Application Note - Spiral Sorter Chip Fluidic 382
Sorting 7 µm and 15 µm Beads
Introduction
Many research applications call for sorting and isolating cells from a heterogeneous cell mixture. This can be for purifying cellsamples to improve efficiency in research applications and is a critical step in many diagnostic and therapeutic practices1. The
expanding need to isolate rare cells such as circulating tumor cells (CTCs) from blood samples increases the demand for cellsorting devices. Cell detection is generally performed using optical methods such as FACS (Fluorescent Activated Cell Sorting).
As it is an automated solution for cell sorting, it was considered a gold standard for decades. FACS has however, significant
limitations, including the need for a large number (millions) of cells. This limits the volume of solutions to be sorted. A labelingstep is required, and the high operating pressures required can affect cell function or viability. FACS systems are also cumbersome, expensive, and require experienced users. As an alternative, microfluidic spiral sorting devices allow one to passively sortcells with high throughput (>1.5 ml/min) without the need for a labeling step.

Why use microfluidics for cell/particle sorting?
As opposed to conventional instrumentation, microfluidic devices are easy to use, smaller, versatile, and affordable. Microfluidic cell sorting can be combined with additional fluidic operations for complete lab-on-a-chip applications, as well as fordiagnostic and therapeutic purposes. These devices make use of a wide range of techniques to sort cells with specific speeds
and efficiencies. The possibility to easily tune the design of the microfluidic device allows for the sorting of cells of different sizes
with throughput (flow rate) specific to the user‘s need.
Table 1: Advantages and disadvantages of cell sorting using FACS and microfluidics
Standard FACS
methods

Spiral sorterbased method

Control on experimental parameters

MediumHigh

Throughput

Very highHigh

Automation

Yes

PossiblePressure in use

High (can affect cells)

Controllable

Price

Very highMediumDevice size

Bulky

Small

Cell sorting is based on determining a specific cell parameter that can differ from one cell type to another, such as cell size,
shape, or density. The device is designed such that cells with different properties experience a different amount of force (thatcould originate from inertia, channel geometry, external source) while similar cells undergo equal forces. The differentiated cells
are subsequently pushed into different streamlines and exit the chip from different outlets.
Contact in Japan: ASICON Tokyo Ltd.
www.asicon-tokyo.com / asicon-tokyo@asicon-tokyo.com

Application Note Spiral Sorter Fluidic 382
The advantages of passive cell sorting systems
Passive cell sorting systems do not require labeling steps nor additional external power (e.g. electrical field), which makes themattractive. Microfluidic devices can sort cells due to specific geometric designs. Channels with spiral shapes are used to separate
particles according to their size based on the Dean forces.
The main benefit of this design is high throughput (>1.5 ml/min) without the need for sheath flow or sequential cell manipulation. Channel dimension, number of spirals, and diameter of the curvature influence the sorting effect. Flow rate is also akey parameter as the Dean and lift forces depend on it2. It is important to use well-designed microfluidic devices and excellent
flow control for sorting experiments. Fluigent and microfluidic ChipShop validated an effective and commercial solution for cellsorting.
Our cell sorting platform consists of a cell sorter device from microfluidic ChipShop and flow controllers from Fluigent to maintain precise flow control. To demonstrate the separation of particle mixtures, a solution containing 7.5 µm and 15 µm diameter
polystyrene particles labeled with FITC and TRITC fluorophores respectively, was used. The particle streams were viewed andcaptured separately using appropriate filter cubes.

Material and Methods
Material: Pressure driven pump
Together with our partner Fluigent, we offer everything you require for a spiral sorter experiment using one channel of apressure-driven pump. The major components are:Microfluidic Pressure Pumps - LineUp Flow EZ™:

LINK:

Flow Units:

All-in-One (A-i-O) control software:• Highly advanced system for pressure-based flow control• Stand-alone unit for use near the microfluidic setup• Flow EZ with 7 bar• Flow sensors that allows real-time flow rate measurements• Enables to switch from pressure control to flow rate control• Guarantee reproducibility of long-term droplet production• Flow Units L and XL are used depending on the targeted flow rate

• Provides connection of LineUp Flow EZ™ series
modules to a PC for software control• Real-time control of pressures and flow rates• Modular interface

P-CAP• Air-tight connector• Allows pressurization of standard lab tubes

Material: Spiral sorter chip Fluidic 382
The spiral sorter chip Fluidic 382 features four individual sorting units. Each sorting unit possesses one inlet to introduce the fluid
stream and six to eight outlets to collect sorted particle fractions. In- and outlets come in Mini Luer format and are interfacedwith Mini Luer fluid connectors. Sorting unit dimensions are given in Table 2. In this application note, sorting units 2 and 3 wereused.

