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Enrichment of live unlabelled cardiomyocytes from heterogeneous cell
populations using manipulation of cell settling velocity by magnetic field
Aarash Sofla, Bojana Cirkovic, Anne Hsieh, Jason W. Miklas, Nenad Filipovic et al.
Citation: Biomicrofluidics 7, 014110 (2013); doi: 10.1063/1.4791649
View online: http://dx.doi.org/10.1063/1.4791649
View Table of Contents: http://bmf.aip.org/resource/1/BIOMGB/v7/i1
Published by the American Institute of Physics.
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Enrichment of live unlabelled cardiomyocytes from
heterogeneous cell populations using manipulation
of cell settling velocity by magnetic field
Aarash Sofla,
1
Bojana Cirkovic,
2
Anne Hsieh,
3
Jason W. Miklas,
1
Nenad Filipovic,
2
and Milica Radisic
1,3,a)
1
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto,
Ontario M5S 3G9, Canada
2
University of Kragujevac, Kragujevac, Serbia
3
Department of Chemical Engineering and Applied Chemistry, University of Toronto,
Toronto, Ontario M5S 3E5, Canada
(Received 12 December 2012; accepted 25 January 2013; published online 13 February 2013)
The majority of available cardiomyocyte markers are intercellular proteins,
limiting our ability to enrich live cardiomyocytes from heterogeneous cell
preparations in the absence of genetic labeling. Here, we describe enrichment of
live cardiomyocytes from the hearts of adult mice in a label-free microfluidic
approach. The separation device consisted of a vertical column (15 mm long,
700 lm diameter), placed between permanent magnets resulting in a field strength
of 1.23 T. To concentrate the field at the column wall, the column was wrapped
with 69 lm diameter nickel wire. Before passing the cells through the column, the
cardiomyocytes in the cell suspension had been rendered paramagnetic by
treatment of the adult mouse heart cell preparation with sodium nitrite (2.5 mM)
for 20 min on ice. The cell suspension was loaded into the vertical column from
the top and upon settling, the non-myocytes were removed by the upward flow
from the column. The cardiomyocytes were then collected from the column by
applying a higher flow rate (144 ll/min). We found that by applying a separation
flow rate of 4.2 ll/min in the first step, we can enrich live adult cardiomyocytes to
93% 62% in a label-free manner. The cardiomyocytes maintained viability
immediately after separation and upon 24 h in culture. V
C2013 American Institute
of Physics.[http://dx.doi.org/10.1063/1.4791649]
I. INTRODUCTION
Chronic cardiovascular disease such as heart failure is increasing to epidemic levels, affect-
ing 1 in 5 persons. The beating heart muscle has no significant ability to regenerate and the via-
ble tissue remaining after an injury, such as myocardial infarction, is often insufficient to main-
tain adequate cardiac output.
1
Heart transplant is often not an available or appropriate option.
Thus, there is a pressing need for alternative interventions
2,3
through innovative therapeutic sol-
utions enabled by tissue engineering. Since cardiomyocytes (CM), the beating cells of the heart,
are terminally differentiated, they cannot be propagated from the heart biopsies of adult
patients. Recent advances in the stem cell field enable derivation of CM from embryonic stem
cells (ESCs)
4
or induced pluripotent stem cells (iPSCs).
5
However, engineering advances are
required to enable label-free separation of these cells from heterogeneous populations in a cost-
effective manner.
To our knowledge, there are no label-free separation techniques to isolate CM at high pu-
rity and yield. Prior to the recent discovery of a signal regulatory protein a(SIRPA) as a CM
a)
Author to whom correspondence should be addressed. Electronic mail: m.radisic@utoronto.ca. Telephone: 416-946-5295.
Fax: 416-978-4317.
1932-1058/2013/7(1)/014110/15/$30.00 V
C2013 American Institute of Physics7, 014110-1
BIOMICROFLUIDICS 7, 014110 (2013)
surface marker
6
all of the available cardiac markers were intracellular proteins, thus antibody
staining or genetic labeling had to be used for cell isolation. The antibody staining of intracellu-
lar markers such as contractile proteins requires cell permeabilization, which unfortunately ren-
ders the cells non-viable and useless for cardiac cell therapy or tissue engineering. Genetic
labeling of cells for clinical applications cannot be performed in humans due to ethical con-
cerns. A mitochondrial die, Tetramethylrhodamine (TMRM), was reported effective in labeling
and enrichment of CM;
7
however, cells labeled with fluorescent probes cannot be used for clini-
cal applications due to the unknown long-term effects of these organic molecules in humans.
Although the newly identified SIRPA
6
acts as a marker of CM derived from human pluripotent
stem cells, the wide applicability of the SIRPA antibody is yet to be determined. In addition,
separation of cells for clinical application using mouse derived antibodies may cause sensitiza-
tion in patients and development of anti-mouse antibodies.
8
Other separation approaches, such
as dielectrophoresis,
9
are yet to be extensively studied with CM due to the fact that electrical
fields can affect these electrically excitable cells. Using engineering principles to achieve high
efficiency label-free separation of CM will have a significant impact even when the CM surface
markers are identified, as expensive antibodies will not be required.
