The Process of Cell Culture

Cell culture known to be 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, since that days animal cell culture became world widely available as a technique for scientist [1].

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″[2]. The methodology of tissue culture was established by Ross Granville Harrison, while he was published results of his research work from 1907-19103″[3].

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 and improving of techniques played a key role that made cell culture widely used technique for most of the scientist[4].

Cell culture system

There are two basic culture systems, which are used for growing cells.

Monolayer culture system; capability of the growing cells to attached to the glass surface or treated plastic flask substrate such as; T-flasks, roller bottles, multiple well plates, or culture chambers.

Suspension culture system; the cells which are grown floating (unattached) free in the medium. The usual flasks used for growing this type of cell culture are:

Magnetically rotated spinner flasks or shaken erlenmeyer flasks.

Stationary culture vessels such as bottles and T-flasks, however cells are not distributed because they are not able to attach to the substrate.

Blood stream cells are naturally live in suspension without attaching to the vessels surface and called ( anchorage-independent). Modified cell lines are able to survive in suspension cultures which allow them to grow into higher density than adherent conditions. Adherent cells need a surface, such as tissue culture plastic, which might be layered with extracellular matrix components to raise adhesion properties and supply other signal required for growth and differentiation. Most cells derived from solid tissues are adherent[5].

Types of cells culture

Cultured cells are frequently expressed based on their functional features or their morphology based on shape and appearance. Three kind of cell morphology are know:

• Epithelial-like: cells that are appended to a substrate and appear compressed and many-sided in shape.
• Lymphoblast-like: cells that are no appended to a substrate but in same time it settled in the suspension with the globular shape ( bloodstream cells).
• Fibroblast-like: cells that are appended to a substrate and appear extended and bipolar, usually in heavy culture it is found to be forming swirls.

Culture conditions plays a key role in determining form that many cell culture are able of demonstrate multiple difference in morphologies. Cell fusion techniques was used to get hybrid cells from dissimilar parents. In 1975 scientist generated cells that have the ability to make custom modified monoclonal antibodies, which called hybridomas cells. Hybridomas cells are created by fusion two unlike although related cells[6].

Cells functional characteristics

Cultured cells can achieve their characteristics from the original origin (heart, liver, etc.) and their settle in to the culture circumstances. The characteristics of some cell can be changed or lost as a consequence of being located in artificial atmosphere. There are two markers can be used to decide if the cells are still carrying their particular functions that they carry out in vivo, its recommended to use biomedical markers and morphological or ultrastructural markers. There are 6 different known cells lines which can be described as:

• Finite cell line: finally those cells discontinue dividing and demonstrate symptoms of aging (figure 1a).
• Continuous cell lines: those cell became everlasting can be continue dividing forever (figure 1b).
• Transformed cell lines: those cells are faster growing cells and known to grow in the suspension as they have abnormal chromosomes caused by viruses, radiation, or abuse drugs (figure 1c) .
• Diploid cell lines: those cell have ordinary amount of chromosomes.
• Aneuploid cell lines: those cells have other than the ordinary amount of chromosomes.
• Neoplastically transformed: those cell considered as tumor cells that have been introduced to animals [7].

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 it the researcher should be aware of this factor [10]:

• suitable temperature ( 37oC). a temprture vary from 18o to 25oC; most mammalian cells need 36o to 37oC to happily grow and that can be achived by proper calibration, frequent checking, and good maintained incubators.
• good quality substrate (Glass and Plastic) for better attachment by using attachment factors (collagen, lamnin, and fibronectin) and excellent cell growth.
• proper culture media and maintained incubator for accurate PH and Osmolality. This factor is playing the key role in cell culturing because its response of making good growth and produce happy environment for the cells.

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 [11].

Nutrition is an important element in the medium and it should consist of adequate vitamins, proper amino acids, significant minerals, and right carbohydrates and all of these help to provide the necessary energy to build a new proteins and metabolism.

Growth factors and hormones are second important elements for regulation and controlling the cells growth and they play a primary function when serum is not added . These are very heterogeneous group consist of steroids, polypeptide, aromatic amino acid compounds, glycoprotein and proteins.

