BIOLOGICAL APPLICATIONS OF SOFT LITHOGRAPHY CONTROL CELL GROWTH by fpe17463

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									BIOLOGICAL APPLICATIONS OF SOFT LITHOGRAPHY:
  CONTROL CELL GROWTH POSITION USING PDMS
                PETRI DISHES




Natalia Arteaga Marrero
Experimental Biophysics Project (Spring 2005)
Supervisors: Jonas Tegenfeldt
            Patrick Carlberg
PURPOSE


       The purpose of this project is to place cells in predetermined locations in an
array with defined shape, size and distance of separation. This arrangement is needed
in order to facilitate the irradiation of living cells at the Lund Nuclear sub-micron
Probe.



INTRODUCCTION


CELLION PROJECT

        The CELLION project is directed towards the studies on cellular response to
targeted single ions. In order to evaluate the response of the cells, it is very important
to avoid procedures that can modify their natural state, i.e., the manipulation of the
cells should be done without disturbing the function of the system.
        The experiment requires analysis of individual cells, the position of every cell
has to be fixed before in order to achieve the irradiation process and after to evaluate
the damage. Consequently, it could be a great advantage if the cells maintain their
specified location facilitating the repeated access to the cell. The fabrication of special
Petri dishes with some kind of limiting structure, where the cells can be confined,
allows us to partially control the growth position of the cells.

        The development of single ion hit facilities using a sub-micron beam line is
done at Lund Nuclear sub-micron Probe using a 3 MeV single-ended Pelletron
electrostatic accelerator. The system is capable to control single ions, thus, several
conditions have to be fulfilled previously. First of all, a wet environment as well as
oxygen supply are important parameters for cell culture in order to keep the cells
alive. The problem is that the beam is destroyed outside the vacuum system due to the
scattering process with air atoms. The beam has to be extracted from the chamber
minimizing the straggling, with that purpose a 200nm Si3N4 layer is used as vacuum
window.
        The current has to be limited by closing slits to approximately 100 ions per
second. A microscope connected to a CCD camera combined with a dedicated
program is used to locate the cells. The program searches regions of interest, i.e. cells,
and gives the coordinates of the centre of the region. Then, the coordinates of the cells
are transferred to the beam positioning system. In the first tests it is not of vital
importance the final position of the ion within the cell, it is enough just to touch the
cell.
        A fast beam deflecting system combined with an after target particle detector
is used to prevent secondary hits; i.e., to limit the dose applied. This means that once
the cell is irradiated the beam is deflected while is moving to the position of the next
cell.




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SOFT LITHOGRAPHY

        Soft lithography techniques have a common characteristic, they use a
patterned elastomer as mask, stamp or mold. Soft lithography techniques that are
currently explored and their resolution are shown in Table 1. The resolution is
referred to the lateral dimension of the smallest structure generated.

                          Table 1: Soft lithography techniques
                            METHOD                          RESOLUTION
            Microcontact printing (µCP)                        35 nm
            Replica molding (REM)                              30 nm
            Microtransfer molding (µTM)                        1 µm
            Micromolding in capillaries (MIMIC)                1 µm
            Solvernt-asistent micromolding (SAMIM)             60 nm


        There are many biophysical applications based on soft lithography, whose
main advantages are that devices can be produced very quickly, inexpensively and
with a high throughput.


PDMS

        PDMS consists of repeating units of –OSi(CH3)2-. PDMS is durable
elastomer, deformable, homogeneous, and isotropic. Its surface is low in interfacial
free energy and chemically inert. The surface properties can be modified by treatment
with plasma followed by the formation of SAMs to give appropriate interfacial
interactions with materials.
        In addition, it is inexpensive, flexible, optically transparent to wavelengths
greater than 230nm, impermeable to water but not to gases, and most important for
our purpose, non-toxic to cells.
        Due to the CH3 groups the PDMS surface is very hydrophobic. Treating the
PDMS surface with oxygen plasma, the surface will become hydrophilic. The oxygen
plasma introduces silanol groups (Si-OH) on the surface by oxidation of methyl
groups (Si-CH3) of PDMS at the plasma/polymer interface. See Figure 1.




