Micro- & nano- fluidic research for chemistry, physics, biology, & bioengineering
2D micro gas chromatography
Continuous sorting of embryoid
Volume 10 | Number 13 | 7 July 2010 | Pages 1633–1740
Volume 10 | Number 13 | 2010
Lab on a Chip
As featured in:
See Ho et al., Lab Chip, 2010, 10,
Registered Charity Number 207890
Featuring research from the Microsystems Laboratory
of Dr Chih-Ming Ho in the Mechanical and Aerospace
Engineering Department at the University of California,
Title: Continuous sorting of heterogeneous-sized embryoid bodies
Embryoid bodies (EBs) are spherical aggregates of spontaneously
differentiating embryonic stem cells and EB-mediated differentiation
efficiency is critically dependent upon EB size. This paper presents a
microfluidic device for sorting EBs into multiple size-dependent groups
with high separation efficiencies. Our proposed separation scheme
utilizes appropriated spaced pillars within a microchannel to alter
the fluid flow pathway, thus allowing particles of defined sizes to be
diverted into size-specific collection reservoirs.
Continuous sorting of heterogeneous-sized embryoid bodies†
Peter B. Lillehoj,aHideaki Tsutsui,aBahram Valamehr,bHong Wuband Chih-Ming Ho*a
Received 5th January 2010, Accepted 12th March 2010
First published as an Advance Article on the web 7th April 2010
This paper presents a microfluidic device for sorting embryoid bodies (EBs) with large dynamic size
ranges up to 300 mm. The proposed separation scheme utilizes appropriately spaced pillars within
a microchannel to alter the fluid flow pathway, thus allowing particles of defined sizes to be diverted
towards specific flow paths. We test the device functionality by separating polystyrene beads 90, 175
and 275 mm in diameter, demonstrating separation efficiencies approaching 100%. We then
demonstrate for the first time on-chip separation of mouse EBs, which were separated into three size
groups. The ability to extract specific size ranges of EBs will greatly facilitate their subsequent
With their developmental potential to differentiate into all three
of the germ layers (endoderm, mesoderm and ectoderm),
embryonic stem (ES) cells provide a unique opportunity to study
lineage commitment and can potentially serve as a source of
specialized cells for regenerative medicine. Among the various
published ES cell differentiation protocols,1–4the formation of
embryoid bodies (EBs), spherical aggregates of spontaneously
differentiating ES cells, is commonly utilized as a critical inter-
mediate step. EBs appear to recapitulate embryonic develop-
ment, facilitating induction of differentiation and commitment
into specific cell types.5There are several conventional methods
to create EBs in vitro, including suspension culture on low-
attachment plates and hanging drop methods; however, these
methods suffer from heterogeneous size distribution or lack of
scalability.6We recently reported that EB-mediated differentia-
tion efficiency is critically dependent upon EB size. EBs between
100 and 300 mm in diameter had the highest survival rate and
greatest expression of genetic markers of differentiation.7,8
Therefore, obtaining EBs of homogeneous size appears to be
a key factor for successful ES cell research.
Microfluidic processes can be an efficient means for the sepa-
ration/sorting of particles andbiological cells. Todate, numerous
techniques have been developed to separate micron-sized
samples including those utilizing acoustic, magnetic, optical, or
electric forces.9–12Although these techniques allow for contin-
uous separation on a miniaturized platform, they require the use
of external force fields. Methods based on gravitational13and
centrifugal forces14have also been demonstrated; however, these
require long processing times or bulky external devices.
Alternatively, work has been done to create microdevices based
on fluorescence (mFACS).15,16Some of these techniques require
particle/cellular modification prior to separation, thereby incur-
ring particle damage and complicated sample collection proce-
dures. To circumvent these problems, researchers have been
developing separation techniques based solely on particle size
and fluidic forces within a microchannel. This strategy offers
improved specimen integrity while simplifying sample prepara-
tion procedures for lab-on-a-chip biological/chemical analysis.
Furthermore, by integrating this technique onto a compact
microfluidic platform, additional advantages of lower produc-
tion costs and reduced reagent consumption are achieved.
