Page 87 - ASME_NEMB_2016_Program

TRACK 6 TRACK 6 Technical Program

collagen I hydrogels with monocultures, co-cultures, or tri-cultures of MDA- perform SCFS, and nano-indentation experiments on single cells, and 3D
MB-231 breast cancer cells, normal human dermal fibroblasts (NHDF), and scaffolds with a need for up to 100 microns pulling length. A side view hold-
telomerase-immortalized microvascular endothelial (TIME) cells of varying er enables a side view on the cantilever tip-sample interaction while AFM
cell seeding densities of 1 million cells/ml, 0.5 million cells/ml, and 50,000 experiment. This setup gives optical access to the contact interface during
cells/cm2 respectively. Both NHDF and MDA-MB-231 cells were seeded force spectroscopy, and provides complementary information without ex-
in 8 mg/ml solubilized collagen and polymerized at 37°C in a cylindrical pensive optical z-stacking function.
fluorinated ethylene propylene (FEP) tube, while TIME cells were seeded in
the center of the channel forming an endothelialized lumen through which A new range of experiments is opened up when light is used as a handle for
flow is introduced. Overall, seven groups of cell cultures with different cells the manipulation of cells or molecules rather than a cantilever. We improved
combinations were studied: MDA, NHDF, TIME, MDA+NHDF, MDA+TIME, our optical tweezers system for more flexible 3D experiment geometry, and
NHDF+TIME, and MDA+NHDF+TIME. The influence of intercellular signaling a force sensitivity down to 0.1 pN. Using AOD steering, and multiplexing we
on angiogenic response was analyzed by performing ELISA and quantitative have investigated the frequency dependent rheology on red bloods, and
PCR for the expression of ANG-1, ANG-2, MMP-9, bFGF, PDGF and VEGF simultaneous up to 8 multiplexed traps.
and immunofluorescence staining for F-actin. Three different shear stresses,
4 dyn/cm2 (normal microvascular wall shear stress), 1 dyn/cm2 (low micro-
vascular wall shear stress), and 10 dyn/cm2 (high microvascular wall shear Nano-contact Printing of Netrin-1 Digital Nano-dot Gradients on
stress) were used to test the angiogenic response. To determine the effect Ultra-soft Substrates: The Impact of Substrate Stiffness on Hap-
of flow on cellular behavior and angiogenic response of cells, the micro-
fluidic tumor platform was compared to a static tumor model without flow. totaxis
In the static model in the MDA+TIME and MDA+NHDF+TIME groups, TIME
cells that were in direct contact with MDA-MB-231 cells underwent anoikis, a Poster Presentation. NEMB2016-6110
process when cells detach from the ECM and undergo apoptosis whereas
this behavior was not present in the microfluidic platform with the flow. In Donald MacNearney, Bernard Mak, David Juncker, McGill Univer-
the flow system, TIME cells proliferated, elongated and formed a confluent sity, Montreal, QC, Canada
layer. Also, the secretion levels for VEGF has a similar trend in both systems
but in the static system, the amount of VEGF expressed was much higher. Introduction:
This study improved the complexity of an existing in vitro tumor platform and Cellular navigation, migration, and motility are vital requirements for life,
revealed signaling between MDA-MB-231, TIME and NHDF cells impact mor- whether it be during the developmental stages of an organism, or through-
phology, viability, expression of angiogenic factors. Overall, we concluded out the life cycle - for example during tissue maintenance and wound
that this system provides a mean to study and better understand the dynam- healing. This process is regulated by continuous integration of multiple
ic interplay between stromal cells and microenvironmental factors such as extracellular cues. Surface bound chemical cues regulate cell navigation in
flow which play an important role in tumor progression and therefore must neutrophils, myoblasts, and developing neurons through a process known
be better understood to create effective anti-cancer therapies. as haptotaxis, while mechanical cues, in the form of substrate stiffness, can
also regulate cell motility, through a process commonly denoted durotaxis.
To properly investigate cellular navigation in a meaningful context, it is nec-
6-4 essary to understand the interplay of these different influences. Here, we
NANO-PHENOMENA IN LIVING SYSTEMS present a technique to investigate cellular migration and navigation via sur-
face bound gradients patterned on ultra-soft substrates (E = 1 kPa), and we
use this method to compare the characteristics of haptotaxis on both hard
Grand Ballroom 5:00pm - 8:00pm and soft substrates.
Methods:
Continuous gradients of surface bound proteins were created by patterning
A force toolkit for cell and molecular research digital nano-dot gradients on glass using lift-off nano-contact printing. The
digital nano-dot gradients consist of 200 nm squares with varying pitch,
from 15 um to 300 nm, resulting in a continuous surface density gradient of
Poster Presentation. NEMB2016-5981
the printed protein. A modification of nano-contact printing was developed,
which uses dissolvable thin films of polyvinyl alcohol as an intermediate
Torsten Mueller, JPK Instruments AG, Berlin,Germany, Randy substrate, and the same protein gradients were patterned on Sylgard 527,
Beaubien, JPK INSTRUMENTS USA INC, Carpinteria, CA, United an ultra-soft formulation of PDMS. A reference surface solution was applied,
States, Heiko Haschke, Torsten Jähnke, JPK Instruments AG, Ber- consisting of 75% poly-ethylene glycol grafted with poly-lysine and 25% po-
lin, Germany ly-D-lysine, to obtain the same background surface chemistry on both sub-
strates. C2C12 myoblasts were cultured on the gradients and live-imaged
Topography, roughness, (bio)chemical cues, and mechanical properties of overnight to observe cell phenotypes and cell migration with respect to the
biomaterials are crucial parameters affecting cell adhesion, morphology, and patterned cues.
differentiation. Atomic Force Microscopy (AFM) is a multipurpose technology
suitable for imaging a wide range of different samples with nanometer scale Results:
resolution under controlled environmental conditions, but also for mapping The novel printing technique is shown to generate the desired gradients
mechanical and adhesive properties of sample systems and tissues. of surface bound cues quickly, accurately and reliably. The patterns were
compared with the same patterns on glass, as well as with the computer
We have developed a new distinct imaging mode called “Quantitative Im- generated design files, and excellent agreement was found in both cases.
aging” which is based on fast force-distance curves with intelligent control, C2C12 myoblasts were observed to respond to patterned nano-dot gradi-
to simultaneously obtaining topographic, nanomechanical, and adhesive ents of Netrin-1, migrating towards higher surface densities of Netrin-1 over
sample properties. Additionally, even more complex data like contact point the course of 15-hour live imaging. Preliminary data suggests a stronger
images, Young´s moduli images, or recognition events can be achieved. To migratory response on the ultra-soft substrate. In addition, myoblast pheno-
demonstrate the capability and flexibility of AFM mode, a variety of soft and type and migration speeds were compared between glass and the ultra-soft
hard samples have been investigated. A comparison between “Quantitative substrate, with significantly rounded phenotypes and increased migration
Imaging”, conventional force spectroscopy and traditional AFM imaging velocities observed on the softer substrate.
modes clearly reveals that supplementary information can be gained. 87
Using Single Cell Force Spectroscopy (SCFS), early cell adhesion phenom- Conclusion:
ena can be quantified. A specialized AFM platform has been improved to We developed a novel printing technique which allowed us to pattern con-