protocol

Synthesis and characterization of well-defined

hydrogel matrices and their application to intestinal
stem cell and organoid culture
Nikolce Gjorevski1 & Matthias P Lutolf1,2
1Laboratory ofStem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences and School of Engineering, École Polytechnique Fédérale de Lausanne
(EPFL), Lausanne, Switzerland. 2Institute of Chemical Sciences and Engineering, School of Basic Science, EPFL, Lausanne, Switzerland. Correspondence should be
addressed to M.P.L. (matthias.lutolf@epfl.ch).

Published online 5 October 2017; doi:10.1038/nprot.2017.095

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Growing cells within an extracellular matrix-like 3D gel is required for, or can improve, the growth of many cell types ex vivo. Here, we
describe a protocol for the generation of well-defined matrices for the culture of intestinal stem cells (ISCs) and intestinal organoids.
These matrices comprise a poly(ethylene glycol) (PEG) hydrogel backbone functionalized with minimal adhesion cues including
RGD (Arg-Gly-Asp), which is sufficient for ISC expansion, and laminin-111, which is required for organoid formation. As such, the
hydrogels present a defined and reproducible, but also tunable, environment, allowing researches to manipulate physical and chemical
parameters, and examine their influence on ISC and organoid growth. Hydrogels are formed by an enzymatic cross-linking reaction of
multiarm PEG precursors bearing glutamine- and lysine-containing peptides. PEG precursors containing either stable or hydrolytically
degradable moieties are used to produce mechanically softening hydrogels, which are used for the expansion of ISCs or the formation of
organoids, respectively. We also provide protocols for immunofluorescence analysis of cellular structures grown within these matrices,
as well as for their dissociation and retrieval of cells for downstream use. Hydrogel precursors can be produced and their mechanical
properties characterized to ascertain stiffness within 5–7 d. Hydrogel formation for ISC expansion or organoid formation takes 1–2 h.
The materials described here can be readily adapted for the culture of other types of normal or transformed organoid structures.

INTRODUCTION
Over the past decade, stem-cell-derived organoids have received
attention as promising models of development and disease, poten-
tial drug-screening platforms and a source of transplantable tis-
sue. These structures have been developed from adult, embryonic
or induced pluripotent stem cells, and organoid counterparts have
been developed for a wide range of organs1.
One common feature for virtually all organoid models
remains the use of animal-derived hydrogels such as Matrigel—a
basement-membrane-like gel secreted by Engelbreth-Holm-
Swarm mouse sarcoma cells—as the 3D scaffold necessary to
grow them. The presence of animal-derived matrices in orga-
noid cultures undermines their applicability in basic research,
drug development and cell-based therapies, owing to its com-
plex, poorly defined and variable composition2–4. Furthermore,
whereas the profound influence of the extracellular matrix (ECM)
on stem-cell fate and tissue morphogenesis has long been recog-
nized5, its role in organoid formation cannot be easily investi-
gated, as Matrigel is not conducive to physical and biochemical
manipulations. Finally, Matrigel is used as a universal organoid
culture matrix, overlooking the possibility that matrices tailored
to specific tissues and organs may push the structural and func-
tional sophistication of organoids even further. To overcome these
limitations, we have been developing well-defined alternatives to
Matrigel, customized for the culture of ISCs and organoids6.
Development of the protocol
Over the past several decades, bioengineers have incorporated
signals found in native tissues into otherwise bioinert, synthetic
polymer-based hydrogels to render them biofunctional7–9 and fit
for 3D cell culture10.
To create well-defined matrices for ISC and intestinal organoid
culture, we customized enzymatically cross-linked PEG hydrogels,
selected for the highly specific cross-linking reaction that occurs
under mild conditions compatible with the growth of cells11, by
incorporating adhesion signals from the small-intestinal base-
ment membrane and optimizing their mechanical and degrada-
tion properties to match the needs of ISCs and organoids6. We
discovered for example that fibronectin- or RGD-based adhe-
sion and a stiffness (shear modulus) of ~1 kPa are sufficient
for the expansion of ISCs in the presence of soluble Wnt and
Notch agonists. On the other hand, laminin-based adhesion and
a shear modulus <200 Pa were indispensable for differentiation,
morphogenesis and organoid formation. This insight enabled
the design of two types of matrices: a fully defined synthetic
gel for ISC expansion (termed PEG RGD) and a well-defined,
softening gel that supports both ISC expansion and subsequent
organoid formation (termed PEG RGD LAM). In the latter, the
sustained softening is afforded by the presence of PEG-acrylate
groups (PEG-Acr), whose ester bonds are cleaved through spon-
taneous hydrolysis.
The hydrogel systems introduced here overcome the multiple
limitations of Matrigel and other animal-derived matrices. The
gel-forming backbone of the matrices is a synthetic polymer,
and we believe that the incorporated bioinstructive signals rep-
resent the minimal set required for ISC expansion (RGD only)
and organoid formation (RGD and laminin-111). The synthetic
basis of the hydrogel and the low number of instructive compo-
nents afford a well-defined, reproducible environment, devoid of
unknown factors that confound the interpretation of basic studies
and preclude clinical use. Indeed, we have observed higher-purity