Recent research on flow-based separators has led to several
techniques, including deterministic lateral displacement,17,18
pinched flow fractionation (PFF),19,20hydrodynamic filtration,21
Brownian ratchet separation,22
separation23and hydrophoretic separation.24These techniques
can achieve continuous particle separation with high efficiencies,
which is crucial for large-scale processing; however, these
methods are designed for small particles and cells less than 25 mm
in diameter. The present device was designed to simultaneously
sort particles within a large size range (from 1–300 mm), allowing
for the separation of a wide variety of samples which differ in
orders of magnitude in size. Compared with smaller sized
particles, the influence of inertial forces is more dominant for
larger sized particles, which is an important concept to consider
for their separation. Based on this device, we demonstrate the
separation of mouse EBs, which vary from tens to hundreds of
microns in size.
Current pipetting methods for separating EBs are time
consuming, inefficient and result in poor size uniformity. Addi-
tionally, the separation of EBs through external force fields, such
as dielectrophoretic (DEP), acoustic or magnetic, may raise
potential issues in damaging the fragile cellular entities, thereby
affecting subsequent cell differentiation. Replacing low-attach-
ment plateswith hydrophobic
homogeneity of the EB size.7Alternatively, several research
groups employed microdevices, such as microwells25and
microchannel compartments,26to physically define the size of
such as poly-
aMechanical and Aerospace Engineering Department, University of
California, Los Angeles, CA, USA. E-mail: firstname.lastname@example.org;
Tel: +1 310-825-9993
bDepartment of Molecular and Medical Pharmacology, David Geffen
School of Medicine, Los Angeles, CA, USA
† Electronic supplementary information (ESI) available: Computation
modeling, materials and methods
preparation, experimental setup, EB viability analysis, COMSOL
simulations for various channel configurations and data on EB
viability. See DOI: 10.1039/c000163e
1678 | Lab Chip, 2010, 10, 1678–1682 This journal is ª The Royal Society of Chemistry 2010
PAPER www.rsc.org/loc | Lab on a Chip
growing EBs. While these latter methods attempted to obtain
homogeneous EBs by manipulating their growth conditions, our
approach has been to separate a population of heterogeneous
EBs into uniform size groups using a novel microfluidic sorting
procedure. Continuous sorting of EBs by size can be a valuable
tool for obtaining large numbers of homogeneous EBs using
either conventional EB forming platforms or the newer culture
methods, potentially facilitating EB-mediated differentiation of
embryonic stem cells.
The proposed method of separation is solely based upon the
modification of a fluid flow pathway within a microchannel
through the strategic positioning and geometric design of micro-
sized pillars. In this paper, computational modeling of the flow
profile is presented to guide the rationale in designing the device.
The separation efficiency of the device was calibrated using
polystyrene beads of three different sizes. Finally, high-
throughput separation of EBs is presented.
Results and discussion
The fluidic network (Fig. 1B) was designed for high separation
efficiency and high throughput while maintaining a compact
profile. The particles are sorted within a narrow separation
region (spanning less than ?1.3 mm) which is located immedi-
ately upstream of the outlet branches. This region is comprised of
bullet-shaped pillars which are strategically placed within the
channel to alter the flow pathway. The modified pathlines allow
for particles to be diverted toward predetermined branches of
specific size groups (0–100 mm, 100–200 mm and 200–300 mm).
A sheath flow is utilized to align the particles along the upper
wall of the channel prior to separation. This sheath flow
produces a normal force to the channel wall to minimize the
vertical displacement caused bythe inertial forces of the particles.
Computation simulations were performed to optimize the sheath
flow for enhanced particle alignment (see Fig. S2 in ESI†).
Simulations were also performed to optimize the pillar
geometry and configuration for optimal separation efficiency
(see Fig. S3 and S4 in ESI†). Various parameters (i.e., pillar
geometry, pillar angle and inter-pillar spacing) were modified to
observe their effects on the response of the system. Based on our
results, the pillars are designed with lengths and widths of 300 mm
and 60 mm respectively and are positioned at a 15?angle with
respect to the upper channel wall. The spacing between the first
and second set of pillars, S1 and S2, are 115 mm and 210 mm
respectively. Pillar angles <15?had a minimal effect on altering
the flow profile, resulting in closely packed pathlines unable to
sufficiently divert particles to their corresponding outlets.
However, angles [15?dramatically obstructed the flow profile,
which would cause particles to veer off their intended pathlines.
