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

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1600380  (1 of 14) © 2016  WILEY-VCH Verlag GmbH &  Co.  KGaA, Weinheim wileyonlinelibrary.com DOI: 10.1002/macp.201600380
Macromolecular
Cell Microencapsulation by Droplet Microfluidic Templating Chemistry and Physics
www.mcp-journal.de

Overall, microgels allow for cells to Torsten Rossow obtained his Ph.D. in organic and polymer chemistry from
be encapsulated, stored, and mani­ the Freie Universität Berlin in 2014. Following this, he took up a postdoc-
pulated in a very controlled manner toral position at Harvard University, USA, in the group of David Mooney
ex-vivo, and subsequently injected working on single-cell encapsulation in polymer microgels. His further
into the body.[9] research interests are supramolecular chemistry and stimuli-responsive
In order to recapitulate the in polymer gels. Recently, he joined the Siemens AG and works in the devel-
vivo milieu as closely as possible opment of polymeric insulation materials for electrical machines.
using droplet-based microfluidics,
Philipp Lienemann is a postdoctoral fellow in the group of Professor
selection of the appropriate input
David J. Mooney at Harvard University. He holds a B.Sc. in Biology
materials and settings regarding cell and an M.Sc. in Biotechnology from the Swiss Federal Institute of
types, cell numbers, biomolecules, Technology (ETH) in Zurich. In his dissertation at École Polytechnique
and ECM properties, is essential. The Fédérale de Lausanne (EPFL) and at University Hospital of Zurich he
design and usage of suitable artifi- used biomaterials to investigate and manipulate the fate of mes-
cial ECM poses a significant technical enchymal stem cells during bone regeneration. He received a Ph.D.
challenge, and great efforts have in Biotechnology and Bioengineering from EPFL in 2014. His current
been undertaken over the last years research interest lies in microfluidic technology for cell encapsulation
towards this goal. The creation of a to probe single cell fates in 3D.
suitable artificial ECM requires not David Mooney is the Pinkas Family Professor of Bioengineering at Harvard
only appropriate mechanical proper- University, and a Core Faculty Member of the Wyss Institute. His labora-
ties (e.g. elasticity), but potentially tory designs biomaterials to make cell and protein therapies effective
also degradation sites, cell adhesion and practical approaches to treat disease. He is a member of the National
sites, and biomolecule affinity sites. Academy of Engineering, and the National Academy of Medicine.
The development of crosslinking
mechanisms that allow for cell
friendly gelation after droplet forma-
tion is also crucial. Polymer-based of synthetic and natural polymers microgels. We begin by introducing
hydrogels offer themselves as a and mixtures thereof.[10] droplet-based microfluidics and dis-
promising type of material that can Herein, we discuss the combina- cussing polymers used as artificial
meet these requirements due to their tion of droplet-based microfluidics ECM in microgels. Next, we review
high degree of modifiability and and hydrogels for creation of artifi- work published after 2012 on cell-
ability to create these from a myriad 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 Figure 2.  Droplet-based microfluidic templating to fabricate uniform microgel particles
particle shell while the core remains that encapsulate cells. Two solutions of functional precursor polymers are injected into
liquid. Droplet microfluidics is based a microfluidic device, along with a suspension of cells. These three fluids meet at the
on the formation of emulsion drop- 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
lets of a cell suspension and func-
fourth fluid that could be an immiscible oil; this produces uniform premicrogel droplets.
tional polymer solution as templates By mixing of the three liquids inside the droplets the precursor polymers crosslink and
for particle synthesis; the drop- cell-laden microgels are formed. Adapted with permission.[21] Copyright 2012, American
lets are formed using micrometer- Chemical Society.
scale channels. Subsequently, the
functional polymers serve for droplet of the fluid streams and down- fluid (continuous phase), as shown
solidification by physical or chemical stream manipulation of the droplets. in Figure 2. In a T-junction micro-
crosslinking. Droplet microfluidics In addition, lithography facilitates fluidic device, the dispersed phase
uses either glass capillary devices[18] mass production of these devices; flows into the junction, where the
or devices made by lithography once a given type of photomask is continuous phase forms a thin film
techniques, commonly consisting of designed, a large number of devices between the dispersed phase and the
poly(dimethylsiloxane) (PDMS).[19] can be replicated.[11] Depending on walls of the device. This leads to an
Glass capillary microfluidic devices the device geometry, microemul- increase of the pressure upstream of
are composed of coaxial assemblies sification can occur via different the emerging droplet and squeezes
of glass capillary tubes with cylin- mechanisms. For cell microencap- the dispersed phase to form the
drical or square cross-sections that sulation the most common devices droplet.[20] In both types of devices,
are glued onto glass slides. While have a flow-focusing or a T-junction the size, shape, and monodisper-
these devices exhibit a robust resist- design. In flow-focusing devices, sity of the droplets is controlled by
ance to organic solvents, the attrac- a stream of functional polymer the dimensions of the microchan-
tive feature of PDMS devices for cell solution and a cell suspension (dis- nels, and the viscosities, polarities,
encapsulation is the ability to design persed phase) is created and its and flow rates of the fluids. The
and fabricate highly complex flow periodic break-up is induced by flow average number of cells per droplet
channels for upstream adjustment focusing with a second, immiscible can be either controlled by the

