Application Note 12Biofabrication of hydrogel encapsulated
and cast micro-structures
Jenna James, Joseph Lloyd, Matt Dunn, Daniel Merryweather, Rosemary Fricker, Paul RoachInstitute for Science and Technology in Medicine, Keele University, United Kingdom

INTRODUCTION

Growing cells in 3D has become important in the development of in vitro fabricated tissue.
Developing so-called ‘organ on a chip’ models allows us to mimic how tissues function in asmaller, well defined engineered tissue construct. Various models have been established toreplicate many different organ and tissue types, although the central nervous system remainsa challenge due to the many different intercommunicating cell types and the dependence of
their complex organisation for normal function. The need to develop such complex functionalmodels, particularly using human-relevant tissues (non-animal models) will be highly
relevant for establishing a better understanding of neurological dysfunction and an enabling
technology for clinical intervention, such as regenerative medicine and cell therapies.
Typical in vitro models of neuronal circuits have so far been achieved by growing cells
in a 2D monolayer, being segregated and directed to form a cellular circuit using microstructures. It is well known that cells grown in 2D do not replicate effectively the activity orfunction of native cells experiencing a 3D environment, lacking many of the intercellularand environmental cues that guide standard metabolic behaviour in vivo. Here we haveestablished a method of introducing the GrowDex® nanocellulose hydrogel into micro(fluidic)
devices fabricated using soft lithography for the trivial conversion of a 2D culture
environment to a 3D construct. These devices present micro-channels into which neuronalcell bodies are too large to enter, but neurites are directed along their length to connect
to neighbouring cells on the opposite side. We have been able to embed and cast microstructures down to sub-5 microns in width and height (aspect ratio of 1) with lengths upwardsof 800 microns.

MATERIALSAND METHODS• Molds for microfluidic devices (Fig. 1) were fabricated using conventional lithographic
processing of SU8 to form an inverse master, which was replicated in poly(dimethylsiloxane) PDMS.
• Devices were then fabricated by pouring well-mixed PDMS over the molds in a 10:1
ratio of polymer to cross-linker.
• After two hours at 50°C the PDMS hardened sufficiently to be peeled from the mold.
• Loading ports were made by coring a small hole above the channels using a 3-gauge
corer.
• If casting and lift off of the PDMS was required, a pre-treatment of hydrophobicsilane was used to cover the PDMS at this stage (e.g. chemical vapor deposition ofhexamethyldisilazane at RT for 1 h). This ensured reduced interaction of the GrowDex
with the PDMS allowing lift-off without damage to the micro-structure. If GrowDex was toleft embedded in the structure then this stage was not needed.
• Wettability was confirmed (WCA > 100°C) using sessile droplet shape• The patterned PDMS was then adhered to a cleaned glass slide, oxygen plasma etchedat 30W for 30 sec in a 300 mtor oxygen atmosphere. This step could be replaced withrepeat washes of the glass in tween, water and isopropanol, being dried under clean
nitrogen immediately prior to contact with the PDMS.
• 1% w/v solution of GrowDex hydrogel was prepared using a DMEM-F12 based
culture medium containing 10% v/v human serum, 1% L-glutamine, and 1% pen/strep.
A suitable volume of media was first loaded into an Eppendorf tube followed by therequired quantity of stock GrowDex solution. It was found that loading in this manner
greatly reduced bubble formation and that the hydrogel set significantly faster in thepresence of media compared to PBS.
• The hydrogel solution was introduced into the devices using an 18G syringe needle andallowed to fill the microfluidic channels via capillary action at room temperature, Fig 1.
• After one hour each device was filled with culture medium and incubated at 37°C for ten
days, replenishing media lost to evaporation on alternating days.
• If cell culture was required within the device, cells were mixed with GrowDex solution
prior to injection.
• If cast features were required, the PDMS was carefully lifted off after gelation to reveal
the hydrogel features standing proud of the glass surface, Fig. 1B.

Figure 1. Microscope images of 1% w/v GrowDex hydrogel A) cast and retained within
micro-channels and B) cast and mould removed, after standing at 37°C for ten days withmedia replenishment on alternating days. Insert in B) shows schematic representation of castgel structures.

RESULTS

CONCLUSIONS

Features cast in GrowDex were stable for long periods both cast within the PDMS device
and presented alone on a glass substrate. Features were well replicated, with porosity of
the hydrogel not presenting an issue at this size scale. A neuronal cell line (SH-SY5Y) andprimary cortical neurons (E16) were cultured within the structures showing good viability
over several days, along with neurite outgrowth where media supplements were used toallow this (retanoic acid for cell lines support this).

We report a protocol for the replication of micro-structures with hydrogel. 1% v/w solutionof GrowDex was suitable to allow its injection into a microfluidic device, to replicate feature
structure and sustain cell culture. Determining the most effective mixing technique (such as
the order in which media and gel were mixed) and the equipment that should be used also
formed a key part of the resulting protocol.

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