Homeostasis allows for cells to maintain a steady state and normal proliferation. Organisms have certain body temperatures at which their cells work best. For survival, cells must find all of the substances required for energy generation and cellular biosynthesis. When creating an artificial environment, the media is provides all essential nutrients in the solution for growth. The two medias used are DMEM and RPMI. Growth factors are proteins, which influence proliferation by naturally occurring regulatory molecules that bind to receptors on the cell. The growth factor used in this study is Fetal Bovine Serum. The present study is performed to observe the effects of RPMI, DMEM, temperature, and FBS concentrations on the growth of RAW 264.7 cells. The cells were treated in 1%, 5%, or 10% FBS at the temperatures 25°C, 37°C, or 40°C. The results are obtained by running the treated plates through the CytoFluor II reader. When observing the effects on the growth of RAW 264.7 cells it is seen that the greatest proliferation occurred when the RAW 264.7 cells were treated with DMEM at FBS concentration of 10% at 37°C. The lowest count was seen in the cells that were treated with RPMI at FBS concentration of 1% at 25°C.
Introduction:
Homeostasis is essential for all living organisms to maintain a healthy balance and normal proliferation and growth. Homeostasis is defined to be any process that living things use to actively maintain fairly stable conditions necessary for survival. (Rodolfo, n.d). Factors that correlate to homeostasis include, but are not limited to, temperature, blood sugar levels, and water content. Homeostasis is achieved by negative feedback mechanisms which help maintain the internal environment as stable as possible. (BBC, 2011). When changes to the normal factors are made, homeostasis is no longer at equilibrium and the results consequently affect the organism in various ways.
To maintain homeostasis, organisms must have certain body temperatures. Each organism requires a temperature growth range, which is determined by the heat sensitivity of its particular enzymes, membranes, ribosomes, and other cell components. For humans, the ideal body temperature at which enzymes work best is 37° C. When temperature is increased, enzymatic activity within the cell is also increased. When temperature is decreased, enzymatic activity is also decreased within the cell. The problem occurs if the temperature is increased or decreased too high. An increase too high of temperature would result in the enzymes and proteins degrading within the cell while a high decrease would result in the stop of enzymatic activity all together. If the temperature does not remain relatively constant, the proteins will denature or ice crystals can form resulting in the death of the cell. (Kelly, Lab #3, 2017).
Each organism must find in its environment all the substances required for energy generation and cellular biosynthesis in order to survive. The chemicals and elements of the environments that are utilized for cell growth are referred to as nutrients or nutritional requirements. When cells are grown in the laboratory, they are placed in a culture media, which is designed to provide all essential nutrients in solution for growth. (Todar, 2008). The two nutrients in media that were focused in the experiment were Dulbecco’s Modified Eagle Medium (DMEM) and Roswell Park Memorial Institute (RPMI). DMEM is unique because it contains four times the concentrations of amino acids and vitamins. DMEM can contain high or low concentrations of glucose and be made with or without sodium pyruvate. The DMEM used in this experiment has a high concentration of glucose and is made with sodium pyruvate. RPMI on the other hand, is unique because it contains the reducing agent, gluthione. RPMI also contains high concentrations of vitamins such as inositol, choline, biotin, B12, and PABA. In both medias, GlutaMax is supplemented into the media to minimize toxic ammonia build up to help cell growth and proliferation. Both DMEM and RPMI utilize a sodium bicarbonate buffer system, which requires 5-10% carbon dioxide environment to maintain physiological pH. (Kelly, Lab #3, 2017).
Growth factors are essentially proteins that influences growth and proliferation of the cell. They are naturally occurring regulatory molecules that bind to receptors on the surface of the cell. The primary goal of growth factors is to activate cellular proliferation and/or differentiation. They stimulate cell and tissue function through influencing cell differentiation by changing their biochemical activity and cellular growth, and regulating their rate of proliferation. (Kelly, Lab #3, 2017). Many growth factors are quite versatile, stimulating cellular division in numerous different cell types; while others are specific to a particular cell-type. (King, 2016). The growth factor used in this experiment is Fetal Bovine Serum (FBS). FBS is the most widely used growth supplement for cell cultures because of its high levels of growth stimulatory factors and low levels of growth inhibitory factors. (Zheng, 2006). FBS provides the necessary growth factors and provides other metabolic requirements to ensure cell survival.
The growth curve of cells shows the growth of a cell over time. There are four phases that influence the curve of a cell’s lifetime. The lag phase is when cells are acclimating to their new environments. Some cells may be proliferating while others are not but it is not yet at a detectable level. The log phase is exponential growth. The cells are now acclimated to the new environment and are proliferating at a high rate due to growth factors. The stationary phase is when there is equal growth and death among cells. The concentration of cells stays constant throughout this phase. The final phase, the death phase is when the cells begin to die at an exponential rate. The death can be due to the media no longer having nutrients, acidity due to waste, or many other factors. (Kelly, Lab #3, 2017).
