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Using microdispensing to manufacture a customized cell dish for microbeam irradiation of single, living cells
Nuclear Instruments and Methods in Physics Research B 267 (2009) 1199–1205
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Using microdispensing to manufacture a customized cell dish for microbeam irradiation of single, living cells E.J.C. Nilsson a,*, M.G. Olsson b, J. Nilsson c, J. Pallon a, A. Masternak a,1, J. Paczesny a,b,1, N. Arteaga-Marrero a, M. Elfman a, P. Kristiansson a, C. Nilsson a, B. Åkerström b a
Division of Nuclear Physics, Department of Physics, Lund Institute of Technology, Lund University, Box 118, S-22100 Lund, Sweden Division of Infection Medicine, Department of Clinical Sciences, Lund University, S-22184 Lund, Sweden c Department of Electrical Measurements and Industrial Electrical Engineering and Automation, Lund Institute of Technology, Lund University, Box 118, S-22100 Lund, Sweden b
a r t i c l e
i n f o
Article history: Received 15 October 2008 Received in revised form 6 February 2009 Available online 16 March 2009 PACS: 87.53. j 07.79 87.17.Rt 87.14.E 87.18.Gh 87.85.J
a b s t r a c t In this paper is described the preparation of patterned cell dishes to be used in studies of low dose irradiation effects on living cells. Using a droplet microdispenser, an 8 lm thick polypropylene cell substrate, to which cells do not naturally adhere, was coated in a matrix pattern with the cell adhesive mussel protein Cell-Tak. Cells were shown to adhere and grow on the protein-coated spots, but not on the uncoated parts, providing for guided cell growth. Cultivation of isolated cell colonies provides an opportunity to study how low doses of ionizing radiation affect neighbouring un-irradiated cell colonies. Ó 2009 Elsevier B.V. All rights reserved.
Keywords: Microdispensing Cell substrate Patterned cell dish Microbeam irradiation Bystander effect HepG2 cells
1. Introduction By guiding and restricting cell growth, using patterned cell substrates of various kinds, information regarding for example cell adhesion, spreading, communication and migration can be obtained. Furthermore, specific applications can be found in the areas of cellular biosensors, tissue engineering and neuronal networks [1]. ‘‘Targeted” irradiation of single cells, i.e. delivering a predetermined number of particles to a selected cell in a culture can be achieved at dedicated microbeam irradiation facilities, using scanning, focused particle beams at very low intensities. Applying this modus of irradiation instead of broad beam irradiation, either
* Corresponding author. Tel.: +46 46 2227630; fax: +46 46 2224709. E-mail addresses: [email protected], [email protected] (E.J.C. Nilsson). 1 Permanent address: Department of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland. 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.02.051
accelerator produced or from radioactive sources, allows for delivery of a well-defined dose (all the way down to a single ion) with a high spatial resolution. The hit inaccuracy is less than the size of a typical cell (about 10 lm) and gives, in combination with counting the number of individual irradiating ions, a high precision in dose calculations. This shall be seen in contrast to the broad beam case, where cells in an irradiated culture receive an average of the applied dose and where (Poisson distributed) statistical variations occur [2]. In addition, by selecting or ‘‘targeting” a subpopulation of the cells in a culture instead of irradiating all cells, indirect effects of radiation can be studied. It is known that not only directly irradiated cells will show responses, but also the neighbouring cells that have not been directly hit by ionizing radiation may still be affected by the irradiation [3]. The effects seen on these neighbour cells are gathered under the generic term ‘‘bystander effects” and they can be induced by at least two different pathways – cell medium-mediated [4] or gap junction-mediated [5].
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E.J.C. Nilsson et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1199–1205
When setting up a microbeam irradiation experiment for studying low dose effects, accurate counting of the ions is an integral part of the system. If the ions are to be detected and counted after having traversed the cells, as is often the case, it must be made sure that the ions still retain sufficient energy to enable detection. Thus, cells must be cultured on a thin substrate, rather than in a standard plastic Petri dish, as the beam stopping in the Petri dish would cause the ions to lose all of their energy, i.e. be completely stopped. Working with a patterned cell dish, where cell growth is restricted to certain isolated, well-defined areas – as opposed to uniform cell growth covering the entire area of the cell dish – will provide an opportunity to study in particular cell-medium mediated bystander effects. Irradiating one isolated island of cells will give the opportunity to observe how the cells on the other islands respond to the irradiation. This way, information can be gained about signals transferred via the cell medium, as there is no direct cell–cell contact between the irradiated island and the non-irradiated islands. Earlier work on patterned substrates has generally sought to turn sections of a cell-unfriendly surface into a suitable cell substrate and has most often involved different lithographical techniques. Examples of this technique include microcontact printing (soft lithography) of poly-L-lysine patterns on a polyethylene glycol substrate [6], but also other methods, such as production of nanogrooves on polystyrene by UV laser irradiation [7], have been reported. An opposite approach has also been used, changing a cell-friendly surface so that it no longer supports cell growth. An example of this approach is implantation of negative silver ions into tissue-culture polystyrene dishes [8]. A patterned cell dish for irradiation of single cells has also been prepared using lithography, more specifically preparation of an SU-8 (an epoxy-based photopolymer) dish by UV lithography [9]. In this paper another technique for manufacturing a patterned cell substrate is presented – namely microdispensing, i.e. spraying minute amounts of fluid, of an adhesive protein on a plastic foil.