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© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
 protocol
ISC cultures within these hydrogels compared with those cultured
in Matrigel, wherein populations of differentiated cells are present
even under expansion conditions6.
Another key advantage of the hydrogels introduced here is their
modularity, i.e., the possibility of independently altering their
biophysical and biochemical properties. By varying the polymer
content, the mechanical properties of the gels can be controlled
over a wide range, and various adhesion and cell–cell interaction
factors can be incorporated at precisely controlled concentrations,
tethered or dispersed throughout the gel. Owing to this tunability,
the effects of various microenvironmental components on stem-
cell fate and organoid formation can be untangled. The proper-
ties of the materials were essential in identifying the mechanical
environment as a regulator of ISC fate and organoid formation
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
and in elucidating the underlying molecular mechanism6. Using
Matrigel to perform such modifications and address similar ques-
tions is currently not possible.
Experimental design
Here, we provide a detailed protocol for the synthesis of PEG RGD
and PEG RGD LAM matrices, as well as for their characterization
and use for ISC and organoid culture. Briefly, to create hydrogel
precursors, 8-arm PEG-VS and PEG-Acr macromers are end-
functionalized with lysine- and glutamine-presenting peptides
that serve as substrates for the activated transglutaminase factor
XIII (FXIIIa)12 (Steps 1–11). The cross-linking of the macromers
and resulting gel formation occur through the FXIIIa-mediated
formation of ε-(α-glutamyl)lysine isopeptide side-chain bridges
between the two peptide substrates. Importantly, we take advan-
tage of the same cross-linking mechanism to conjugate RGD
to the hydrogel backbone, presenting it to encapsulated cells
as an attachment cue (Fig. 1a). For the expansion of ISCs and
their routine culture (Step 26A), mechanically stable hydrogels
with a stiffness of ~1 kPa and RGD content of 1 mM are used
(Fig. 1a). These matrices (termed PEG RGD) also support the
establishment of ISC culture from freshly isolated mouse intestinal
crypts. When the formation of organoids starting from dissoci-
ated ISCs is the objective (Step 26B), a softening matrix contain-
ing RGD and laminin-111 is used (referred to as PEG LAM RGD)
(Fig. 1b). Cells comprising ISC colonies or organoids can be
retrieved from the gels for subculturing or downstream analyses
(Step 27A, C). Immunofluorescence or immunohistochemical
analysis can similarly be performed in-gel (Step 27B).
Potential future applications and extensions of the protocol
There is the potential to engineer the matrices described here
to respond to cell-produced proteases. Cell-inducing proteolytic
remodeling of the ECM plays crucial roles in tissue develop-
ment, disease and regeneration13. Recently, we demonstrated
that the proteolytic sensitivity of the matrix controls multiple
aspects of ISC behavior, including colony formation, polariza-
tion, ISC fate and organoid formation6. The degradation proper-
ties of the materials can be modulated by incorporating peptide
sequences that are sensitive or insensitive to cell-produced pro-
teases, such as matrix metalloproteinases, into the backbone of
the PEG hydrogels14.
The newly introduced system also has limitations that should be
overcome in future design iterations. A main caveat to the PEG-
based hydrogel systems for ISC and organoid culture as compared
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Gln-RGD
a PEG-VS-Gln
PEG-VS-Lys
Intestinal crypts
ISC colonies
Or
4 d, ENRCV
PEG RGD
+
Lgr5 ISCs
Laminin-111
b PEG-Acr-Gln
PEG-Acr-Lys
Intestinal crypts
Intestinal
Or organoids
4 d, ENRCV + 2 d,
ENR
PEG RGD LAM
+
Lgr5 ISCs
Figure 1 | Overview of ISC and intestinal organoid culture in PEG-based
hydrogels. (a) Mechanically stable, 1-kPa gels functionalized with RGD are
used for the expansion of dissociated ISCs or freshly isolated intestinal
crypts. (b) Intestinal organoids are formed by embedding ISCs into
mechanically dynamic matrices functionalized with RGD and laminin-111,
which are initially stiff enough to support ISC expansion but subsequently
soften to permit differentiation and morphogenesis.
with use of Matrigel is that there is generally a lower efficiency
of ISC colony and intestinal organoid formation in PEG-based
hydrogels. The minimal nature of these matrices, while rendering
them reproducible and well-defined, also leads to a lower initial
survival of embedded dissociated ISCs. Thus, the use of Matrigel
may be preferable for research endeavors in which routine ISC
and organoid culture in vitro is the main objective, and matrix
tunability, a well-defined environment and downstream clinical
use are not envisioned or required. However, we have previously
found that the addition of other putative ECM components of
the ISC niche (e.g., collagen IV, hyaluronan or perlecan) can sub-
stantially improve ISC survival and self-renewal in PEG-based
hydrogels6. We envision that enriching the current gels with some
of these or other ECM components, either singly or in combina-
tion, will further improve ISC and organoid culture efficiencies.
The softening hydrogels used for intestinal organoid formation
currently contain mouse-derived laminin-111. Thus, although
they are substantially more defined and controlled compared with
Matrigel, they are not fully defined or artificial. Nonetheless, we
believe that this matrix, which allows for controlled biochemical
and mechanical manipulations, can still be very useful in basic
research. We are actively working toward replacement of the
animal-derived laminin in the gels with a recombinant version.
The clinical significance of the system is not compromised by the
presence of mouse laminin within the organoid-forming matrix,
as the PEG RGD system used for the expansion of ISCs is both
synthetic and defined. Complete absence of animal-derived prod-
ucts is most important in clinical settings, and cell-based treat-
ment strategies may involve transplantation of pure populations
of stem cells, rather than organoids, which are composed mainly
of short-lived differentiated cells. Moreover, we show that the
clinically relevant human intestinal organoids, which are only

