(447e) Noninvasive Manipulation of Cells and Chemicals within Live Cultures Via Addressable Microfluidics

text-align:justify">Introduction: According to the U.S.
Department of Health & Human Services, nearly 115,000 people in the U.S
needed a lifesaving organ transplant in 2018, while only ~10% of them have
received it. Yet, almost no artificial products are commercially available
today – three decades after the inception of tissue engineering. The two major bottlenecks restricting the progress of
recreating complex organs and tissues in vitro are: 1) product size limitation,
due to inability to deliver nutrients to inner pore space of large scaffolds; and
2) product variability, due to the lack of access and control over cells post
seeding.  In this
study, we hypothesize that in order to overcome these obstacles, an ideal
scaffold should be composed of the following elements:  (1) Active pores capable of delivering
nutrients, oxygen and chemical signals throughout the scaffold’s pore space,
(2) Transparent material for real-time microscopy observation of cell behavior
and tissue development, (3) Targeted localized cell seeding/chemical delivery and/or
sampling to enable tissue patterning and non-invasive monitoring at different
locations within the scaffold  (4)
Interactive, continuous, closed-loop spatial and temporal control of the
biology occurring in the scaffold throughout the whole culturing process.  We further hypothesize that these goals can be achieved by merging microfluidic and scaffold
technologies.  To that end, our
proof-of-concept device utilizes addressable microfluidic plumbing in order to
perform noninvasive cell and chemical manipulations within the live culture.

Materials and Methods: Multilayered PDMS devices were fabricated using soft photolithography.
The microfluidic plumbing was actuated pneumatically via
external solenoids, driven by a Wago 750 PLC
controller programmed with a custom Matlab code.  Directed migration of mouse embryo NIH/3T3 fibroblasts
and normal human dermal fibroblasts was induced via a timed
release of the platelet-derived growth factor – BB (PDGF-BB) chemoattractant.  Cytoskeleton-altering drugs (Blebbistatin, cytochalasin
D, and Nocodazole) and osteogenic differentiation
factors (dexamethasone, b-glycerophosphate, 1,25-Dihydroxyvitamin D3, ascorbate and BMPs) were used for
patterning the morphology and the lineage of human mesenchymal stem cells, respectively.  A non-invasive alizarin red assay was performed by sampling the patterned culture by reversing
the flow through the selected microfluidic ports. Time-lapse phase-contrast
imaging of the cells behaviors were performed using a
fully automated Olympus IX83 microscope operated by custom-written software.

Results and Discussion: We created a proof-of-concept automated microfluidic
platform, which uses a combination of micro-sized channels and valves in order
to actuate XY ports (aka “addresses”) independent of each other (see Figure 1a for operation).  We then used these ports to
perform the following manipulations:  1)
Seeding cells in pre-determined patterns, by delivering them through selected
addresses; 2) Nourishing them by continuously renewing the culture media; 3) Patterning
tissue via delivery of differentiation factors to specified locations within
the device; 4) Modifying cell morphology via delivery of cytoskeleton-altering
drugs to selected cells; 5) Controlling cell migration by guiding the cells via
chemoattractant delivery in front them (see normal">Figure 1b).  Finally,
we demonstrated the device’s ability to sample a living culture non-invasively
by picking up and, sending off for analysis, effluents from different locations
above the cell culturing layer.

Conclusions and Future Work: We have presented a microfluidic
platform that can control the spatial delivery/sampling of chemical for manipulation
of single-cell behavior in the XY plane.  In the
near future, we will extend this concept to XYZ manipulations within
transparent, biocompatible and biodegradable 3D scaffolds. It is our hope that
the technology will ultimately resolve the bottlenecks plaguing tissue engineering technologies today, and ultimately enable
computer-driven tissue engineering through coupling with automation electronics.  This would, in turn, allow
machines to culture tissue reproducibly, and without the need to train hospital
staff onsite.