Preparing Cell Cultures of Human Embryonic Kidney Cells: Clone 293T Submitted by Devin Mollegard, Lab Partner: Megan Wydner 4 November 2015 Abstract Biology is the complex study of life which studies time scales of millions of years to small second by second changes. The one consistency in biology is the presence of cells. Cells are studied by biologists by growing cell cultures. Cell cultures serve to demonstrate how particular conditions can affect cell growth. The purpose of this study was to formulate a growth curve by culturing cells and counting their populations each day. While cells were the focus, a growth curve could potentially be established with any kind of population, which is why growth curves are so important to know how to make. The final growth curve ended up being of a similar shape to the standardized growth curve for HEK 293T. A few minor errors were made, but nothing that could not be accounted for during calculations. The largest impact to the growth curve was the inability to count cells for all 9 days. This created gaps in the data and the growth curve. Still, using the right techniques, the final product ended up being a completed growth curve. A growth curve serves the purpose of displaying the growth pattern of our cells. The cells were counted for nine days to create this graph. Introduction
Mollegard 2 Learning a particular science requires a great deal of attention to detail. In Biology, the smallest detail available to our observation is called the cell. Cells are the building blocks of all life. There is no life on earth that is not comprised of at least one cell. Therefore, every kind of biologist needs to know about cells and how they work. Many scientists work with different kinds of cells their whole lives. Studying cells has led to the production of medicines that can heal good cells, or chemicals that can destroy harmful ones. In this laboratory exercise, the goal was to create a cell growth curve. A cell growth curve is a graphical representation of the change in population of a cell, using time on the x axis in units of days, and population on the y axis in units of cells per dish (see Figure 1 ). Growth curves are useful, because we can test which conditions are optimal for a cell. We can also use growth curves to see how a particular drug or medicine affects certain cells. By establishing the typical growth curve for a specific kind of cell, there is a standard that future cultures can be compared to. This is especially useful to scientists, because cells are always being tested with various chemicals. Learning how to prepare a cell culture and create a growth curve is a necessary skill set for any aspiring biologist. It was the goal of our class to establish these skills by creating our own cell growth curve.
Mollegard 3 Materials and Methods For this experiment, we required access to a sterile laboratory hood. The aseptic technique was used by applying ethanol to wipe all surfaces and a small gas flame to burn the tip of the aspirator. Our cells were HEK 293T (Human Embryonic Kidney cells clone 293). These cells are immortal and also kept in a culture media. The culture media consisted of inorganic salts, amino acids, vitamins, glucose, glutamine, phenol red, which was used as a ph indicator, and 10% fetal bovine serum, which was used as a growth factor. The media also had a 50:50 ratio of Hams F10: Dulbecco s modified Eagle s medium. To begin, we observed our cells under a microscope to record confluence. Observing confluence was a step followed subsequently with every cell dish that was counted. Confluence is the estimated proportion of cells in a culture to empty space which is typically represented as a percentage (see Figure 2 in Results ). Next, cell dishes were aspirated such that all media was removed. The serum within the media partly consisted of some trypsin inhibitors. It was necessary to rinse the cell dish of these inhibitors by using phosphate buffered saline (PBS). A quantity of 2.0 5.0 ml of PBS sufficed to rinse the dishes accordingly. Using the aspirator, the PBS was removed. Note: The volume of PBS is not necessary to record each time, so long as there is enough to completely wash the dish of trypsin inhibitors. Following the removal of PBS, 2.0 5.0 ml of trypsin EDTA is added to the dish. Immediately, the trypsin was removed after being added to the dish. Cells are then incubated for 5 15 minutes. The trypsin serves to suspend the cells by breaking their bonds to the dish, and takes time to complete. Note: The volume of trypsin is not necessary to record each time, so
Mollegard 4 long as there is enough to rinse all cells in the dish. Note: Trypsin can be damaging to some cells, so minimal time between application and removal is recommended. Next, the cells were counted as follows. Only on Day 0, the dishes for the following days needed to be created, and so these next steps were carried out. A quantity of 11.0 ml of media was added to the dish. An automatic pipettor was used to mix up the cell suspension by pipetting the dish media up and down several times, with the cells still suspended in the media. This ensured that cells were spread equally throughout the dish. Next, 1.0 ml of the dish media was removed and added to an empty dish. This was repeated nine times such that ten new dishes containing 1.0 ml of dish media were produced, with the original dish now containing 1.0 ml. 9.0 ml of new media was added to each dish, and the new dishes were stored for incubation. The original dish remained for counting after being mixed. The new dishes would be removed on subsequent days for their own rinsed and counting. Finally, counting began by taking two test tubes of 20 microliters per dish by using a P20 micropipette. 20 microliters of trypan blue (0.4%) was added to each sample, so that the resulting proportion of media to dye was 1:1, or a dilution of 2X. From each tube, 9 microliters was removed using the P20 micropipette and inserted between a hemocytometer and its glass slide, at the notched area. The notch and the pressure of the glass slide allow the hemocytometer to spread the mixture via capillary action. Using a microscope and a hand tally counter, cells were counted per box in the counting grid. The volume of each box was 0.0001ml. Living cells were colored as clear or green, and dead cells appeared blue. We only counted living cells. A microscope with at least a 20X lense is required to properly distinguish between cells. Again, the Day 0 consisted of one dish being counted, and two dishes for all the subsequent days. It is
Mollegard 5 important to remember that students did not have access to the lab every day, and so some days cells were not counted, but the scale of time is still maintained in the growth curve. To account for all procedures, the following formula is used to estimate the cell count: C = ( N/V ) X D Wherein C is the cell count in cells per ml, N is the number of cells counted in the counting area, V is the volume of the counting area, and D is the dilution factor. For this experiment, D = 2X and V = 0.0001 ml. As C results in a unit of cells per ml, and our objective was to count cells per dish, we equate these two values by multiplying C by the total mls per dish, which is equal to 10 mls. This results in a final dish count for one dish. By averaging the counts of two dishes each day (excluding Day 0), we arrive at an average cell count per dish that can be graphed over time.
