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Summary: The design of this experiment was conducted in order to analyse the effect of tissue culture plastic surface and establish the optimal tissue culture plastic surface for growing the Human Fibrosarcoma HT 1080 cell line, which is still lacking even this human cell line is commonly used in vitro studies and it is strongly recommended as a gold standard for the Lentivirus titration. In this context it appears especially interesting to which extent the HT1080 cell line proliferation depends on which type of tissue culture plastic plates were used in any experiment. In this study we optimized the growth and the proliferation of the HT1080 cell line by growing them in three different 96 wells tissue culture plates including Falcon, Corning and Greiner, and study the cells proliferation using XTT assay Roche based. Thus we considered the HT 1080 cell line proliferation curve obtained on 2 weeks time and we investigated the proliferation influence of this cell line seeded in 3 different plastic plates. We found that falcon tissue culture plastic were could be more widely considered as a potential plastic ware tool for growing HT 1080 cell line from proliferation curves obtained under 3 different experimental plastic plates, Falcon 96 well tissue culture plate was more likely suitable plastic plate to be used in seeding the HT 1080 cell line.
Cell culture known to be a complex process by removal of tissue or cells from plants, animals, microbes (such as bacteria and viruses), and fungi process them by growing them in specific conditions and atmospheres. In the 19th century scientist discovered the way of maintaining live cell lines taken from the animals tissue .
Principle of tissue culture was established by Wilhelm Roux In 1885, he removed a part of the medulla oblongataHYPERLINK "http://en.wikipedia.org/wiki/Medullary_plate" dish of an embryonic chicken and preserved it in a warm saline for some days2". The methodology of tissue culture was established by Ross Granville Harrison, while he was published results of his research work from 1907-19103".
In 1950s Cell culture techniques were progressed significantly in virology research, which helped in manufacture of vaccines. Development of antibiotics helped tissue culturing to be success, as it made it easy to avoid tissue culture contaminations.
Types of tissue culture
There are two types of tissue culture used for growing cells, adherent and suspension cultures. Adherent cells are known to be anchorage-dependent and attachment to a solid surface is a requirement for proliferation. Generally, the cells grow as an adherent monolayer and discontinue dividing when they reach a density that they touch each other. The majority of cells are adherent as they derived from solid tissues. Cells cultured from bone marrow, spleen or blood adhere poorly if at all to the culture dishes. These cells in the body naturally live in suspension or they are loosely adherent. Adherent cells need a solid phase like tissue culture plastic, which might be layered with extracellular matrix components to raise adhesion properties and supply other signal required for differentiation and growth.
Suspension cultures are easier to spread, since subculture requires only dilution with medium. Moreover, cultures with cells growing attached to each other or to a solid phase have to be treated by a protease to break the bond between the cells and solid surface. Trypsin is the most commonly enzyme used. Obviously, freely suspended cultures do not require trypsinization. Thus, they are easier to harvest.
Maintain cells in culture
Different cell types need different environments to survive in the culture. Environment means is to allow the cells to increase in number by mitosis (cell division). To achieve that, suitable temperature ( 37oC) is required as cells need it to grow happily and that can be achieved by proper calibration, frequent checking, and good maintained incubators. Second, good quality substrate (Glass and Plastic) for better attachment by using attachment factors (collagen, lamnin, and fibronectin) and excellent cell growth. Finally, proper culture media and maintained incubator for accurate pH and osmolality.
Cell culture medium
The culture medium should got the proper nutrition of the cells requirement, growth factors, control the osmolality and pH, and present vital O2 and CO2 gases . The culture medium provides necessary nutrients that are included into dividing cells, such as fatty acids, amino acids, sugars, vitamins and carbohydrates and all of these help to provide the necessary energy to build a new proteins and metabolism. The pH of the medium can be control by buffer which is usually a CO2 based or an organic buffer (e.g HEPES) to maintain the pH level in suitable range 7.0 - 7.4. Sodium Bicarbonate usually used in most cultures media as a standard buffer. Furthermore, Phenol Red is usually added as pH indicator in media, which change when if pH 7.4 decreased. The osmotic pressure adjust the regulation of the substances flow inside and outside of the cell, which is managed by adding salt to the medium. Supplement such as fetal serum enhance the cells growth when it is added to media as it consist of high growth factor concentration and low antibiotics concentration. In addition, serum protein when added to media it acts as nutrition and it undertakes transporter function via cell membrane and combines toxic metabolic products. Antibiotics and antimycotics must be added to the culture media as it suppress the bacterial and fungus growth.