                        Figure 1: Treatment with oxygen plasma



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       If the PDMS is left on air after the oxygen plasma treatment, the hydrophilic
surface tends to revert to hydrophobic in approximately 30 minutes. In order to
maintain the hydrophilicy of the PDMS surface the PDMS has to be introduced into
water.

        PDMS presents some technical problems. Gravity, adhesion and capillary
forces exert stress on the elastomeric features and cause them to collapse and generate
defects in the formed pattern. There is a risk of structural collapse if the aspect ratio of
the PDMS structure is far away from the unit. If the aspect ratio of the relief structures
is too large, the PDMS microstructures fall under their own weight or collapse owing
to the forces typical of inking or printing of the stamp [Figure 2]. If the aspect ratios
are too low, the relief structures are not able to withstand the compressive forces
typical of printing and the adhesion between the stamp and the substrate; these
interactions result in sagging [Figure 3]. PDMS shrinks by a factor of about 1% upon
curing and can be readily swelled by nonpolar solvents such as toluene and hexane
[Figure 4].




                                     Figure 2: Pairing




                                     Figure 3: Sagging




                                    Figure 4: Shrinking




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EXPERIMENTAL PART



1.- Device design

        The design of the device was made taking into account the cells size and the
undesired effects observed in previous works [11]. The cells used are V79 or chinese
hamster and has a size between 10 and 20µm. The designed pattern was 5x5mm and
consists of circles with 20µm of diameter and the separation between circles of 10µm.
If the gap between holes is not big enough, the cells can make a bridge and cross from
one region to the next one. Sometimes this “bridges” can be very useful biologically
because it allows us to study the effect of the damage cells in the surrounding ones.
Since only the cells in the holes are targeted and it is assumed that there is any kind of
communication between cells, the cells in the surrounding regions can show an
anomalous behaviour. If the depth of the holes is big enough, the bridge effect can be
avoided but another problem can appear like more than one cell occupying the same
hole.


2.- Mask fabrication

       Chromium mask on a glass was fabricated specially for this project.


3.- Master creation

        The master was produced on a 1’’ standard silicon wafer. The silicon wafer is
spin coated with a photocurable negative tone resist SU-8, 2010 and 2050. This
formulations produce resists structures on top of the wafer of heights 20 and 50µm,
depending on the spin velocity which the photoresist is applied onto the wafer. After
this procedure, the substrate is soft baked on a hot plate. The wafer is then exposed to
UV-light through the chrome mask. Following, the post exposure substrate is baked
again.
        In order to complete the master, the uncured photoresist has to be removed
using an etching solution, after that, the substrate is washed in isopropyl alcohol and
deionised water and dried using nitrogen gas. Following the substrate is hard baked in
a convection oven at 200°C for 30 minutes.
        Finally, the surface of the master is silanized minimizing the elastomer
adhesion during modeling. The silanization process consists of exposing the master to
the vapours of chlorosilane.
        Two masters were made, one of them produces holes of 50µm and the other
one 20µm. The diameter of the holes was 20µm, but the separation between holes was
not 10µm. During the etching process the distance between holes for the 20µm depth
Petri dish was diminished. Probably the master was exposed to the etching solution
for a long time. Thus, the holes in the 20µm depth Petri dish are connected.