Mollegard 6 Results Table 1: The final data Day Dish Confluence Cells Per Dish 0 A 80% 4,000,000 1 A 10% 525,000 1 B 10% 405,000 2 A 20% 893,000 2 B N/A N/A 3 No lab access 4 No lab access 5 A 50% 2,626,000 5 B 50% 3,200,000 6 A 90% 6,260,000 6 B N/A N/A 7 No lab access 8 No lab access 9 A 95% 12,900,000 9 B 95% 7,290,000 The data in Table 1 shows cell confluency and the cell count per dish. Also noted is the missing data for days without lab access. Data represented by N/A is due to having no dish for that day, or the dish
Mollegard 7 being ruined. Our confluency seems to follow a pattern of 10% being about equal to about 500,000 cells, until the final day. Also demonstrated is the differences in population by dish. After several days, the population differences between dishes rises from about 100,000 to several million cells. Figure 3: The final growth curve for HEK 293T Above, Figure 3 shows the final growth curve for our cell cultures. The number of cells listed is the average of the population for any given day. Where data is missing, lab access was not available. Below, Figure 3 shows the standard growth curve for HEK 293T.
Mollegard 8 Figure 4: The standard HEK 293T growth curve Discussion The final growth curve for HEK was as we expected. Comparing our growth curve ( Figure 3 ) to the standardized HEK 293T growth curve ( Figure 4 ), there are definite similarities. Both cell cultures experience a lag phase for approximately one day, during which cell replication stops. Our own cell culture experienced a short lag phase. The cell count only rose from 400,000 to 465,000. However, the cells began growing exponentially in several days. There is no data for days 3 and 4, as lab access was not available. For these days, a line of best fit was used to fill the gap. The graph shows an increase in cells at this point, similar to the standard curve. When cell counting continued on days 5 and 6, there was a clear jump in population. There was no counting for days 7 and 8, so a line of best fit is used to fill in the gaps. The line here shows a large increase in cells. The standard graph shows the point of
Mollegard 9 senescence at around days 8 10. Our own graph cannot indicate this change, because we are missing the cell counts for days when the lab was closed. However, the increase in cells from day 5 to 6 is about 3 million cells, compared to an increase of about 4 million cells from day 6 to 9. If the cells were still replicating, we should see an exponential growth pattern, meaning larger subsequent increases to the population. Instead, the overall rate for the missing last couple days seems to be lower, perhaps suggesting that cell replication was slowing down. There were a couple errors during cell counting, such as diluting the cells by a factor of 11X instead of 2X, but this error was accounted for during calculations by replacing the variable D. Also, two dishes were lost: one on day 2 and one on day 6. The data for these dishes is represented by N/A in Table 1. Moving forward, a better growth curve would result from more time spent in the lab. The best way to accurately display the growth pattern is to have points to base that pattern on. In total, we spent almost half of our days not counting cells. Also, having experienced different levels of confluency, it will be easier to judge a cell suspension properly. Procedurally, there are many rookie mistakes that were made, that would most likely be avoided going forward. Over diluting, spills, and improper counting all had an effect on the data. In the future, losing less dishes would also be a goal. To conclude, cells were cultured and counted for a period of nine days to establish a growth curve. This growth curve still appears similar in shape to the standard growth curve for HEK 293T. Building a growth curve like this is essential for biologists to learn, because it accurately depicts how changes in cell conditions affect certain cells. A few laboratory errors were corrected while making final calculations. Some days were unavailable for cell counting,
Mollegard 10 and so the growth curve is not entirely complete. In the gaps of data, lines of best fit were used to represent expected patterns. The final growth curve is a detailed rendition of how life can grow in a dish.
Mollegard 11 References João Pedro De Magalhães. "Welcome to Senescence.info!" Senescence, Longevity and the Biology of Aging. N.p., n.d. Web. 31 Oct. 2015. "Select Your Mirus Solution." Overview of Transfection. N.p., n.d. Web. 31 Oct. 2015. Raritan Valley Community College. Cellular and Molecular Biology Laboratory. N.p.: Raritan Valley Community College, 2014. Print. "(GMA 099) Transferrin Monoclonal:GMA 099." (GMA 099) Transferrin Monoclonal:GMA 099. N.p., n.d. Web. 31 Oct. 2015.