Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

Relationships between Electric Impedance and Orientation of Biological Cells:
Control by Micro-stripes Grooves In Vitro
Shigehiro HASHIMOTO, Kento KIMURA, Kazuyuki ABE
Biomedical Engineering, Department of Mechanical Engineering,
Kogakuin University, Tokyo, 163-8677, Japan
shashimoto@cc.kogakuin.ac.jp
http://www.mech.kogakuin.ac.jp/labs/bio/

ABSTRACTThe orientation of cells in the target area might relate to theelectric impedance of the area, which might be applied toestimate the orientation of cells. In the present study, theimpedance has been measured over several elongated cells
between the surface electrodes, of which orientation iscontrolled on the scaffold surface with the several parallel lines
of micro rectangular grooves.

The relationships between the electric impedance and theorientation of biological cells have been studied in vitro. To
control the orientation, parallel lines of micro rectangulargrooves (1 μm depth, 3 μm width and interval) have been madebetween a pair of surface titanium electrodes on the scaffold of
glass by the photolithography technique. C2C12 (mouse
myoblast cell line) was cultured on the micro-patterned scaffold.
The electric impedance (z) between electrodes was measured
every 24 hours with the sinusoidal electric waves (1 kHz <frequency < 10 MHz; amplitude, ±0.05 V,). The orientation
was evaluated by the orientation index (P) calculated by themean value of the angle between the longitudinal direction ofgrooves and the major axis of each cell. The crossing index(Nn) was also calculated by the number of cells, which cross thestraight-line connecting between electrodes. The experimental
result shows that z relates to Nn, which relates to P. The
present study shows that the measurement of the electric
impedance is effective to estimate the orientation of cells
perpendicular to the electric line of force.

2.

Stripe Pattern for Orientation of Cell
A scaffold of solid surface, which has both micro patterns andsurface electrodes (Fig. 1), has been designed to measureelectric signals of oriented cells. Several parallel lines of
micro grooves have been made in the square area of 1 mm × 1
mm at the surface of the scaffold. The depth, the width, andthe interval of the rectangular grooves are 1 μm, 3 μm, and 3
μm, respectively. Two types of arrangements of groove lines
are designed at the micro ridges: parallel, and perpendicular to
the electric line of force (Fig. 2).

Keywords: Biomedical Engineering, Electric Impedance,
Micro-machining, C2C12 and Orientation.

1.

METHODSElectric field
Micro pattern

Electrode

INTRODUCTION

The biological tissue includes a lot of cells. The biologicalcell has the shell of the membrane. The membrane consists of
the lipid-bilayer. The other part of the tissue is filled with theelectrolyte solution. The electrolyte solution is conductive,
although the lipid is not conductive. The membrane plays as acondenser when the electric current flows through the tissue [1].

ElectrodeFig. 1: Micro pattern (right) between electrodes (left).
Electrode

The direct electric current may occur electrolysis in the
biological tissue. The alternating electric current, on the other
hand, can sense the distribution of the element of the lipid in the
biological tissue [2]. The impedance depends on the numberof cells. The electric property of the biological tissue also
depends on the frequency of the alternating electric current.

A

The elongated cell has the orientation. The myoblast elongateson the scaffold of the cell culture in vitro. The orientation ofmyoblast can be controlled by the micro pattern on the surfaceof the scaffold plate [3–5]. Myoblasts differentiate into
myotubes, and make muscles. The orientation of myotubes
governs the function of muscles [6]. The orientation ofmyoblasts governs the orientation of myotubes.

C

Electrode

B

C

Electrode
ElectrodeFig. 2: Micro stripe pattern between electrodes: parallel (left),
perpendicular (right): A, on micro pattern; B, between
electrodes; C, control area.

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Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

sub-confluent state. Cells were observed with an inverted
phase-contrast microscope every 24 hours.
The number of cells crossing the electric line of force (Ns) was
counted by the following method. On the microscopic image,parallel 25 straight lines connecting between T-shape ofelectrodes were drawn at regular intervals (Fig. 3). The
contour of each cell was traced at “Image J” to count theposition of each cell. The total number of cells (Nc), whichmakes crossing with the straight lines, was counted. The mean
numbers (Ns) of cells, which makes crossing with the straightlines, was defined by Eq. (1).
Ns = Nc / k(1)

0.2 mmNs increases with numbers of cells in the area between
electrodes (B in Fig. 2). The crossing index (Nn) of cells wascalculated by Eq. (2), where n is the number of cells in thetarget area.