Isolation methods that rely on inherent physical properties of cells have been used for
label-free separation of cells. For example, Murthy et al. used a sieve-like microfluidic device
to
10
demonstrate the feasibility of enriching fibroblasts from CM on the basis of size. The high
purity enrichment of CM from other cell types such as fibroblasts remains challenging because
of the dimensional similarity of the suspended CM to other cell types including large fibroblasts
present in native heart isolates.
11
Another set of techniques relies of differential adhesion prop-
erties. In native heart isolates, CM are a cell type that takes the longest time to attach to the
surfaces of tissue culture plates. Non-myocytes, such as fibroblasts, take significantly less time.
These characteristics form a basis of an enrichment technique called pre-plating. The native
heart isolate is incubated in a tissue culture plate for 1 h, during which fibroblasts preferentially
adhere to the surface of the tissue culture plate and CM remain in suspension. If these steps are
sequentially repeated, enrichment of CM to over 80% can be achieved.
12
Although simple, this
technique is non-specific and leads to the loss of cell viability as many pre-plating steps are
repeated in sequence.
Amongst all inherent physical properties of cells, perhaps, magnetic properties are the most
relevant for isolation of CM. CM contain a high amount of iron due to the presence of myoglo-
bin. Under physiological conditions, myoglobin contains Fe
2þ
. Upon treatment of CM with
molecules such as NaNO
2
, the cells can be rendered transiently paramagnetic by oxidation of
myoglobin to metmyoglobin. Metmyoglobin consists of a backbone of eight helices wrapping
around a central pocket containing a prosthetic protoheme group, a stable compound of ferric
iron, Fe
3þ
.
13
Metmyoglobin is paramagnetic from the isolated coordination complex with an
unpaired d-electron.
14
In principle, the realignment of the unpaired electrons in the ferric iron
in the direction of an externally applied magnetic field should direct the paramagnetic CM to
move along the magnetic field gradient.
Magnetic separation has been successfully implemented for the identification and isolation
of red blood cells (RBCs) by taking advantage of the high level of iron in hemoglobin. Zbor-
owski et al. used a magnetic field of 1.4 T, and mean gradient of 0.131 T/mm and showed that
separation based on the magnetic properties of RBCs is possible.
15
Han and Frazier, through a
series of papers, reported magnetophoretic separation of RBCs,
16,17
while Huang et al. devel-
oped a multi-step microfluidic system for the isolation of nucleated RBCs from blood of preg-
nant women.
18
The first module in their device depleted the non-nucleated RBCs. The magnetic
module of their device, then, separated the white blood cells from nucleated RBCs with purity
of over 99.90%. Interesting approaches, such as capture of beads by magnetotactic bacteria, to
directly and actively transport the beads along the magnetic field lines were reported.
19
How-
ever, this approach is not applicable to CM since they have limited surface markers and cannot
be selectively attached to magnetophoretic bacteria.
Other magnetophoretic devices, described above, also cannot be directly applied to the sep-
aration of CM from heterogeneous cell preparations since the amount of iron per volume of a
014110-2 Sofla et al. Biomicrofluidics 7, 014110 (2013)
CM is 1000 times less than the amount of iron in RBCs. Thus, it was thought in the past that
CM cannot be isolated from heterogeneous cell populations using their magnetic properties and
that only RBCs are suitable for this kind of separation.
20
Here, it is first demonstrated that the
non-magnetic CM can be rendered paramagnetic by treating the cells with a solution of
NaNO
2
. We then report enrichment results obtained from a microfluidic device that relies on
the manipulation of the settling velocity of the cells by magnetic force (Figure 1).
II. THEORETICAL ANALYSIS: FORCE BALANCE ON CARDIOMYOCYTES IN THE
SEPARATION COLUMN
The separation mechanism is based on a high gradient magnetic separation method.
21
We
used magnetic force effects in conjunction with the settling velocity of the paramagnetic cells
in the separation column in order to affect the paramagnetic cell trajectory and achieve capture
of the target cells in the column. In our design, the column was vertical (Figure 1). The media
flowed from the bottom to the top and pushed the cells upward. However, the magnet attracted
CM toward the boundary layer of the flow, where the flow velocity was less than in the central
parts of the column (Figure 5(a)). By adjusting the flow rate such that the settling velocity of
CM due to gravity and the magnetic field remained larger than the upward velocity of CM
within the boundary layer of the fluid flow resulted in CM being trapped within the channel,
while the rest of the cells (non-myocytes) were carried out with the flow (Figure 1). The
desired cell type, CM, was then released from the column in the second step by increasing the
rinsing flow rate.
In this study, it was necessary to generate a strong magnetic field and magnetic gradient at
the vertical column wall. Calculation of an average flow rate and applied magnetic force for the
paramagnetic particles inside the column was performed in order to clarify the effect of mag-
netic field gradient on the accumulation of the paramagnetic CM in the column.
