Factors that Influence or Inhibit 9HTE Lung Cell Growth

Keithen B Cast TA: Ben Gamache

Abstract:
The entire spectrum of biological life is governed by the ability cells have to grow and reproduce. The cell cycle encompasses all growth and reproduction through a variety of separate phases. This lab studied the growth rate of 9HTE human lung cells. cells were specifically grown in varying concentrations of growth serum and also a death-inducing protein called Camptothecin. The hypothesis was that cells grown in lower concentrations of growth serum/or serum with the protein Camptothecin would have low cell confluency and high cell death. The results show that cells in 2% serum had very low confluency and poor health; cells grown in 10%+Camptothecin had a significantly higher degree of confluency, but also a much higher death count.

Introduction:
All biological organisms must grow and divide in order to survive. The cell theory states that 1) all organisms are composed of cells, 2) the cell is the basic unit of life, 3) and that cells can only arise from pre-existing cells (DiCicco-Skinner, 2012) . The cell theory explains that all cells come from pre-existing cell, thus meaning that cellular division must occur. Cellular division in eukaryotic cells occurs in the cell cycle. The cell cycle contains two distinct parts. The cycle contains interphase and mitosis. Mitosis is when the cell undergoes a complex process that involves replication of all internal parts and DNA. During this phase the cell splits and separates into two new daughter cells. Interphase is the time between mitosis and the M phase. Interphase is separated into three different processes. Immediately after mitosis occurs, cells enter G1 phase of interphase. The Gap 1 of G1 phase is usually considered to be a cellular resting phase. However the cell in the G1 phase is not resting; in this phase the cell is carrying out normal everyday functions such as protein synthesis, protein secretion, cellular respiration, and Photosynthesis (plants). Cells that do not

receive certain signals to further divide arrest in the cell cycle at the G1 checkpoint. As a result these cells are in the G0 phase. Cells can remain in G0 for long amounts of time before re-entering the cell cycle to undergo cellular division. The next phase is the S phase; this phase is characterized by the replication of nucleic DNA. If replication is hindered or not completed the cell will arrest in the S phase and not be able to further continue to the G2 checkpoint. The G2 or gap 2 phase is very similar to G1. The G2 phase the cell carries out normal functions dependant on the type of cell. At the end of the G2 phase the cell is ready to undergo cellular division which occurs in mitosis. Mitosis is subdivided into four different phases: prophase, metaphase, anaphase, telephase. In prophase the nuclear envelope starts to break down, while chromatin condenses into chromosomes and the mitotic spindle forms. After the completion of these vital functions the cell enters metaphase. In metaphase the cellular chromosomes begin to line up in the middle of the cell. The chromosomes are on the equator of the mitotic spindle. In anaphase the sister chromatids begin to move towards opposite sides of the cell. This causes the cell to stretch and begin actual division. In telephase the cell is separating apart and the nuclear envelope begins to reform and chromosomes de-condense into chromatin (DiCicco-Skinner, 2012). There are many factors that influence or inhibit the speed of the cell cycle. For many organisms for example yeast, can replicate and completely move through the entire cycle in merely 90-120 minutes (DiCicco-Skinner, 2012). However for many animals this is not the case. Cell replication is foremost dependant on the type of cell/organism. In the human body liver cells only divide around once a year, while intestinal cells may divide twice a day. The duration of the cell cycle is primarily based upon the length of time the cells remain in G0 phase of interphase. Environmental factors also affect the cell cycle. All cells carry out chemical metabolism and synthesis by means of enzymes. These enzymes all have an optimum Ph, temperature, and concentration to function properly. Any shift from optimum

environmental conditions will affect the efficiency and furthermore time of division. An example of this is anthrax; anthrax can remain in dormant spore form for years but once in a suitable environment cell growth and division will occur. In this lab human 9HTE lung cells will be used for culture and cell growth procedures. Each culture of cells was grown with different concentrations of RPMI growth media. The following concentrations were used: 2%, 5%, 10%, and 10%+Camptothecin. These cells will undergo a variety of different procedures in order to obtain a count of cells, confluency percentage, and health observations. Through the procedures explained in the materials and methods section cell data will be observed and recorded. The first purpose of this lab is to learn how to perform aseptic techniques using human 9HTE lung cells. The second is to understand some factors that affect the rate of cell growth and division. I hypothesize that the 2% and 10%+C will have the least overall confluency and also poor cell health.

