technical
challenge, and great efforts have
been undertaken over the last years
towards this goal. The creation of a
suitable artificial ECM requires not
only appropriate mechanical proper-
ties (e.g. elasticity), but potentially
also degradation sites, cell adhesion
sites, and biomolecule affinity sites.
The development of crosslinking
mechanisms that allow for cell
friendly gelation after droplet forma-
tion is also crucial. Polymer-based
hydrogels offer themselves as a
promising type of material that can
meet these requirements due to their
high degree of modifiability and
ability to create these from a myriad
Torsten Rossow obtained his Ph.D. in organic and polymer chemistry from
the Freie Universität Berlin in 2014. Following this, he took up a postdoc-
toral position at Harvard University, USA, in the group of David Mooney
working on single-cell encapsulation in polymer microgels. His further
research interests are supramolecular chemistry and stimuli-responsive
polymer gels. Recently, he joined the Siemens AG and works in the devel-
opment of polymeric insulation materials for electrical machines.
Philipp Lienemann is a postdoctoral fellow in the group of Professor
David J. Mooney at Harvard University. He holds a B.Sc. in Biology
and an M.Sc. in Biotechnology from the Swiss Federal Institute of
Technology (ETH) in Zurich. In his dissertation at École Polytechnique
Fédérale de Lausanne (EPFL) and at University Hospital of Zurich he
used biomaterials to investigate and manipulate the fate of mes-
enchymal stem cells during bone regeneration. He received a Ph.D.
in Biotechnology and Bioengineering from EPFL in 2014. His current
research interest lies in microfluidic technology for cell encapsulation
to probe single cell fates in 3D.
David Mooney is the Pinkas Family Professor of Bioengineering at Harvard
University, and a Core Faculty Member of the Wyss Institute. His labora-
tory designs biomaterials to make cell and protein therapies effective
and practical approaches to treat disease. He is a member of the National
Academy of Engineering, and the National Academy of Medicine.
of synthetic and natural polymers microgels. We begin by introducing
and mixtures thereof.[10] droplet-based microfluidics and dis-
Herein, we discuss the combina- cussing polymers used as artificial
tion of droplet-based microfluidics ECM in microgels. Next, we review
and hydrogels for creation of artifi- work published after 2012 on cell-
cial cellular microenvironments in laden microgels based on covalent

Figure 1.  Recapitulating the natural cellular microenvironment in artificial extracellular matrix (ECM)-based microgels using droplet based
microfluidics. (A) The natural cellular microenvironment is composed of neighboring cells, ECM, and biomolecules such as growth factors.
Signals received from these components determine a cell's fate. (B) Droplet-based microfluidics allows for versatile and high throughput
creation of microgels mimicking the natural cellular environment. By mixing defined amounts of selected cells, ECM, and biomolecules,
microenvironments can be designed in a bottom-up approach with defined properties including matrix elasticity, matrix degradation sites,
biomolecule binding sites, cellular adhesion sites, and cell numbers.

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or physical polymer crosslinking.

For work published before 2012 the
reader is referred to an excellent
review article by Kumacheva and
coworkers[7] about microfluidic cell
encapsulation in polymer microgels.
Finally, we describe current technical
challenges in the field and provide
perspectives for further development
and potential future applications
of technologies for cell encapsula-
tion in microgels by droplet-based
microfluidics.

2. Droplet Microfluidics

For microgel production, droplet-

based microfluidics has emerged
as a powerful and versatile tech-
nique,[11–17] that allows microgels to
be formed either with a uniform or
a capsular core–shell morphology. In
the first type of microgel, the polymer
network spans the whole particle
and cells can be entrapped within its
mesh, whereas in the second type,
the polymer network forms only the
particle shell while the core remains
liquid. Droplet microfluidics is based
on the formation of emulsion drop-
lets of a cell suspension and func-
tional polymer solution as templates
for particle synthesis; the drop-
lets are formed using micrometer-
scale channels. Subsequently, the
functional polymers serve for droplet
solidification by physical or chemical
crosslinking. Droplet microfluidics
uses either glass capillary devices[18]
or devices made by lithography
techniques, commonly consisting of
poly(dimethylsiloxane) (PDMS).[19]
Glass capillary microfluidic devices
are composed of coaxial assemblies
of glass capillary tubes with cylin-
drical or square cross-sections that
are glued onto glass slides. While
these devices exhibit a robust resist-
ance to organic solvents, the attrac-
tive feature of PDMS devices for cell
encapsulation is the ability to design
and fabricate highly complex flow
channels for upstream adjustment
Figure 2.  Droplet-based microfluidic templating to fabricate uniform microgel particles
that encapsulate cells. Two solutions of functional precursor polymers are injected into
a microfluidic device, along with a suspension of cells. These three fluids meet at the
first cross-junction, where they form a laminar coflowing stream in the microchannel. In
the second junction, periodic break-up of this stream is induced by flow focusing with a
fourth fluid that could be an immiscible oil; this produces uniform premicrogel droplets.
By mixing of the three liquids inside the droplets the precursor polymers crosslink and
cell-laden microgels are formed. Adapted with permission.[21] Copyright 2012, American
Chemical Society.
of the fluid streams and down- fluid (continuous phase), as shown
stream manipulation of the droplets. in Figure 2. In a T-junction micro-
In addition, lithography facilitates fluidic device, the dispersed phase
mass production of these devices; flows into the junction, where the
once a given type of photomask is continuous phase forms a thin film
designed, a large number of devices between the dispersed phase and the
can be replicated.[11] Depending on walls of the device. This leads to an
the device geometry, microemul- increase of the pressure upstream of
sification can occur via different the emerging droplet and squeezes
mechanisms. For cell microencap- the dispersed phase to form the
sulation the most common devices droplet.[20] In both types of devices,
have a flow-focusing or a T-junction the size, shape, and monodisper-
design. In flow-focusing devices, sity of the droplets is controlled by
a stream of functional polymer the dimensions of the microchan-
solution and a cell suspension (dis- nels, and the viscosities, polarities,
persed phase) is created and its and flow rates of the fluids. The
periodic break-up is induced by flow average number of cells per droplet
focusing with a second, immiscible can be either controlled by the

