Factors that Affect Cell Spreading of B16F1 cells using Poly-L-Lysine and Laminin

Kelsey Matika Group 5 March 18, 2013

Abstract Cell adhesion is how well a cell can bind to the surface of a cell, the extracellular matrix, or other cells. They are able to do this with the use of selectins, cadherions, and more importantly integrins. These are transmembrane receptors that ultimately link the cell to the extracellular matrix or the basement membrane by cellular adhesions. They are made up of an alpha and beta subunits and are activated by a ligand binding on the inside or the outside of the membrane and causing a response on the opposite side (Alberts et al., 2008). B16F1 melanoma cancer cells from mice were used to study how well cells are able to spread on different substrates and what factors affect the cells ability to spread and survive. Two experiments were done on the B16F1 cells. They were cultured in the tissue culture room using PBS, culture medium, and trypsin. The culture medium contains sources of sugars, salts, a buffer for pH, and specific amino acids. Trypsin was used to cut the cell attachments and leave round cells for the use of the experiment. B16F1 were counted using a hemocytometer and then plated on 35mm dishes with coverslips that had been submerged in laminin or poly-L-lysine (PLL). The cells were incubated at 37C for either 30 minutes or 120 minutes respectively. They were then fixed using 4% formaldehyde and rinsed with PBS, and viewed under a microscope to determine the percent spread of cells on each substrate. The amount of spread cells on the laminin increased as the incubation time increased in both experiments from 21% to 59% and 61% to 93%. PLL was the opposite though with the percent spread decreasing in between 30 minutes and 120 minutes. The numbers showed 66% to 21% and 56% to 39% respectively (Figures 1 and 2). Laminin and Poly-L-Lysine are both proteins that create different effects on cells and their ability to attach and spread. When laminin is present in high concentrations and is glycosylated the cells will spread better than if they are present in low concentrations. However, when laminin is

unglycosylated the cells will adhere to the surface but will not be able to spread due to the lack of a necessary saccharide group. Poly-L-Lysine was the opposite of laminin, when it is present in low concentrations the cells are able to spread better, however as the amount of PLL increases the levels become toxic and inhibited the cells ability to spread. This shows how different types of proteins can cause the cell to spread differently, which in the end effects whether or not the cell will be able to survive. Results The B16F1 cells were mounted on slides and counted across at least 10 different fields for approximately 100 cells overall, they were observed on the basis of spread versus not spread. The spread cells had a greyish appearance, they were elongated instead of round and some had visible lamellipodia. In general, they are thinner than the non-spread cells. The non-spread were round and seemed to radiate, meaning they had a bright appearance rather than grey. They were smaller than the spread cells and no lamellipodia was visible. The cells were placed on laminin and PLL coated coverslips. After 30 minutes the cells on the laminin were counted and the first experiment was found to have 23 spread, 84 not spread giving a total of 107 cells and 21% spread (Figure 1). The second experiment was 63 spread, 40 not spread for a total cell count of 103 cells giving the result of 61% spread (Figure 2). The PLL at 30 minutes had 66 spread and 33 not spread for a total of 99 cells and 66% spread (Figure 1). No cells were present on the original slide so data was taken from group 2.The second time there were 58 spread and 46 not spread for a total of 104 cells and 56% spread (Figure 2). However, the cells on the slide were hard to differentiate even when the microscope was in focus, it was determined that most of the not spread cells were very blurry since they did not radiate as well as the other cells.

The same procedure was used to count the cells at 120 minutes. The cells on the laminin for the first experiment were 98 spread and 7 not spread for a total of 105 cells giving 93% spread (Figure 1). In the second experiment 70 were spread and 49 were not spread for 119 cells overall and 59% spread (Figure 2). PLL for the first experiment was 24 spread and 77 not spread, giving 101 cells and 21% spread (Figure 1). The second experiment had 42 spread and 66 not spread giving 108 cells and 39% spread (Figure 2). Overall, the cells on the laminin and the PLL coverslips were consistent at each time point for their own comparison. However, The laminin had more spread at the 120 minute mark than it did at the 30 minute mark in both experiments (Figures 1 and 2). This trend was the opposite for the PLL which had many more spread cells at the 30 minute mark than at the 120 minute mark. This held true for both experiments as well (Figures 1 and 2). Discussion There are many different factors that affect cell adhesion and then the ability the cell has to spread, two proteins, PLL and laminin were used to view this phenomenon. Poly-L-Lysine is a synthetic positively charged amino acid chain that is water-soluble and can be used to promote cell attachments in specific cell types. It is generally seen in the adhesion of plastics and glass, because it can proliferate and spread easily. Certain cell types can secrete proteases that digest PLL and decrease its ability to attach (Poly-L-Lysine, 2011). This is evidence to show why there were less spread cells in B16F1 because their proteases start to degrade the interactions that occur between the cell and the substrate. Also, in an experiment using mesenchymal stem cells along with the use of specific watersoluble polymers like PLL to determine how well cells attach to different concentrations based off of the three different polymers specific properties. The results for PLL showed that the higher the concentration the more rounded the cells were and the lower the concentration the more spread the cells become. The concentrations of 10ug/ml were used for the more highly concentrated cells and