maintained under conditions that favor stem-cell expansion 15,
can be cultured in the fully defined PEG RGD matrices. It should
be noted, however, that the materials described here were designed
specifically for the growth of mouse ISC colonies and organoids,
whereas applications for routine culture of human organoids
will probably require further optimization and customization.
Indeed, although freshly isolated human crypts survived in matri-
ces optimized for mouse organoid culture, and were successfully
maintained after at least two passages, the frequency of organoid
formation and the rate of growth were lower, compared with their
mouse-derived counterparts.
Although we used enzymatically cross-linked hydrogels, other
hydrogel systems and cross-linking chemistries16–18 can be used
for the application described here, provided that the requisite bio-
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
logical features—adequate mechanical, adhesive and degradation
properties—are preserved. Namely, hydrogels can be covalently
cross-linked—formed, for example, via ‘bioclick chemistry’ such as
Michael additions, azide–alkyne cycloadditions or photoinitiated
MATERIALS
REAGENTS
• TG-Gln: H-NQEQVSPL-ERCG-NH2 (GL Biochem, custom synthesis)
• TG-NDG-Lys: Ac-FKGG-GDQGIAGF-ERCG-NH2 (GL Biochem,
custom synthesis)
• TG-DG-Lys: Ac-FKGG-GPQGIWGQ-ERCG-NH2 (GL Biochem,
custom synthesis)
• TG-FDG-Lys: AcFKGG-VPMSMRGG-ERCG-NH2 (GL Biochem,
custom synthesis)
• 8-Arm PEG-VS, 40 kDa (NOF, custom synthesis)
• 8-Arm PEG-Acr, 40 kDa (Creative PEGWorks, cat. no. PSB-824)
• Triethanolamine (TEA; Sigma, cat. no. 90278)
• Tris(hydroxymethyl)aminomethane (Sigma-Aldrich,
cat. no. 252859)
• CaCl2 (Sigma-Aldrich, cat. no. C1016)
• Lys-RGD: Ac-FKGGRGDSPG-NH2 (GL Biochem, custom synthesis)
• Fibrogammin 1250 (CSL Behring) or FXIII (Zedira, cat. no. T007)
• Thrombin (Sigma-Aldrich, cat. no. T1063)
• Advanced DMEM/F12 (Gibco, cat. no. 12634-010)
• Glutamax (Gibco, cat. no. 15630-056)
• HEPES (Gibco, cat. no. 35050-038)
• Penicillin–streptomycin (Gibco, cat. no. 15140-122)
• B27 supplement (Thermo Fisher Scientific, cat. no. 17504044)
• N2 (Thermo Fisher Scientific, cat. no. 17502048)
• N-acetylcysteine (Sigma-Aldrich, cat. no. A9165-5G)
• EGF (produced by the EPFL Protein Expression Core Facility (PECF);
commercial source: R&D Systems, cat. no. 236-EG)
• R-spondin (produced by the EPFL PECF; commercial source: R&D
Systems, cat. no. 4645-RS/CF)
• Noggin (produced by the EPFL PECF; commercial source: R&D
Systems, cat. no. 6057-NG)
• CHIR99021 (Calbiochem, cat. no. 361559)
• Valproic acid (Sigma-Aldrich, cat. no. P4543)
• Thiazovivin (Stemgent, cat. no. 04-0017)
• Laminin (Gibco, cat. no. 23017015)
• 1× PBS (Gibco, cat. no. 10010015)
• Sigmacote (Sigma-Aldrich, cat. no. SL2-100 ML)
• Y27632 (Stemgent, cat. no. 04-0012)
• TrypLE (Thermo Fisher Scientific, cat. no. 12605028)
• DNAse I (Sigma-Aldrich, cat. no. 10104159001)
• Triton X-100 (Sigma-Aldrich, cat. no. X100)
• Goat serum (Thermo Fisher Scientific, cat. no. 16210064)
• Freshly isolated mouse intestinal crypts (obtained as described in Sato et al.22)
• Mouse ISC colonies (obtained as described in Yin et al.23)
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.
protocol
thiolene reactions19. Furthermore, so long as the final matrix
stiffness is in the range required for ISC expansion and integrin
ligands are incorporated at the appropriate concentration, hydro-
gels can also be formed via reversible covalent bonds—including
Schiff base (imine) bonds, reversible hydrazone bonds, oxime
bonds and disulfide bonds—or weak interactions—including
hydrophobic interactions, hydrogen bonds, van der Waals forces,
electrostatic interactions and host–guest interactions20.
Similarly, whereas we opted for gel softening based on the
hydrolysis of resident ester groups, alternative mechanisms for
gel softening can readily be used, including photo-induced or
enzymatic gel degradation21. Indeed, we believe that the necessity
for gel softening in the current system originates from the need
to alleviate the deleterious compressive stresses that accumulate
upon colony growth within a linearly elastic environment6. Hence,
we anticipate that organoid formation can similarly be achieved
in materials that have a constant stiffness but exhibit viscoelastic,
i.e. stress-dissipative, mechanical behavior.
EQUIPMENT
• 50-ml Falcon tube (Corning, cat. no. 352070)
• SnakeSkin dialysis tubing (Thermo Fisher Scientific, cat. no. 68100)
• Plastic clamps (Spectrum Laboratories, cat. no. 142174)
• Dialysis float buoys (Thermo Fisher Scientific, cat. no. 66430)
• Kimwipes (Kimtech, cat. no. 7552)
• 0.22-µm filter (Sarstedt, cat. no. 83.1826.001)
• Glass microscope slides (Thermo Scientific, cat. no. J3800AMNZ)
• Parafilm (Parafilm, cat. no. PM999)
• Rheometer (Bohlin, model no. CVO 120)
• Lyophilizer (Labconco, cat. no. 7750020)
• Temperature-controlled water bath (Fisher Scientific, model no. Isotemp 210)
REAGENT SETUP
0.3 M TEA buffer, pH 8.0 To prepare 100 ml of 0.3 M TEA buffer, mix
3.98 ml of TEA with 80 ml of autoclaved Milli-Q water. Use 5 M HCl to adjust
the pH to 8.0 and fill the remaining volume (up to 100 ml) with Milli-Q water.
Store the TEA buffer at room temperature up to 1 month.
11× Buffer To prepare 100 ml of 11× buffer (550 mM Tris, 1.65 M NaCl and
550 mM CaCl2), dissolve 6.05 g of Tris(hydroxymethyl)aminomethane,
8.76 g of NaCl and 5.80 g of CaCl2 in 70 ml of autoclaved Milli-Q water.
Adjust the pH to 7.4 using 5 M HCl, and bring the volume to 91 ml with
autoclaved Milli-Q water. Store the buffer at 4 °C for up to 1 year.
Activation of FXIII Dissolve Fibrogammin, as provided by BSL Behring,
using 6.25 ml of autoclaved Milli-Q water to make a 200 U/ml solution. To
activate the FXIII solution, add 625 µl of thrombin (20 U/ml). Incubate at
37 °C for 30 min, shaking every 10 min. Divide the activated FXIII (FXIIIa)
into 10- and 20-µl aliquots and store them at −80 °C for up to 3 years.
Base medium To 500 ml of advanced DMEM/F12, add 5 ml of Glutamax,
5 ml of HEPES and 5 ml of penicillin–streptomycin. Base medium (BM) can
be stored at 4 °C for 1 month.
BM N2B27 medium To 50 ml of BM, add 0.5 ml of N2, 1 ml of B27
and 50 µl of N-acetylcysteine (1 mM stock). BM N2B27 medium can be
stored at 4 °C for 1 week.
Expansion medium To prepare expansion medium (ENRCV), add BM
N2B27 medium, supplemented with EGF (50 ng/ml), Noggin (100 ng/ml),
R-spondin (500 ng/ml), CHIR99021 (3 µM), valproic acid (1 mM)
and thiazovivin (2.5 µM). Use the medium immediately after preparation.
Organoid formation medium To prepare organoid formation medium
(ENR), add BM N2B27 medium, supplemented with EGF (50 ng/ml),
Noggin (100 ng/ml) and R-spondin (500 ng/ml). Use the medium
immediately after preparation.
nature protocols | VOL.12 NO.11 | 2017 | 2265
 protocol

PROCEDURE
Synthesis of gel precursors
1| Allow the peptides TG-Gln, TG-DG-Lys and TG-NDG-Lys to reach room temperature, and weigh appropriate amounts of
them needed to functionalize 1 g of PEG-VS or PEG-Acr, ensuring a 1.2 molar excess of peptide to PEG (see Box 1 for an
example calculation).
 CRITICAL STEP Be sure to take into account the purity of the peptides, along with their functionality, i.e., the amount of
free cysteines present, when calculating the mass of peptides to include in the reaction.

