Cell Migration in Shear Field: Comparative Study between
MC3T3-E1 and 3T3-L1
Shigehiro HASHIMOTO, Yuji ENDO, Hiromi SUGIMOTO
Yusei KACHI, Sora TOMA, Ryota MATSUZAWA
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
http://www.mech.kogakuin.ac.jp/labs/bio/
ABSTRACT
Migration of a cultured cell exposed to Couette type of shear
flow has been observed at the incubator microscope in vitro.
The shear flow was made between a rotating disk and astationary disk, which are in parallel position each other. Eachcell adhered on the disk was observed under the steady shear
flow by an inverted phase contrast microscope (shear stresslower than 2 Pa). The migration of cells was evaluated at thetime lapse images (every five minutes for 24 hours). Twokinds of cells were used in the test: MC3T3-E1 (mouse
osteoblast precursor cell line), 3T3-L1 (mouse fat precursor
cells). The experiments show the following results. Under
the shear stress of 2 Pa, 3T3-L1 tends to be rounded andmigrate to the downstream. MC3T3-E1, on the other hand,migrates to every direction.
Keywords: Biomedical Engineering, Shear Stress, MC3T3-E1,
3T3-L1, Couette Flow and Migration.

cone-plate type is one of the famous Couette flow devices[10-11].
In the present study, an experimental system of the Couette typeflow with a rotating disk has been designed to apply wall shear
stress quantitatively on the cell during incubation at the
microscopic observation in vitro, and the effect of the shear
flow on the migration of the cell has been studied.

2. METHODS
Rotating Parallel Disk System
A rotating parallel disk system is used to make Couette type offlow. The fluid is sheared between a rotating disk and astationary disk. The stationary disk is the bottom of theculture dish (diameter 60 mm, Iwaki, Japan) (Fig. 1a).
In the system, the shear rate (γ) is calculated by Eq. 1.
γ=rω/d(1)

1. INTRODUCTION
A biological cell makes several responses in the force field [1].
The effect of the cyclic stretching on alignment andproliferation of osteoblasts has been studied in the previous
study [2]. The flow can be used to apply a stress field to a
specimen. The cells exposed to the flow directly receive theshear stress in the shear flow. Endothelial cells are exposed to
the wall shear stress by blood flow in vivo. The flow direction
affects orientation of endothelial cells [3]. The effect of shear
stress on the response of endothelial cells were studied in theprevious studies: deformation [4], orientation [5], proliferation[6], and tissue formation [7]. The high shear flow might
deform a cell, peel off a cell from the scaffold, and inhibit
proliferation as well as tissue formation. The mild shear flow
under the threshold of the wall shear stress, on the other hand,might accelerate migration, proliferation, and orientation.
These mechanotransduction was analyzed in vitro bymicrofluidics in the previous study [8].
In the previous study, cells were exposed to the shear flow in adonut-shaped open channel [9], and the effect of flow
stimulation on cultured cells has been studied in vitro. Whenthe flow has a free surface, it is difficult to estimate the shear
stress in the fluid. Couette type of flow, on the other hand, isconvenient to realize the uniform shear field [10-15]. A

In Eq. 1, ω is the angular velocity [rad s－1], and d is the
distance [m] between the moving wall and the stationary wall(Fig. 1b). In the rotating parallel disk system, the shear rate (γ[s－1]) increases in proportion to the distance (r [m]) from therotating axis.
The shear rate varies with the rotating speed, which iscontrolled by the stepping motor, which makes variation ofangular velocity ω between 7 rad/s and 33 rad/s. The position
for the observation has the variation on r (the distance from therotating axis) between 12 mm and 18 mm.
The distance d (0.5 mm < d < 0.6 mm) is estimated from thepositions of the focus of the walls at the microscope. The
shear rate (γ) generates the shear stress (τ [Pa]) in a viscous
fluid.
τ=ηγ(2)In Eq. 2, η is the viscosity of the fluid [Pa s]. The fluid is themedium of cell culture in the present study. When theviscosity of the fluid η is 0.002 Pa s (at 310 K), the shear stressτ varies between 0.5 Pa and 2 Pa.

stress is observed with the microscope. The system allows
observation of cells during exposure to the shear flow.

