1

Magnetic Separation of Undifferentiated Mouse Embryonic Stem (ES) Cells from Neural Progenitor Cultures using a microfluidic

device.

Sousa, A.F.1,2,3, Diogo, M.M.2,3, Freitas, P.P1,3. 1INESC-MN, 2BERG-IBB, 3Instituto Superior Técnico

ABSTRACT

Embryonic Stem (ES) cells are cell lines derived directly from the pre-implantation embryo. The generation of pure populations of neural

progenitors from ES cells and their further differentiation into neurons, astrocytes and oligodendrocytes allows the potential use of these

cells for the cure of neurodegenerative diseases and for neural drugs testing. An integrated device based on magnetophoresis, including

microfluidic channels and incorporated high magnetic field gradients, was used to control the motion of cells, labeled with magnetic

particles (MPs), through a biochip. This will result in a device capable of high-throughput separation at low cost. The separator was

fabricated in polydimethylsiloxane (PDMS) comprising an inlet channel for cells 200 µm wide and inlet channel for buffer solution 700 µm

wide. This device allows high separation efficiency of MP’s even when using inlet laminar fluid velocities up to 30 mm/s, by using a 15 mm

wide and 60 µm thick separation chamber. The permanent magnet used was the W-12-Nfrom Supermagnete® made from an alloy of

neodymium, iron and boron (Nd2Fe14B).In this work, the magnetophoretic device has been used for depletion of tumorogenic pluripotent

stem cells from 46C mouse ES cell cultures by the specific recognition and labeling of the stage specific embryonic antigen 1 (SSEA-1).

Purity degrees ranging from 95% to 99% were obtained and determined by flow cytometry analysis. These results raise the possibility of

using this micro-device for the purification of human neural progenitor cultures from tumurogenic pluripotent stem cells.

Key words: Magnetophoresis, Magnetization, Microfluids, ES cells, Flow Cytometry, SSEA-1.

1. INTRODUCTION

he miniaturization of analytical and practical

techniques has become today a leading area of

investigation [1]. This trend encloses various fields

since many laboratories are interested in creating

novel microfabricated structures. This interest is

driven by the need to reduce costs by reducing

consumption of expensive reagents and by the high

throughput analysis that can be achieved.

Generally, microfluidics refers to devices or flow

configurations that have the smallest design feature

on the scale of a micron or smaller. Frequently, this

means rectangular channels with cross-sectional

dimensions on the order of tens or hundreds of

microns [2]. Besides the traditional advantages

conferred by the miniaturization described above,

the understanding of the microscale phenomena,

can be used to perform techniques and

experiments not possible on the macroscale,

allowing new functionality and experimental

paradigms [3]. Some examples of devices

considered microfluidic are gene chips, chip based

capillary electrophoresis and magnetophoresis cell

sorters [4-6]. The latter is the main concern of this

paper.

Flow (Q) is the quantity of material passing a given

area in a unit period time. Regardless of the scale

(macroscopic or microscopic) working with, a fluid

flow is said to be laminar whenever the viscous

forces dominate inertia [7]. The laminar flow

regime is characterized by high moment diffusion

and low momentum convection. For pipe flow, the

critical Reynolds number above which turbulence

may exist is about 2000 [8]. Such flow regimes are

important for the purpose of this work since a

controlled environment with defined microfluidic

fluid flows is required, as explained next.

T

2

1.1. MAGNETOPHORESIS

Free-flow Magnetophoresis is a method that allows

the separation of magnetic particles (MPs) in

continuous flow. It consists in a microfluidic device

with a separation chamber in which laminar flow is

generated in the x-direction. Perpendicularly to the

laminar flow, that is in the y-direction a magnetic

field gradient is applied (figure, 2). By doing so,

superparamagnetic particles usually composed by

iron oxide material are deflected from their

trajectory. Magnetophoresis can be used to control

the motion of labeled cells with MP’s [9]. This is

obtained by labeling the cells with specific

antibodies that are previously immobilized in

magnetic particles with different sizes, and so

different magnetic momentum – this allows

for immunomagnetic separation of different cells in

an heterogeneous population [10]

Magnetophoresis in lab-on-a-chip devices is

currently undergoing a rapid development, being a

strong tool for biological research. Most of the

biological samples under study are non-magnetic

and are neither affected nor destroyed by the

presence of a weak magnetic field gradient.

