Micromolding of shape-controlled, harvestable cell-laden hydrogels
Introduction
Tightly regulated and controlled in situ microenvironments are comprised of cells, soluble factors, and extracellular matrix molecules. [1]. Mimicking the in vivo microenvironment can be useful for a variety of applications such as tissue engineering [2], cell-based assays [3], and directed stem cell differentiation [4]. Hydrogels, are three-dimensional (3D) crosslinked networks of hydrophilic polymers that, resemble the physical characteristics of extracellular matrices [5] and are often used to encapsulate cells. They can be tailored to exhibit high permeability to oxygen, nutrients, and other water-soluble metabolites [6]. Cell encapsulating hydrogels can be used for the generation of 3D tissue engineering structures [7] and immunoisolation microcapsules [8], [9] as well as for use in scalable bioreactors [10].
Typically, cells are encapsulated within hydrogels through mixing a cell suspension with hydrogel precursors followed by crosslinking of the network. The crosslinking reaction may be controlled by a variety of environmental factors such as temperature, pH and the addition of chelating ions. In addition, hydrogels can be photopolymerized in the presence of photoinitiators via exposure to ultraviolet (UV) light [11]. Both biological hydrogels (e.g. fibrin [12], hyaluronic acid (HA) [13], agarose [8]) and synthetic hydrogels (e.g. poly(ethylene glycol) (PEG) [14], [15]) have been used to encapsulate cells. For example, photopolymerized PEG diacrylate hydrogels, have been explored for the transplantation of islets of Langerhans for development of a bioartificial endocrine pancreas [16], [17], [18]. Similarly, photopolymerized hyaluronic acid hydrogels have been investigated as potential implantable/injectable cell delivery vehicles for cartilage regeneration [19].
The encapsulation of cells within hydrogels has been proposed as a method of enabling the scalable expansion of anchorage dependant cells within stirred bioreactors. However, the immobilization of cells within larger structures decreases the viability of cells in the center of these structures due to the lack of appropriate levels of oxygen and nutrients [16], [20]. Spherical microcapsules with high surface area to volume ratios and coated annuli of cells immobilized within polymers have therefore been generated to overcome transport difficulties [8], [19]. Currently most approaches to generate such structures have been based on spherical structures because of the available technologies to generate microscale spheres based on emulsification [10] or shear-induced droplet formation from syringes [8]. These approaches have been shown to be capable of forming spherical cell-laden (microscale hydrogels) microgels of controlled sizes; however, they are not amenable for the generation of other well-defined shapes. Thus, the development of approaches to generate cell-laden hydrogels with controlled sizes and shapes in a homogeneous manner may be of benefit.
Recently, photolithography [21], [22], [23] and soft lithography [24] have been used to encapsulate live cells within microscale polymeric hydrogels (i.e. microgels) anchored onto two-dimensional (2D) surfaces, which offer great potential for diagnostics and cell screening applications. Alternatively, cell encapsulation within free floating microgels (i.e. in suspension) may be advantageous not only for immunoisolation and bioreactor applications where long term cell culture is imperative, but also for tissue engineering. Such systems allow for the creation of micron sized units of tissue that can be potentially assembled to create tissue engineering constructs of controlled microscale structural and architecture. For such applications, controlling the size and shape of cell-laden microgels is important for minimizing diffusion limitations and for exhibiting control over the macroscopic engineered tissue. Micromolding of hydrogels provides a potentially powerful method for fabricating micro- and nanostructures [25], [26]. Micromolding approaches are compatible with soft lithographic technology and therefore greatly minimize the need for costly photolithographic equipment and clean room facilities.
