There is significant interest in developing new approaches for culturing mammalian cells in a three-dimensional (3D) environment due to the fact that it better recapitulates the in vivo environment. The goal of this work was to develop thiol-acrylate, biodegradable hydrogels that possess highly tunable properties to support in vitro 3D culture. Six different hydrogel formulations were synthesized using two readily available monomers, a trithiol (ETTMP 1300 [ethoxylated trimethylolpropane tri (3-mercaptopropionate) 1300]) and a diacrylate (PEGDA 700 [polyethylene glycol diacrylate 700]), polymerized by a base-catalyzed Michael addition reaction. The resultant hydrogels were homogeneous, hydrophilic, and biodegradable. Different mechanical properties such as gelation time, storage modulus (or the elasticity G'), swelling ratio, and rate of degradation were tuned by varying the weight percentage of polymer, the molar ratio of thiol-to-acrylate groups, and the pH of the solution.Cytocompatibility was assessed using two model breast cancer cell lines by both 2D and 3D cell culturing approaches. The hydrogel formulations with a thiol-to-acrylate molar ratio of 1.05 were found to be optimal for both 2D and 3D cultures with MDA-MB-231 cellular aggregates found to be viable after 17 days of 3D continuous culture. Finally, MCF7 cells were observed to form 3D spheroids up to 600 μm in diameter as proof of principle for the thiol-acrylate hydrogel to function as a scaffold for in vitro 3D cell culture. A comparison of the different mechanical properties of the six hydrogel formulations coupled with in vitro cell culture results and findings from previously published hydrogels conclude that the thiol-acrylate hydrogels have significant potential as a scaffold for 3D cell culture.
Microfluidic gradient
generators have been used to study cellular
migration, growth, and drug response in numerous biological systems.
One type of device combines a hydrogel and polydimethylsiloxane (PDMS)
to generate “flow-free” gradients; however, their requirements
for either negative flow or external clamps to maintain fluid-tight
seals between the two layers have restricted their utility among broader
applications. In this work, a two-layer, flow-free microfluidic gradient
generator was developed using thiol-ene chemistry. Both rigid thiol-acrylate
microfluidic resin (TAMR) and diffusive thiol-acrylate hydrogel (H)
layers were synthesized from commercially available monomers at room
temperature and pressure using a base-catalyzed Michael addition.
The device consisted of three parallel microfluidic channels negatively
imprinted in TAMR layered on top of the thiol-acrylate hydrogel to
facilitate orthogonal diffusion of chemicals to the direction of flow.
Upon contact, these two layers formed fluid-tight channels without
any external pressure due to a strong adhesive interaction between
the two layers. The diffusion of molecules through the TAMR/H system
was confirmed both experimentally (using fluorescent microscopy) and
computationally (using COMSOL). The performance of the TAMR/H system
was compared to a conventional PDMS/agarose device with a similar
geometry by studying the chemorepulsive response of a motile strain
of GFP-expressing
Escherichia coli
.
Population-based analysis confirmed a similar migratory response of
both wild-type and mutant
E. coli
in
both of the microfluidic devices. This confirmed that the TAMR/H hybrid
system is a viable alternative to traditional PDMS-based microfluidic
gradient generators and can be used for several different applications.
Culturing cancer cells in a three-dimensional (3D) environment
better recapitulates
in vivo
conditions by mimicking
cell-to-cell interactions and mass transfer limitations of metabolites,
oxygen, and drugs. Recent drug studies have suggested that a high
rate of preclinical and clinical failures results from mass transfer
limitations associated with drug entry into solid tumors that 2D model
systems cannot predict. Droplet microfluidic devices offer a promising
alternative to grow 3D spheroids from a small number of cells to reduce
intratumor heterogeneity, which is lacking in other approaches. Spheroids
were generated by encapsulating cells in novel thiol–acrylate
(TA) hydrogel scaffold droplets followed by on-chip isolation of single
droplets in a 990- or 450-member trapping array. The TA hydrogel rapidly
(∼35 min) polymerized on-chip to provide an initial scaffold
to support spheroid development followed by a time-dependent degradation.
Two trapping arrays were fabricated with 150 or 300 μm diameter
traps to investigate the effect of droplet size and cell seeding density
on spheroid formation and growth. Both trapping arrays were capable
of ∼99% droplet trapping efficiency with ∼90% and 55%
cellular encapsulation in trapping arrays containing 300 and 150 μm
traps, respectively. The oil phase was replaced with media ∼1
h after droplet trapping to initiate long-term spheroid culturing.
The growth and viability of MCF-7 3D spheroids were confirmed for
7 days under continuous media flow using a customized gravity-driven
system to eliminate the need for syringe pumps. It was found that
a minimum of 10 or more encapsulated cells are needed to generate
a growing spheroid while fewer than 10 parent cells produced stagnant
3D spheroids. As a proof of concept, a drug susceptibility study was
performed treating the spheroids with fulvestrant followed by interrogating
the spheroids for proliferation in the presence of estrogen. Following
fulvestrant exposure, the spheroids showed significantly less proliferation
in the presence of estrogen, confirming drug efficacy.
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