Contamination and cell culture
contamination consider to be a serious problem as it can end an experiment to misidentified or lead to wrong outcome. Recent, studies propose 15-20% of the time researchers been a victim of contamination. There are two types of cell culture contamination, biological and chemicals. Biological contamination caused by fast growing yeast, bacteria and fungi. This type of contamination changes the turbidity of the medium and have observable effects on the cell culture. On the other hand, there are other types of biological contamination which are very difficult to detect such as; mycoplasmas and viruses. Chemical contamination caused by many different agents involve metal irons, plasticizers and Endotoxins.
Different plastic wares used in cell culture
The use of disposable plastic materials for tissue culture has become popular, and in many laboratories plastic cell culture vessels have completely replaced glassware. For example, multiwell plastic plates are used for comparing different growth conditions, plastic wares, media, growth factor, sera and cytotoxines.
Untreated plastic surfaces (usually made of polystyrene) are generally unsuitable for the culture of vertebrate cells, because they do not permit ready attachment and spreading of cells(19). Thus the polystyrene must be subjected to a surface treatment to make the plastic surface suitable for cell attachment(21).Chemical methods, such as sulfuric acid-sodium carbonate rinses (4) and alcohol rinses (5), have been proposed to modify plastic surfaces so that cell attachment occurs.
Cell viability and proliferation
The quantification of cellular growth, including viability and proliferation and, is essential to optimize the cell culture conditions. Measurements of cell viability assess the number of healthy living cells and dead cells, whereas measurement of cells proliferation is used to assess the response of cells to a specific stimulus or toxin quantitantion of culture growth. Cells proliferation is significant in steering maintenance as it is an essential element for controlling the stability of the culture and identifying the superlative time for the optimum dilution, sub culturing, and the estimated platting competence at various cell densities. Proliferation rate is a key quantitative parameter to be estimated when studying the dynamic behaviour of a cell population, measure of cells growth and obtain the cells growth curve.
Fibrosarcoma cell line (HT1080)
HT-1080 cell line is mainly human fibrosarcoma adherent cell line (15). It was instigated from a biopsy of a fibrosarcoma obtained from the acetablum of a 35 year old male in July 1972 the patient had never received radiation or chemotherapy therapy. A fine piece of the tumor tissue was cultured into plastic flasks and dishes were sheltered with Eagle's minimum essential medium with 10% fetal bovine serum and antibiotics . ''Quick trypsinization '' and ''picking'' procedures were used to reduce fibroblasts from the cultures(16).
The human fibrosarcoma cell line HT1080 is strongly recommend as the "gold standard" for reproducibly titrating Lentivirus. Lentiviruses belong to Retooviridae Family, which are the most multitalented of retroviruses since they are capable to infect, transduce and maintain expression in approximately any mammalian cell. Lentiviral vectors obtained from the human immunodeficiency virus (HIV-1) have become main apparatus in mammalian cells for gene delivery. The beneficial characteristic of Lentiviral vectors is the capability to mediate competent transduction, mixing and long-term expression into non-dividing and dividing cells both in vivo and in vitro. The most commonly used cell lines for titrating are adherent cells which show a replication time in the range of 18-25 hours. The human fibrosarcoma HT1080 cell line is able to give more accurate viral titres because these cells are easily transduced and very efficiently by recombinant Lentiviruses . To produce reliable transduction results using a known multiplicity of infection (MOI), it is essential to titrate Lentivirus stocks, and that can be determined by infecting HT1080 cells with serially diluted supernatants produced using control vector containing an easily detectable receptor gene (e.g. Lac Z and fluorescent protein). Furthermore, titration values will depend heavily on the cell type and method used for titration, so there may be significant differences between titres determined in cells typically used for titration and the number of target cells that are ultimately transduced. However titrations are important for determining the relative virus content of stocks prepared from different vectors , Confirming the viability of virus stocks, Determining the optimal transduction conditions, Adjusting the MOI to control the viral copy number of transduced cells, Determining the maximum number of cells that can be infected by a virus stock.
