Importance of Cell Cultures

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 1900s 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 environme nt which includes pH and osmotic pressure, as well as temperature. Temperature is kept at 37Â °C, CO2 levels at 5% and humidity at 95%. 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 dont 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 arent 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 Cell Line Original Cells Example paper Henrietta Lack (HeLa) cell line Cervical cancer cells from a biopsy from Henrietta Lacks, first immortalised cell line (Turner, 2012) COS-7 cell line Fibroblast-like cells from African Green Monkey kidney tissue (Vacante et al., 1989) SH-SY5Y cell line Neuroblastoma cells from a biopsy of a 4-year old female TO FIND AND ENTER!!! Hep G2 cell line Hepatocellular carcinoma cells from a biopsy of a 15-year old males liver (Mersch-Sundermann et al., 2004) Jurkat cell line T-lymphocyte cells in the blood of a 14-year old male leukaemia patient (Wang et al., 2012) 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. 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 1980s, 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 Parkinsons 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 globins 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 Dulbeccos minimum essential medium (DMEM) and Hams F-12 nutrient medium along with 10% foetal bovine serum at 5% CO2 atmosphere. The culture was maintained at 37Â °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 p revents 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 2ÃŽ ¼g 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 isnt 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.

Synesthetic Reality - Is our notion of reality the only truth? :: Synthesia Research Science Papers

Synesthetic Reality - Is our notion of reality the only truth? "What's first strikes me is the color of someone's voice. [V--] has a crumbly, yellow voice, like a flame with protruding fibers. Sometimes I get so interested in the voice, I can't understand what's being said." --From Synesthesia: A Union of the Senses by Robert E. Cytowic. What would you make of the preceding account? Would you think the speaker was...crazy?...on drugs?...making a play for attention? Would you be skeptical if the speaker told you it was her natural way of perceiving the world? In truth, it is an example of the way in which about one in every 25,000 people observes the world (1). The term (I hesitate to use "scientific term" for reasons I'll discuss further on) given to the condition-- synesthesia--derives from the Greek roots syn, meaning together, and aesthesis, to perceive, and conveys the principle features of the synesthete's perceptual state (2). In synesthetic perception, stimuli activate not just one sense, but several. An oral stimulus isn't a taste alone--it may also be a taste, a shape, a color, a movement (1). For example, a synesthete might explain that the taste of "squid produces a large glob of bright orange foam, about four feet away, directly in front of me" (3). Such joint perceptions are automatic and involuntar y, just as is usual perceptual experience, and, unlike imaginary images or ideas, synesthetic perception is not only vividly real, but "often outside the body, instead of imagined in the mind's eye" (2). Though accounts of synesthetic experience are receiving increased study and documentation, the many in the scientific community remain partially unconvinced, if not wholly dismissive. Lacking sufficient empirical, objective data to depict the synesthetic experience, synesthetes and researchers of the condition have had to combat doubt, disregard, and ridicule in defense of the condition's reality and validity. The question raised by synesthesia then becomes: Why does science discount first-person evidence to such an extent? If a condition has little to no "objective" or empirical "proof," does that mean it can't exist? If researchers can produce no computer read-out, no resonance imaging, no technologically-generated chart, should the scientific community turn up its nose? The existence of synesthesia has been questioned and discussed for nearly 300 years, and it received the most enthusiastic investigation between 1869 and 1930 (12).

Bus401 Mini Case Chapter 9

Percentage of future financing Type of financing Bonds (8%, $1,000 par, 16- year maturity38% Preferred stock (5,000 shares outstanding $50 par, $1. 50 dividend15% Common equity47% Total100% A. Market prices are $1,035 for bonds, $19 for preferred stock, and $35 for common stock. There will be sufficient internal common equity funding (i. e. , retained earnings) available such that the firm does not plan to issue new common stock. Calculate the firm's weighted average cost of capital. BondsPreferred stockCommon Stock 1035-15% (155. 25) = 879. 75 1. 50/(19-2. 01) 16. 99 = 8. 83% 2. 65/35 + . 06 = 13. 57% 9. 9% 9. 49% (1-. 34) = 6. 26% WeightsAfter tax captialProduct Bond 0. 38X6. 26%=2. 3788 Preferred Stock0. 15X8. 83%=1. 3245 Common Stock0. 47X13. 57%=6. 3779 10. 08% B. In part a we assumed that Nealon would have sufficient retained earnings such that it would not need to sell additional common stock to finance its new investments. Consider the situation now when Nealon's retained ear nings anticipated for the coming year are expected to fall short of the equity requirement of 47% of new capital raised. Consequently, the firm foresees the possibility that new common shares will have to be issued.To facilitate the sale of shares, Nealon's investment banker has advised management that they should expect a price discount of approximately 7%, or $2. 45 per share. Under these terms, the new shares should provide net proceeds of about $32. 55. What is Nealon's cost of equity capital when new shares are sold, and what is the weighted average cost of the added funds involved in the issuance of new shares? Common Stock 2. 65/32. 55 + . 06 = 14. 14% WeightsAfter tax captialProduct Bond 0. 38X6. 26%=2. 3788 Preferred Stock0. 15X8. 83%=1. 3245 Common Stock0. 47X14. 14%=6. 6458 10. 35%