The present study is performed to observe the effects that RPMI, DMEM, temperature, and FBS concentrations have on the growth of RAW 264.7 cells. The RAW 264.7 cells were treated in FBS concentrations of 1%, 5%, and 10% at the temperatures 25° C, 37° C, or 40° C. The cells were grown in either DMEM or RPMI media. Each of the factors that are being tested on the RAW 264.7 cells plays a crucial role in the maintenance of homeostasis throughout the cells. When altering what the cell is used to, imbalances occur and patterns and trends can arise giving justification to changes in its environment. It is expected that the most cell growth will occur at the 37° C temperature with 5% FBS because that is the normal quantities in order for the cells to maintain homeostasis. It is also expected that the media in which the cells are placed in will not play a large role in cell quantification because both will provide essential cell nutrients and regulations. When the temperature is dropped or increased, cell growth will be adversely affected regardless of the FBS concentration because the RAW 264.7 cells will no longer be in homeostasis. To analyze the data received, the consequent treated plates will be run through the CytoFluor II plate reader and fluorescent units and relative fluorescent units are obtained to quantify cell count and understand potential cell proliferation or death patterns and trends. (Kelly, Lab #3, 2017)
Methods and Materials:
RAW 264.7 cells are first counted and quantified. Next, cells at a concentration of 100,000 cells/mL are placed into six 24-count plate with the corresponding concentrations of FBS and incubated at 25° C, 37° C, and 40° C for 48 hours. After the 48 hours, the media is aspirated and monolayers are washed away using 1 mL of phosphate buffered saline (PBS) and aspirated. Then, 1 mL of OptiMEM is added for 24 hours. After the 24 hours, OptiMEM is then aspirated and monolayers are washed with 1 mL of PBS and aspirated. Next, the calcein-AM working solution must be made, per plate. For each well there must be 24 mL of OptiMEM and 24 uL of calcein-AM. Once the solution is made, add 1 mL of it and incubate at the corresponding temperature for 20 minutes. Once the 20 minutes have passed, aspirate the working solution, wash it with 1 mL of PBS, and aspirate completely. The plates should then be read on the CytoFluor II plate reader at an excitation of 485 nm, emission of 530 nm, and a gain of 70. (Kelly, Lab #3, 2017)
To analyze the data obtained through the CytoFluor II plate reader, the fluoresecent units and relative fluorescent units must be obtained. The fluorescent units are obtained by averaging the wells and the relative fluorescent units are obtained by subtracting the background from the averaged fluorescent units. The background fluorescence for a 24-well plate is 800. (Kelly, Lab#3, 2017)
Results:
Figure 1 – The figure above shows the quantification of cells during each temperature and concentration when affected with DMEM.
Figure 2 – The figure above shows the quantification of cells during each temperature and concentration when affected with RPMI
Figure 3 – The figure above shows the quantification of cells during each temperature and concentration when affected with RPMI.
Discussion:
The present study is performed to observe the effects that RPMI, DMEM, temperature, and FBS concentrations have on the growth of RAW 264.7 cells. The RAW 264.7 cells were treated in FBS concentrations of 1%, 5%, and 10% at the temperatures 25° C, 37° C, or 40° C. The cells are grown in either DMEM or RPMI media to see whether or not the media will affect cell quantification. Each of the factors that are being tested on the RAW 264.7 cells plays a crucial role in the maintenance of homeostasis throughout the cells. When altering what the cell is used to, imbalances occur and patterns and trends can arise giving justification to changes in its environment. It is expected that the most cell growth will occur at the 37° C temperature with 5% FBS because that is the normal quantities in order for the cells to maintain homeostasis. It is also expected that the media in which the cells are placed in will not play a large role in cell quantification because both will provide essential cell nutrients and regulations. When the temperature is dropped or increased, cell growth will be adversely affected regardless of the FBS concentration because the RAW 264.7 cells will no longer be in homeostasis. (Kelly, Lab #3, 2017).
To analyze the data received, the consequent treated plates will be run through the CytoFluor II plate reader and fluorescent units and relative fluorescent units are obtained to quantify cell count and understand potential cell proliferation or death patterns and trends. The results can be seen in Figures 1-3. Figure 1 compared the results of the cells, which were given DMEM, Figure 2 compared the results of the cells, which were given RPMI, and Figure 3 combined both graphs together to better analyze the comparisons and potential correlation between the two graphs. When looking at Figure 1, cell proliferation occurred best at 10% FBS at 37° C. The least amount of cell growth in Figure 1 occurred at 1% FBS at 25° C. Figure 2 shows the greatest cell proliferation at 10% FBS at 37° C and the least cell growth at 1% FBS at 25° C. When looking at Figure 3 and comparing both DMEM and RPMI results, the greatest cell proliferation occurred at DMEM 10% FBS at 37° C. The least cell growth occurred at RPMI 1% FBS at 25° C.
