ABSTRACT Biomolecular signaling is of utmost importance in governing many biological processes such as the patterning of the developing embryo where biomolecules regulate key cell-fate

decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in

time and space, long-term (stem) cell culture at the micro-scale is often far from ideal due to medium evaporation, limited space for cell growth or shear stress. To overcome these

challenges, we here introduce a concept based on hydrogel microfluidics for decoupling conventional, macro-scale cell culture from precise biomolecule delivery through a gel layer. We

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2023 INTRODUCTION Spatiotemporally variable signaling cues control cellular and multicellular behavior in many crucial biological systems. A case in point is the concentration- and

time-dependent display of biomolecules termed morphogens that locally control key cell-fate decisions such as lineage commitment during the patterning of the early embryo1. Insights gained

from developmental biology studies have also spurred advances in differentiating pluripotent stem cell such as embryonic stem cells (ESC) into various specialized cell types2. However,

typical ESC differentiation protocols, relying on the formation of cell aggregates termed embryoid bodies (EB), expose cells to bulk culture conditions that crudely recapitulate the

intricate biomolecule display found during embryogenesis. Although this method allows at least to some extent the temporal recapitulation of embryonic gene expression patterns in vitro,

developing EBs lack an embryo-like spatial organization3. The delivery of graded biomolecules by traditional methods such as micropipettes or the Boyden chamber is rather limited in

mimicking natural biomolecule presentation. The emergence of microfluidic technology has revolutionized the generation of in vitro model systems by affording very precise, picoliter-scale

fluid handling, parallelization of experiments and minimization of reagent consumption4,5. Microfluidic chips also offer unprecedented means to systematically probe the role of

microenvironmental signals on stem cell fate in high-throughput6 and with exquisite spatiotemporal resolution7,8. Application of such microsystems have for example allowed to polarize single

EBs9 or direct neural progenitor differentiation by soluble gradients10. However, state-of-the-art microfluidic culture systems are often not ideal for long-term (stem) cell culture11,12.

Firstly, since cells are continuously exposed to fluid flow in most microsystems, the constant removal of autocrine signals and shear stress may be problematic13, even though more complex

shear-free systems have been reported14,15,16,17,18,19. Secondly, microsystems are typically made of poly(dimethylsiloxane) (PDMS), a material that, despite its many advantages for

microfabrication and its excellent gas permeability20, suffers from susceptibility to liquid evaporation, protein adsorption from the medium21, leaching of non-reacted compounds and

hydrophobic recovery22,23. Thirdly, it is not trivial to functionalize PDMS surfaces with biomolecules in order, for example, to mimic some of the natural interactions of stem cells with

their microenvironment that are often critical in maintaining stem cell function24. To overcome some of these issues, researchers have started to incorporate hydrogels into PDMS microfluidic

chips20, for example to generate gradients within 3D scaffolds25 or shield cells from flow and shear stresses26,27. In some cases, PDMS has been entirely replaced by hydrogels to generate

‘biomicrofluidic’ networks3,20,28 or scaffolds for tissue engineering21,29,30. However, despite these advances, key challenges related to micro-scale cell culture remain: The limited space

available for cell growth and tissue development or the difficulty to perform routine cell culture manipulation such as passaging cells or remove cells from chips for downstream analyses

have not yet been overcome31. We reasoned that a simple hybrid system combining traditional cell culture in multiwell plates with microfluidic biomolecule delivery might offer a powerful

means to tackle these challenges. We consequently designed a microfluidic hydrogel chip comprising an engineered culture substrate that could be used for ‘macro-scale’ cell culture, as well

as embedded microchannels that could be used for the precise spatiotemporal biomolecule delivery through the gel layer ( FIG. 1 ). Here we report the design and characterization of this open

access hydrogel microfluidic system for both adherent or aggregate (_i.e._ spheroid)-based cell culture and provide proof-of-principle for its application for the spatiotemporally

controlled manipulation of mouse ESCs fate via delivery of a morphogen gradient. Since the microchip can be fabricated from different hydrogel types whose bulk and surface properties can be

engineered, our hydrogel chip technology should be amenable to probe and manipulate the fate of multiple cell types. RESULTS AND DISCUSSION DESIGN AND FABRICATION OF THE HYDROGEL MICROCHIP

To reduce the concept shown in FIGURE 1 to practice, we first created a PDMS mold by standard photo- and soft-lithography processes ( FIG. 2A and ESI FIG. S1A,B , scheme 1) allowing the

fabrication of molds of various shapes, sizes and types of micro-scale features (e.g. microchannels or micropillars). In the simplest prototype, each chip comprises a cell culture surface (h

