Versatile And Multiplex Cell Migration Experiment: Patterns And Processes

Discuss about the Versatile and Multiplex Cell Migration.

The wound healing experiments are important in the determination of the patterns in which the cells migrate and interact with one another. When there is a wound, the monolayers of the cells respond to the cell to cell contact disruptions (Elong et al.,2014). This leads to an increase in the number as well as the concentration of the growth factors on the margins of the wound. This process involves the migration of the cells which finally results in the complete healing of the wound. This process therefore demonstrates the manner in which individual as well as layers of cells behave, following a damage to the tissue (Wolf et al., 2013). In this experiment, a healing assay was conducted in an effort to determine the patterns of migration of cells (Gibbs et al., 2013) . The scratch assay is therefore intended to study the process via which re-epithelialization of tissues occur invitro.

Confluent PC-3 epithelial cells were grown in a three culture dishes  until they reached confluence (100%).

Using a 5ml pipet, a continuous scratch was made on the line of cells in order to mimic the development of a wound.

Each culture dish was exposed to varying treatment conditions to determine the roles played by microtubules and microfilaments in wound healing.

25ml of dimethylsulfoxide solution was put in the culture media containing the PC- 3 cells to act as the control of this experiment.

Before the Paclitaxel and cytochalasin D were added to the cells, DMSO was added to them.

Paclitaxel was added to one of the culture dishes at a concentration of 100nM.to the third culture dish, cytochalasin D was added at a concentration of 100nM

Upon the establishment of the three treatment conditions, the culture dishes were then incubated for 48 hours to give time of the re-epithelialization to take place. After incubation, each of the culture dishes was fixed using 10% neutral and buffered formalin for preservation purposes.

Each of the fixed culture dishes was stained by use of eosin and hematoxylin stains.

Re-epithelialization of the wound was monitored over time by observing the migration of the cells.

This was measured by measuring the change in the thickness of the wound from the time a wound was introduced to the ultimate healing.

The total change in the thickness of the wound was calculated against time.

Observations

The organization of the microfilaments and was also determined by exposing the cells to varied conditions.

To achieve this, the f-actin was labelled fluorosly by use of FITC- conjugated phalloidin.

The network of the microfilaments was then observed by use of the fluorescence microscope.

It was observed that as time passed by, the epithelial cells within the margin of the wound migrated into the area of the wound. This was in an effort to replace the cells that had been scratched away as a result of tissue injury. Moreover, it was observed that the cells that remained in the margin of the wound underwent increased proliferation to replace the cells that had migrated to the wounded area of the tissues. With time, the thickness of the injured area decreased.

The addition of dimethyl sulfoxide as a control in this assay was in order to account for the possibility of any nonspecific effect of this solution on the wound healing. Paclitaxel was added to a different culture dish in order to stabilize the microtubules in the cells by interfering with the division and migration of cells (Cai et al., 2014). The addition of cytochalasin D was in order to find out the roles played by microfilaments in healing of wounds. This is because cytochalasin D blocks the reorganization of microfilaments and hence it affects proliferation and cell migration in a negative way (Kamimura et al., 2015). This experiment or assay clearly gives an indication of the way in which the cells behave upon injury. The healing of the wound occurred in a stereotyped way, that is, the cells polarized in a negative way to the wound area. The polarized cells then initiated the protrusion and migration and finally the wound became closed. The time based microscopy provides an efficient tool via which the events of cell migration and healing of the wound occurs in a sequential manner. Some of the practical examples of this assay are the proliferation of cells and deposition of the extracellular matrix, and an increase in inflammatory cells. In another approach, this assay is important in explaining the way in which a disease is distributed in the body from an initial infected organ through metastasis.  The PC-3 cells were stained in order to increase the contrast during observation under the fluorescent microscope.it is therefore important to consider the methods of staining because some methods can damage the cells being studied. Some staining methods require the dyes to penetrate deep into the cell, something which is not possible in live cells. In order to curb this and make it important to store the stained cells for future reference, fixation, which involves killing of the cells while maintaining their structure and composition is carried out before staining (Vasicova et al., 2016). Fixation can be done by use of the chemicals like the buffered solution containing formaldehyde. This chemical is known to maintain the integrity and structure of organelles and structure of the cells while at the same time blocking the decomposition of cells by enzymes. Since this chemical is carcinogenic, it needs to be handled with great caution (Kuzmin et al., 2014). The physical methods of fixation involves either drying of freezing of cells. The choice of the fixation methods to be used depends on a number of factors such as types of cells being used and the intention of the experimental set up. In most cases however, the formaldehyde is used for fixation purposes. The preservation of cells using this chemical is made possible through a process which forms cross bridges between the nucleic acids and the amines on the proteins (Andreeva et al., 2016). The eosin and hematolxylin were used for staining purposes to be able to observe the cells in details. In the oxidized state, the hematolxylin stain is blue or purple in color and easily binds to the nucleic acids in the nucleus. On the other hand, since the eosin stain is negatively charged, it gets attracted to the amino acids which are negatively charged and found in the cytoplasm and stains pink color. Cell migration in this assay is likely to be inhibited by several cell processes such as the inhibitors of metabolism or translation of proteins (to form amino acids).