2. Cell dish Several different types of cell dishes are used in microbeam irradiation experiments, as well as in experiments using radioactive sources, to study low dose effects. Depending on the experimental set-up, the use of a standard plastic Petri dish may not be possible for practical reasons, for example when using a post-cell detector to detect the ions. In this case, the ions will be completely stopped in a standard Petri dish and will never reach the detector, meaning that the ions cannot be counted so as to achieve a well-defined low dose experiment. Common choices of cell substrates are Si3N4 windows [10], or dishes with a Mylar film [11] or a polypropylene film [12]. Depending on whether or not the substrate is naturally cellfriendly, additional treatment of the substrate prior to use may be necessary to make the cells attach, grow and form the desired monolayer. A cell-unfriendly substrate has to be used to make patterns, and thus Si3N4 wafers are not suitable, as they are naturally cell-friendly. The choice of a suitable substrate and coating was based on a literature survey, which revealed that a common way of preparing cell-friendly foils is by coating a thin polypropylene film with the mussel protein Cell-Tak, BD Biosciences [13]. This procedure has been reported previously [14–16]. Other alternatives are Cell-Tak on Kynar [17], Cell-Tak on Mylar [18] or polypropylene coated by poly-L-lysine [19]. Polypropylene, a non-fluorescent, hydrophobic plastic, was considered to be a more suitable alternative than Mylar, which is fluorescent, as fluorescence is intended to be used during biological investigations of the irradiated cells. Also, the fluoropolymer Kynar
(C2F2) was less suitable, as Particle Induced X-ray Emission (PIXE) was planned to be used as a way of imaging the patterned foils and analyse the cell composition. Thus, a foil containing fluorine would not be a suitable choice, as PIXE analysis of specimens containing fluorine will produce gamma rays, which will lead to a high background in the PIXE spectrum. An 8 lm thick polypropylene foil, Goodfellow [20], was chosen as cell substrate and the adhesive protein Cell-Tak as coating of the surface. 3. Preparation of dishes The cell dishes were prepared by attaching the polypropylene foil to acrylic glass frames (area = 28 40 mm2) with a circular opening. The foil was fixed to the frame in a heat treatment process. The cell dishes were then sterilized with ethanol and washed with distilled water to avoid staining of the foil. The cell dishes, see Fig. 1, have an area of cell growth of 1.54 cm2 and can hold a cell medium volume of approximately 400 ll. 4. Microdispensing technique: equipment and procedure The micromachined flow-through dispenser was designed and developed at the Department of Electrical Measurements, Lund Institute of Technology in 1999 [21,22]. Using this technique, droplets (typically on the order of 100 pl) can be dispensed onto a substrate in a well-defined and accurate fashion. Fluids of a wide range of viscosities can be dispensed. Applications of the technique are found in e.g. biochemistry and analytical chemistry, where small sample volumes and rapid sample handling are desired features [23,24]. The microdispensing principle is shown in Fig. 2. Setting up the dispensing equipment takes 15–30 min and the production of the pattern on a substrate takes from less than a minute up to a few minutes, depending on the number of droplets that need to be dispensed. The typical dispensing frequency used in this application is 50 droplets/second. To create the patterned cell substrate the procedure is as follows: The silicon microdispenser, filled with Cell-Tak, is controlled using an in-house developed electronic control unit, which enables simple, button triggered, dispensing in one point at a time. The sample stage, where the cell dish is placed, is controlled via dedicated software (LabView, National Instruments Corporation, Austin, TX, USA), which allows for more complex, externally triggered, dispensing sequences, producing patterns. Patterns consisting exclusively of ordered rows and columns, i.e. matrices of varying dimensions, can be defined directly in the software. Customized patterns may be programmed beforehand using a spreadsheet software like Microsoft Excel and loaded into the software as
Fig. 1. The polypropylene cell dish, before patterning.
E.J.C. Nilsson et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1199–1205
the pulse shape (pulse height, pulse length and rise time) to achieve single, satellite-free droplets also have to be set. A satellite droplet is an undesired second droplet, formed together with the main droplet and following immediately after that droplet in time but usually following a trajectory that deviates from that of the main droplet. The satellite droplet results in a smaller, subsidiary area on the substrate in the immediate vicinity of the area formed by the main droplet.
piezoactuated pushbar
microdispenser nozzle
5. Results of protein dispensing on polypropylene
orifice
satellite droplet
Y substrate
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X
Fig. 2. The microdispensing principle, including an illustration of an unwanted satellite droplet. The substrate can be moved during the dispensing, to create the matrix pattern.
text files. The desired matrix, defining the number of spots to be produced as well as the distance between the spots, needs to be specified along with the number of protein droplets per spot. Additional parameters, such as the dispensing frequency and the delay that is needed to stop the sample stage from moving to the next position prematurely, as well as the applied voltages that define
Different cell culturing conditions can be achieved by changing the distance between spots as well as the number of protein droplets per spot. The limit in terms of the spacing between the spots between achieving closely spaced but still separate, clearly defined spots and spots which bleed into each other forming a larger cohesive surface has been found to lie between 150 and 200 lm centerto-center, when the spots have a diameter of approximately 100 lm. A spacing of 150 lm between the centers of two adjacent spots will result in a large number of protein spots starting to run into each other, forming larger protein coats with an area at least twice to three times larger than that of a single spot, whereas a spacing of 200 lm is sufficient to maintain discrete spots. To assure no cell growth between spots the spacing should be increased further. Using a distance of 450 lm between protein spots will ensure that no cells will manage to grow between the spots. The number of spots needed for homogenous cell growth depends on the concentration of the protein and will in turn influence the size of the protein-coated spot. The diameter of the protein droplets is 55 lm, giving a droplet volume of about 90 pl. 5 droplets/spot give a spot diameter of about 100 lm and 20 droplets/ spot give a spot diameter of just below 150 lm. Two different batches of protein, with different concentrations – 1.36 mg/ml and 2.4 g/ml – have been used. With a concentration of 1.36 mg/ ml 20 drops/spot are needed to achieve homogenous cell growth and with a concentration of 2.4 mg/ml 10 drops/spot are sufficient. This will result in a density of about 20 lg/cm2 of Cell-Tak coating per surface area, which is higher than the recommendation from the manufacturer of 3.5 lg/cm2. A lower density of protein, 5– 10 lg/cm2 (in some instances as low as 2 lg/cm2) is enough to create a cell-friendly surface, but the cell growth will in that case fol-
Fig. 3. (a) Protein spots on polypropylene – discrete, distinct spots with a diameter of approximately 100 lm have been obtained. (b) Protein spots on polypropylene – substantially larger protein-coated areas have been formed.
