Importance of Cell Cultures

Introduction

Cell culture is an extremely widely used process by which cells are removed from their natural environment and grown artificially under controlled and monitored conditions. It occurs in vitro, or in glass, more specifically in multicellular eukaryotic cells. The cells may be removed from their habitat directly and disaggregated with enzymes or mechanically before harvesting, or they may be a derivative of a cell line that has been created previously. It was adapted from a practice used in the early 1900’s and since then it has expanded and advanced research and scientific knowledge enormously. The conditions required for each culture vary, however the artificial environments conditions are consistent. It must consist of a suitable vessel which contains a medium that provides vital nutrients such as amino acids, vitamins, carbohydrates and minerals. Growth factors and hormones are also needed, as well as oxygen and carbon dioxide. It must monitor and regulate physico-chemical environment which includes pH and osmotic pressure, as well as temperature. Temperature is kept at 37°C, CO₂ levels at 5% and humidity at 95%.

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Cell cultures are an extremely important tool for healthcare scientists. They provide a model system for physiology and biochemistry of selected cells to be studied. By examining their physiology their aging pathway can be studied and their biochemistry allows processes such as metabolic rate to be observed. The cells interaction with drugs could also be observed which proves a useful tool for drug screening programs, clinical trials and pharmaceutical companies. Whatever the purpose for using cell cultures, it is an extremely consistent and reliable process that has good reproducibility of results that can be obtained using a batch of clonal cells.

Primary cell cultures are cultures that grow and maintain cells dissociated from their parental tissue via mechanical or enzymatic methods. They can be either adherent or suspension cells. Adherent cells are also known as anchorage dependent cells because they require attachment for growth. These cells are usually derived from organs such as the kidney where they are immobile and implanted into connective tissue. Suspension cells are the opposite and don’t require attachment to the culture vessel for growth. These types of cells are anchorage independent cells. They are cells that derive from the blood, where they aren’t attached to anything but are still suspended e.g. in plasma like lymphocytes. A secondary culture is a primary culture that has been sub-cultured. The sub-culture (passage) occurs when the cells are transferred from a culture vessel to another. This provides fresh nutrients and space for continued growth, because a primary culture has a finite life span. Common primary and secondary lines can be found in Table 1.

After the first sub-culture, the culture becomes known as the cell line. Cells only undergo a finite number of replication cycles before cell death. This means that some cell lines will be finite cell lines. However, some cells undergo transformation. This can occur spontaneously but can also be virally induced in vitro. Undergoing transformation gives the cell the ability to divide infinitely, such as HeLa cells. The HeLa line is the oldest and most commonly used continuous cell line. Cervical cancer cells biopsied from Henrietta Lacks in 1951 show that they are remarkably durable and prolific. In 2012, Turner published a paper documenting its importance in the development of the polio vaccine.

Table 1: Summary comparison table of cell line examples, their uses and origins

The COS-7 cell line is a line derived from African green monkey kidney tissue. It is used in research against SV40, a cancer causing virus that was hidden in the polio vaccine (Vacante et al., 1989). The Hep G2 cell line is another continuous cell line of hepatocellular carcinoma. It plays a vital part in the research of human liver diseases by being a model for intracellular trafficking (Mersch-Sundermann et al., 2004). Jurkat cells, another continuous line, are a line of lymphocyte cells used to study leukaemia, T-cell signalling and HIV (Wang et al., 2012). This review will explore the use of cell lines in the laboratory and their applications. SH-SY5Y will be a particular focus, and will explore the application and importance of the cell line as one of the only lines used to study neuronal function and differentiation.

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SH-SY5Y cell line

SH-SY5Y cells are a derivative cell line used majorly in scientific research. SH-SY5Y originally was cloned from a biopsy of bone marrow derived line called SK-N-SH, and then named as SH-SY. The biopsy was from a 4-year old female with neuroblastoma. This was subcloned again to make SH-SY5 and subcloned once more to form SH-SY5Y. Because this cell line has been derived from a primary source, it is a secondary culture. There is new, fresh growth medium in which the cells are suspended not attached, making them anchorage-independent cells in the cell line. They have been widely used since the 1980’s, due to their ability to express dopaminergic markers and neuronal function such as neurodegenerative processes. Because of these characteristics, they play a major role in the research of Parkinson’s disease.