2| Prepare 5× 50-ml Falcon tubes, each containing one of the following mixes: PEG-VS and TG-Gln; PEG-VS and
TG-DG-Lys; PEG-VS and TG-NDG-Lys; PEG-Acr and TG-Gln; and PEG-Acr and TG-NDG-Lys.

3| Add 40 ml of 0.3 M TEA buffer to each tube to dissolve the powder.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

4| Incubate the tubes in a 37 °C water bath for 2 h, with occasional (~every 30 min) mixing by tube inversion.
 CRITICAL STEP This step is the coupling reaction between the vinyl sulfone groups or acrylate of the PEG and the thiol
groups of the peptides.

5| Transfer the contents of each tube to separate SnakeSkin dialysis tubings, rinsing the tube with additional 10 ml of
0.3 M TEA. Before transferring the liquid, clamp one side of the tubing with a ‘heavy’ plastic clamp. After filling the tubing,
close it with a ‘light’ plastic clamp. Attach a dialysis buoy to the top side of the tubing (the side with the light clamp), and
suspend each tube in a separate 5-liter glass beaker filled with ultrapure water and containing a magnetic stirrer.

6| Dialyze the contents for 4 d at 4 °C with stirring, changing the water three times per day (morning, noon and evening)
to remove the unreacted peptides and TEA.

7| Separate the contents of each dialysis tube into four 50-ml Falcon tubes (~14 ml per tube). Close the tube with a
rubber-band-fastened Kimwipe.

8| Freeze the samples by keeping them immersed for 5 min in a tank of liquid nitrogen fitted with a tube rack.
Transfer the frozen samples to a lyophilizer and dry them for 2 d.

9| Combine the lyophilates of the same product in a single preweighed 50-ml Falcon tube, and reweigh to determine the
weight of the product. Add the appropriate volume of autoclaved Milli-Q water to make a 13.33% (wt/vol) PEG stock solu-
tion of each of the five precursors: [PEG-VS]-DG-Lys, [PEG-VS]-NDG-Lys, [PEG-VS]-Gln, [PEG-Acr]-NDG-Lys, and [PEG-Acr]-Gln.
Filter the solutions using a 0.2-µm filter (see Box 1 for a sample calculation).
 CRITICAL STEP Take care to account for the density of PEG when calculating the volume of water for precursor reconstitution.

10| Make 13.33% (wt/vol) premixes of the complementary macromers (glutamine-containing macromer with a lysine-con-
taining macromer) by mixing them at the volumetric ratios listed below (see Box 2 for a sample calculation).

Species Ratio

[PEG-VS]-DG-Lys to [PEG-VS]-Gln 1.05

[PEG-VS]-NDG-Lys to [PEG-VS]-Gln 1.04

[PEG-VS]-NDG-Lys to [PEG-Acr]-Gln 1.04

[PEG-Acr]-NDG-Lys to [PEG-Acr]-Gln 1.04

11| To make a 75% PEG-Acr premix, to be used for the formation of mechanically dynamic, softening hydrogels, first
mix [PEG-Acr]-Gln with [PEG-VS]-NDG-Lys at the appropriate volumetric ratios to make a 50% (mole %) PEG-Acr mix, and
then mix [PEG-Acr]-Gln with [PEG-Acr]-NDG-Lys to make a 100% (mole %) PEG-Acr mix. Mixing equal volumes of 50%
(mole %) PEG-Acr and 100% (mole %) PEG-Acr gives a 75% PEG-Acr precursor solution, with a 13.33% (wt/vol) overall
polymer content (see Box 2 for a sample calculation).
 PAUSE POINT The premixes can be stored at −80 °C for several years, at −20 °C for up to a year and at 4 °C for several weeks.

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 protocol

Box 1 | Example calculation

The calculation below exemplifies the synthesis of [PEG-VS]-Gln and [PEG-VS]-NDG-Lys gel precursors, starting from 1 g of 8-arm
PEG-VS in each case
m8-armPEG-VS = 1,000 mg (mass of PEG-VS we wish to functionalize)
MW8-arm PEG-VS = 40,661 Da (molecular weight of 8-arm PEG-VS; provided by vendor)
8 = number of VS groups within an 8-arm PEG-VS molecule
0.925 = typical efficiency of PEG-VS functionalization (provided by the vendor)
MWTG-GLn = 1358.5 Da (molecular weight of TG-Gln; provided by the vendor)
MWTG-NDG-Lys = 1639.8 Da (molecular weight of TG-NDG-Lys; provided by the vendor)
PCTG-Gln = 0.808 (peptide content fraction; provided by the vendor)
PCTG-NDG-Lys = 0.838 (peptide content fraction; provided by the vendor)
ρPEG-VS = 1.128 g/ml (density of PEG)

1. Determine moles of vinyl sulfone groups (nVS) contained in 1,000 mg of 8-arm PEG-VS:
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

m8 −armPEG− VS
nVS = ⋅ 8 ⋅ 0.925 = 0.18199 mmol
MW8 −armPEG− VS

2. Determine the peptide masses (mTG-Gln and mTG-NDG-Lys) needed to functionalize 1 g of 8-arm PEG-VS, ensuring a 1.2 molar access:
n ⋅ 1.2 ⋅ MWTG−Gln
mTG−Gln = VS = 367.18 mg
PC TG−Gln

nVS ⋅ 1.2 ⋅ MWTG−NDG−Lys

mTG−NDG−Lys = = 443.22 mg
PC TG−Gln

3. Determine the volume of water for reconstitution of the reaction products to a final concentration of 13.33% (wt/vol)
(133.3 mg/ml):
m[PEG− VS]−Gln = 1332.0 mg (typical yield)
m[PEG− VS]−Gln  mg  m[PEG− VS]−Gln
Vwater = ⋅3  − = 8.819ml
133  ml  rPEG− VS

m[PEG− VS]−NDG−Lys 1226.0 mg (typical yield)

m[PEG− VS]−NDG−Lys  mg  m[PEG− VS]−NDG−Lys
Vwater = ⋅3  − = 8.110 ml
133  ml  rPEG− VS

Gel formation and mechanical characterization

12| Render the glass microscope slides hydrophobic by immersing them in Sigmacote for several seconds (the reaction is
nearly instantaneous).

13| Allow the slides to air-dry in a chemical hood, rinse them with water and dry them.

