FU Yi （傅怡）， WU Jian-hua （吳建華）， WU Jie （吳潔）， SUN Ren （孫仁）， DING Zu-rong （丁祖榮），DONG Cheng，3

1. MOE Key Laboratory of Hydrodynamics and School of Naval Architecture， Ocean and Civil Engineering，Shanghai Jiao Tong University， Shanghai 200240， China

2. School of Bioscience and Bioengineering， South China University of Technology， Guangzhou 510006， China

3. Department of Biomedical Engineering， The Pennsylvania State University， University Park， PA 16802， USA

Micro-PIV measurements of the flow field around cells in flow chamber*

FU Yi （傅怡）1， WU Jian-hua （吳建華）2， WU Jie （吳潔）1， SUN Ren （孫仁）1， DING Zu-rong （丁祖榮）1，DONG Cheng1，3

1. MOE Key Laboratory of Hydrodynamics and School of Naval Architecture， Ocean and Civil Engineering，Shanghai Jiao Tong University， Shanghai 200240， China

2. School of Bioscience and Bioengineering， South China University of Technology， Guangzhou 510006， China

3. Department of Biomedical Engineering， The Pennsylvania State University， University Park， PA 16802， USA

The velocity profile around cells in a flow chamber coated with the immobilized protein and the endothelial cells is studied using the micro particle image velocimetry （PIV）. The main purpose is to study the effect of the endothelial cells on the local hydrodynamic environment and the local shear rates above a single polymorphonuclear neutrophil （PMN） and a melanoma cell when they adhere to different immobilized protein substrates. Micro-PIV images are taken in the top-view and the side-view under10X and40X objective lens and the ensemble correlation method is used to analyze the data. The results show that the endothelial monolayer has changed the patterns of the flow velocity profile of the side-view flow on the chamber bottom， and also increased the wall shear rates. The melanoma cells adhered on the immobilized fibrin disturb the local flow more than those adhered on the immobilized fibrinogen， but one sees no significant difference between the local shear rates above the PMNs adhered on the immobilized fibrinogen and those above the PMNs adhered on the immobilized fibrin.

micro-PIV， flow chamber， velocity profile， local shear rates

Introduction

At the end of the 20th century， when the studies of micro fluid were of great interest and many scientific techniques were developed， the conventional particle imaging velocimetry （PIV） was applied to the visualization and measurement of micro fluid， which is named the micro-PIV. Santiago et al.［1］used the epifluorescent illumination technique to record the discrete particle images of fluorescent particles and to analyze the velocity of the particles by the correlation method in a micro flow with a resolution length of less than 10 μm. Meinhart et al.［2］then used a similar method to measure the flow field in a micro flow chamber with a dimension of 13.6 μm in length， 0.9 μm in height， 2.2 μm in width， and 27 μm3in volume. Except observing the fluid field with the help of a microscope， Raffel et al.［3］pointed out， comparing with the conventional PIV， there are three major differences in the micro-PIV： （1） the diameter of tracer particles is small， which is comparable with the wavelength of the illumination light， （2） the light source is a volumetric illumination instead of a light sheet， （3） the tracer particle is so small that the Brownian motion should be considered.

With the rapid progress of the experimental techniques and analysis methods， the micro-PIV technique was widely used in various research areas for micro fluid measurements. Currently， more than 5 000 micro-PIV journal papers were published to date， as is estimated based on the statistics of Google Scholar search. Sugii et al.［4］measured the velocity profile of red blood cells in the arteriole in the rat mesentery by using an intravital microscope and a high-speed digitalvideo system and Jeong et al.［5］applied the micro-PIV to study the lipsomes movement in rat mesenteric blood vessels. Poelma et al.［6］measured the wall shear stress profile in vitelline network， and Hove et al.［7］obtained the velocity profile in the embryonic zebrafish heart. Besides， for in vivo micro bio-flow measurements， the micro-PIV was also used in lab-on-chip and microfluidic devices［8］. Combining with the stereo-imaging technique and the confocal imaging， the 3-D velocity profile can be obtained with the micro-PIV［9］. The Micro-PIV technique was also used to study two-phase flows and bubbles［10］.