Materials and Methods:

The group first obtained four Petri dishes of cells from the TA. The plates originally were plated with 400,000 cells 24 hours previous to the lab. Each of the plates contained different concentrations of growth media. Plate A had cells grown in 2% RPMI serum. Plate B had 5% RPMI Growth media. Plate C had 10%RPMI growth media. Plate D had 10% RPMI + Camptothecin. All cells were grown at 37 degrees Celsius and %5 CO2. After cells were obtained they were labelled with the group name and lab section. Next, each plate was observed under the microscope in order to check confluency and health. Both confluency and health observations were recorded in lab notebooks. Using a technique called passing cells; cells were transferred using the following steps. First the TA handed out four 1.5mL micro centrifuge tubes. Each tube was labelled corresponding with the cells in the plates. With a

5mL pipette media was removed from each of the growth plates and placed in designated waste containers. A new pipette head was used for each removal. Next 3mL of PBS was administered to each growth plate. The plate was rocked in order to thoroughly wash the cells in PBS. After washing the PBS, it then was removed with a 5mL pipette. Next, using a p-200 pipetman, 200uL of trypsin was added to each plate. Each dish was thouroghly rocked back and forth. Cells were observed underneath the microscope and notes were written in the lab notebook. Next, in a hood, 500uL of RPMI + 10% FBS was added to each plate. The 500uL of RPMI + 10% FBS was pipette up and down five times to ensure that cells are suspended in the fluid. 50uL of each plate was transferred to its respective micro centrifuge 1.5mL tube. To each tube 50uL of Trypan Blue dye was added and pipetted up and down to ensure proper mixing. A sample from each micro centrifuge tube was added to the hemocytometer chamber and then viewed under the microscope for counting. Cell counts were written in the lab notebook for later calculations. Cell calculations were done using Dr. Ds formula on the blackboard. After calculations the appropriate amount of cells from respective centrifuge tubes were added to a Petri dish. 7mL of 10% FBS growth media was added to all new plates of cells and then put into the incubator. Later in the week Dr.D switched cells back into 2%, 5%, 10%, and 10%+Camptothecin. Results: The plates containing cells in 2%, 5%, 10%, and 10%+ C were viewed under the microscope and the following observations were made about cell confluency and health. In plate A 2% RPMI growth serum cells appeared to have very low growth. The cells had almost no confluency. 1-3% confluency was recorded; cells appeared to be in very poor health and most were dead. All dead cells were suspended in the growth media. Plat B in 5% RPMI growth serum were 33-35% confluent. Cells in plate B were in much better health than A, yet there was still moderate cell death suspended in the growth media. Plate C, containing

10% RPMI growth serum, cells was highly confluent. Estimated confluency was between 7580%. Cell health was better than any other plate, however cell death was observed in the media. Using the hemocytometer, cells were counted for replating. In the 2%, cells ranged between 0-3 cells per quadrant. The total count for 2% was 5 cells. In 5%, there were 7-10 cells per quadrant totalling to equal 34 cells. In 10%, there were 27-44 cells per quadrant and total cell count was 137. In 10%+ C, there were between 2-8 cells in each quadrant, total cell count was 17. After cells were counted, data was put through an equation to figure out how many cells were in 1mL. Using this information it was possible (as seen below in sample calculations) figure out how much fluid from the micro centrifuge (50uL cells+50uL dye) needs added to new plates. the whole sample in the 2% and 10%+C was added to new plates. 0.6mL of 5% and 0.15mL of 10% were added to their respective plates. Table 1, Cell confluency and Health. 2% 5% 10% 10%+C Confluency 1-3% 33-35% 75-80% 5% Health Very Poor Moderate Good Very Poor Table 1 shows interpreted observations from each of the four plates. note that each plate was observed by all four group members and a common confluency and health observation was suggested. Confluency contains a ranging number directly due to where on the plate was observed. Table 2, Cell Count from Hemocytometer 2% 5% 10% 10%+C Quadrant A 1 7 27 3 Quadrant B 1 9 35 4 Quadrant C 3 8 44 8 Quadrant D 0 10 31 2 Total 5 34 137 17 Table 2 contains raw data observations concluded from one group member. Note that if a cell was on the boarder/line of a different quadrant the cell was only counted once.