14| To form PEG hydrogels with a range of polymer contents, starting from the desired 13.33% (wt/vol) PEG premix, prepare
the solutions listed in Table 1 by mixing the components on ice, in the order in which they are listed.

15| To prepare PEG hydrogel discs for mechanical characterization, cast 50-µl droplets of the liquid,
non-cross-linked gel mix onto a Sigmacote-treated slide. One standard microscope slide can typically
accommodate three droplets.

16| Carefully place 1 cm × 1 cm × 1 mm plastic spacers on both ends of the slide, and sandwich the droplets by
resting a second Sigmacote-treated slide on top of the spacers. Secure the slides by clamping them with standard
binder clips on both sides (Fig. 2).

17| Allow gel formation by leaving the slides for 30 min at 37 °C.

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 protocol

Box 2 | Preparation of premixes

The procedure below exemplifies the preparation of 13.33% (wt/vol) premixes of the complementary PEG precursors used for
ISC expansion or organoid formation
1. Prepare a 13.33% (wt/vol) [PEG-VS]-Gln/[PEG-VS]-NDG-Lys premix, used to form gels suitable for ISC expansion:
A typical synthesis of Gln- and Lys-conjugated PEG hydrogel precursors yields 7–9 ml of 13.33% (wt/vol) solution of each precursor
species. To achieve a stoichiometrically balanced mixture of [PEG-VS]-NDG-Lys and[PEG-VS]-Gln precursors within the premix, they
must be balanced at a volumetric ratio [PEG − VS] − NDG − Lys . Thus, mix 7.000 ml of 13.33% (wt/vol) [PEG-VS]-Gln solution
= 1.04
[PEG − VS] − Gln

with 7.280 ml of 13.33% (wt/vol) [PEG-VS]-NDG-Lys solution to generate 14.708 ml of 13.33% (wt/vol) [PEG-VS]-Gln /
[PEG-VS]-NDG-Lys premix.
2. Prepare a precursor premix used to form gels suitable for ISC expansion. The overall PEG content of this premix is 13.33% (wt/vol).
Of the total PEG macromers, 75% contain acrylate groups (PEG-Acr) and the remaining 25% contain vinyl sulfone groups (PEG-VS):
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

• Mix 7.000 ml of 13.33% (wt/vol) [PEG-Acr]-Gln solution with 7.280 ml of 13.33% (wt/vol) [PEG-Acr]-NDG-Lys to generate
14.280 ml of precursor A. The overall PEG content of precursor A is 13.33% (wt/vol). Of the total PEG arms in precursor A,
100% are functionalized with an acrylate group.
• Mix 7.000 ml of 13.33% (wt/vol) [PEG-Acr]-Gln solution with 7.280 ml of 13.33% (wt/vol) [PEG-VS]-NDG-Lys to generate
14.280 ml of precursor B. The overall PEG content of precursor B is 13.33% (wt/vol). Of the total PEG arms in precursor B,
50% are functionalized with an acrylate group, and 50% with a vinyl sulfone group.
• Mix 7.000 ml of precursor A with 7.000 ml of precursor B to form 14.000 ml of precursor C. The overall PEG content of precursor
C is 13.33% (wt/vol). Of the total PEG arms in precursor C, 75% are functionalized with an acrylate group, and 25% with a vinyl
sulfone group.

? TROUBLESHOOTING

18| Inject 2 ml of autoclaved Milli-Q water into the space between the slides to prevent gel damage during
decasting. Gently detach the bottom and top surfaces of the gel discs from the glass slides with a flat and thin
metal spatula. Unclamp the binder clips, remove the top slide and carefully transfer each gel to a separate well
of a 12-well plate filled with 1.5 ml of autoclaved Milli-Q water per well. Allow the gels to swell overnight at 37 °C.
Swelling of acrylate-containing gels is performed in complete cell culture medium instead of water.

19| Perform small-strain oscillatory shear measurements on a rheometer, using the ‘parallel-plate’ rheometer
measuring setup with a spacing of 800 µm during measurement. Perform measurements at a constant strain
of 5% and a range of frequencies (0.1–10 Hz). Figure 3 shows typical stiffness values of gels containing
1.5–3% (wt/vol) PEG.
 CRITICAL STEP PEG RGD LAM gels will undergo substantial softening overnight, and, to ascertain the initial
stiffness, rheometric characterization should be performed on the day of gel formation after 3 h of swelling in
full cell culture medium.
 PAUSE POINT The PEG RGD gels are mechanically stable for at least 4 d, and the time of measurement is flexible.

Cell dissociation and embedding

20| Prepare the dissociation solution: 1 ml of TrypLE
containing 1,000 U/ml DNAse I, 1 mM N-acetylcysteine a b
and 10 µM Y27632.
 CRITICAL STEP Freshly prepare the solution before each
cell dissociation. c
21| Resuspend freshly isolated crypts21 or ISC colonies that
have been pre-expanded in Matrigel for 3–4 d22 in
the dissociation solution. Figure 2 | Preparation of PEG gels for rheometric characterization. (a) Items
required: two Sigmacote-treated glass microscope slides, two plastic spacers
and two metal clamps. (b,c) Gel droplets (b, dashed lines) are cast on top
22| Incubate the crypts or ISC colonies for 8 min in a 37 °C of one slide, sandwiched and secured, as shown, to generate gel discs to be
water bath. used for rheometric measurements (c). Scale bars, 8 mm.

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 protocol

23| Triturate the crypts or ISC colonies gently with a 1-ml 2,000

pipette, dilute with 9 ml of BM (advanced DMEM/F12

containing 1× Glutamax, 1× HEPES and 1× penicillin–
streptomycin) and pass through a 40-µm cell strainer. 1,500

Storage modulus (Pa)

24| Pellet the cells by centrifugation (250g, 4 min, 4 °C).
1,000

25| Resuspend the cell pellet in 1 ml of BM and count the

cells using a hemocytometer.
500
26| Encapsulate and expand ISCs (option A) or organoids
(option B) in the prepared hydrogels.
(A) ISC expansion in fully defined hydrogels
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