Tumor cell metastasis is a complicated procedure，including the cell adhesion and the cell extravasation in blood vessel， in which the chemokine and protein factors play an important role［11］. Dong et al. developed a “two step theory” that the PMNs could facilitate the tumor cell adhesion on the endothelial cells in blood. The studies show that the melanoma cells adhesion on the endothelial cells is significantly enhanced under the flow conditions when the PMNs are added［12］. In their early studies， the in vitro parallel flow chamber， the flow migration chamber and the cone-plate viscometer were used to investigate the impact of different shear conditions. Before the development of the micro-PIV， most experiments were conducted in microscale geometries consisting of bulk flow measurements， such as the flow rate， the pressure drop and the thrust. With the development of the application of the micro-PIV to the blood flow， Leyton-Mange successfully combined the micro-PIV with a side-view imaging technique to study the local hydrodynamic environment around a single adherent Jurkat cell under different flow conditions［13］， which was further improved by Fu to measure the velocity profile around a pair of adherent cells［14］. In the present study，we apply our side-view micro-PIV technique to measure the local hydrodynamic environment around cells in a flow chamber and study the effect of different substrates.

1. Experimental setup and micro-piv method

1.1Experimental setup

The study was specifically approved by The Pennsylvania State University Institutional Review Board （IRB）， and written consents were obtained from all participants. The transfected fibroblast L-cells （EI cells， kindly provided by Dr. Simon S.， University of California Davis， Davis， CA） with a stable human E-selectin and the ICAM-1 expression and the metastatic melanoma cells Lu1205 （generously provided by Dr. Robertson G.， Penn State Hershey Medical Center， Hershey， PA） were cultured and treated following previous studies. The separation and the preparation of the PMNs from health human blood were following the Pennsylvania State University IRB approved protocol （No. 19311）［12］. The fibrinogen and the fibrin （Sigma， St Louis， MO） of desired concentration were prepared as described［15］.

A flow chamber consists of two microslide with the smaller one （VitrotubesTM#5005， Vitrocom，Mountain Lakes， NJ） being inserted into the bigger one （VitrocellsTM#8270， Vitrocom， Mountain Lakes，NJ） to form a dimension of 700 μm （width） × 550 μm（height）. The smaller microslide is coated with the EI cells or the fibrin（ogen） as the substrate in advance. Two needles and tubings are used to assemble the chamber. A Harvard syringe pump （Harvard Apparatus，Holliston， MA） is used to generate the flow at desired volumetric flow rates， 91.25 μl min-1and 292 μl min-1，which are equivalent to the wall shear rates of 62.5 s-1and 200 s-1， respectively. The calculations are as follows

whereμis the fluid molecular viscosity，Qis the volume flow rate，Wis the width of the chamber，H is the height of the chamber； andF is the correction factor for a rectangular channel with a finite aspect ratio.F is equal to 1.45 when the aspect ratio is 1.27［16］， and the viscosity is measured to be 1.5 cP at room temperature by a RotoViscoI cone-plate viscometer （ThermoMC； Madison， WI）.

1.2Micro-PIV setup and data analysis

The micro-PIV system consists of a Basler A504k high speed camera， a Olympus inverted fluorescent microscope， and a computer with two custom 45ohighly reflective mirrors （Red Optronics， Mountain View， CA） which are put close to both sides of the chamber［14］. Orange fluorescent micro beads （excitation/emission： 540/560 nm， Invitrogen， Carlsbad，CA） of 1 μm in diameter are chosen to be the tracer particles. 2 000 raw images are taken by the camera in 2 s under each desired conditions. After dividing the raw images with a time interval 1 ms into group A and group B， certain images are used to generate the average background images for each group. The background images are then subtracted from the raw images. Each group is further divided into 20 smaller groups of 50 images each and the median and the math min filter are calculated in the image processing. 50 images in each small group are then overlapped into a single image to increase the particle density and the NI-IMAQ （National Instruments， Austin， TX） is applied to compensate the missing dynamic boundary. After the above image processing， there are 20 images in group A and group B， separately， which are ready for the velocity profile calculation. The JPIV， an opensource PIV analysis software is used for the correlation calculations （http：//www.jpiv.vennemann-online. de）. As the flow in the micro flow chamber could be treated as a low Reynolds number Stokes flow， a novel correlation method different from the conventional PIV and called the ensemble correlation or the correlation averaging is chosen to minimize the errors due to the Brownian motion， the low particle density and low quality images， and the accuracy and the reliability are verified by the experimental results of Devasenathipathy， Meinhart and Wereley［17，18］.

2. Experimental results and discussions

Top-view （XY plane） and side-view （XZ plane） fluorescent images are recorded （Fig.1） by Basler high speed camera under both10X and40X objective lens and the ensemble correlation method is imported to obtain the velocity profiles.