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low a clear ‘‘coffee stain” pattern, i.e. the cells will tend to grow in a circle around the circumference of the spot, with very little cell growth in the center. To achieve a more evenly coated surface, two protein droplet matrices – each with a spacing between spot
centers of 150–200 lm – can be dispensed in the same location on the cell substrate – the second matrix having a starting point offset in the horizontal as well as the vertical direction of 100 lm with respect to the starting point of the first matrix.
Fig. 4. (a) Cell growth in discrete spots – cf. Fig. 3(a); (b) cell growth over a larger area – cf. Fig. 3(b); (c) cell growth in a standard plastic Petri dish; (d) cell growth following a ‘‘coffee stain” pattern – the distance between spots is 500 lm; (e) cell growth on a 5 month old protein coating.
E.J.C. Nilsson et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1199–1205
In Figs. 3(a) and (b) pictures of cell dishes with protein but without cells can be seen. In Fig. 3(a) the spacing between spot centers is 200 lm and the coating density is 21 lg/cm2. In Fig. 3(b) the combination of two matrices, each with a center-tocenter spacing of 150 lm, with a small offset between them has been used. The result is a number of larger cohesive areas of protein coating. The smallest protein spots, with a diameter on the order of 100 lm, are well suited for microbeam irradiation experiments, whereas larger structures, with an area on the order of 1 mm2, can just as well be used for irradiation experiments using a well-collimated radioactive source, e.g. the alpha-emitter 228 Th. 6. Cell culturing Cells from the human hepatoma cell line HepG2 were used in these studies. HepG2 cells are tumour cells and are normally
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quite tolerant to culture under different growth conditions. Since our experiments present rather harsh growth conditions, we chose to work with these cells. The cells were cultured in a Petri dish (Sarstedt, Nümbrecht, Germany) using RPMI1640 with GlutaMAX culture medium, suspended with 10% FBS and 100 lg/ml antibiotics and antimycotics. The cells were seeded onto the protein-coated substrates and cultured on the foils for a maximum of 3–5 days, which is expected to be sufficient for the foreseen irradiation experiments. In Fig. 4(a) and (b) pictures of cultured cell dishes with growing, attached cells can be seen (the same dishes as shown in Fig. 3(a) and (b), before adding cells). In Fig. 4(b), cells are growing quite evenly over the surface – they tend to follow the outlines of the protein coating, but some cells actually manage to overcome the channels of uncoated surface, thereby connecting two coated regions. Fig. 4(c) shows cell growth in a standard plastic Petri dish, for comparison. Some differences between the cultures in Fig. 4(a)–(c) can
Fig. 5. (a) PIXE image of chlorine – a protein coating, 21 lg/cm2, in discrete spots. Image size is 384 384 lm2. (b) PIXE image of chlorine – a larger area protein coating. Image size is 384 384 lm2. (c) Traverse of the protein-coated spots in (a).
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be seen. Mitotic cells with a rounded shape seem to be more frequent in the Cell-Tak cultures (Fig. 4(a) and (b)) compared to the Petri dish-cultures (Fig. 4(c)). In addition, approximately 20–25% of the surface in the Petri dish was still not covered with cells. The explanation for these differences may be that Cell-Tak cultures were deliberately grown to 100% confluency to ensure that the protein-coated areas were completely covered, while the standard procedure (maximally 75–80% confluency) was employed for the Petri dish-cultures. However, a more detailed comparison of the growth behaviour on the two surfaces, using a more thorough analysis of cell biological markers, should be performed. It is also important to remember that these are tumour cells and as such more tolerant to harsh growth conditions. It is possible that when using a different cell strain, for instance primary cell cultures, they would not be as tolerant. Therefore, the proposed culture conditions need to be optimized for each cell strain. Fig. 4(d) shows an example of cell growth on a ‘‘coffee stain” spot, i.e. when the amount of protein coating was too low to form a sufficiently homogenous surface and instead dried in a circular pattern, resulting in cell growth that follows this protein circle. The Cell-Tak protein itself, as well as the protein-coated dishes, has to be stored in a refrigerator, according to the manufacturer. Under these conditions (2–8 °C) the protein is stable for a minimum of 3 months, whereas coated surfaces can be stored for approximately 2 weeks [25]. Coated dishes that had been kept refrigerated for a few months were investigated to assess the stability of the protein spots. The protein matrix pattern is still visible to the naked eye, as well as with PIXE analysis, for at least 6 months. Culturing cells on protein-coated dishes, 5 and 6 months after coating, revealed that at least some growth can be seen in a 5 month old dish, see Fig. 4(e). Some protein spots are still cellfriendly enough to make cells adhere and grow, but the matrix pattern of growing cells cannot be reproduced. No cell growth was found in a 6 months old dish. 7. Element mapping of the Cell-Tak protein PIXE (Particle Induced X-ray Emission) was used to analyse the content of the Cell-Tak protein and map the elemental distribution in the protein spots. In Fig. 5(a) and (b) PIXE images of the chlorine content of two different foils are shown – in Fig. 5(a) an image of smaller spots and in