In terms of pillar configuration, inter-pillar spaces much larger
than their corresponding particle size would allow for the
passage of smaller particles, thus lowering the separation
The width of the main channel is 640 mm and the widths of the
upper (B1), middle (B2) and lower (B3) outlet branches are
120 mm, 220 mm, and 320 mm respectively. The height of all the
channels is 340 mm. The widths of the outlet branches are
designed not only to minimize particle clogging, but also to
correspond with the spacing between the respective pillars so as
to maximize the flow of the particles into their respective reser-
voirs. The reservoirs are 4 mm in diameter and are open to the
atmosphere which allows for the sorted particles to be easily
collected using a pipette. The overall size of the PDMS chip is
16 mm ? 16 mm (Fig. 1A), which greatly minimizes the footprint
of this system and enables for large batch fabrication.
Separation of polystyrene beads
Initial experiments were performed to separate polystyrene beads
into 0–100 mm, 100–200 mm and 200–300 mm groups. The flow
rates ofthe sample and buffersolutions wereadjusted to align the
particles along the upper wall of the channel. Essentially, the flow
rates were modified to produce an optimal velocity shear, in
which the force normal to the particle path could push the
particle against the upper channel wall. A flow rate of 5 mL min?1
with a ratio of 4 : 1 (buffer : sample) was sufficient to maintain
proper flow focusing. The smallest particles (#100 mm) could be
easily focused alongside the upper wall throughout the channel
and could follow their corresponding pathlines. Due to the exact
spacing between the pillars, these smaller particles were able to
move through inter-pillar space S1 between pillars P1 and P2 and
remain on the flow path leading towards branch B1 (Fig. 2A).
Particles between 100 and 200 mm were also focused along the
upper channel wall as they encountered the pillars; however, they
were too large to enter space S1 between pillars P1 and P2.
Following their corresponding pathlines, these particles flowed
past P2 and entered into space S2 between pillars P2 and P3,
leading towards branch B2 (Fig. 2B). Similarly, the largest
particles (200–300 mm) flowed past pillar P3 and were guided
toward branch B3 (Fig. 2C). By observing the simulation results
of pathlines in the separation region (Fig. S1 in ESI†), it is
device is roughly 16 mm ? 16 mm and is filled with dye for enhanced
visualization of the fluidic network. (B) Schematic illustration labeling
the relevant sections with an enlarged view of the separation region.
(A) Photograph of the proposed sorting device. The overall
This journal is ª The Royal Society of Chemistry 2010Lab Chip, 2010, 10, 1678–1682 | 1679
apparent that the experimental results follow very closely with
Based on this approach, only three pillars are needed for
separation into three size ranges, which is a much simpler design
than previous techniques. Such a simplified design greatly
reduces the complexity of the system, especially for multiple size
separation. Another advantage of this approach is the high
separation efficiencies that are achievable. Fig. 3 shows the size
distribution of particles in each reservoir for the sorting of
a mixed population of particles. Reservoirs R1 (0–100 mm), R2
(100–200 mm) and R3 (200–300 mm) exhibited purities of 100%,
92% and 92% respectively based on a population of several
hundred particles. The careful design of the separation region,
i.e., the precise spacing between the pillars and specific channel
dimensions, enables for such high separation specificity to be
achieved. Additionally, the 100% purity of particles in Reservoir
R1 is due to larger particles being unable to enter the upper
branch. Generally, while larger particles were unable to enter
reservoirs intended for smaller particles, it was possible for
smaller particles to enter incorrect reservoirs, which is evident by
the lower purities of Reservoirs R2 and R3. However, particles
will enter their respective reservoirs if they are properly focused
prior to entering the separation region. Based on this principal,
we determined that the separation efficiency is greatly affected by
the quality of particle focusing and the ability of the particles,
particularly smaller ones, to remain in their intended pathlines.
By operating in the microscale regime, this can be achieved by
maintaining laminar flow and precisely controlling the experi-
mental flow rates.
In addition to proper particle focusing, the distribution of
particles within the microchannel is another important factor
that was observed to affect the operation of the device. Sample
solutions with high particle concentrations tended to form
particle clusters that occluded the inter-pillar spaces, thus
inhibiting separation. Clumped particles were also poorly
focused, causing them to veer outside of their intended pathlines
and flow toward an incorrect reservoir. Additionally, grouped
particles will naturally follow unique pathlines since their
centroids have been altered. To account for these issues, beads
were diluted to a concentration of approximately 103beads per
mL during sample preparation. Sample dilution ensured that
particles were adequately spaced within the microchannel to
minimize particle clustering and alleviate device clogging.
However, excessive sample dilution reduced device throughput.
In order to address this concern, experiments were performed at
higher flow velocities to increase the sorting rate. Increasing the
flow rate to 20 mL min?1did not significantly affect separation
efficiency. At this flow rate, the beads could be separated at a rate
of 5 s?1.