Fig.1 The schematic diagram of flow chamber

Fig.2X -velocity profile along Y-direction of flow chamber with immobilized fibrin （ogen） under10Xobjective lens

2.1The velocity profile of flow over a substrate coated with fibrin （ogen）

The top-view velocity profile of the immobilized fibrin（ogen） substrate inXY plane under10Xobjective lens shows that the velocity in the middle is higher than that on the sides. One column ofX-velocity profile is picked up and plotted againstYcoordinates as shown in Fig.2. It is indicated that the velocity under the high shear flow condition （292 μl/min） is about 3 times of that under the low shear flow condition （91.25 μl/min）. The discrete shape of the velocity profile may be due to the lack of resolution and the near wall effect.

Fig.3X -velocity profile along Y-direction of flow chamber with immobilized fibrin（ogen） under40Xobjective lens

If the objective lens is switched to40X， there is no significant velocity gradient observed in the calculated velocity profile. TheX -velocity profile along Yaxis in Fig.3 is in a very discrete shape and shows no regular pattern， which might be caused by the small region of interest and the amplifying wall effect of the flow chamber bottom. Moreover， as shown in Fig.3（b）， the maximum velocity appears not in the middle but on one side. That is because the region of interest is not placed in the middle of the flow chamber.

When the view moves from the top to the side，the velocity profile inXZplane could be measured and c alcula ted. In Fig .4， under both l ow andhigh shearflowconditions，the X -velocityprofilealongZ axis under 10Xobjective lens shows a parabolic trend with the maximum velocity appearing in the middle and the minimum velocity appearing on the sides，as is consistent with the theoretical velocity profile.

Fig.4X -velocity profile along Z-direction of flow chamber with immobilized fibrin（ogen） under10Xobjective lens

Fig.5X -velocity profile along Z-direction of flow chamber with immobilized fibrin（ogen） under40Xobjective lens

Since under40Xobjective lens， only part of the chamber could be included in the region of interest，the region near the flow chamber bottom is of interest. It is indicated in Fig.5 that X -velocity profile along Zaxis shows a linear trend with a significant gradient. The wall shear rates are calculated to be 59.23 s-1and 197.77 s-1under low shear flow conditions（91.25 μl/min） and high shear flow conditions（292 μl/min）.

Fig.6X -velocity profile along Y-direction of flow chamber with EI under10Xobjective lens

2.2The velocity profile of flow over a substrate grown with EI cells

If the bottom of the flow chamber is coated with the EI cells， would the velocity profile show a similar trend？ The EI cells are adherent cells， which would form a monolayer. The shape of a single EL cell is long and narrow on sides and the cell height is about 1 μm-2 μm［16］. The EI monolayer looks like a rough mountain with “peaks” and “valleys”. In both Fig.6 and Fig.7， no regular pattern is observed in X-velocity profile alongYaxis either under10Xobjective lens or40Xobjective lens.

However， when the view moves from the top to the side，X -velocity profile along Z axis displays aparabolic trend under10Xobjective lens （Fig.8） and a linear trend under40Xobjective lens （Fig.9）. Comparing with Fig.4 and Fig.5， the change of the substrate does not dramatically change the pattern ofX-velocity profile alongZaxis. The wall shear rates in Fig.9 are calculated to be 64.15 s-1and 203.1 s-1under low shear flow conditions （91.25 μl/min） and high shear flow conditions （292 μl/min）. It is noted that the wall shear rates of the EI substrate are higher than those of the fibrin （ogen） substrate. The reason might be that the thickness of the EI monolayer reduces the height of the flow chamber， which results in an increase of X -velocity gradient near the wall.

Fig.7X -velocity profile along Y-direction of flow chamber with EI under40Xobjective lens

Fig.8X -velocity profile along Z-direction of flow chamber with EI under10X objective lens

Fig.9X -velocity profile along Z-direction of flow chamber with EI under40Xobjective lens

Many researchers applied the micro-PIV to the shear flow mechanical stimulation of endothelial cells［19，20］. Rossi et al.［21］reconstructed the 3-D wall shear stress above a single endothelial cell and reported that the maximum shear stress appeared at the“peak” of the cell. Since the height of a single endothelial cell is small， we are more interested in the effect of the EI monolayer on the local hydrodynamic environment. The micro-PIV measurement of the velocity profile in the flow chamber with different substrates shows that the EI monolayer disturbs the flow field in twoways：（1）X -v elocity profile in XYpla ne becomesmorediscrete，（2）X -velocityprofileinXZplane does not change much， but the wall shear stress is increased. Moreover， other studies show that the morphology of the endothelial cell monolayer would grow to be more regular under the flow condition of the physiological blood flow. However， no shear flow is applied to the EI cells when they are cultured， leading to irregular morphology， which might be an explanation for the discrete X -velocity profile inXY plane.