Fig. 1: Schematic drawing of spiral sorter Fluidic 382 with four
sorting units

Fig. 2: Sorting unit 4 connected with tubing using Mini Luer fluid
connectors (red: inlet; blue: outlets)

Fig. 3: Close up of spiral sorting unit 4 with detailed view of the“sorting claw”, feeding individual outlet channels

Application Note Spiral Sorter Fluidic 382
Table 2: Sorting unit dimensions Fluidic 382
Sorting
unit

Number ofturns

Number ofoutlets

Channel width[µm]Channeldepth [µm]Channel length[mm]

1

48500

12082

28830080

16639615070

19541268050

270Material: Reagents & Optics
Fluorescently labeled polystyrene particles purchased from Bangs Laboratories. 7.3 µm (+/- 0.53 µm) with red fluorescenceand 15.25 µm (+/- 0.669 µm) with green fluorescence.
The surfaces are monitored using an inverted microscope (Eclipse Ti-U, Nikon) in bright field or in fluorescence mode. For theacquisition of the fluorescent beads, we use the software NIS-Elements with standard Nikon filter cubes for FITC and TRITC.

Protocol for micrometer beads size sorting
The system for cell sorting is shown in the Figure 4. The 7.3 µm and 15.25 µm fluorescent microbeads are mixed and diluted
in DI water before testing to reach a concentration of ~1x105 beads/ml. The sample is homogenized using a vortex mixer.
The Flow EZ is connected to the reservoir containing the mixture using a P-CAP. The reservoir is connected to the inlet of themicrofluidic device using 1/32“ and 1/16“ outer diameter (OD) tubing of 500 µm inner diameter (ID). 1/32“ and 1/16“ tubingare connected using a MicroTight adapter. Tubing passes through the Flow Unit to control and monitor flow rate. Tubing isconnected to the outlets of the microfluidic device to recover the beads. The flow rate is set using the Flow EZ. Here, sorting units
2 and 3 with flow rates of respectively 1.5 ml/min and 150 µl/min are used. The particle streams were viewed and captured
separately using TRITC and FITC filter cubes. The individual images were superimposed to create a composite image to display
the formation of two separate focused particle streams.

b)

Fig. 4 a) Complete system b) Schematic of the system c) Close up of the microfluidic chip

Table 3: Flow rates related to sorting units
Flow rate

Sorting unit 2

Sorting unit 3

1.5 ml / min

150 µl / minApplication Note Spiral Sorter Fluidic 382
Working principle of cell sorting using spiral channels
Particles that flow in a spiral microchannel with rectangular cross-section experience a combination of Dean drag forces and an
inertial lift. Dean drag forces are a consequence of fluids experiencing centrifugal acceleration. This acceleration leads to theformation of rotating vortices known as Dean vortices in the top and bottom halves of the channel (Figure 5a). The net lift force
originates from shear-induced and wall-induced lift forces. Additional information can be found in some research papers2,3. The
position at which particles of different sizes equilibrate depends on the ratio of lift and Dean drag forces. This results in several
particle streams that are size-dependent, and that can be extracted by designing appropriate outlets (Figure 5b).

Fig. 5 a) Channel cross-section and illustration of the effects of lift (FL) and Dean forces (FD). The position at which particles of different sizes equilibrate depends on the ratio FL/FD. More details about lift andDean forces equations can be found in the literature2 b) Schematic drawing of spiral sorter Fluidic 382. The randomly dispersed particles equilibrate at different equilibrium positions along the inner wall of thespiral microchannel.