Separation of EBs
The resultsfrom EB separation experiments matched very closely
with those from the bead experiments. EBs could be properly
focused utilizing a flow rate ratio of 4 : 1 (buffer : sample) and
maintained their streamline positions within the microchannel.
As previously mentioned, the proposed separation scheme
enables for simultaneous separation, in which specimens of
different sizes can be processed in a concurrent manner. Shown
in Fig. 4 is the simultaneous sorting of three mouse EBs of 90,
160 and 300 mm in diameter. Similar to the separation of poly-
styrene beads, high separation efficiencies could also be achieved
with EBs (Fig. 5). Reservoirs 1 (0–100 mm), 2 (100–200 mm) and 3
(200–300 mm) exhibited purities of 100%, 86% and 81% respec-
tively based on a population of several hundred EBs. The effects
of flow rate on EB separation were also studied and it was found
that altering the flow rates did not have a negative impact on the
separation efficiency. However, flow velocities >20 mL min?1
damaged the EBs, causing them to break apart. Unlike poly-
styrene beads, EBs are comprised of individual ES cells packed
together in a spherical mass and high flow velocities were detri-
mental to their structure. An operational flow rate #16 mL min?1
ensured EB integrity, preventing cell disassociation.
To evaluate the effects of the proposed microfluidic separation
technique on cell viability, a flow cytometry analysis was
microchannel:(A) 90 mm in size,(B)175 mm in size and(C) 275 mm in size.
Images were generated by superimposing video frames.
Trace of polystyrene beads showing their trajectory within the
Experiments were performed at a flow rate of 5 mL min?1. Error bars
represent the standard deviation of sorted beads from several indepen-
Size distribution of particles in each of the three reservoirs.
1680 | Lab Chip, 2010, 10, 1678–1682 This journal is ª The Royal Society of Chemistry 2010
populations. Cell viability, measured by the percentage of
a population negative to 7-AAD staining, showed minimal and
temporal impacts on cell viability (see Fig. S5 in ESI†). The issue
of clogging that was observed during bead experiments was more
prevalent during EB separation. EBs exhibited a higher degree of
clustering than beads, even at low cell concentrations. Addi-
tionally, current methods for EB formation lack size and shape
controllability, resulting in nonspherical-shaped cell formations.
As previously mentioned, specimen uniformity plays a crucial
role in the proper operation of this device. EBs heterogeneous in
shape were likely to shift toward different pathlines or obstruct
the device. As a result, the separation efficiencies for EB exper-
iments were slightly lower compared with bead experiments.
While it is not required for EBs to be perfectly spherical in shape,
it is important that they maintain a certain degree of homoge-
neity to follow the separation principals of the device. The ability
to form homogenous EBs is possible through the study of their
growth and formation procedures.25,26,27
onboth sortedand control (unsorted)EB
A microfluidic device capable of separating EBs and polystyrene
beads is presented. The unique design employed in this device
allows for separation into size-dependent groups with a high
degree of uniformity. Compared with conventional microfluidic
devices employing similar separation principals, this method
enables for continuous separation of large particles and cells
(#300 mm) in a compact device, while still achieving the same
functional purposes for particle separation. Furthermore, this
technique can be employed for the separation of specimens which
are too large for current microfluidic sorting methods and are
sensitive to external force fields. Such a device will greatly
improve the sorting and collection processes of vulnerable bio-
logical species, such as EBs, for further downstream studies and
provide ES cell researchers with healthy and homogenous
populations of cells.
This work was funded by the following agencies: National
Institutes of Health (NIH) through the Roadmap for Nano-
medicine Research (PN2EY018228) and NASA National Space
Biomedical Research Institute (NSBRI) (NCC 9-58-317). The
authors thank Dr T. S. Wong and Dr E. P. Lillehoj for their
useful comments in reviewing the manuscript.
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ration of mouse EBs 90, 160 and 300 mm in size. This experiment was
performed at a flow rate of 5 mL min?1. Time stamps are located at the
bottom left corners of each frame.
Sequential video frames demonstrating the simultaneous sepa-
are based on continuous cell separation, excluding clogging events.
Experiments were performed at a flow rate of 5 mL min?1. Error bars
represent the standard deviation of sorted EBs from several independent
Size distribution of EBs in each of the three reservoirs. The results
This journal is ª The Royal Society of Chemistry 2010 Lab Chip, 2010, 10, 1678–1682 | 1681