UNIVERSITY OF SHEFFIELD

Cell Culture
MAT2530: Biology and Chemistry of Living Systems II
Mta10ad 14/11/2011

Cell Culture Write Up The main point of a cell culture is to create a suitable environment for cells to grow and develop. These in vitro procedures create the understanding of an artificial controlled environment and would therefore allow development into conditions in which a cell or tissue can survive/grow. The primary goal of the practical was to determine the proliferation of cells on 4 different materials. This is done by examining the protein concentration after the cells have cultured. Any living cells would adhere to the material whereas the dead cells could be cleared out. Experimental Procedure Anything that went into the recirculating cabinet had to be cleaned with 70% Ethanol, in order to remove any contamination from the apparatus/containers. Using 100% Ethanol is far too volatile to be used. The first required step was to create a cell culture medium in a recirculating cabinet. The medium consisted of; 46ml of Dulbeccos Modified Eagless Medium (DMEM) which was basically sugars and Vitamins, 2.5ml of Foetal Calf Serum (Protein), 0.5ml of L-Glutamine, 0.5ml of Penicillin (100g/ml) in order to remove any bacteria based contaminations and 125l of AmphotericinB (Fungizone) to reduce any chance of a fungus based infection. The combined mixture was then stored in a 37C tissue culture incubator. The four materials that were used in the investigation were: Polyvinyl Chloride (PVC), Perspex, Tissue Culture Plastic (Polystyrene) and Delrin. These materials were in the form of a disc format apart from the TCP as it is the tissue culture plate itself. The 3 discs of each material were then placed in separate wells in the culture plate. The next procedure was to count number of cells using a haemocytometer which is basically a glass slide with a measured scale in the background in order to obtain cell count per area/volume. 10l of a fibroblast stock cell was added to the haemocytometer, and then placed under a microscope to count the cells. The volume of the medium mixture combined with the fibroblast cell suspension was calculated to obtain a total cell count of 5000 cells. This volume was then added to each of the wells containing discs (3 discs for each material as well as 3 empty wells). 1ml of the cell culture medium was then added to each well. The 24-well plate was then covered up and then placed in the tissue culture incubator for 7 days. After 3 days in the culture, the cells were given more medium. After the 7 days of cell culture, a protein assay test was conducted on the samples to test for existence of any healthy cells in the sample. The protein assay reagent was made up from the Pierce BCA reagent assay kit, with 10ml of Reagent A and 200l of Reagent B and was then mixed by inverting the container. The protein standard solution was then made using 20mg of Bovine serum Albumin (BSA) along with 10ml of distilled water. This was used as the protein reference standard for the practical. A cell

extraction solution was also made; this consisted of 100mg of sodium dodecyl sulphate (SDS) and 20ml of distilled water. Neither the BSA standard or the SDS solutions were shaken to be mixed. The cultured cells were then removed from the incubator and then all the used DMEM was extracted using a pipette and disposed off into dilute bleach. This DMEM would contain all the dead uncultured cells and would therefore make it easier to detect any living cells. 1ml of prewarmed phosphate buffered saline was added to wash the cells in each well, to remove any extra medium and then washed 2 more times. The solution is a buffer and is used to keep the pH constant and so the cells dont shrink or swell. 200l of 0.5% SDS (cell extraction solution) was added to each well and then placed in the incubator for 20 minutes, due to time constraints this was slightly quicker than the originally proposed 30 minutes. During the 20 minute waiting time, the BSA solution can be prepared in the 96-well plate. Protein Standard Water (l) Solution (l) 0.0 10.0 2.0 8.0 4.0 6.0 6.0 4.0 8.0 2.0 10.0 0 Table 1 BSA standard solution concentrations. Protein Concentration (l/l) 0 0.4 0.8 1.2 1.6 2.0 Protein Amount per well (g) 0 4 8 12 16 20

The 1.0 l protein standard solution could not be added to the BSA standard curve due to the pipette not being small enough to accurately measure out that volume. After the 20 minutes, the SDS solution was then circulated around the individual well several times in order to make sure that there is complete dissolution of the cells. If there is any frothing of the SDS solution during the pipetting , this will alter the readings as there will be scattering of light during the absorption process. 10l of each sample was then transferred to a 96-well plate and was in this arrangement: BSA Protein Standard (l) 0 0 2 2 4 4 6 6 8 8 10 10 Samples