A small paramagnetic particle that travels in a fluid stream in the presence of a wrapped
magnetized wire experiences magnetic, hydrodynamic, and gravitational forces. The balance of
forces which describes the particle motion is given by the following equation:
FIG. 1. Mechanism of action for cell separation. (a) Cell suspension is loaded into a vertical column placed in a magnetic
field. (b) Paramagnetic cells are attracted to the column wall where magnetic field is concentrated due to the circumferen-
tially positioned nickel wire. (c) Non-paramagnetic cells are rinsed out of the column by application of the flow at the bot-
tom inlet to the column.
014110-3 Sofla et al. Biomicrofluidics 7, 014110 (2013)
~
F¼~
Fmþ~
FDþ~
Fgþ~
Fb;(1)
where Fmis the magnetic force, Fgthe gravitational force, Fbbuoyancy, and FDthe drag force.
The magnetic force Fmacting on a magnetic particle is proportional to the applied mag-
netic field Hand magnetic field gradient rH
Fm¼l0VpvpHrH;(2)
where l0is the magnetic permeability of vacuum, Vpis the particle volume, and vpis the par-
ticle magnetic susceptibility. In order to examine the value of magnetic force Fmand the
boundary fluid flow required for accumulation of paramagnetic particles in the column, the tra-
jectory of a paramagnetic particle moving as a result of flow in the vertical column under the
magnetic field gradient was calculated.
The general configuration of the particle motion problem and a 2D schematic of the parti-
cle control system utilized for modeling the targeting of the paramagnetic particle by the mag-
netic force arising from the magnet placed outside the column are represented in Figure 2(a).
21
Fmr ¼l0kVpMsa2
r3
Msa2
r2þH0cos 2h
;(3)
Fmh¼l0kVpMsH0a2
r3sin 2h;(4)
Fg¼qpgVp;(5)
FIG. 2. Microfluidic device schematics and forces acting on the paramagnetic particle in the column. (a) Typical dimen-
sions of the vertical column are: length of 15 mm and diameter of 700 lm. The nickel wire diameter was 69 lm. Trajectory
of the paramagnetic particle in the vertical column is determined by the balance of the gravitational force (F
g
), drag (F
D
),
buoyancy (F
b
), and magnetic force (F
mr
,F
mh
). (b) The column is placed vertically in the field generated by four permanent
nickel-iron-boron magnets on each side of the column.
014110-4 Sofla et al. Biomicrofluidics 7, 014110 (2013)
Fb¼qfgVp;(6)
FD¼6pcgðvfvpÞ;(7)
where k¼vpvfis the difference in magnetic susceptibility of the particles and media at
room temperature, ris the distance of a particle from the nickel wire, ais the radius of the
nickel wire, Mssaturation magnetization of wire, H0external magnetic field strength, g
dynamic viscosity of fluid, and vf;vprepresent the velocity of the fluid and the particle. In addi-
tion, q
p
is the density of the particle, q
f
is the density of the fluid media, V
p
is the volume of
the CM, gis the dynamic viscosity of the media, and cis the effective radius of the CM.
The trajectory of the paramagnetic particle, positioned in the center at the bottom of the
column, was calculated using Eqs. (3)–(7) assuming that the particle is at the same level as the
nickel wire (h¼0Þ. All calculations and plotting were performed using MATLAB. The initial ve-
locity of the paramagnetic particle in the cylindrical column at an initial location S0½X0;Y0was
set as v0¼vðS0Þ. Particle acceleration, A0¼AðS0Þat the S0½X0;Y0position was calculated
using the following equation:
~
A¼~
F
mp
;(8)
where mpis the mass of the paramagnetic particle. At the second and, generally, nth position,
velocity and acceleration are calculated using the following equations:
Sn¼Sn1þVn1tþ1
2An1t2;(9)
Vn¼Vn1þAn1t;(10)
An¼AðSnÞ:(11)
III. MATERIALS AND METHODS
A. Neonatal and adult mouse heart cells
All animal experimental procedures were approved by the Animal Care Committee of the
University of Toronto following the Guide of Care and Use of Laboratory Animals. Ventricular
CM were obtained from 8- to 12-week-old adult yellow fluorescent protein (YFP) transgenic
mice (129-Tg 7AC5Nagy/J, Jackson Laboratory) of either sex using an isolation procedure
described previously.
22
Mice were heparinized (10 IU/g body weight) and 5min later, anesthe-
tized with 2.5% isoflurane as confirmed by the absence of pedal reflexes. Hearts were excised via
a midline thoracic incision and transferred to ice-cold Ca
2þ
-free Tyrode’s solution ((mmol/l) 137
NaCl, 5.4 KCl, 1.0 MgCl
2
,0.33NaH
2
PO
4
,10D-glucose, 10 4-(2-hydroxyethyl)-1-piperazineetha-
nesulfonic acid (HEPES), pH 7.4). The hearts were then mounted on a 20-gauge blunted stainless
steel cannula and retrogradely perfused via aorta with 37 C, oxygenated Ca
2þ
-free Tyrode’s so-
lution for 5 min, followed by collagenase (1 mg/ml, Worthington) for 8 min. Ventricular tissues
were dissected out and transferred to and stored in Krebs-bicarbonate solution ((mmol/l) 120 po-
tassium glutamate, 20 KCl, 20 HEPES, 1.0 MgCl
2
,10D-glucose, 0.5 K-Ethylenediaminetetraace-
tic acid (EDTA), and 0.1% bovine serum albumin, pH 7.4) at 4C, with gentle trituration to dis-
sociate the cells. The cell suspension was then subjected to separation as described below.