Labeling those species with MP’s provides a

versatile physical handle for manipulation of

biological samples.

Superparamagnetic particles are available with

carboxyl groups or amino groups on their surface.

Biomolecules such as DNA strands or antibodies can

then easily be attached to the particle surface [11].

When coated with, for example, an antibody, the

particles can attach themselves to particular

targeted cells (figure, 3).

Depending on their magnetic susceptibility

magnetic particles are deflected from the path, in

the presence of a permanent magnet. Non

susceptible cells keep their trajectory. The

deflection of the magnetic particles can be

described as the sum of two vectorial components.

The magnetically induced flow, umag, is the ratio of

the magnetic force, Fmag (in N), exerted on the

particle by the magnetic field to the viscous drag

force:

where η is the viscosity of the medium (in kg/m/s)

Cell

Fig. 3 - Diagram of an immunomagnetically labeled cell. Note that the monoclonal antibodies are binding only to the specifically targeted cell surface molecules (antigens).

Fig. 2 – Concept of free-flow magnetophoresis. A heterogeneous population of non-labbeled and labbeled cells with MP are pumped into a laminar flow chamber; a magnetic field is applied perpendicular to the direction of flow. Cells deviate from the direction of laminar flow according to their magnetic susceptibility and are thus separated from each other and from nonmagnetic material.

Fig. 1 - Fluid flowing through microfluidic channels. a) Low velocity profile induces laminar fluid flow even in a presence of an obstacle. b) As the velocity increases, and so the Reynolds number, the flow becomes turbulent and the creeping fluid flow regime is achieved.

3

a)

b) Fig. 4 – Sparation system using a permanent magnet of 500 mT.

There are two inlets converging to a PDMS separation chamber where cells labeled with MPs from inlet 1 are deflected by magnetic attraction into outlet 2. a) top view of the microfluidic device in the presence of the permanent magnet. b) Perspective view of the structure comparing the dimension of the permanent magnet with the PDMS channels.

and r the particle radius (in m). The magnetic force,

is proportional to the magnetic flux density, B (in

tesla) and the gradient of the magnetic field,

(in tesla m-1

) of the externally applied field. The

magnetic force is proportional to the particle

volume Vp (m3) and the difference in magnetically

susceptibility between the particle and the fluid, ∆χ

(dimensionless):

where μ0 is the permeability of a vacuum (in H m-1).

When inserting eq. 3 into eq. 2, it can be seen that,

for a given magnetic field and a given viscosity, the

magnetic velocity, umag, is dependent on the size

and the magnetic characteristics of the particle. It is

proportional to the square of the particle radius and

to the magnetic susceptibility of the particle:

,

So, when having a heterogeneous culture of cells on

which some are labeled with MPs, these will be

defected from their initial trajectory and will then

be separated from the rest of the culture, allowing

the purification of a given sample.

2. MATERIALS AND METHODS

utoCad, a software that allows the design of

microfluidic structures, was used in this work.

Microfluidic structure was designed in order to have

a large separation chamber with enough height so

cells could pass freely without colliding with the

Polydimethylsiloxane (PDMS) walls. The chamber

was also designed to have two inlets; one for the

solution containing the cells and the other one to a

buffer solution of PBS 0,1 M, pH 7.3. After several

designs (see annex I), a final microfluidic structure

was designed with a separation chamber 2mm

wide, a inlet for the cell solution 200μm wide and a

buffer solution inlet channel 700μm wide. The

whole structure had 60μm thickness and it is

represented in figure 4. Such a big microfluidic

structure represents several advantages as:

- Lower cell aggregation, allowing much more

efficient separation;

- By using a separation chamber with 60 μm

thickness the cells will more probably be located in

the central part of the channel and won’t get so

easily stuck in the walls lowering the percentage of

cell loss.