Fig. 5(b) an image of a larger area. The images are of the size 384 384 lm2. In Fig. 5(c) a traversal profile of the image in Fig. 5(a) is shown, to illustrate the homogeneity of the spots. As can be seen from the figure, the protein spots are well-defined and well-separated, with no chlorine content present between the two spots, however, the thickness and evenness of the coating does vary to a certain extent. The spots are about 125 lm in diameter and separated by almost 100 lm of protein-free surface. Apart from chlorine, the spots contain small amounts of sulphur and zinc. 8. Summary and outlook A patterned cell substrate, suitable for irradiation experiments using microbeam irradiation or a well-collimated radioactive source, has been successfully developed using microdispensing equipment. The technique is uncomplicated – it can be carried out in a regular laboratory, no clean-room is required and no complicated techniques such as lithography need to be employed – and the preparation of one foil takes a few minutes. The technique allows for preparation of rectangular structures, but also arbitrarily shaped patterns can be manufactured depending on
one’s needs. Various sizes of the protein-coated surfaces, from spots with a diameter of 100 lm to larger areas on the order of 1 mm2, can be achieved to suit different purposes. Cells were shown to adhere to and grow well on the substrate in a satisfactory fashion. The protein coats in the cell dishes were shown to stable for a longer period of time than guaranteed by the manufacturer. These dishes are intended to be used in future microbeam irradiation experiments, in which low dose irradiation-induced bystander effects will be studied. These experiments will be facilitated by the fact that the cells growing in separated islands under no circumstances can come in physical contact with each other. Another appealing idea would be to use this island structure to mix different cell types on different islands, e.g. irradiate one cell type and try to find out if the overall behaviour, or response, in the cell dish depends on the mixture. References [1] C.S. Chen, M. Mrksich, S. Huang, G.M. Whitesides, D.E. Ingber, Micropatterned surfaces for control of cell shape, position and function, Biotechnol. Prog. 14 (1998) 356. [2] S. Gerardi, G. Galeazzi, R. Cherubini, Single-ion microbeam as a tool for lowdose radiation effects investigations, J. Phys.: Conf. Ser. 41 (2006) 282. [3] K.M. Prise, M. Folkard, B.D. Michael, A review of the bystander effect and its implications for low-dose exposure, Radiat. Protect. Dosim. 104 (2003) 347. [4] C. Mothersill, C. Seymour, Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells, Int. J. Radiat. Biol. 71 (1997) 421. [5] S.A. Lorimore, M.A. Kadhim, D.A. Pocock, D. Papworth, D.L. Stevens, D.T. Goodhead, E.G. Wright, Chromosomal instability in the descendants of unirradiated surviving cells after alpha particle irradiation, Proc. Natl. Acad. Sci. USA 95 (10) (1998) 5730. [6] A. Ruiz, L. Ceriotti, L. Buzanska, M. Hasiwa, F. Bretagnol, G. Ceccone, D. Gilliland, H. Rauscher, S. Coecke, P. Colpo, F. Rossi, Controlled micropatterning of biomolecules for cell culturing, Microelectron. Eng. 84 (2007) 1733. [7] H.W. Lu, Q.H. Lu, W.T. Chen, H.J. Xu, J. Yin, Cell culturing on nanogrooved polystyrene petri dish induced by ultraviolet laser irradiation, Mater. Lett. 58 (2003) 29. [8] H. Tsuji, H. Satoh, S. Ikeda, S. Ikemura, Y. Gotoh, J. Ishikawa, Negative-ion beam surface modification of tissue-culture polystyrene dishes for changing hydrophilic and cell-attachment properties, Nucl. Instr. and Meth. B 148 (1999) 1136. [9] N. Arteaga-Marrero, V. Auzelyte, M.G. Olsson, J. Pallon, A SU-8 dish for cell irradiation, Nucl. Instr. and Meth. B 263 (2007) 523. [10] R. Ugenskiene, J. Lekki, W. Polak, K.M. Prise, M. Folkard, O. Veselov, Z. Stachura, W.M. Kwiatek, M. Zazula, J. Stachura, Double strand break formation as a response to X-ray and targeted proton-irradiation, Nucl. Instr. and Meth. B 260 (2007) 159. [11] M. Folkard, K.M. Prise, A.G. Michette, B. Vojnivic, The use of radiation microbeams to investigate the bystander effect in cells and tissues, Nucl. Instr. and Meth. A 580 (2007) 446. [12] Ph. Barberet, A. Balana, S. Incerti, C. Michelet-Habchi, Ph. Moretto, Th. Pouthier, Development of a focused charged particle microbeam for the irradiation of individual cells, Rev. Sci. Instr. 76 (2005) 015101. [13] BD Biosciences Europe, URL:. [18] C. Shao, V. Stewart, M. Folkard, B.D. Michael, K.M. Prise, Nitric oxide-mediated signalling in the bystander response of individually targeted glioma cells, Cancer Res. 63 (2003) 8437. [19] M.R. Folkert, J.R. Albritton, A. Dart, R.J. Ledoux, C.M. Luo, K.D. Held, J.C. Yanch, Design, characterization and application of a charged-particle microslit for subnuclear irradiation, Radiat. Res. 161 (2004) 100. [20] Goodfellow, URL:
E.J.C. Nilsson et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1199–1205 [22] J. Nilsson, J. Bergkvist, T. Laurell, Optimization of the droplet formation in a piezo-electric flow-through microdispenser, in: A. VandenBerg, P. Bergveld, W. Olthuis (Eds.), Micro Total Analysis Systems 2000, Proceedings, Mesa Monographs, 2000, p. 75. [23] P. Gustavsson, F. Johansson, M. Kanje, L. Wallman, C. Eriksson Linsmeier, Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser, Biomaterials 28 (2007) 1141.
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[24] S. Gaspar, M. Mosbach, L. Wallman, T. Laurell, E. Csoregi, W. Schuhmann, A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser, Anal. Chem. 73 (2001) 4254. [25] BD Cell-TakTM Cell and Tissue Adhesive – Instructions for Use, BD Biosciences, URL:.