The PH of the medium also play a initial role in cell growing and that can be control by buffer which is usually a CO2 based or an organic buffer e.g HEPES, which is used 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 to yellow or orange when the Ph decreased and remains red at pH7.4.

The CO2 – bicarbonate based buffer is a required to adjust the quantity of CO2 melted in the medium and that can be achieved by using a special CO2 environment incubator with 2% – 10% CO2.

The osmotic pressure also known to be an important element in the cell culturing because it adjust the regulation of the substances flow inside and outside of the cell. It is managed by adding or subtracting salt in the culture medium . The salts also supply the media with trace elements and important minerals such as sodium chloride, iron nitrate, potassium chloride, calcium chloride or zinc sulphate.

Supplement such as fetal serum enhance the growth action 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. Also it combines toxic metabolic products .

Vitamins can not be produced by the cells in the media, so they are an essential element of culture media. They are acting as co-substrate or co-enzymes in cell metabolism.

Antibiotics and antimycotics must be added to the culture media as it suppress the bacterial and fungus growth.

MEM Minimal Essential Media (or EMEM for Eagle’s MEM)

In this experiment, I have used MEM with Earle’s Salts without L-Glutamine catalogue number (E15-024). And this type of medium is used with cultivation of many cells grown in monolayers and most their features are (Modification of the eagle medium and it contains a high concentration of amino acids). MEM is a basal medium consisting of vitamins, amino acids, salts, glucose and a pH indicator. It contains no proteins or growth promoting agents. Therefore, it requires supplementation to be a “complete” medium. It is most commonly supplemented with 5-10% Fetal Bovine Serum (FBS). MEM utilizes a sodium bicarbonate buffer system (2.5 g/L) and therefore requires 5% CO2 to maintain the required pH. When exposed to ambient levels of CO2, the sodium bicarbonate in the medium will cause MEM to become basic very rapidly. This why one will often observe a half used bottle of media appears purple, indicating a rise in pH[12] .

Contamination and cell culture

contamination consider to be a serious problem for scientists, researchers and companies to create cell-base parenteral. Such contamination problem 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[9].

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, traces of chemical disinfectants and Endotoxins[1].

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Endotoxins and cell cultures

There is rising facts that endotoxins be able to produce a range of problems for researchers in cell culturing technique. Endotoxins is a mixture of lipopolysaccharide (LPS) which is a most important element of the external membrane of the majority gram negative bacteria. Bacteria excrete Endotoxins into their surrounding atmosphere in little quantity while they are actively growing, and in a big quantities after they die. LPS composed of a very extremely hydrophobic lipid group (A) covalently bound to a long complex polysaccharide tale[9] (see figure2).

Sources of Endotoxins in cell culture

Water: high clarity water is necessary in every cell culture labs, not just for making solution and media, although for glassware rinsing(13). And some of those reason why water considers as a source of Endotoxins as followed:

inadequately preserved water system in the research labs such as ion exchange resins system.

Water storage conditions in the research labs following water purified procedure.

N.B water endotoxins problem can be fixed by purchasing nonpyrogynic water for injection (WFI) to prepare media.

Sera: in the early days sera such as (fetal bovine sera) (FBS) used to be a critical source of Endotoxins. But nowadays this have been improved because of manufacture awareness. the majority of FBS manufactures provide a high quality FBS with low endtoxins (<1 ng/ml) however its more expensive than the standard type of FBS and its not recommended for all kind of cell line(14).

Media and additives: nowadays, the media are prepared and shipped to all research labs through commercial manufactures and that reduced the contamination about 90% and they declared that their media contains less than 0.1 ng/ml of endotoxins . However some research labs still preparing their own stock of media and this can be a cause of contamination because of water been used to dissolve the components of media.

Glassware: Endotoxins can be attached to the glassware even after washing procedure done. However today’s modern research labs uses autoclaves to avoid this problem by heating their glassware for 3 hours at 180oC.

Plasticware: this type of Endotoxins source can occurred after molding the plastics resins during handling and packing procedure.

