Page 36 - ASME_NEMB_2016_Program

Technical Program TRACK 3

Andreas Kourouklis, University of Illinois Urbana-Champaign, Ur- microfluidic device with independently addressable ports and in-vitro testing
bana, IL, United States, Kerim Kaylan, University of Illinois, Urbana, of the device for 2D surface stimulation of explanted rat retinas.
IL, United States, Gregory Underhill, University of Illinois Urba-
na-Champaign, Urbana, IL, United States The preliminary multiport microfluidic device features on-chip reservoirs for
storing chemicals on one side and a 3x3 array of delivery ports (10 microns
Recent efforts have utilized microfabricated platforms such as high-through- dia) on the other, with each port connected to a separate reservoir via a
put cellular microarrays to examine combinations of microenvironmental sig- microchannel. The device (1 cm sq. and 1.3 mm thick) is a composite of two
nals that determine cell functions. To date, the majority of these approaches layers – a silicon layer containing the delivery ports and microchannels and
a glass layer containing the reservoirs – that were independently microfabri-
have focused on the biochemical properties of extracellular matrix (ECM)
or soluble growth factors, and have yet to address the vast number of bio- cated and then anodically bonded. The reservoirs were filled with glutamate
physical cues presented by cellular microenvironments. Here, by integrating (1 mM) and connected to an 8-channel pressure injector that permits inde-
a cellular microarray platform with defined substrates of modular stiffness, pendent actuation of each reservoir via tubing. The device was interfaced
we sought to systematically investigate the combinatorial effects of ECM with an explanted WT rat retina placed over a multi-electrode array (MEA)
with the RGC side contacting the electrodes and photoreceptor side con-
composition and mechanical stiffness on the differentiation of liver progeni-
tor cells. Although several pathways have been suggested to regulate liver tacting the device delivery ports. The RGC responses to the chemical and
progenitor fate decisions, a potential role for biophysical signals had not light stimuli were recorded using the MEA and the recorded data was pro-
previously been explored. Cellular microarrays were fabricated through the cessed using custom Matlab scripts.
seeding of liver progenitor cells onto defined islands of ECM proteins sup- Using short duration (10-30 ms) and low pressure (0.1-3 psi) pulses, nanoliters
ported by polyacrylamide gel substrates. Independently presented islands
of 5 distinct ECM proteins, including their pair combinations, were created of glutamate were injected into the retina through either single or multiple
by means of a contact microarrayer. To control the biophysical stimuli, the ports in various patterns. The glutamate injections stimulated a total of 408
elastic modulus of the polyacrylamide gels was tuned between 4, 13 and 30 unique RGCs across 9 retinas. Most injections produced purely excitatory
kPa by changing the cross-linking density. Progenitor cells were induced to RGC responses but a subset (35%) elicited both inhibitory and excitatory
responses suggesting differential stimulation of the OFF and ON pathways
differentiate within the array of protein islands, and at distinct time points,
both hepatocyte (e.g. albumin) and biliary (e.g. osteopontin) markers were in the inner retina. Stimulations corresponding to single port injections were
quantitatively evaluated using immunostaining and a custom imaging anal- spatially localized with a median spread of 155 microns (equivalent to a
ysis pipeline. The results of these microarray studies suggest that substrate LogMAR value of 1.5), comparable to those of current generation electrical
stiffness influences liver progenitor fate decisions in a manner dependent prostheses and our previous works with micropipettes. Stimulations corre-
sponding to multiple simultaneous injections in simple horizontal/vertical line
on the protein composition of the ECM. In particular, biliary differentiation,
was broadly reduced on 4 kPa substrates compared to stiffer 30 kPa sub- patterns as well as more complex shapes elicited strongly corresponding
strates. However, our results demonstrate that in contrast to type I collagen RGC responses.
and fibronectin, type IV collagen supports biliary differentiation independent
of the elastic modulus of the supporting gel substrate. To further examine If the chemical-evoked neural patterns could be translated to blind human
subjects, the combination of simple patterns such as these might enable
the physical interactions associated with this combinatorial ECM signaling,
we developed an approach for interfacing traction force microscopy with the text recognition similar to the current generation electrical prostheses.
cellular microarrays. The systematic assessment of cell-derived traction forc- Furthermore, the first ever demonstration of biomimetic differential stimu-
es illustrated that the degree of cell traction was consistent with the biliary lation of the inner retinal neurons with exogenous glutamate indicates the
marker expression data, which indicated that liver progenitor differentiation possibility of achieving more effective and naturalistic means of conveying
visual information to the brain with a subretinal chemical-based prosthesis
is force-correlated and further modulated by the protein composition of the
ECM. By merging traction force microscopy with defined biochemical micro- than an electrical prosthesis. These preliminary data supporting localized
environments, we have established a unique approach for investigating cell- and patterned chemical stimulation of the retina suggest that not only a mi-
ECM interactions. Continued efforts focused on the processes guiding liver crofluidic-based chemical retinal prosthesis is feasible, but also it could be
differentiation would form the foundation for the optimization of stem cell a better alternative to electrical prosthesis for people who are affected by
photoreceptor degeneration diseases.
differentiation protocols and the development of cell-based therapies.
The work was supported by the National Science Foundation NSF-EFRI
4:20pm Demonstration of a Multiport Microfluidic Chip with grant number 0938072.
Independently Addressable Ports towards the Development of a
Chemical Retinal Prosthesis 4:40pm Uniform Electric Field Generation in Circular Dishes for
Cell Stimulation by A 3D Designed Polymeric Insert
Technical Presentation. NEMB2016-6119
Technical Presentation. NEMB2016-5946
Corey M Rountree, Ashwin Raghunathan, University of Illinois at
Chicago, Chicago, IL, United States, John B. Troy, Northwestern Hsieh-Fu Tsai, Okinawa Institute of Science and Technology Grad-
University, Evanston, IL, United States, Laxman Saggere, Univ Of uate University, Kunigami kun, Okinawa, Japan, Ji-Yen Cheng,
Illinois/Chicago, Chicago, IL, United States Academia Sinica, Taipei,Taiwan, Amy Shen, Okinawa Institute of
Science and Technology Graduate University, Okinawa, Japan
Photoreceptor degeneration diseases cause irreversible loss of vision for
millions worldwide and no cure exists to restore vision for those affected. This work reports a CAD design principle to fabricate a polymeric insert to
Retinal prostheses based on electrical stimulation are being developed as a create uniform electric field (EF) in circular petri dishes or multi-well plates
restorative aid, but current electrical-based prosthesis technology is limited for cell stimulation.
in its ability to provide high resolution natural vision. To circumvent these lim-
itations, we propose a microfluidic-approach wherein the retina is biomimet- Conventional application of uniform EF in vitro in the physiological range
ically stimulated using native neurotransmitters. We have recently demon- has been achieved in rectangular shaped microfluidic channel. However,
strated the feasibility of this approach by focally injecting 1 mM glutamate existing in vitro electrical stimulation systems have several disadvantages
into wild-type (WT) rat retinas from both the epiretinal and subretinal sides including complicated device preparation procedure, low usage of cell
via a micropipette and eliciting physiological responses from retinal ganglion culture area, low cell yield and low cellular product recovery. The effective
36 cells (RGC). To advance this approach further, we are currently exploring stimulation area percentage to total area reported to date ranged from 4%
2D spatial stimulation of the retina via simultaneous multisite injections of to 45.4%. Using only two-dimensional geometry, it is very difficult to create
glutamate. Here, we present, for the first time, the development a multiport
uniform EF in a circular area from two electric potentials due to different