Results and Discussion
As previously outlined, a homogeneous mixture of 7.3 µm and 15.25 µm fluorescent microbeads are injected into the central
inlet of the microfluidic chip using flow rates of 1.5 ml/min for unit 2 and 150 µl/min for unit 3 Figure 6a and 6b show
composite images of the microfluidic channels in a part of the spiral and at the outlet of the chip using bright field, green,
and red fluorescence. Figure 6a clearly indicates the formation of two distinct particle streams, confirming that 7.3 and 15 µm
microbeads are well separated. In addition, as predicted2, we observe the larger particles (green fluorescence, 15 µm) closer
to the inner channel. We can observe in figure 6b that the green fluorescence particles are in great majority collected at thefirst and second outlets of the microfluidic device, while red fluorescence particles are collected at the fourth outlet of the same
sorting unit. This result confirms that 7.3 µm and 15 µm particles are well separated and subsequently sorted by the microfluidic
device with very high throughput.

a)b)Fig. 6: Composite images illustrating the position of the 15 µm (green fluorescence), and 7.3 µm (red fluorescence) diameter particles in the channel section a) prior to the outlet, and b) at the outlet of unit 2of the microfluidic device. Flow rate: 1.5 ml/min.

Application Note Spiral Sorter Fluidic 382
It can also be of interest to sort cells using lower flow rates, as some cell types can be affected by the shear stresses generated
by high flow rates. We repeated the experiment using sorting unit 3 of the Fluidic 382, which has smaller channel dimensions
compared to unit 2. Figure 7 shows composite images of the microfluidic channel in a part of the spiral of the channel ofunit 3. We clearly observe two well-separated streamlines, confirming that 7.3 and 15 µm microbeads are also well separated
at 150 µl/min using the appropriate combination between channel geometry and flow rate.

Fig. 7: Composite images illustrating the position of the 15 µm (green fluorescence), and 7.3 µm (red fluorescence) diameter particles in the channel section prior to the outlet of unit 3 of the microfluidic device.
Flow rate: 150 µl/min.

Conclusion
In this application note, we introduced a commercially-available microfluidic system to perform passive size separation of a
microparticle mixture. The system includes a spiral-shaped microfluidic device from microfluidic ChipShop and pressure-based
flow controllers from Fluigent. Tuning Dean and lift forces induced by spiral microchannels allow to obtain distinct particle
streams and subsequently sort particles according to their sizes. Beads with diameters of 15 and 7.3 µm were successfully sorted
using flow rates of 150 µl/min and 1.5 ml/min. We provide an easy-to-use, versatile, and cost-effective setup for particle size
sorting.

Related Products
microfluidic ChipShop provides everything you need for your successful spiral sorting experiment. Please get in contact with our
products specialists at inquiries@microfluidic-ChipShop.com to determine your complete setup containing a Fluigent pump,chips and accessories. Chips and accessories are also available for individual purchase.
Product Codefor Fluidic 382

LidThickness [µm]Material

10000159

175

PMMA

10000160

188Zeonor

Product CodeDescription of Accessories

Material

Quantity

10000116

Male Mini Luer fluid connector

TPE - opaque

10 pcs / pack

10000280

Male Mini Luer plugs – Low volume displacement

PP - Red

10 pcs / pack

10000031

Silicone tube, ID: 0.76 mm, OD: 1.65 mm

Silicone

1m10000032

Micro tubes, PTFE, ID: 0.5 mm, OD:1.0 mm

PTFE

1mReferences
1. Wyatt Shields IV, C., Reyes, C. D. & López, G. P. Microfluidic cell sorting: A review of the advances in the separation of cells from debulking to rare cellisolation. Lab Chip 15, 1230–1249 (2015).
2. Kuntaegowdanahalli, S. S., Bhagat, A. A. S., Kumar, G. & Papautsky, I. Inertial microfluidics for continuous particle separation in spiral microchannels. Lab
Chip 9, 2973–2980 (2009).
3. Bhagat, A. A. S., Kuntaegowdanahalli, S. S. & Papautsky, I. Continuous particle separation in spiral microchannels using dean flows and differentialmigration. Lab Chip 8, 1906–1914 (2008).

Contact in Japan: ASICON Tokyo Ltd.
www.asicon-tokyo.com / asicon-tokyo@asicon-tokyo.com