Additionally, the human fibrosarcoma cell line HT-1080 has been used widely to study the consequence of anti-inflammatory agents such as glycocortiodis on the gene expression of inflammatory mediator(17) and in the study of the extracellular matrix proteins involved in attachment, invasion and metastasis. It is also has been involved in assessment the function of the Ras-oncogenes in the altered phenotype and the function of the expression of the rentiblastoma gene product in the cellular response to therapy(18).
In most studies that used the Human Fibrosarcoma HT1080 cell line in their studies there is inconsistencies regarding the growth of this cell line with different media, different serum and different tissue culture plastic surface. Thus, it remains mainly descriptive and not quantify the relative influence of the underlying type of media, serum or plastic surface on cell growth and proliferation.
Aim of this study
In order to find the optimal plastic tissue culture plates for this cell line, I aimed to optimize the growth and the proliferation of the Human Fibrosarcoma HT1080 cell line by growing them in different tissue culture plastic plates including Falcon, Greiner and Corning plates and study their proliferation using colorimetric assay(XTT based, Roche).
Materials and methods
Tissue culture ( pre-experiment to obtain growth curve of HT1080 cell)
Tissue culture of HT1080 cell line
Experiment was performed using Human fibrosarcoma cell line HT1080 (ATCCCCL121) epithelial cells. This cell line was obtained from ECACC European collection of animal cell cultures. Cells were grown in HPA culture collections facilities (catalogue number :85111505).
Routinely, cells were grown in complete medium composed of Dulbecco's Modified Eagle Medium (DMEM supplemented with high glucose liquid without phenol red, without L-Glutamine + 1% Non Essential Amino Acids (NEAA) + 10% Foetal Bovine Serum (FBS) (all were purchased from PAA). The bench surface were cleaned before starting tissue culture, to avoid any contamination The cells were Cultured and grown in 75 cm2 falcon flask which was kept in a humidified atmosphere with 5% CO2 at 37oC for 24 hours to obtain the optimum growth condition. The cells were subculture every 3 days which helped to achieve 0.45-1.0X 106 cells/ml. all the culturing and sub-culturing procedure were done in class II safety cabinet.
The cells were twice a week checked for any contamination before cell count done, the media should be in a good condition, and healthy clear yellow color should be observed. HT1080 cells were counted by using counting chamber (haemocytometer) as it shown below.
(A, B, D, and C) as shown above contain 16 small squares of volume 0.1mm3 or 10-4 cm3 which means (length x width x height). 10ul of HT1080 cells were placed between the counting chamber and cover slips. After cells settle the haemocytometer were fixed under light microscope with X40 magnification. The cells were counted and equation was used to find out the total cells number and then it was divided by 4 to obtain an average X104 cells/ml. Cells can be sub-cultured in fresh supplemented media.
Actual concentration = Dilution factor required
Sub- Culture of HT1080
All cells were detached by using tryptirization by trypsine/EDTA (0.1% /0.02%) solution, average of 60X104 cells/ml of HT 1080 cells were reseed into new labeled flask (75 cm2). Then 20 ml of media was added to the same flask. Finally it were kept in the incubator for 24 hour at 37oC in 5% CO2 atmosphere.
obtain growth curve of HT1080 cell
Briefly, 1ml from the cell suspension were mixed with 480ul of MEM media (PAA). Then 100ul of the tissue culture medium equally distributed to all wells of 96 tissue culture plastic multiwells plate except the first row as 200ul of the cell suspension mixture was added to whole first row. Then Serial dilution was performed by taking 100ul from the first row of 96 wells and transferred to the second row wells ( shown in the figure below). The last 100ul were discarded after doing serial dilution in the sixth well (each dilution included four wells) Finally the 96 wells tissue culture micro plate were kept for incubation (at 37Co, 5% CO2) for 24 hours.