Table 1 Local shear rates above an adherent cell on immobilized fibrin（ogen）

2.3The velocity profile around cells adhered on immobilized fibrin（ogen）

Although the melanoma cells could not adhere to the EI monolayer under flow conditions， the fibrin（ogen） acts as a bridge between the melanoma cells and the EI cells. The adhesion of the melanoma cells on the immobilized fibrin（ogen） attracts our interest. Two concentrations are chosen to be 0.25 mg/ml and 2.5 mg/ml for coating the flow chamber substrate. A column of X-velocity profile is put above the top of an adherent cell to two cells high， then it is picked up and the local shear rate is calculated. As shown in Table 1， the local shear rates above the tumor cells（TCs） adhered to the immobilized fibrin are smaller than those above the TCs adhered to the immobilized fibrinogen under both low and high shear conditions（91.25 μl/min and 292 μl/min respectively）. In previous studies， we found that for a single adherent cell，its height affects the local shear rate most and a negative correlation is shown. A deformed cell with smaller height has less effect on the local hydrodynamic environment［14］. The deformation of a cell would be affected by the substrate and the bulk shear flow condition. Under the same shear flow condition， the melanoma cells may form a firmer adhesion on the immobilized fibrin than on the immobilized fibrinogen when the concentration is low.

When the concentration increases， could we observe a similar phenomenon？ Table 1 indicates that the TCs adhered on the immobilized fibrin disturb the local flow more than those adhered on the immobilized fibrinogen as well. However， one sees no significant difference between the local shear rates above PMNs adhered on the immobilized fibrinogen and those above the PMNs adhered on the immobilized fibrin. For the same substrate， both adherent PMNs and TCs show larger deformation under high shear flow conditions （cell dimension measurement data are not shown here）.

The fibrin（ogen） could form bonds with the ICAM-1 expressed on the melanoma cell surface. Moreover， the fibrin could form another bond with the αvβ3integrin expressed on the melanoma cell surface，which may promote the adhesion of the melanoma cells on the immobilized fibrin. Studies show that the affinity between the integrinαvβ3and the fibrin may be higher than that between the ICAM-1 and the fibrin［22］. That may explain why the melanoma cells experience larger deformation when adhered to the immobilized fibrin than to the immobilized fibrinogen. Nevertheless， the fibrin（ogen） could form bonds with the Mac-1 expressed on the PMNs surface， and one sees no difference between the dissociation rate constant of the fibrinogen-Mac-1 bond and that of the fibrin-Mac-1. Once the PMN forms a firm adhesion with the immobilized fibrin（ogen）， the shape of the PMNs would not change too much and the PMNs would hardly detach from the immobilized protein surface.

3. Conclusion

In this study， we use the micro-PIV technique to measure and analyze the velocity profile around cells in a flow chamber coated with the immobilized protein or the EI cells. It is found that the existence of the EI monolayer affects the X -velocity profile in XY andXZ planes， especially for the flow velocity profile pattern inXY plane. The wall shear rates in the flow chamber with the immobilized protein surface are calculated to be 59.23 s-1（under low shear flow condition） and 197.77 s-1（under high shear flow condition）， while those in the flow chamber with the EI monolayer substrate are calculated to be 64.15 s-1（under low shear flow condition） and 203.1 s-1（underhigh shear flow condition）. The velocity profiles around a single PMN or a melanoma cell adhered on the immobilized fibrin（ogen） are then measured and analyzed by the micro-PIV coupled with the side-view technique. The results indicate that the melanoma cells form a firmer adhesion on the immobilized fibrin and display a larger deformation， which results in less disturbance to the local hydrodynamic environment. However， one sees no significant difference between the adhesion of the PMNs on the immobilized fibrinogen and the fibrin， so the local shear rates above the adherent PMNs are comparable. In the future， the micro-PIV may be used to measure not only the local hydrodynamic environment change during the process of the PMNs facilitated tumor cell adhesion on the endothelial cells， but also the velocity of the moving cells at the same time.

Acknowledgement

The authors thank Dr. Robertson G. （Penn State University Hershey Medical Center） for providing Lu1205 melanoma cells， Dr. Simon S. （University of California Davis， Davis， CA） for providing EI cells.

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（October 16， 2014， Revised January 12， 2015）

* Project supported by the National Institute of Health（NIH， USA， Grant No. CA-125707）， the National Science Foundation （NSF， USA， Grant No. CBET-0729091）， the National Natural Science Foundation of China （Grant Nos. 11302129， 11432006 and 31170887） and the Fellowship from Chinese Scholarship Council.

Biography： FU Yi （1983-）， Female， Ph. D.

DONG Cheng，

E-mail： cxd23@psu.edu