Rubber belt
Motor

Bearing
Rotating disk

50 mm
Dish
Fig. 1a: Cell culture device with rotating disk.

To make adhesion of cells to the bottom of the dish, the cells
were cultured for 24 hours in the incubator without flow
stimulation (without rotation of the disk). After the incubationfor 24 hours, the cells were sheared with the rotating disk for 24hours in the incubator (Fig. 2).

ωrRotating Disk

Microscopic Observation
The time-lapse image was taken every five minutes during thetest. The direction of the flow adjacent to the culture plate wastraced by the floating particle in the medium at the video image,and defined as the x axis in Figs. 5 & 6.

dγ

Cell Culture
Two kinds of cells were used in the experiment: MC3T3(osteoblast precursor cell line derived from Mus musculus(mouse) calvaria; passage between fourth and ninth) -E1, and
3T3-L1 (mouse fat precursor cells, a cell line derived from cellsof mouse 3T3; passage between third and ninth). D-MEM(Dulbecco’s Modified Eagle Medium) containing 10%
decomplemented FBS (Fetal Bovine Serum) and 1% penicillin/
streptomycin was used for the medium.

Culture DishFig. 1b: Couette flow between rotating disk and stationary disk
(culture dish).

At the microscopic image, the contour of each cell was traced,
and the apparent adhesion area (S) of each cell to the scaffold
was calculated. The centroid of each cell image was used to
trace the migration of the cell.
The contour of each cell was approximated to the ellipsoid (Fig.
3), and the shape index (R) was calculated by Eq. 3.
R = 1 – (b / a)Fig. 2: Experimental protocol: resting (static), and flow
(stimulation).

(3)

In Eq. 3, b is length of the minor axis, and a is length of the
major axis of the ellipsoid. R is zero at the circle (a = b). R
approaches to unity, when the major axis (a) increases as theelongation of the cell. At each cell, the angle (θ) between the
major axis and the flow direction was measured. The angle (θ)
is zero degree, when the major axis is parallel to the flow
direction.

3. RESULTSab0.05 mmFig. 3: Contour of cell (yellow, left) is approximated toellipsoid (right), and major (a) and minor (b) axes are calculated(red).

The rotating disk system is mounted on the stage of the inverted
phase contrast microscope mounted in the incubator(LCV-110SK, Olympus Co., Ltd., Tokyo). In the incubator,
both the temperature and the partial pressure of carbon dioxide
are maintained at 310 K and 5 percent, respectively. The
behavior of cells adhered on the stationary wall under shear

Fig. 4 exemplifies cells, which are adhered on the bottom
surface of the culture dish, exposed to Couette type of the shear
flow at the shear stress of 2.0 Pa: MC3T3-E1 (a), and 3T3-L1(b). The arrow shows the direction of the flow. The left
image shows cells at the beginning of exposure to the shear
flow, and the right image shows cells after 24 hours exposure to
the shear flow. The most of cells of 3T3-L1 are exfoliatedafter 24 hours of exposure to the shear flow, while the most ofcells of MC3T3-E1 remains adherence on the plate.
Figs. 5 & 6 exemplify migration of twenty cells for 24 hours.
In Figs. 5 & 6, the direction of the flow is from left to rightalong the x axis. The y axis shows the direction of the centerof the rotation. The wall shear stress is proportional to thewall shear rate, so that the wall shear stress decreases with ycordinate. The original position of each cell is adjusted to theorigin of the coordinate. To tarce the migration of each cell,
the image of cell every one hour has been selected from thetime lapse image. Each polygonal line, which consists of theconnected line segments, shows the locus of coordinates of thecentroid of each cell traced every hour (Figs. 5 & 6). Without
flow, each cell migrates on the culture plate (on the scaffold) torandom direction (Figs. 5a & 6a). MC3T3-E1 migrates at
random including to the perpendicular direction against the floweven at 2 Pa (Fig. 5). 3T3-L1, on the other hand, migrates tothe downstream direction under the shear stress higher than 1 Pa
(Figs. 6c-6e). Especially at 2 Pa, the larger migration of
3T3-L1 occurs to the downstream direction.
Fig. 7 exemplifies tracings of two parameters of a cell (3T3-L1)
every hour: the area (Fig. 7a) and the migration (Fig 7b),
simultaneously. When the area increases, the migration tends
to decrease.