In this paper, we present a micromolding approach for generating cell-encapsulating 3D hydrogels of controlled shapes and sizes in the form of harvestable free units. Cells were suspended in a hydrogel precursor solution containing photoinitiator, deposited onto hydrophilic poly(dimethylsiloxane) (PDMS) patterns, crosslinked under UV radiation, and retrieved upon hydration. Two common photocrosslinkable hydrogel materials, methacrylated hyaluronic acid (MeHA) and poly(ethylene glycol) diacrylate (PEGDA), were tested using this technique, yielding shape-controlled microgels with homogeneous cell distribution at various viable cell densities. Hyaluronic acid is a natural component of the extracellular matrix known for its biodegradable, bioresistant properties [27] and its role in facilitating cellular functions such as adhesion, proliferation, and migration [28], while PEG is an inert, non-biofouling synthetic material often used as templates for immobilizing cells on 2D surfaces [25], [26] or within microfluidic channels [25]. These two diverse materials were both shown to be compatible with this micromolding approach, suggesting the versatility of this technique and the feasibility of developing it further for tissue engineering applications in mimicking the architectural intricacies of physiological cell–cell interactions.
Section snippets
Cell culture
All cells were manipulated under sterile tissue culture hoods and maintained in a 95% air/5% CO2 humidified incubator at 37 °C. NIH-3T3 mouse embryonic fibroblast cells were maintained in Dulbecco's modified Eagle media (DMEM) supplemented with 10% FBS. Confluent dishes of NIH-3T3 cells were passaged and fed every 3–4 days. Murine embryonic stem (ES) cells (R1 strain) were maintained on gelatin treated dishes with media comprised of 15% ES qualified FBS in DMEM knockout medium. ES cells were fed
Microgel fabrication
The procedure illustrated in Fig. 1 was used to fabricate homogenous microgels of specific shapes and sizes. In this process cells were suspended in a hydrogel precursor solution and molded using a PDMS stamp. Subsequently, the hydrogel precursor solution was induced to photocrosslink to form a gel. The mold was then removed to generate an array of micromolded hydrogels that could be harvested into the solution using a simple wash. The techniques can be used to generate microgel suspensions of
Conclusion
In summary, a micromolding technique for encapsulating live cells in microscale photocrosslinkable hydrogels of controlled 3D shapes was developed. The distribution of cells successfully encapsulated in both MeHA and PEGDA hydrogels was found to be homogeneous, and the technique was shown to be amenable for various cell densities. This simple method may be a potentially useful tool for tissue engineering applications that require controlled spatial distributions of cells.
Acknowledgements
The authors would like to acknowledge funding from NIH (HL60435, DE16516), the Institute for Soldier Nanotechnology (DAAD 19-02-D-2002), the Coulter Foundation, CIMIT, and the Draper Laboratory. JMK is supported by an NSERC postdoctoral fellowship. We also acknowledge helpful discussions with Dr. Jason Burdick and Dr. Junji Fukuda.
References (33)
- et al.
Photopolymerizable hydrogels for tissue engineering applications
Biomaterials
(2002) - et al.
Delivery of osteoinductive growth factors from degradable PEG hydrogels influences osteoblast differentiation and mineralization
J Control Release
(2002) - et al.
Interfacial photopolymerization of poly(ethylene glycol)-based hydrogels upon alginate-poly(l-lysine) microcapsules for enhanced biocompatibility
Biomaterials
(1993) - et al.
Layer-by-layer deposition of hyaluronic acid and poly-l-lysine for patterned cell co-cultures
Biomaterials
(2004) - et al.
Photoinitiated crosslinked degradable copolymer networks for tissue engineering applications
Biomaterials
(2003) - et al.
Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells
Biomaterials
(2005) - et al.
Microscale technologies for tissue engineering and biology
Proc Natl Acad Sci USA
(2006) - et al.
Tissue engineering
Science
(1993) - et al.
Probing heterotypic cell interactions: hepatocyte function in microfabricated co-cultures
J Biomater Sci Polym Ed
(1998) - et al.
An extracellular matrix microarray for probing cellular differentiation
Nat Methods
(2005)
Conformal coating of mammalian cells immobilized onto magnetically driven beads
Tissue Eng
Agarose enhances the viability of intraperitoneally implanted microencapsulated L929 fibroblasts
Cell Transplant
Efficiency of embryoid body formation and hematopoietic development from embryonic stem cells in different culture systems
Biotechnol Bioeng
Photopolymerization fundamentals and applications
Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering
Faseb J
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