Cytocompatible click-based hydrogels with dynamically tunable properties through orthogonal photoconjugation and photocleavage reactions

Article abstract of DOI:10.1038/nchem.1174

To provide insight into how cells receive information from their external surroundings, synthetic hydrogels have emerged as systems for assaying cell function in well-defined microenvironments where single cues can be introduced and subsequent effects individually elucidated. However, as answers to more complex biological questions continue to be sought, advanced material systems are needed that allow dynamic alteration of the three-dimensional cellular environment with orthogonal reactions that enable multiple levels of control of biochemical and biomechanical signals. Here, we seek to synthesize one such three-dimensional culture system using cytocompatible and wavelength-specific photochemical reactions to create hydrogels that allow orthogonal and dynamic control of material properties through independent spatiotemporally regulated photocleavage of crosslinks and photoconjugation of pendant functionalities. The results demonstrate the versatile nature of the chemistry to create programmable niches to study and direct cell function by modifying the local hydrogel environment.

Full text of DOI:10.1038/nchem.1174

ARTICLES
PUBLISHED ONLINE: 23 OCTOBER 2011 | DOI: 10.1038/NCHEM.1174
Cytocompatible click-based hydrogels with
dynamically tunable properties through orthogonal
photoconjugation and photocleavage reactions
1
1,2
Cole A. DeForest and Kristi S. Anseth
*
To provide insight into how cells receive information from their external surroundings, synthetic hydrogels have emerged
as systems for assaying cell function in well-defined microenvironments where single cues can be introduced and
subsequent effects individually elucidated. However, as answers to more complex biological questions continue to be
sought, advanced material systems are needed that allow dynamic alteration of the three-dimensional cellular environment
with orthogonal reactions that enable multiple levels of control of biochemical and biomechanical signals. Here, we seek to
synthesize one such three-dimensional culture system using cytocompatible and wavelength-specific photochemical
reactions to create hydrogels that allow orthogonal and dynamic control of material properties through independent
spatiotemporally regulated photocleavage of crosslinks and photoconjugation of pendant functionalities. The results
demonstrate the versatile nature of the chemistry to create programmable niches to study and direct cell function by
modifying the local hydrogel environment.
1
ince its conception by Sharpless in 2001 , the concept of click masked light is projected directly onto a sample, enable photoreac-
chemistry has been rapidly adopted by many disciplines, tions to be confined to specific regions within a sample as defined by
S
perhaps most notably in materials science, with annual publi- a two-dimensional mask pattern, while focused laser light (either
cation numbers continuing to increase exponentially2–5. The click single- or multiphoton) provides full three-dimensional control
philosophy idealizes reactions that enable researchers to covalently over where a specific reaction occurs within the volume of a
link two reactants in a straightforward, modular and high-yielding material. Although the effects of attenuation and scattering must
manner. The copper(I)-catalysed azide-alkyne cycloaddition be carefully taken into consideration, light-based chemistries have
(CuAAC) is frequently billed as the quintessential example in become a powerful tool for materials synthesis and spatial modifi-
meeting these criteria. Although each click reaction has a number cation, owing to their ease of implementation and the fact that inex-
of desirable properties, their true benefit lies in the orthogonality pensive light sources are readily available, and have become
of these reactions with respect to many common reactive groups indispensible in the formation and subsequent modification
6
(
for example, amines, alcohols and acids) . Reaction orthogonality of biomaterials.
enables independent control over multiple functional groups in a
So far, biocompatible light-based chemistries have enabled
single system, opening the door to the synthesis of materials with control in space and time of either the gel degradation or gel chem-
ever-increasing complexity for an ever-expanding list istry of synthetic cell culture systems. By introducing chemical func-
of applications.
tionalities in user-defined patterns within the material, cell
There is also a growing interest in chemical reactions that can be spreading and migration have been explicitly controlled in three
6
8–13
performed in the presence of live cells . These reactions must dimensions . Also, cell outgrowth and stem cell fate have been
1
4–18
proceed mildly in an aqueous medium and defined chemical directed by altering the structural properties of the gel
.
environment (5% CO and atmospheric O levels), under regulated Independent control over both the physical and chemical make-
2
2
pH (7.4), temperature (37 8C) and osmolarity (ꢀ300 mOs M), and up of the material in three dimensions, as well as in time, would
involve non-toxic reactive moieties and (by)products. The require- allow dynamic tailoring of the microenvironment of a cell, but as
ment that reactions proceed under physiological conditions is a yet this has not been demonstrated. Such four-dimensional
stringent constraint and presents severe limitations on reaction control of material properties would be tremendously advantageous
selection. In particular, there are few chemistries that proceed in a in a number of biomaterial applications, including three-dimen-
specific manner while limiting side reactions with the plethora of sional cell culture, stem cell expansion, cancer metastasis and
functional groups that are found in biological systems. These elite tissue regeneration. Of further importance and novelty, the ability
reactions are considered bio-orthogonal and are necessary for for the experimenter to control gel properties at any point in
chemically probing and directing biological function.