= 5 mm, ø = 6 mm) and two embedded individually addressable microchannels (h = 400 μm, w = 200 μm). The hydrogel chip was formed by injecting a reactive gel precursor solution into the PDMS

mold ( FIG. 2A step 1 and ESI FIG. S1A,B , scheme 2), resulting in a hydrogel chip upon crosslinking ( FIG. 2A and ESI FIG. S1A,B , scheme 3). Upon completion of cross-linking, the hydrogel

chips were retrieved from the mold, placed at the bottom of a standard multi-well plate and immersed in water. Hydrogel chips were stored at 4°C for at least a day prior to use. ESC CULTURE

ON THE HYDROGEL CHIP SURFACE To target adherent cell culture applications, in particular those that include pluripotent stem cells, we reasoned that gelatin-based hydrogels might provide an

adequate substrate. Most mammalian cells express integrins that bind gelatin domains (e.g. integrin αvβ3 binding RGD sites32) and pluripotent stem cells are routinely expanded on

gelatin-coated substrates where they form adherent colonies. We therefore molded a gelatin-based hydrogel chip by cross-linking a 10% (w/v) solution of porcine gelatin using bacterial

transglutaminase (TG)28,33 ( FIG. 2B ). This enzymatic cross-linking scheme was chosen to stabilize the physically cross-linked and temperature-sensitive gelatin network, avoiding

destruction of the relatively fragile features during demolding. Indeed, TG-cross-linked gelatin gels sustained mouse ESC adhesion and self-renewal under standard culture conditions, as

demonstrated by efficient colony formation and maintenance of Oct4 expression ( FIG. 2C ). To target in vitro culture applications that are based on cell aggregates such as EBs or

neurospheres (_i.e._ free-floating colonies of neural stem/progenitor cells34), we sought to generate a surface topography that could be used to enhance, for example, homogeneity of EB

differentiation or bias lineage commitment depending on EB size35. To this end, an AggreWellTM chip (STEMCELL Technologies) served as a template to fabricate the desired topography on a PDMS

cylinder that could then be inserted into the mold ( ESI FIG. S1B ). A microwell array containing 100 microwells per chip was successfully molded into an agarose hydrogel surface ( FIG. 2D

). Highly uniformly sized ESC aggregates were obtained upon gentle centrifugation of cell suspensions of variable seeding density (ranging from 100 to 4000 cells) per aggregate loaded on the

surface of the hydrogel chip ( FIG. 2E,F and ESI FIG. S2 ). These aggregates were cultured over several days on the hydrogel chip surface ( ESI FIG. S2 ) and can be easily removed for

downstream assays such as differentiation ( ESI FIG. S2 ). Therefore, our hydrogel chip surface is well suited for ESC expansion cultures. HYDROGEL MICROFLUIDICS: CHARACTERIZATION OF

BIOMOLECULE DELIVERY THROUGH THE HYDROGEL CHIP We next characterized the delivery of biomolecules from microchannels embedded in the hydrogel slab ( FIG. 3 ). In a first approach, simple

pipette tips inserted into the channel inlets and outlets were used as reservoirs to deliver biomolecules through the chip by hydrostatic pressure ( FIG. 3A ). Solutions of fluorescein

isothiocyanate (FITC) and red-fluorescent bovine serum albumin (DsRED-BSA) as model biomolecules were loaded at 0.1 mgmL−1 into the reservoirs. Time-lapse fluorescence microscopy showed that

the molecules initially filling the microchannels rapidly diffuse outwards until depletion. This resulted in the generation of anti-parallel overlapping gradients ( FIG. 3B,C ). Intensity

profiles show transient gradients with decreasing intensity over time in good accordance with a finite element analysis ( ESI FIG. S3A ). In a second approach, to ensure continuous

replenishment of factors, we used commercial syringe pumps to perfuse the hydrogel microchips with the model molecules at a flow rate of 0.01 μLmin−1 ( FIG. 3D ). These experiments showed

rapid diffusion and the generation of gradients of increasing intensity over time ( FIG. 3E and ESI FIG. S4 ), again in accordance with predictions from simulations ( ESI FIG. S3B ).

Importantly, by dispensing biomolecules in a pulsatile fashion, stable long-term gradients were also generated. Intensity profiles initially increase over a period of three hours before

remaining stable over extended periods of time ( FIG. 3F ). Together, these data show that by fluid handling in microchannels embedded in the hydrogel chip, biomolecules can be delivered to

build up transient or stable gradients. SPATIOTEMPORALLY CONTROLLED NEURAL DIFFERENTIATION OF ESCS VIA GRADED RETINOIC ACID DELIVERY To validate this versatile cell culture platform, we

tested the spatiotemporally controlled induction of neurogenic differentiation of ESC under serum-free culture conditions (N2B27 medium36) by delivery of a gradient of the morphogen retinoic