Neonatal mouse CM and fibroblasts were dissociated according to a standard isolation pro-
tocol.
23
Briefly, neonatal mice (YFP transgenic, 129-Tg(CAG-EYFP)7AC5Nagy/J; Jackson Lab-
oratories) were first euthanized. The hearts were removed and quartered. Quartered hearts were
digested in 0.06% (w/v) solution of trypsin in Ca
2þ
and Mg
2þ
free Hank’s balanced salt solu-
tion (HBSS), overnight at 4 C. Then, collagenase II (Worthington, USA 220 units/ml) in
HBSS was used to further digest the hearts at 37 C in series of five 4-8 min digestions. After
014110-5 Sofla et al. Biomicrofluidics 7, 014110 (2013)
the last digestion step, the cells were centrifuged at 500 rpm for 5 min, which ensured that car-
diac cells were pelleted, while red blood cells remained in the suspension. The cells were pre-
plated in T75 flasks for 1 h in an incubator in cardiac culture medium. The cells that remained
unattached after 1 h of pre-plating were CMs and used for quantification of myoglobin as
described below. The attached cells, cardiac fibroblasts, were expanded for up to 1 week and
used as a negative control. The CM culture medium and fibroblast culture medium were
identical in composition, consisting of Iscove’s modified Dulbecco’s medium (IMDM) with
L-glutamine and 25 mM HEPES, 10% certified fetal bovine serum (FBS), 100 U/ml penicillin,
and 100 lg/ml streptomycin.
B. Myoglobin quantification by enzyme-linked immunosorbent assay
Cell lysates were prepared with NP40 lysis buffer ((mmol/l) 50 Tris-HCl, pH 7.4, 150
NaCl, 40 NaF, 0.5 Na
3
PO
4
, 1% NP40) supplemented with complete protease inhibitors without
EDTA and 200 lMNa
3
PO
4.
To normalize the amount of myoglobin per number of CM, the
cells in suspension were counted prior to lysis. Since myocardium is composed of a heterogeneous
cell population, total CM number in the sample lysis was obtained by analyzing the percentage of the
CM. Neonatal myocardium cells collected from enzymatic digestion were all spherical and vaguely
distinguishable by shape or size distribution; therefore, the percentage of neonatal CMs in the cell
suspension was determined by immunostaining of paraformaldehyde fixed cells for a cardiac marker
Troponin I and flow cytometry as we have described previously.
12
The collected adult CMs were
morphologically distinguishable rod shaped cells in bright field microscopy. Thus, their percentage
was quantified using hemocytometer by counting the rod shaped cells (ratio of length to width >3).
Myoglobin determination by sandwich enzyme-linked immunosorbent assay (ELISA) was performed
according to the manufacturer’s protocol (Life Diagnostics). Briefly, the test samples were placed into
microtiter wells pre-immobilized with monoclonal antibody directed against the myoglobin molecule.
The polyclonal anti-myoglobin antibody conjugated to horseradish peroxidase (HRP) was added in so-
lution. The test samples were allowed to react simultaneously with the two antibodies, resulting in the
myoglobin sandwiched between the solid phase and the enzyme-linked antibodies. After 60 min of
incubation at room temperature, the wells were thoroughly washed to remove unbound HRP labeled
antibodies. HRP substrate, tetramethyl-benzidine (TMB), was added to allow a development of color,
with the intensity directly proportional to the concentration of myoglobin. The absorbance was meas-
ured spectrophotometrically at 450 nm using a plate reader (Apollo LB911, Berthold Technologies).
C. Metmyoglobin evaluation using spectrophotometry
UV-vis measurements were made in cells lysed with NP40 lysis buffer for 30 min on ice,
with cell debris spun down at 13 000 rpm for 10 min at 4 C. The lysates were collected and
mixed with NaNO
2
solution (2.5 mM for up to 1 h on ice), while NaNO
2
-free solutions were
used as controls. ND-1000 Nanodrop spectrophotometer (Nanodrop Technologies, Inc., Wil-
mington, DE, USA) was used to collect absorption spectra from 200 to 700 nm.