space and time enables opportunities for unique experiments,
In many instances, an ideal cytocompatible reaction would not such as the ability to dynamically introduce a cell ligand or allow
only be selected by its bio-orthogonality, but also by its capacity cell–cell interactions at specified locations. These programmable
to be controlled in both time and space. Accordingly, photochemi- cell culture niches would facilitate the ability to perform newfound
cal reactions are widely regarded for their spatiotemporal control, experiments and answer questions about the dynamic exchange of
where the reaction of interest is defined by when and where light information between a cell and its niche. In this Article, we
7
is delivered to the system . Photolithographic techniques, where present one such system where multiple wavelengths of light are
1
2
Department of Chemical and Biological Engineering, University of Colorado, UCB Box 424, Boulder, Colorado 80309-0424, USA, Howard Hughes Medical
Institute, University of Colorado, UCB Box 424, Boulder, Colorado 80309-0424, USA. *e-mail: kristi.anseth@colorado.edu
NATURE CHEMISTRY | VOL 3 | DECEMBER 2011 | www.nature.com/naturechemistry
925
©
2011 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1174
ARTICLES
a
O
Photoreactive group
for patterning (visible light)
F
F
H2N
HN
NH
F
F
HN
O
O
O
O
Photodegradable
backbone (UV light)
HN
NH
Poly(ethylene glycol)
TetraDIFO3
O
H
H
N
M ª10,000
O
O
O
n
N3
N
HN
N
NH
O
n
n
H
H
O
O
N
O
NH2
O
O
O
=
O
N3
H2N
NH
NH
O
O
HN
NH
n
n
HN
O
O
NO2
F
F
F
O
F
c
b
Gel formation—SPAAC
Gel chemical patterning—thiol-ene
d
Gel mechanical patterning—photodegradation
O
R2
F
N
+
N
HS
O
R1
+
R1
+
R2
–
O
N
R1
F
O
NO2
Aqueous, 37 °C
Visible light (single or multiphoton)
UV light (single or multiphoton)
R2
F
O
O
S
O
R1
O
+
R1
F
R2
HO
NO
R1
N
N
N
e
g
f
Idealized 3D gel network
Chemically patterned gel
Photodegraded gel
Figure 1 | Synthesis, photocoupling and photodegradation for tuning chemical and physical properties of click-based hydrogels. a,b, Click-functionalized
macromolecular precursors (PEG-tetraDIFO3 and bis(azide)-functionalized polypeptides) form a three-dimensional ideal hydrogel structure (a) by means of a
step-growth polymerization mechanism via the SPAAC reaction (b). c, In the presence of visible light (l ¼ 490–650 nm or 860 nm), thiol-containing
biomolecules are covalently affixed to pendant vinyl functionalities throughout the hydrogel network via the thiol-ene reaction. d, A nitrobenzyl ether moiety
within the backbone of the polymer network undergoes photocleavage in the presence of single or multiphoton ultraviolet light (l ¼ 365 nm or 740 nm) that
results in photodegradation of the network. e–g, Schematics of the formed SPAAC-based idealized gel (e), the network after thiol-ene functionalization (f)
and the material after photodegradation (g).
used to independently control the functionality and architecture of a strain-promoted azide-alkyne cycloaddition (SPAAC) reaction
hydrogel network formed by means of a copper-free alkyne-azide between terminal difluorinated cyclooctyne (DIFO3) and azide
reaction (Fig. 1). Each of the reactions is cytocompatible, and (2N ) moieties with 1:1 stoichiometry at 10 wt% total macromer
3
both photoconjugation and photocleavage reactions are used to concentration to form an idealized three-dimensional network
spatiotemporally regulate material properties, including the presen- with minimal local defects. The ring strain and electronegative flu-
tation of integrin-binding motifs and network erosion through clea- orine substituents of DIFO3 enable the SPAAC reaction to proceed
1
9
vage of crosslinking moieties. This platform allows gel parameters to rapidly, without a catalyst, and in the presence of cells . Network
be tuned in real time, and the results demonstrate how spatiotem- gelation occurs ꢀ2 min after mixing, as estimated by the crossover
′
′′
poral regulation of material properties can be used to direct the point of the rheological storage (G ) and loss (G ) moduli
function of embedded cells.
(Supplementary Fig. S1). By including a synthetic polypeptide in
the gel formulation, the precise chemical make-up of the material
is tailored readily by choice of the amino-acid sequence, allowing
Results and discussion
A four-arm poly(ethylene glycol) (PEG) tetracyclooctyne (M ≈ one to tailor the biofunctionality (for example, enzymatic degrad-
n
1
0,000 Da) was reacted with a bis(azide) di-functionalized polypep- ability, integrin-binding ligands, protein affinity binding sites) and
tide (Azide–RGK(alloc)GRK(PLazide)–NH ) via a copper-free, introduce bio-orthogonal reactive moieties (for example, vinyl
2
9
26
NATURE CHEMISTRY | VOL 3 | DECEMBER 2011 | www.nature.com/naturechemistry
2011 Macmillan Publishers Limited. All rights reserved.
©

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1174
ARTICLES
a
c
b
x–z
x–y
3D
1
0
0
.0
.5
.0
10µM
5
µM
2.5µM
1
0
.0
.5
y–z
0.0
0.5
1.0
1.5
2.0
0
1,000 2,000 3,000 4,000 5,000
Position ( m)
Exposure time (min)
µ
Figure 2 | Biochemical patterning within preformed click hydrogels using visible light. Upon swelling thiol-containing biomolecules into pre-formed gels,
pendant functionalities are affixed to the hydrogel backbone via the thiol-ene reaction on exposure to visible light (l ¼ 490–650 nm). a, The final patterned
concentration of a fluorescent RGD peptide (AF4 –AhxRGDSC–NH ) depends on the amount of photoinitiator present (2.5, 5 and 10 mM eosin Y), as well
88
2
22
as the exposure time to visible light (0–2 min, 10 mW cm ). Error bars represent the standard deviation of experiments performed in triplicate. b, Network
functionalization with pendant fluorescently labelled peptides is confined to user-defined regions within pre-formed gels using photolithography (0.5, 1 or
22
2
min exposure with increased patterning concentration for increased exposure time, 10 mW cm , 10 mM eosin Y). c, The photocoupling reaction is
controlled in three dimensions by rastering the focal point of multiphoton laser light (l ¼ 860 nm) over defined volumes within the gel, affording
micrometre-scale resolution in all spatial dimensions. The patterning process can be repeated many times to introduce multiple biochemical cues within the
same network, as demonstrated by the red- and green-labelled patterned peptides within the same gel. Images represent confocal projections and
three-dimensional renderings. Scale bars, 400 mm (b) and 100 mm (c).