- Higher flow rates at the inlets, while maintaining

low velocities in the separation chamber, around

4mm/s. This way, the magnetic field gradient used

will be sufficient to deviate the magnetically

susceptible cells in order to be collected in the

outlet 2. This is an important characteristic of such a

device, since it may be necessary to process

solutions of cells in the range of milliliters.

2.1. THE PROCESS FOR FABRICATION OT THE

MICROFLUIDIC DEVICE

The microfluidic structure was made of PDMS. This

polymeric organosilicon compound presents many

A

4

Fig. 5 – Schematic representation of the entire PDMS structure fabrication process.

advantages, namely the easiness of fabrication,

good physical properties and the low price of this

material [12]. PDMS is optically clear, non-toxic,

non-flammable and inert material, and it is known

for its unusual rheological (or flow) properties.

There are many methods for the fabrication of

PDMS based microfluidic devices. The method used

during this work is the most commonly used and is

schematically represented in figure 5. All this

process is presumed to be done in a clean

environment.

2.2. CELL LINE

For this work, the 46C mouse ES cell line [13],

established at the laboratory of Professor Austin

Smith, Wellcome Trust Centre for Stem Cell

Research, University of Cambridge, England, UK,

was used as the model cells. This cell line can be

induced to undergo neural commitment in serum

free conditions [14]. 46C mouse ES cells were kept

cryopreserved in liquid nitrogen until further use.

2.3. SSEA-1 EXPRESSION DETERMINATION BY

FLOW CYTOMETRY

After in vitro expansion of 46C ES cells, the

percentage of SSEA-1+ cells was evaluated by flow

cytometry. The same procedure was used after cell

separation using the different procedures (batch

and microfluidic-based). For that purpose, cells

were collected and ressuspended in 100 μL of PBS.

The antibody was then added to the cell suspension

(IgM, PE mouse/human monoclonal conjugated

anti-SSEA1 (BD), 1:10 dilution in PBS), followed by

incubation for 15 min at room temperature in the

dark. Negative Isotype controls (γ1- PE) were used

in every experiment in order to exclude non-specific

binding of the antibody to the cells surface.

2.4. BSA BLOCKING STEP AND CALCULATION

OF NON-SPECIFIC ADSORPTION OF CELLS TO

MP’S

A first experiment was designed in order to

evaluate the degree of non-specific adsorption (non

antibody-based) of cells to these MP’ and in order

to test the feasibility of using an incubation with

bovine serum albumin (BSA) to block this

adsorption. For that purpose, approximately 7×108

MP’s were added to a 100 µL solution of PBS 0,1M

pH 7.2 and incubated with 5×105 46C ES cells

containing 76,9% of SSEA-1+ cells. The mixture

containing the cells and MP’s was then left to

incubate for one hour at room temperature with

constant mixing. Three different studies were

performed in parallel with increasing BSA

concentrations (%w/v) of 1%, 2% and 5%. The cell–

MP complex pellet was then isolated from the

supernatant by placing the eppendorfs near a

magnet in a magnetic particle separator Magna-

SepTM

. The remaining pellet of cells-MPs complex

was ressuspended in 100µL of PBS 0,1M. The

number of cells in each one of the phases

(supernatant and pellet) was then counted using a

5

Fig. 6 - Experimental setup for the separation in microfluidic device. a) Cubic Permanent Magnet. b) Inlet 2 for buffer solution c) Inlet 1 for cells-MPs complex containing solution d) microfluidic separation chamber e) glass surface f) fluorescence microscope ocular g) outlet 2 for SSEA-1+ cells h) outlet 1 for SSEA-1- cells. Note that the entire structure is mounted in an inverted position due to technical properties of the microscope.

hemocytometer. The percentage of cells that were

adsorbed to the MP’s by non-specific adsorption

(NSA) (non-based in antibody interaction) was

calculated as shown below:

ells dsorbed of cells pellet

of total cells pellet supernatant)

