Cell Response To Different Concentrations Of Growth Sera Biology Essay

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Culture of mammalian cells in vitro is essential for the study of mammalian viruses and the course of their replication and virulence, as well as in cytogenetic, biochemical, and molecular research and for diagnostics1. Generally, Eukaryotic cells are more difficult to grow than prokaryotic cells due their complex media requirements and susceptibility to contamination.2 Mammalian cells must also be incubated at an appropriate temperature and gas mixture, of 37ËšC and 5% CO2, for most mammalian cell lines.

Usually, primary cell cultures have a limited lifespan and after a certain number of rounds of mitosis, known as the Hayflick Limit, cells senesce due to the shortening of telomeres to a critical threshold, which prevents further division of cells. Established cell lines, on the other hand, can proliferate indefinitely in the correct culture conditions, either as a result of spontaneous mutation or through deliberate modification. Such cell lines may be derived from carcinoma cells, where the activity of telomerase, the enzyme which prevents successive shortening of telomeres, is altered. Cells from the original tissue can be grown and passaged repeatedly to give rise to a relatively stable cell line. The process of passaging, or sub-culturing cells enables an individual grow cells in culture for extended periods of time, allowing their growth response to specific conditions to be assayed.1

Cells are passaged by removing cell monolayers from the surface of the primary culture vessel by trypsinization or by mechanical means and the cell suspension is then subdivided into fresh cultures. Secondary cultures are monitored for growth and changes in conditions and may be further passaged. The time between passaging cells depends on the growth rate and varies with the cell line. The growth media used contains supplemented factors, often en derived from animal blood, such as foetal calf serum. It is vital to select the tissue culture medium suitable for the type of cells to be cultured and type of treatment they are used for. Media are available commercially in liquid form or in a powdered form, or alternatively, specific media may be prepared. Contamination is a major issue in the culture of mammalian cells, and contamination almost exclusively occurs from handling of cultures, reagents and materials, thus good aseptic technique must be maintained. All materials that come into contact with cultures must be sterile and care must be taken to keep the work environment equally clean. The media of choice may then be supplemented with antibiotic and/or fungicide to prevent growth of contaminants.1, 2

The objectives of this experiment were to carry out the protocols for passaging mammalian two cell lines: HeLa and baby hamster kidney cells (BHK), monitor them by observing their appearance and collecting samples in order to assess the cells' growth response to different serum concentrations in the culture medium. The basis of this experiment is the fact that the amount of protein released from each cell upon lysis can be used to calculate the number of cells in each culture, and thus the time taken for the cells to double.

Materials and Methods

The Experiment was carried out according to the practical procedure described in the laboratory manual, pages 11-17.Over the course of five days, two cell lines, BHK and HeLa, were each cultured in two specific media containing different concentrations of Foetal Calf Serum. Each culture was passaged four times to give a total of sixteen dishes. One culture of each cell type and serum concentration was analysed over the following four days and samples collected for analysis of protein concentration at a later date. The concentration of each culture was examined.

For step 4(c) on page 11/12For the determination of cell concentration in the original cell suspensions, the cells were diluted 1/5 in versene i.e. to give a suitable cell concentration for counting, of approximately 6 x 105 cells/ml. For step 2. On page 16, the standard protein was diluted to 0.1-0.9 mg/ml protein with increments of 0.1 mg/ml to give a dilution series in order to obtain a standard curve.

Cells were washed in media +2% serum, as per step 8; p. 13 of the laboratory manual, before re-plating the cells in appropriate medium in order to remove the concentrated (approximately 16.67%) FCS used in step 3(d) on page 11 of the laboratory manual. This concentration of serum is far higher than the optimal concentration and is only required in the removal of primary cultures from the plate. Omitting this step may result in rapid growth that may be impossible to monitor and early death of cells due to overcrowding and build-up of toxic metabolic products.

The Standard Cell pellets were washed in Phosphate Buffered Saline as per page 13, step 8 in the lab manual before freezing to remove any remaining media and to maintain the ionic concentration and thus the osmolarity of the cell pellets to prevent cell death after thawing.

Results

The results are presented with hours as the measure of time, since the working in terms of days would have made the results less accurate. However, the allocated time for working in the labs was not the same on each day and the exact time of harvesting of each sample was not recorded. Therefore the results are presented at intervals of 24 hours, even though this is slightly inaccurate.