groups, azides). Ultimately, the timescale and mechanism of and exposure time (Fig. 2a). Specifically, patterning concentrations
the SPAAC reaction permits high viability (.95%) during encapsu- between 0 and 1 mM were obtained with short light exposures of
lation of both established cell lines, as well as primary cell types only a few minutes. By irradiating through a photomask, patterning
8
(
Supplementary Fig. S11) .
was confined to specific locations throughout the gel, as demon-
strated by the transfer of a 400-mm-wide line pattern through the
Biochemical control via thiol-ene photoconjugation. Incorporated depth of the sample (Fig. 2b, Supplementary Fig. S4).
into the synthetic peptide is the commercially available Multiphoton initiation techniques (l ¼ 860 nm) were also used to
lysine(allyloxycarbonyl) (alloc) amino acid, in which the alloc create elaborate, user-defined, three-dimensional biochemical pat-
protecting group is stable to standard solid-phase peptide terns within the hydrogel (Fig. 2c). Here, a 300 × 400 × 400 mm
synthesis methods and contains a vinyl functionality that is interconnected three-dimensional structure composed of multiple
readily photocoupled to thiol-containing compounds, such as shapes and two distinct peptides was created. The resolution that
cysteine, via the thiol-ene reaction. Although a number of we achieve with multiphoton-based patterning is ꢀ1 mm in the
reactions can be controlled with light, including the CuAAC by a x–y plane and ꢀ3–5 mm in the z plane, values that are typical of
20
photogenerated copper(I) catalyst , the radical-mediated thiol-ene multiphoton imaging methods and represent a limitation of
addition has emerged as a versatile click reaction that can be the optics and not the chemistry. The photocoupling process can
photochemically initiated2 . This reaction, which involves the be repeated many times over, with each cycle requiring a timescale
catalytic propagation of a thiyl radical across an olefin (2C¼C), of a few hours for introduction and removal of the signal via
and subsequent chain transfer from the resulting carbon radical to diffusion (depending on the gel dimensions). Thus, multiple
a thiol (2SH), has gained recent interest as an approach to signals can be incorporated with micrometre-scale patterning
1,22
8
,23,24
functionalize systems with biomolecules
, control dendrimer resolution on time and size scales that are relevant for many cell
formation , as well as synthesize other complex materials26,27. culture experiments.
25
The propagating thiyl radical is readily generated in the presence
of both cleavage type, as well as hydrogen-abstracting Biophysical control via photodegradation. The utility of a peptide
2
8
photoinitiators , enabling thiol-containing molecules to be linker enables desired sequences, as well as desired functionalities, to
physically linked to vinyl-functionalized moieties over a variety of be precisely incorporated in a modular fashion. In addition to the
light conditions including in the visible range. This reaction can pendant alloc vinyl functionality, the peptide includes
a
be used with peptides, thiolated full proteins, and small molecules photodegradable nitrobenzyl ether moiety (PLazide) within its
that are individually capable of diffusing throughout the hydrogel backbone, enabling photocleavage of the crosslinks upon exposure
(
Supplementary Figs S2, S3), although peptides that contain free to ultraviolet light (either l ¼ 365 nm for single-photon or l ¼
14,29
thiols in their bioactive domain may exhibit reduced biological 740 nm for multiphoton ). Specifically, the irreversible
effect upon thiol-ene coupling. The reaction is regarded as photocleavage of an o-nitrobenzyl ether moiety into nitroso- and
cytocompatible (Supplementary Fig. S11), as well as bio- acid-terminated by-products permits a previously intact chemical
orthogonal, facilitating its use in biological systems , and enables linkage to be cleaved photolytically. The photolabile group
materials to be functionalized dynamically with specific molecules degrades under cytocompatible irradiation conditions, including
21
2
9
30
of interest at any given location and time.
365 nm light , and has been used for the uncaging of proteins ,
3
1
After gel formation, fluorescently labelled thiol-containing bio- to cleave peptides from a solid support , as well as to control cell
9
,32
molecules were swollen into the network together with a small adhesion . Functionality has also been incorporated into
amount of eosin Y photoinitiator (2.5–10 mM), which was followed materials to produce networks that are capable of degrading in
1
4,33,34
by visible light irradiation (l ¼ 490–650 nm) at low intensities the presence of light
, allowing the effects of physical
10 mW cm ) and short durations (0.5–2 min). The extent of material cues on cell function to be probed35–37.
photocoupling was visualized and quantified using confocal Based on kinetic nuclear magnetic resonance (NMR) as well as
microscopy and was controlled by photoinitiator concentration photorheometry studies, the photoscission of the PLazide moiety
2
2
(
NATURE CHEMISTRY | VOL 3 | DECEMBER 2011 | www.nature.com/naturechemistry
2011 Macmillan Publishers Limited. All rights reserved.
927
©

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1174
ARTICLES
a
800
c
b
0mWcm–2
0mWcm–2
mWcm–2
x–z
3D
2
1
5
6
4
2
00
00
00
0
6
4
2
00
00
00
0
x–y
y–z
0
10
20
30
40
50
0
1,000 2,000 3,000 4,000 5,000
Position ( m)
Exposure time (min)
µ
Figure 3 | Biophysical patterning within pre-formed click hydrogels using ultraviolet light. In the presence of ultraviolet light (l ¼ 365 nm), photolabile
functionalities within the hydrogel crosslinks undergo an irreversible cleavage, thereby decreasing total network connectivity, resulting in local material
degradation and removal of the fluorescent hydrogel material. a, In optically thick samples, the depth of photodegradation is directly related to the incident
22
light intensity (5, 10 and 20 mW cm ), as well as the exposure time to visible light (0–45 min). Error bars represent the standard deviation of experiments
performed in triplicate. b, Using mask-based photolithographic techniques, network degradation was confined to user-defined regions within fluorescently
labelled gels (10, 15, 20, 30 and 45 min, feature height increasing with exposure time), as measured by profilometry. c, The photodegradation reaction is
controlled in three dimensions with micrometre-scale resolution in all dimensions using focused multiphoton laser light (l ¼ 740 nm). Images represent
confocal projections and three-dimensional renderings. Scale bars, 400 mm (b) and 100 mm (c).
