Printing of biochemically-patterned slides with the InnoStamp40 for deterministic cell immobilization.

Transcription

1 Printing of biochemically-patterned slides with the InnoStamp40 for deterministic cell immobilization. Jean christophe CAU 1 1 Innopsys, Parc activestre, Carbonne France contact@innopsys.fr Introduction Mastering the adhesion landscape of living cells on a surface is emerging as a new tool for investigating many fundamental mechanisms of cell biology such as shape control, differentiation, division, polarity or motility [1 12]. The common point between all these studies is the production of micro patterns of various shapes and dimensions along which adherent cells are immobilized in a deterministic way. The control of cell adhesion through this technology allows the biologists to design specific scenari and record how various living cell types (neural, epithelial, tumoral and stem cells) respond to them. Because this technology allows reproducing identical precise patterns along well arranged periodical arrays, these experimental observations can be made systematically over large population of cells thus reaching a high level of representativeness. The micro contact printing of proteins of the Extra Cellular Matrix (ECM) on conventional glass slides or coverslips turned out to be an efficient method for fabricating these micro patterned cell culture substrates. However, when this technological process is made manually, it fails to generate reproducible patterns over large surfaces thus limiting the actual potential of this new approach. In order to bring this process to a standard level in cell biology, we propose to automate the printing process of ECM proteins on cell culture substrates. As a matter of fact, a fine control of the stamping parameters is a sine qua none condition to be able to print in one step reproducible patterns of different shape, size and pitch. In this note, we present a dedicated study in which we screen different pattern shapes and pattern sizes for the deterministic immobilization of cancer cells. With the perspective in mind of remote production of these patterned cell culture supports, we have also investigated the conservation potential over time of the printed slides before cell seeding. We have used a patterned stamp designed with four different classes of features (disc, line, square, and triangle) of different sizes (from 10 to 50µm) and pitches (10 to 100µm). The samples were seeded with Prostate Cancer 3 (PC3) cells. Then, we have quantified the spreading of the immobilized cells on these different patterns and the selectivity of the immobilization on the adhesive patterns compared to the antifouling background. 1 Cellular growth on patterned samples fabricated with a fully automated microcontact printer: the InnoStamp40. The workflow of this study is based on three steps (figure 1). The first one is the fabrication of ECM (Extra Cellular Matrix) protein micro

2 patterns on glass slides. For that, we used the automatic microcontact printer named InnoStamp40. It is based on magnetic field assisted microcontact printing [13]. This technology allows for: Figure 1: the workflow of Cellular growth on patterned samples An automation of the full process of microcontact printing, A fine control of the pressure applied during the printing step, An alignment of the patterns on the sample, A high reproducibility of the process. Three elements are handled in the InnoStamp40 ( A PDMS stamp with the micropatterns, A sample (in this study: a glass slide), The ink (In this study, it is a 100µg/mL fibronectin solution with PBS 1X). This equipment automatically generates micropatterns of fibronectin on the glass slide according to the layout of figure 2. Four different shapes of patterns with five different sizes (from 10µm to 50µm) and four different gaps (from 10µm to 100µm) are printed and arranged into periodic arrays. Each shape array is repeated at least one time on a stamp. The second step is the backfilling of the fibronectin micro patterns with an antifouling layer (figure 1). For that, we incubated the printed sample in a 100µg/mL solution of Pll g PEG during 1h. After drying, the space between fibronectin patterns is filled with Pll g PEG. The third step is the PC3 GFP cells seeding (figure 1). The PC3 GFP cells are seeded during 3h at 37 C with a concentration of cells/cm². Then, they are washed with PBS and dehydrated with 3 baths of 50%, 75% and 100% of ethanol. The images are acquired with a standard fluorescence microscope.

3 more general manner, we observe that the higher the spatial confinement produced by the patterns is, the lower the selectivity is. Selectivity (inside/outside %) Triangle 75 Square 85 Round 91 Line 99 Figure 2: layout of the micropatterns. There are 4 different cells with different shapes: disc, line, square, and triangle. Their size varies between 10µm to 50µm and the gap between the patterns varies between 10 to 100µm. 2 Different shapes of the biochemical patterns. The cells are selectively captured on the micropatterned slides. We can observe on figure 3 that the PC3 GFP cells in adhesion with the surface of the sample decorate the printed fibronectin patterns. The PC3 GFP cells which are selectively immobilized on the molecular patterns spread on and fill them while the very few cells immobilized outside the patterns are circular and do not spread. We quantify the selectivity of the immobilization as the number of cells counted inside the printed patterns divided by the number of cells counted in the PLL g PEG background. Table 1 displays the selectivity observed for the four different shapes. We find a good selectivity for all the shapes (higher than 75%). With triangles, the selectivity is lower than the line patterns. In a Figure 3: fluorescence images of PC3 GFP cells immobilized on ECM micro patterns of various shapes (the patterns are depicted by dashed lines). 3 Quantification of the area of a cell trapped onto a biochemical pattern. After shape investigation, we studied the minimal surface needed to trap a single PC3 GFP cell and the area occupied by captured cells. To achieve that, we selected a region near the interface between two areas (figure 4). In order to distinguish and well identify the different areas (one with 50µm and the other one with 40µm triangle patterns), we have also printed a dashed line pattern composed of 20µm*10µm rectangles (violet arrow). We first estimated the mean area of the cells trapped into triangles by dividing the triangle