Fibrosarcoma cell line (HT1080)

HT-1080 cell line is mainly human fibrosarcoma cell line used in studies(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 (figure 3). ”Quick trypsinization ” and ”picking” procedures was used to reduce fibroblasts from the cultures. The main tumor biopsy was very cellular with little intercellular substance and assaultd the acetablum cartilage(16).

Even though, the most important cell type in the tumor was an extended tumor cell with only a small population of rounded cells. Moreover, this cell line was modified constant approximately totally of rounded cells. 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). The human fibrosarcoma cell line HT-1080 has also been used in the study of the extracellular matrix proteins involvedved in attachment, invasion and metastasis. Human fibrosarcoma cell have 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). It was establish that Human HT-1080 fibrosarcoma cells voluntarily agree to transfected gene carried by a range of vector. as a result, this cell line has been valuable in the study of gene over expression and possible gene therapy strategies. Human fibrosarcoma cell line HT-1080 has been also helpful in the study of therapy associated concerns in the treatment of fibrosarcoma.

This review aims to find out the best culture plasticware (Falcon, Griner and Corning) can be used to grow human fibrosarcoma cell line HT1080 which is recommended as a gold standard for reproducibly tittering lentivirus, this cell line is adherent and the growth catachrestic change depending on media formulation, plastic used and sources of serum.

Using plastic wares 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. Untreated plastic surfaces (usually made of polystyrene) are generally unsuitable for the culture of vertebrate cells, however, because they do not permit ready attachment and spreading of cells(19). 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. However, it has long been known that the polystyrene must be subjected to a surface treatment to make the micro plate suitable for cell attachment(21). Polystyrene surfaces, as pressed by the manufacturer, are unsuitable for cell attachment.

Multiwall plate are suitable for comparing different growth conditions, plastic wares, media, growth factor, sera and cytotoxines. In this review I have compared 3 different types of tissue culture plasticwares;

• Greiner.
• Falcon.
• Corning.

All these products from the three different companies are sterile and authorized free of endotoxins(<0.06EU/ml). In addition, all these different multiwall plates are non pyrogenic, RNAase and DNAase free and free of DNA polymerase inhibitors. A buried space between wells on the multiwall plates give improved environment control as it provides a convoluted pathway for gas exchange cross the plate. All lids of the cell culture plates include rings to extensively minimize cross-contamination and reduce evaporation.

Surface treatment

Consistent growth surfaces guarantee reliable cell performance. Treatments of all tissue-culture make polystyrene hydrophilic and consequence in the assimilation of a diversity of anionic efficient groups that hold up cell culture(19).

There are different surface treatment available adopted by different manufacturing companies such as:

surface modified using either corona discharge or gas-plasma . These processes generate highly energetic oxygen ions which graft onto the surface polystyrene chains (Figure 7) so that the surface becomes hydrophilic and negatively charged when medium is added. The more oxygen that is incorporated on to the surface the more hydrophilic it becomes and the better it is for cell attachment and spreading(22).

Using special physical method as Greiner company adopted. This treatment directs to polar groups, such as carboxyl and hydroxyl groups, being cooperated into the plastic surface

N.B hydrophilic: This considerably enhances the cells adhesion and proteins binding to the plastic surface.

Conclusion

In this literature review, I aimed to show the optimized way growth and proliferation of HT1080 cells by growing them in different treated plasticware and study their proliferation using colorimetric assay (XTT based Roche). Measurement of cell proliferation rates are often used to determine the response of cells to a particular stimulus or toxin quantitantion of culture growth is also important in routing maintenance as it is a crucial 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 cell densities. Testing medium, serum, new culture vessels or substrate, and so forth, all require quantitative assessment. From this literature review I assume that corning and falcon plasticwares are more likely to be the better than greiner plasticwares and that is because of the quality material and the way of manufacture of corning and falcon plasticware for better cell adhestion and cell attachment. However I would like to mention it was impossible to find any articles or previous literature reviews on this based topic made it difficult to find out the best and the accurate plastic which will help me to get standards and excellent results.