(Supplementary Fig. S5) was found to follow a first-order degra- conditions using model compounds (Fig. 4b). To illustrate the
dation with a rate constant k that can be expressed as
orthogonality of the reactions within the same material system, a
buffalo logo was first photocoupled within the three-dimensional
network using visible light, and user-defined letters (CU) were
eroded within the fluorescent logo at a later time (Fig. 4c).
w1I
k =
N hn
A
where w is the quantum yield (determined to be 0.020), 1 is the Photodegradation was confirmed to be confined only to the areas
21
21
molar absorptivity of the sample (4,780 M cm for PLazide at of interest by bright-field microscopy (Fig. 4d), as well as with the
l ¼ 365 nm), I is the intensity of light, N is Avogadro’s number, disappearance of the fluorescently labelled reporter peptide,
A
h is Planck’s constant, and n is the frequency of the associated elec- indicating that cues can be spatially coupled and subsequently
tromagnetic wave (Supplementary Figs S6, S7, S8). For typical removed with orthogonal reactions.
2
2
exposure conditions (l ¼ 365 nm, 10 mW cm ), k was determined
To provide a demonstration of the potential utility of these two
2
3 21
to be 2.9 × 10
s
, and correlates well with other photodegrad- photoreactions for advanced three-dimensional cell culture, Fig. 5
1
4,31
able moieties . Physical channels were eroded downward from presents an approach where one might assay the specific effects of
the surface of optically thick samples, with the depth of photodegra- a variety of biomolecular cues on cell function within an otherwise
dation directly related to the total light intensity (5, 10 and uniform gel culture platform. Here, human mesenchymal stem cells
22
2
0 mW cm ), as well as the exposure time of ultraviolet light (hMSCs) were encapsulated in gels synthesized by means of SPAAC
(
0–45 min) (Fig. 3a, Supplementary Fig. S9) and the total light chemistry, and their cellular microenvironment was patterned using
dosage delivered to the material (Supplementary Fig. S10). As the thiol-ene photocoupling reaction with perpendicular lines
with the photocoupling reaction, the photocleavage reaction was (width ¼ 200 mm) of integrin-binding peptide ligands, RGD and
confined to regions of interest within the sample using photolitho- PHSRN (ꢀ1 mM each). These peptide sequences are both derived
graphic processes to create channels of varying depths (ꢀ150–600 mm) from sequences found in fibronectin and are known to elicit some
3
8
(
Fig. 3b), as well as multiphoton patterning approaches to erode degree of synergy on cell adhesion . The patterning created a
precisely defined three-dimensional regions of interest with repeating array of four distinct biochemical culture conditions (no
user-defined shapes and connectivity (Fig. 3c, l ¼ 740 nm). Each cue, RGD alone, PHSRN alone, or both RGD and PHSRN)
process affords a high level of patterning fidelity, similar to that within the same gel (Fig. 5a). At a later time point, the photodegra-
obtained by the photocoupling reaction.
dation reaction was exploited to capture cells in spatially defined
regions of interest by exposing the gel to a given condition of
Orthogonal photoreactions for advanced three-dimensional cell light to induce erosion and liberate cells from their three-dimen-
culture. The utility of the photocoupling and photocleavage sional culture environment (Fig. 5b). This process can be repeated
reactions ultimately stems from their ability to be performed many times at different time points and locations to collect cells
orthogonally, such that both network mechanical and chemical that have been exposed to either the same or different biochemical
make-up are controlled independently. The peak values of conditions, demonstrating full spatiotemporal control over cell sub-
absorbance for the photolabile group and the visible population sampling (Fig. 5c). The released cells are readily col-
photoinitiator were found to be ꢀ350 and ꢀ520 nm, respectively, lected, subsequently plated, expanded and made available for
with relatively little overlap of the absorbance spectra (Fig. 4a). As additional biological assays, including those that may be more diffi-
eosin Y also has a low absorbance at l ¼ 365 nm, photocoupling cult to perform on encapsulated cells. hMSCs remained viable
during photodegradation was readily prevented by performing (.95%) throughout the entire process (Supplementary Fig. S11).
degradation only in the absence of a photoinitiator. Alternatively, We plated the released hMSCs and visualized their cytoskeletal
photocoupling was initiated first with visible light (l ¼ 490–650 nm) organization using a fluorescent phalloidin, which stains for
and photodegradation was commenced with subsequent F-actin (Fig. 5d).