Table 3, Results from Calculations Amount needed for 400,000 cells 2% 4mL 5% 0.6mL 10% 0.15mL 10%+C 1.18mL Table 3 shows calculated data of amount of each solution needed to replate cells. All calculations were done by one group member. If the amount calculated was greater than the total amount of the solution in the tube, the entire solution was replated. Table 4, Total amount of dead and alive cells Alive Dead 2% 25000 cells/mL 4000 cells/mL 5% 170000 cells/mL 10000cells/ml 10% 685000 cells/mL ------------------------------------10%+C 85000 30000 cells/mL Table 4 shows the amount of dead and alive cells per mL of solution. Numbers were calculated via equation 1 in sample calculations. The 10% data was lost, therefore there are no calculations done for the dead cell total Sample Calculations: Cells per mL= ((total cells counted)/4) 210^4 Amount of solution to replate= 100000/ (cells/ml) Equation 1 Equation 2

Formula= (total cells/4) 210^4= cells/mL 100,000/ (cells/mL) =amount to add 2% solution (5/4) 210^4=25000 100,000/25000=4m Discussion: The only difference between all four cell cultures was growth media; this suggests that any change in cell health is directly based upon growth media concentration. Because certain organic and biological molecules present in the blood serum in the growth media facilitated the growth of the cells, it is likely the cells with a higher percentage of blood

serum exhibited a higher degree of growth, replication, and overall healthiness because of this fact. The data collected corroborated with this prediction. The plates with higher percentage of serum generally had more growth and overall better health, Table 1 & 2. However there was an exception in the plate containing 10%+Camptothecin. This plate had high growth from the 10% serum, but Camptothecin had a direct influence on cell health. In this plate there was significantly higher cell death than in any other plate, figure 4. This suggests that Camptothecin protein is more essential to cell growth and division than the serum concentration. There are many other factors than can effect cell growth and division. For example temperature directly affects speed of cellular metabolism, thus indirectly slowing cell growth. Temperature would also influence reproduction rates. If temperature can inhibit cell metabolism, it is reasonable to conclude that temperature can slow or stop the cell cycle. an additional factor that could manipulate cell growth is oxygen levels in the environment grown. Dependent on the organism, O2 is needed for respiration and metabolism. Since human lung cells were used in this lab O2 levels has a direct effect on the growth rate and health of the cells. in environments with low concentrations of O2 gas it is likely that the cells would arrest the cell cycle at one of the G phases. It is likely that the cell would arrest the cycle in a G phase, in order to focus on the normal functions the cell carries out. Stopping the cycle would allow the cell to preserve energy (need O2 for respiration) normally used to divide, thus allowing the cell to still survive (yet grow very slow). Lastly, the enzyme trypsin is an example that is not beneficial for cellular growth and division. Trypsin breaks down proteins into smaller polypeptides and with sufficient time trypsin could start to destroy the proteins on the cell membrane. If trypsin was left on the cells for too long it would definitely affect overall health, therefore a factor for cellular reproduction (Kastern, 2009).

Floating cells were representative of dead cells. however once trypan blue was added to the solution most of the floaters did not appear to be stained. The reasoning behind this is that trypan blue only stains dead cell (floating), yet most of the cells suspended in the mixture were trypsinized previously. After attached cells go through the trypsinization process the living cells become suspended in the mother liquid (Kastern, 2009). If trypan blue was added subsequent to trypsinization the cells in suspension would be dead and therefore dyed blue. If originally suspension cells were used both living and dead cells would be in the serum. Thus if the cells were healthy there would be hardly any blue dyed cells, but if cell death was prevalent significant amounts of blue cells would appear. In conclusion, results match the hypothesises made previous to the lab. The results showed that with increasing concentration of growth serum the cells had greater growth and less death form starving. The 10%+C had moderate growth, but had very high cell death because of Camptothecin(Ning,2010) . This suggests that Camptothecin must be necessary for cells to move throughout the cell cycle in good health.

References: DeCicco-Skinner, Katie. 2012. Investigating the effect of environmental factors on 9HTE lung cell growth. Biology 300 Lab Manual. Kastern, H Fredrick, Yip, K Dominic.2009. Reanimation of Cultured Mammalian Myocardial Cells During Multiple Cycles of Trypsinization-Freezing-Thawing In Vitro, 9: 246-252. Ning, Wu, Wu Xi-Wei, Keli Agama, et al. 2010. "A Novel DNA Topoisomerase I Inhibitor with Different Mechanism from Camptothecin Induces G2/M Phase Cell Cycle Arrest to K562 Cells." Biochemistry 49, no. 47: 10131-10136.