MEM media distributed 100ul
Figure 1: shows the serial dilution
The cells proliferation was studied by using XTT proliferation assay (Cat. No. 11465015001)by Roche. First, the XXT solution was prepared by thawing the XTT labeling reagent and the electron-coupling reagent, respectively in a water bath at 37oC. Mix each vial thoroughly to obtain a clear solution. Then XTT labeling mixture was prepared by mixing 4ml of XTT labeling reagent with 80ul of electron-coupling reagent, to prepare the XTT labeling mixture. Finally, 50ul of the XTT prepared mixture was added to all wells of 96 wells tissue culture plates wells after the incubation period of 24 hours and the plate was incubated in the incubator in humidified atmosphere with 5% CO2 at 37oC for 6 hours .
Reading the tissue culture multiwell plate
After 6 hours of the incubation period, the plate was kept in the ELISA spectrophotometer reader (Tecan sunrise colorimeter) to measure the absorbance of HT1080 cells at 450nm and obtain the cells growth curve.
Tissue culture of HT1080 cell line (Actual experiment)
Growing the HT1080 cell line in 3 different tissue culture plastic 96 wells plate including Greiner, Falcon and Corning.this experiment conducted within two weeks.
From the previous obtained graph of growth curve of HT1080 cell line the seeding density of all our future subculture fixed to 60X104 cells/ml. First of all, the tissue cultured flask checked under the microscope to check the cell growth confluence. Then cell counting was performed to seed all the 3 different 96 wells tissue culture plastic plates at cell density of 60X104 cells/ml and that was possible after finding out the dilution factor by applying an equation (see below). Usually we used dilution factor 1:19.
Actual concentration = Dilution factor required
After preparing the correct dilution of HT1080 cell line, plates were seeded with 60X104 cells/ml ,100ul/well. Each plates was divided to four parts for four days to run the experiment, for example the first part was labeled as day 1, second part day 2, third part day 3 and the last part day 4 with 6 wells for each day along with 2 blank, total of 8 wells daily. Then the tissue culture plates were incubated in humidified atmosphere with 5% CO2 at 37oC for 24 hours. ( see figure 2).
Figure 2: Cells suspension +proliferation reagent Blank: media only
Cells proliferation assay
After the incubation period of 24 hours, the XTT solutions were prepared as explained previously and added to the first part of each different tissue culture plates each part was included 6 wells. Then the tissue culture plates were incubated again in the incubator at 37oC in 5% CO2 for 24 hours. After 6 hours of the incubation period, the platse were kept in the ELISA spectrophotometer reader (Tecan sunrise colorimeter) to measure the absorbance of HT1080 cells at 450nm. Then the plates were returned to the same incubator and used for the rest of days. The same procedure of preparing proliferation reagent and reading the plates was performed to obtain the results and check the cells proliferation for the rest of days .
This investigation was done to rule out the growth density of the Human Fibrosarcoma HT1080 cell line to be seeded in three different 96 wells plastic tissue culture plates to study the growth and the proliferation of this cell line. HT1080 cells were seeded as described in 96 wells plastic plates and incubated for 24 hours in media. For cell proliferation, XTT mixture reagent was added after the incubation period . Briefly, 96 well plates of from each different plastics plates used were seeded with HT1080 cells(6X103 cells/ml, 100 ul/well) and incubated for 24 hours in media. After 6 hours of incubation, the HT1080 cell line proliferation rate were measured by using Tecan sunrise colorimeter at 450nm and the following growth curve was being obtained.
Figure 1: shows a growth curve of the human fibrosarcoma HT 1080 cell line by using XTT assay. Measurement of the HT 1080 cell line proliferation incubated in the 96 well plate on culture medium alone for 24 hours and allowed to adhere. After adding XTT reagent cells were incubated for 6 hours. Then the cells proliferation was analyzed by Tecan sunrise colorimeter at 450 nm, the log phase were determined as 6X103.
From the obtaining HT 1080 cell line growth curve (figure 1) the initial Lag phase where the cells were growing very slow started from 1562 cells /ml to approximately 6000 cells/ml. then the HT1080 cell growth starts to accelerate into the exponential phase which represents the period when the cells are growing most rapidly. This phase continued till the number of cells reached 25000 cells/ml which may due one or more nutrients became limited, oxygen became depleted and or metabolic by - products accumulate to toxic level. After that the cells were Decelerated (Declined). This was followed by a Stationary phase, during which there was no discernible change in cell concretion. Finally if the cells were kept more time we may observe of cell death and lysis which results in a decrease of cells number. The cell growth density was determined by observing the ht1080 cell line growth proliferation curve and it was decided to be 6000 cells/ml as our standard density for this experiment.