Staining Methods

This assay is therefore important in the determination of the molecules that can be able to inhibit the migration of cells in a wounded area. For instance, if there is an inhibition by the chemical, cytochalasin D, then is possible to get the inhibitors of cell migration.

The cell migration refers to an important process which is critical for the development and maintenance of cells in organisms it is important especially during immune responses upon injury, embryonic responses and healing of wounded areas. If an error occurs in the process of cell migration, then there is a possibility of series effects occurring which include development of tumors, intellectual capability and metastasis. When this process is clearly understood, it can be important for the development of novel drugs for instance in the control of invasive cancers.

The eukaryotic cell cycle is a series of growth and development which enables cells to move from the birth until they form the mother cell which eventually forms the daughter cells.  The stages of cell cycle include the interphase, cytokinesis and mitosis.in the interphase, cells grow in size in G1 phase, synthesize new copies of DNA in S phase and synthesizes organelles and proteins in the G 2 phase. During the mitosis stage, the nuclear DNA condenses to form chromosomes, pulled apart to form spindles (composed of microtubules) in prophase, metaphase, anaphase and telophase phases (Yanagida & Pines, 2015). During cytokinesis stage, the cytoplasm of the cell splits into two, forming two daughter cells from one parent cell. In cell division, the microtubules controlled the movement of the chromosomes while the microfilaments forms a contracting ring which splits during cytokinesis to form two daughter cells (Akhshi et al., 2014).

References

Akhshi, T. K., Wernike, D., & Piekny, A. (2014). Microtubules and actin crosstalk in cell migration and division. Cytoskeleton, 71(1), 1-23.

Andreeva, N. V., Leonova, O. G., Popenko, V. I., & Belyavsky, A. V. (2016). Controlled formaldehyde fixation of fibronectin layers for expansion of mesenchymal stem cells. Analytical Biochemistry, 514, 38-41.

Cai, D., Chen, S. C., Prasad, M., He, L., Wang, X., Choesmel-Cadamuro, V., & Montell, D. J. (2014). Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration. Cell, 157(5), 1146-1159.

Elong Edimo, W. S., Derua, R., Janssens, V., Vanderwinden, J. M., Waelkens, E., & Erneux, C. (2014). SHIP2 controls focal adhesion size and affects cell migration in a glioblastoma cell model.

Gibbs, S., Spiekstra, S., Corsini, E., McLeod, J., & Reinders, J. (2013). Dendritic cell migration assay: a potential prediction model for identification of contact allergens. Toxicology in vitro, 27(3), 1170-1179.

Kamimura, M., Scheideler, O., Shimizu, Y., Yamamoto, S., Yamaguchi, K., & Nakanishi, J. (2015). Facile preparation of a photoactivatable surface on a 96-well plate: a versatile and multiplex cell migration assay platform. Physical Chemistry Chemical Physics, 17(21), 14159-14167.

Kuzmin, A. N., Pliss, A., & Prasad, P. N. (2014). Changes in biomolecular profile in a single nucleolus during cell fixation. Analytical chemistry, 86(21), 10909-10916.

Vasicova, P., Rinnerthaler, M., Haskova, D., Novakova, L., Malcova, I., Breitenbach, M., & Hasek, J. (2016). Formaldehyde fixation is detrimental to actin cables in glucose-depleted S. cerevisiae cells. Microbial Cell, 3(5), 206-214.

Wolf, K., Te Lindert, M., Krause, M., Alexander, S., Te Riet, J., Willis, A. L. & Friedl, P. (2013). Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J Cell Biol, 201(7), 1069-1084.

Yanagida, M., & Pines, J. (Eds.). (2015). Mitosis. Cold Spring Harbor Laboratory Press.