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4.- PDMS device

        The PDMS devices were prepared by replica molding by casing the liquid
prepolymer of an elastomer, in this case PDMS, against the master that has a
patterned relief structure in its surface.
        The elastomer is prepared by mixing PDMS base and curing agent in the
weight ratio 9:1. During the mixing process some air bubbles are formed, in order to
remove them the PDMS was degassed introducing it in a vacuum chamber until no
bubbles were visible. Then, the PDMS was poured over the master and placed into a
convection oven at 80°C during 45 minutes. After the baking process the PDMS was
allowed to cool and removed from the master.
        PDMS surface is highly hydrophobic. Thus, it is assumed that the cells cannot
attach easily to the surface. However, both hydrophilic and hydrophobic Petri dishes
were tested. In order to make PDMS surface hydrophilic, the PDMS was exposed to
oxygen plasma for 60 seconds to activate PDMS surface for bonding and make it
hydrophilic. Hydrophilic stability is needed, and hence, the Petri dish is introduced in
a water bath to maintain the hydrophilicity for a longer period.


5.- Cell culture

           The cell medium is a solution of DMEM (Dulbecco’s modification of
Eagle’s Medium), fetal calf serum, penicillin and glucose. The quantity of the cell
medium that should be added depends on the incubation time required. This
parameter is important because if it is not enough the cells can be dry and die.
           The medium contains approximately 3000 cells/µl. In this case 10µl have
been added, i. e., roughly 30000cells per Petri dish. The cells were cultivated for
about 17 hours. It must be noticed that the cell population is doubled approximately
every 10 hours.
          There are no additional methods available to sterilize the Petri dishes, thus,
the procedure to cultivate the cells consists of adding enough quantity of cell medium
above each Petri dish and 10µl of cell solution. Then, the Petri dishes are introduced
into the incubation system.




RESULTS


        Usually, the cells are cultivated in a commercial plastic Petri dish. The
concentration of cells in the cell medium can be modified according to the purpose.
The size and the quantity of cells are related with the incubation time. Generally the
cell size is between 10 and 20µm as can be seen in Figure 5.




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                       Figure 5: Cells in ordinary Petri dish (plastic)




       Regarding the cell attachment, in order to see the differences between the
ordinary Petri dish and the PDMS ones, a picture was taking at the interface of both
surfaces. In Figure 6 and Figure 7, a diagonal can be seen; this line is the separation
between the plastic (below) and the PDMS (above). The incubation time was longer
than 17 hours, thus, many cells can be observed. In these two figures, the difference
between the hydrophilic and hydrophobic surfaces can be notice that there are no cells
attached to the hydrophobic surface.




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Figure 6: Hydrophilic Petri dish outside the pattern




Figure 7: Hydrophobic Petri dish outside the pattern




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         Figure 8 shows the hydrophilic Petri dish with 20µm holes. Several results can
be extracted from this picture.
    i.      Cells attach to the surface hydrophilic PDMS surface. Usually the cells
            migrate to the location where the attachment is favourable, in this case,
            they prefer the position at the bottom of the holes.
    ii.     “Bridge” effect is not avoided using 20µm holes, the cells can cross
            between holes without problem. However, the separation between holes
            does not seem 10µm, it can be due to the etching process during the
            fabrication of the master.
    iii.    Coincidence of several cells in the same hole position can be observed in
            several holes.




                       Figure 8: Hydrophilic Petri dish - 20µm depth




        Figure 9 shows the hydrophilic Petri dish with 50µm holes. The results are
better with this depth. In this case, “bridge” effect as well as coincidence of cells in
the same hole, have been avoided. However, the quantity of attached cells is small but
it could be because the concentration of cells used was not enough. A magnification
image of this pattern has been taken in order to show how the cell is attached, see
Figure 10.




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Figure 9: Hydrophilic Petri dish - 50µm depth




Figure 10: Hydrophilic Petri dish - 50mm depth




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       Finally, the hydrophobic Petri dish with 50µm holes is shown in Figure 11. It
can be seen that there are no cells attached to this surface.




