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Magnetic Separation of Undifferentiated Mouse Embryonic Stem    (ES) Cells from Neural Progenitor Cultures using a microfl...
Magnetophoresis           in      lab-on-a-chip     devices        is                                                     ...
and r the particle radius (in m). The magnetic force,                the purification of a given sample.is proportional to...
Fig. 5 – Schematic representation of the entire PDMS structure fabrication process.advantages, namely the easiness of fabr...
hemocytometer. The percentage of cells that were                at room temperature. To each eppendorf, a                 ...
cells in 2                     antibody. However, from the control run (no                                         Separat...
it is possible to say that some of the non-specific        same values after batch separation since they                  ...
and a control run where no antibody was used was                                                                     also ...
≈4 sec, as they travel at ≈4mm/s and the                             the      graphics       decreases        as     the  ...
solution of cells several times in the microfluidic      conclude that this procedure of purification can bedevice since t...
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Microfluidic device for cell purification

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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.

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  1. 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. 1 INESC-MN, 2BERG-IBB, 3Instituto Superior TécnicoABSTRACTEmbryonic Stem (ES) cells are cell lines derived directly from the pre-implantation embryo. The generation of pure populations of neuralprogenitors from ES cells and their further differentiation into neurons, astrocytes and oligodendrocytes allows the potential use of thesecells for the cure of neurodegenerative diseases and for neural drugs testing. An integrated device based on magnetophoresis, includingmicrofluidic channels and incorporated high magnetic field gradients, was used to control the motion of cells, labeled with magneticparticles (MPs), through a biochip. This will result in a device capable of high-throughput separation at low cost. The separator wasfabricated in polydimethylsiloxane (PDMS) comprising an inlet channel for cells 200 µm wide and inlet channel for buffer solution 700 µmwide. 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 mmwide and 60 µm thick separation chamber. The permanent magnet used was the W-12-Nfrom Supermagnete® made from an alloy ofneodymium, iron and boron (Nd2Fe14B).In this work, the magnetophoretic device has been used for depletion of tumorogenic pluripotentstem 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 ofusing 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. allowing new functionality and experimental1. INTRODUCTION paradigms [3]. Some examples of devicesT he miniaturization of analytical and practical techniques has become today a leading area ofinvestigation [1]. This trend encloses various fields considered microfluidic are gene chips, chip based capillary electrophoresis and magnetophoresis cell sorters [4-6]. The latter is the main concern of thissince many laboratories are interested in creating paper.novel microfabricated structures. This interest is Flow (Q) is the quantity of material passing a givendriven by the need to reduce costs by reducing area in a unit period time. Regardless of the scaleconsumption of expensive reagents and by the high (macroscopic or microscopic) working with, a fluidthroughput analysis that can be achieved. flow is said to be laminar whenever the viscousGenerally, microfluidics refers to devices or flow forces dominate inertia [7]. The laminar flowconfigurations that have the smallest design feature regime is characterized by high moment diffusionon the scale of a micron or smaller. Frequently, this and low momentum convection. For pipe flow, themeans rectangular channels with cross-sectional critical Reynolds number above which turbulencedimensions on the order of tens or hundreds of may exist is about 2000 [8]. Such flow regimes aremicrons [2]. Besides the traditional advantages important for the purpose of this work since aconferred by the miniaturization described above, controlled environment with defined microfluidicthe understanding of the microscale phenomena, fluid flows is required, as explained next.can be used to perform techniques andexperiments not possible on the macroscale, 1
  2. 2. 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 Fig. 1 - Fluid flowing through microfluidic channels. a) biological samples. Low velocity profile induces laminar fluid flow even in a Superparamagnetic particles are available with presence of an obstacle. b) As the velocity increases, and so the Reynolds number, the flow becomes carboxyl groups or amino groups on their surface. turbulent and the creeping fluid flow regime is achieved. Biomolecules such as DNA strands or antibodies can1.1. MAGNETOPHORESIS then easily be attached to the particle surface [11].Free-flow Magnetophoresis is a method that allows When coated with, for example, an antibody, thethe separation of magnetic particles (MPs) in particles can attach themselves to particularcontinuous flow. It consists in a microfluidic device targeted cells (figure, 3).with a separation chamber in which laminar flow is Depending on their magnetic susceptibilitygenerated in the x-direction. Perpendicularly to the magnetic particles are deflected from the path, inlaminar flow, that is in the y-direction a magnetic the presence of a permanent magnet. Nonfield gradient is applied (figure, 2). By doing so, susceptible cells keep their trajectory. Thesuperparamagnetic particles usually composed by deflection of the magnetic particles can beiron oxide material are deflected from their described as the sum of two vectorial components.trajectory. Magnetophoresis can be used to control ⃗ ⃗ ⃗the motion of labeled cells with MP’s [9]. This is The magnetically induced flow, umag, is the ratio ofobtained by labeling the cells with specific the magnetic force, Fmag (in N), exerted on theantibodies that are previously immobilized in particle by the magnetic field to the viscous dragmagnetic particles with different sizes, and so force:different magnetic momentum – this allows ⃗for immunomagnetic separation of different cells inan heterogeneous population [10] where η is the viscosity of the medium (in kg/m/s) CellFig. 2 – Concept of free-flow magnetophoresis. A heterogeneous population of non- Fig. 3 - Diagram of an immunomagneticallylabbeled and labbeled cells with MP are pumped into a laminar flow chamber; a labeled cell. Note that the monoclonalmagnetic field is applied perpendicular to the direction of flow. Cells deviate from the antibodies are binding only to thedirection of laminar flow according to their magnetic susceptibility and are thus specifically targeted cell surface moleculesseparated from each other and from nonmagnetic material. (antigens). 2
  3. 3. and r the particle radius (in m). The magnetic force, the purification of a given sample.is proportional to the magnetic flux density, B (in 2. MATERIALS AND M ETHODStesla) and the gradient of the magnetic field, -1 AutoCad, a software that allows the design of(in tesla m ) of the externally applied field. The microfluidic structures, was used in this work.magnetic force is proportional to the particle 3 Microfluidic structure was designed in order to havevolume Vp (m ) and the difference in magnetically a large separation chamber with enough height sosusceptibility between the particle and the fluid, ∆χ cells could pass freely without colliding with the(dimensionless): Polydimethylsiloxane (PDMS) walls. The chamber was also designed to have two inlets; one for thewhere μ0 is the permeability of a vacuum (in H m-1). solution containing the cells and the other one to aWhen inserting eq. 3 into eq. 2, it can be seen that, buffer solution of PBS 0,1 M, pH 7.3. After severalfor a given magnetic field and a given viscosity, the designs (see annex I), a final microfluidic structuremagnetic velocity, umag, is dependent on the size was designed with a separation chamber 2mmand the magnetic characteristics of the particle. It is wide, a inlet for the cell solution 200μm wide and aproportional to the square of the particle radius and buffer solution inlet channel 700μm wide. Theto the magnetic susceptibility of the particle: whole structure had 60μm thickness and it is ⃗ , represented in figure 4. Such a big microfluidicSo, when having a heterogeneous culture of cells on structure represents several advantages as:which some are labeled with MPs, these will be - Lower cell aggregation, allowing much moredefected from their initial trajectory and will then efficient separation;be separated from the rest of the culture, allowing - 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 a ) 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 b solutions of cells in the range of milliliters.Fig. 4 – Sparation system using a permanent magnet of 500 mT. )There are two inlets converging to a PDMS separation chamberwhere cells labeled with MPs from inlet 1 are deflected by 2.1. THE PROCESS FOR FABRICATION OT THEmagnetic attraction into outlet 2. a) top view of the microfluidic MICROFLUIDIC DEVICEdevice in the presence of the permanent magnet. b) Perspectiveview of the structure comparing the dimension of the permanent The microfluidic structure was made of PDMS. Thismagnet with the PDMS channels. polymeric organosilicon compound presents many 3
  4. 4. Fig. 5 – Schematic representation of the entire PDMS structure fabrication process.advantages, namely the easiness of fabrication, (IgM, PE mouse/human monoclonal conjugatedgood physical properties and the low price of this anti-SSEA1 (BD), 1:10 dilution in PBS), followed bymaterial [12]. PDMS is optically clear, non-toxic, incubation for 15 min at room temperature in thenon-flammable and inert material, and it is known dark. Negative Isotype controls (γ1- PE) were usedfor its unusual rheological (or flow) properties. in every experiment in order to exclude non-specificThere are many methods for the fabrication of binding of the antibody to the cells surface.PDMS based microfluidic devices. The method usedduring this work is the most commonly used and is 2.4. BSA BLOCKING STEP AN D CALCULATION OF NON-SPECIFIC ADSORPTION OF CELLS TOschematically represented in figure 5. All this MP’Sprocess is presumed to be done in a clean A first experiment was designed in order toenvironment. evaluate the degree of non-specific adsorption (non antibody-based) of cells to these MP’ and in order2.2. CELL LINE to test the feasibility of using an incubation withFor this work, the 46C mouse ES cell line [13], bovine serum albumin (BSA) to block thisestablished at the laboratory of Professor Austin 8 adsorption. For that purpose, approximately 7×10Smith, Wellcome Trust Centre for Stem Cell MP’s were added to a 100 µL solution of PBS 0,1MResearch, University of Cambridge, England, UK, 5 pH 7.2 and incubated with 5×10 46C ES cellswas used as the model cells. This cell line can be + containing 76,9% of SSEA-1 cells. The mixtureinduced to undergo neural commitment in serum containing the cells and MP’s was then left tofree conditions [14]. 46C mouse ES cells were kept incubate for one hour at room temperature withcryopreserved in liquid nitrogen until further use. constant mixing. Three different studies were2.3. SSEA-1 EXPRESSION DETERMINATION BY performed in parallel with increasing BSAFLOW CYTOMETRY concentrations (%w/v) of 1%, 2% and 5%. The cell–After in vitro expansion of 46C ES cells, the MP complex pellet was then isolated from the +percentage of SSEA-1 cells was evaluated by flow supernatant by placing the eppendorfs near acytometry. The same procedure was used after cell magnet in a magnetic particle separator Magna-separation using the different procedures (batch TM Sep . The remaining pellet of cells-MPs complexand microfluidic-based). For that purpose, cells was ressuspended in 100µL of PBS 0,1M. Thewere collected and ressuspended in 100 μL of PBS. number of cells in each one of the phasesThe antibody was then added to the cell suspension (supernatant and pellet) was then counted using a 4
  5. 5. hemocytometer. The percentage of cells that were at room temperature. To each eppendorf, a 5adsorbed to the MP’s by non-specific adsorption heterogeneous mixture of 3×10 46C ES cells + -(NSA) (non-based in antibody interaction) was containing both SSEA-1 and SSEA-1 cells was addedcalculated as shown below: and again left for incubation for one hour at room of cells pellet temperature. The total solution in each eppendorf ells dsorbed of total cells pellet supernatant) was ressuspended in 300μL of PBS 0,1 M, ph 7.32.6. SEPARATION OF CELLS USING IGM, solution and fed into a 1mL syringe located in theBIOTIN ANTI-MOUSE/HUMAN SSEA -1 NE-1000 Programmable Syringe Pump from NAI –ANTIBODY IN BATCH PR OCEDURE Wis Biomed with a constant fluid flow of 20μL/minThe main purpose was to place a magnet near the in inlet 1. The buffer solution was injected into inleteppendorfs and once the magnetically susceptible 2 with a constant fluid flow of 100μL/min. Each runcells were attracted to the eppendorfs walls, the was performed during 15min approximately. Thesupernatant was decanted to new eppendorfs and outlets fluid flows were finally collected inthe number of cells were counted again. In this eppendorfs connected to the separation chambercase, 2 µL of 3 nm MP’s were added to a 100µL by polymer tubes. In order to differentiate fluidsolution of PBS 0,1 M pH 7.2, containing final flows through inlet 1 and 2 during the separation,antibody concentrations of 0,125µg/mL 0,25µg/mL FITC diluted 1:1000 was added to the bufferand 0,375µg/mL in different eppendorfs. A negative solution and visualized under a fluorescencecontrol run with no antibody added was also microscope (figure, 6). Prior to the injection of theperformed. The number of cells in each eppendorf 5 5 cells, the entire microfluidic device as well a s all thevaried between 1,4×10 - 3,5×10 since it was not accessories such as syringes, microfluidic tubes andpossible to collect the same number of cells from eppendorfs that would contact with the cells-MP’sthe initial cell suspension. complex were coated with a blocking solution +2.7. SEPARATION OF CELLS USING containing 2% (w/v) BSA. The percentage of SSEA-1MICROFLUIDIC DEVICE negatively selected cells in the microfluidic device isThe fabricated microfluidic device was used for the calculated as: +negative separation of SSEA-1 cells from of aheterogeneous 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 µLof PBS 0,1M pH 7.2, with antibody at finalconcentrations of 0,250µg/mL 0,375µg/mL and0,625µg/mL in different eppendorfs.A negative control run with no antibody was alsoperformed. All the mixtures were left to incubate Fig. 6 - Experimental setup for the separation in microfluidic device. a) Cubic Permanent Magnet. b) Inlet 2 for buffer solutionfor one hour. The 300µL solution was then c) Inlet 1 for cells-MPs complex containing solution d) microfluidic separation chamber e) glass surface f) fluorescenceressuspended in the same volume of BSA blocking microscope ocular g) outlet 2 for SSEA-1+ cells h) outlet 1 for SSEA-1- cells. Note that the entire structure is mounted in ansolution 2%(w/v) and left to incubate for one hour inverted position due to technical properties of the microscope. 5
  6. 6. cells in 2 antibody. However, from the control run (no Separation cells total antibody used) results it can be concluded that aBeing Ncells total, the sum of the number of cells high percentage of cells (42,4%) was captured duecounted in outlet 1 and outlet 2. to non-specific adsorption. In order to evaluate the efficiency of this batch3. RESULTS AND DISCUSSION + system concerning the capture of SSEA-1 cells, the +3.1. SEPARATION OF CELLS USING IGM, percentage of SSEA-1 cells, in the supernatant wasBIOTIN ANTI-MOUSE/HUMAN SSEA -1 determined by flow cytometry analysis (figure, 8).ANTIBODY IN BATCH PR OCEDURE Only after this analysis it will be possible to evaluateAfter the results presented in previous works (data if the antibody-MP’s complex is efficiently bindingnot shown) several considerations were taken in to the specif ic antigen in the cells surface. The firstaccount and it was decided to perform the batch plot [figure 8, a)] represents the negative control ofprocedure using an antibody already biotinylated – the flow cytometry analysis by using an IgG γ1IgM biotin anti-mouse/human SSEA-1. In this case, antibody with a linked fluorophore – PE – butthe biotin group is labeled to the antibody in a without a specific affinity for the ES cells.different area than the antigen binding-site (Fab This negative control is used namely to account forRegion), this way the specific recognition of the + the non-specific adsorption of the fluorophore toSSEA-1 cells by the antibody is not affected. Since the ES cells.this antibody is concentrated, each run will have a By making this negative control run it is possible tolow cost because a low monoclonal antibody 0 define a peak with fluorescence between 10 andvolume is used. In order to perform this 1 5 10 of the PE fluorescence (FL2-H intensity scale).experiment, 3×10 46C ES cells containing 53,4% of + This way, only ES cells with fluorescence intensitySSEA-1 cells were used. 1 (FL2-H) higher than 10 will be considered toAs can be observed in figure 7, an increasing express the SSEA-1 antigen. After performing thepercentage of captured cells were obtained when + negative control run, the percentage of SSEA-1increasing the concentration of biotinylated cells was determined in 46C ES cells feed to theantibody. The maximum percentage of captured batch system, before purification, using the SSEA1cells (91,1%) was obtained when using a antimouse/human IgM PE antibody [figure 8, b)].concentration of 0,375ug/mL of the biotinylated Next, in figures 8, c), d), e) and f) it is possible to Capture of Cells 100 91,1 90,7 observe the flow cytometry analysis of the 90 Separation Percentage 80 75,5 supernatants after the batch procedure when 70 antibody concentrations of 0 μg/mL, 0,25 μg/mL, 60 50 42,4 0,25 μg/mL, 0,375 μg/mL were used. As can be 40 observed in figure 8, c), when no antibody was used 30 , 25μg/mL ,25μg/mL ,375μg/mL control in the batch separation procedure, a percentage of Antibody concentrationFig. 7 - Results for the capture percentage of 46C ES cells labeled + 36,4% of SSEA-1 cells was obtained in thewith a specific biotinylated antibody (in differentconcentrations) immobilized into MPs and using the batch supernatant. Looking at the separation value ofprocedure. 42,4% [figure 7, Control run with no antibody used] 6
  7. 7. it is possible to say that some of the non-specific same values after batch separation since they -interaction of the antibody-MPs complex with the represent the number of SSEA-1 cells beforecells is promoting depletion of SSEA-1+ cells from (columns on the left of each cluster) and afterthe 46C ES cells heterogeneous population. (columns on the right of each cluster) theNext, after the separation protocol using 0,125μ/mL separation in batch procedure.of antibody, the percentage of 46C ES cells Thus, the first conclusion that can be taken fromexpressing the SSEA-1 antigen was also determined this study is that the batch procedure for the +by flow cytometry. The M region denotes the separation of SSEA-1 46C ES cells is promoting highpercentage of ES cells expressing SSEA-1 antigen, levels of non-specific interactions either betweenand for this run that percentage decreased to 15,6% cells and between cells and the eppendorfs, during[figure 8, d)] leading to conclude that the separation a)protocol while using specific antibodies was 6,1%purifying the initial solution of 46C ES cells. Onceincreasing the antibody concentration to 0,250μg/mL and 0,375 μg/mL, the percentage of cells b)expressing the SSEA-1 antigen in the supernatant 53,4%decreased to 4,1% and 1,2% respectively [figure 8,e) and f)]. After performing flow cytometry analysisit was possible to calculate the purity degree of the c)supernatant after the batch procedure (Table 1) is: 36,4%Purity Degree = 100% - % SSEA-1 positive cells in the supernatantIt is important to notice that although the obtained dpurity degree is high (table 1), the loss of cells is ) 15,6%also high (see figure 9). Since the percentage of cellsexpressing the SSEA-1 antigen before the batchseparation is 53,4% the number of cells that are e)attracted to the magnet in the batch procedure 4,1%should not overcome that value, otherwise thisseparation is not only based on specific cell-antibody interactions. Since the separation f)percentages are higher than that value (as can be 1,2%seen in figure 7) this means that cells are beingattracted to the magnet placed near the eppendorf Fig. 8 – Flow cytometry analysis of SSEA-1 positive 46C ES cellsby non-specific interactions. The percentages of cell when using the batch procedure: a) Flow cytometry negative - control run, b) %SSEA-1 positive cells before separation; c) Controlloss (SSEA-1 cells non-specifically attracted to the run, where no antibody was used for the selective separation ofmagnet) have values around 60-70% (figure 9). In cells. %SSEA-1 positive cells in the supernatant after the batch procedure while using d) , 25μg/mL, e) , 25μg/mL and f)this figure the white areas should maintain the ,375μg/mL of SSEA1 specific antibody. 7
  8. 8. 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: cells in 2 Separation of cells in outlet 2 cells total Table 1 - Resume table for the purity degree of Where Ncells total, is the total number of cells each solution after the separation procedure. 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.Fig. 9 - Four different clusters of results, each one representingthe use of a different antibody concentration during batch Indeed, in this case, only 10,3% of the 46C ES cellsseparation. In each cluster, the left column represents the totalnumber of cells prior to the separation, and the right column were attracted to O2 via non-specific interactionsrepresents the total number of cells in the supernatant afterthe separation. In blue is the number of ES cells expressing the (figure, 10). This value is much lower than theSSEA-1 antigen. obtained in the batch procedure which presents athe time the solution remains in batch. Perhaps the major advantage of the microfluidicformation of aggregates between the antibody-MP- procedure.Indeed, in this case, all the cells in thecell is contributing for the non-specific adsorption separation device are in constant motion due to theof cells. However, more studies about this aspect fluid flow, decreasing the amount of interactionwould be needed to understand this phenomena. between all the species and preventing cells fromTaking these results into consideration, a different + forming aggregates. In fact, cell aggregates aremethod for the depletion of SSEA-1 cells from 46C more difficult to separate based on the expressionES cells should be attempted. of surface antigens. The cells labeled with MPs are in constant movement, while in the separation3.2. SEPARATION OF SSEA-1 + 46C ESCELLS USING THE MICROFLUIDIC DEVICE chamber they get subjected to the magnetic field +After performing SSEA-1 cell separation using the generated by the permanent magnet. This exposurebatch procedure, the protocol was adapted for the to the magnetic field occurs for only a few secondsseparation in the microfluidic device. Looking at the Cell separation Outlet 2 50 42,0 42,9microfabricated structure, there are two inlets, one 39,4 40 Percentage of Separaionfor the solution containing the cells (inlet1) and the 30other for the buffer solution of PBS 0,1M pH 7.3. 20The presence of the permanent magnet will induce 10,3 10cells labeled with MPs to deviate from their path to 0outlet 2, while non-susceptible cells will continue ,25μg/mL ,375μg/mL ,625μg/mL control Antibody concentrationalong their path to outlet 1. Three different Fig. 10 – Results for the capture percentage of cells labeled withconcentrations of antibody were again evaluated, specific antibody biotinylated (in different concentrations) immobilized into MPs in the microfluidic structure. 8
  9. 9. ≈4 sec, as they travel at ≈4mm/s and the the graphics decreases as the antibodyseparation chamber is 15mm wide), and that is concentration gets higher [figure 11, d) e) and f)],sufficient to alter the cells trajectory according to denoting again that the antibody is recognizing theresults shown in figure 10. cells to be deviated from their path with the use ofIn these experiments, the microfluidic system was the permanent magnet. By other words, most of +injected with a cell suspension containing 42,4% of the SSEA-1 ES cells (and not other cells) which wecells expressing the SSEA-1 antigen [figure 11, b)]. want to deplete from the initial cell solution areHowever, as can be seen in figure 11, c) and in exiting the structure through outlet 2, resulting in aopposition to the batch procedure [fig ure 7, c)], the negative selection separation procedure in a novelseparation of cells by non-specific adsorption in this working microfluidic structure with low cost perdynamic protocol is negligible, which means that a run. Another aspect is the purity degree that washigh specificity is achieved in cell binding to MP’s- obtained using this microfluidic procedure. Theantibody complex. Besides that, the M region on results obtained are presented in table 2. The purity degree is not as high as in the batch procedure, a) however it is highly comparable, once standard 1,25% errors are considered. The advantages of using a microfluidic device, is the ease and low cost of the b) procedure. Besides, in this case, each solution of 42,4% 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 c) than in batch procedure. The results achieved here 44,6% could potentially be improved by passing the d) 10,3% Table 2 - Resume table for the purity degree of each 46C ES cells solution after the separation procedure in the microfluidic e) structure. 5% f) 3,7%Fig. 11 – Flow cytometry analysis of SSEA-1 positive 46C ES cellswhen using the microfluidic separation device: a) Flowcytometry negative control run, b) %SSEA-1 positive cells before Fig. 12 - Four different clusters of results, each one representingseparation; c) Control run, where no antibody was used for the the use of a different antibody concentration. In each cluster,selective separation of cells. %SSEA-1 positive cells in the the left column represents the total number of cells prior to thesupernatant after the batch procedure while using d) separation, and the right column represents the total number of , 25μg/mL, e) , 25μg/mL and f) ,375μg/mL of SSE specific cells out of outlet 1 after the separation. In blue is the numberantibody. of ES cells expressing the SSEA-1 antigen. 9