This investigation was designed to study the proliferation of Human Fibrosarcoma HT1080 cell line in 3 different 96 well plastic tissue culture plates including Falcon, Corning and Greiner. Briefly, 96 well plates from each different plastics plates used were seeded with HT1080 cells(6X103 cells/ml, 100 ul/well) and incubated for 24 hours in media. For each investigation sample were set up in 6 wells. After incubation, the HT1080 cell line proliferation rate were measured by using Tecan sunrise colorimeter at 450nm and the following result were being obtained.
n = 2
Figure 2: Determine comparison of the HT1080 cells proliferation by growing them in three different 96 wells plastic microplates (Falcon, Greiner and Corning) by using XTT assay for 5 days in MEM media . Each experiment includes 6 duplicate reading . The graph represent the average of three independent experiments data mean, while the errors bars represent the standard deviation of the data.
The results that obtained from this experiment revealed that, the Human fibrosarcoma HT1080 cell line showed different growth proliferation rate depends on the plastic wares including Falcon, Greiner and Corning that have been used in this study (figure 2). The same cells seeding density that was obtained previously from the HT1080 cells growing curve(6X103) were applied to all the 96 wells plastic tissue culture plates. Falcon, Greiner and corning plastic plates showed varies proliferation in the mean (±SD) number of HT1080 cells. From our graph the cells that were grown in Greiner plate proliferate at slower rate in comparison to the other two types of plastic plates were used. On the other hand, the cells showed good proliferation in Corning plate with double increased in the mean (±SD) number of HT1080 cells compared to the proliferation which was obtained in Greiner plate. In contrast the cells, which were seeded in Falcon plate showed the best proliferation of HT1080 cells from the first day of the experiment till the last day and reached a peak at day 4. Moreover, from the obtained data it was clear that we can see HT 1080 cell that were seeded in Falcon plate were proliferating two times more than the mean (±SD) number of HT1080 cells in Corning plate and three times more than the mean (±SD) number of HT1080 cells in Greiner this continued with same significant proliferation rate till the last day of our experiment .
Our results confirm that the plastic wares have a major influence on the Human Fibrosarcoma HT1080 cell line adherence, growth and proliferation. It was very clear from our obtained data that Falcon tissue culture plastic plates shown to be the best plastic ware to optimize the growth and the proliferation of the Human Fibrosarcoma HT 1080 cell line between the other two plastic Corning and Greiner that were used in this experiment. Although the three types of plastic surface treatment almost the same but these cells were growing with different proliferation rate on these plastic wares surface.
The human fibrosarcoma HT1080 cell line are extensively accepted as the standard target cell for titrating Lentivirus because these cells are transduced very efficiently by recombinant Lentiviruses. The health of HT1080 cells at the time of transfection has a significant effect on the success of Lentivirus production. Use of "unhealthy" cells will negatively affect the transfection efficiency, resulting in production of a low titre Lentiviral stock. For optimal Lentivirus production (i.e. producing Lentiviral stocks with the expected titers) the cells should be healthy and greater than 90% viable.
Furthermore, the growth characteristics of this cell line HT1080 changes depending on media formulations, plastic ware used and sources of serum used. Generally, cell attachment, growth, and cell-to-cell contacts on a surface or extracellular matrix substrate are extremely complex proceedings involving cell adhesion molecules. An additional factor leading the growth of cells is the composition of the culture medium, especially serum which supplies the essential nutrients for cells and influences the cell attachment. As it contains numerous extracellular matrix proteins . However, there are known limitations to serum in culture medium. Apart from being expensive, it can interfere with specific assays and introduce variability due to inconsistencies and the presence of indeterminate components.