2.6. SEPARATION OF CELLS USING IGM,

BIOTIN ANTI-MOUSE/HUMAN SSEA-1

ANTIBODY IN BATCH PROCEDURE

The main purpose was to place a magnet near the

eppendorfs and once the magnetically susceptible

cells were attracted to the eppendorfs walls, the

supernatant was decanted to new eppendorfs and

the number of cells were counted again. In this

case, 2 µL of 3 nm MP’s were added to a 100µL

solution of PBS 0,1 M pH 7.2, containing final

antibody concentrations of 0,125µg/mL 0,25µg/mL

and 0,375µg/mL in different eppendorfs. A negative

control run with no antibody added was also

performed. The number of cells in each eppendorf

varied between 1,4×105 - 3,5×10

5 since it was not

possible to collect the same number of cells from

the initial cell suspension.

2.7. SEPARATION OF CELLS USING

MICROFLUIDIC DEVICE

The fabricated microfluidic device was used for the

negative separation of SSEA-1+ cells from of a

heterogeneous mixture of 46C ES cells containing

both SSEA-1+ and SSEA-1

- cells. For that purpose,

2µL of 3 nm MP’s particles were added to 3 µL

of PBS 0,1M pH 7.2, with antibody at final

concentrations of 0,250µg/mL 0,375µg/mL and

0,625µg/mL in different eppendorfs.

A negative control run with no antibody was also

performed. All the mixtures were left to incubate

for one hour. The 300µL solution was then

ressuspended in the same volume of BSA blocking

solution 2%(w/v) and left to incubate for one hour

at room temperature. To each eppendorf, a

heterogeneous mixture of 3×105 46C ES cells

containing both SSEA-1+ and SSEA-1

-cells was added

and again left for incubation for one hour at room

temperature. The total solution in each eppendorf

was ressuspended in 300μL of PBS 0,1 M, ph 7.3

solution and fed into a 1mL syringe located in the

NE-1000 Programmable Syringe Pump from NAI –

Wis Biomed with a constant fluid flow of 20μL/min

in inlet 1. The buffer solution was injected into inlet

2 with a constant fluid flow of 100μL/min. Each run

was performed during 15min approximately. The

outlets fluid flows were finally collected in

eppendorfs connected to the separation chamber

by polymer tubes. In order to differentiate fluid

flows through inlet 1 and 2 during the separation,

FITC diluted 1:1000 was added to the buffer

solution and visualized under a fluorescence

microscope (figure, 6). Prior to the injection of the

cells, the entire microfluidic device as well a s all the

accessories such as syringes, microfluidic tubes and

eppendorfs that would contact with the cells-MP’s

complex were coated with a blocking solution

containing 2% (w/v) BSA. The percentage of SSEA-1+

negatively selected cells in the microfluidic device is

calculated as:

6

Separation cells in 2

cells total

Being Ncells total, the sum of the number of cells

counted in outlet 1 and outlet 2.

3. RESULTS AND DISCUSSION

3.1. SEPARATION OF CELLS USING IGM,

BIOTIN ANTI-MOUSE/HUMAN SSEA-1

ANTIBODY IN BATCH PROCEDURE

After the results presented in previous works (data

not shown) several considerations were taken in

account and it was decided to perform the batch

procedure using an antibody already biotinylated –

IgM biotin anti-mouse/human SSEA-1. In this case,

the biotin group is labeled to the antibody in a

different area than the antigen binding-site (Fab

Region), this way the specific recognition of the

SSEA-1+ cells by the antibody is not affected. Since

this antibody is concentrated, each run will have a

low cost because a low monoclonal antibody

volume is used. In order to perform this

experiment, 3×105 46C ES cells containing 53,4% of

SSEA-1+ cells were used.

As can be observed in figure 7, an increasing

percentage of captured cells were obtained when

increasing the concentration of biotinylated

antibody. The maximum percentage of captured

cells (91,1%) was obtained when using a

concentration of 0,375ug/mL of the biotinylated

antibody. However, from the control run (no

antibody used) results it can be concluded that a

high percentage of cells (42,4%) was captured due

to non-specific adsorption.