  10. 10. solution of cells several times in the microfluidic conclude that this procedure of purification can bedevice since this would enhance the final purity repeated several times in order to obtain higherdegree [16]. However, in this case, clearance rates final purity degrees. Each run can be performed infor undifferentiated ES cells would decrease with less than 8 hours using a simple protocol. +decreasing amounts of undifferentiated SSEA-1 ES The microfluidic device was used for thecells [16]. purification of a population of mES cells from cells expressing the SSEA-1 antigen, a marker for4. CONCLUSIONS AND FUTURE WORK pluripotent and tumorogenic cells. Such model canBefore using the microfluidic device for the be used as a proof of concept of this technology formagnetophoresis based separation all the biological the direct application in the purification of a neuralprotocols needed optimization. The immobilization progenitors population of cells from tumorogenicof the 300nm particles from Ademtech® to the cells. A high-throughput separation system with lowspecific antibodies used, needed to be engineered cost per run was developed in this MSc project.from the beginning with constant improvementsand changes. The final protocols were adequate for 5. REFERENCES + 1.Figeys, D. and D. Pinto, Lab-on-a-chip: a revolution in biologicalthe specific and correct labeling of SSEA-1 cells. and medical sciences. Anal Chem, 2000. 72(9): p. 330A-335A.The batch procedures performed lead ultimately to 2.Stone, H.A. and S. Kim, Microfluidics: Basic issues, applications, and challenges. AIChE Journal, 2001. 47(6).good results for the purification of a heterogeneous 3.Beebe, D.J., G.A. Mensing, and G.M. Walker, Physics and applications of microfluidics in biology. Annu Rev Biomedpopulation of cells. Final purity degrees obtained in Eng, 2002. 4: p. 261-86. 4.Dolnik, V., S. Liu, and S. Jovanovich, Capillary electrophoresis onbatch separation ranged from 96,9% to 99,5% while microchip. Electrophoresis, 2000. 21(1): p. 629-43. 5.Heller, C., Principles of DNA separation with capillaryusing well defined concentrations of IgM, Biotin anti electrophoresis. Electrophoresis, 2001. 22(4): p. 629-43. 6.Adams, J.D., U. Kim, and H.T. Soh, Multitarget magneticmouse/human SSEA1 antibody. However, the activated cell sorter. Proc Natl Acad Sci U S A, 2008. 105(47):values for the cells loss percentage were also high p. 18165-70. 7.Krantz, W.B., Applications in Fluid Dynamics. Scaling Analysis inand observed between 60% and 70%. Modeling Transport and Reaction Processes. 2006: John Wiley & Sons, Inc. 19-144.As for the magnetophoresis in a dynamic 8.Eckhardt, B., Introduction. Turbulence transition in pipe flow: 125th anniversary of the publication of Reynolds paper.microfluidic device the use of a permanent magnet, Philosophical Transactions of the Royal Society A: Mathematical, Physical and Engineering Sciences, 2009.the W-12-N: Cube 12mm from Supermagnete® 367(1888): p. 449-455. 9.Pamme, N. and A. Manz, On-Chip Free-Flow Magnetophoresis: made from an alloy of neodymium, iron and boron Continuous Flow Separation of Magnetic Particles and Agglomerates. Analytical Chemistry, 2004. 76(24): p. 7250-to form the Nd2Fe14B proved to be sufficient for the 7256. + 10.McCloskey, K.E., J.J. Chalmers, and M. Zborowski, Magneticdeviation of labeled SSEA-1 cells with 300nm Cell Separation:  Characterization of Magnetophoreticparticles from Ademtech®. The final purification Mobility. Analytical Chemistry, 2003. 75(24): p. 6868-6874. 11.Pamme, N., Magnetism and microfluidics. Lab on a Chip,degrees obtained in this procedure ranged from 2006. 6(1): p. 24-38. 12.Friend, J. and L. Yeo, Fabrication of microfluidic devices using92,3% to 98,5% in a single step run which in terms polydimethylsiloxane. Biomicrofluidics, 2010. 4(2). 13.Ying, Q.-L., et al., BMP Induction of Id Proteins Suppressesof results is comparable to those obtained in the Differentiation and Sustains Embryonic Stem Cell Self- Renewal in Collaboration with STAT3. Cell, 2003. 115(3): p.batch procedure if laboratorial errors are taken in 281-292. 14.Diogo, M., D. Henrique, and J. Cabral, Optimization and neuralaccount. As for the percentages of cells loss, while commitment of mouse embryonic stem cells. Biotechnol Appl Biochem, 2008. 49: p. 105–112.using the microfluidic device, the values obtained 16.Schriebl, K., et al., Stem cell separation: A bottleneck in stem cell therapy. Biotechnology Journal, 2010. 5(1): p. 50-61.were at most, 5%. This characteristic leads to 10

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