PVC1 PVC1 PVC1

PVC2 PVC2 PVC2

PVC3 PVC3 PVC3

Perspex1 Perspex2 Perspex3 Perspex1 Perspex2 Perspex3 Perspex1 Perspex2 Perspex3 TCP1 TCP1 TCP1 TCP2 TCP2 TCP2 TCP3 TCP3 TCP3

Delrin1 Delrin2 Delrin3 Delrin1 Delrin2 Delrin3 Delrin1 Delrin2 Delrin3

Fig. 1 Diagram of 96-well plate.

The main reason for the 3 samples of each disc was due to any accidental contamination and just set to minimise errors. 200l of the BCA reagent (Protein Assay) was then added to each well that contained the samples or the BSA protein standards and then the plate was covered and then set into the incubator for 30 minutes. The plate with the culture samples were then placed in 540nm scanning plate reader and the spectrophotometric absorbances were read. Results

Fig. 2 Absorbance values of the 96-well plate. The absorbance values highlighted in yellow in Fig. 2 is the BSA protein standard, the orange represents the PVC, the blue represents the Delrin, the purple represents Perspex and the green shows the TCP. Anomalies can be observed quite easily. The empty wells come out to an average of 0.038.

Protein Standards
0.500 0.450 0.400 0.350 0.300 Average 0.250 Absorbance 0.200 0.150 0.100 0.050 0.000 0.0 0.5 1.0

y = 0.2021x + 0.049

1.5

2.0

2.5

Protein Concentration (g/l)

Fig. 3 Graph of Calibration Standard curve.

All values used were subtracted by the value of absorbance of the blank well.

Based on the absorbance readings, following results were calculated with the average results (ignoring any serious anomalies) Average Absorbance Value 0.071 0.059 0.123

Material PVC Perspex Delrin

TCP 0.051 Table 2 Shows the average absorbance value for each material From Fig.3, a trend line and equation had been added to the graph to make it easier to calculate the protein concentrations for each material. Equation: y = 0.2021x + 0.049 Substituting the average absorbance value of each material into y and solving for x would end up with the protein concentration for that particular material.

Based on the equation, the protein concentrations would be as following: Protein Concentration (g/l) 0.10886 0.04948 0.36616

Material PVC Perspex Delrin

TCP 0.00990 Table 3 Shows the protein concentration calculated for each material A clear distinction can be seen from TCP (control surface) having the lowest protein concentration with 0.01g/l, followed by Perspex with 0.049g/l. PVC has 10 times the concentration of the control surface with 0.11g/l and the material with the highest protein concentration is Delrin with 0.37g/l.

T-test Based on the equation:

All with respect to the TCP control surface (C) and T being each of the 3 material values. Calculated t-value for PVC: 0.505, Perspex: 0.201, Delrin: 2.501 and respectfully a degree of freedom of 15, 14 and 14. Using these values, the p-value can be obtained from a t-table and therefore effectively describing the statistical significance of the reading obtained. PVC, which turns out to have a p-value of 0.6209 and by conventional criteria, this difference between the PVC value and the control surface (TCP) value would be considered not to be statistically significant. Perspex also has a similar p-value of 0.8436 and is also considered to be statistically insignificant with respect to the TCP value. Delrin, on the other hand, has been seen to have a p-value of 0.0255, which conventionally implies that the difference is considered to be statistically significant. Discussion The growth and proliferation of cells required specific and certain conditions to optimise the rate of growth. Temperature, pH, osmotic conditions, bacterial contamination, etc, the conditions used were all based in a controlled environment. These parameters directly affect growth and optimising the proliferation is very important in the cell culture procedure. Nutrients are a key for cell growth as in with living systems, every organism requires nutrients to grow. This was the primary reason to use DMEM, this medium contains sugars, vitamins, iron and amino acids which is a key factor involving the growth of cells. Time is also an important condition for cell proliferation; an error that could be mentioned is that the cells were cultured for 7 days rather than the optimum 4 days required for growth. There was over-densification of the cells due to growth and this would cause the cells to become claustrophobic, resulting in the death of the cells. With regards to temperature, all the cells that were cultured were placed in a 37C tissue culture incubator which was the optimum temperature for fibroblast growth due to having mesophile properties. If the temperature was set higher, chances are that the fibroblast cells would denature and if the temperature was much lower, it would inhibit any cell growth due to the dormant cell phase If there is any bacterial or fungus based contamination during any of the processes, the Penicillin and AmphotericinB would aim to reduce as much infection as possible in order to keep the sample sterile. The bioincubator may encourage growth but it would also induce infection if there was a contamination. The phosphate buffered saline used in the experiment was the primary purpose for pH and osmotic conditions as it is a water based salt with a constant pH. The 4 surface materials used in the cell culture all had different results, but each one of them adsorbed a certain rate of cell growth. Tissue culture plastic made from modified polystyrene has a hydrophilic and ionic binding interface, which allows adsorption of the cells. Based on the results, a relatively low concentration of protein can be seen (0.01g/l) but nevertheless still some inhibition of growth was observed. The process that converts untreated polystyrene into TCP involves high energetic oxygen ions which bond with

