Praktikum III: Experiment B

Transcription

1 Praktikum III: Experiment B Cell Patterning Using Micro Contact Printing Fall Term 2009 Amanda Hüsler, Katja Fröhlich, Philippe Knüsel, Schwarzenberger Michael Participants: Amanda Hüsler, Katja Fröhlich, Philippe Knüsel, Schwarzenberger Michael Date: Assistant: huesleam@student.ethz.ch frkatja@student.ethz.ch pknuesel@student.ethz.ch michschw@student.ethz.ch Ang Li, Isabel Gerber

2 Abstract The main goal of this experiment was to understand how proteins interact with surfaces and how cells interact with proteins. First, a substrate was treated with a polymer and a protein in order to create a protein-patterning to which cells could adhere. Secondly cells were seeded onto the pattern. After staining the cell nuclei, the cells and the patterns were analyzed under the fluorescence microscope. There we could see pattern and distinguish the cell nuclei and plasma. The images show that the cells didn t spread everywhere. Nonetheless, the cell patterning was not successful because distribution is similar to the one in the control sample. 1. Introduction 1.1 Goals This experiment gives a short introduction to proteins, cells and extra cellular matrix (ECM). Typical surface modification strategies were shown for better understanding of the interactions of proteins with surfaces. The main goal was to produce a pattern with an extracellular matrix protein and to investigate the cell adhesion to this pattern. A basic introduction to cell culture techniques and safety aspects defined the second part of the experiment. 1.2 Theory Proteins are based on 20 different types of amino acids which are linked to each other by peptide bonds. Some proteins tend to deposit on synthetic surfaces. One of them is fibronectin (Fn). Fibronectin is an essential protein which can exist in soluble as well as insoluble form. Fn can only be assembled into fibrils on the surface of cells. It is responsible for linking other proteins to the cell. Bacteria and cells locate well on fibronectin. Poly-(ethylene glycol) (PEG) illustrates the contrary because of its bad adsorption of cells and bacteria. After adding cells to a matrix of Fn and PEG, areas of adhering and of non-adhering cells can be found under the microscope. 1/12

3 2. Materials und Methods 2.1 Micro contact printing The first step (A), producing a master with a pattern by photolithography, was already prepared by our assistant. Our task was to produce a substrate on which the cells would adhere on certain places, but not on others. Before exposing to a fibronectin solution (B), the PDMS master was cleaned first with a nitrogen jet and afterwards by treating it with air plasma (Harrick Scientific Corporation, pressure 0.1 mbar) for 30 sec in order to remove dust and assure a hydrophilic surface. The incubation we used was made of a fluorescently labeled fibronection and had a concentration of 50 µg/ml. The master was exposed for one hour and then dried with a nitrogen jet (C). While the master was treated with the fibronection solution, we also treated the substrate with the air plasma cleaner for 30 sec (0.1 mbar). The stamp was then placed on the substrate. We put a 5 gram weight on the stamp for 30 sec (D). Afterwards, the stamp was removed and the substrate was treated with a PLL-g-PEG solution (0.1 mg/ml) for one hour (F) and then rinsed (G). Before using the substrate, we checked the quality of the pattern without PLL-g-PEG incubation with a fluorence microscope. A cell solution was then placed on the substrate and put into the incubator. As the cells can adhere on the fibronectin surface but not on the PLL-g-PEG, we expected the same pattern of cells that we printed with the stamp (H). This was examed with a microscope at the end of the experiment. Fig. 1: Overview of the different steps of the microcontact printing process. 2/12