Contents lists available at ScienceDirect
Nuclear Instruments and Methods in Physics Research B journal homepage: www.elsevier.com/locate/nimb
Using microdispensing to manufacture a customized cell dish for microbeam irradiation of single, living cells E.J.C. Nilsson a,*, M.G. Olsson b, J. Nilsson c, J. Pallon a, A. Masternak a,1, J. Paczesny a,b,1, N. Arteaga-Marrero a, M. Elfman a, P. Kristiansson a, C. Nilsson a, B. Åkerström b a
Division of Nuclear Physics, Department of Physics, Lund Institute of Technology, Lund University, Box 118, S-22100 Lund, Sweden Division of Infection Medicine, Department of Clinical Sciences, Lund University, S-22184 Lund, Sweden c Department of Electrical Measurements and Industrial Electrical Engineering and Automation, Lund Institute of Technology, Lund University, Box 118, S-22100 Lund, Sweden b
a r t i c l e
i n f o
Article history: Received 15 October 2008 Received in revised form 6 February 2009 Available online 16 March 2009 PACS: 87.53. j 07.79 87.17.Rt 87.14.E 87.18.Gh 87.85.J
a b s t r a c t In this paper is described the preparation of patterned cell dishes to be used in studies of low dose irradiation effects on living cells. Using a droplet microdispenser, an 8 lm thick polypropylene cell substrate, to which cells do not naturally adhere, was coated in a matrix pattern with the cell adhesive mussel protein Cell-Tak. Cells were shown to adhere and grow on the protein-coated spots, but not on the uncoated parts, providing for guided cell growth. Cultivation of isolated cell colonies provides an opportunity to study how low doses of ionizing radiation affect neighbouring un-irradiated cell colonies. Ó 2009 Elsevier B.V. All rights reserved.
Keywords: Microdispensing Cell substrate Patterned cell dish Microbeam irradiation Bystander effect HepG2 cells
1. Introduction By guiding and restricting cell growth, using patterned cell substrates of various kinds, information regarding for example cell adhesion, spreading, communication and migration can be obtained. Furthermore, specific applications can be found in the areas of cellular biosensors, tissue engineering and neuronal networks [1]. ‘‘Targeted” irradiation of single cells, i.e. delivering a predetermined number of particles to a selected cell in a culture can be achieved at dedicated microbeam irradiation facilities, using scanning, focused particle beams at very low intensities. Applying this modus of irradiation instead of broad beam irradiation, either
* Corresponding author. Tel.: +46 46 2227630; fax: +46 46 2224709. E-mail addresses: [email protected], [email protected] (E.J.C. Nilsson). 1 Permanent address: Department of Chemistry, Adam Mickiewicz University, Grunwaldzka 6, 60-780 Poznan, Poland. 0168-583X/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.nimb.2009.02.051
accelerator produced or from radioactive sources, allows for delivery of a well-defined dose (all the way down to a single ion) with a high spatial resolution. The hit inaccuracy is less than the size of a typical cell (about 10 lm) and gives, in combination with counting the number of individual irradiating ions, a high precision in dose calculations. This shall be seen in contrast to the broad beam case, where cells in an irradiated culture receive an average of the applied dose and where (Poisson distributed) statistical variations occur [2]. In addition, by selecting or ‘‘targeting” a subpopulation of the cells in a culture instead of irradiating all cells, indirect effects of radiation can be studied. It is known that not only directly irradiated cells will show responses, but also the neighbouring cells that have not been directly hit by ionizing radiation may still be affected by the irradiation [3]. The effects seen on these neighbour cells are gathered under the generic term ‘‘bystander effects” and they can be induced by at least two different pathways – cell medium-mediated [4] or gap junction-mediated [5].
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E.J.C. Nilsson et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1199–1205
When setting up a microbeam irradiation experiment for studying low dose effects, accurate counting of the ions is an integral part of the system. If the ions are to be detected and counted after having traversed the cells, as is often the case, it must be made sure that the ions still retain sufficient energy to enable detection. Thus, cells must be cultured on a thin substrate, rather than in a standard plastic Petri dish, as the beam stopping in the Petri dish would cause the ions to lose all of their energy, i.e. be completely stopped. Working with a patterned cell dish, where cell growth is restricted to certain isolated, well-defined areas – as opposed to uniform cell growth covering the entire area of the cell dish – will provide an opportunity to study in particular cell-medium mediated bystander effects. Irradiating one isolated island of cells will give the opportunity to observe how the cells on the other islands respond to the irradiation. This way, information can be gained about signals transferred via the cell medium, as there is no direct cell–cell contact between the irradiated island and the non-irradiated islands. Earlier work on patterned substrates has generally sought to turn sections of a cell-unfriendly surface into a suitable cell substrate and has most often involved different lithographical techniques. Examples of this technique include microcontact printing (soft lithography) of poly-L-lysine patterns on a polyethylene glycol substrate [6], but also other methods, such as production of nanogrooves on polystyrene by UV laser irradiation [7], have been reported. An opposite approach has also been used, changing a cell-friendly surface so that it no longer supports cell growth. An example of this approach is implantation of negative silver ions into tissue-culture polystyrene dishes [8]. A patterned cell dish for irradiation of single cells has also been prepared using lithography, more specifically preparation of an SU-8 (an epoxy-based photopolymer) dish by UV lithography [9]. In this paper another technique for manufacturing a patterned cell substrate is presented – namely microdispensing, i.e. spraying minute amounts of fluid, of an adhesive protein on a plastic foil.
