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

Sousa A.F.1,2, Loureiro J.2, Diogo M.M.1, Cabral J.M.S., Freitas P.P.2

1 IBB (Instituto de Biotecnologia e Bioengenharia) and 2INESC-MN Microssistemas & NanotecnologiasInstituto Superior Técnico, Av. Rovisco Pais, 1049-001 Lisbon, Portugal

Presenting: António Filipe Gomes Teixeira de Sousa

March 2011

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Embryonic stem (ES) cells for Regenerative Medicine

Controlling microliter volumes –

Microfluidics

Culturing and differentiation of embryonic stem cells – Stem Cell Bioengineering

1.Motivation and Goals

Human ES cells Human ES (embryonic stem) cells, are very important for

human cell therapy.

Mouse ES cells culture in vitro

Expansion of mES cells in vitro

Differentiation of mES cells in vitro into neural

progenitors.“... A selective and quantitative removal of undifferentiated ES cells from a pool of differentiated and undifferentiated cells is essential before cell therapy...” Schriebl, K., et al. (2009)

The model system, 46C ES

cells

Mouse ES cells that were used for separation efficiency

studies as a model system – Proof of Concept

Differentiated cells (Neural progenitors) - no specific antigen marker available

Undifferentiated cells (ES cells)

Stage specific embryonic antigen SSEA-1 is a specific

marker for these cells.

2.Introduction3

Fig – Cell surface antigens interact with magnetic particle-labelled antibodies.

Fig – Concept of free-flow magnetophoresis.

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Magnetophoresis, cells labeled with

magnetic particles can be separated

from other non-labeled cells by

attractive magnetic forces.

Laminar flow is generated in the x-

direction. Perpendicularly to the laminar

flow, in the y-direction a magnetic

field gradient is applied.

𝑣𝑑𝑒𝑓𝑙=�⃗�𝑚𝑎𝑔+�⃗�h 𝑦𝑑

𝑣𝑚𝑎𝑔∝𝑟2∆ χ

2.Introduction

a)

b)

Fig - Separation system using a permanent magnet of 500 mT.

- Lower cell aggregation, allowing much more efficient separation;

- Cells located in the central part of the

channel and won’t get stuck in the PDMS walls lowering the percentage of cell loss.

- Higher flow rates at the inlets.

This Design Reported in bibliography

Fluid Flow (Q) at Inlet (μL/h) 3600-7200 50-200 [1]--- [2]

Fluid Velocity inside the device (mm/s)

2-6 0,1-0,4 [1]<1 [2]

3. Materials and Methods

53.1 The Design of the structure

The structure was microfabricated in PDMS - Non-reactant with biological samples and transparent.

[1] Pamme, N. and A. Manz, On-Chip Free-Flow Magnetophoresis:  Continuous Flow Separation of Magnetic Particles and Agglomerates. Analytical Chemistry, 2004. 76(24): p. 7250-7256.[2] Pamme, N. and C. Wilhelm, Continuous sorting of magnetic cells via on-chip free-flow magnetophoresis. Lab on a Chip, 2006. 6(8): p. 974-980.

Inlet 1 – Cells

Inlet 2 – Buffer Solution

Outlet 2 – Positive Selected Cells (SSEA1+)

Outlet 1 – Negative selected Cells (SSEA1-)

Permanent Magnet

+ BSA blocking Solution 2% (w/v)

Different antibodies concentrations: 2,5μg/mL3,75 μg/mL6,25 μg/mL0 μg/mL – Control

Inlet flows:Q(I1)=20uL/minQ(I2)=100uL/min

Ncells total for each run = 1×105 - 5×105

% Separation =Ncells   in  O 2Ncells   total

×100

3. Materials and Methods3.2 Separation using microfluidic device

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Fig - Use of fluorescence proved to be important for the fluid flow control inside the separation chamber of the device.

Inle

t 1In

let 2

Separation Chamber

0,25μg/mL 0,375μg/mL

0,625μg/mL

control0

20

40

60

80

100

39.4 42.0 42.9

10.3

Cell separation

Outlet 2

Antibody concentration

Perc

enta

ge o

f S

epara

ion

Fig - Results for the capture percentage of cells labeled with specific antibody.

42,4%

44,6%

3,7%

Fig - Flow cytometry analysis of SSEA-1 positive 46C ES cells when using the microfluidic separation device.

a)

b)

c)

a) SSEA1 expression of 46C cells prior to the separation in batch b) SSEA1 expression of cells after separation procedure (control run).c) SSEA1 expression of cells after separation procedure (0,625μg/mL of antibody);

High specificityNon-Specific Adsorption is low

4. Results and Discussion7

4.1 Separation using microfluidics

Fig.17 – Four different clusters of results, each one representing the use of a different antibody concentration

Purity Degree = 100% - % SSEA-1 positive cells in the supernatant/Outlet1

- Purity degree varies in the same way for the batch and the microfluidic procedure; Purity Degree Antibody Concentration

4. Results and Discussion8

4.2 Purity Degree

- Left columns represents the total number of cells prior to the separation, and the right columns represents the total number of cells the separation. In blue is the number of ES cells expressing the SSEA-1 antigen.

Percentages of cell loss values around 1%-5%.

Percentages of cell loss values around 60-70%, in classical technique of separation (batch procedure).

4. Results and Discussion9

4.3 Cells Yield

  Batch

Microfluidics

Purity degree achieved

+++ ++

Separation Specificity

+ +++

Cells yield - +++Time of protocol ≈6h ≈8h

Fig.17 – Four different clusters of results, each one representing the use of a different antibody concentration

4. Conclusions and Future work

- mES cells worked as a model system for a higher objective of purifying a neural progenitor culture after differentiation

- Low percentage of cell loss using microfluidics;

- Higher separation efficiency while using microfluidic device;

- Sterile environment for cells;

- Miniaturization with possible integration in Lab-on-a-chip devices. Low cost and simple use;

- A single step with 99% purity can be done in less than 8hours;

According to the aim of the project, the objective was accomplished successfully!

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High-throughput technology

for pure ESC sorting

Macroscale Quantity

Microscale specificity

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Professor Paulo Freitas Professor Joaquim Cabral Doctor Margarida Diogo Professor João Pedro Conde Project Colleagues INESC-MN and BERG/IBB Family and Friends

Acknowledgments