                     Figure 11: Hydrophobic Petri dish - 50mm depth




CONCLUSIONS


        PDMS Petri dishes have been made in order to control the growth and
attachment of cells. Untreated PDMS, i. e., hydrophobic surface, inhibited cell
growth. However, hydrophilic PDMS allows cells to attach and grow.
        Gap dimensions between holes have been shown to be critical regarding the
cell spread and movement. Changing this parameter, “bridge” effect can be allowed or
avoided depending on the purpose. Coincidence of cells in the same hole position can
be prevented increasing the depth of the holes. Consequently, three-dimensional
structures can be used to guide the cell growth because high aspect ratio
microstructures can be used to encourage or discourage cell attachment.
        Petri dishes with 50µm holes seem to achieve better results, but it can be due
to an error in the etching process during the master fabrication. The gap between
holes is not 10µm in both cases.
        The PDMS dish in a future will be used to positioning cells during single ion
damage analysis in CELLION project at Lund Nuclear Probe facility.




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ACKNOWLEDGMENTS


        I would like to thank my supervisors, Jonas Tegenfedt and Patrik Carlberg for
their help regarding soft lithography procedures.
        I also would like to thank Johan Christensson, at the Department of Medical
Radiation Physics at Lund University Hospital, for his help during cell culturing.




BIBLIOGRAPHY


[1] Soft lithography. Younan Xia and George M. Whitesides. Angew. Chem. 37, 550-
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[2] Effect of high-aspect-ratio microstructures on cell growth and attachment.
Mengyan Li; Glawe J.D.; Green H.; Mills D.K.; McShane M.J.; Gale B.K..
Microtechnologies in Medicine and Biology, 1st Annual International, Conference,
531-536 (2000)

[3] Engineering Cell Shape and Function. Rahul Singhvi; Amit Kumar; Gabriel P.
Lopez; Gregory N. Stephanopoulos; Daniel I. C. Wang; George M. Whitesides;
Donald E. Ingber. Science. 3-264, 696-698 (1994)

[4] Cell Culture in 3-Dimensional Microfluidic Structure of PDMS
(polymethilsiloxane). Leclerc, Eric; Sakai, Yasuyuki; Fujii, Teruo. Biomedical
Microdevices, 5, 109 – 114 (2003)

[5] Biological surface engineering: a simple system for cell pattern formation.
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Frankel, Douglas A. Lauffenburger, George M. Whitesides, Alexander Rich.
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[6] Micropatterning of micro/nanospheres on PDMS by using layer-by-layer self-
assembly. Al H.; Lvov Y.M.; Mills D.K.; Jones S.A. Microtechnologies in Medicine
& Biology 2nd Annual International IEEE-EMB Special Topic Conference, 144-147
(2002)

[7] Low-Contact-Angle Polydimethil Siloxane (PDMS) Membranes for Fabricating
Micro-Bioarrays. Gillmor, S.D.; Larson, B.J.; Braun, J.M.; Mason, C.E.; Cruz-Barba,
L.E.; Denes, F.; Lagally, M.G. Microtechnologies in Medicine & Biology 2nd Annual
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[8] Poly(dimethilsiloxane) thin films as biocompatible coatings for microfluidic
devices: Cell culture and flow studies with glial cells. Peterson, Sophie L.; McDonald,
Anthony; Gourley, Paul L.; Sasaki, Darryl Y. Journal of Biomedical Materials
Research, 72A, 10-18 (2005)



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[9] Control of Mammalian Cell and Bacteria Adhesion on Substrates Micropatterned
with Poly(ethylene glycol) Hydrogels. Koh, Won-Gun; Revzin, Alexander; Simonian,
Aleksandr; Reeves, Tony; Pishko, Michael. Biomedical Microdevices, 5, 11-19
(2003)

[10] Patterning proteins and cells using soft lithography. Kane, R.S.; Takayama, S.;
Ostuni, E.; Ingber, D.E.; Whitesides, G.M. Biomaterials, 20, 23-24 (1999)

[11] Micropatterned Surfaces for Control of Cell Shape, Position, and Function. Chen
C. S.; Mrksich M.; Huang S.; Whitesides G. M.; Ingber D. E. Biotechnology progress,
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[12] Microfluidics for a lab-on-a-chip applications. Håkan Jönsson Diploma Thesis




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