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Cell Microencapsulation by Droplet Microfluidic Templating
concentration of the cells in the pre-
cursor solution, or by the flow rate of
the cell containing fluid if a device
with two separate inlets for the
inner phases is used. After droplet
solidification by physical or chem-
ical crosslinking, the pre-microgel
droplets retain their size, shape, and
monodispersity, thereby yielding
cell-laden microgel particles with
precisely controlled morphology,[21]
as also illustrated in Figure 2. For
long-term cell culture, the micro-
gels can be transferred from the oil
phase into aqueous culture medium,
wherein the microgels may swell
to a certain degree depending on
their composition. One limitation
of such flow-focusing microfluidic
approaches is the emulsification of
precursor polymer solutions that can
crosslink on a very short time scale
(e.g., a few seconds). In this case, the
thread of the droplet phase forms
nodules and does not break-up into
droplets;[13,22] finally, the microflu-
idic device can get clogged. To solve
these problems, the crosslinking
reactions have been slowed[23–25]
or sophisticated microfluidic tech-
niques have been applied.[26–28]
To ensure high viability of the
encapsulated cells, the process of
microemulsification itself should
be carefully considered, in addi-
tion to the polymeric precursor
materials and crosslinking chemis-
tries. The non-polar liquid forming
the continuous phase as well as
the surfactant need to be highly
biocompatible. In this regard,
fluorocarbon oils and fluorosur-
factants[29] have turned out to be
very useful[30,31] and are the subject
of current research.[32] Moreover,
the microgel work-up, the transfer
of the microgels into the aqueous
phase, need to be performed as
gently as possible; high shear
forces created by centrifugation or
pipetting, as well as non-cytocom-
patible chemicals that are used
to break the emulsion, can harm
the encapsulated cells.[31] On-chip
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extraction of microgels presents
an elegant approach to minimize
the time microgels are suspended
in oil and allows for their gentle
transfer to the aqueous phase
without the need for pipetting and
centrifugation.[33,34]
Microgels can serve as model sys-
tems for 3D cell culture of single
cells. For this purpose, the number of
cells per particle needs to be exactly
controlled. This can be achieved by
controlling the concentration of cells
in the cell suspension or by adjusting
the size of the droplets, since micro-
fluidic encapsulation of cells is a
stochastic process following Poisson
statistics with
λ ne − λ  (1)
f ( λ ; n) = ,
n!
where f(λ;n) is the number of
droplets that contain n cells in
the droplet and λ is the average
number of cells per droplet. This
means for single cell encapsulation
in droplets with typical diameters
of 50 to 100 µm, approximately 86
of 100 droplets are empty if only 1
drop should contain more than one
cell.[24,35,36]
To characterize the mechanical
properties of microgels, atomic force
microscopy has become the state
of the art method. Several variants
of this technique have been used
such as nanoindentation, which pro-
vides information about mechanical
response on the nanoscale[37,38] or
the colloidal probe method, which
provides information on the micro-
scale.[36,39] Kumacheva, Walker and
coworkers presented an AFM method
to characterize the average Young’s
modulus and the stress relaxation time
of entire microgel particle using tipless
cantilevers.[40] The method was applied
for the examination of the temporal
variation in mechanical properties of
cell-laden and cell-free agarose micro-
gels. The values of Young’s moduli for
both types of microgels decreased
by around 30% on day 3; afterwards
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no significant change in the Young’s
moduli was observed following 7-days
of cell culture.
3. Cell-Laden Microgels
Microgels containing cells can be
formed from both synthetic and
natural precursor polymers. Biopoly-
mers that have been used for the
preparation of cell-laden microgels
using droplet microfluidic tem-
plating are alginate[41] and its copoly­
mers,[42] agarose,[43,44] collagen,[45]
gelatin,[46] hyaluronic acid,[47] and
peptides.[48] Synthetic microgels
have been formed by crosslinkage
of macromolecular precursors, often
referred to as macromonomers, such
as poly(ethylene glycol),[49] poly­
glycerol,[50] or copolymers of poly(N-
(2-hydroxypropyl)-methacrylamide)
(PHPMA) and poly(hydroxyethyl
methacrylate) (PHEMA) grafted with
poly(N-isopropylacrylamide) (PNI-
PAAm) side chains.[51] While the
polymerization of monomers is not
typically of relevance for the syn-
thesis of cell-laden microgels due to
their cytotoxicity, the crosslinking of
macromonomers affords the oppor-
tunity to exactly control the microgel
composition and properties by the
precursor concentration, molecular
weight, and functionalization, along
with the chemistry of crosslinking.
An overview of commonly used syn-
thetic and natural macromonomers
used to form cell-laden microgels is
given in Scheme 1.
In the following sub-sections,
the features and properties of the
most frequently used synthetic and
natural precursor polymers will
be briefly discussed and the most
recent approaches to form cell-
laden microgels will be presented
in detail. Both covalently and physi-
cally crosslinked microgels will be
reviewed. Table 1 summarizes the
strategies for the functionalization
and crosslinking of the different
precursor polymers.
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A HO H H O be non-immunogenic, non-toxic, and

