Cellular Immunology 265 (2010) 44–49

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Cellular Immunology

journal homepage: www.elsevier .com/ locate/yc imm

A single cell tracking system in real-time

Yeon Hwa Kwak *, Sung Min Hong, Soon Sup ParkConvergence Sensor & Device Research Center, Korea Electronics Technology Institute, Seongnam-si 463-816, Republic of Korea

a r t i c l e i n f o

Article history:Received 17 August 2009Accepted 2 July 2010Available online 7 July 2010

Keywords:Real-timeCell trackingChemotaxis

0008-8749/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.cellimm.2010.07.001

* Corresponding author. Fax: +82 31 789 7279.E-mail address: yhkwak@keti.re.kr (Y.H. Kwak).

a b s t r a c t

We describe here a novel real-time cell tracking system which can measure cell migration routes undercell culture condition. This system includes a mini incubator which controls temperature and CO2 gasflow and a PDMS (polydimethylsiloxane) chip for chemotaxis measurement. The main differences fromprevious ones are real-timely long-term (P24 h) tracking for single cell quantitatively, simple and inex-pensive constitution of optical parts for illumination and imaging, and compatible to commercial wellplate. The tracking principle is to trace cell images for each 0.2 s by converting the live cell images to bin-ary images of black and white. Migration results of HUVEC and NCI-H23 cells are obtained respectivelyusing this system. The results are single cell path (x, y) during migration, cell size, migration distance,migration speed, real-time pictures and so on. This system is applicable to all kinds of researches relatedto cell migration such as cell angiogenesis, chemotaxis, and moreover cancer metastasis.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

The most important area of cancer research is recently how can-cer metastasis happens and how it is prevented [1,2]. Therefore,chemotactic migration and invasion researches of cells have beenincreasing either in vivo [3] or ex vivo. Moreover, quantitative anal-ysis [4] is the key aspect in cell research.

Measurements of cellular chemotaxis and invasion in vitro havebeen historically made in Boyden-type chambers which haveupper and lower compartments in between polycarbonate mem-branes coated with reconstituted basement membrane mixturessuch as Matrigel�; cells are added on the upper compartmentand a chemoattractant on the lower one for a defined length oftime, and those that migrate to the lower well are stained andcounted. Modifications of this assay have been made using 24-wellor 96-well plates fitted with membrane inserts [5,6]. Although thisstaining approach is straight forward, it has several disadvantages.First, it is impossible to check the status of cell migration in thechamber in real-time due to the chamber’s vertical structure. Sec-ond, quantification can be subjective and labor intensive since cellsare counted microscopically. Third, stained cells cannot be manip-ulated further to test for specific markers of interest.

Recently, 2D-type systems for cellular chemotaxis measurementhave been appearing to overcome shortcomings of Boyden-typechambers [7–9]. Kanegasaki et al. [10] developed EZ-TAXIScan™including a horizontal chemotaxis chamber called KK chamber. It

ll rights reserved.

is a real-time chemotactic assay device which consists of the siliconsubstrate, the stainless steel holder, CCD cameras, and the softwarefor monitoring and analysis. However, this system has a few limita-tions to adherent cell measurement due to short-term culture struc-ture and cell deformation induced during measurement. Bothtemperature and CO2 flow are not controlled for cell culture andmigration measurement in the chamber. In addition, concentrationgradient of chemoattractant is not stable for more than 24 h and thisis not appropriate to adherent cells which show relatively slowmigration compared to suspension cells. Lastly, cell migrationresults are displayed with the number of cells migrated to the oppo-site compartment, therefore individual cell migration path is nottraced.

Here, we suggest a new cell tracking system which makes up forall drawbacks mentioned above. The 2D chip with the mini incuba-tor is also possible to be used independently on a general cellobservation microscope because material of the substrate is trans-parent glass and PDMS which are somewhat inexpensive com-pared to silicon for only one time use. This chip can be replacedby the commercial well plate or culture dish if the experimentalpurpose is only cell culture related.

2. Material and methods

2.1. PDMS chip

The chip to measure cell chemotaxis was made up of transpar-ent glass and PDMS. It consisted of two wells to load media andattractant, and one channel to create a concentration gradient.

Fig. 2. A mini incubator.