The concentration and number of cells in the undiluted suspension, found using Neubauer counting chamber, were as follows:

HeLa cell line: 330 cells counted in total

Concentration of cells = 3.90 x 105 cells/ml

Number of cells in suspension = 3.90 x 106 cells.

BHK cell line: 175 cells counted in total

Concentration of cells = 4.38 x 106 cells/ml

Number of cells in suspension = 4.38 x 107 cells.

The expected number of HeLa cells was approximately 8 x 106, around half the value calculated. The expected number of BHK cells was approximately 1-2 x 107, and the value calculated was approximately double the upper limit of the expected number of cells.

Figure 1 shows that the estimates of percentage coverage of dishes as observed with an inverted light microscope. Cells of the same line covered approximately the same surface percentage after 24 hours of incubation. The BHK cells in GMEM/10% FCS grew at the greatest rate and had reached the highest percentage confluency by 96 hours after passaging. Judging by the gradient of the computerised line of best fit, the HeLa cells in DMEM/10% FCS grew at approximately the same rate, though after 24 hours, the percentage coverage was 5% less, at 20%. The HeLa cells in DMEM/2% FCS and BHK cells in GMEM/2% FCS also grew at approximately the same rate as each other, but a slower rate than the cells in 10% FCS. The BHK cells in GMEM/2% FCS also covered a larger percentage than the HeLa cells in DMEM/2% FCS at all time points.

Since the Lysis Buffer that was used as a blank in the spectrophotometer was contaminated, the first two results of the lowest dilutions of serum were negative. Zero was set as the baseline for these values and 0.015 was added to each HeLa reading and 0.035 added to each BHK reading to account for this. Thus, no values were recorded for each of the cell lines in 2% FCS. The A590 values of the HeLa in DMEM/10% FCS harvested at 72 and 96 hours were higher than range of standard graph (shown in blue), so the solution was diluted 1 in 2 and the A590 re-measured. This dilution was accounted for by doubling the concentration of protein read from the standard curve. The corrected and diluted (in the case of BHK 10% 72 and 96 h) values were used to calculate the amount of protein released from each cells and the total cell number in each extract.

Table 2: A590 Cell extracts

Cell Line in Media/serum

Length of Incubation (hours)

Standard Cell Pellets

24

HeLa DMEM/ 2% FCS

-

HeLa DMEM/10% FCS

0.081

0.182

0.374

0.728

BHK GMEM/2% FCS

-

0.353

0.183

0.430

BHK: 0.439

BHK GMEM/10% FCS

0.031

0.345

(Undiluted) 1.186

(Diluted 1 in 2) 0.439

(Undiluted) 1.270

(Diluted 1 in 2) 0.730

The spectrophotometric assay was calibrated with standard protein solution to produce a standard curve of protein concentration (Figure 2, see appendix). The concentration of protein in each culture was estimated from Figure 2.

Table 3: Concentration of protein estimated from Figure 2 (Appendix) (mg/ml)

Cell Line in Media/serum

Length of Incubation (hours)

24

48

72

96

HeLa DMEM/ 2% FCS

-

0.004

0.030

0.074

HeLa DMEM/10% FCS

0.009

0.018

0.036

0.072

BHK GMEM/2% FCS

-

0.032

0.016

0.037

BHK GMEM/10% FCS

0.002

0.031

0.056

0.140

As described above, no values were recorded for each of the cell lines in 2% FCS. The concentration of protein in the HeLa culture in DMEM/ 2% FCS rose steadily between 48 and 72 hours and more rapidly between 72 and 96 hours in the incubator. The concentration of protein in the HeLa in DMEM/10% FCS exactly doubled over the course of the assay. There was a large drop in protein concentration after 72 hours of incubation of the BHK culture in GMEM/2% FCS, and after 96 hours the concentration had increased to little more than the value at 48 hours. The concentration of protein in the BHK culture in GMEM/10% FCS rose very rapidly after the first 24 hours, rose at a slower rate until 72 hours and then rose very steeply between 72 and 96 hours.

The Concentration of protein from the standard curve (Table 3, appendix) and the Original Volume of the culture extracts (Table 4, appendix) were used to calculate the amount of protein released from each cell (Table 5), and the total cell number per culture (Table 7, Appendix and Figure 2). Figure 2 shows the logged values of the cell numbers. Details of calculations are included in the appendix.