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concentration of the cells in the pre- extraction of microgels presents no significant change in the Young’s
cursor solution, or by the flow rate of an elegant approach to minimize moduli was observed following 7-days
the cell containing fluid if a device the time microgels are suspended of cell culture.
with two separate inlets for the in oil and allows for their gentle
inner phases is used. After droplet transfer to the aqueous phase
solidification by physical or chem- without the need for pipetting and 3. Cell-Laden Microgels
ical crosslinking, the pre-microgel centrifugation.[33,34]
droplets retain their size, shape, and Microgels can serve as model sys- Microgels containing cells can be
monodispersity, thereby yielding tems for 3D cell culture of single formed from both synthetic and
cell-laden microgel particles with cells. For this purpose, the number of natural precursor polymers. Biopoly-
precisely controlled morphology,[21] cells per particle needs to be exactly mers that have been used for the
as also illustrated in Figure 2. For controlled. This can be achieved by preparation of cell-laden microgels
long-term cell culture, the micro- controlling the concentration of cells using droplet microfluidic tem-
gels can be transferred from the oil in the cell suspension or by adjusting plating are alginate[41] and its copoly­
phase into aqueous culture medium, the size of the droplets, since micro- mers,[42] agarose,[43,44] collagen,[45]
wherein the microgels may swell fluidic encapsulation of cells is a gelatin,[46] hyaluronic acid,[47] and
to a certain degree depending on stochastic process following Poisson peptides.[48] Synthetic microgels
their composition. One limitation statistics with have been formed by crosslinkage
of such flow-focusing microfluidic of macromolecular precursors, often
approaches is the emulsification of λ ne − λ  (1) referred to as macromonomers, such
f ( λ ; n) = ,
precursor polymer solutions that can n! as poly(ethylene glycol),[49] poly­
crosslink on a very short time scale glycerol,[50] or copolymers of poly(N-
(e.g., a few seconds). In this case, the where f(λ;n) is the number of (2-hydroxypropyl)-methacrylamide)
thread of the droplet phase forms droplets that contain n cells in (PHPMA) and poly(hydroxyethyl
nodules and does not break-up into the droplet and λ is the average methacrylate) (PHEMA) grafted with
droplets;[13,22] finally, the microflu- number of cells per droplet. This poly(N-isopropylacrylamide) (PNI-
idic device can get clogged. To solve means for single cell encapsulation PAAm) side chains.[51] While the
these problems, the crosslinking in droplets with typical diameters polymerization of monomers is not
reactions have been slowed[23–25] of 50 to 100 µm, approximately 86 typically of relevance for the syn-
or sophisticated microfluidic tech- of 100 droplets are empty if only 1 thesis of cell-laden microgels due to
niques have been applied.[26–28] drop should contain more than one their cytotoxicity, the crosslinking of
To ensure high viability of the cell.[24,35,36] macromonomers affords the oppor-
encapsulated cells, the process of To characterize the mechanical tunity to exactly control the microgel
microemulsification itself should properties of microgels, atomic force composition and properties by the
be carefully considered, in addi- microscopy has become the state precursor concentration, molecular
tion to the polymeric precursor of the art method. Several variants weight, and functionalization, along
materials and crosslinking chemis- of this technique have been used with the chemistry of crosslinking.
tries. The non-polar liquid forming such as nanoindentation, which pro- An overview of commonly used syn-
the continuous phase as well as vides information about mechanical thetic and natural macromonomers
the surfactant need to be highly response on the nanoscale[37,38] or used to form cell-laden microgels is
biocompatible. In this regard, the colloidal probe method, which given in Scheme 1.
fluorocarbon oils and fluorosur- provides information on the micro- In the following sub-sections,