In order to evaluate the efficiency of this batch

system concerning the capture of SSEA-1+ cells, the

percentage of SSEA-1+ cells, in the supernatant was

determined by flow cytometry analysis (figure, 8).

Only after this analysis it will be possible to evaluate

if the antibody-MP’s complex is efficiently binding

to the specif ic antigen in the cells surface. The first

plot [figure 8, a)] represents the negative control of

the flow cytometry analysis by using an IgG γ1

antibody with a linked fluorophore – PE – but

without a specific affinity for the ES cells.

This negative control is used namely to account for

the non-specific adsorption of the fluorophore to

the ES cells.

By making this negative control run it is possible to

define a peak with fluorescence between 100 and

101 of the PE fluorescence (FL2-H intensity scale).

This way, only ES cells with fluorescence intensity

(FL2-H) higher than 101 will be considered to

express the SSEA-1 antigen. After performing the

negative control run, the percentage of SSEA-1+

cells was determined in 46C ES cells feed to the

batch system, before purification, using the SSEA1

antimouse/human IgM PE antibody [figure 8, b)].

Next, in figures 8, c), d), e) and f) it is possible to

observe the flow cytometry analysis of the

supernatants after the batch procedure when

antibody concentrations of 0 μg/mL, 0,25 μg/mL,

0,25 μg/mL, 0,375 μg/mL were used. As can be

observed in figure 8, c), when no antibody was used

in the batch separation procedure, a percentage of

36,4% of SSEA-1+ cells was obtained in the

supernatant. Looking at the separation value of

42,4% [figure 7, Control run with no antibody used]

75,5

90,7 91,1

42,4

30

40

50

60

70

80

90

100

, 25μg/mL ,25μg/mL ,375μg/mL control

Sep

arat

ion

Pe

rce

nta

ge

Antibody concentration

Capture of Cells

Fig. 7 - Results for the capture percentage of 46C ES cells labeled with a specific biotinylated antibody (in different concentrations) immobilized into MPs and using the batch procedure.

7

6,1%

53,4%

36,4%

15,6%

4,1%

1,2%

Fig. 8 – Flow cytometry analysis of SSEA-1 positive 46C ES cells when using the batch procedure: a) Flow cytometry negative control run, b) %SSEA-1 positive cells before separation; c) Control run, where no antibody was used for the selective separation of cells. %SSEA-1 positive cells in the supernatant after the batch procedure while using d) , 25μg/mL, e) , 25μg/mL and f) ,375μg/mL of SSEA1 specific antibody.

a)

b)

c)

d)

f)

e)

it is possible to say that some of the non-specific

interaction of the antibody-MPs complex with the

cells is promoting depletion of SSEA-1+ cells from

the 46C ES cells heterogeneous population.

Next, after the separation protocol using 0,125μ/mL

of antibody, the percentage of 46C ES cells

expressing the SSEA-1 antigen was also determined

by flow cytometry. The M region denotes the

percentage of ES cells expressing SSEA-1 antigen,

and for this run that percentage decreased to 15,6%

[figure 8, d)] leading to conclude that the separation

protocol while using specific antibodies was

purifying the initial solution of 46C ES cells. Once

increasing the antibody concentration to 0,250

μg/mL and 0,375 μg/mL, the percentage of cells

expressing the SSEA-1 antigen in the supernatant

decreased to 4,1% and 1,2% respectively [figure 8,

e) and f)]. After performing flow cytometry analysis

it was possible to calculate the purity degree of the

supernatant after the batch procedure (Table 1) is:

Purity Degree = 100% - % SSEA-1 positive cells in the

supernatant

It is important to notice that although the obtained

purity degree is high (table 1), the loss of cells is

also high (see figure 9). Since the percentage of cells

expressing the SSEA-1 antigen before the batch

separation is 53,4% the number of cells that are

attracted to the magnet in the batch procedure

should not overcome that value, otherwise this

separation is not only based on specific cell-

antibody interactions. Since the separation

percentages are higher than that value (as can be

seen in figure 7) this means that cells are being

attracted to the magnet placed near the eppendorf

by non-specific interactions. The percentages of cell

loss (SSEA-1- cells non-specifically attracted to the

magnet) have values around 60-70% (figure 9). In

this figure the white areas should maintain the

same values after batch separation since they

represent the number of SSEA-1- cells before

(columns on the left of each cluster) and after

(columns on the right of each cluster) the

separation in batch procedure.