2. Cell dish Several different types of cell dishes are used in microbeam irradiation experiments, as well as in experiments using radioactive sources, to study low dose effects. Depending on the experimental set-up, the use of a standard plastic Petri dish may not be possible for practical reasons, for example when using a post-cell detector to detect the ions. In this case, the ions will be completely stopped in a standard Petri dish and will never reach the detector, meaning that the ions cannot be counted so as to achieve a well-defined low dose experiment. Common choices of cell substrates are Si3N4 windows [10], or dishes with a Mylar film [11] or a polypropylene film [12]. Depending on whether or not the substrate is naturally cellfriendly, additional treatment of the substrate prior to use may be necessary to make the cells attach, grow and form the desired monolayer. A cell-unfriendly substrate has to be used to make patterns, and thus Si3N4 wafers are not suitable, as they are naturally cell-friendly. The choice of a suitable substrate and coating was based on a literature survey, which revealed that a common way of preparing cell-friendly foils is by coating a thin polypropylene film with the mussel protein Cell-Tak, BD Biosciences [13]. This procedure has been reported previously [14–16]. Other alternatives are Cell-Tak on Kynar [17], Cell-Tak on Mylar [18] or polypropylene coated by poly-L-lysine [19]. Polypropylene, a non-fluorescent, hydrophobic plastic, was considered to be a more suitable alternative than Mylar, which is fluorescent, as fluorescence is intended to be used during biological investigations of the irradiated cells. Also, the fluoropolymer Kynar
(C2F2) was less suitable, as Particle Induced X-ray Emission (PIXE) was planned to be used as a way of imaging the patterned foils and analyse the cell composition. Thus, a foil containing fluorine would not be a suitable choice, as PIXE analysis of specimens containing fluorine will produce gamma rays, which will lead to a high background in the PIXE spectrum. An 8 lm thick polypropylene foil, Goodfellow [20], was chosen as cell substrate and the adhesive protein Cell-Tak as coating of the surface. 3. Preparation of dishes The cell dishes were prepared by attaching the polypropylene foil to acrylic glass frames (area = 28 40 mm2) with a circular opening. The foil was fixed to the frame in a heat treatment process. The cell dishes were then sterilized with ethanol and washed with distilled water to avoid staining of the foil. The cell dishes, see Fig. 1, have an area of cell growth of 1.54 cm2 and can hold a cell medium volume of approximately 400 ll. 4. Microdispensing technique: equipment and procedure The micromachined flow-through dispenser was designed and developed at the Department of Electrical Measurements, Lund Institute of Technology in 1999 [21,22]. Using this technique, droplets (typically on the order of 100 pl) can be dispensed onto a substrate in a well-defined and accurate fashion. Fluids of a wide range of viscosities can be dispensed. Applications of the technique are found in e.g. biochemistry and analytical chemistry, where small sample volumes and rapid sample handling are desired features [23,24]. The microdispensing principle is shown in Fig. 2. Setting up the dispensing equipment takes 15–30 min and the production of the pattern on a substrate takes from less than a minute up to a few minutes, depending on the number of droplets that need to be dispensed. The typical dispensing frequency used in this application is 50 droplets/second. To create the patterned cell substrate the procedure is as follows: The silicon microdispenser, filled with Cell-Tak, is controlled using an in-house developed electronic control unit, which enables simple, button triggered, dispensing in one point at a time. The sample stage, where the cell dish is placed, is controlled via dedicated software (LabView, National Instruments Corporation, Austin, TX, USA), which allows for more complex, externally triggered, dispensing sequences, producing patterns. Patterns consisting exclusively of ordered rows and columns, i.e. matrices of varying dimensions, can be defined directly in the software. Customized patterns may be programmed beforehand using a spreadsheet software like Microsoft Excel and loaded into the software as
Fig. 1. The polypropylene cell dish, before patterning.
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the pulse shape (pulse height, pulse length and rise time) to achieve single, satellite-free droplets also have to be set. A satellite droplet is an undesired second droplet, formed together with the main droplet and following immediately after that droplet in time but usually following a trajectory that deviates from that of the main droplet. The satellite droplet results in a smaller, subsidiary area on the substrate in the immediate vicinity of the area formed by the main droplet.
piezoactuated pushbar
microdispenser nozzle
5. Results of protein dispensing on polypropylene
orifice
satellite droplet
Y substrate
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X
Fig. 2. The microdispensing principle, including an illustration of an unwanted satellite droplet. The substrate can be moved during the dispensing, to create the matrix pattern.
text files. The desired matrix, defining the number of spots to be produced as well as the distance between the spots, needs to be specified along with the number of protein droplets per spot. Additional parameters, such as the dispensing frequency and the delay that is needed to stop the sample stage from moving to the next position prematurely, as well as the applied voltages that define
Different cell culturing conditions can be achieved by changing the distance between spots as well as the number of protein droplets per spot. The limit in terms of the spacing between the spots between achieving closely spaced but still separate, clearly defined spots and spots which bleed into each other forming a larger cohesive surface has been found to lie between 150 and 200 lm centerto-center, when the spots have a diameter of approximately 100 lm. A spacing of 150 lm between the centers of two adjacent spots will result in a large number of protein spots starting to run into each other, forming larger protein coats with an area at least twice to three times larger than that of a single spot, whereas a spacing of 200 lm is sufficient to maintain discrete spots. To assure no cell growth between spots the spacing should be increased further. Using a distance of 450 lm between protein spots will ensure that no cells will manage to grow between the spots. The number of spots needed for homogenous cell growth depends on the concentration of the protein and will in turn influence the size of the protein-coated spot. The diameter of the protein droplets is 55 lm, giving a droplet volume of about 90 pl. 5 droplets/spot give a spot diameter of about 100 lm and 20 droplets/ spot give a spot diameter of just below 150 lm. Two different batches of protein, with different concentrations – 1.36 mg/ml and 2.4 g/ml – have been used. With a concentration of 1.36 mg/ ml 20 drops/spot are needed to achieve homogenous cell growth and with a concentration of 2.4 mg/ml 10 drops/spot are sufficient. This will result in a density of about 20 lg/cm2 of Cell-Tak coating per surface area, which is higher than the recommendation from the manufacturer of 3.5 lg/cm2. A lower density of protein, 5– 10 lg/cm2 (in some instances as low as 2 lg/cm2) is enough to create a cell-friendly surface, but the cell growth will in that case fol-
Fig. 3. (a) Protein spots on polypropylene – discrete, distinct spots with a diameter of approximately 100 lm have been obtained. (b) Protein spots on polypropylene – substantially larger protein-coated areas have been formed.