ultraviolet irradiation. The orthogonality of these reactions was
To further demonstrate how these reactions can be used to
confirmed by solution NMR studies where photocleavage was manipulate cellular functions in a spatiotemporally regulated
quantified under both visible and ultraviolet light initiation manner, a cell-laden (3T3 fibroblasts) fibrin clot was encapsulated
9
28
NATURE CHEMISTRY | VOL 3 | DECEMBER 2011 | www.nature.com/naturechemistry
2011 Macmillan Publishers Limited. All rights reserved.
©

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1174
ARTICLES
a
10,000
80,000
b
1
0
0
0
.0
.8
.6
.4
8
6
4
2
,000
,000
,000
,000
0
6
4
2
0
0,000
0,000
0,000
490–650nm
365nm
0.2
0.0
300
400
500
600
0
30
60
Exposure time (min)
90
120
Wavelength (nm)
c
d
Figure 4 | Orthogonality of photocoupling and photodegradation reactions. a, Peak absorbances for the photoinitiator (red) and the photolabile group (blue)
are well separated (ꢀ520 and ꢀ350 nm, respectively), thus enabling photocoupling and photodegradation reactions to be performed independently from
one another using different light sources (illustrated with coloured bars). b, NMR studies indicate that the photodegradable moiety cleaves readily in the
22
presence of ultraviolet light (l ¼ 365 nm, 10 mW cm ), but remains intact when exposed to the visible light used to initiate the photocoupling reaction
2
2
(
l ¼ 490–650 nm, 10 mW cm ). Error bars represent the standard deviation of experiments performed in triplicate. c, Multiphoton visible light was first
used to couple a fluorescently labelled peptide within the centre of the hydrogel in a user-defined three-dimensional pattern (top, buffalo), and the network
was subsequently degraded locally with multiphoton ultraviolet light, thereby removing the peptide from selected regions (bottom, CU and horn). d, Bright-
field microscopy confirms that photocleavage is confined only to user-defined locations within the gel, and that the photocoupling light conditions do not give
rise to undesired degradation. Images in c represent three-dimensional renderings of confocal z-stacks. Scale bars, 100 mm.
a
b
c
d
Figure 5 | Culture and recovery of hMSCs from hydrogel microenvironments. CellTracker Orange-labelled hMSCs were encapsulated within the click
6
hydrogel formulation at 5 × 10 cells/ml. a, At 24 h post-encapsulation, perpendicular 200-mm-wide lines of ꢀ1 mM RGD and PHSRN were patterned
throughout the hydrogel via thiol-ene photocoupling to create an array of four distinct biochemical conditions (no cue, RGD, PHSRN, RGD and PHSRN).
b, Four hours later, channels of user-defined shape (cylindrical) were eroded down from the surface of the hydrogel to capture entrapped cells exposed to a
specific cue. c, This process was repeated 1 h later to release entrapped cells within a different location of the material and a different shape (star-shaped
cylinder). d, The released cells were isolated by centrifugation, cultured in a 96-well plate for 48 h, and their cytoskeletons visualized with fluorescent
phalloidin. In a–c, RGD is shown in green, PHSRN in red and hMSCs in orange. Images represent single confocal slices within the three-dimensional gel.
In d, F-actin is shown in green and nuclei in blue. Image represents inverted fluorescence micrograph. Scale bars, 200 mm (a–c), 50 mm (d).
within the click hydrogel formulation. After 2 h, physical channels photon patterning techniques, cell outgrowth was explicitly directed
were eroded radially from the spherical clot by multiphoton photo- in all three spatial dimensions (Fig. 6b). This directed outgrowth can
degradation of the network to direct collective cell migration. Only be performed in the presence of other encapsulated cells or with
specific regions of the gel were functionalized with RGD via the combinations of cell types. For example, hMSCs were encapsulated
thiol-ene photocoupling reaction. Cells were found to leave the in the gel surrounding the 3T3-fibroblast-laden clot, and the fibro-
clot and migrate into the patterned hydrogel channels, but only blasts were directed into the surrounding hMSC microenvironment
when eroded migration channels were present and their surfaces in a manner controlled by changes in the local gel environment. The
decorated with the RGD adhesive ligand (Fig. 6a). Using two- patterned 3T3 fibroblasts were found to create complex structures in
NATURE CHEMISTRY | VOL 3 | DECEMBER 2011 | www.nature.com/naturechemistry
2011 Macmillan Publishers Limited. All rights reserved.
929
©

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1174
ARTICLES
a
b
c
Figure 6 | Directed three-dimensional cell motility within patterned hydrogels. a, A fibrin clot containing 3T3 fibroblasts was encapsulated within the click
hydrogel formulation. Chemical channels of RGD, a cell-adhesive fibronectin motif, as well as physical channels of user-defined shape were created radially
out of the roughly spherical clot. The combination of having physical space to spread into as well as chemical moieties to bind to was required for collective
cell migration. By day 10, cells were found to migrate only down the physical channel functionalized with RGD. b, By creating three-dimensional functionalized
channels, cell outgrowth was controlled in all three spatial dimensions, with the image inset illustrating a top-down projection. c, Outgrowth of 3T3 fibroblast
cells was controlled in the presence of encapsulated hMSCs and confined to branched photodegraded channels functionalized with RGD. The regions of RGD
functionalization are depicted by the dashed polygons in a and c. Hydrogel is shown in red, F-actin in green and cell nuclei in blue. Scale bars, 100 mm.
the presence of encapsulated hMSCs (Fig. 6c). These photoreactions DE Voyage) using a-cyano-4-hydroxycinnamic acid matrix (Sigma): calculated
þ
þ
(
[M þ H] 1,288.4); observed ([M þ H] 1,288.1) (Supplementary Fig. S14).
For the synthesis of fluorescently labelled adhesive ligand, H–AhxRGDSC–NH2
0.5 mmol) was synthesized and modified with Alexa Fluor 488 carboxylic acid,
are included to demonstrate how one might engineer complex,
multicellular structures, ultimately expanding the potential for
engineering tissue constructs with spatially varying cellularity in
advanced bioreactors or culture systems.
As presented, this work makes use of two novel photoreaction
schemes to combine and exploit features of previously mutually
exclusive technologies. Namely, the physical and chemical proper-
(
2
,3,5,6-tetrafluorophenyl ester (2 mg, Invitrogen) in DMF overnight at room
temperature. The peptide was cleaved from resin, precipitated and lyophilized to give
a yellow solid (denoted AF488–AhxRGDSC–NH ). AF –AhxPHSRNC–NH was
2
633
2
synthesized in a similar manner.