A Brief Note On Alzheimer s Disease ( Ad ) Essay

Introduction Alzheimer’s disease (AD) is a neurological disorder commonly found in elderly individuals. It affects about 6-8% of people over the age of 65, and this number is expected rise in the coming decades (Gà ©linas et al., 1999). AD is a neurodegenerative disease that cause the deterioration of higher mental functions, this includes the deterioration of memory, cognitive tasks, and eventually motor and sensory skills. However, motor and sensory skills decrease much later in the development of AD. This is because AD is a hierarchical deterioration, attacking the first the complex functions before moving on to the simpler ones (Gà ©linas et al., 1999). Because of this, AD is one of the most frequent causes of dementia, about 70% of dementia patients developed the disorder from AD (Gà ©linas et al., 1999). Alzheimer’s disorder occurs in several stages (Roses, 1996). The first stage, Anticipating Metabolism, is in which patients report mild memory complaints. The next stag e is the Trigger, in which patients get diagnosed, but only after several visits to a specialist for the aforementioned memory complaints. In the third stage, Progression, memory problems and cognitive deterioration increases as plaques and other biological changes accumulate in the brain. Finally, the Terminal Neuropathology, in which further brain lesions occur (Roses, 1996). AD is a slow developing disease. The changes do not show themselves month-to-month, but rather manifest themselves whenShow MoreRelatedA Brief Note On Alzheimer s Disease ( Ad ) Essay882 Words   |  4 PagesBackground Alzheimer’s Disease (AD) is the most common type of dementia. AD is initially presented as short term memory loss, forgetfulness, and decrease ability to learn and retain new information. It is a slow and progressive disease. There are two types of AD, sporadic AD which is more common in patients who are 65 or older and familial AD. Amyloid precursor protein, presenilin-1 and presenilin-2 mutations are associated familial AD. Causes A cholesterol transport protein Apolipoprotein E is linkedRead MoreA Brief Note On Alzheimer s Disease ( Ad )2624 Words   |  11 PagesReview Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder affecting approximately 5.1 Americans, and has become one of the most common chronic diseases in developed countries. Consequently, a more comprehensive understanding of the etiology of the disease and possible treatment for AD has become a public health concern. An increasing body of evidence supports a strong and causal association between cardiovascular disease (CVD), its risk factors, and AD, along with its precursor, cognitiveRead MoreSymptoms Of Alzheimer s Disease2043 Words   |  9 PagesAlzheimer’s disease. It took an emotional toll on me and my family members. I believe Alzheimer is much harder on me and my loved ones than on my grandfather; because he was unaware of what was going on. It’s painful watching my grandfather degrade and get worse each and every day. The worst thing is that he doesn’t even know who I am anymore. In this paper I will discuss certain risk factors, background of Alzheimer’s, Background of Alzheimer’s Alzheimer’s disease was first identifiedRead MoreTreatments for Alzheimers Disease Essay1893 Words   |  8 PagesAlzheimer’s disease (AD) was named after Dr. Alois Alzheimer who described the symptoms in a woman in Germany in the 1907 but it was not until the 1970’s that AD was considered to be a major disorder and AD continues to be a major health concern worldwide (Reger, 2002). The onset of symptoms is usually between 40 and 90 years of age, although onset before 65 years of age is considered to be the early onset form of the disease and onset at 40 is very rare (Reger, 2002). Characteristics of AD are extracellularRead MoreThe Marketing Research of Brainquiry33782 Words   |  136 Pagesresearch provides detailed information related to Belgian psychologists that were found. The same counts for golf clubs. Extra information about Belgium includes Word documents containing information about Belgian ADHD support groups and Alzheimer s disease support groups, additional to this PDF files include information about patients with Mental Health problems in Belgium. These .pfd files are taken from Belgian federal statistics website. Unfortunately, not much information was not found