(i) Encapsulate the dissociated ISCs, organoids or freshly 1.5 2.0 2.5 3.0
PEG content % (wt/vol)
isolated crypts in 1–1.5 kPa PEG RGD gels, formed
from [PEG-VS]-Gln and [PEG-VS]-NDG-Lys precursors, Figure 3 | Typical results of rheometric characterization of PEG hydrogels.
and containing 1 mM Lys-RGD. The PEG polymer Plot shows the mechanical properties (storage modulus) of PEG hydrogels
formed from [PEG-VS]-Gln/ [PEG-VS]-NDG-Lys precursors, as a function
content needed to afford this stiffness range is of overall PEG content. Data are represented as mean values (bars) and
determined by rheometry, as described in Steps individual points (circles).
12–19, and it typically falls in the range of 2–3%
(wt/vol). An example recipe for gels suitable for ISC
expansion is provided in Table 2. Cast 15- to 20-µl
droplets of the cell–gel mix on the bottom of a
24-well plate. Allow gelation to occur for 30 min at 37 °C.
? TROUBLESHOOTING
(ii) Overlay the gels with 600 µl of ENRCV. Replenish the growth factors and small molecules, except for thiazovivin,
every 2 d, and replace the full medium every 4 d.
 CRITICAL STEP To ensure homogeneous cell distribution throughout the gel, flip the plate between the upright and
upside-down positions every minute during the first 5 min of gelation. Use caution to prevent displacement of the
liquid droplets from the center of the wells and onto the well walls during the flipping process.
? TROUBLESHOOTING
(iii) Allow the colonies to form and expand over 4–6 d, then fix for immunofluorescence analysis or retrieve the cells from
the gels for subculturing or downstream analyses (see below).
(B) Organoid formation in well-defined hydrogels
(i) Encapsulate the dissociated ISCs, organoids or freshly isolated crypts in 75% PEG-Acr gels (see Step 11 and Box 2)
with an initial stiffness of 1–1.5 kPa, containing 1 mM Lys-RGD and 0.2 mg/ml laminin-111. The PEG polymer content
needed to afford this stiffness range is determined by rheometry as described in Steps 12–19. An example recipe for
gels suitable for organoid formation is provided in Table 2.
(ii) Cast 15- to 20-µl droplets of the mix onto the bottom of a 24-well plate, and allow gelation to occur (30 min, 37 °C).
? TROUBLESHOOTING
(iii) Overlay the gels with 600 µl of ENRCV, and allow for ISC colonies to form over 4 d.
? TROUBLESHOOTING

Table 1 | Recipe for preparing PEG gels (100 µl per condition) with 1.5–3% (wt/vol) polymer content.

Component 1.5% 2.0% 2.5% 3.0% Final concentration

11× buffer 9.1 9.1 9.1 9.1 1×

PEG premix (13.33% (wt/vol)) 11.3 15.0 18.8 22.5 1.5–3.0% (wt/vol)

Lys-RGD (10 mM) 10.0 10.0 10.0 10.0 1 mM

Base medium 54.7 50.9 47.2 43.4

FXIIIa 200 (U/ml) 5.0 5.0 5.0 5.0 10 U/ml

Note that the concentration of the 11× buffer and FXIIIa is held constant, whereas the remaining parameters are varied to form gels with the desired mechanical or biochemical properties. Numbers indicate
volumes in microliters.

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 protocol

(iv) Wash once with 1× PBS and overlay with 600 µl of ENR. Buds should emerge within 24–48 h, and organoids should
fully form by day 3 after medium change.
? TROUBLESHOOTING

Downstream analyses
27| To release ISCs for passage and downstream analysis, follow option A. To fix ISCs and analyze with immunofluorescence,
follow option B. To fix organoids for immunofluorescence analysis, follow option C. To examine the effect of proteolytic
matrix degradability on ISCs and organoids, follow option D.
(A) ISC release for passaging and downstream analysis
(i) Aspirate the medium, gently detach the gel from the bottom of the well using an autoclaved metal spatula bent at a
90° angle and transfer the gel to a 15-ml Falcon tube containing 1 ml of dissociation solution (1 ml of TrypLE
containing 1,000 U/ml DNAse I, 1 mM N-acetylcysteine and 10 µM Y27632).
 CRITICAL STEP Freshly prepare the solution before each cell dissociation.
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

(ii) Transfer the tube with the floating gel to a 37 °C water bath.
(iii) Incubate the tube for 4 min and triturate the contents gently using a 1-ml pipette with a tip that has been truncated
with sterilized scissors.
(iv) Incubate the tube in the water bath for an additional 4 min and triturate the contents gently with a 1-ml pipette.
(v) Incubate the tube in water for an additional 1–2 min. By this time, most of the gel and ISC colonies should be
dissociated.
(vi) Dilute the dissociation solution with 10 ml of ice-cold BM (advanced DMEM/F12 containing Glutamax, HEPES,
penicillin–streptomycin) and pass it through a 40-µm strainer. Centrifuge the tube at 250g for 4 min at 4 °C.
(vii) Aspirate the medium, resuspend the cell pellet in 10 ml of ice-cold BM and centrifuge the tube (250g,
4 min, 4 °C).
(viii) Re-embed the cells in a new gel for further culture or use them for downstream molecular analyses (e.g., RT-qPCR,
western blot and flow cytometry).
(B) Immunofluorescence analysis of ISCs in-gel
(i) Remove the cell culture medium and replace it with 600 µl of 1× PBS. Incubate the tube for 15 min at 37 °C.
(ii) Fix the samples with 4% (wt/vol) paraformaldehyde (PFA) (30 min, room temperature).
(iii) Wash them with 600 µl of 1× PBS (15 min, room temperature, with shaking) and add 600 µl of 1× PBS.
 PAUSE POINT Samples can be stored for several weeks at 4 °C.
(iv) Aspirate the PBS and gently detach the gels from the bottom of the plate using a metal spatula, bent at a 90° angle.
Transfer the gels to individual wells of a 12-well plate.
 CRITICAL STEP Performing the staining steps on an attached gel would result in reduced staining quality, owing to
antibody transport limitations. We thus recommend moving the gel to wells of a different tissue culture plate.
(v) Permeabilize the gels by adding 1.5 ml of 0.2% (vol/vol) Triton X-100 in PBS (1 h, room temperature, with shaking)
per well of a 12-well plate.
 PAUSE POINT Samples can be stored for several weeks in the refrigerator.
(vi) Block the samples by adding 1.5 ml of 10% (vol/vol) goat serum in PBS + 0.01% (vol/vol) Triton X-100 per well (be-
tween 3 h and overnight, 4 °C, with shaking).

Table 2 | Typical recipes for forming gels (100 µl per condition) suitable for ISC expansion and organoid formation.