acid (RA) ( ESI FIG. S5 ). To probe induction of neurogenic differentiation _in situ_, we used a Sox1-GFP reporter ESC line36. Uniform EBs composed of circa 400 ESCs were exposed to RA

gradients for two, six, eight or twelve hours and kept in culture for an additional three days ( FIG. 4 ). Bright-field and fluorescent images were acquired in mosaïque mode at day four of

differentiation. Image analysis was performed to quantify Sox1 expression in individual EBs on the entire microwell array. Stitched images show a striking position- and thus

concentration-dependent effect of RA on EB size and Sox1-expression with EBs close to the RA source showing strongest neural induction ( FIG. 4A–D and ESI FIG. S6 ). EBs close to the RA

source are significantly larger (216 ± 41 μm) than far away from the source (124 ± 42 μm) ( ESI FIG. S7 ). The difference in size is likely due to differential cell proliferation that

precedes commitment. The GFP expression profiles along the RA gradient was found to transition from a relative flat line for short exposure times (two and six hours, FIG. 4E,F ), whereas the

profile became increasingly steep for longer exposures (eight and twelve hours, FIG. 4G,H ). Statistical analysis revealed a significant dose dependency of GFP expression for RA gradients

delivery for six hours or longer. These data suggest that the temporal modulation of RA delivery influences neurogenic induction in our in vitro model. It is well known that morphogen

signaling in vivo is highly complex, not only spatially but also temporally1. For example, recent in vivo visualization of RA in zebrafish embryos via genetically encoded probes for RA

allowed for the first time to detect short lived (< six hours) RA gradients (ranging from two to six nM) during embryogenesis37. Although the RA gradient ranges (0.01 to 100 nM) and

exposure times tested here are different, species differences could explain the requirement of higher RA concentration and longer exposure times for neurogenic commitment of mouse ESC.

CONCLUSIONS We present a simple yet powerful concept to deliver biomolecules in a tightly controlled fashion to stem cells cultured in a conventional, macro-scale format. Our chip design

allows the decoupling of cell culture from microfluidic manipulation, which we think solves a key problem related to microfluidic long-term cell culture. By selecting different hydrogel

systems for the chip fabrication, we demonstrate that adherent and spheroid-type ESC culture is possible. For example, the introduction of a surface topography allows the formation of an

array of uniformly sized EBs that can be cultured over up to at least a week, not different from conventional assays. We believe that the direct access to cells on the substrate is important

because cells can be easily retrieved to perform downstream analyses such as flow cytometry or PCR. The fluid processing within the embedded microchannels affords precise spatiotemporal

delivery of biomolecules comparable to other microfluidic systems. The possibility to fabricate virtually any microchannel layout can be exploited to generate more complex dynamic

microenvironments reminiscent of the morphogen display during embryogenesis. We believe that this approach will for example be useful for testing the effect of the dose and timing of

morphogens, singly or in combination, on stem cell fate, for example to model in vitro more complex developmental processes. The simplicity of the platform and the interface with traditional

multiwell plates will hopefully enable wide adoption in biology laboratories. METHODS MICROFABRICATION Standard photo- and soft lithography processes were used to fabricate customized PDMS

molds ( FIG. 2A , scheme 1). In short, the bottom piece of the PDMS molds were fabricated as follow: (i) a PDMS bearing the desired microchannel layout was fabricated by injection molding

against a SU8 on silicon master (made in advance in a clean room, alternatively stacked layers of tape could be cut to create the desired microstructure). Note the custom-made PMMA mold we

use to fabricate these chip comprise a cylinder (h = 5 mm, ø = 6 mm) to fabricate a macro-well on the opposite side of the microstructures. (ii) This PDMS chip was plasma treated for two

minutes, immersed in Sigmacote® (Sigma) for two minutes and dried overnight. (iii) Uncured PDMS was then poured onto the Sigmacoted PDMS chip for PDMS/PDMS replica molding. (iv) After baking

the PDMS chip was removed from the larger PDMS slab that will become the bottom part of our mold for hydrogel chip fabrication. For microwell array fabrication, PDMS pieces from an

AggreWellTM (Stem Cell Technologies) were plasma-treated, immersed in Sigmacote® (Sigma) for two minutes, dried overnight and used for PDMS/PDMS molding. The resulting PDMS pieces were

punched (ø = 6 mm) and inserted into the top part of the PDMS mold (the PDMS chip mentioned above). HYDROGEL MICROFLUIDIC CHIP FABRICATION A detailed fabrication procedure can be found in

the electronic supplementary information (ESI), FIGURE S1. Briefly, for adherent cell culture, the PDMS mold was assembled ( FIG. 2A , Scheme 1) and a gelatin solution (10% w/v, porcine high

strength, Fluka) supplemented with bacterial transglutaminase (1 UmL−1 TG, Zedira) was injected with a syringe. The mold was then placed in a humidified incubator at 37°C for five hours (