Gel formation. Hydrogels were created by mixing 10 wt% total macromer
ties of the network can be controlled independently with orthogonal photodegradable, photocouplable click gel formulation in media between azide-
functionalized (Supplementary Fig. S15) and Rain-X-treated glass slides spaced
at a known distance (typically 500 mm), and reacted for 30 min at 37 8C.
The slides were separated, and the gel remained covalently attached to the
azide-functionalized slide.
light-based chemistries, allowing for real-time manipulation of cell
function within a simplified synthetic microenvironment. These
reactions are performed dynamically with full spatiotemporal
control, enabling full user direction over these programmable cell
niches. The cytocompatibility of the reaction processes should
enable newfound opportunities for experiments to test basic
hypotheses about critical events regulating cell–materials inter-
Biochemical patterning. Hydrogels were swollen in phenol red-free media
2
1
containing 3 mg ml patterning agent AF488–AhxRGDSC–NH and eosin Y
2
(10 mM) for 1 h. For photolithographic-based experiments, gels were exposed to
collimated visible light (l ¼ 490–650 nm), achieved with an Acticure (EXFO) high-
actions at multiple time and size scales and, with this knowledge, pressure mercury lamp equipped with an internal band-pass filter (350–650 nm)
improve strategies for stem cell culture, biomaterial design, three- and an external 490 nm long-pass filter (Edmund Optics), through a patterned
chrome photomask. Alternatively, three-dimensional patterning was obtained using
dimensional cell culture assays and tissue regeneration.
two-photon techniques where subvolumes within the hydrogel were selectively
22
exposed to pulsed laser light (l ¼ 860 nm, power ¼ 350 mW mm , scan speed ¼
Methods
2
2
1
.27 ms mm ) at 1 mm z-plane increments on a 710 LSM NLO confocal microscope
Synthesis of click-functionalized macromolecular precursors. For the synthesis of
PEG-tetraDIFO3, DIFO31
9,39
stage (Carl Zeiss) equipped with a ×20/0.8 Plan-Apochromat objective (NA ¼ 1.0).
Unreacted patterning agent and initiator were swollen into fresh media as the sample
was gently agitated on an orbital shaker (2 h), yielding the final patterned hydrogel.
In both the photolithographic and multiphoton patterning techniques,
photocoupling of the peptide was confined to volumes exposed to light within the
material and was visualized by fluorescent confocal microscopy.
(121 mg, 0.6 mmol; Supplementary Fig. S12) and
2-(1H-7-azabenzotriazol-1-yl)–1,1,3,3-tetramethyl uronium hexafluorophosphate
methanaminium (HATU, 225 mg, 0.6 mmol, Anaspec) were dissolved in minimal
dimethylformamide (DMF, 5 ml) with N,N-diisopropylethylamine (DIEA, 210 ml,
1.2 mmol) and reacted for 5 min at room temperature. This solution was then added
to four-arm PEG tetraamine (M ≈ 10,000 Da, 1 g, 0.4 mmol NH , JenKem) and
n
2
stirred overnight, concentrated, dissolved in dH O, dialysed (molecular weight
2
Biophysical patterning. Gels containing 0.125 mM Alexa Fluor 594 azide
cutoff, MWCO ≈ 2 kDa, SpectraPor), filtered and lyophilized to yield a white
(Invitrogen) were patterned using photolithographic techniques, where hydrogels
1
powder (1.03 g, 96%). Functionalization was confirmed to be .95% by H-NMR.
were exposed to collimated ultraviolet light (l ¼ 365 nm) from an Omnicure S1000
For the synthesis of bis(azide)-functionalized photodegradable peptide
crosslinker, the allyl-ester containing peptide H-RGK(alloc)GRK(dde)-NH was
(
(
EXFO) high-pressure mercury lamp equipped with an internal band-pass filter
365 nm). Three-dimensional patterning was obtained using two-photon techniques
2
synthesized (Protein Technologies Tribute peptide synthesizer) through Fmoc solid-
phase methodology and HATU activation. 4-Azidobutanoic acid (Supplementary
Fig. S13) was coupled to the N-terminal amine with HATU, the 1-(4,4-dimethyl-2,6-
dioxacyclohexylidene)ethyl (Dde) group was removed with 2% hydrazine
monohydrate (Sigma) in DMF (3 × 10 min), and 4-(4-(1-(4-
where regions of interest (x–y control) within the hydrogel were selectively exposed
22
to pulsed laser light (l ¼ 740 nm, power ¼ 670 mW mm , scan speed ¼
22
1
.27 ms mm ). Photodegraded monomer was swollen into fresh media, yielding the
final patterned hydrogel.
azidobutanoyloxy)ethyl)-2-methoxy-5-nitrophenoxy)butanoic acid (PLazide;
Supplementary Fig. S13) was coupled to the 1-amino group of the C-terminal lysine.
Resin was treated with trifluoroacetic acid/triisopropylsilane/water (95:2.5:2.5) for
Cell culture. NIH 3T3s (mouse) were cultured in high-glucose Dulbecco’s modified
Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (Invitrogen),
1% penicillin/streptomycin (Gibco), 0.2% fungizone and 0.4% gentamicin. hMSCs
2
h and precipitated in and washed (2×) with ice-cold diethyl ether. The crude
were cultured in low-glucose DMEM with 10% fetal bovine serum, 1%
penicillin/streptomycin, 0.2% fungizone and 0.4% gentamicin. All cells were
peptide was purified using semi-preparative reversed-phase high-performance liquid
chromatography (RP-HPLC) (Waters Delta Prep 4000) using a 70 min linear
gradient (5–95% of acetonitrile and 0.1% trifluoroacetic acid) and lyophilized to give
maintained in 5% CO at 37 8C. Cells were used between passages P4 and P6.