D. Microfluidic device fabrication
A schematic of the microfluidic column that was used for the separation studies is shown
in Figure 2. In the developed device, nickel wire was wrapped around the circumference of a
separation column (Figure 2(a)). The column was placed in a vertical position between the per-
manent magnets with the flow inlet at the bottom of the column (Figure 2(a)).The effects of
gravity, hydrodynamic conditions, and magnetic properties of the cells were combined in the
device in such a way that upon application of the magnetic field, paramagnetic CM remained in
the column, while fibroblasts and other non-myocytes traveled out of the column. The columns
were fabricated by embedding a core inside polydimethylsiloxane (PDMS). The core was made
by wrapping a nickel wire of thickness 69 lm in diameter around a stainless steel rod. The rod
was coated by an anti-sticking Teflon spray (Dupont) before the wrapping of the nickel wire.
The rod length was 30 mm and the diameter varied from 300 to 1200 lm for different designs.
014110-6 Sofla et al. Biomicrofluidics 7, 014110 (2013)
PDMS was prepared by a 10:1 ratio of monomer to crosslinker according to the manufac-
turer’s instructions. The mold was then degassed in a dessicator and placed in a 65 C convec-
tion oven for 2 h, followed by overnight curing at room temperature. After curing, the rod was
pulled out. Tygon tubing was inserted to either end of the column and sealed with 5-min epoxy.
To generate a uniform magnetic field, four neodymium-iron-boron permanent magnets of cubic
shape (12.7 mm 12.7 mm 12.7 mm) were separated from four similar magnets by means of
two spacers. The field strength within the space between two magnets was measured to be
1.23 60.05 T. The separation column was then placed inside the space between the magnets
and the spacers as shown in Figure 2(b). In initial studies, it was observed that the column di-
ameter of 700 lm resulted in the enrichment of adult CM and was selected as the column diam-
eter thereafter. The column length within the space between the magnets was 15 mm.
E. Cell separation
For the enrichment tests, adult mouse heart cells were mixed with primary neonatal mouse
fibroblasts at different ratios ranging from 15% to 85%, to study the effect of initial CM per-
centage on final enrichment. Cell concentration was determined using a hemocytometer. The
concentration of the cell sample also varied from low, 0.2 10
6
/ml, to high 5 10
6
/ml to track
the effect of sample concentration on the enrichment results. The cells were treated with
NaNO
2
in the concentration from 2.5 mM to 50 mM for 20 min on ice using media consisting
of Ca
2þ
and Mg
2þ
free Hank’s balanced salt solution with 30% FBS. Sodium nitrite was kept
in solution during the entire separation process to ensure that metmyoglobin is present in the
cells during separation. Initial studies demonstrated that 0.2 10
6
cells/ml and 2.5 mM of
NaNO
2
resulted in the highest enrichment, thus they were pursued in all subsequent studies.
To load the cells into the column, 20 ll of the sample was withdrawn by a pipette tip from
the Eppendorf tube. The tip was then extracted from the pipette and placed on the top of the
column. The mixture was either withdrawn into the column by a syringe pump that was con-
nected to the bottom of the column or just by settling of the cells into the column as a result of
gravity. Upon settling, the flow of 4.2 ll/min was applied from the bottom of the column to
rinse the fibroblasts out, while keeping CM in the column. A pipette tip was inserted into the
top of the column to collect the rinsing flow. After 200 ll, the flow was stopped, a new pipette
tip was inserted and the flow rate was increased to 144 ll/min. As a result, the trapped cells
inside the column were pushed out, into the pipette tip.
After collecting 200 ll of media into the pipette tip, the tip was removed and emptied into
a well of a 12-well plate that was already coated with a solution of 10% Matrigel and 90%
plating media for adult CM. Plating media, 2 ml, was added to the well and suspended few
times. The fluid was then split into two volumes of 1 ml each in new wells. One well was used
to count the enrichment of the cells and to study the cell viability right after the test, while the
other well was incubated for 24 h to study the viability of the cells upon separation and culture.
Bright field imaging was used to count the number of CM and fibroblasts, where rod shaped
cells were considered CM.
F. Cell viability
Viability of adult mouse CM upon treatment with NaNO
2
(2.5 mM for 20 min on ice) and
upon enrichment within the microfluidic device was determined using trypan blue exclusion
with untreated cells used as a control. The cells were emptied into one well of a 24-well plate
and an equal volume of trypan blue was added to each well. After 1 min, 1 ml of media was
added to each well and the images were taken to determine the viability with dark blue cells
being considered dead and transparent cells alive.
The cell viability after 24 h of culture was determined by live/dead staining. Following the
incubation, 500 ll of supernatant from each well was removed. In each well, 0.5 ll of carboxy-
fluorescein diacetate and 37 ll of propidium iodide were added. The plate was then incubated
at 37 C and 5% CO
2
for 30 min. After 30 min, 500 ll of M199 media with 1% penicillin/strep-
tomycin was added to each well and imaging was done to assess viability. The size of adult
014110-7 Sofla et al. Biomicrofluidics 7, 014110 (2013)
CM was determined from 5-Carboxyfluorescein Diacetate (CFDA) stained images using IMAGEJ,
by determining the long and the short axes of each cell.