factants[29] have turned out to be scale.[36,39] Kumacheva, Walker and the features and properties of the
very useful[30,31] and are the subject coworkers presented an AFM method most frequently used synthetic and
of current research.[32] Moreover, to characterize the average Young’s natural precursor polymers will
the microgel work-up, the transfer modulus and the stress relaxation time be briefly discussed and the most
of the microgels into the aqueous of entire microgel particle using tipless recent approaches to form cell-
phase, need to be performed as cantilevers.[40] The method was applied laden microgels will be presented
gently as possible; high shear for the examination of the temporal in detail. Both covalently and physi-
forces created by centrifugation or variation in mechanical properties of cally crosslinked microgels will be
pipetting, as well as non-cytocom- cell-laden and cell-free agarose micro- reviewed. Table 1 summarizes the
patible chemicals that are used gels. The values of Young’s moduli for strategies for the functionalization
to break the emulsion, can harm both types of microgels decreased and crosslinking of the different
the encapsulated cells.[31] On-chip by around 30% on day 3; afterwards 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 often exhibit a fibrous structure facili- the encapsulation of cells, not all of
ranges from 100 to 8 000 kDa. Due to tating cell migration, cell-cell contact, such reactions have turned out to be
its excellent biocompatibility it is a and receptor-ligand clustering.[74] equally effective. Most widely used
frequently used biopolymer in tissue Further advantages of protein-based is the free-radical crosslinking of
engineering as well as in regen- hydrogels are their inherent bio- acrylates or methacrylates, because
erative medicine, and formulations logical properties, such as specific these functionalities can be easily
have been approved by the FDA.[47] amino acid sequences serving as cell introduced to hydroxy-functionalized
adhesion sites, degradation sites, or polymers via ester-bonding; by this
3.1.3. Proteins growth factor binding sites.[69] How- means, even bifunctional linear poly­
ever, due to their natural origin, big mers can be radically crosslinked
Proteins are widely used for the for- variations can exist between dif- to form polymer networks. Radical
mation of hydro- and microgels, ferent batches of protein-based poly- crosslinking has been used exten-
especially, fibrinogen, collagen and mers, and since they are not bioinert, sively for the synthesis of cell-laden
its irreversibly hydrolyzed form, gel- unintentional biological effects might microgels and can occur either by
atin.[69] Polymerization of fibrinogen be induced. thermally-generated radicals or by
is performed enzymatically by the photoinitiators and exposure to UV
actions of the protease thrombin and light. Haag, Seiffert, and colleagues
3.2. Covalent Microgels
the plasma transglutaminase factor employed the first method and pre-
XIII, resulting in a fibrin mesh resem- In contrast to several biopolymers pared yeast-cell-laden microgels
bling that found in blood clots.[70] Col- that can be used to form microgels composed of hPG and PEG.[60] How-
lagen and gelatin can be physically even without chemical modifica- ever, despite the biocompatibility and
polymerized by thermal gelation or tion, synthetic precursor polymers favorable elasticity of the hPG–PEG
covalently crosslinked by chemical usually need to be functionalized polymer matrix, the presence of free
or enzymatic strategies, resulting in a post-polymerization step to radicals during gel formation was
in mechanically and thermally sta- make them serviceable in a subse- detrimental for cell viability. Recently,
bilized gels.[71–73] As opposed to the quent crosslinking reaction. This Huck and coworkers applied photo-
typically nanoporous structure of crosslinking can occur by many crosslinking to produce fibroblast-
polyether- and polysaccharide-based different reaction types, but if the laden collagen–gelatin beads with
hydrogels, protein-based hydrogels microgels are anticipated to serve for tunable mechanical properties in the