Table 5: Concentration of protein released from each cell (mg/ml)

Cell Line in Media/serum

Length of Incubation (hours)

24

48

72

96

HeLa DMEM/ 2% FCS

-

0.0192

0.1515

0.3552

HeLa DMEM/10% FCS

0.0432

0.0855

0.1800

0.3456

BHK GMEM/2% FCS

-

0.1552

0.0808

0.1832

BHK GMEM/10% FCS

0.0098

0.1473

0.2800

0.6720

The gradient of the trend line of each data set was used to calculate the T1/2 of each culture.

Table 6: Cell Doubling Times calculated from Figure 2

Cell Line in Media/serum

Doubling Time (hours)

HeLa DMEM/ 2% FCS

11.43

HeLa DMEM/10% FCS

24.00

BHK GMEM/2% FCS

205.7

BHK GMEM/10% FCS

11.74

Figure 2 shows that the HeLa culture in DMEM/ 2% FCS and the BHK culture in GMEM/ 10% FCS had similar gradients and therefore similar doubling times were calculated, though the HeLa culture had a slightly lower doubling time, meaning that he cells grew faster. The HeLa culture in DMEM/ 10% FCS had a doubling time of exactly 24 hours, which was expected, since the A590 and concentration of protein in each successive extract was double the extract harvested 24 hours prior. The BHK culture in DMEM/ 2% FCS had a much higher doubling time, since the gradient from Figure 2 is very gradual due to the much lower A590 (and therefore cell number) reading at 72 hours, and the fact that the cell number at 96 was not much higher than the cell number at 48 hours. Therefore, the overall cell number increases very little over the course of the experiment. The first 24 hours of growth were not recorded, but this was when growth was fastest, judging by the fact that this is the highest data point at 48 hours. It must also be noted that no data were collected at 24 hours for each cell line in 2% FCS for reasons stated above, and therefore there are only three time points for these cultures. The implications of this are explored in the discussion section.

The data of the BHK cells in GMEM/2% FCS does not correlate with the percentage coverage of the plastic plates (Figure 1), which increased linearly over the course of the experiment. The last data point from this cell culture had the lowest cell number (Figure 3), but the second lowest percentage cover after 92 hours. The percentage coverage (Figure 2) of cell cultures in 10% FCS correlated more closely with the cell number (Figure 1). The percentage coverage of the BHK cells in GMEM/10% FCS was lowest at all time points in Figure 1, conversely, the cell number on Figure 2 is much lower than the number of cells in the other cultures at 48 hours and is only slightly lower than the cell number of the other HeLa culture at 76 hours and around the same at 96 hours.

Discussion

The original data collected could not be used, due to contamination of materials and reagents, including the lysis buffer that was used as a blank for the spectrophotometric assays. Although this may have meant that the A590 reading of the extracts were inaccurate, this should have been negated by the fact that the blank was also contaminated, although the source and type of contamination was unknown.

The first data collected in this experiment was the percentage cover of cells in each of the samples over 96 hours. The BHK cultures had covered a greater percentage of the surface by the end of the experiment. The cells in 10% FCS grew at a slightly faster rate. However, these figures may be misleading because the BHK cells were longer and thinner, and thus may have appeared to cover more surface are than the smaller HeLa cells. It was also difficult to estimate the percentage coverage, since the cells were not evenly distributed over the surface of the dishes in any of the cultures, with the centre of the dishes being more confluent than the outer edges. This is due to inadequate mixing of material and distribution cell suspension in the dishes. The percentage of cell coverage did correlate in some cases to the cell number, but the BHK cells in media containing 2% and 10% FCS, they did not correlate as closely, especially the BHK cells in 2% FCS.

It must be noted that the doubling times calculated are highly inaccurate due to very small number of data points collected, especially for HeLa and BHK 2% FCS. This meant that the anomalous data skewed the T1/2 measurements. In order to collect more accurate data, more samples would need to be collected over the course of the experiment i.e. using more time points at smaller intervals e.g. every 12 hours instead of every 24 hours. However, this could only be achieved by collecting data early in the morning and then in the evening, which may be impossible due to time constrains.

The BHK cells in GMEM/2% FCS produced some unusual spectrophotometer reading, since the reading at 72 hours was around half the value at 48 hours and the value read at 96 hours was also low. This affected the protein concentration in the culture read from Figure 3 (see appendix) and thus the calculated values for the amount of protein released from each cell, total cell numbers and T1/2. The T1/2 was very high, at 205.7 hours compared to the rest of the cultures, making it difficult to compare this culture against the other cultures with any degree of accuracy. Reasons for the low cell number calculated may include error in measurement of reagents used during preparation of the culture or during the harvesting of the culture, resulting in denaturation of protein and thus produce a lower absorbance. Other possible explanations are contamination of one or more of the samples of this culture or extensive protein denaturation due to the freezing process in the 72 hour extract. The only way this could have been rectified would have been to repeat the passing and incubation of this particular culture but time constraints and lack of materials did not allow for this.

