Cell migration plays an important role in physiology and disease (Gardel,
Schneider et al. 2010). Cells fail to migrate to the right position during embryogenesis
may cause severe development disorders. Cancer cells might change the extra cellular
matrix (ECM) and induce a series of events to help them migrate to other organs, the
metastasis. For tissue engineering, constructing an appropriate microenvironment and
guiding the cells to the right place are critical for cells to function as they did in the
original organs. Therefore, understanding the fundamental mechanisms of cell
migration is crucial to create strategies for disease treatment and regenerative
medicine.
Biochemical and biomechanical cues are two major stimulation types to guide
cell migration, and clarifying how these signals affect cell migration is an important
research field. Microfluidic systems are capable to generate stable linear or
logarithmic chemical gradients and therefore are widely used to study chemotaxis,
which describe how chemical factors attract or repel cell migrate along the direction
of chemical gradient. Human neutrophils migrate to the place with the highest
concentration of interleukin-8 (IL-8) and demonstrate different behaviors to different
gradient patterns (Li Jeon, Baskaran et al. 2002). Venular and arterial endothelial cells
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show different reactions to the gradient of fibroblast growth factor 2 (FGF2) and
different gradient strengths of vascular endothelial growth factor A 165 (VEGFA165)
could induce human umbilical vein endothelial cells (HUVECs) to generate different
chemotaxis responses (Barkefors, Le Jan et al. 2008).
Astrocyte has been observed to migrate toward hypotonic region under an
osmotic gradient (Saadoun, Papadopoulos et al. 2005), and that observation might be
involved with the water flow through aquaporins (AQPs) on the cell membrane
(Papadopoulos, Saadoun et al. 2008) Some other experiments also show that the
osmolarity play a role in controlling cell motility (Loitto, Karlsson et al. 2009). A
theoretical study proposes that the osmotic gradient can induce water flux to facilitate
the cell motility (Jaeger, Carin et al. 1999). Although the mechanism is still not clear,
the osmotic gradient might be a fundamental mechanical stimulation to guide cell
migration.
Substrate stiffness is another important mechanical stimulation that has been
shown to control the differentiation of mesenchymal stem cells (Engler, Sen et al.
2006) and cell migration. 3T3 fibroblast (Lo, Wang et al. 2000), vascular smooth
muscle cells (Wong, Velasco et al. 2003; Isenberg, DiMilla et al. 2009) and
macrophages (Nemir, Hayenga et al. 2010) have been demonstrated that they would
migrate to stiffer region from softer region of the substrata. This phenomenon is
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durotaxis, and the most common material to study how cell response to substrata
biophysical signals is polyacrylamide (PA) gel (Gribova, Crouzier et al. 2011). The
preparation and characteristics of polyacrylamide gel are well established and studied
(Tse and Engler 2010).
Although these studies have shown deep insights to explain the relation between
one specific signal and cell migration, there are still few have built an environment
providing dual or multiple gradient cues simultaneously. Understanding the cell
behaviors under a more in vivo-like situation will further extend our knowledge to cell
migration and have more reliable information for medical application. However, this
idea is mainly limited by the nature that one method can usually generate only one
well-controlled signal. One of the approaches to overcome this challenge with
microfluidics is designing a functional channel-geometry to generate shear stress
gradients in one cell culture chamber, thus it can cooperate with chemical gradient to
guide cell migration at the same time (Park, Yoo et al. 2009). Despite their many
benefits, there are a number of small, but important, problems. For example, the two
stimulations could not be controlled independently since the chemical gradient is also
influenced by the flow profile. Another weakness is that the real flow velocity is very
difficult to be measured for the flow direction is not simply straight. Moreover, the
properties of substrata still could not be changed while these signals are also critical to
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guide cell migration.
In this study, we propose a new approach that integrates two different systems
into a dual cues device which can generate substrate stiffness and osmotic gradients
simultaneously. The two systems in this device are: (1) a gradient-compliant PA gel
which is responsible for the first mechanical cue, the substrate stiffness gradient, and
(2) a microfluidic system which can generate the second mechanical cue, the osmotic
gradient. Sucrose is selected here to adjust the osmolarity of the cell culture media,
and it has been used before to study the effect of hypertonic stress on C2C12 cells
(Alfieri, Bonelli et al. 2006). The design of the microfluidic system can be divided
into two sides: (1) the source side which continues to provide cell culture media with
highest sucrose concentration to the cell culture chamber in the microfluidics, and (2)
the free side which allows the cell culture chamber to access the bulk solution without
sucrose. The gradient is built through the diffusion of sucrose from source side to free
side and can be maintained for long term cell culture studies. To develop our dual
cues device, we have created two new steps to embed the PA gel in the microfluidic
system and protect the gel from damages during the conventional
poly(dimethylsiloxane) (PDMS) microfluidics fabrication process.
The C2C12 mouse myoblast is an appropriate candidate to be investigated with
osmotic and stiffness gradients. This cell line has been shown that the expression of
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AOP5 is regulated by differentiation and hypertonic stress (Hwang, Lee et al. 2002).
The morphology of C2C12 cells is found to depend on substrate stiffness (Engler,
Griffin et al. 2004). However, the effects of these mechanical cues on C2C12 cell
migration are still not clear.
We hypothesize that C2C12 cell migration can be influenced by both stiffness
and osmotic gradients and the two signals can facilitate with each other to guide cell
migration. So far, we have successfully performed C2C12 cell culture in our device.
We also cultured C2C12 cells with a high osmolarity cell culture medium in the
culture dish, and analyzed cell migration on a gradient-compliant PA gel. These
results provide important information for further experiments to test our hypotheses.
The device presented in this study might be the first one to embed a
gradient-compliant PA gel in a microfluidic system, and this device has the potential
to be used to study cell migration under two different stimulations which is more
close to the in vivo system. The first stimulation is stiffness gradient, and the second
one can be osmotic gradients or theoretically, chemical gradients if the solute is
replaced with chemoacttractants, thus making the device more flexible to be adopted
for other studies.
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