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low a clear ‘‘coffee stain” pattern, i.e. the cells will tend to grow in a circle around the circumference of the spot, with very little cell growth in the center. To achieve a more evenly coated surface, two protein droplet matrices – each with a spacing between spot
centers of 150–200 lm – can be dispensed in the same location on the cell substrate – the second matrix having a starting point offset in the horizontal as well as the vertical direction of 100 lm with respect to the starting point of the first matrix.
Fig. 4. (a) Cell growth in discrete spots – cf. Fig. 3(a); (b) cell growth over a larger area – cf. Fig. 3(b); (c) cell growth in a standard plastic Petri dish; (d) cell growth following a ‘‘coffee stain” pattern – the distance between spots is 500 lm; (e) cell growth on a 5 month old protein coating.
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In Figs. 3(a) and (b) pictures of cell dishes with protein but without cells can be seen. In Fig. 3(a) the spacing between spot centers is 200 lm and the coating density is 21 lg/cm2. In Fig. 3(b) the combination of two matrices, each with a center-tocenter spacing of 150 lm, with a small offset between them has been used. The result is a number of larger cohesive areas of protein coating. The smallest protein spots, with a diameter on the order of 100 lm, are well suited for microbeam irradiation experiments, whereas larger structures, with an area on the order of 1 mm2, can just as well be used for irradiation experiments using a well-collimated radioactive source, e.g. the alpha-emitter 228 Th. 6. Cell culturing Cells from the human hepatoma cell line HepG2 were used in these studies. HepG2 cells are tumour cells and are normally
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quite tolerant to culture under different growth conditions. Since our experiments present rather harsh growth conditions, we chose to work with these cells. The cells were cultured in a Petri dish (Sarstedt, Nümbrecht, Germany) using RPMI1640 with GlutaMAX culture medium, suspended with 10% FBS and 100 lg/ml antibiotics and antimycotics. The cells were seeded onto the protein-coated substrates and cultured on the foils for a maximum of 3–5 days, which is expected to be sufficient for the foreseen irradiation experiments. In Fig. 4(a) and (b) pictures of cultured cell dishes with growing, attached cells can be seen (the same dishes as shown in Fig. 3(a) and (b), before adding cells). In Fig. 4(b), cells are growing quite evenly over the surface – they tend to follow the outlines of the protein coating, but some cells actually manage to overcome the channels of uncoated surface, thereby connecting two coated regions. Fig. 4(c) shows cell growth in a standard plastic Petri dish, for comparison. Some differences between the cultures in Fig. 4(a)–(c) can
Fig. 5. (a) PIXE image of chlorine – a protein coating, 21 lg/cm2, in discrete spots. Image size is 384 384 lm2. (b) PIXE image of chlorine – a larger area protein coating. Image size is 384 384 lm2. (c) Traverse of the protein-coated spots in (a).
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be seen. Mitotic cells with a rounded shape seem to be more frequent in the Cell-Tak cultures (Fig. 4(a) and (b)) compared to the Petri dish-cultures (Fig. 4(c)). In addition, approximately 20–25% of the surface in the Petri dish was still not covered with cells. The explanation for these differences may be that Cell-Tak cultures were deliberately grown to 100% confluency to ensure that the protein-coated areas were completely covered, while the standard procedure (maximally 75–80% confluency) was employed for the Petri dish-cultures. However, a more detailed comparison of the growth behaviour on the two surfaces, using a more thorough analysis of cell biological markers, should be performed. It is also important to remember that these are tumour cells and as such more tolerant to harsh growth conditions. It is possible that when using a different cell strain, for instance primary cell cultures, they would not be as tolerant. Therefore, the proposed culture conditions need to be optimized for each cell strain. Fig. 4(d) shows an example of cell growth on a ‘‘coffee stain” spot, i.e. when the amount of protein coating was too low to form a sufficiently homogenous surface and instead dried in a circular pattern, resulting in cell growth that follows this protein circle. The Cell-Tak protein itself, as well as the protein-coated dishes, has to be stored in a refrigerator, according to the manufacturer. Under these conditions (2–8 °C) the protein is stable for a minimum of 3 months, whereas coated surfaces can be stored for approximately 2 weeks [25]. Coated dishes that had been kept refrigerated for a few months were investigated to assess the stability of the protein spots. The protein matrix pattern is still visible to the naked eye, as well as with PIXE analysis, for at least 6 months. Culturing cells on protein-coated dishes, 5 and 6 months after coating, revealed that at least some growth can be seen in a 5 month old dish, see Fig. 4(e). Some protein spots are still cellfriendly enough to make cells adhere and grow, but the matrix pattern of growing cells cannot be reproduced. No cell growth was found in a 6 months old dish. 7. Element mapping of the Cell-Tak protein PIXE (Particle Induced X-ray Emission) was used to analyse the content of the Cell-Tak protein and map the elemental distribution in the protein spots. In Fig. 5(a) and (b) PIXE images of the chlorine content of two different foils are shown – in Fig. 5(a) an image of smaller spots and in Fig. 5(b) an image of a larger area. The images are of the size 384 384 lm2. In Fig. 5(c) a traversal profile of the image in Fig. 5(a) is shown, to illustrate the homogeneity of the spots. As can be seen from the figure, the protein spots are well-defined and well-separated, with no chlorine content present between the two spots, however, the thickness and evenness of the coating does vary to a certain extent. The spots are about 125 lm in diameter and separated by almost 100 lm of protein-free surface. Apart from chlorine, the spots contain small amounts of sulphur and zinc. 8. Summary and outlook A patterned cell substrate, suitable for irradiation experiments using microbeam irradiation or a well-collimated radioactive source, has been successfully developed using microdispensing equipment. The technique is uncomplicated – it can be carried out in a regular laboratory, no clean-room is required and no complicated techniques such as lithography need to be employed – and the preparation of one foil takes a few minutes. The technique allows for preparation of rectangular structures, but also arbitrarily shaped patterns can be manufactured depending on
one’s needs. Various sizes of the protein-coated surfaces, from spots with a diameter of 100 lm to larger areas on the order of 1 mm2, can be achieved to suit different purposes. Cells were shown to adhere to and grow well on the substrate in a satisfactory fashion. The protein coats in the cell dishes were shown to stable for a longer period of time than guaranteed by the manufacturer. These dishes are intended to be used in future microbeam irradiation experiments, in which low dose irradiation-induced bystander effects will be studied. These experiments will be facilitated by the fact that the cells growing in separated islands under no circumstances can come in physical contact with each other. Another appealing idea would be to use this island structure to mix different cell types on different islands, e.g. irradiate one cell type and try to find out if the overall behaviour, or response, in the cell dish depends on the mixture. References [1] C.S. Chen, M. Mrksich, S. Huang, G.M. Whitesides, D.E. Ingber, Micropatterned surfaces for control of cell shape, position and function, Biotechnol. Prog. 14 (1998) 356. [2] S. Gerardi, G. Galeazzi, R. Cherubini, Single-ion microbeam as a tool for lowdose radiation effects investigations, J. Phys.: Conf. Ser. 41 (2006) 282. [3] K.M. Prise, M. Folkard, B.D. Michael, A review of the bystander effect and its implications for low-dose exposure, Radiat. Protect. Dosim. 104 (2003) 347. [4] C. Mothersill, C. Seymour, Medium from irradiated human epithelial cells but not human fibroblasts reduces the clonogenic survival of unirradiated cells, Int. J. Radiat. Biol. 71 (1997) 421. [5] S.A. Lorimore, M.A. Kadhim, D.A. Pocock, D. Papworth, D.L. Stevens, D.T. Goodhead, E.G. Wright, Chromosomal instability in the descendants of unirradiated surviving cells after alpha particle irradiation, Proc. Natl. Acad. Sci. USA 95 (10) (1998) 5730. [6] A. Ruiz, L. Ceriotti, L. Buzanska, M. Hasiwa, F. Bretagnol, G. Ceccone, D. Gilliland, H. Rauscher, S. Coecke, P. Colpo, F. Rossi, Controlled micropatterning of biomolecules for cell culturing, Microelectron. Eng. 84 (2007) 1733. [7] H.W. Lu, Q.H. Lu, W.T. Chen, H.J. Xu, J. Yin, Cell culturing on nanogrooved polystyrene petri dish induced by ultraviolet laser irradiation, Mater. Lett. 58 (2003) 29. [8] H. Tsuji, H. Satoh, S. Ikeda, S. Ikemura, Y. Gotoh, J. Ishikawa, Negative-ion beam surface modification of tissue-culture polystyrene dishes for changing hydrophilic and cell-attachment properties, Nucl. Instr. and Meth. B 148 (1999) 1136. [9] N. Arteaga-Marrero, V. Auzelyte, M.G. Olsson, J. Pallon, A SU-8 dish for cell irradiation, Nucl. Instr. and Meth. B 263 (2007) 523. [10] R. Ugenskiene, J. Lekki, W. Polak, K.M. Prise, M. Folkard, O. Veselov, Z. Stachura, W.M. Kwiatek, M. Zazula, J. Stachura, Double strand break formation as a response to X-ray and targeted proton-irradiation, Nucl. Instr. and Meth. B 260 (2007) 159. [11] M. Folkard, K.M. Prise, A.G. Michette, B. Vojnivic, The use of radiation microbeams to investigate the bystander effect in cells and tissues, Nucl. Instr. and Meth. A 580 (2007) 446. [12] Ph. Barberet, A. Balana, S. Incerti, C. Michelet-Habchi, Ph. Moretto, Th. Pouthier, Development of a focused charged particle microbeam for the irradiation of individual cells, Rev. Sci. Instr. 76 (2005) 015101. [13] BD Biosciences Europe, URL:
E.J.C. Nilsson et al. / Nuclear Instruments and Methods in Physics Research B 267 (2009) 1199–1205 [22] J. Nilsson, J. Bergkvist, T. Laurell, Optimization of the droplet formation in a piezo-electric flow-through microdispenser, in: A. VandenBerg, P. Bergveld, W. Olthuis (Eds.), Micro Total Analysis Systems 2000, Proceedings, Mesa Monographs, 2000, p. 75. [23] P. Gustavsson, F. Johansson, M. Kanje, L. Wallman, C. Eriksson Linsmeier, Neurite guidance on protein micropatterns generated by a piezoelectric microdispenser, Biomaterials 28 (2007) 1141.
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[24] S. Gaspar, M. Mosbach, L. Wallman, T. Laurell, E. Csoregi, W. Schuhmann, A method for the design and study of enzyme microstructures formed by means of a flow-through microdispenser, Anal. Chem. 73 (2001) 4254. [25] BD Cell-TakTM Cell and Tissue Adhesive – Instructions for Use, BD Biosciences, URL: