Cell Array Fabrication by Plasma Nanotexturing

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on nanostructured surfaces is currently a hot topic throughout the ..... [6] Knight, C.G., Morton, L.F., Onley, D.J., Peachey, A.R., Messent, A.J., Smethurst, P.A., et al. ... R.G., Gadegaard , N., McMurray, R.J., Affrossman, S., Wilkinson, C.D.W., et al.,.
Cell Array Fabrication by Plasma Nanotexturing Dimitrios Kontziampasisa,*, Athanasia Bourkoulab, Panagiota Petroub, Angeliki Tserepia, Sotirios Kakabakosb, Evangelos Gogolidesa a IMEL, N.C.S.R. “Demokritos”,15310, Aghia Paraskevi, Greece; bIRRP, N.C.S.R. “Demokritos”,15310, Aghia Paraskevi, Greece ABSTRACT Cell behavior (i.e. attachment, proliferation, etc.) on nanostructured surfaces is currently a hot topic throughout the scientific community. However, studies often show diverging results due to differences in cells, local surface chemistry, and nanotopography fabrication methods. In this study, we use Oxygen plasma etching to both randomly nanotexture a PMMA surface and change its surface chemistry. We find that 3T3 cells behave quite differently on flat PMMA surfaces as compared to nanotextured ones, showing an on-off attachment behavior. Work is under progress to exploit this effect allowing selective cell capturing, and creation of cell arrays in adjacent plasma-nanotextured/smooth areas using a stencil mask during etching. Keywords: surface modification, plasma etching, cell attachment, nanotexturing, PMMA.

1. INTRODUCTION Development of nanobiotechnology requires a better understanding of how cells and surfaces interact in the nanoscale. Recently, advances in nanoscale patterning as well as in detection methods have allowed the fabrication of appropriate substrates and the study of cell-cell and cell-substrate interactions. Several reviews have thus appeared on topographic and chemical patterning for bioengineering purposes [1–3]. Nanotopography can be sensed by the cell usually as a barrier to its movement, or as a deformation of its surface. Nanotopography most commonly influences focal adhesions, filopodia development and extension, and receptor clustering [4-10]. Several studies have suggested that for nanostructures 10-30 nm high, the smaller the size of nanotopography the more it affects cells [11-15]. According to these studies, the minimum detectable by cells topography size is slightly less than 10 nm. It is proposed that surface nanotopography is sensed as a discontinuity by the cell, and that it affects the formation of focal adhesions, as it changes the ability of integrins to cluster on the nanostructures. Furthermore, studies on the influence of nanotopography shape (e.g. grooves, columns, pits, protrusions) show that the shape affects cell behaviour as well, by affecting focal adhesions and philopodia [16-24]. Moreover, the spacing between adjacent nanofeatures also plays a role in cell behaviour since it affects not only the distance between focal adhesions, but also the cell membrane, which needs to bend between nanofeatures [25-26]. The influence of nanofeature organization was studied by Dalby et al. using stamps made by electron beam lithography [27-29]. Our group has been using plasmas to etch, chemically modify and roughen polymer surfaces and this modification has been named “plasma nanotexturing”. Plasma nanotexturing has been used for controlling the wetting of polymer surfaces and for creating intense and sensitive protein arrays [30]. In this work, we evaluated the effect of oxygen plasma nanotexturing of PMMA on cell adhesion, viability, morphology, and cytoskeleton organization and compared to untreated PMMA surface (flat PMMA).

2. MATERIALS, INSTRUMENTS AND PROCESSES 2.1 PMMA Surface Preparation and Characterization Poly(methyl methacrylate)/PMMA polymer with molecular weight of 120 kDa was purchased from Aldrich and dissolved in Propylene Glycol Methyl Ether Acetate/PGMEA solvent for creating 700 to 30.000 nm thick films by spin Bio-MEMS and Medical Microdevices, edited by Angeliki Tserepi, Manuel Delgado-Restituto, Eleni Makarona Proc. of SPIE Vol. 8765, 87650B · © 2013 SPIE · CCC code: 1605-7422/13/$18 · doi: 10.1117/12.2017894

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coating on Silicon wafers. Molecular sieves were planted inside the solvent before using it to remove humidity, which could lead to film peeling-off from the substrates. Spin coating was performed at speeds ranging from 1000 to 5000 rpm for 30 s with acceleration of 300 rpm/s2. A two-step baking follows, initially at 90 oC for 20 min and then at 150 oC for 1 h. The etching experiments have been conducted in a high density helicon plasma reactor (MET system from AlcatelAdixen). Oxygen gas at pressures ranging from 0.5 to a few Pa (typically 0.75 Pa) was used to etch/nanotexture the PMMA films. The samples were etched typically at 50-70 oC, with bias voltages ranging from 0 to -100 V, (to find the value of ion energy the plasma potential of 15-25 V should be added to the absolute value of the bias voltage, e.g. 0V+15V, or 100V+15V). Temperature was controlled by helium backside cooling of a carrier wafer on which samples were glued with thermal paste. Etching time was typically 1 minute, while the etching rate varied between 500-1500 nm/min depending on the bias voltage used in each case. The AFM measurements were conducted on a CP-II instrument from Veeco. All AFM measurements have been performed in (non-contact) tapping mode, with the use of PPP-NCHR-50 tips (Tip characteristics: n+-Silicon, resistivity 0.01-0.02 Ωcm, coated with 30 nm Aluminum reflex located on the detector side of the AFM, thickness 4.0 ± 1 μm, length 125 ± 10 μm, width 30 ± 7.5 μm, height is 10 – 15 μm, radius of curvature (of the tip) less than 10 nm, resonance frequency 204 to 497 kHz, force constant 10-130 N/m). To characterize surface morphology and therefore define the nanotexture mathematically, first we extract the x-y-ztopography of the surface from the AFM image. We use a homemade software SURFANALYSIS [31] and estimate several roughness metrics characterizing both vertical and spatial (frequency) aspects of surface morphology. In this paper, we focus on the following vertical roughness parameters: 

The root mean square (rms) which shows statistically the average height of the nanotexturing on the surface.



The skewness which indicates the symmetry of the surface height distribution. It is zero for surfaces with fluctuations symmetrical around their mean value (e.g. for a Gaussian distribution), whereas it is larger (smaller) than 0 for surfaces with peaks (holes).



The kyrtosis which describes the “peakedness” of the surface: Higher kyrtosis values mean that more surface fluctuations are the result of extreme deviations from the mean value, as opposed to frequent modestly sized deviations. For a Gaussian distribution the kyrtosis is equal to 3.

As regards the spatial aspects of surface roughness, we calculate the Power Spectral Density (PSD), which is the square of the amplitude of the Fourier transform, versus spatial frequency. Actually, Fourier transform and consequently PSD is a 2D function of the spatial frequency vector k = (kx,ky). Here, we calculate the 1D radial average of Fourier transform and present its square (1D PSD) versus the amplitude of k (√kx2+ky2). In case of the presence of periodic structures on a surface, the PSD exhibits one or more well-defined peaks. The wettability of all treated samples was characterized by water contact angle measurements using a Digidrop Contact Angle Measurement System from GBX. The system allowed automatic loading of single droplets on the surfaces under investigation and measurements of static contact angles were performed through observation of the droplet (and its reflection) at nearly right angles with respect to the sample surface. For static water contact angle measurements in this work, the droplets volume was adjusted to 4 μl, in order to minimize the effect of gravity on droplet shape. 2.2 Cell adhesion and cell morphology evaluation experiments The mouse immortalized fibroblasts 3T3 cell line derived from Swiss mouse embryo tissue was used in our experiments. 3T3 fibroblasts were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, 2 mM L-glutamine, and 1% (v/v) penicillin/streptomycin. The cells were cultured in tissue culture dishes at 37 oC in a water-saturated atmosphere of 5% CO2 humidified incubator until the cell culture attained 70-80 % confluence. For plating, cells were treated with a 0.25% solution of trypsin-EDTA. For cell culture experiments all nanotextured PMMA surfaces (0 Volt/1 min, -50 Volt/1 min, -100 Volt/1 min) were left to age for at least 20 days prior to use, in order to stabilize the surface chemistry (see section 3.1). The treated samples, including the flat untreated PMMA, underwent sterilization by exposure to ultraviolet light for 20 min. The surfaces to be tested were placed in 6-well plates and seeded with 4x104 and 1.3x104 3T3 cells/mL, for the 1- and 3-day culture, respectively. Cells were cultured as described above. After incubation for 1 and 3 days, respectively, all samples were washed with 10 mM phosphate buffered saline, pH 7.4 (PBS), in order to remove the non-adhered dead cells, and then

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the live cells were imaged using light microscopy (AxioImager A1m, Carl Zeiss). The number of cells in seven random fields per substrate was counted, averaged and recorded as cells/cm2. The cells attached on the surfaces were also characterized with respect to their morphology by immunocytology. In particular, after 1 and 3-day culture of mouse fibroblasts on the different substrates, surfaces were rinsed with PBS and then the adhered cells were fixed by incubating with a 4% (w/v) paraformaldehyde solution (PFA) in PBS for 20 min. After fixation, cells were rinsed 3 times with PBS, and their membrane was permeabilized by treatment with a Triton X100 solution (0.1%, v/v, in PBS) for 5 min. After gentle washing with PBS, the cells were incubated with 50 mg/ml BSA solution in PBS (blocking solution) at room temperature for 1 h. Then, the fixed 3T3 fibroblasts were incubated with a 150 nM Phalloidin Atto 488 (Sigma) solution in PBS for 1 h, to visualize cytoskeleton (F-actin), followed by 3X washing with PBS. Afterwards, cells nuclei were stained by incubating the cells with a 50 ng/mL 4',6-diamidino-2phenylindol solution (DAPI) in PBS for 5 min and washed as previously. Finally, the surfaces with the cells were covered with anti-fading solution containing p-Phenylenediamine (PPD), and coverslips are placed on top prior to examination under an epifluorescence microscope (Axioscop 2, Carl Zeiss). Experiments were performed three times in duplicate. Twenty random individual cells per substrate were selected and their nucleus length and width was measured using the Image Pro Plus software (Media Cybenetics Co.).

3. RESULTS AND DISCUSSION 3.1 Oxygen Plasma Nanotextured PMMA Surfaces with Increasing Roughness In this section, a series of experiments has been conducted using different Bias Voltages, with all other parameters kept constant, in order to increase roughness and sharpness of the nanotexturing of the PMMA film surface. The selected Bias Voltages are 0, -50 and -100 Volts. In Figure 1, the morphologies of these PMMA films are shown. All images show a 2 μm x 2 μm area of the surface and have been taken with the AFM as described in section 2.1. The morphological characteristics of those surfaces are shown on Table 1, and give us a clear view of the difference in height, shape and size of the nanotexturing in each value of the Bias Voltage.

4,0x108 3,5x108 3,0x108

PSD

2,5x108 2,0x108 1,5x108

1 µm

µm

1,0x108 5,0x107 0,0

1 µm

1E-3

0,01

Spatial Frequency

0 µm 0 µm

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0,1

1,6x1010 1,4x1010

PSD (nm)4

1,2x1010 1,0x1010

1 µm

8,0x109 6,0x109 4,0x109 2,0x109

m

0,0 1 µm

1E-3

0,01 0,1 Spatial Frequency (nm)-1

0 µm 0 µm

3,0x1011

PSD (nm)4

2,5x1011

1 µm

2,0x1011 1,5x1011 1,0x1011 5,0x1010 0,0 1E-3

0,01 0,1 Spatial Frequency (nm)-1

2 µm 1 µm 0 µm 0 µm

Figure 1: On the left, we show AFM images of surfaces with increasing height and sharpness in their nanotexturing after Oxygen plasma treatment with different bias voltages. Conditions: 1900 W top power, 0.75 Pa, 65o C, etching time 1 min, bias voltage 0, -50 and -100 V. On the right, we present the Power Spectral Density Vs Spatial Frequency plots of each AFM measurement. All AFM images are 2x2 μm in size and their analysis is 512x512 pixels. Each surface is characterized mathematically as shown in the next table.