On O
n inert to protein and cell interactions.[58]
Poly(ethylene glycol) O O
H O O H Hyperbranched polyglycerol
n n
(PEG) (hPG)[59] is structurally similar to
O
O H PEG and exhibits analogous phys-
n
Tetra-arm PEG ical properties, but offers multiple
hydroxy functionalities that allow for
OH
HO postmodification (e.g., to acrylates,[60]
O OH
HO O alkynes and azides[61]) while the
OH polymer remains water-soluble. By
O OH
O subsequent crosslinking reactions,
O nH HO O OH
hydro-,[62] micro- and nanogels[60,63]
H O O H O O
O n O n OH for biomedical applications have
6 O O O OH
O O been prepared that have proven to
Octa-arm PEG O OH OH
be protein[64] and cell resistant, and
O O
O even less cytotoxic than PEG.[65]
HO O OH
O OH HO O
OH
HO OH 3.1.2. Polysaccharides
HO O
Alginate is the most commonly used
HO OH biopolymer for the preparation of
hyperbranched Polyglycerol physical crosslinked hydrogels. It
(hPG) is derived from brown algae with
molecular weights ranging from 50 to
B OH OH 100 000 kDa and is composed of man-
HO OH O H O
O HO O nuronic and guluronic acid residues
O O HO O OH
O O OH that are covalently linked in different
O OH NH
H OH sequences or blocks.[66] The blocks are
OH O
n
n either similar or strictly alternating,
Agarose Hyaluronic Acid and the exact composition depends
on the origin of the alginate. Alginates
OH
OH O OH can be physically crosslinked to form
O O hydrogels by complexation of their car-
H O HO O HO OH
n boxy groups to divalent metal ions such
O OH m
as Ba2+ or Ca2+. The mechanical proper-
Alginate ties of the gels can be adjusted by the
Scheme 1.  Overview of commonly used (A) synthetic and (B) natural precursor polymers concentration of the crosslinking metal
for the formation of cell-laden microgels. ions, the polymer molecular weight, or
by the ratio of guluronic and mannu-
3.1. Polymer Precursors for Microgel homo- and heterobifunctional linear ronic acid in the polymer used.[67] Algi-
Formation PEG, as well as multi-arm PEGs and nate hydrogels are typically considered
3.1.1. Polyethers PEG dendrimers can be synthe- to be non-immunogenic, bio-inert and
sized.[52] Postmodification of hydro­ highly biocompatible. Besides physical
Poly(ethylene glycol) (PEG) is a com- xy-terminated PEG provides access crosslinking, very recently a method
mercially available synthetic polyether to a large variety of functionalities to covalently crosslink alginate by
backbone polymer that is prepared (e.g., acrylates,[53] vinyl sulfones,[54] bioorthogonal click chemistry has
by the well controllable anionic thiols,[55] and alkynes and azides).[56] been introduced.[68]
ring-opening polymerization of eth- These functional groups can serve to Hyaluronic acid is composed of
ylene oxide. By this means, a wide form polymer hydro- and microgels alternating units of d-glucuronic acid
range of molecular weights is acces- by using linear PEG-acrylates and and d-N-acetylglucosamine. It is a
sible from 300 to 10 000 000 g mol–1 applying free-radical crosslinking[57] or normal component of the human
along with narrow molecular weight by mixing functional linear PEGs with body, particularly in the ECM of car-
distributions. By choosing the appro- complementary multiarm-PEGs. PEG tilage, and it is involved in cell sign-
priate initiator, monofunctional, hydrogels are typically considered to aling and matrix organization. The

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Table 1.  Representative strategies for the functionalization and crosslinking of synthetic and natural precursor polymers for the formation
of cell-laden microgels. (A) Covalent and (B) physical crosslinked microgels.

Polymers Part A Ref.

Functionalization X-Linking
Gelatin Methacrylate Free-radical (photoinduced) [77]
Gelatin, Collagen / Hydrogen bonding + free-radical (photoinduced) [39]
PEG Maleimide Thiol-Michael with dithiothreitol [85]
PEG Acrylate Free-radical (photoinduced) [86]
PEG Peptide Enzymatic with FXIIIa [87]
PEG, Hyaluronic Acid Vinylsulfone, Thiol Thiol-Michael [36]
PHPMA, PHEMA, PNIPAAm Azide, Cyclooctyne SPAAC [51]
Polyglycerol, PEG Acrylate, Thiol Thiol-Michael [21]
Polyglycerol, PEG Acrylate Free-radical (thermally induced) [60]
Polyglycerol, PEG Azide, Cyclooctyne SPAAC [84]
Part B
Alginate / Ca2+-complexation [23–26,88–91,93]
Collagen / Hydrogen bonding (+covalent) [92]
PEG Bipyridine Fe2+-complexation [27]
PEG Peptide Non-covalent with oligosaccharides [94]