When growing any cells, one of the first thing is to optimize all culture conditions. Generally people know about Media, FCS/FBS, CO2 concentration, split ratios etc, but very few ever think about TC Plastic....corning, costar, nunc, greiner, falcon, tpp etc will all support cell growth, but optimizing your conditions can save time and money in the long run. Culture environmental conditions influence the proliferative characteristics of cells, while this environment is not fully controlled. Plastic is one of the most important things to know about and understand. It can have a major influence on cell adherence and growth and can therefore ultimately influence the experimental results.
Most of studies devoted to the analysis of HT1080 cell line growth relies on using different types of tissue culture plastic surface leading to inconsistencies regarding the growth of HT1080 in different plastic . Thus, optimal plastic surface for HT1080 cell line long term growth is usually unknown. For example, a study has been conducted by shalinsky et al for the modulation of dipyridamole (DPM) to act synerglstically with vinblastine (VBL) in the HT 1080 cell line, they have used corning plastic micro-plates to seed the cells and ran their experiment. The Human Fibrosarcoma HT1080 cell line used in the studies of the extracellular matrix proteins involved in attachment, invasion and metastasis as the HT1080 cells must attach to and spread underlying matrix in order to carry out normal metabolism, proliferation and differentiation. One of these studies is done by Miyake and colleges same corning 96 well micro-plates were used but it was coated with ECM and they found that HT1080 had better proliferation while cultured with coated microplate rather than uncoated and that can be explained due to capability of extracellular matrix (ECM) to hold the HT1080 cells and provide a highly organized lattice within cells can migrate and interact with each other. In addition, they found that HT 1080 cell line secret a large amount of extracellular matrix on the microplate surface, then HT1080 cell attach rapidly and they leave the underlying ECM intact and firmly attached to the plastic.
Additionally, Ohizumai et al, have another choice of the plastic ware in their experiment as they have used falcon 96 well microplate to grow HT1080 cells and processed their study. In other study was done by Markus and Richard they have seeded the HT 1080 cell line in standard treated uncoated Falcon 24 well microplate and they have found that the HT1080 cell migration increased compared to other cell line such as HT29 and MCF7 cell lines, which confirms that different cell needs different types of plastic microplates to get the optimum growth and proliferation.
Another study where HT1080 cells have been grown on different plastic ware type done by Simpson et al, in their study (combination of afusogenic Glycoprotein, producing Activation and oncolytic Herpes Simplex virus for Enhanced local tumor control). They have used coated Greiner plastic ware with lamim and they are of thousands of researchers who prefer to use coated plastic ware as its more effective and more significant while using plastic ware that are coated with ECM.
In this context, the study of HT1080 cell line proliferation by growing the cells in different tissue culture plastics plates appears necessary for cell proliferation performance within different tissue culture plastic surfaces as well as cell response to such environmental changes
In this study we have optimized the growth and the proliferation of the Human Fibrosarcoma HT1080 cells by growing them in different plastic ware including Falcon, Greiner and Corning plastic wares and study their proliferation using colorimetric assay(XTT based, Roche). The XTT assay method is based on the reduction of the tetrazolium salt XTT by viable cells in the presence of an electron coupling reagent. The reaction produces a soluble formazan salt. The XTT assay is sensitive, quantitative, reliable and automated methods led to the development of standard assays. Cell proliferation and viability assays are of particular importance for routine applications. Tetrazolium salts MTT and XTT are especially useful for assaying the quantification of viable cells. In this case variability of proliferation rates would more likely reflect plastic surface variability than real variations of inherent cell proliferation capabilities. Moreover, Measurement of HT1080 cell proliferation rates is used to determine the response of the cells growth as it is a significant element for monitoring the consistency of the culture and knowing the best time to subculture the optimum dilution, and the estimated platting efficiency at different HT1080 cell densities. Testing medium, serum, new culture vessels or substrate, and so forth, all require quantitative assessment. One of the difficulties in growing cells in vitro using conservative tissue culture techniques is that the cells rest on plastic rather than on their natural biological support and can only be nourished with media from their apical side.
To explain my results it is important to know the plastic surface treatment of each type of plastic used in this study. Normal TC plastic has a net negative charge. TC treatment cross links carboxyl and amine groups and gives the plastic its net negative charge. TC surface modification is usually done by ionizing radiation or other physio-chemical methods ( F. Grinnell 1978 Int. Rev.Cytol 43. p.65 ).