The cell numbers of the rest of the cultures increased over the course of the experiment, as expected, and produced doubling times that lay within the time limits of the experiential protocol.

HeLa cells in DMEM/2% FCS: The final two data points, of the samples harvested at 72 and 96 hours correlated with the percentage coverage data, though the point at 48 hours was much lower. This anomalous value is one of the lowest cell numbers calculated, which indicates that there was a mistake made during preparation of the culture for incubation or harvesting. This has skewed the gradient, making it steeper than it otherwise might have been and resulted in the highest doubling time.

HeLa cells in DMEM/10% FCS correlated to the corresponding graph on Figure 1, although the gradient is steeper than that in Figure 1. The difference may be due to the culture not being evenly distributed and therefore an inaccurate estimate of estimated percentage cover being recorded. As the cells were incubated over the course of the experiment, the cell numbers increased.

BHK cells in GMEM/10% FCS: to the corresponding graph on Figure 1, and has a similar gradient, although the value at 48 hours is slightly higher than the trend line. The first time point is the lowest on figure 1, and the points at 72 and 96 hours are the highest. This culture had the second highest doubling time, only slightly lower than that of the HeLa cells in DMEM/2% FCS. Due to the fact that the HeLa culture had fewer time points and an anomalous result that was more considerable that the anomaly in the BHK culture in 10% FCS can be considered to have the highest rate of growth due to a larger overall increase in cell number.

From the cell number of each culture, it can be concluded that the cells of both cell lines grown in 10% Foetal Calf Serum reached higher total cell numbers, but that the HeLa cell line in 10% FCS grew with the fastest doubling time. The BHK cells in 2% FCS grew very quickly during the first 48 hours and reached the greatest cell number of all the cultures at 48 hours. Taking into account the appearance of the data on Figure 2 and the slow T1/2, as well as the fact that the cells grew at the fastest rate before data was taken, it is difficult to reach a conclusion about this data in relation to the other cell cultures. . Had the value at 48 hours not been unexpectedly high and a value collected at 24 hours, the T1/2 would very likely be much lower and closer to that of the HeLa culture in 2% FCS. However, it is clear that both cell lines incubated in 2% FCS reaches lower total cell numbers than the cultures of the same cell line in 10% FCS. Thus, it can be suggested that, since the cells grown in 2% serum have a lower concentration of nutrients, and are not able to proliferate to produce cell numbers as high as cells grown in 10% serum. The BHK cells in 10% serum have the fastest doubling time.

According to Figure 1, the numbers of BHK cells incubated in both serum concentrations increase at a greater rate than the HeLa cells, resulting in greater percentage coverage at the end of the experiment. Figure 2 reveals that the numbers of BHK cells incubated in 10% serum increase at a similar rate to that recorded on Figure 1. Taken together, this evidence indicates that the BHK cells are more prolific in cell culture. Given the nature of the cell types, this would be expected. Baby Hamster Kidney Fibroblasts are spindle-shaped cells that are responsible for synthesising fibrillar extracellular matrix and collagen and thus have a relatively high metabolic rate and proliferate rapidly due to the need for high turnover of ECM in the kidney3. HeLa cells, on the other hand, are transformed cervical carcinoma cells that proliferate rapidly, and are a commonly used cell line in biological research. However, even though they are carcinomas and thus proliferate at a greater rate than many cell types, cervical cells do not proliferate as rapidly as fibroblasts.

According to the doubling times collected, the optimal serum concentration for HeLa cells is 2% and for BHK cells, 10% since these result in the shortest doubling times. It may be that HeLa cells do indeed proliferate at the optimum rate in 2% serum since 10% serum is too concentrated for this particular cell line. However, since the results are so flawed due to experimental error, it is difficult to reach a conclusion about the affect of serum concentration on the growth of cells in culture.

The serum concentration greatly affected the total cell number over the course of the experiment, in that at greater serum concentration, the cultures reached a greater total cell number. However, the cultures incubated in lower concentrations of serum appeared to double in number more rapidly, but not reach cell numbers as high as the cultures grown in a higher concentration of serum.

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