molecular weight of hyaluronic acid
ranges from 100 to 8 000 kDa. Due to
its excellent biocompatibility it is a
frequently used biopolymer in tissue
engineering as well as in regen-
erative medicine, and formulations
have been approved by the FDA.[47]
3.1.3. Proteins
Proteins are widely used for the for-
mation of hydro- and microgels,
especially, fibrinogen, collagen and
its irreversibly hydrolyzed form, gel-
atin.[69] Polymerization of fibrinogen
is performed enzymatically by the
actions of the protease thrombin and
the plasma transglutaminase factor
XIII, resulting in a fibrin mesh resem-
bling that found in blood clots.[70] Col-
lagen and gelatin can be physically
polymerized by thermal gelation or
covalently crosslinked by chemical
or enzymatic strategies, resulting
in mechanically and thermally sta-
bilized gels.[71–73] As opposed to the
typically nanoporous structure of
polyether- and polysaccharide-based
hydrogels, protein-based hydrogels
often exhibit a fibrous structure facili-
tating cell migration, cell-cell contact,
and receptor-ligand clustering.[74]
Further advantages of protein-based
hydrogels are their inherent bio-
logical properties, such as specific
amino acid sequences serving as cell
adhesion sites, degradation sites, or
growth factor binding sites.[69] How-
ever, due to their natural origin, big
variations can exist between dif-
ferent batches of protein-based poly-
mers, and since they are not bioinert,
unintentional biological effects might
be induced.
3.2. Covalent Microgels
In contrast to several biopolymers
that can be used to form microgels
even without chemical modifica-
tion, synthetic precursor polymers
usually need to be functionalized
in a post-polymerization step to
make them serviceable in a subse-
quent crosslinking reaction. This
crosslinking can occur by many
different reaction types, but if the
microgels are anticipated to serve for
the encapsulation of cells, not all of
such reactions have turned out to be
equally effective. Most widely used
is the free-radical crosslinking of
acrylates or methacrylates, because
these functionalities can be easily
introduced to hydroxy-functionalized
polymers via ester-bonding; by this
means, even bifunctional linear poly­
mers can be radically crosslinked
to form polymer networks. Radical
crosslinking has been used exten-
sively for the synthesis of cell-laden
microgels and can occur either by
thermally-generated radicals or by
photoinitiators and exposure to UV
light. Haag, Seiffert, and colleagues
employed the first method and pre-
pared yeast-cell-laden microgels
composed of hPG and PEG.[60] How-
ever, despite the biocompatibility and
favorable elasticity of the hPG–PEG
polymer matrix, the presence of free
radicals during gel formation was
detrimental for cell viability. Recently,
Huck and coworkers applied photo-
crosslinking to produce fibroblast-
laden collagen–gelatin beads with
tunable mechanical properties in the

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range of 1–10 kPa.[39] In this approach
the authors take advantage of micro-
fluidics to achieve efficient mixing
of two different polymer solutions in
the microdroplets by internal recircu-
lation. The homogeneity of collagen–
gelatin mixtures was determined by
the relative gelling speed of collagen
and gelatin. Low concentrations of the
precursor polymers prevent gelling of
both, while cooling facilitates gelatin
gelling and slows down collagen gel-
ling. By using a maximum collagen
loading of 0.18 wt%, homogeneity
of the beads on the sub-micro­meter
length-scale could be achieved.
Almost 90% of the encapsulated cells
were viable after 24 h, and about
70% of the cells were viable after
1 week. Although the initial high cell
viability indicates that the droplet
and gel bead formation processes are
not acutely detrimental for the cells,
and despite the simplicity of radical
induced crosslinking, UV irradiation
and the radicals themselves could be
potentially cytotoxic.[75,76] Weitz and
coworkers also used photo-crosslink-
able gelatin to microencapsulate
bone marrow-derived mesenchymal
stem cells (BMSCs) and growth fac-
tors.[77] The authors demonstrated
that the gelatin microspheres can
support cell spreading, migration,
and proliferation and that BMSCs
encapsulated in gelatin microspheres
show enhanced osteogenesis in vitro
and in vivo, associated with a signifi-
cant increase in mineralization. How-
ever, the initial viability of the BMSCs
one day after encapsulation was only
about 60%, perhaps suggesting detri-
mental effects of the radicals gener-
ated during crosslinking.
To enhance the viability of encap-
sulated cells, mild crosslinking reac-
tions are desired that are orthogonal
to the functional groups of the cell
membrane, non-cytotoxic, and fast
at 37 °C. One class of chemistry
that fulfills these requirements is
termed ‘bioorthogonal’ click chem-
istry[78] and was introduced by Car-
olyn R. Bertozzi in 2003 as chemical
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reactions that can occur inside of
living systems without interfering
with biological processes.[79,80]
For this purpose, the substrates of
bioorthogonal reactions must react
selectively with each other at fast
rates under physiological conditions,
and they must be inert to the func-
tionalities found in vivo. One reac-
tion type that fulfills many of these
attributes is the thiol-Michael addi-
tion to electron-deficient carbon–
carbon double bonds such as vinyl
sulfones, acrylates, and maleim-
ides;[81,82] a side reaction of these
substrates with thiols in the cell
membrane could occur and there-
fore the thiol-Michael addition is
also called a pseudo-click reaction.[83]
The thiol-Michael addition was com-
bined with droplet microfluidic tem-
plating to fabricate monodisperse,
cell-laden microgel particles wherein
encapsulated lymphoblast and
fibroblasts exhibited viability up to
90%.[21] In this approach, microdro-
plet gelation was achieved via the
nucleophilic addition of dithiolated
PEG to acrylated hPG building blocks.
The mechanical properties of the gels
could be varied by the concentration
and molecular weight of the pre-
cursor polymers and their influence
on the viability and proliferation
of encapsulated cells was probed.
Degradation of the microgel parti-
cles through hydrolysis of the ester
bond close to the thioether bond was
observed on a timescale of several
weeks, but was not be precisely con-
trolled. To overcome this limitation,
another microgel construction kit
was introduced that uses the strain-
promoted azide–alkyne cycloaddi-
tion, another type of bioorthogonal
reaction, for crosslinking along with
acid-labile benzacetal linkers for the
programmed release of encapsulated
cells, as shown in Figure 3A and
B.[84] Variation of the substitution
pattern of the benzacetals allowed
the microgel degradation kinetics
to be exactly controlled in the inter-
esting pH range between 4.5 and 7.4
Macromol. Chem.  Phys. ,  ,  1600380
T. Rossow et al.
(Figure 3C). The encapsulated fibro-
blast cells could be cultured inside
the microgels with full retention
of their viability, and subsequent
microgel degradation had no detri-
mental effect on the encapsulated
and released cells, as also shown in
Figure 3C.
The same bioorthogonal
crosslinking chemistry was applied
to prepare hybrid microgels with
thermo-tunable elasticity composed
of poly(N-(2-hydroxypropyl)-meth-
acrylamide) and poly(hydroxyethyl
methacrylate) grafted with thermo-
responsive poly(N-isopropylacryla-
mide) side chains.[51] Upon change
of the temperature from 32 to 37 °C,
these hybrid microgels can reversibly
expel water and thereby reversibly
increase their elastic modulus, the
extent of which can be tuned by the
precursor polymer chain length and
grafting density. As the viabilities of
encapsulated fibroblasts and stem
cells was at least 80%, this microgel
construction could serve as a versa-
tile material platform to investigate
mechanotransduction.
García and coworkers used the
thiol-Michael addition for stem
and islet cell microencapsula-
tion.[85] Their approach was based
on crosslinking tetra-arm PEG
maleimide with the small molecule
dithiothreitol (DTT). Functionaliza-
tion with a cell adhesive GRGDSPC
peptide occurred by reaction of the
maleimide groups in the precursor
polymer with the cysteine-thiol in
this peptide prior to cell encapsula-
tion. By using a flow-focusing micro-
fluidic device, human pancreatic
islets were encapsulated into micro-
gels with viability of ≈90% after 1
and 8 days of culture. The microgels
exhibited size distributions ranging
from 300–800 µm; large clusters
of human islets make the produc-
tion of monodisperse particles chal-
lenging. To evaluate the function of
the islet cells after encapsulation, a
glucose-stimulated insulin secretion
(GSIS) assay was performed. This
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 Macromolecular
Cell Microencapsulation by Droplet Microfluidic Templating Chemistry and Physics
www.mcp-journal.de