G. Statistical analysis
Statistical significance was determined using one-way ANOVA in conjunction with
Tukey’s test. Normality and equality of variance were tested. p <0.05 were considered signifi-
cant. A minimum of 3 samples were used per data point.
IV. RESULTS AND DISCUSSION
A. Myoglobin quantification and paramagnetic properties of cells
The amounts of myoglobin in the cells were confirmed by ELISA (Figure 3). While non-
contractile fibroblasts consistently tested below myoglobin detection levels for the ELISA kit,
this protein was detected in both neonatal mouse CM, as well as in the adult CM with signifi-
cantly higher concentration in the adult CM compared to the neonatal CM. Consistent with
maturation of the cells, the amounts of myoglobin per cell increased more than 5 times in adult
mouse CM compared to the neonatal mouse CM.
The presence of metmyoglobin was also confirmed by spectrophotometric measurements in
the samples treated with NaNO
2
(Figure 4). We assessed the extent of induction of metmyoglobin
from the total myoglobin upon treatment with 2.5 mM NaNO
2
, conditions used for cell separation
in this study (Figure 4). Since myoglobin derivatives differ in their absorbance spectra, the ratio
of the absorbance peak for metmyoglobin at 635nm to the isobetic point for myoglobin, oxymyo-
globin, and metmyoglobin at 525 nm contains information on the amount of metmyoglobin as a
fraction of the total myoglobin.
24
We observed that the maximum amount of myoglobin induc-
tion upon treatment with 2.5mM NaNO
2
occurred 45min after nitrite was introduced to the cells,
motivating our decision to load the cells into the column 30 min after exposure, to ensure cardio-
myocytes are rendered paramagnetic at maximal levels during separation.
B. Calculation of the trajectory of a paramagnetic CM in a cylindrical tube
The parameters in Eqs. (3)–(7) and the values for a typical experimental condition with a
700 lm diameter column are described in Table I.
The size of the adult CM was determined by image analysis. These cells were rod/elliptical
shaped with the average long axis of 77 614 lm and the short axis of 25 63lm(N¼10). The
area of each cell was calculated using the area of the ellipse formula, based on the measured
long and short axes. For each calculated area, a radius of the circle occupying the same area
FIG. 3. Myoglobin quantification in cells by ELISA. m-FB, neonatal mouse fibroblasts; m-neo CM, neonatal mouse
cardiomyocyte; m-adult CM, adult mouse cardiomyocyte.
014110-8 Sofla et al. Biomicrofluidics 7, 014110 (2013)
was calculated. This radius was termed effective cell radius. For the purpose of mathematical
modeling, the effective cell radius determined as described above was 22 lm. The ratio of mag-
netic force to the gravitational force applied on a representative sodium nitrite treated paramag-
netic CM that is at the same level as the nickel wire (h¼0Þis plotted in Figure 5(b) by substi-
tuting values from Table Iin the equations above. The theoretical analysis shows that at the
central areas of the column (farther than 100 lm from the column wall), the magnetic force is
much smaller than the gravitational force due to the low magnetic susceptibility of the CM.
These results indicate that the resulting velocity of the cells toward the magnet is much smaller
than the cell’s typical settling velocity, which is 64.4 lm/s for a cell with effective radius of
22 lm.
25
Therefore, the flow rate in the column must be slow to prevent the washout of the
desired cell type.
In our batch separation approach, the heterogeneous cell population was loaded into the
column. Once in the column, the paramagnetic CMs were attracted towards the column wall as
a result of the magnetic field gradient generated by the nickel wire and the presence of the
permanent magnet. The theoretical analysis showed that the flow rate from the bottom of
the column could be adjusted in such a way to keep the CM in the column and rinse out the
FIG. 4. Metmyoglobin induction as a function of NaNO
2
treatment in cardiomyocytes. Treatment of adult cardiomyocytes
with 2 mM NaNO
2
indicates maximal induction of metmyoglobin after 45 min.
TABLE I. Experimental and mathematical model parameter values.
Variable Name Value Unit
l0Vacuum permeability 4p10
7
H/m
kpMagnetic susceptibility of CM 7.11 10
8
kfMagnetic susceptibility of fluid 0.9 10
5
aNickel wire radius 34.5 lm
MSSaturation magnetization of nickel 486 10
3
A/m
rDistance of cell from the wire 0–200 lm
B0External magnetic field 1.23 T
gAcceleration of gravity 9.98 m/s
2
HAngle to define the location of cell vs wire Rad
gDynamic viscosity of fluid 0.001 Ns/m
2
qpDensity of CM 1060 kg/m
3
qfFluid density 1000 kg/m
3
cEffective radius of adult CM 22 lm
014110-9 Sofla et al. Biomicrofluidics 7, 014110 (2013)
FIG. 5. Mathematical modeling results of hydrodynamic and magnetic force effects on a paramagnetic particle trajectory
in the vertical separation column. (a) Theoretical velocity profile of the fluid flow at 4:2ll=min within a 700 lm diameter
vertical column. (b) Balance of magnetic and gravitational forces in the vertical separation column as a function of radial
position in the column. (c) Trajectories of a paramagnetic particle initially placed in the center at the bottom of the column
as a function of fluid flow rate (4.2, 9.5, or 10 ll/min). Column length of 15 mm is assumed.