When considering the results from the three figures from above, it is seen that DMEM is the ideal media to present an artificial environment for the RAW 264.7 cells. DMEM differs from RPMI in because it contains four times the concentrations of amino acids and vitamins. DMEM can contain high or low concentrations of glucose and be made with or without sodium pyruvate. The DMEM used in this experiment has a high concentration of glucose and is made with sodium pyruvate. RPMI on the other hand, is unique because it contains the reducing agent, gluthione. RPMI also contains high concentrations of vitamins such as inositol, choline, biotin, B12, and PABA. (Kelly, Lab #3, 2017). Both medias provided the essential nutrients and physiological and environmental regulations in order for the cells to thrive but when considering quantification, it was greater when DMEM was used over RPMI. In a study, titled “Effects of DMEM and RPMI 1640 on the biological behavior of dog periosteum-derived cells” the effects of DMEM and RPMI on cell growth and cell differentiation of PDCs in vitro were studied. The results obtained showed that DMEM was the optimal growing media for the PDCs. DMEM was the better media because it induced alkaline phosphatase activity and strongly stimulated matrix mineralization in cell culture while RPMI did not. (Xiaohong, 2009). Although the cells used in this study were not the same, the correlation between DMEM being the better growing media due to alkaline phosphatase activity can be considered.
Temperature is a huge factor is homeostasis. Each organism must maintain a certain body temperature in order for the cells to function correctly. When temperature is increased, enzymatic activity also increases. When temperature decreases, enzymatic activity also decreases. Problems begin to occur when the temperature is either too high or too low. When the temperature is too high, degradation begins to occur due to high enzymatic activity. When the temperature is too low ice crystals begin to form causing the cells to die. (Kelly, Lab #3, 2017). It was expected that the cells would proliferate best at 37° C. When looking at Figures 1 and 2, it is seen that the highest points are all at when the temperature was set to be 37° C. When the temperature was brought down to 25° C, cell numbers were low. When the temperature was increased to 40° C, the numbers were also low. When comparing the two extremes, 25° C and 40° C, the cells did better when exposed to the warmer temperature than the cold one.
The final factor, in the influence of RAW 264.7 quantification is FBS concentrations. FBS is a growth factor, which is essentially a protein that influences growth and proliferation of the cell. FBS provides the necessary growth factors and provides other metabolic requirements to ensure cell survival. (Kelly, Lab #3, 2017). When looking at Figures 1 and 2, 10% FBS concentrations ensured the greatest number of cells in both DMEM and RPMI. The 10% FBS groupings did better in each temperature. The higher concentration of FBS ensured extra influences in growth and proliferation. It is understood that the greater the growth factor, the greater growth there will be. When FBS concentrations are at 10%, no harm occurs to the RAW 264.7 cells but it is important to consider at which point would the potential rift occur for cell count to decrease from overexposure to FBS. In a study done titled “Proteomic analysis for the assessment of different lots of fetal bovine serum as a raw material for cell culture,” the quality of different types of FBS solutions and the different concentrations was tested. The researches hypothesized that FBS had varying concentrations of proteins such as growth stimulatory factors, growth inhibitory factors, and/or other proteins that may correlate with cellular growth rate. It was found that time played a crucial role in the increase of protein concentrations in the FBS. Time also resulted in the degradation of additives in the FBS. The greater the concentration of FBS and the longer it is used, the greater growth will occur. (Zheng, 2006).
When observing the effects that RPMI, DMEM, temperature, and FBS concentrations have on the growth of RAW 264.7 cells it is seen that the greatest proliferation occurred when the RAW 264.7 cells were treated with DMEM at an FBS concentration of 10% at 37° C. The lowest cell count was seen in the cells that were treated with RPMI at an FBS concentration of 1% at 25° C. DMEM is found to be the better media than RPMI. Although results between the two medias were not abnormally large, they are significant enough to note that DMEM has greater cell quantification. The most successful FBS concentration was at 10% and the least successful concentration was at 1%. Temperature also provided significant results. The ideal temperature for growth for the RAW 264.7 cells was 37° C and the least ideal temperature was 25° C. Homeostasis is essential for all living organisms to maintain a healthy balance and normal proliferation and growth. Changes in the environment will change the homeostasis of the organism resulting in an imbalance that will cause consequent effects.