Thus, the first conclusion that can be taken from

this study is that the batch procedure for the

separation of SSEA-1+ 46C ES cells is promoting high

levels of non-specific interactions either between

cells and between cells and the eppendorfs, during

8

Separation of cells in outlet 2 cells in 2

cells total

the time the solution remains in batch. Perhaps the

formation of aggregates between the antibody-MP-

cell is contributing for the non-specific adsorption

of cells. However, more studies about this aspect

would be needed to understand this phenomena.

Taking these results into consideration, a different

method for the depletion of SSEA-1+ cells from 46C

ES cells should be attempted.

3.2. SEPARATION OF SSEA-1+ 46C ES

CELLS USING THE MICROFLUIDIC DEVICE

After performing SSEA-1+ cell separation using the

batch procedure, the protocol was adapted for the

separation in the microfluidic device. Looking at the

microfabricated structure, there are two inlets, one

for the solution containing the cells (inlet1) and the

other for the buffer solution of PBS 0,1M pH 7.3.

The presence of the permanent magnet will induce

cells labeled with MPs to deviate from their path to

outlet 2, while non-susceptible cells will continue

along their path to outlet 1. Three different

concentrations of antibody were again evaluated,

and a control run where no antibody was used was

also performed. The separation percentage is given

by a similar formula to that presented before:

Where Ncells total, is the total number of cells

counted in outlet 1 and outlet 2. Cells that went

through outlet 2 are magnetically susceptible and

presumed to be labeled with the 300nm particles

from Ademtech®. The percentage of separation

using the microfluidic procedure is lower than that

obtained in batch procedure. One important result

is the one of the control run, where no antibody

was used.

Indeed, in this case, only 10,3% of the 46C ES cells

were attracted to O2 via non-specific interactions

(figure, 10). This value is much lower than the

obtained in the batch procedure which presents a

major advantage of the microfluidic

procedure.Indeed, in this case, all the cells in the

separation device are in constant motion due to the

fluid flow, decreasing the amount of interaction

between all the species and preventing cells from

forming aggregates. In fact, cell aggregates are

more difficult to separate based on the expression

of surface antigens. The cells labeled with MPs are

in constant movement, while in the separation

chamber they get subjected to the magnetic field

generated by the permanent magnet. This exposure

to the magnetic field occurs for only a few seconds

Table 1 - Resume table for the purity degree of each solution after the separation procedure.

Fig. 9 - Four different clusters of results, each one representing the use of a different antibody concentration during batch separation. In each cluster, the left column represents the total number of cells prior to the separation, and the right column represents the total number of cells in the supernatant after the separation. In blue is the number of ES cells expressing the SSEA-1 antigen.

39,4 42,0 42,9

10,3

0

10

20

30

40

50

,25μg/mL ,375μg/mL ,625μg/mL control

Pe

rce

nta

ge o

f Se

par

aio

n

Antibody concentration

Cell separation Outlet 2

Fig. 10 – Results for the capture percentage of cells labeled with specific antibody biotinylated (in different concentrations) immobilized into MPs in the microfluidic structure.

9

Table 2 - Resume table for the purity degree of each 46C ES cells solution after the separation procedure in the microfluidic structure.

Fig. 12 - Four different clusters of results, each one representing the use of a different antibody concentration. In each cluster, the left column represents the total number of cells prior to the separation, and the right column represents the total number of cells out of outlet 1 after the separation. In blue is the number of ES cells expressing the SSEA-1 antigen.

≈4 sec, as they travel at ≈4mm/s and the

separation chamber is 15mm wide), and that is

sufficient to alter the cells trajectory according to

results shown in figure 10.