the outer layer of the polystyrene chain. The surface becomes hydrophilic when the medium is added. One reason why the reading was relatively low could be not old due to errors, but also a lower amount of oxygen fused to the surface, thus decreasing the hydrophilic properties and hence lowering the ability for cell attachment. Untreated polystyrene is hydrophobic and does not directly inhibit cell growth. Some cell culture plates are made from untreated polystyrene, which could be the case with the plates used and hence therefore resulting in a significantly low protein concentration. With reference to medical applications, crystal polystyrene is made into lab ware and in this example tissue culture.

Fig. 4 Untreated polystyrene

Fig.5 Standard tissue Culture Surface

Perspex or also known as Poly(Methyl Methacrylate) is a transparent polymer and has several medical application. The materials showed 5 times the adsorption of the control surface (TCP) but still relatively low. PMMA is most commonly used for contact lenses due to the optimum optical properties. It is also used as bone cement for joint prostheses fixations. As the material is used directly within the body, it is clearly said to be extremely biocompatible. Polyvinyl chloride (PVC) is typically has a hydrophobic surface and in theory should have a fairly low protein concentration; however this isnt the case based on the results (0.11g/l). Perhaps there may have been some contamination, or the medium inhibited cell growth on the PVC disk surface. In terms of medical application, PVC is used primarily for tubes and solution storage bags as well as surgical packaging. PVC has been tested to be cytotoxic, and therefore an assumption can be made that there has been a certain coating either due to the medium or a contamination that created a hydrophilic property, and thus allowing cell growth. Delrin or chemically known as polyoxymethylene (POM) has been seen to have the highest measured protein concentration from the experiment (0.37g/l) and shows a clear property to induce cell adhesion onto the material surface. POM is used for several applications in the medical field, including insulin pens, surgical implant materials and more recently, for tissue engineering bioreactors. A particular journal (Laluppa et al 1997) suggests an unsuitability for POM to be used within a cell culture as it contains stabilisers which would be released into the medium causing an adverse affect to the cells in practice. The 4 phase process that occurred to the cultured cells began with the G1 phase which was growth and synthesis, in which the cell usually replicates the DNA, and then moves onto the second step which is the Synthesis phase, where the DNA is synthesised and replicated completely. The reason why some protein concentrations were low might have been due to abnormal DNA due to a failure in the S-phase. The G2 phase is the preparatory phase for Mitosis. In the final stage, (M stage) Mitosis occurs forming a complete replicated cell.

Conclusion The use of the protein assay for the experiment was very useful and was considered to be a valid investigative method as it determined the presence of health cells. Within cell culture experiments, the understanding of the importance of sterility when it comes to equipment and apparatus was very important as contamination of the medium and culture wells was imminent without the ethanol spray. From the calculations, Delrin had the highest cell inhibition, followed by PVC and then Perspex, leaving the Tissue Culture Control (Polystyrene) with the lowest protein concentration.

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