As mentioned before, the cells are subcloned. This process of sub-culturing is also known as cell passaging. Cell passaging is where a new microbiological culture is created by transferring a sample, or all, of a cell culture to a different growth medium. This process prolongs the life of the organism, renews depleted nutrient levels and also increases the concentration of cells in the culture. Cells cannot be held in their primary culture indefinitely because continual cell activity means there will be a gradual rise in toxic metabolites. For SH-SY5Y cells, there is a recommended limit of cell passaging. Passage numbers can affect cell physiology and morphology, protein expression and transfection efficiency, so the limit has been set to 20 to prevent unreliable and irreproducible results being collected.

Use of SH-SY5Y cell line in research

As conferred, SH-SY5Y is one of the only cell lines that can be used as a model system for neuronal function investigation. It is particularly good for investigating the effective of oxidative stress on neuronal cell lysis. Reactive oxygen species (ROS) at specific concentration are essential for standard cell function however over exposure to ROS is harmful to cells. There are 2 globin’s whose functions are still unclear. Neuroglobins (NGBs) and cytoglobins (CYGB) role has been suggested to involve detoxifying the effects of over exposure. Excessive ROS has been known to cause cell lysis after ischaemic strokes. By investigating the correct levels and limit levels of ROS it can have an enormous clinical impact on stroke recovery and treatment. Forde et al. investigates the effect of NGB and CYGB on the detoxification of ROS. The influence of cell lysis of surplus ROS is the primary focus, more explicitly hydrogen peroxide.

SH-SY5Y cells were cultured at a ratio of 1:1 of Dulbecco’s minimum essential medium (DMEM) and Ham’s F-12 nutrient medium along with 10% foetal bovine serum at 5% CO₂ atmosphere. The culture was maintained at 37°C in a humidified 95% atmosphere. L-glutamine provided an energy source and sodium bicarbonate acts as a pH buffer. Growth factors and non-essential amino acids (NEAA) are also present and standard factors. In the culture, penicillin and streptomycin are the selected antibiotics used. The pathogen cell membranes are broken down to prevent infection. In cell lines cross contamination can be rife, so using antibiotics prevents this and induced recombinant protein expression. Apart from preventing the obvious infection risk, if there is contamination there will be unreliable and inaccurate results. However, antibiotic resistance means that there may always be a level of low contamination. The prolonged use means antibiotics are only used where absolutely necessary so that it prevents these problems, such as in initial cell lines to prevent contaminated cells being carried on in sub cultures and protecting stock solutions.

Methods and Materials

After SH-SY5Y had been cultured, they then were transfected. NGB and CYGB plasmids were transfected with SH-SY5Y by nucleofectin. Nucelofectin is a transfection method that requires the use of electrode force to administer specific voltage. Reagents and electrodes produce the conditions required for transfection, which increases the permeability of the target cell. This allows the genetic material present in the culture to transfect into the globin plasmids. This is a reliable mechanism and produces good rates of success.

After transfection, the globins were fused with the GFP gene by PCR-amplification. The NGB to CYGB region was amplified and digested with restriction enzymes. Ligation was then performed directly after in to PEGFP-N1 vectors. The culture cells were briefly re-suspended in nucelofactor solution and nueclofected with 2μg of plasmid DNA, producing a final result of NgbN1-pEGFP and CygbN1-pEGFP fusion proteins. These produce a yield of 40% eFP positive cells. The PCR identified the expression

To examine the success of the transformation, PCR determined the expression of the globins. PCR measures the expression by recording the amount of mRNA present before and after amplification. For reactions involving GFP, fluorophene is added to act as a marker and signal upon excitation. Upon examination, over a 12 hour period there was upregulation 12 hours after transfection, meaning the globins were transfected successfully.

This examination isn’t thorough enough to provide evidence of success. A western blot was performed to ensure thorough examination. Protein expression can be detected by electrophoresing the proteins through a 10% polyacrimide gel. The proteins were transferred on to a western blot by being electroblotted to an Immobilon P membrane. After staining with primary polyclonal antibodies they were incubated with a secondary antibody, and probed for antibodies upon completion with Supersignal West Pico Chemiliminescent substrate. Figure 1 displays the result of the western blot.