Component ISC expansiona Organoid formationb Final concentration

11× buffer 9.1 9.1 1×

PEG premix (13.33% (wt/vol)) 18.8 35.6 2.5; 4.75% (wt/vol)

Lys-RGD (10 mM) 10.0 10.0 1 mM

Laminin-111 (1.2 mg/ml) 0.0 16.7 0; 0.2 mg/ml

Cell stock (105 cells per ml) 10.0 10.0 1,000 cells per ml

Base medium 30.5 13.6

FXIIIa 200 (U/ml) 5.0 5.0 20 U/ml

Numbers indicate volumes in microliters. aFor ISC expansion, use [PEG-VS]-Gln/[PEG-VS]-NDG-Lys premix. bFor organoid formation, use 75% PEG-Acr premix, i.e. precursor C described in Box 2.

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 protocol

(vii) Transfer the gels to wells of a 24-well plate. Incubate the gels with primary antibody dissolved at the
appropriate concentration in a blocking buffer to obtain a 500-µl total volume (36 h, 4 °C, with shaking).
(viii) Transfer the gels to the wells of a 12-well plate. Wash the gels with 1.5 ml of 1× PBS per well for ~8 h,
replacing the PBS with fresh PBS every hour (room temperature, with shaking).
(ix) Incubate the gels (overnight, 4 °C, shaker) in 1.5 ml of secondary antibody solution (1:1,000,
in blocking buffer).
(x) Wash the gels with 1.5 ml per well of 1× PBS for the entire next day, replacing the PBS every hour
with fresh PBS (room temperature, with shaking).
 PAUSE POINT Samples can be stored for several weeks in the refrigerator.
(xi) Transfer the gels to a glass slide for confocal imaging.
(C) Recovery of organoids from 75% PEG-Acr gels and immunofluorescence analysis
(i) On day 7 or 8 of culture, aspirate the cell culture medium.
 CRITICAL STEP This step should be performed with care to avoid aspiration of the very soft gel and organoids along
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

with the medium.

(ii) Add 1 ml of ice-cold 1× PBS, and triturate the gel using a 1-ml pipette to break up the remaining gel. Transfer the
organoid suspension to a 15-ml Falcon tube. Centrifuge the tube (110g, 3 min, 4 °C).
(iii) Resuspend the pelleted organoids in 1 ml of 4% PFA, and fix them for 20 min at room temperature.
Centrifuge the tube (110g, 3 min, 4 °C).
(iv) Wash the organoids by resuspending them in 1 ml of DI water. Centrifuge the tube (110g, 3 min, 4 °C).
(v) Resuspend the tissues in 20 µl of autoclaved Milli-Q water, spread them on glass coverslips and allow them to dry at
room temperature (45 min–1 h). Immediately after drying, rehydrate the tissues by adding PBS.
 CRITICAL STEP Do not resuspend them in 1× PBS before the drying step, as drying will result in salt-crystal
formation around the organoids.
? TROUBLESHOOTING
 PAUSE POINT Samples can be stored for several weeks in the refrigerator.
(vi) Remove the PBS and transfer the coverslips with organoids to the wells of a six-well plate. Permeabilize the organoids
by adding 2 ml of 0.2% (vol/vol) Triton X-100 in PBS and incubating the plate for 1 h at room temperature (20–25 °C)
with shaking.
 PAUSE POINT Samples can be stored for several weeks at 4 °C.
(vii) Block the sample by adding 2 ml of 10% (vol/vol) goat serum in PBS + 0.01% (vol/vol) Triton X-100 (for between 3 h
and overnight, at 4 °C, with shaking).
(viii) Prepare a primary antibody solution in blocking buffer at the appropriate concentration. Make 90-µl droplets on the
Parafilm, and place the coverslips on top of the droplets, exposing the attached organoids to the antibody solution
(overnight, at 4 °C).
(ix) Transfer the coverslips with organoids back to the six-well plate and wash them with 2 ml of 1× PBS for 3 h,
replacing the PBS every hour with fresh PBS (room temperature, with shaking).
(x) Incubate the plate in 2 ml of secondary antibody dissolved at the appropriate concentration in blocking buffer
(overnight, 4 °C, shaker).
(xi) Wash the plate with 2 ml of 1× PBS for 3 h, replacing the PBS every hour with fresh PBS (room temperature, with
shaking).
 PAUSE POINT Samples can be stored for several weeks at 4 °C.
(D) Examination of the effect of proteolytic-matrix degradability on ISCs and organoids
 CRITICAL For this analysis, you must have synthesized lysine-presenting PEG precursors using peptides with increasing
proteolytic sensitivity: TG-NDG-Lys, TG-DG-Lys and TG-FDG-Lys, as well as embedded ISCs, intestinal crypts or organoid
fragments into PEG gels containing TG-NDG-Lys, TG-DG-Lys and TG-FDG-Lys sequences.
(i) Analyze the processes of interest by bright-field immunofluorescence microscopy, or by cellular and molecular
analyses of cells retrieved from the matrices.
 CRITICAL STEP To isolate the effects of matrix proteolytic sensitivity on cell behavior, other important matrix
properties, including adhesion cues and mechanical properties, must be matched between the hydrogels harboring
the separate peptide sequences. More specifically, gels of variable proteolytic sensitivities should have comparable
stiffness values (achieved by adjusting the polymer content of the gels) and should contain the same adhesive
signals (ECM proteins and ECM-derived peptides or proteoglycans), at comparable concentrations.
 CRITICAL STEP Often, the effect of proteolysis is context-dependent, and it varies in response to the
mechanical and adhesive properties of the matrix. Hence, we suggest probing the influence of proteolytic sensitivity
within a range of matrix stiffness values and in the presence of several different ECM-derived proteins or peptides.

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 protocol

 CRITICAL STEP The presence of PEG-Acr within the gels introduces another variable—temporal changes in
the mechanical properties of the gel, owing to hydrolytic degradation of the gel—which is likely to complicate
dissecting the effect of cell-mediated proteolytic degradation of the matrix. Thus, unless users are specifically
interested in coupling proteolytic degradation with sustained gel softening in an experiment, we would suggest using
PEG-VS backbones to form gels meant to explore the effects of proteolysis.

? TROUBLESHOOTING
Troubleshooting advice can be found in Table 3.

Table 3 | Troubleshooting table.