2
6
the product (Azide–RGK(alloc)GRK(PLazide)–NH ) as a fluffy, yellow solid.
Fibrin clot encapsulation. Cells were suspended at 10 × 10 cells/ml in a fibrinogen
2
2
solution (10 mg ml in PBS, Sigma) containing thrombin (5 U ml , Sigma) and
1
21
Peptide purity was confirmed with analytical RP-HPLC and matrix-assisted laser
desorption–ionization time-of-flight mass spectrometry (Applied Biosystems
reacted for 30 min at 37 8C. The formed cell-laden clots were suspended in a 10 wt%
9
30
NATURE CHEMISTRY | VOL 3 | DECEMBER 2011 | www.nature.com/naturechemistry
©
2011 Macmillan Publishers Limited. All rights reserved.

NATURE CHEMISTRY DOI: 10.1038/NCHEM.1174
ARTICLES
total macromer photodegradable, photocouplable click gel formulation sandwiched
between azide-functionalized (Supplementary Fig. S10) and Rain-X-treated
glass slides spaced at 1 mm, and reacted for an additional 30 min at 37 8C.
The slides were separated, and the gel remained covalently attached to the
azide-functionalized slide. After 2 h in media, physical channels were patterned
into the network with two-photon patterning (l ¼ 740 nm). The media was then
20. Adzima, B. J. et al. Spatial and temporal control of the alkyne-azide cycloaddition
by photoinitiated Cu(II) reduction. Nature Chem. 3, 258–261 (2011).
21. Hoyle, C. E. & Bowman, C. N. Thiol-ene click chemistry. Angew. Chem. Int. Ed.
49, 1540–1573 (2010).
22. Dondoni, A. The emergence of thiol-ene coupling as a click process for materials
and bioorganic chemistry. Angew. Chem. Int. Ed. 47, 8995–8997 (2008).
23. Polizzotti, B. D., Fairbanks, B. D. & Anseth, K. S. Three-dimensional
biochemical patterning of click-based composite hydrogels via thiolene
photopolymerization. Biomacromolecules 9, 1084–1087 (2008).
24. DeForest, C. A., Sims, E. A. & Anseth, K. S. Peptide-functionalized click
hydrogels with independently tunable mechanics and chemical functionality for
3D cell culture. Chem. Mater. 22, 4783–4790 (2010).
21
supplemented with Ac–RGDSC–NH (3 mg ml ) and eosin Y (10 mM),
2
equilibrated for 1 h, and selected regions within the gel were biochemically
decorated with RGD (l ¼ 860 nm). On day 10, the hydrogels were fixed in
formalin for 1.5 h, followed by cell permeabilization with 0.5% Triton X-100 (Fisher)
in PBS for 2 h. The samples were blocked with 3% bovine serum albumin (BSA,
Sigma) in PBS for 1 h and rinsed with PBS. F-actin was visualized using Alexa Fluor
21
4
88 Phalloidin Conjugate (5 U ml , Invitrogen), and nuclei were stained with
25. Killops, K. L., Campos, L. M. & Hawker, C. J. Robust, efficient, and orthogonal
synthesis of dendrimers via thiol-ene ‘click’ chemistry. J. Am. Chem. Soc. 130,
5062–5064 (2008).
DAPI (500 nM, Invitrogen), each for 2 h. The samples were washed with PBS before
confocal visualization.
2
2
2
2
6. Fairbanks, B. D. et al. A versatile synthetic extracellular matrix mimic via thiol-
norbornene photopolymerization. Adv. Mater. 21, 5005–5010 (2009).
7. Gupta, N. et al. A versatile approach to high-throughput microarrays using
thiol-ene chemistry. Nature Chem. 2, 138–145 (2010).
8. Uygun, M., Tasdelen, M. A. & Yagci, Y. Influence of type of initiation on
thiol-ene ‘click’ chemistry. Macromol. Chem. Phys. 211, 103–110 (2010).
9. Alvarez, M. et al. Single-photon and two-photon induced photocleavage for
monolayers of an alkyltriethoxysilane with a photoprotected carboxylic ester.
Adv. Mater. 20, 4563–4567 (2008).
Received 21 June 2011; accepted 13 September 2011;
published online 23 October 2011
References
1.
Kolb, H. C., Finn, M. G. & Sharpless, K. B. Click chemistry: diverse chemical
function from a few good reactions. Angew. Chem. Int. Ed. 40,
2004–2021 (2001).
2.
3.
4.
5.
6.
7.
Kolb, H. C. & Sharpless, K. B. The growing impact of click chemistry on drug
discovery. Drug Discov. Today 8, 1128–1137 (2003).
Moses, J. E. & Moorhouse, A. D. The growing applications of click chemistry.
Chem. Soc. Rev. 36, 1249–1262 (2007).
Hawker, C. J. & Wooley, K. L. The convergence of synthetic organic and polymer
chemistries. Science 309, 1200–1205 (2005).
Barner-Kowollik, C. et al. ‘Clicking’ polymers or just efficient linking: what is the
difference? Angew. Chem. Int. Ed. 50, 60–62 (2011).
Sletten, E. M. & Bertozzi, C. R. Bioorthogonal chemistry: fishing for selectivity in
a sea of functionality. Angew. Chem. Int. Ed. 48, 6974–6998 (2009).
Bowman, C. N. & Kloxin, C. J. Toward an enhanced understanding and
implementation of photopolymerization reactions. AIChE J. 54,
3
3
0. Deiters, A. Principles and applications of the photochemical control of cellular
processes. ChemBioChem 11, 47–53 (2010).
1. Holmes, C. P. Model studies for new o-nitrobenzyl photolabile linkers:
substituent effects on the rates of photochemical cleavage. J. Org. Chem. 62,
2370–2380 (1997).