FIG. 2A , scheme 2), cooled to 4°C and the cross-linked chips carefully recovered from the mold and placed in a well plate ( FIG. 2A , scheme 3). The wells containing the chips were filled

with PBS, placed at 65°C for 30 minutes to arrest the enzymatic reaction and then stored at 4°C before use in cell culture ( FIG. 2A , scheme 4–5). Chips for spheroid culture were fabricated

similarly (details in ESI FIG. S1B ). The custom-made mold was assembled to bear the micropillar array and a solution of agarose (2% w/v) injected with a syringe. The mold was placed for

ten minutes at 4°C before recovery from the PDMS mold. To ensure tight microchannels, a thin slab of agarose (2% w/v) was heat-bonded (3 sec at 71°C) at the bottom of the agarose chip. After

cooling the construct to room temperature for 10 minutes, it was placed in a well plate, immersed in PBS and stored at 4°C before use in cell culture. CHARACTERIZATION OF BIOMOLECULE

DIFFUSION Fluorescent time-lapse microscopy (Zeiss Axio Observer, Metamorph) was used to characterize gradient generation and evolution over time. Depletion gradients of model biomolecules

(FITC and DsRED-BSA, 0.1 mg mL−1) were obtained by filling (10 μL) custom-made reservoirs (pipette tips inserted in inlets and outlets) with gel-loading tips. In the case of connection to a

syringe pump, 1 mL syringes (Omnifix) were used with tygon tubings (ø = 1.35 mm). The tubings were directly inserted into the inlets of the hydrogel microfluidic chips. Another open tube was

inserted at each outlet to remove excess liquid waste. ESC CULTURE Mouse ESCs were maintained in standard feeder-free culture condition (DMEM, Gibco, sodium pyruvate, non-essential amino

acids, beta mercaptoethanol and supplemented with 15% fetal bovine serum, Hyclone and Leukemia inhibitory factor). FORMATION OF CELL AGGREGATES Centrifugation of cells in microwell array was

used to generate uniformly sized cell aggregates. ESC were passaged and resuspended in N2B27 medium36. Cell suspension containing 2 × 106, 8 × 106, 2 × 107 and 8 × 107 cells per mL were

gently dispensed (50 μL) onto the central cell culture area of the chips to yield 100, 400, 1000 or 4000 cells per well, respectively. The plates were centrifuged at 1000 rpm for three

minutes and placed overnight in a humidified incubator at 37°C. Microscopy (Zeiss Axio Observer) and image analysis (Metamorph) was used to quantify the resulting cell aggregates. INDUCTION

OF EB DIFFERENTIATION BY RA GRADIENTS Mouse ESC aggregates were formed as mentioned above to yield ca. 400 cells per aggregate. The plate was incubated overnight. Reservoirs (pipette tips)

were inserted into the inlets and outlets of the chips. A solution of retinoic acid (25 μL, 10−6 M) was injected into the reservoirs with a gel-loading tip. This solution was exchanged every

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A. Visualization of an endogenous retinoic acid gradient across embryonic development. Nature 496, 363 (2013). Article  CAS  ADS  Google Scholar  Download references ACKNOWLEDGEMENTS We

thank Austin Smith for providing the Sox1-GFP knock-in (46C) reporter mouse ESC line. We thank Yannick Devaud for testing hydrogels for microfluidic fabrication and Sonia Hallen for help

with optimizing ESC aggregate culture and differentiation. This work was funded by the EU FP7 Large-scale integrating project ‘PluriMes’ (http://www.plurimes.eu/) as well as a EURYI award to

M.P.L. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * School of Life Sciences, Institute of Bioengineering and Laboratory of Stem Cell Bioengineering, Ecole Polytechnique Fédérale de

Lausanne (EPFL), CH-1015, Lausanne, Switzerland S. Cosson & M. P. Lutolf Authors * S. Cosson View author publications You can also search for this author inPubMed Google Scholar * M. P.

Lutolf View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.C. and M.P.L. conceived the study, interpreted results and wrote the manuscript.

S.C. performed all experiments. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. ELECTRONIC SUPPLEMENTARY MATERIAL SUPPLEMENTARY INFORMATION

Supplementary Info RIGHTS AND PERMISSIONS This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported license. To view a copy of this license, visit

http://creativecommons.org/licenses/by-nc-nd/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Cosson, S., Lutolf, M. Hydrogel microfluidics for the patterning of

pluripotent stem cells. _Sci Rep_ 4, 4462 (2014). https://doi.org/10.1038/srep04462 Download citation * Received: 28 January 2014 * Accepted: 10 March 2014 * Published: 25 March 2014 * DOI:

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