014110-10 Sofla et al. Biomicrofluidics 7, 014110 (2013)
non-paramagnetic fibroblasts. At the given column radius and the magnitude of the magnetic
field, the percentage of CM trapped in the column would depend on the applied flow rate and
the length of the column. In our theoretical analysis, we assumed that the paramagnetic cell
was initially present at the centerline at the bottom of the column. We calculated the trajectory
of such a paramagnetic cell inside the column at different flow rates. According to our analysis,
the paramagnetic cell would travel upward in the column and towards the wall where the nickel
wire was circumferentially positioned, reaching the wall of the column after a certain distance
was traveled in the z-direction. Due to the no slip boundary condition at the column wall, we
assumed that cells would be trapped inside the column once they reached the wall. Solving
Eqs. (3)–(7) and plotting the cell trajectories (Figure 5(c)), we observed: (1) at 4.2 ll/min, the
cell would fall on the column wall after 1.475 mm, and percentage of trapped cells was 100%
for a column described in this paper, which was 15 mm long; (2) at 35 ll/min, the cell would
fall on the column wall after 14.817 mm and percentage of trapped cells was 100%; (3) at
36 ll/min, the cell would leave the column and the percentage of trapped cells was 75%.
C. Microfluidic enrichment results
Cell separation experiments with paraformaldehyde fixed adult CM showed that the highest
enrichment was achieved at a flow rate of 4.2 ll/min consistent with our theoretical analysis
above. The average enrichment in a single pass for the fixed cell population was 93% 64%
(N ¼8); thus, 4.2 ll/min was selected for all experiments shown in Figure 6. The initial cell
concentration was consistently maintained at 0.2 10
6
/ml, as higher concentrations tested (up
to 5 10
6
/ml), resulted in lower enrichment due to overcrowding and formation of heterogene-
ous cell clumps, consisting of both CM and FB that hindered the separation process.
The velocity entrance length was calculated for the 700 lm diameter column at 4.2 ll/min
according to the following formula:
26
Lv ¼Rcð1:18 þ0:112ReÞ;(12)
where R
c
is the column radius and Re is Reynolds number. The calculated velocity entrance
length of 0.42 mm indicated that the flow was laminar and fully developed over most of the
column length validating our theoretical analysis at the given experimental conditions as pre-
sented above (Figure 5).
FIG. 6. Correlation between input cardiomyocyte percentage and output percentage in the cell population during the sepa-
ration at 4.2 ll/min. Application of magnetic field during separation enables consistently high enrichment efficiently
between 90% and 100%.
014110-11 Sofla et al. Biomicrofluidics 7, 014110 (2013)
To confirm the enabling contribution of the magnetic effects and rule out the possibility
that the enriched population was only due to the difference in size, density, or the difference in
settling velocities alone of the adult mouse CM and neonatal fibroblasts, several tests were con-
ducted in the absence of the magnet. For these control tests, similar to the tests with magnet,
mixtures of live or fixed CM with fibroblasts were treated with NaNO
2
. The same media and
flow rate was used for the control tests to ensure that the viscosity of the fluid was the same. In
the absence of a magnet, most of the cells were rinsed out of the column, as expected.
Next, the separation tests with live cells at the flow rate of 4.2 ll/min were performed.
Live adult mouse CMs were mixed with live pre-plated neonatal fibroblasts at different ratios.
The enrichment efficiency of the live cells was averaged at 93% 62%. No significant differ-
ence was observed between the results of fixed and live cells (Figure 6).
Viability of adult mouse CM after separation and subsequent culture for 24 h was not com-
promised by the treatment with NaNO
2
or passage through the device (Figure 7). In both cases,
FIG. 7. Viability of adult mouse cardiomyocytes (a) after separation and (b) subsequent culture for 24 h. All values are nor-
malized to the average cell viability of the control group, at the appropriate time point. The control was kept on ice during
separation and was not treated with NaNO
2
(no treatment).
014110-12 Sofla et al. Biomicrofluidics 7, 014110 (2013)
cell viability was comparable to that of the control cell population that was not treated with
NaNO
2
and not separated. The treatment of cells by NaNO
2
is not expected to decrease cell vi-
ability; in fact, the protective effects of NaNO
2
on myocardium were reported.
27,28
Each molecule of myoglobin contains a single iron atom which may bind reversibly to one
molecule of O
2
when the iron is in the ferrous state (Fe
2þ
), to act as an oxygen storage and
enable efficient oxygen supply under conditions of hypoxia and ischemia. Myoglobin undergoes
oxidation to the inactive ferric state (Fe
3þ
), known as metmyoglobin upon treatment with
NaNO
2
. Studies have shown that at the concentrations required for this conversion of myoglo-
bin to metmyoglobin, nitrite did not affect the respiration of isolated aerobic heart mitochon-
dria, or glycolysis, and that the levels of adenosine triphosphate remained constant in both
cases.