Figure 3.  Cell encapsulation and release. (A) Hyperbranched polyglycerol-azide precursors P1–P3 with acid-cleavable linkers that had dif-
ferent hydrolysis kinetics and PEG-dicyclooctyne were used to prepare NIH3T3 cell-laden microgels; crosslinking of the precursors was
achieved by the strain-promoted azide–alkyne cycloaddition. (B) The viability of cells encapsulated was at least 94%, as determined by
fluorescence-based live–dead assays, in which living cells were stained green and dead cells were stained red. (C) Incubation of the micro-
gels M1–M3 at pH 7.4, 37 °C, and 5% CO2 led to complete particle degradation of M1 after three days and release of the cells with unchanged
residual viability of 96%. Under these incubation conditions microgel M2 showed incomplete degradation and no release of cells for an
observation period of two weeks. Incubation of microgel M2 at pH 6.0, 37 °C, and 5% CO2 led to complete particle degradation after three
days and release of the cells with an unchanged residual viability of 94%. Microgel M3 showed no degradation between pH 6.0–7.4. Repro-
duced with permission.[84]

demonstrated no significant differ-
ence in insulin secretion between
encapsulated and non-encapsulated
islets. This finding suggests that
microfluidic-based encapsulation
in tetra-arm PEG-maleimide has no
detrimental effect on human islet
function, and that mass transfer
of molecules relevant to islet func-
tion and therapeutic application is
not significantly affected by this
microencapsulation.
In a recent approach, Huck and
coworkers used the thiol-Michael
addition for single cell encapsula-
tion of human mesenchymal stem
cells (hMSCs) into monodisperse
fibrinogen-containing hyaluronic
acid microgels.[36] For this purpose,
the authors prepared linear PEG-divi-
nylsulfone and thiol-modified hyalu-
ronic acid; fibrinogen that provides
natural binding sites for cell sur-
face integrins was incorporated by
specific hyaluronic acid–fibrinogen
interactions. The number of cells
in each droplet roughly followed a
Poisson distribution; approximately
30% of the microgels contained a
single cell while the rest of the beads
contained either no cells or multiple
cells. The hMSC viability was deter-
mined to be 70% after 24 hours of
culture and almost 100% after long
term culture of 2 weeks, likely due
to disintegration of initially dead
cells. Although fibrinogen was used
to provide cell adhesion sites, hMSCs
embedded in the microgels displayed
an overall rounded morphology and
no spreading independent of matrix
mechanics ranging from 0.9–9.2 kPa.
This finding suggests a lack of matrix
degradation in these microgels.
Characterization of the multipotency
and differentiation potential of the
hMSCs demonstrated the preserva-
tion of hMSC multipotency during
fabrication and 3D culture. After
14 days of culture in osteo/adipo
bipotential differentiation medium,
the microgels supported a pref-
erential differentiation of hMSCs
into adipocytes even at the highest
explored modulus of 9.2 kPa. How-
ever, a clear heterogeneity in the cell
population can be observed in the 3D
cell culture samples which reveals
the importance of deepening the
understanding of stem cell fate at
the single cell level.[36]
Complementary to the single cell
encapsulation work is an approach
used by Bhatia and coworkers.[86]
These authors took advantage of the
stochasticity of cell encapsulation
at high cell densities to generate
multi­ple populations of cell numbers
from a single microfluidic experi-
ment. First, fluorescently labeled
tumor cells were microfluidically
encapsulated or co-encapsulated
with stromal cells into PEG micro-
gels (Figure 4A). In the second step, a
large-particle flow analyzer was used