4 2.2 Cell experiments Because of the biological safety aspects, all work had to be done in Class 2 safety cabinets. All waste had to be collected in special bottles and boxes and autoclaved afterwards. We did not do all experiments but the 105 cells/ml with BSA. For the cell cultures, we first had to rinse the 25 cm2 flask about 2 to 3 times with 5 ml PBS. Then 1.5 ml 0.25% trypsin-edta was added and then the cells had been incubated for 5 min at 37 C in the incubator. Afterwards, 3 ml of α-mem supplemented with 10% FCS (α 10) was added to the solution and gently pipetted for 5 times up and down. So the cell suspension was mixed and then transferred into a 50 ml centrifuge tube. It was centrifuged for 5 min at 1100 rpm. After this procedure, a small pellet at the bottom of the tube was visible. This consisted of cells and the supernatant was aspirated. The cells were resuspended in 5 ml α10. This solution was centrifuged again and 5 ml of α-mem supplemented with 0.1 % BSA was added. This suspension was gently mixed with pipetting up and down 5 times. It had to be paid attention not to produce foam. Now 50 µl of the cell suspension could be mixed with 50 µl trypanblue in a 1.5 ml Eppendorf tube. So the dead cells had a blue color. After cleaning the haemocytometer and positioning a small glass plate on the little crosses on the surface, we could fill the blue cell suspension in the clearance between plate and haemocytometer. Now the cells could be counted under a microscope. For that, we had to examine 4 fields or till we had about 100 counted cells. The dead ones mustn t be counted. With the result, the cell number was calculated: # 2 10 =# Now we had to dilute our solution with corresponding medium down to 10 5 cells/ml. =1: We wanted to have 10 ml solution. Calculation of the part of the cell suspension: 10 = 10 = h So we transferred the calculated volume of the cell suspension in a little bottle and added the corresponding medium, until we had our 10 ml. The cells were incubated for 2.5 h at 5% CO2 and 37 C in the incubator. 3/12

5 Then the cell nuclei were stained with Hoechst by diluting the stock solution 1: ml per well was added. After 10 min they were washed with 2 ml HBSS. The cells were not fixed. After all this treatments, we were able to do the analysis by Fluorescence Microscopy. The different parts were differently colored: Red Blue: Green: cell membranes nuclei Fn matrix 4/12

6 3. Results 3.1 Mirco Contact Printing The pattern quality was examed by the fluorescence microscopy (fig. 2). Fig. 2: The fluorescently fibronectin (green) show the pattern of Fn 3.2 Cell Experiments Cell Culture Table 1 shows the number of the cells in 4 squares ( mm 2 ) on the haemocytometer grid with the calculated cell number. # cells in mm 2 cell number [cells/ml] Transferred suspension [ml] Amanda Katja Philippe Michael Tab. 1: number of cells in 4 squares on the haemocytometer grid and the calculated cell number. 5/12

7 3.2.2 Analysis by Fluorescence Microscopy The following figures 3-12 were made with the fluorescence microscope. They show three samples. The 5x and 20x objectives were used. The red colour identifies the cell membranes. The nuclei are blue. Fig. 3: phase contrast with 5x objective, sample 1 Fig. 4: fluorescence image with 5x objective, sample 1 6/12

8 Experiment B Fall Term 2009 Cell Patterning Fig. 5: phase contrast with 20x objective, sample 1 Fig. 6: fluorescence image with 20x objective, sample 1 7/12

9 Fig. 7: phase contrast with 20x objective, sample 2 Fig. 8: fluorescence image with 20x objective, sample 2 8/12

10 Fig. 9: phase contrast with 20x objective, sample 3 Fig. 10: fluorescence image with 20x objective, sample 3 9/12

11 Fig. 11: phase contrast with 20x objective, control group Fig. 12: fluorescence image with 20x objective, control group In order to make statements about the success of the patterning, the figures have to be compared to those of the control group. 10/12

12 When looking at figures 3 and 4, the cells seem to have adhered with a certain regularity. But the distribution is not as good as in figure 2. Figures 7 and 8 show that the cells did not adhere everywhere and that there is a patterning of the cells. By comparing the figures 9 and 10 to the control group (fig. 11 and 12) it can be seen that the distribution of the cells is similar. 11/12

13 4. Discussion 4.1 Micro Contact Printing The figure 2 shows that the pattern was successfully transferred to the glass substrate. The green islands are the pattern of Fn. The pattern is regularly ordered and shaped. 4.2 Cell Experiments Cell Culture All calculated cell numbers in table 1 are quite equal. Therefore the cell cultures grew almost identically. That is logical because they had all the same conditions Analysis by Fluorescence Microscopy The images of our samples are similar to the control images (fig. 11/12). Furthermore, the cell-patterning in our samples (fig. 3 and 4) is not very similar to the Fnpatterning in figure 2. This leads to the conclusion, that the something in the experiment went wrong for unknown reasons. 5. References Laboratory course instruction Cell Patterning Using Micro Contact Printing, Materials Science BSc, ETH Zurich 12/12