29
Under normoxic conditions, in single cell suspension, such as during the microfluidic
cell separation, myoglobin is of little functional significance. Post separation and upon NaNO
2
removal, myoglobin can be restored freely based on the studies that reported observed reduction
of metmyoglobin in isolated perfused rat and sea raven hearts following treatment with
NaNO
2
.
30
This reduction involves enzymes normally present in the cells such as endogenous
metmyoglobin reductase.
31
Here, a microfluidic approach for label-free magnetic separation of CM was presented. The
basis for the separation was the higher amount of iron in cardiomyocytes found in myoglobin
when compared to the iron level of other cardiovascular cells. After collagenase digestion, the
heart isolate contained CM, fibroblasts, smooth muscle cells, and endothelial cells. The ratio of
these cells in the native neonatal rat heart after collagenase digestion was 47% cardiomyocytes,
48% fibroblasts, 3% smooth muscle cells, and 2% endothelial cells as we
32,33
and others
34
reported. Myoglobin is found in both CM and smooth muscle cells. However, the myoglobin
content of smooth muscle cells (0.2 mg/g wet weight) is significantly smaller than that of CM
(2.6-5.4 mg/g wet weight), due to the high demand for oxygen in contracting CM.
35
A fraction of the non-CMs, which were found adjacent to the vertical walls of the column
at the beginning of separation, as a result of random distribution of cells in the column, would
remain inside the column during the rinsing stage. These non-CM represent the impurity which
is reflected in the experimental results.
The yield of the separation process in experiments presented in this paper is limited by the
capacity of the column. Loading the cells with too concentrated cell mixture would result in the
rinsing of CM out of the column, and consequently losing the target cells. It was observed that
maximum yield of over 80% was achieved when the total cell number that was loaded into the
column was less than 10 000. In each experiment described here, between 3000 and 5000 adult
CM were collected. Using only one column and given that each experiment takes 30 min, the
through-put of the experiments in this study was between 6000 and 10 000 collected CM/h.
Future studies will focus on improving the yield of the separation process by using multiple
columns in parallel.
Alternatively, the yield of the separation process could be improved by switching to a con-
tinuous separation process, with several interesting examples described in the recent literature.
In one case, the authors used a U shaped device with permanent magnet located at one arm of
the separation column. As the cells passed through the U turn, and entered the other arm of the
U shaped column, their trajectory was affected as a function of particle size, bulk flow rate in
the column, and the magnetic properties of the particles and the surrounding fluid, thus enabling
cell separation without the use of the sheath fluid.
36
In another approach, the cell separation
through-put was enhanced in the microfluidic device by utilizing two electromagnets with anti-
parallel current flow, placed around the separation channel. The two magnetic fields acted in
such a way to push the magnetically tagged cells into the center of the channel, where the flow
rate of the sheath fluid was the fastest.
37
Additionally, the use of electromagnets vs. permanent
magnets in this configuration enabled simple and fast tuning of the magnetic field strength on
the same chip.
37
The trajectory of magnetic particles in a microfluidic channel could also be
affected by an array of stripes, whose orientation, width, and spacing, together with the posi-
tioning and strength of the external magnets, would determine the particle trajectory, potentially
enabling continuous separation.
38
The contaminating fibroblasts could also potentially be
014110-13 Sofla et al. Biomicrofluidics 7, 014110 (2013)
removed from the final cell preparation by techniques akin to resistive pulse sensing in tunable
or non-tunable pores.
39
In this paper, we achieved enrichment of the adult mouse CM from a heterogeneous cell
population. This is not a target cell type for regenerative medicine applications due to the non-
human species and the fact that adult CMs are terminally differentiated. We used this cell type
here in order to demonstrate the proof of concept that live CM can be enriched using magneto-
phoresis in a label-free manner. Adult CM have the highest myoglobin content compared to
other developmental states (Figure 3), thus simplifying the separation problem. In future stud-
ies, the column will be further refined (e.g., smaller diameter) to enable enrichment of neonatal
rodent CM as well as CM derived from human pluripotent stem cells. The CM derived from
human embryonic stem cells or induced pluripotent stem cells represent a true target cell type
for applications in regenerative medicine, tissue engineering, and drug testing.
V. CONCLUSIONS
We developed here a new method to enrich live CM in a label-free manner starting from a
heterogeneous cell population containing CM and fibroblasts. CM were rendered transiently
paramagnetic by application of NaNO
2
. Manipulation of CM settling velocity in a vertical col-
umn by application of permanent magnetic field and field concentration to the column walls via
a nickel wire enabled enrichment of live CM up to 93%. The separation process did not com-
promise cell viability immediately after separation as well as after 24 h in culture.
ACKNOWLEDGMENTS
This work was supported by an NSERC I2I, NSERC Discovery Grant and NSERC Discovery
Accelerator Supplement to M.R. The authors would like to thank Moniba Mirkhani, Samy Makary,
and Dr. Peter Backx for help with isolation of adult cardiomyocytes.
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