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Chemistry and Physics
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Figure 4.  3D tumor microenvironment
screening platform. (A) Fluorescently
labeled tumor cells were encapsulated
or co-encapsulated with stromal cells
into PEG microgels. (B) The microtis-
sues produced were rapidly interrogated
in multiple fluorescent channels using
large-particle flow analysis. (C) Cytom-
etry-like flow sorting separated and
defined microtissues with controlled
levels of homotypic and heterotypic
interactions. (D) Sorted microgels were
collected in tissue-culture wells, wherein
which they were cultured over several
days and treated with soluble growth fac-
tors or drugs. The extent of cell prolifera-
tion within individual microtissues was
then detected by flow analysis (B) to col-
lect population-level data for responses
to microenvironmental conditions. Repro-
duced with permission.[86] Copyright 2013,
The Royal Society of Chemistry.
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to initially characterize freshly gener-
ated cell-laden microgels in multi­ple
channels of embedded-cell fluores-
cence (Figure 4B). Then, defined pop-
ulations of microgels were selected
and sorted by tumor and/or stromal
cell density (Figure 4C). In the next
step, sorted microgels were col-
lected in tissue-culture wells and
cultured over several days; during
this time, they could be treated
with soluble growth factors or drugs
(Figure 4D). At the desired time point,
treated microgels were collected and
reanalyzed by flow cytometry for
changes in overall fluorescence of
the embedded cells (Figure 4B). After
the analysis step, each microgel
population could be re-collected for
additional culture periods and sub­
sequent analysis, thereby allowing
the evolution of the cell population
to be studied over time.
Lutolf and coworkers demon-
strated by the successful microencap-
sulation of several mammalian cell
types, including fibroblasts, embry-
onic stem cells, and cancer cells, that
enzymatic crosslinking can serve as
a promising alternative to bioorthog-
onal click chemistry.[38,87] For this
purpose, octa-arm PEG-vinylsulfone
precursor polymers were modified
to display peptide substrates for
the activated transglutaminase Fac-
torXIII (FXIIIa). Moreover, the gels
could be rendered enzymatically
degradable by inserting a matrix
metalloproteinase (MMP) sequence
into one of the peptides. Cells could
be encapsulated within microgels of
500 and 1000 Pa stiffness with ini-
tial cell viabilities of 90% by using
a microfluidic flow-focusing device.
However, after a few days of culture,
cell escape from the microcapsules
was observed independent of the gel
degradability, which has also been
an issue for other cell-laden micro-
gels.[39] To address this problem,
microgels were co-encapsulated
within a 200 µm thick gel film or
microgel layer of higher stiffness
by again employing microfluidic
Macromol. Chem.  Phys. ,  ,  1600380
T. Rossow et al.
templating; however, it was unclear
if there was interpenetration of the
outer and inner gel layers. By this
method, cell escape could be signifi-
cantly reduced without significantly
affecting cell viability much, as long
as the outer gel layer did not exceed
a modulus of 40 kPa.
3.3. Physical Microgels
Alginate is one of the most widely
used biopolymer for cell microencap-
sulation due to its typical biocom-
patibility and the simplicity with
which it forms gels by mixing with
divalent cations, such as Ca2+. The
physical crosslinking occurs imme-
diately upon contact of alginate
polymers with calcium ions. As a
result, conventional microemulsifica-
tion can cause clogging of the device
along with non-uniform drop forma-
tion.[7] To solve these problems, drop
formation and gelation have been
separated in time and location by
dispersing calcium carbonate nano-
particles in the alginate solution and
by using acidic conditions to dissolve
these after drop formation.[23,88,89]
In other techniques the crosslinking
process is initiated by dissolution
of Ca2+-salts in the oil phase and by
diffusion of Ca2+ into the emulsion
droplet after its formation.[22,90] Weitz
and coworkers developed a technique
that works in the opposite way. In
this approach, a glass capillary device
was used to fabricate double emul-
sions comprised of an alginate drop
surrounded by a mineral oil shell, as
shown in Figure 5A.[26] As the algi-
nate drop separated from the mineral
oil shell, it contacts with Ca2+ ions in
the continuous phase and gelation
occurred (Figure 5B). By this means,
microgels could be prepared with
diameters ranging from 60 to 230 µm
and viability of encapsulated yeast
cells of 65% after one week of culture.
All these techniques, however,
result in rather heterogeneous
microgel particles exhibiting regions
of low and high crosslink density. To
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 Macromolecular
Cell Microencapsulation by Droplet Microfluidic Templating Chemistry and Physics
www.mcp-journal.de

Figure 5.  Preparation of cell-laden alginate microgel particles. (A) Optical image showing the formation of alginate/mineral oil double-
emulsions. Reproduced with permission.[26] (B) Optical images demonstrating the separation of the inner alginate drop from the mineral
oil shell. Reproduced with permission.[26] (C) Scheme of a non-planar microfluidic flow-focusing device for the one-step generation of
core–shell microcapsules from two aqueous fluids. Reproduced with permission.[91] Copyright 2013, The Royal Society of Chemistry. Scale
bars in (A) and (B) denote 100 µm.

overcome these limitations, Weitz
and coworkers developed another
approach, wherein calcium ions
were delivered in the form of water-
soluble calcium–ethylenediamine-
tetraacetic acid complexes.[24] By
this means, a highly homogenous
mixture of alginate and the chelated
calcium ions can be microemulsi-
fied, followed by the dissociation of
the complex and release of calcium
ions after drop formation initiated
by addition of acetic acid to the con-
tinuous phase. The freed Ca2+ ions
react with the alginate chains in a
highly controlled fashion and form
alginate microgels with good struc-
tural homogeneity, as shown by flu-
orescence microscopy. This approach
has been used for single-cell encap-
sulation of mesenchymal stem cells
that were cultured for 15 days with
resultant cell viabilities of around
70%; encapsulated cells also demon-
strated proliferation.
The previously described
approaches all fabricate microbeads
with a cell-containing, solid-like
hydrogel core. In 2011, Kang and
coworkers instead demonstrated the
encapsulation of embryonic carci-
noma cells in microcapsules with
a liquid core and alginate hydrogel
shell using a microfluidic device.[25]
Recently, He and coworkers used a
non-planar (3D) microfluidic flow-
focusing device to achieve one-step
generation of core–shell microcap-
sules with an alginate hydrogel
shell of controllable thickness and
an aqueous liquid core of embryonic
stem cells.[91] This core–shell archi-
tecture mimics in certain ways a
pre-hatch embryo. To produce such
microgels a 1% cellulose solution
containing the cells, a 2% sodium
alginate solution, and mineral oil
infused with calcium chloride were
injected through the core, shell, and
oil channels, respectively, as shown
in Figure 5C. At the non-planar flow-
focusing junction, the shell flow
was driven by the crossing oil flow
to fold over the parallel core flow,
and both the aqueous core and shell
flows were broken into droplets
by the crossing oil flow. To achieve
this, a non-planar flow-focusing
design with varied channel depth
(Figure 5C) is necessary. The viability
of the encapsulated stem cells was
found to be higher than 92% right
after microencapsulation, and the
cells proliferated to form a single
aggregate in each microcapsule
within 7 days. By delivery of cardiog-
enol C, a cardiogenic inductor, the
aggregated cells could be efficiently
differentiated into beating cardio-
myocytes. Another type of core–
shell architecture was introduced
by Takeuchi and coworkers.[92] They
encapsulated HepG2 cells within
collagen microgels and then seeded
fibroblasts onto the microgel sur-
face. By this means, the authors were
able to generate a 3D hierarchic co-
culture system of two different cell
types. Recently, Weitz and coworkers
prepared core–shell microgels by
using water–water–oil double emul-
sions.[93] The microgels consist of a
liquid core of hepatocytes and an
alginate shell containing fibroblasts,
and could serve as a 3D liver model.
Synthetic polymers with side
or end groups functionalized with
supramolecular crosslinkable motifs
provide an alternative to biopoly-
mers for the formation of physically
crosslinked microgels. Werner, Zhang
and coworkers used this method-
ology to prepare cell-laden microgels
from peptide-functionalized tetra-
arm PEG and oligosaccharides by a
non-covalent crosslinking mecha-
nism.[94] The microgels were stable
in various buffer conditions and in
cell culture media, and showed no
degradation over a period of months.
Survival rates of embedded fibro-
blasts up to 98% were observed after
7 days in standard cell culture condi-
tions. After freezing and storing the
beads at –196 °C in liquid nitrogen
for six weeks, around 86% of the
encapsulated cells were viable, dem-
onstrating the potential of this
approach for preserving cells in 3D.
Moreover, the authors showed the
potential for protein-expression by

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encapsulating eGFP-secreting insect
cells; the protein expression profile
of insect cells embedded in microgels
was similar to that of cells in suspen-
sion. Linear PEG precursor polymers
functionalized with bipyridines on
both chain ends that can be gelled
by complexation to iron(II) ions pro-
vides another system for the fabrica-
tion of physically crosslinked micro-
gels.[27] By using PEG precursors of
different molecular weights at dif-
ferent concentrations, the microgel
elasticity could be controlled, and
the viability of encapsulated lymph-
oblast and breast cancer cells was
optimized to exceed 90%. The micro-
gels were degradable by addition of
EDTA, a competitive ligand to bipy-
ridine, within 1.5 h under very mild
conditions and with no apparent
detrimental effect on the released
cells. However, this approach is not
suitable for long-term applications
due to microgel auto-degradation
within timespans of several hours.
4. Summary, Challenges, and
Perspectives
4.1. Summary
Droplet-based microfluidics enables
the creation of picoliter-sized droplets
that can serve as miniaturized vessels
for short-term (hours) culture and
straightforward analysis of small cell
numbers, down to individual cells.
Through addition and crosslinking of
ECM mimicking synthetic or natural
polymers to these vessels, fabrication
of microgels becomes possible. Cells
encapsulated in microgels can be cul-
tured for days, and the biochemical
and biophysical properties of the
microenvironment can be adjusted
to mimic the physiological environ-
ment. This conjunction of hydrogels
and droplet-based microfluidics for
fabrication of microgels has opened
up new possibilities in basic as well
as applied biological research. Over
the last years, fabrication and culture
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of microgels went from proof of con-
cept studies to recapitulating, in a
high throughput manner, complex
tissue architectures that address
significant problems. The impact of
these systems can be expected to
multiply in the near future.
4.2. Challenges
Over the last decade, great strides
were made in combining droplet-
based microfluidics with advanced
biomaterials for 3D cell culture. How-
ever, we believe that there are still
technical challenges that currently
limit advances in the technology and
its suitability for broad use. Three of
the major technical challenges we
see:
Strategies to evade the poisson
distribution: Current methods to
encapsulate cells into microgels
are rather inefficient. Typically,
in order to prevent cell aggrega-
tion and to control the number of
cells in a microgel, cells are highly
diluted and are then randomly dis-
tributed into droplets based on a
poisson distribution.[95] This results
in numbers of empty microgels far
exceeding the numbers of cell-laden
microgels, especially if one aims for
encapsulation of individual cells
using a highly diluted cell solution.
In this case a typical ratio would
be approximately 6.6:1 in favor of
empty microgels while ensuring that
only every 100th microgel contains
two or more cells.[35] This makes
downstream processing and analysis
a laborious venture. We therefore
believe that simple technologies
that allow for controlled encapsula-
tion of an individual cell or defined
numbers of multiple cells in every
microgel would be greatly benefi-
cial, in regards to throughput and
simplicity. This could for example
be achieved by selective gelation
only of microgels that contain cells,
or by loading cells into droplets in a
controlled manner by prearranging
them in the microfluidic device like
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T. Rossow et al.
has been done for solid particles
using close-packed ordering.[95]
Strategies to prevent cell egress: For
an artificial ECM to serve as a tissue
mimetic, it is often desirable for
cells to be capable of remodeling the
ECM and potentially migrate within
it. However, due to cells following
nutrient gradients from the cul-
ture medium or just due to random
migration this might lead to cells
escaping the microgel.[87] This often
may not be a limitation, and may
be desirable in certain situations.
For example, in studies assessing
biological processes on a time scale
of hours, this is most likely not an
issue. However, in certain situations
one may want to avoid cell escape.
Lutolf et al. proposed elegant tech-
nologies for preventing cell egress
by encapsulating microgels in larger
non-degradable microgels either
through reinjection into a microflu-
idic device or by placing microgels
in non-degradable bulk gels.[87] Fur-
ther strategies that would allow for
placing a dense shell around micro-
gels without the need for the techni-
cally challenging reinjection into a
microfluidic device, or the loss of the
ability to manipulate with a pipette
would be highly advantageous. Also,
precisely positioning cells at the
center of the microgel may help to
reduce cell egress by increasing the
distance to the edge of the microgel.
Strategies for monitoring indi-
vidual microgels in long-term cul-
ture: Droplet-based microfluidics
can be used to create a myriad of
hetero­geneous microenvironments
differing in ECM components, cell
types, or cell numbers.[86] To ascribe
changes that are observed during
culture periods to different starting
compositions, it is crucial to be able to
follow individual microgels. We see
great room for advancements in the
creation of microfluidic traps that
allow for immobilizing and culturing
microgels in specified locations, as
were developed for culturing single
cells.[96,97] Ideally, such arrays would
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Cell Microencapsulation by Droplet Microfluidic Templating
allow not only for monitoring cells
during culture, but also for on chip
staining and analysis.
4.3. Perspectives
Cell encapsulation by droplet-based
microfluidics offers novel and versa-
tile approaches for addressing bio-
medical challenges. We see future
applications of these technologies in
the following broad areas:
Single cell drug testing: Due to
the possibility to unify advan-
tages of 3D cell culture and single
cell analysis, we see great room for
applying droplet-based microflu-
idics to assess the response of single
cells to selected drugs or for drug
screening with consideration of the
mechanical and biological properties
of the cellular microenvironment.
This might be of particular impor-
tance, for example, in finding new
drugs specifically targeting cancer
stem cells, which are hypothesized
to be the subpopulation of a tumor
responsible for its reoccurrence
after chemotherapy and for forming
metastases.[98] The findings that
solid tumors within various tissues
exhibit alterations in the ECM prop-
erties, and that tumor progression
may be attributed to changes in ECM
stiffness and composition support
the relevance of ECM properties for
drug testing in vitro.[99,100]
Deciphering stem cell niches: Adult
stem cells reside within the human
body within specific tissue niches,
consisting of specific neighboring
cells and ECM composition.[101] These
niches provide a microenvironment
for stem cells to maintain their
stemness. The ability to systemati-
cally reconstruct stem cell niches in
vitro could have great implications
for both basic and translational
research. For example, this could
allow for stem cell in vitro expan-
sion without loss of stemness. In
addition, combining cell encapsula-
tion in microgels with technologies
for controlled assembly of individual
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microgels, such as optical tweezers,
might allow for bottom up engi-
neering of stem cell niches.
Stem cell therapy: The therapeutic
delivery of stem cells in hydrogels
may be useful in a range of situa-
tions, including immunomodula-
tion and tissue regeneration.[102,103]
However, such approaches are often
limited by poor biomolecule trans-
port, leading to impaired viability
of transplanted stem cells as well
as impaired delivery of biomol-
ecules secreted by the transplanted
stem cells. Microgels may provide
ideal candidates for therapeutic cell
delivery due to the possibility to
reduce the hydrogel to cell volume to
a minimum and thereby improving
nutrient transfer without losing the
protective and functional properties
of a hydrogel microenvironment.
Furthermore, the size of microgels
may allow for minimally invasive
delivery through needles.
Acknowledgements: T.R. and P.S.L.
contributed equally to this work. This
work has been supported by grants
from the German Research Foundation
(DFG) to T.R. (GZ: RO 5138/1-1), the Swiss
National Science Foundation (SNSF) to
P.S.L. (Grant P2ELP3_161850) and the
National Institutes of Health (NIH) to
D.J.M. (Grant RO1EB014703).
Received: August 4, 2016; Revised:
September 21, 2016; Published online:
November 7, 2016; DOI: 10.1002/
macp.201600380
Keywords: cell encapsulation; microfluidics;
microgels; tissue engineering
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