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

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

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to initially characterize freshly gener- templating; however, it was unclear


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

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

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encapsulating eGFP-secreting insect of microgels went from proof of con- has been done for solid particles
cells; the protein expression profile cept studies to recapitulating, in a using close-packed ordering.[95]
of insect cells embedded in microgels high throughput manner, complex Strategies to prevent cell egress: For
was similar to that of cells in suspen- tissue architectures that address an artificial ECM to serve as a tissue
sion. Linear PEG precursor polymers significant problems. The impact of mimetic, it is often desirable for
functionalized with bipyridines on these systems can be expected to cells to be capable of remodeling the
both chain ends that can be gelled multiply in the near future. ECM and potentially migrate within
by complexation to iron(II) ions pro- it. However, due to cells following
vides another system for the fabrica- nutrient gradients from the cul-
4.2. Challenges
tion of physically crosslinked micro- ture medium or just due to random
gels.[27] By using PEG precursors of Over the last decade, great strides migration this might lead to cells
different molecular weights at dif- were made in combining droplet- escaping the microgel.[87] This often
ferent concentrations, the microgel based microfluidics with advanced may not be a limitation, and may
elasticity could be controlled, and biomaterials for 3D cell culture. How- be desirable in certain situations.
the viability of encapsulated lymph- ever, we believe that there are still For example, in studies assessing
oblast and breast cancer cells was technical challenges that currently biological processes on a time scale
optimized to exceed 90%. The micro- limit advances in the technology and of hours, this is most likely not an
gels were degradable by addition of its suitability for broad use. Three of issue. However, in certain situations
EDTA, a competitive ligand to bipy- the major technical challenges we one may want to avoid cell escape.
ridine, within 1.5 h under very mild see: Lutolf et al. proposed elegant tech-
conditions and with no apparent Strategies to evade the poisson nologies for preventing cell egress
detrimental effect on the released distribution: Current methods to by encapsulating microgels in larger
cells. However, this approach is not encapsulate cells into microgels non-degradable microgels either
suitable for long-term applications are rather inefficient. Typically, through reinjection into a microflu-
due to microgel auto-degradation in order to prevent cell aggrega- idic device or by placing microgels
within timespans of several hours. tion and to control the number of in non-degradable bulk gels.[87] Fur-
cells in a microgel, cells are highly ther strategies that would allow for
diluted and are then randomly dis- placing a dense shell around micro-
4. Summary, Challenges, and tributed into droplets based on a gels without the need for the techni-
Perspectives poisson distribution.[95] This results cally challenging reinjection into a
in numbers of empty microgels far microfluidic device, or the loss of the
4.1. Summary exceeding the numbers of cell-laden ability to manipulate with a pipette
microgels, especially if one aims for would be highly advantageous. Also,
Droplet-based microfluidics enables encapsulation of individual cells precisely positioning cells at the
the creation of picoliter-sized droplets using a highly diluted cell solution. center of the microgel may help to
that can serve as miniaturized vessels In this case a typical ratio would reduce cell egress by increasing the
for short-term (hours) culture and be approximately 6.6:1 in favor of distance to the edge of the microgel.
straightforward analysis of small cell empty microgels while ensuring that Strategies for monitoring indi-
numbers, down to individual cells. only every 100th microgel contains vidual microgels in long-term cul-
Through addition and crosslinking of two or more cells.[35] This makes ture: Droplet-based microfluidics
ECM mimicking synthetic or natural downstream processing and analysis can be used to create a myriad of
polymers to these vessels, fabrication a laborious venture. We therefore hetero­geneous microenvironments
of microgels becomes possible. Cells believe that simple technologies differing in ECM components, cell
encapsulated in microgels can be cul- that allow for controlled encapsula- types, or cell numbers.[86] To ascribe
tured for days, and the biochemical tion of an individual cell or defined changes that are observed during
and biophysical properties of the numbers of multiple cells in every culture periods to different starting
microenvironment can be adjusted microgel would be greatly benefi- compositions, it is crucial to be able to
to mimic the physiological environ- cial, in regards to throughput and follow individual microgels. We see
ment. This conjunction of hydrogels simplicity. This could for example great room for advancements in the
and droplet-based microfluidics for be achieved by selective gelation creation of microfluidic traps that
fabrication of microgels has opened only of microgels that contain cells, allow for immobilizing and culturing
up new possibilities in basic as well or by loading cells into droplets in a microgels in specified locations, as
as applied biological research. Over controlled manner by prearranging were developed for culturing single
the last years, fabrication and culture them in the microfluidic device like cells.[96,97] Ideally, such arrays would

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allow not only for monitoring cells microgels, such as optical tweezers, [7] D. Velasco, E. Tumarkin,
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A. R. Gascon, R. Calafiore,
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from the German Research Foundation J. R. Anderson, D. T. Chiu, H. Wu,
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stiffness and composition support
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