Falcon plastic ware showed better growth and proliferation of this cell line more than greiner and corning. The HT1080 cells were proliferating with Falcon plastic tissue culture plate two times more than with Greiner plastic plate and double its proliferation with Corning plastic plate. Falcon surface treatment is more advanced than Greiner plastic ware as Falcon Standard Tissue Culture (TC) surfaces exposed to vacuum-gas plasma or corona discharge treatment that create a number of negatively charged functional groups on the polystyrene surface and make it hydrophilic. Falcon company is believed to facilitate direct cell attachment and indirectly support attachment, spreading, and growth by binding serum proteins to the plastic surface. Each lot of Falcon plastic products is gamma irradiated to produce a sterile product and from the obtained results it was proved that Falcon is the best plastic substrate for the Human Fibrosarcoma HT1080 cell line growth and proliferation as the cells were proliferating increasingly till the last day.
In contrast the HT1080 cells with Corning were growing and proliferating with gradual increase from the first day till the last day of experiment but their proliferation lower than the cells proliferation with Falcon. Although the Standard Corning polystyrene cell culture plastic wares have the same treatment of Falcon surface treatment. In addition from the results that we have obtained it seems to be that HT1080 cells were growing and proliferating more than when comparing their growth and proliferation with Greiner microplate.
On the other hand, Greiner plastic ware are using different method to treat their tissue culture plastic wares, they are using a "physical" modification to make their TC-treated plates rather than chemical modification and that may explained results obtained from this study, the steady increased growth proliferation of Human Fibrosarcoma HT1080 cell line. As Greiner products for adherent cells culture are treated using particular physical method, this treatment leads to polar groups, such as carboxy and hydroxyl groups being in cooperated into the plastic surface. Thus making the plastic surface hydrophilic. In addition, all Greiner cell culture relevant in products are checked for freedom of cytotoxicity. This significantly improves the adhesion of cells and the binding of proteins to the plastic surface. Hence, the growth proliferation of HT1080 cell line shows steady increase of the cell line till the last day of the experiment but did not show that growth progresses seen while growing these cells on Falcon and Corning microplates.
As most of research have some limitation also this research do, since no previous research was conducted to optimize the growth and the proliferation of this cell line and find out the best plastic, media or serum to grow the cells in, thus it was difficult to collect information to help us in this study or to compare our study with other literature reviews. Secondly, we have used only standard treated plastics which were uncoated as we have seen most of researches including HT1080 cells have been used coated plastic for better cells attachment and growth as in it is recommended for adherent cell lines.
In this study, we have used three different types of plastic wares to optimize the growth and the proliferation of the Human HT 1080 cell line and find out the optimum plastic ware to be use while growing this cell line. However, further study should be carried on while repeating this study by using different types of treated plastics such as plastics coated with ECM ,as HT 1080 of prime importance for various researches.
To investigate the reason behind the different results of HT1080 cells growth and proliferation while growing them on the three different plastic wares that have been used in this experiment, Cell viability assay should be carried on. Measurements of cell viability assess the number of healthy living cells and dead cells in a sample. Additionally, increasing cell viability in a culture indicates cell growth, whereas a decrease in cell viability could be understood as a result of toxicity. Cell viability can be measured by Counting the number of living cells directly by using 0.4% of trypan blue dye or o.4% nigrosin dye to determine the number of healthy viable cells. Dead cells will be stained with the dye whereas living cells will be resistant to the dye (24).
In this study we proceed the experimental work and repeated it within two weeks , in future it will be more useful to be proceeded with much longer time, more than two weeks to confirm the results and get more reliable results.
In future it will be more functional if the growth and proliferation of the Human Fibrosarcoma cell line optimized by incubating with different CO2 percentage and different levels of pH.
This study was able to show the difference in the HT1080 cell line growth and proliferation by using three different types of tissue culture plastic plates. It also reveals that Falcon plastic ware was the best to optimize the growth and proliferation of this cell line. Though the other two types of plastic wares (Greiner and Corning) showed good growth and proliferation of these cells but as good as growing them on Falcon. Hence, our finding suggest that the Falcon plastic tissue culture is the best plastic surface to be used for growing the Human Fibrosarcoma cell line.