3
3
3
3
2. Ohmuro-Matsuyama, Y. & Tatsu, Y. Photocontrolled cell adhesion on a surface
functionalized with a caged arginine-glycine-aspartate peptide. Angew. Chem.
Int. Ed. 47, 7527–7529 (2008).
3. Johnson, J. A., Baskin, J. M., Bertozzi, C. R., Koberstein, J. T. & Turro, N. J.
Copper-free click chemistry for the in situ crosslinking of photodegradable star
polymers. Chem. Commun. 3064–3066 (2008).
4. Wong, D. Y., Griffin, D. R., Reed, J. & Kasko, A. M. Photodegradable hydrogels
to generate positive and negative features over multiple length scales.
Macromolecules 43, 2824–2831 (2010).
5. Kloxin, A. M., Tibbitt, M. W. & Anseth, K. S. Synthesis of photodegradable
hydrogels as dynamically tunable cell culture platforms. Nature Protoc. 5,
2775–2795 (2008).
8.
DeForest, C. A., Polizzotti, B. D. & Anseth, K. S. Sequential click reactions for
synthesizing and patterning three-dimensional cell microenvironments. Nature
Mater. 8, 659–664 (2009).
9
1
1
.
Luo, Y. & Shoichet, M. S. A photolabile hydrogel for guided three-dimensional
cell growth and migration. Nature Mater. 3, 249–253 (2004).
1867–1887 (2010).
36. Kloxin, A. M., Benton, J. A. & Anseth, K. S. In situ elasticity modulation with
dynamic substrates to direct cell phenotype. Biomaterials 31, 1–8 (2010).
37. Frey, M. T. & Wang, Y. L. A photo-modulatable material for probing cellular
responses to substrate rigidity. Soft Matter 5, 1918–1924 (2009).
3
0. Aizawa, Y., Wylie, R. & Shoichet, M. Endothelial cell guidance in 3D patterned
scaffolds. Adv. Mater. 22, 4831–4835 (2010).
1. Lee, S. H., Moon, J. J. & West, J. L. Three-dimensional micropatterning of
bioactive hydrogels via two-photon laser scanning photolithography for guided
8. Redick, S. D., Settles, D. L., Briscoe, G. & Erickson, H. P. Defining fibronectin’s
cell adhesion synergy site by site-directed mutagenesis. J. Cell Biol. 149,
3D cell migration. Biomaterials 29, 2962–2968 (2008).
1
1
1
2. Hoffmann, J. C. & West, J. L. Three-dimensional photolithographic patterning
of multiple bioactive ligands in poly(ethylene glycol) hydrogels. Soft Matter 6,
5
21–527 (2000).
9. Sims, E. A., DeForest, C. A. & Anseth, K. S. A mild, large-scale synthesis of
,3-cyclooctanedione: expanding access to difluorinated cyclooctyne for
copper-free click chemistry. Tetrahedron Lett. 52, 1871–1873 (2011).
3
5056–5063 (2010).
1
3. Seidlits, S. K., Schmidt, C. E. & Shear, J. B. High-resolution patterning of
hydrogels in three dimensions using direct-write photofabrication for cell
guidance. Adv. Funct. Mater. 19, 3543–3551 (2009).
4. Kloxin, A. M., Kasko, A. M., Salinas, C. N. & Anseth, K. S. Photodegradable
hydrogels for dynamic tuning of physical and chemical properties. Science 324,
Acknowledgements
The authors thank A. Kloxin and M. Tibbitt for useful discussions regarding
photopatterning, C-C. Lin for advice on cell outgrowth experiments, A. Aimetti and
P. Hume for communication on general experimental design, and C. Kloxin for insightful
feedback on the written manuscript. Fellowship assistance to C.A.D. was awarded by the US
Department of Education’s Graduate Assistantships in Areas of National Need. This work
was made possible by financial support from the National Science Foundation (DMR
59–63 (2009).
1
5. Khetan, S., Katz, J. S. & Burdick, J. A. Sequential crosslinking to control cellular
spreading in 3-dimensional hydrogels. Soft Matter 5, 1601–1606 (2009).
6. Khetan, S. & Burdick, J. A. Patterning network structure to spatially control
cellular remodeling and stem cell fate within 3-dimensional hydrogels.
Biomaterials 31, 8228–8234 (2010).
1
1006711) and the Howard Hughes Medical Institute.
1
7. Sarig-Nadir, O., Livnat, N., Zajdman, R., Shoham, S. & Seliktar, D. Laser
photoablation of guidance microchannels into hydrogels directs cell growth in
three dimensions. Biophys. J. 96, 4743–4752 (2009).
8. Ilina, O., Bakker, G. J., Vasaturo, A., Hofmann, R. M. & Friedl, P. Two-photon
laser-generated microtracks in 3D collagen lattices: principles of mmp-
dependent and -independent collective cancer cell invasion. Phys. Biol. 8,
Author contributions
C.A.D. and K.S.A. developed the material concept. C.A.D. and K.S.A. designed the
experiments. C.A.D. carried out the experiments. C.A.D. and K.S.A. wrote the manuscript.
1
Additional information
015010 (2011).
The authors declare no competing financial interests. Supplementary information
accompanies this paper at www.nature.com/naturechemistry. Reprints and permission
information is available online at http://www.nature.com/reprints. Correspondence and
requests for materials should be addressed to K.S.A.
1
9. Codelli, J. A., Baskin, J. M., Agard, N. J. & Berozzi, C. R. Second-generation
difluorinated cyclooctynes for copper-free click chemistry. J. Am. Chem. Soc.
1
30, 11486–11493 (2008).
NATURE CHEMISTRY | VOL 3 | DECEMBER 2011 | www.nature.com/naturechemistry
2011 Macmillan Publishers Limited. All rights reserved.
931
©