1ug/ml for the less concentrated cells (Chen et al., 2009). The evidence from this experiment supports the results using B16F1 cells. Figures 1 and 2 both show that as the incubation time for PLL increases the less spread the cells become. At 30 minutes there is a lower concentration of PLL allowing the cells to better attach to the surface and spread. While at the 120 minute mark the concentration of PLL has increased causing the cells to remain round or not spread. Laminin is an extracellular matrix protein that makes a heterotrimer. It is the major protein that is found in the basal lamina, which makes the basement membrane. It plays a huge factor in cell differentiation, cell migration and more prominently cell adhesion (Alberts et al., 2008). Laminin can come in many forms; a major distinction between all of the forms is whether it is glycosylated or unglycosylated. In an experiment dealing with how cells spread across different surfaces, kifunensinederived laminin in mouse melanoma cells was used. The differing factor was whether the laminin was glycosylated or not because the cells that survived had the same amount present, but on glycosylated laminin the cells spread which was unapparent on the unglycosylated laminin. The experimenters went further by then testing if concentrations on unglycosylated made the cells spread, but once again they did not. This proved that saccharides play an important role in cell spreading and need to be present on laminin in order to allow the cell to spread (Chandasekaran et al., 1993). Another factor that plays a role in the spreading on laminin is the concentration. Experimenters He, Ito, Kosaka, Maruyama, Sogo, Wang, and Ye tested a laminin-apatite versus an albumin-apatite on titanium using a supersaturated calcium phosphate solution. As the concentration increased of laminin so did the cells and their ability to spread. On the albumin-apatite composite the cells spread but not as well or efficiently as in the laminin (2013). Both these experiments support the results with the B16F1 cells because as the incubation time increased the more spread cells appeared; also it shows that the laminin must be glycosylated which allows the cells to spread in the first place.

In conclusion, PLL and laminin are both proteins that affect the way the cell adheres to surfaces and then spreads. However, both can cause very different responses and in the long run affect how well the cells attach to the surface or the extracellular matrix and whether or not they are going to spread. This is true for all factors involved in cell spreading, making the cell a very complex organism to move and get the jobs it needs done.

Tables and Figures Figure 1

Cell Adhesion Experiment A

120 min, 59%
Lamin

30 min, 21%

120 min, 24%

PLL

30 min, 66%

0%

10%

20%

30%

40%

50%

60%

70%

Percentage Attached

Figure 2

Cell Adhesion Experiment B

120 min, 93%
Lamin

30 min, 61%

120 min, 39%

PLL

30 min, 56%

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

Percentage Attached

Works Cited Advanced Biomatrix. (2011). Poly-L-Lysine. Retrieved from http://www.purecol.nu/dfu_Poly-L Lysine.pdf. Alberts, B. et al.. 2008. Moleculat Biology of the Cell, 5th Edition. Garland Science, New York. Chandrasekaran, S., Giniger, M.S., Tanzer, M.L. 1994. Oligomannosides Initiate Cell Spreading of Laminin-adherent Murine Melanoma Cells. The Journal of Biological Chemistry. 269: 3356-3366. Chen, G., Guo, L., Kawazoe, N., Lu, H., and Tateishi, T. 2009. Effects of poly (L-lysine), poly (acrylic acid) and poly (ethylene glycol) on the Adhesion, Proliferation and Chondrogenic Differentiation of Human Mesenchymal Stem Cells. Journal of Biomaterial Science: Polymer Edition. 20: 577589. He, F., Ito, A., Kosaku, R., Maruyama, O., Sogo, A., Wang, X., Ye, J. 2013. Improvement in Endothelial Cell Adhesion and Retention Under Physiological Shear Stress Using a Laminin-apatite Composite Layer on Titanium. Journal of Royal Society/ Interface. 10.