Cell differentiation by mechanical stress

Questions: 1. Compare and contrast primary cells and cell lines. In your answer, make sure to address the following points? 2. Describe how cell lines are maintained in the lab. In your answer, make sure to address the following points? 3. Describe the process of differentiation. In your answer make sure to address the following points? Answers: 1. a. The preparation of primary cells for cell culture. i. The organ is isolated and selected (Stacey, Doyle, Alan, Ferro, Margherita, 2002).ii. The organ is dissected to remove fat and necrotic cellsiii. The cells are subjected to enzymatic degradation (Stacey et al., 2002).iv. Trypsin or collagenase is addedv. Re-suspend and seed the medium Conditions needed for cell to survive include; gas mixture and temperature of 5% CO2 and 37 degrees Celsius respectively, appropriate PH, glucose and nutrients (Stacey et al., 2002). Advantages of primary cells over in vivo i. Use of primary cell is cost effective ("Primary Cell Culture | Sigma-Aldrich," n.d.). ii. Primary cells use avoids ethical objections. Disadvantages of primary cells i. Primary cells take longer to grow ii. Culture and isolations costs for primary cells are prohibitive and high c. Cell lines originate from a cell culture made from a single cell. The main distinguishing features of cell lines from primary cells include, cell lines have minimal variability while primary cells have variability existing between preparations and the donor cells ("Primary Cell Culture | Sigma-Aldrich," n.d.). d. Advantages of using cell lines over primary cells in experimentation i. Cell lines take less time to grow unlike primary cells ii. Cell lines have minimal variability unlike in primary cells. Disadvantages of using cell line over primary cells in experimentations i. They provide less relevant results as compared to primary cells ("Primary Cell Culture | Sigma-Aldrich," n.d.). 2. i. Confluence is the approximate number of the adherent cells in the flask or dish of the cell culture (Mather, 2008). ii. The procedure of cell line passaging starts with the removal of the medium of the HEK 293 cell flask completely (Masters, Stacey, 2007). This is then followed by addition of 3ml trypsin to the medium. The next step is placing the flask containing the medium to an incubator at a temperature of 37 degrees Celsius for 5 minutes (Masters, Stacey, 2007). Then rock the flask and wash down the cells by addition of medium and pipette to separate cell clusters into single cell lines. Add desirable volume of re-suspended cell mix into new plates. Finally, shake the plates or the flask in gentle manner to allow proper mixing then place them back to CO2 incubator at temperature of 37 degrees Celsius (Masters, Stacey, 2007).Passing cell lines helps to evade senescence. Passaging a cell line too many times results in cells having little in common with original reference strain. Preparation of cell lines for cryopreservation i. Harvesting cells in the log growth phase (Stacey, Hawkins, Fleck, 2011).ii. Bring both the semi-adherent and adherent cells into suspension by the use of EDTAiii. Remove a small aliquot of the cells and do cell quantification iv. Perform centrifugation on the remaining culture for approximately 5 minutesv. Re-suspend the cells in the freeze medium vi. Then pipette one aliquot of the cells vii. Place the ampoules in a freezer whose rate is controlled viii. The medium is then shifted to a freezer for cryopreservation 3. i. Differentiation is a process that happens in multicellular organisms where a cell with less specialization matures so that it becomes more discrete in its functions and form (Altman, 2001). The major importance of differentiation in multicellular organisms is that it provides them with a variety of cell specialized in performing specific functions. ii. Somatic cells differentiation starts with the chromatin for both plants and animals. Chromatin reorganization is the first steps that occurs which in turn lead to activation of the pluripotent genes including Oct3/4 in animals and NAM in plants, establishing pluripotentiality (Nakagawa, M., Yamanaka, 2010). There are a variety of activities that lead to chromatin modelling induced pluripotentiality that may include activity of the thrombin in serum, cell wall removal and viral infections. Basically, the presence of these stimuli signals the cells to differentiate thus that are how the cells know when to differentiate. iii. Stem cells have the following characteristics: Ability to divide and renew themselves Basic and unspecialized cells (Miki et al., 2005). Give rise to specialized cells through differentiation (Mather, 2008). According to Baker (2008) stem cells are appealing to the medical community because of their therapeutic potential. References Altman,G.H. (2001). Cell differentiation by mechanical stress. The FASEB Journal. doi:10.1096/fj.01-0656fje Baker,M. (2008). Cancer and stem cells: Beckman conference. Nature Reports Stem Cells. doi:10.1038/stemcells.2008.47 Masters,J.R., Stacey,G.N. (2007). Changing medium and passaging cell lines. Nature Protocols, 2(9), 2276-2284. doi:10.1038/nprot.2007.319 Mather,J.P. (2008). Stem cell culture. London: Elsevier/Academic Press. Miki,T., Lehmann,T., Cai,H., Stolz,D.B., Strom,S.C. (2005). Stem Cell Characteristics of Amniotic Epithelial Cells. Stem Cells, 23(10), 1549-1559. doi:10.1634/stemcells.2004-0357 Nakagawa,M., Yamanaka,S. (2010). Reprogramming of Somatic Cells to Pluripotency. Advances in Experimental Medicine and Biology, 215-224. doi:10.1007/978-1-4419-7037-4_14 Primary Cell Culture | Sigma-Aldrich. (n.d.). Retrieved from https://www.sigmaaldrich.com/technical-documents/articles/biology/primary-cell-culture.html Stacey,G., Doyle, Alan, Ferro, Margherita. (2002). Cell Culture Methods for in Vitro Toxicology. Springer Verlag. Stacey,G.N., Hawkins,R., Fleck,R.A. (2011). Cryopreservation and Banking of Cell Lines. Animal Cell Culture, 185-203. doi:10.1002/9780470669815.ch6