4 area by the number of trapped cell. We found a mean area of 420µm². The area of these triangles is 450µm², which is close to the 420µm² area found previously. Several cells can sometimes be observed on these patterns because the pattern area is a bit larger than 420µm² and also due to cell size heterogeneity. Put together, these results indicate that, for patterns of area close to 200µm 2, single cells can be deterministically immobilized but their shape is not constricted by the shape of the patterns, while for patterns of area close to 400 µm2, single cells are selectively immobilized and shaped by the printed patterns. Figure 4: A selected region at the interface between two areas. The upper part is made of 50µm triangles and the lower part is made of 40µm triangles. At the interface, rectangles of 20µm*10µm are printed (yellow dashed line). We can observe in figure 4 that single cells decorate the rectangular patterns of the printed dashed line. However, they do not follow exactly the contours of the rectangle but instead they overfill the patterns. The area of these patterns is 200µm². Compared to the cell trapped into triangles, we assume that a 200µm² rectangular area is enough to trap single cells but not enough to make the contours of the cell match the contours of the printed features. This is why the PC3 GFP cells on this kind of patterns exhibit circular shapes and not rectangular ones. The interplay between PC3 GFP cells and 200µm² rectangles of fibronectin enables to achieve a deterministic immobilization of single cells on the patterns but do not conform the cell shape to the contours of the pattern. The circular shape of these cells could also be demonstrated by the interplay of the protruding parts of the cells (outside of the patterns) with the antifouling layer. Single cells trapped on the 30µm triangles (figure 5) seem to be shaped by the patterns. Figure 5: fluorescence image of PC3 GFP cells immobilized on 30µm printed triangles (scale bar 40µm). 4 Aggregates of cells near the molecular patterns. We have also investigated the interplay between cells according to their positions into the patterns. We observe on figure 6 the cell spreading on lines of different widths: 10, 20 and 40µm. On 10µm lines (figure 6.A), the PC3 GFP cells spread on the entire length of the lines and create a pattern similar to a cell rosary. If there are more cells across, PC3 GFP cells reduce their spreading in order to cohabit within the line. Cells found outside the patterns exhibit circular shapes and they hang on to the spread ones. For 20µm and 40µm wide lines (figure 6.B and C), the cells interplay is similar. However, due to a lower spatial confinement, PC3 GFP cells are less stressed and, thus, can shape the lines with two or three cells across. On figure 7, PC3 GFP cells shape discs of 40µm diameter but some additional cells hang on to

5 them. We observe the same shape difference between cells inside (spread) and outside (circular) the patterns. This effect being visible for different shape and size patterns, we assume that it is due to some cell aggregates present in the seeding solution. A Figure 7: fluorescence images of PC3 GFP cells immobilized on 40µm circular patterns. Scale bar: 40µm. 5 Study of the patterned slides shelf time Figure 6: fluorescence images of PC3 GFP cells trapped immobilized on 10µm (A), 20µm (B) and 40µm (C) wide printed lines. Scale bar: 40µm. C B To conclude this study, we have investigated if these printed slides could be used days after the manufacturing process. Indeed, in order to evaluate this technology, it could be interesting to send to researchers some printed slides, raising the question: what is the shelf time of the printed slide?. To answer this question, we compared cell culture on a fresh printed slide and cell culture operated 12 days after printing in different conditions of conservation. The temperature in the conservation phase was changed (room temperature, 4 C or 20 C) and two media of conservation were tested (ambient air or PBS1X). We could not notice any difference across these conditions. This is why we present the results after 12 day conservation at room temperature, ambient air. We are now using these parameters to send some samples to interested customers.

6 A these patterns. Single cell arrays can be generated. The shape of these individual cells can be dictated by the shape of the patterns, if the area of each pattern is smaller than the mean area of a cell in adhesion on a nonspatially confined zone. To complete the study, we have characterized the conservation of the patterned slides before cell seeding and have proven that functional micro patterned slides are compatible with standard shipping. Acknowledgment B This work was done within the framework of a joint laboratory named BIOSOFT ( between LAAS CNRS and. Author wants to acknowledge Julie FONCY, Charline BLATCHE, Emmanuelle TREVISIOL and Christophe VIEU. References [1] McBeath R et al. Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell Apr;6(4): Figure 8: comparison between a fresh printed slide (A) and a 12 day stored patterned glass slide (B). On figure 8, we can observe no significant differences between freshly printed slides and conserved ones. The selectivity is better after 12 days but the number of cells per pattern is higher. This trend could be explained by a small variation in the cell concentration of the seeding solution. Conclusion Patterned slides with ECM proteins can be routinely and reproducibly produced using the InnoStamp40 microcontact printer. Adherent cells can be immobilized deterministically on [2] Tseng Q et al. Spatial organization of the extracellular matrix regulates cell cell junction positioning. Proc Natl Acad Sci U S A Jan 31;109(5): [3] Gao L et al. Stem cell shape regulates a chondrogenic versus myogenic fate through Rac1 and N cadherin. Stem Cells Mar 31;28(3): [4] Mrksich M et al. Using microcontact printing to pattern the attachment of mammalian cells to self assembled monolayers of alkanethiolates on transparent films of gold and silver. Exp Cell Res Sep 15;235(2): [5] Huang S et al. Shape dependent control of cell growth, differentiation, and apoptosis: switching between attractors in cell regulatory networks. Exp Cell Res Nov 25;261(1):

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