As we can observe in Figure 1, the nanotexturing pattern has already appeared after 1 min etching time for 0 V Bias Voltage. The shape of the features is mount-like. Inhibitors coming from the reactor dome [30] cause local micromasking, leading to random nanomount formation during etching. We observe that as the applied Bias Voltage increases the nanotexturing becomes taller and sharper. This can be attributed to the fact that the more Bias Voltage we apply, the more energy the ions have and thus etching gets faster and more anisotropic leading to taller mounts. By measuring the etch rates we can see that it triples as we reach -100 V Bias Voltage compared to 0 V Bias.

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Table 1: Metrological Characteristics of the nanotextured PMMA surfaces shown on Fig.1

Bias Voltage (V) Rms (nm) Skewness Kyrtosis Correlation Length (nm) Etch Rate (nm/min) 0

5.2

0.5

2.3

13.0

650

-50

16.1

0.7

3.2

33.9

1050

-100

41.6

1.1

4.8

70.5

1500

In Table 1, we can evaluate the differences of the three nanotextured surfaces. RMS height increases successively, and more than doubles, as the Bias Voltage increases from 0 to -50 and then to -100V. Skewness increases also in the case of -100 V showing a more mount-like morphology. The increase in kyrtosis shows that more sharp structures are created as we increase the Bias Voltage. Finally, the correlation length shows that the size of the structures becomes 4-5 times bigger. This way we have achieved to nanotexture a PMMA surface by Oxygen plasma etching and create three different morphologies characterized by increasing height, size and sharpness of features.

Table 2: Contact angle values of O2 plasma treated PMMA surfaces vs. time after plasma treatment [The contact angle value of the untreated PMMA sample was 62o (0.5)].

Contact Angles (degrees) [Mean value (sd); n=5] Bias Voltage (V) 0 days

4 days

10 days

20 days

30 days

0

30 (0.6)

60 (1.2)

60 (1.5)

60 (1.2)

60 (0.8)

-50

10 (0.1)

30 (0.8)

62 (1.3)

63 (1.1)

62 (1.0)

-100

0 (0)

7 (0.2)

30 (1.1)

65 (0.7)

65 (0.3)

As it is shown in Table 2, where the contact angle values of the different surfaces are provided in the time course after plasma treatment, right after the oxygen plasma the surfaces appear to be extremely hydrophilic, especially for the higher Bias Voltages, while all of them revert to the value of the untreated PMMA (~60o) 20 days after treatment. As it is also shown, the higher the Bias Voltage used during treatment, the lower the initial contact angle and the longer the time period required for the contact angle to return to the value of the untreated surface. To avoid any possible effect of surface wettability and focus only on the effect of surface nanostructure on cell adhesion, we used samples that have been aged for at least twenty days for the cell attachment studies. In the next section we will describe the effect of these surfaces on 3T3 cell attachment behavior and morphology. 3.2 Cell Attachment on Oxygen Plasma Nanotextured PMMA Surfaces Oxygen plasma nanotextured PMMA surfaces were used as culture substrates for 3T3 fibroblasts. After 1 and 3 days of cell plating and culture under standard conditions, live cells were imaged as described above using light microscopy. From Figure 2, it can be clearly seen that after 1 day of culture, fewer cells attach on flat rather than on the nanotextured PMMA surfaces, with the higher number of cells per surface observed on the rougher surfaces. On the other hand, after 3 days of culture, cells show the opposite behavior and more cells adhere on flat PMMA or less nanotextured surfaces compared to more nanotextured ones. More specifically, for short culture times (1 day), the number of cells per area of plasma treated surfaces (Table 3) is approximately 5-6 times higher to that determined for the flat PMMA surface, indicating that the nanotextured surfaces strongly favor cell adhesion. Despite this finding, the results obtained after 3 days of culture indicated that the cells remained attached and proliferated on the flat and the less nanotextured surfaces (0 and -50 V), whereas on the highly nanostructured surfaces (-100 V bias), cell proliferation was negatively affected.

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1day culture

3days culture

b

i

t A

Flat .c IJ

.a

p ir-N,

3

w aka

P

. 0 Volt 1 min

f

\: f

"Ti

\fr »

*

k

\

/

.

\ Ñf I -' ,% ,i,

=

50 Volt 1 min

Æ-



\ \_

,.

_

}

-

E

a

.7

~ \. ,§

Figure 2: Dark field images of 3T3 mouse fibroblasts after 1 day of culture on untreated-flat and oxygen plasma nanotextured PMMA surfaces (a, c, e, g) and after 3 days (b, d, f, h). The magnification of the optical microscope is 40X. Notice that for 1 day culture cells seem to adhere well and proliferate on the rough surface, while the opposite happens for a 3 day culture.

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Table 3: Number of attached cells per surface after 1 and 3 days of culture. Cell numbers are displayed as mean value ± SD.

Cells/cm2 1 day

3 days*

Flat

1662 ± 428

4113 ± 466

0 Volt 1 min

8221 ± 1010

6891 ± 809

-50 Volt 1 min

10912 ± 1372

5110 ± 884

-100 Volt 1 min

10456 ± 1216

2776 ± 853

*It should be noted that the number of cells seeded for the 3-day culture was three times less than the number of cells used for the 1-day culture.

In order to evaluate the effect of surface nanotexturing on the morphology of both the whole cells and their nuclei, cell cytoskeleton and nuclei were fluorescently stained. The images obtained after staining of F-actin were used to determine the shape of cell cytoskeleton while the images of cell nuclei obtained through staining with DAPI were used to determine the nucleus elongation. Nucleus elongation was defined as the ratio of the length to the width of each nucleus.

1 day culture

3 days culture

Figure 3: Representative fluorescence microscope images obtained after F-actin (green) and nucleus (blue) staining of surface adherent 3T3 fibroblasts after 1-day (a, c) and 3-day culture (b, d) on untreated flat (a, b) and on oxygen-plasma nanotextured PMMA surfaces with -100V Bias. Notice that for 1 day culture cells seem to adhere well and proliferate on the rough surface, while the opposite happens for a 3 day culture. (c, d).

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As it is demonstrated from the fluorescence images shown in Figure 3, after 1-day culture of cells on both flat and nanotextured substrates, adherent cells growing on PMMA flat surface (image a) do not spread as well as on PMMA nanotextured surface where the cells connect to each other with filopodia-type extensions (image c). On the other hand, after 3-day culture, cells growing on the flat PMMA surface (image b) are clearly more spread than on rough PMMA surface (image). In general, the cytosceleton of the cells corresponding to images b and c resembles that of cells cultured on standard culture plates whereas the morphology of cells corresponding to images a and d deviates significantly from that of cell grown on standard culture substrates. Regarding the nucleus elongation in different substrates no statistically significant change is observed (Table 4).

Table 4: Nucleus elongation of 3T3 mouse fibroblasts after 1 and 3 days of culture on flat and oxygen-plasma treated PMMA surfaces.Values are displayed as mean ± SD

Nucleus elongation 1 day

3 days

Flat

1.26 ± 0.11

1.39 ± 0.11

0 Volt 1 min

1.26 ± 0.09

1.40 ± 0.11

50 Volt 1 min

1.31 ± 0.12

1.40 ± 0.12

100 Volt 1 min

1.31 ± 0.11

1.28 ± 0.11

4. CONCLUSIONS It has been reported that cells are sensitive to substrate nanotopography and therefore cells respond specifically to substrates with different roughness and sharpness. Changes in nanotopography induce changes in cells that affect not only the cell morphology (e.g., the cytoskeleton) but also important cellular processes such as apoptosis, growth, proliferation and differentiation [1]. For the type of cells used in the present study, it was found that the topography of the culture substrate affected their attachment behavior and morphology. In particular, for short culture periods (1 day), surface roughness enhanced the adhesion of cells as compared to the flat surface. Concerning cell proliferation, plasma treated surface with a 40-nm rms roughness affected negatively cells proliferation and morphology. Based on these results, selective cell capturing on surfaces for the creation of cell arrays in adjacent plasma-

nanotextured/smooth areas using a stencil mask during etching is foreseen.

ACKNOWLEDGMENT: We thank Dr Vassilios Constantoudis for helping with the surface analysis and Dr Katerina Tsougeni and Kosmas Ellinas for helping with contact angle measurements.

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