Y.H. Kwak et al. / Cellular Immunology 265 (2010) 44–49 45

The PDMS microchannel was fabricated by peeling hardened PDMS(Sylgard 184, Corning Co.) off from the Si mold which is fabricatedby deep RIE etching after photolithography as shown in Fig. 1(a).The chip is completed by attaching the fabricated PDMS structureon the glass substrate by O2 plasma. This fabrication method givesus the least chip to chip variation because PDMS microchannelstructures are casted from the same Si mold. The fabricated chan-nel of the chip has 15 mm length, 100 lm depth as Fig. 1(b), andgradually narrowed (4 mm to 10 lm) width to maintain more than1–2% concentration gradient over 24 h. There have been previousreports that the concentration gradient should be maintained atleast 1�2% for cells to migrate to a specific direction [11,12]. Weconfirmed that the microchannel has maintained more than 2%concentration gradient for 24 h. The gradient generation and howthe gradual narrowing of the fabricated channel leads the total2% concentration gradient over 24 h are studied in our previouswork [13]. In the work, we focused on concentration gradient whenadhered cell size is 50 lm, because cell sizes are generally about50 lm when cells loaded in the chip are adhered on the surface.The concentration gradient was proved by using fluorescence-tagged chemoattractant (EGF-Cy3) [13].

2.2. Mini incubator

It is necessary that the 37 �C temperature and 5% CO2 flow arekept to culture cells for a long time. To optimize cell culture condi-tions, we made a mini incubator (see Fig. 2) to control temperatureand CO2 flow possible with the size of 5 cm diameter. The incuba-tor consists of a thermocouple, a heater, CO2 injection holes and acontroller. The PDMS chip is inserted in the mini incubator, andwater is injected around the PDMS to prevent media evaporationwith no higher than PDMS height. This is also good to keep 80%of humidity in the chamber. A rubber ring seals the chip to preventwater from leaking. We verified the temperature control by com-paring temperature by the thermocouple with a commercial ther-mometer. And we verified the control of CO2 flow by comparingmedia pH in the mini incubator with a commercial incubator. AsFig. 3(a) shows, the thermocouple reading in the mini incubatorand the thermometer reading have been coincident from 5 minlater. The pH readings each in the fabricated mini incubator, inthe commercial CO2 incubator, and in the air (see Fig. 3(b)) wereinvestigated and compared. The fabricated incubator showedalmost the same pH value as the commercial one.

Fig. 1. PDMS chip fabrication; (a) fabrication proces

2.3. Tracking software

Cell tracking software consists of a live imaging window, aparameter setting window, a cell tracing coordinate window, anda cell information window as Fig. 4(a) shows. The tracking princi-ple is to trace cell images for each 0.2 s by converting the live cellimages to binary images of black and white. User can select cells ina few groups or one by one to check migration, the total measure-ment time, and the time intervals. Each cell tracing results are re-corded including the measured time, cell sizes, cell coordinates,migration distances, and migration velocities.

2.4. Light source and device constitution

A white LED as a light source is used to create binary imagesclearly because the LED is known as a rather stable light source.A 4� lens and a CCD camera (Sony, XC-ES30CE) are located underthe incubator to get cell images. This is a simple and inexpensiveconstitution compared to the phase-contrast microscope which isnormally used to observe cell images. Instead, it is very importantto make a parallel light path without any gradient of light intensityover the incubator. To make a successful light illumination, weintroduced 3-axis adjustable column. The total hardware constitu-tion is shown in Fig. 4(b).

s, (b) photograph of a fabricated 2D structure.

Fig. 3. Incubator operating test; (a) temperature changes of the DMEM, (b) pH changes of the media.

Fig. 4. Cell tracking system; (a) software window, (b) hardware constitution.

46 Y.H. Kwak et al. / Cellular Immunology 265 (2010) 44–49

2.5. Cell culture and sample preparation

HUVEC (Human Umbilical Vein Endothelial Cell) and NCI-H23(Lung Adenocarcinoma) cells which were cultured for three pas-sages each in EGM-2 (Cambrex, CC-3162) and RPMI1640 (GIBCO,22400-089) were used to check cell migration. Each cell line wascollected by trypsin–EDTA (GIBCO, 25200-056) and washed withDPBS (JBI, LB001-02). The prepared cells were resuspended in eachculture media and counted by the hemocytometer. Cells wereloaded on each fibronectin (Sigma-Aldrich, F0895) coated PDMSchip and ready to be used.

3. Results and discussion

The prepared chip is deposited in the commercial incubator toadhere cells on the substrate for overnight. Then, the chip is loadedin the mini incubator, and then a kind of drug (chemoattractant,etc.) is injected in the chip to see the drug effect. The mini incuba-tor including the chip is loaded on the cell tracking system andthen the temperature/CO2 controller is connected to it. 37 �C tem-perature and 5% CO2 flow are set from the controller, and then 24 hperiod and 5 min interval are set from cell tracking screen. Duringrunning the software, the cell migration data and the cell images

are stored. Cell migration distances and velocities are also dis-played in real-time.

Without any drug, HUVEC and NCI-H23 migrations using thecell tracking system were checked and compared. HUVEC wastraced using this system and the migration paths for 24 h were re-corded. In this result, HUVEC showed relatively more active move-ment and the total distance was average 760 lm of 17 cells for24 h. Fig. 5(a) and (b) shows the chemotaxis results of HUVECincluding cell images and cell routes. HUVEC measurement wasdone several times in different chips and the average migration re-sults were almost 760 lm. Each time we measured, the number ofcells was different but the total measured was almost 100. NCI-H23 cells showed less active movement compared to HUVEC. Theaverage movement distance of 23 cells for 24 h was 317 lm whichwas almost half of HUVEC. And this measurement was also triedfor totally around 100 cells and the average movement was closeto 317 lm. It was found that each cell center of gravity has beencontinuously changed though the cells seemed to be almost fixedat each position (see the enlarged view of Fig. 5(c)). To confirmthe movement amount measured in real-time by this software,the HUVEC tracing results of 290 images (to get picture-takingevery 5 min for 24 h) were compared with the debugging results.The HUVEC migration distance in between the adjacent frameswas each measured using the debugging mode of the software

Fig. 5. Cell tracking results; (a) HUVEC images at each 0 (frame 1), 8 (frame 97), 16 (frame 193), 24 (frame 290) h, (b) HUVEC tracking paths for 17 cells for 24 h, (c) NCI-H23cell tracking paths for 23 cells for 24 h.

Table 1HUVEC tracking results.

Cell number Real-time route (lm) Debugging route (lm) Error rate (%)

1 527.132 534.856 1.42 810.647 814.970 0.53 1480.947 1360.228 8.14 668.678 611.919 8.45 667.675 672.726 0.76 730.050 685.382 6.17 1051.263 1128.914 7.38 443.864 448.984 1.19 817.488 825.996 1.0

10 617.546 586.647 5.011 771.583 721.913 6.412 770.101 693.972 9.813 876.293 840.724 4.014 794.514 740.750 6.715 417.430 328.029 2.516 873.919 834.811 4.417 604.136 566.035 6.3Average 760.780 734.093 4.6 Fig. 6. HUVEC chemotaxis versus temperature when the concentration of VEGF is

120 ng/ml.

Y.H. Kwak et al. / Cellular Immunology 265 (2010) 44–49 47

from the 1st frame image to the 290th. This value was average734 lm, which was compared to the value measured in real-time.The error rate between them was about 4.6% as in Table 1. Thus,the cell kinetics using the cell tracking system was quantitativelymeasured and reliable while the cells have moved for 24 h.

With the chemoattractant VEGF as a drug, the HUVEC migrationusing the cell tracking system was checked [13]. To maximizethe effect of chemoattractant existence, the starvation process(the cells are incubated in the media in which all growth factorsare removed for 6 h) was performed before injecting VEGF. As

Fig. 7. Drug test results; (a) dead NCI-H23 cells depending on the time by 50 lM Doxorubicin, (b) dead NCI-H23 cells depending on the time by 0.8 lM daunorubicin.

48 Y.H. Kwak et al. / Cellular Immunology 265 (2010) 44–49

VEGF concentration was increased to 100 ng/ml, not only themigration distance towards chemoattractant direction but alsothe total cell migration distance was grown. The total migrationamount and migration amount towards attractant are related tocell activity and cell chemotaxis, respectively. The number of cellsmigrated towards attractant was increased up to 50 ng/ml VEGFand maintained at 100 ng/ml concentrations. The percentage ofmigrated cells towards the attractant is increased when VEGF con-centration is increased, and almost maintained as approximately80% at more than 50 ng/ml of VEGF.

Fig. 8. Cell images during drug test; (a) NCI-H23 cells with Doxorubicin 50 lM, (

In addition to the VEGF concentration, we verified variation ofcell migration in accordance with the incubation temperature.We controlled the temperature of the incubator and checked HU-VEC activity and chemotaxis when 120 ng/ml of VEGF is used asa chemoattractant. As Fig. 6 shows, cell activity and chemotacticmigration seemed to be the largest at 37 �C temperature. This re-sult shows the measurement possibility of cell kinetics accordingto an environment of cell culture using this system.

As one more application of this system, we have tested antican-cer drug effect for NCI-H23 by Doxorubicin and Daunorubicin

b) NCI-H23 cells with Daunorubicin 0.8 lM, (c) NCI-H23 cells with no drug.

Y.H. Kwak et al. / Cellular Immunology 265 (2010) 44–49 49

drugs and compared with the result of the general MTS assay. First,we have obtained the IC50 (half-maximal inhibitory concentration)values for 24 h by MTS assay and they were each 50 lM for Doxo-rubicin and 0.8 lM for daunorubicin. We have injected each drugwith these concentrations to NCI-H23 and measured cells’ behav-ior under cell tracking system. We could count the number of deadcells depending on the time as an effect of anticancer drug sincethis system lets us measure each cell’s physical variation includingcell migration. As Fig. 7(b) shows, the effect of daunorubicin toNCI-H23 was almost identical with the result of IC50 of MTS assay.However, the effect of Doxorubicin to NCI-H23 seemed to be differ-ent from MTS assay since IC50 by this system appeared within 12 hat 50 lM as Fig. 7(a). To solve the cause of the difference, we ob-served the images measured depending on the time shown inFig. 8. As Fig. 8(a) shows, cells treated with Doxorubicin for 24 hlooked almost dead. Therefore, we could conclude that the MTS as-say results could be wrong. Generally it is known that MTS assaycannot show the exact data because the number of live cells isjudged by media color. Therefore, by adding the measurement re-sults using this system, we could improve the accuracy of the drugtest and conclude possibilities as anticancer drugs.

Not only the above discussion mentions strong points of thissystem but also one more advantage exists. That is what we canuse a commercial dish or well plate as a substrate of cell culture.We can use a commercial dish to culture cells instead of PDMS chipif the purpose of experiments is only culture related, and we canalso make diameter of the incubator more than 5 cm to use com-mercial well plates in it.

In addition to the above-mentioned cell migration results, weneed to try to test more tumor cell lines to convince cancer metas-tasis or anticancer drug effect and so on. This is our future projectand we are now in progress to test HEP-G2 (human hepatocellularliver carcinoma cell line) and MDAMB-231(breast cancer cell line)with new drugs to see the effect as anticancer drugs.

4. Conclusions

This study showed a novel-type cell tracking system which canquantitatively measure cell migration path to analyze cell kinetics.A mini incubator with temperature and CO2 controller was testedby comparing with a commercial incubator. Cell tracking is runby measuring each cell center of gravity from binary images of it.To prove successful operation of this system, HUVEC migration

for angiogenesis and NCI-H23 for cancer metastasis were mea-sured, respectively.

Not only the measurement of cell migration but also the celldeath as an effect on chemicals can be measured using this system.From this application, the effect of cancer cells on anticancer drugsand the effect of microorganisms on antibiotics can be measured.By this work, we might recognize the differences between cancercells and normal cells and the drug effect of cells. This is our futureproject. The cell tracking system will be more applicable to thenew drug developments, cancer researches and so on.

Acknowledgments

This research was supported by Ministry of Commerce, Industryand Energy (MOCIE), Korea as a project, ‘‘TechnologiesDevelopment for Future Home Appliance” and also by Ministry ofEducation, Science and Technology (MEST), Korea as a project‘‘Nano-Diagnostics by Continuous Monitoring.”

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