In these experiments, the microfluidic system was

injected with a cell suspension containing 42,4% of

cells expressing the SSEA-1 antigen [figure 11, b)].

However, as can be seen in figure 11, c) and in

opposition to the batch procedure [fig ure 7, c)], the

separation of cells by non-specific adsorption in this

dynamic protocol is negligible, which means that a

high specificity is achieved in cell binding to MP’s-

antibody complex. Besides that, the M region on

the graphics decreases as the antibody

concentration gets higher [figure 11, d) e) and f)],

denoting again that the antibody is recognizing the

cells to be deviated from their path with the use of

the permanent magnet. By other words, most of

the SSEA-1+ ES cells (and not other cells) which we

want to deplete from the initial cell solution are

exiting the structure through outlet 2, resulting in a

negative selection separation procedure in a novel

working microfluidic structure with low cost per

run. Another aspect is the purity degree that was

obtained using this microfluidic procedure. The

results obtained are presented in table 2. The purity

degree is not as high as in the batch procedure,

however it is highly comparable, once standard

errors are considered. The advantages of using a

microfluidic device, is the ease and low cost of the

procedure. Besides, in this case, each solution of

cells can be injected more than once into the

separation device. In a better look, cell loss values

obtained was around 1%-5% (figure, 12), better

than in batch procedure. The results achieved here

could potentially be improved by passing the

42,4%

44,6%

10,3%

5%

3,7%

1,25%

Fig. 11 – Flow cytometry analysis of SSEA-1 positive 46C ES cells when using the microfluidic separation device: a) Flow cytometry negative control run, b) %SSEA-1 positive cells before separation; c) Control run, where no antibody was used for the selective separation of cells. %SSEA-1 positive cells in the supernatant after the batch procedure while using d) , 25μg/mL, e) , 25μg/mL and f) ,375μg/mL of SSE specific antibody.

a)

b)

c)

d)

f)

e)

10

solution of cells several times in the microfluidic

device since this would enhance the final purity

degree [16]. However, in this case, clearance rates

for undifferentiated ES cells would decrease with

decreasing amounts of undifferentiated SSEA-1+ ES

cells [16].

4. CONCLUSIONS AND FUTURE WORK

Before using the microfluidic device for the

magnetophoresis based separation all the biological

protocols needed optimization. The immobilization

of the 300nm particles from Ademtech® to the

specific antibodies used, needed to be engineered

from the beginning with constant improvements

and changes. The final protocols were adequate for

the specific and correct labeling of SSEA-1+ cells.

The batch procedures performed lead ultimately to

good results for the purification of a heterogeneous

population of cells. Final purity degrees obtained in

batch separation ranged from 96,9% to 99,5% while

using well defined concentrations of IgM, Biotin anti

mouse/human SSEA1 antibody. However, the

values for the cells loss percentage were also high

and observed between 60% and 70%.

As for the magnetophoresis in a dynamic

microfluidic device the use of a permanent magnet,

the W-12-N: Cube 12mm from Supermagnete®

made from an alloy of neodymium, iron and boron

to form the Nd2Fe14B proved to be sufficient for the

deviation of labeled SSEA-1+ cells with 300nm

particles from Ademtech®. The final purification

degrees obtained in this procedure ranged from

92,3% to 98,5% in a single step run which in terms

of results is comparable to those obtained in the

batch procedure if laboratorial errors are taken in

account. As for the percentages of cells loss, while

using the microfluidic device, the values obtained

were at most, 5%. This characteristic leads to

conclude that this procedure of purification can be

repeated several times in order to obtain higher

final purity degrees. Each run can be performed in

less than 8 hours using a simple protocol.

The microfluidic device was used for the

purification of a population of mES cells from cells

expressing the SSEA-1 antigen, a marker for

pluripotent and tumorogenic cells. Such model can

be used as a proof of concept of this technology for

the direct application in the purification of a neural

progenitors population of cells from tumorogenic

cells. A high-throughput separation system with low

cost per run was developed in this MSc project.

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