Step Problem Possible reason Solution

26 Embedded population of ISCs contains Insufficient dissociation Increase the dissociation time by 1–2 min. Note:
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

both single cells and cell clusters excessive dissociation will reduce cell viability

If embedding only single cells is critical, sort

cells by flow-cytometry-based approaches

26A(ii), Colony formation within the synthetic Prolonged exposure to tryptic Reduce dissociation time by 30 s or 1 min.
26B(iii) matrices is low (<10%) enzymes A shorter dissociation time may lead to an
increased number of cell clusters but will improve
the viability and colony-formation potential of
the embedded cells

Delay in availability of Reduce the time between cell embedding and

soluble factors owing to pro- medium overlay to 15 min
longed gelation

17, 26A(i), After precursor synthesis, gels do not Low content of reactive thiol Using Ellman’s reagent, determine the content
26B(ii) form, or form only at a high polymer groups within the peptides of free thiols within the peptides. Account for
content (>2% (wt/vol)) results in inefficient func- the actual thiol content when performing the
tionalization of the PEG Michael-type coupling of the PEG macromers and
macromers peptides. In general, we deem 80% to be the
lower acceptable limit of thiol-end functionality
for any of the peptides used

26B(iv) ISC colonies sink to the bottom of PEG gels soften too quickly; Increase the overall PEG content within the gels
the dish, depolarize and fail to form softening is too extensive or reduce the acrylate content of the gels
organoids

26B(iv) ISC colonies become dense, but remain PEG gel softening is too slow Decrease the overall PEG content within the gels,
27C(v) round and fail to form organoids or insufficient or increase the acrylate content of the gels

Number of organoids on coverslips seems Organoids were lost through Pretreat the tube with 1% (wt/vol) BSA/PBS for 1
lower than that observed in the gels attachment to the Falcon tube h at room temperature

● TIMING
Steps 1–5, synthesis of gel precursors: 2–3 h
Step 6, synthesis of gel precursors: 4 d
Step 7, synthesis of gel precursors: 1 h
Step 8, synthesis of gel precursors: 2 d
Steps 9–11, synthesis of gel precursors: 2–3 h
Steps 12 and 13, gel formation and mechanical characterization: 3 h
Steps 14–17, gel formation and mechanical characterization: 1–2 h
Step 18, gel formation and mechanical characterization: 3 h–overnight
Step 19, gel formation and mechanical characterization: 3 h
Steps 20–25, cell dissociation and embedding: 1.5–2 h
Step 26A, ISC expansion in fully defined hydrogels: 4–6 d
Step 26B, organoid formation in well-defined hydrogels: 6–9 d

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 protocol

Step 27A, ISC release for passaging and downstream analysis: 1–2 h
Step 27B(i–v), immunofluorescence analysis of ISCs in-gel: 2.5 h
Step 27B(vi), immunofluorescence analysis of ISCs in-gel: 3 h–overnight
Step 27B(vii), immunofluorescence analysis of ISCs in-gel: 36 h
Step 27B(viii), immunofluorescence analysis of ISCs in-gel: 8 h
Step 27B(ix), immunofluorescence analysis of ISCs in-gel: overnight
Step 27B(x–xi), immunofluorescence analysis of ISCs in-gel: 5–8 h
Step 27C( i–vi), recovery of organoids from 75% PEG-Acr gels and immunofluorescence analysis: 3 h
Step 27C(vii), recovery of organoids from 75% PEG-Acr gels and immunofluorescence analysis: 3h–overnight
Step 27C(viii), recovery of organoids from 75% PEG-Acr gels and immunofluorescence analysis: overnight
Step 27C(ix), recovery of organoids from 75% PEG-Acr gels and immunofluorescence analysis: 3 h
Step 27C(x), recovery of organoids from 75% PEG-Acr gels and immunofluorescence analysis: overnight
Step 27C(xi), recovery of organoids from 75% PEG-Acr gels and immunofluorescence analysis: 3 h
© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

Step 27D, examination of the effect of proteolytic matrix degradability on ISCs and organoids: variable
Box 2, preparation of premixes: 2 h

ANTICIPATED RESULTS a b
Intestinal crypts or dissociated ISCs embedded within PEG
RGD gels are expected to form colonies within 24 h, with an
efficiency of 20–25%. Within 4 d, the cells should continue
expanding, ultimately giving rise to large (50–200 µm in
diameter) lumenized, spherical colonies featuring columnar
and polarized ISCs that express Lgr5 (Fig. 4a,b). When
dissociated and re-embedded in PEG RGD, the cells should
continue forming colonies and expanding. Quantitative c d
reverse-transcription (qRT)-PCR indicated that ISC colonies
cultured in PEG RGD expressed high levels of Lgr5, compara-
ble to those of cells cultured in Matrigel, whereas markers of
differentiated cells are not expressed at detectable levels6.
Dissociated ISCs embedded in PEG RGD LAM are expected Mucin-2
to form colonies with an efficiency of 20–30%. Upon E-cadherin
DAPI
replacing the expansion medium (ENRCV) with organoid
formation medium (ENR) on day 4, ISC colonies are expected Figure 4 | ISC colonies and organoids formed in PEG-based
to transform into intestinal organoids, containing both hydrogels. (a) Bright-field image of day 4 ISC colonies in PEG RGD.
cycling Lgr5-expression ISCs and differentiated intestinal (b) Immunofluorescence image of day 4 ISC colonies in PEG RGD, showing
cells (Fig. 4c). Gene expression analysis by qRT-PCR indicat- the expressing of Lgr5-EGFP. (c) Bright-field image of organoids formed in
PEG RGD LAM matrices. (d) Immunofluorescence image of an organoid formed
ed that organoids cultured within PEG RGD LAM express Lgr5
in PEG RGD LAM, showing mucin-2-expressing goblet cells. The organoid is
and markers of Paneth cells, goblet cells (Fig. 4d), stained for DNA (1:5,000; DAPI (blue)), E-cadherin (1:100; Thermo Fisher
enteroendocrine cells and enterocytes at levels comparable Scientific, cat. no. 13-1900 (green)) and mucin-2 (1:50; Santa Cruz,
to those of organoids cultured in Matrigel6. cat. no. sc-15334 (red)). Scale bars, 50 µm.

Acknowledgments We thank D. Hacker and the EPFL Protein Expression Core
Facility (PECF) for the production of R-spondin, Noggin and EGF; D. Pioletti for
rheometer use; and N. Brandenberg and A. Negro for technical assistance.
AUTHOR CONTRIBUTIONS N.G. conducted the experiments. M.P.L. and N.G. wrote
the manuscript.
COMPETING FINANCIAL INTERESTS The authors declare competing financial
interests: details are available in the online version of the paper.
Reprints and permissions information is available online at http://www.nature.
com/reprints/index.html. Publisher’s note: Springer Nature remains neutral with
regard to jurisdictional claims in published maps and institutional affiliations.
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