Fig. 3: Parallel straight lines connecting between T-shape ofelectrodes are drawn at regular intervals.

Nn = Ns / √(n)After plasma ashing, the positive photoresist material of
OFPR-800 was coated with the spin coater. The pattern was
drawn on the photoresist with a laser drawing system. The
photoresist was developed with tetra-methyl-ammoniumhydroxide (NMD-3) for one minute. The glass disk wasetched with the plasma gas using a reactive ion etching system(RIE-10NR). The pattern of the surface of the scaffold wasconfirmed by the laser microscope.

(2)

Electric Measurement
The sinusoidal electric waves (frequency, 1 kHz < f < 10 MHz;amplitude, v2 = ±0.05 V) were generated with an electric
stimulator. The stimulator was connected to the electrodes,
and the sinusoidal electric waves were applied to the medium ofcells. An electric resistance (R) of 10 kΩ is serially inserted
between the electrode and the stimulator. The electric voltages(v1, v2) were monitored by an oscilloscope during measurementof impedance of the medium. The values v1 and v2 are thevoltages between the terminals of the resistance, and of theelectric stimulator, respectively.
The impedance of thespecimen (z) is calculated by the voltage (v) between electrodes
divided by electric current (i) (Eq. (3)).

Electrode with Micro Pattern
After machining the micro-stripes grooves, titanium of 0.2 μm
thickness was deposited on the surface of the glass in theelectron beam vapor deposition apparatus. The positive
photoresist material of OFPR-800 was coated with the spincoater. The photomask of the shape of the electrodes was
mounted on the surface of OFPR-800, and the photoresist was
exposed to the ultraviolet (UV) light through the mask in themask aligner. The photoresist was developed with NMD-3.
The titanium layer on the surface was etched with the plasma
gas using RIE-10NR.

z=v/i

(3)

The voltage (v) is calculated by the difference between v2 and v1.
The electric current (i) is calculated v1 divided by R. The
phase difference (α) between v and i is counted on the displayof oscilloscope. One period corresponds to 360 degrees of thephase.

To insulate the residual area of the surface electrode, thetitanium was covered with the coating of the negative
photoresist material (SU-8). SU-8 was coated with the spincoater. The coating area was exposed to the UV light throughthe mask in the mask aligner. The photoresist was developedwith SU-8 Developer.

Orientation Index
The orientation index (P) was defined by the Eq. (4).

The wall of culture dish is made of the donut-ring (10 mm innerdiameter, 15 mm outer diameter, 10 mm height) of
Polydimethylsiloxane (PDMS). The donut-ring of PDMS wasadhered on the disk with the surface electrodes with the extra
paste of PDMS. The lead wire was connected to the surface
electrode with the crimp style terminals.

In Eq. (4), n is he number of cells in the target area, and θ is theangle (0 degree < θ < 90 degree) between the longitudinal
direction of the approximated ellipsoid of each cell and thelongitudinal direction of the scaffold stripe pattern of the micro
grooves (Fig. 4). P becomes 1, when every cell aligns along
the longitudinal direction of the stripe pattern (θk = 0). P
becomes 0.5, when the alignment of each cell has the uniform
distribution to every direction.

Cell Culture
C2C12 (Mouse myoblast cell line originated with cross-striatedmuscle of C3H mouse; passage between ninth) was used in the
test.
D-MEM (Dulbecco’s Modified Eagle Medium)
containing 10% FBS (Fetal Bovine Serum) and 1% penicillin/
streptomycin was used for the medium. After the oxygen
plasma ashing by FA-1, cells were seeded on the scaffold withthe density of 6×103 cells/cm2. Cells were cultured in the
incubator at 310 K with 5% CO2 for three days before the

3.

RESULTS

The mean values of P at the area of A (on micro pattern), B(between electrodes), and C (control) (Fig.2) were 0.78, 0.75,

26Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

and 0.51, respectively.
Fig. 5 exemplifies cells cultured on striped-micro pattern
between surface electrodes for 24 hours. The microscopic
images show that cells make orientation along the striped micro
pattern: parallel (Fig. 5a), and perpendicular (Fig. 5b) to theelectric line of force between electrodes.
The number of cells increased day by day during cell culture.
The impedance increases with the number of cells between
electrodes (n) (Fig. 6).
Fig. 7 exemplifies the relationship between the impedance andthe frequency. The impedance shows the minimum value atthe frequency between 0.01 MHz and 0.1 MHz. At thefrequency lower than 0.1 MHz, α is larger at the orientation
perpendicular to the electric line of force than at the orientation
parallel to the electric line of force (Fig. 8). The difference of
α between at the perpendicular orientation and at the parallelorientation, however, is smaller at the higher frequency than 1
MHz. The phase difference α does not depend on Ns.

Fig. 5b: Cells on micro pattern (perpendicular) between
electrodes.
Impedance (z) [kΩ]

Fig. 9 shows the orientation index (P). The mean values of theorientation indexes were 0.5 during cell culture for 48 hours on
the scaffold surface with no pattern, which correspond to therandom alignment of cells. The value of P is higher at the area
A than at the area B, which correspond to the regulararrangement of cells on the pattern. The high value of P on
the stripe pattern decreases with time owing to interaction
between cells at the higher density of cells.
Number of cells between electrodes (n)θMicro grooves

Fig. 6: Impedance z related to number of cells between
electrodes (n) at 10 kHz: perpendicular (circle), and parallel(triangle).

Cell

Impedance (z) [kΩ]

Fig. 4: Angle (θ) between longitudinal direction of each celland stripe pattern of grooves.

0.001

0.01

0.1
1
Frequency (f) [MHz]10

Fig. 7: Impedance z related to frequency f.

Fig. 10 shows relationships between impedance (z) and thecrossing index (Nn). At the frequency of 1 kHz (Fig. 10a), thevalues of impedance are split each other between parallel (circle
in Fig. 10a) and perpendicular (triangle in Fig. 10a) orientationof cells. The impedance at 1 kHz tends to increase with thenumber of cells between electrodes (with Nn). At thefrequency of 10 MHz, on the other hand, data were normalized
between parallel and perpendicular orientations of cells, and theimpedance tends to decrease with the crossing index (Nn).

Fig. 5a: Cells on micro stripe pattern (parallel) between
electrodes.

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Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

Fig. 11 shows relationships between Nn and P in the area
between electrodes (B in Fig. 2). At the perpendicularorientation of cells (Fig. 11b), Nn tends to increase with P. At
the parallel orientation of cells (Fig. 11a), on the other hand,relationship between Nn and P is not clear.

z [kΩ]

Phase difference (α)[degree]NnFig. 10a: z (1 kHz) vs. Nn: circle (r = 0.84), parallel; triangle (r= 0.63), perpendicular: dotted line (regression line): r,correlation coefficient.

Number of cells oriented between electrodes(Ns)
Fig. 8:Phase difference (α) related to number of cells oriented
between electrodes (Ns) at 1 kHz: perpendicular (circle), andparallel (triangle).

z [MΩ]

Orientation Index (P)Time [hour]NnFig. 10b: z (10 MHz) vs. Nn: circle, parallel; triangle,
perpendicular: dotted line (regression line), r = −0.51.

Fig. 9a: Orientation index (P) vs. culture time (mean ± standard
deviation (bar), 67 < n < 415) at parallel pattern: circle (A),triangle (B), square (C): n, number of samples.

Nn

Orientation Index (P)PTime [hour]Fig. 11a: Nn vs. P (16 < n < 50) parallel: dotted line (regressionline), r =0.14.

Fig. 9b: Orientation index vs. culture time (mean ± standard
deviation (bar), 13 < n < 148) at perpendicular pattern: circle(pattern), triangle (between electrode), square (no pattern).

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Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

The effect of the membrane on the electric impedance (z)depends on the number of cells (Ns) at the crossing electric lineof force (Fig. 12). Ns relates to orientation, and density ofcells. Ns of the perpendicular orientation of cells is larger thanNs of the parallel orientation of cells (Fig. 12). The electric
impedance might increase with Ns, especially at the higher
frequency of the alternating electric current. The orientation isrepresented by the orientation index (P). The value of P is thehighest in the pattern area (A in Fig. 2), where cells makealignment along the micro-stripes grooves. Ns related to theorientation of cells. In the confluent state, Ns of the elongatedcells is higher at the orientation perpendicular to the electric lineof force. At the lower frequency, the impedance increases
with the density of cells, which is proportional to the crossing
index (Nn) (Fig. 10a). The lower frequency (< 0.1 MHz) of
electric current is effective to monitor the orientation of cells by
the phase difference θ related to the membrane. Ns is sensitive
to the orientation of cells, when the electric line of force isperpendicular to the orientation of cells (Fig. 12). Ns, which
depends on n, is normalized to Nn. The crossing index (Nn)has proportional relationship to the orientation index (P) of cells
in the perpendicular direction. The electrodes had better beplaced to apply the electric field in the perpendicular direction
to the orientation of cells for detection. Several cells show
elongated shape on the scaffold. The orientation of cells in thetissue is related to the property of the tissue.
The
micro-topography of the surface of the scaffold is available tocontrol the orientation of cell [4, 5, 8, 24]. To minimize theeffect of the topography of the surface of the scaffold on the
impedance of the substance between electrodes, the depth of the
microgroove is set within 1 μm in the present study [3].

NnP
Fig. 11b: Nn vs. P (59 < n < 256): perpendicular: dotted line
(regression line), r = 0.51.

4.

DISCUSSION

In previous studies, the electric impedance was used to monitor
cell behaviors [7–10]: cell adhesion [11, 12], spreading [13],shape change [14], proliferation [15], differentiation [16], cell
growth [17], cytotoxicity [17], metastatic cells [18], activity ofcell [19, 20]. Some of them are using evaporated gold
electrodes [21], microelectrode array [6, 22], or vibrating
electrode [23]. The sensitivity depends on the morphology ofthe surface electrodes [24]. The lipid-bilayer at the membraneof the cell governs the frequency response to the alternating
electric current [1, 2]. The optical impedance is also applied
to measure the confluent cell culture [25]. The orientation ofcell depends both on the micro pattern [26] and on the electricfield [27]. The orientation of floating cells is measured by the
optical technique [28, 29].

5.

CONCLUSION

The relationships have been studied between the electric
impedance (z) and the orientation of cells in vitro. The
orientation was evaluated by the orientation index (P)calculated by the mean value of the angle between thelongitudinal direction of micro-strips grooves and the major
axis of each cell. The crossing index (Nn) was also calculatedby the number of cells, which cross the straight-line connecting
between electrodes. The experimental result shows that z
relates to Nn, which relates to P. The phase difference
between voltage and current is larger at the arrangement ofmyoblasts oriented in perpendicular position than that inparallel position.
The present study shows that themeasurement of the electric impedance is effective to estimate
the orientation of cells perpendicular to the electric line offorce.

Electrode

Cell
Electrode
Electrode

REFERENCES[1] S. Hashimoto and K. Abe, “Monitoring of Orientation ofCells by Electric Impedance: Test on Oriented Cells Using
Micro Striped Grooves Pattern by Photolithography”, Proc.
19th IEEE International Conference on Bioinformatics
and Bioengineering, 2019, pp. 557–562.
[2] S. Hashimoto, “Impedance between Micro-electrodes of a
Pair of Concentric Cylinders for Estimation of Local Cell
Configuration”, Proc. 19thIEEE
International
Conference on Bioinformatics and Bioengineering, 2019,
pp. 800–803.
[3] H. Hino, S. Hashimoto and F. Sato, “Effect of Micro Ridges

CellElectrodeFig. 12: Orientation of cells and impedance: parallel (left), andperpendicular (right); number of cells increase from upper tolower; arrow (electric field).

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Proceedings of The 11th International Multi-Conference on Complexity, Informatics and Cybernetics (IMCIC 2020)

on Orientation of Cultured Cell”, Journal of Systemics
Cybernetics and Informatics, Vol. 12, No. 3, 2014, pp.
47–53.
[4] F. Wu, J. Zheng, Z. Li and M. Liu, “Halloysite Nanotubes
Coated 3D Printed PLA Pattern for Guiding Human
Mesenchymal Stem Cells (hMSCs) Orientation”, Chemical
Engineering Journal, Vol. 359, 2019, pp. 672–683. 0.1 mm[5] A.I. Teixeira, G.A. Abrams, P.J. Bertics, C.J. Murphy and P.F.
Nealey, “Epithelial Contact Guidance on Well-defined
Micro- and Nanostructured Substrates”, Journal of Cell
Science, Vol. 116, No. 10, 2003, pp. 1881–1892.
[6] S. Ahadian, J. Ramo´n-Azco´n, S. Ostrovidov, G.
Camci-Unal, V. Hosseini, H. Kaji, K. Ino, H. Shiku, A.
Khademhosseini and T. Matsue, “Interdigitated Array of Pt
Electrodes for Electrical Stimulation and Engineering of
Aligned Muscle Tissue”, Lab on a Chip, Vol. 12, No. 18,
2012, pp. 3491–3503.
[7] C.M. Lo, C.R. Keese and I. Giaever, “Impedance Analysis of
MDCK Cells Measured by Electric Cell-Substrate
Impedance Sensing”, Biophysical Journal, Vol. 69, 1995,
pp. 2800–2807.
[8] I. Giaever and C.R. Keese, “Monitoring Fibroblast Behavior
in Tissue Culture with an Applied Electric Field”,
Proceedings of the National Academy of Sciences of theUnited States of America, Vol. 81, No. 12, 1984, pp.
3761–3764.
[9] H.R. Siddiquei, A.N. Nordin, M.I. Ibrahimy, M.A. Arifin,
N.H. Sulong, M. Mel and I. Voiculescu, “Electrical
Cell-substrate Impedance Sensing (ECIS) Based Biosensor
for Characterization of DF-1 Cells”, Proc. International
Conference on Computer and Communication
Engineering (ICCCE 2010), 2010, pp. 1–4.
[10] A.R.A. Rahman, C.M. Lo and S. Bhansali, “A Detailed
Model for High-Frequency Impedance Characterization ofOvarian Cancer Epithelial Cell Layer Using ECIS
Electrodes”
IEEE
Transactions
on
Biomedical
Engineering, Vol. 56, No. 2, 2009. p. 485–492.
[11] Y. Qiu, R. Liao and X. Zhang, “Real-Time Monitoring
Primary Cardiomyocyte Adhesion Based on Electrochemical
Impedance Spectroscopy and Electrical Cell-Substrate
Impedance Sensing”, Analytical Chemistry, Vol. 80, No. 4,
2008, pp. 990–996.
[12] I.H. Heijink, S.M. Brandenburg, J.A. Noordhoek, D.S.
Postma, D-J. Slebos and A.J.M. van Oosterhout,“Characterisation of Cell Adhesion in Airway Epithelial Cell
Types Using Electric Cell–Substrate Impedance Sensing”,
European Respiratory Journal, Vol. 35, No. 4, 2010, pp.
894–903.
[13] J. Wegener, C.R. Keese and I. Giaever, “Electric
Cell–Substrate Impedance Sensing (ECIS) as a Noninvasive
Means to Monitor the Kinetics of Cell Spreading to Artificial
Surfaces”, Experimental Cell Research, Vol. 259, No. 1,
2000, pp. 158–166.
[14] C. Tiruppathi, A.B. Malik, P.J. Del Vecchio, C.R. Keeseand I. Giaever, “Electrical Method for Detection ofEndothelial Cell Shape Change in Real Time: Assessment ofEndothelial Barrier Function”, Proceedings of the National
Academy of Sciences of the United States of America, Vol.
89, No. 17, 1992, pp. 7919–7923.
[15] L. Vistejnova, J. Dvorakova, M. Hasova, T. Muthny, V.
Velebny, K. Soucek and L. Kubala, “The Comparison of
Impedance-based Method of Cell Proliferation Monitoringwith Commonly Used Metabolic-based Techniques”,
Neuroendocrinology Letters, Vol. 30, Suppl. 1, 2009, pp.
121–127.

[16] I. Park, Y. Hong, Y.H. Jun, G.Y. Lee, H.S. Jun, J.C. Pyun,
J.W. Choi and S. Cho, “Electrical Impedance Monitoring of
C2C12 Myoblast Differentiation on an Indium Tin Oxide
Electrode”, Sensors, Vol. 16, No. 12, 2016, 2068, pp. 1–11.
[17] C. Xiao and J.H.T. Luong, “On-Line Monitoring of Cell
Growth and Cytotoxicity Using Electric Cell-Substrate
Impedance Sensing (ECIS)”, Biotechnology Progress, Vol.
19, No. 3, 2003, pp.1000–1005.
[18] C.R. Keese, K. Bhawe, J. Wegener and I. Giaever,“Real-time Impedance Assay to Follow the Invasive
Activities of Metastatic Cells in Culture”, Biotechniques,Vol. 33, No. 4, 2002, pp. 842–850.
[19] Y.H. Yun, Z. Dong, Z. Tan and M.J. Schulz, “Developmentof an Electrode Cell Impedance Method to Measure
Osteoblast Cell Activity in Magnesium-Conditioned Media”,
Analytical and Bioanalytical Chemistry, Vol. 396, No. 8,
2010, pp. 3009–3015.
[20] V. Bucher, B. Brunner, C. Leibrock, M. Schubert and W.
Nisch, “Electrical Properties of a Light-addressable
Microelectrode Chip with High Electrode Density for
Extracellular Stimulation and Recording of Excitable Cells”,
Biosensors and Bioelectronics, Vol. 16, 2001, pp. 205–210.
[21] I. Giaever and C.R. Keese, “Use of Electric Fields toMonitor the Dynamical Aspect of Cell Behavior in Tissue
Culture”, IEEE Transactions on Biomedical Engineering,Vol. BME-33, No. 2, 1986, pp. 242–247.
[22] L. Giovangrandi, K.H. Gilchrist, R.H. Whittington and
G.T.A. Kovacs, “Low-cost Microelectrode Array with
Integrated Heater for Extracellular Recording of
Cardiomyocyte Cultures Using Commercial Flexible Printed
Circuit Technology”, Sensors and Actuators, B: Chemical,
Vol. 113, No. 1, 2006, pp. 545–554.
[23] S. Hashimoto and H. Otani, “Measurement of Mechatronic
Property of Biological Gel with Micro-Vibrating Electrodeat Ultrasonic Frequency”, Journal of Systemics
Cybernetics and Informatics, Vol. 6, No. 5, 2008, pp.
93–98.
[24] S. Hashimoto, H. Hino, T. Yamamoto, T. Tamura and K.
Abe, “Design of Surface Electrode for Measurement ofElectric Impedance of Arrangement of Cells Oriented on
MicroStripedPattern”,
Proc.
22nd
World
Multi-Conference on Systemics Cybernetics andInformatics, Vol. 2, 2018, pp. 114–119.
[25] B.F. DeBlasio, M. Laane, T. Walmann and I. Giaever,“Combining Optical and Electrical Impedance Techniquesfor Quantitative Measurement of Confluence in MDCK-I
Cell Cultures”, Bio Techniques, Vol. 36, No. 4, 2004, pp.
650–662.
[26] H. Hino, S. Hashimoto, T. Tamura, Y. Takahashi, T.
Sugimoto and T. Yasuda, “Electric Measurement of Cultured
Myoblast Oriented on Scaffold with Micro-pattern”, Proc.
21st World Multi-Conference on Systemics Cybernetics
and Informatics, Vol. 2, 2017, pp. 209–214.
[27] S. Hashimoto, H. Hino, Y. Takahashi and A. Hiraoka,“Design of Comb-shaped Surface Electrode to Measure
Signal from Tissue Cultured with Electric Stimulation”,
Proc. 20th World Multi-Conference on Systemics
Cybernetics and Informatics, Vol. 2, 2016, pp. 99–104.
[28] M. Bitbol, “Red Blood Cell Orientation in Orbit C=0”
Biophysical Journal, Vol. 49, No. 5, 1986, pp.1055–1068.
[29] M.R. Loken, D.R. Parks and L.A. Herzenberg,“Identification of Cell Asymmetry and Orientation by Light
Scattering”, The Journal of Histochemistry andCytochemistry, Vol. 25, No. 7, 1977, pp. 790–795.

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