y [μm]

x [μm]

Fig. 5b: Migration of twenty cells (MC3T3-E1). (0.48 Pa < τ <0.51 Pa)y [μm]

Fig. 8 shows the accumulated migration of each cell related tothe shear stress: MC3T3-E1 (Fig. 8a), and 3T3-E1 (Fig. 8b).
The mean value of accumulated migration of each cell isapproximately 0.5 mm regardless of the shear stress condition(< 2 Pa). Only the value of accumulated migration of 3T3-L1 at
2 Pa is significantly higher.
Fig. 9 shows the area (S) of each cell related to the shear stress.
Each datum shows the value, which is calculated as the meanvalue from data every hour for 24 hours (Figs. 9-11). The
mean value is highest at 1.5 Pa at both kinds of cells:
MC3T3-E1 (Fig. 9a), and 3T3-L1 (Fig. 9b).

x [μm]

Fig. 5c: Migration of twenty cells (MC3T3-E1). (1.04 Pa < τ <1.10 Pa)y [μm]

x [μm]
0.2 mm

0.2 mmFig. 4a: MC3T3-E1 at 2 Pa; 0 h (left), 24 h (right), flow
direction (arrow), bar 0.2 mm.

Fig. 5d: Migration of ten cells (MC3T3-E1). (1.41 Pa < τ <1.58 Pa)y [μm]

0.2 mmx [μm]

0.2 mmFig. 4b: 3T3-L1 at 2 Pa; 0 h (left), 24 h (right), flow direction(arrow), bar 0.2 mm.
y [μm]

Fig. 5e: Migration of ten cells (MC3T3-E1). (1.92 Pa < τ < 2.07
Pa)y [μm]

x [μm]

Fig. 5a: Migration of twenty cells (MC3T3-E1) without flow.

x [μm]

Fig. 6a: Migration of ten cells (3T3-L1) without flow.

y [μm]x [μm]

Fig. 6b: Migration of ten cells (3T3-L1). (0.48 Pa < τ < 0.52
Pa)

Fig. 7b: Migration of 3T3-L1.

y [μm]

x [μm]

Fig. 6c: Migration of ten cells (3T3-L1). (0.97 Pa < τ < 1.03 Pa)y [μm]
Fig. 8a: Accumulated migration of each cell (MC3T3-E1)
related to shear stress: each bar shows mean value.

x [μm]

Fig. 6d: Migration of ten cells (3T3-L1). (1.42 Pa < τ < 1.57
Pa)
y [μm]
Fig. 8b: Accumulated migration of each cell (3T3-L1) related
to shear stress: each bar shows mean value.
xFig. 6e: Migration of ten cells (3T3-L1). (1.93 Pa < τ < 2.10 Pa)

Fig. 9a: Area of each cell (MC3T3-E1) related to shear stress:
each bar shows mean value.

Fig. 7a: Area of 3T3- L1.

Fig. 9b: Area of each cell (3T3-L1) related to shear stress: eachbar shows mean value..

Fig. 10a: Shape index of each cell (MC3T3-E1) related to shear
stress: each bar shows mean value.

Fig. 10 shows the shape index (R) of each cell related to theshear stress: MC3T3-E1 (Fig. 10a), and 3T3-E1 (Fig. 10b).
The shape index of 3T3-L1 slightly decreases under the shear
stress (Fig. 10b), which corresponds to get rounded.
Fig. 11 shows the angle (θ) between the major axis of each celland the flow direction related to the shear stress: MC3T3-E1(Fig. 10a), and 3T3-E1 (Fig. 10b). On MC3T3-E1, thedispersion of θ increases under shear flow, which shows that the
major axis of MC3T3-E1 tilts to the random direction
regardless of the flow direction. On 3T3-L1, on the other hand,the angle θ concentrates to zero degree, which shows that the
major axis of 3T3-L1 tilts to the flow direction.

Fig. 10b: Shape index of each cell (3T3-L1) related to shear
stress: each bar shows mean value.

4. DISCUSSION
The Couette type of flow is convenient to estimate the shear
stress in the flow with the uniform shear rate between themoving wall and the stationary wall, which is also available tonon-Newtonian fluid [10-15]. Many kinds of the devices
including pulsatile type flow [12, 13] were designed forquantitative experiments of biological fluid in the previous
studies.
The “cone and plate type” device has the uniform shear field in
the entire space between the rotating cone and the stationary
plate [10, 11]. The clot formation was quantitatively studied
between the rotating cone and the stationary plate [10], andbetween the rotating concave cone and the stationary convex
cone [12]. The erythrocyte destruction was studied between
the rotating concave cone and the stationary convex cone [13].
A parallel disks system does not have the uniform shear field in
the entire space between rotating disk and the stationary disk.
The parallel-disks system, on the other hand, has several
advantages: stability of the rotating motion of the disk, stabilityof the optical path for the microscopic observation,
morphologic preciseness of the plane of the disks, andsimultaneous observation over the range of variation of shear
rate proportional to the radius from the rotational axis [15].
The erythrocyte deformation was observed between counterrotating parallel discs [14]. In the present study, the rotating
parallel disk system is selected to make Couette type of flow
instead of the cone and plate system. The range of the shear
stress lower than 2 Pa has been selected in the present study.
That is a typical value for the wall shear stress of the bloodvessels in vivo estimated by the vessel diameter and the flowrate in Poiseuille flow.

Fig. 11a: Angle of each cell (MC3T3-E1) related to shear
stress: each bar shows mean value.

Fig. 11b: Angle of each cell (3T3-L1) related to shear stress:
each bar shows mean value.
At the constant angular velocity of 31 rad s－1 (d = 0.5 mm), theshear rate (γ) increases from 0.73×103 s－1 to 1.12×103 s－1,
when the distance from the axis (r) increases from 12 mm to 18
mm in the observation area (Eq. 1). The gradient of shear
stress (0.6 Pa / 0.006 m = 102 Pa m－1) enables the simultaneous

observation of the behavior of cells related to variation of theshear stress (1.1 Pa < τ < 1.7 Pa) in the same view [15]. In thepresent study, the migration speed has been slower than 1 mm /24 hour = 10－8 m/s. The steady actual flow direction adjacentto the scaffold surface of cell culture has been confirmed by thestream line traced by the direction of exfoliation of the cell andof the moving particle adjacent to the surface.
A biological cell shows passive and active behaviors in anenvironment. While the flow might enhance the cell migration
to the downstream, a cell migrates to adapt the shear field.
While the strong stimulation above the threshold damages thecell, the stimulation below the threshold remains in the cell as amemory for the response in the next step [1, 2]. The hysteresis
effect governs the active behavior of the cell. The interaction
between cells also governs the behaviou of each cell [16]. The
seeding density is selected not so high to trace the image ofeach cell in the present study. The migration of the cell mightalso depend on the morphology and the mechanical property ofthe scaffold [17].
In the present study, each cell migrates independently to every
direction includes the counter direction of the flow. The effectof shear flow on migration of the cell depends on the kind ofcells, which might be applied to the cell sorting technology. In
the present comparative study, MC3T3-E1 and 3T3-L1 areselected for the test to observe the response of the cell to theshear flow. The response of 3T3-L1 adhesion on the scaffold
with the micro-pattern was different from that of the other typesof cells in the previous study [18]. MC3T3-E1, on the other
hand, is one of the typical adhesive cells, which are exposed tothe mechanical force field in vivo.

5. CONCLUSION
Migration of a cultured cell exposed to Couette type of theshear flow has been observed at the incubator microscope invitro. Migration might decrease, when affinity between thecell and the scaffold increases. The shear flow has been madebetween a rotating disk and a stationary disk, which are inparallel position each other. The migration of cells has been
evaluated at the time lapse images for 24 hours. Two kinds ofcells has been used in the test: MC3T3-E1 (mouse osteoblast
precursor cell line), 3T3-L1 (mouse fat precursor cells).
Under the shear stress of 2 Pa, 3T3-L1 tends to be rounded, totilt to the flow direction, and to migrate downstream.
MC3T3-E1, on the other hand, migrates to every direction.

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