Mammalian+Cell+Culture

= The Mammalian Cell, Cell Types and Tissues =

What is a cell?
The Cell is the basic building block of all living organisms on Earth. Most living organisms are unicellular, comprised of only one individual cell; others (like humans, cats and dogs) are multi-cellular and contain trillions of living cells. All cells are a result of cellular division. A process in which a cell clones itself, duplicating its DNA and other cellular components, and splits into two cells. Just as our bodies function by unified organ systems, cells also contain their own intricate communication systems. These systems are referred as organelles.

There are two main cell types: eukaryotic and prokaryotic cells.
 * Prokaryotic cells are single celled organisms, most commonly bacteria. Prokaryotic cells are simple, they have no nucleus or membrane bound organelles. The contain a membrane and a cell wall, and due to their unicellular nature often have different forms of locomotion.[[image:http://upload.wikimedia.org/wikipedia/commons/thumb/5/5a/Average_prokaryote_cell-_en.svg/494px-Average_prokaryote_cell-_en.svg.png caption="external image 494px-Average_prokaryote_cell-_en.svg.png"]]
 * Eukaryotic cells are plant and animal cells and are almost always multicellular. They are more complex than prokaryotic cells, they contain a nucleus and membrane bound organelles. They contain a cell wall only in plant cells and the cells are semi-permeable in order to allow substances in and out as necessary.[[image:http://evolution.berkeley.edu/evosite/lines/images/cells.gif caption="external image cells.gif"]]

Most commonly in cellular biology, the mammalian cell is the subject of interest. Research and testing done on mammalian cells leads to greater understanding out human cellular physiology and leads to new advance s in science and technology. In order to understand the cell and how it works, the basic subunits of the cell must be well understood before advancing into a deeper understanding of the cells functionality: 
 * Cytoplasm - is made up of cytosol, it contains the organelles and allows for the transport of nutrients and proteins. It is Jelly-like and is composed of about 80% water. Cytoplasm contains a lattice structure that supports other the other solid structures in the cell called the cytoskeleton. Cytoplasm is constantly flowing, the nucleus usually travels with it, and this allows for effective transport of nutrients.

> Intermediate filaments are the second thinnest filaments on the cytoskeleton. > Microtubules are the thickest filament in the cytoskeleton. They make up the structure of the cell and the internal structure of cilia and flagella as well as provide a track for organelle movement and aid in cell division. 
 * Cytoskeleton - The cytoskeleton is a series of intercellular proteins that help with shape, support, and movement. It has 3 main structural components:microfilaments, intermediate filaments and microtubules.Microfilaments are the thinnest filaments. They are relatively strong and flexible.
 * Endoplasmic Reticulum - The endoplasmic reticulum (commonly referred to as the ER) is a series of sacs and tubules that extend throughout the cytoplasm of plant and animal cells. They are connected by a single continuous membrane which means that this organelle has a very large and complex lumen. This often takes up more than 20% of the cells space. The ER membrane allows for the selective transport of molecules between the lumen and the cytoplasm. Most of the proteins and molecules are destined to leave the ER, however there are a few proteins that are destined to stay in the ER called ER resident proteins, which are necessary for the ER to carry out its specific functions. They do things such as help identify proteins that have been improperly built.


 * Rough Endoplasmic Reticulum: The surface of this part of the ER is covered with ribosomes and has a bumpy appearance when viewed with microscope. Involved mainly in the production and processing of proteins that will be exported. The ribosomes assemble amino acids into proteins that will be further processed later.
 * Smooth Endoplasmic Reticulum: Most proteins exit the ER by budding off of the smooth ER. It is less extensive than the rough ER. It is involved in producing lipids and detoxifying drugs and poisons, therefore the smooth ER much more extensive in organs like the liver.
 * Golgi Apparatus - The Golgi packages molecules that are to be transported outside of the cell. The golgi apparatus, or golgi body is comprised of 5 to 8 cup-shaped, membrane-covered sacs called cisternae and they resemble stacks of deflated balloons. Because it is so big it was one of the first organelles ever observed. It modifies proteins and lipids from the ER. The golgi apparatus has two distinct ends, the cis face where the molecule enters and the trans face where the molecule exits.
 * Lysosome - The lysosome r ecycles cell components that are worn out in a process called autophagy. It breaks down cellular waste products, fats, carbs, proteins, and other macromolecules into simple cell-building materials. It uses about 40 different types of hydrolytic enzymes which are manufactured in the ER and modified in the Golgi apparatus. They are round shaped organelles contained in a single membrane layer. This membrane protects the cell from the harsh enzymes within the lysosome and the are most numerous in disease fighting cells like white blood cells.


 * Mitochondria - They are known as the "power generators" of the cell. Mitochondria convert nutrients into energy that the cell can actually use. They convert oxygen and nutrients into adenosine triphosphate (ATP) through a process called Aerobic respiration. Without this organelle, higher animals would not likely exist since cells would only be able to survive off of anaerobic respiration. Mitochondria allow a cell to develop up to 15 times more ATP than a cell could on its own. mitochondria contains its own set of genetic material, this is due to the idea that mitochondria was once a prokaryote (endosymbiotic theory).


 * Nucleus - The nucleus is known as "the command center" of the cell which controls how and when the cell will grow, the intermediary metabolism, protein synthesis and reproduction (cell division). The nucleus is only found in plant and animal cells and it occupies about 10% of the cell volume. It has a double layered membrane. The nuclear envelope separates the nuclear contents from the cytoplasm, and it has many pores to allow specific types and sizes of molecules to travel back and forth. The liquid matrix in the nucleus is the nucleoplasm. Within it contains the less condensed form of the genetic material called chromatin. The nucleus also contains anucleolus which is a membrane-less organelle that manufactures ribosomes.


 * Plasma Membrane - Both eukaryotic and prokaryotic cells have a plasma membrane that encloses their contents and serves as a semi porous barrier, (it is permeable only to specific molecules). Small molecules like oxygen are allowed to pass freely, while amino acids and sugars are regulated by proteins. The current accepted theory of the structure of the plasma membrane is the fluid-mosaic model. The membrane is a bilayer membrane since it is composed of two lipid layers. Lipids in the bilayer are known as phospholipids which contain a hydrophilic and hydrophobic end.

Connective Tissue
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Connective tissue is a fibrous tissue. It is made up of cells separated by non-living material known as the extracellular matrix. Connective tissue gives shape to organs and holds them in place. As the name implies, these support and bind other tissues. Unlike epithelial tissue, connective tissue typically has cells scattered throughout an extracellular matrix. <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">ex. blood and bone are examples of connective tissue.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 1.1em;">Muscle Tissue
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Muscle tissue is composed of muscle cells, and this muscle tissue can stretch and contract in order to produce motions and force. There are three types of muscle tissue:
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Smooth muscle which lines the inside of organs.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Skeletal muscle which is attached to bone in order to allow for movement of limbs.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Cardiac muscle which is found in the heart which allows for the pumping of blood.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 1.1em;">Nervous Tissue
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">The cells that make up the central nervous system and peripheral nervous system are classified as neural tissue. In the central nervous system, neural tissue forms the brain and spinal cord and, in the peripheral nervous system forms the cranial nerves and spinal nerves, inclusive of the motor neurons. Nervous tissue transmits messages in form of an impulse, this is how the body can move and respond to outside stimuli.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 1.1em;">Epithelial tissue
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">The epithelial tissue is formed by cells that cover the organ surfaces such as the surface of the skin, the airways, the reproductive tract, and the inner lining of the digestive tract. These cells are linked via semi-permeable, tight junctions; therefore, this tissue provides a barrier between the outside environment and the organ. Epithelial tissue may also be specialized to function in secretion and absorption. Epithelial tissue helps to protect against microorganisms, injury, and fluid loss. Examples of epithelial tissue are:
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">the cells that form the outer layer of skin.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">lining of mouth.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">help in the absorption of water and nutrients.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">helps in elimination of wastes.

<span style="font-family: Arial,Helvetica,sans-serif; font-size: 1.1em;">Meristematic tissue
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Meristematic tissue consists of actively dividing cells,found at the growing end of plants. The primary growth of a plant occurs only in specific regions, such as in the tips of stems or roots. The cells of meristematic tissues are similar and have thin and elastic primary cell wall made up of cellulose which helps aid in the growth process. <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Examples include:
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Apical Meristem - It is present at the growing tips of stems and roots and increases their length.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Lateral Meristem - This meristem mainly divides in one direction and causes the plant to increase in diameter. Lateral meristems usually occur beneath the bark of the tree (Cork Cambium).
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Intercalary Meristem - This meristem is located in between permanent tissues. It is usually present at the base of node, inter node and on the base of the leaf.

<span style="font-family: Arial,Helvetica,sans-serif;">Permanent Tissue
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">This tissue loses its ability to divide, this process is called cellular differentiation. Cells of meristematic tissue differentiate to form different types of permanent tissue. There are 3 types of permanent tissues:
 * <span style="font-family: Arial,Helvetica,sans-serif;">simple permanent tissues - These tissues are described as homogenous, since the constituent cells are identical in their structure.
 * <span style="font-family: Arial,Helvetica,sans-serif;">complex permanent tissues - These tissues are characterised by the presence of dissimilar cells.
 * <span style="font-family: Arial,Helvetica,sans-serif;">special or secretory tissues - <span style="color: #333333; font-family: Georgia,Cambria,'Times New Roman',Times,serif; font-size: 14px;">The tissues which are concerned with special functions like secretion or excretion of different kinds of substances like resins, latex, gums, nectar etc.

Mammalian Cell Culture Background
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Mammalian cell cultures have led to an unimaginable number of advancements in the cellular biology field. At its inception in the early twentieth century and was applied to the study of tissue fragment in culture. New growth in culture was limited to the cells that migrated out from the initial tissue fragment. Tissue culture techniques evolved rapidly, and since the 1950’s culture methods have allowed the growth and study of dispersed cells in culture Mammalian cell tissue culture has come a long way in a relatively short period of time in the scientific world, but the study of cellular physiology in depth is still relatively new. The cell culture technique can now be used incytogenetic, biochemical, and molecular laboratories for diagnostic and research studies. Cells may be grown in a culture for days or weeks as needed in order to obtain enough cells for analysis. Maintenance of cells long term requires strict adherence to aseptic techniques in order to avoid contamination of the culture and loss of a valuablecell line. As cells reach a stage of confluency they must be subcultured or passaged. This means is that some of the cells are taken from the culture and moved to a fresh growth medium. If this is not done cells will reach a reduced mitotic index and this will eventually lead to cell death.

<span style="font-family: Arial,Helvetica,sans-serif;">Methods


<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">The first step to subculturing these monolayers is to detach cells from the surface of the primary culture vessel by trypsinization (using trypsin to dissociate adherent cells) or by mechanical means. The resulting cell suspension is then subdivided into fresh cultures. These secondary cultures are then monitored for growth, fed periodically and if need be, subculturing is repeated to create tertiary cultures and so on. The time between subcultures varies depending on the cells growth rate and cell line. <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">One factor in cell culture is the choice of tissue culture medium. There are many different recipes for cell cultures mediums that are available, depending on the needs of the cell culture and experiment, and it can be obtained in a sterile, ready to use liquid form or a powdered form that must be prepared. These mediums often contain antibiotics,fungicides, or both to help prevent contamination.

<span style="font-family: Arial,Helvetica,sans-serif;">Why is this necessary?
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">Cell Culture helps greatly with our basic knowledge and understanding of how different tissues function, and today services as a fundamental necessity to manufacture viral vaccines. Cell cultures produce products such as enzymes, synthetic hormones, immunobiologicals, and anticancer agents, and certain complex molecules and proteins can only be produced by animal cells, such as erythropoietin. Research in tissue engineering, stem cells and molecular biology requires the use of two-dimensional cell culture, which was first developed by Wilhelm Roux in 1885. Advances in cell culture techniques have also led to the discoveries of stem cell self-renewal, cancer cell phenotypes, lineage specification and fibrosis; all discoveries in cellular physiology that may eventually lead to new understandings and treatments of diseases. One recent and phenomenal new discovery is the capability to culture one’s cells and then put them into a drip over the protein structure of an organ, in order to “grow” a new organ for transplant with no chance of rejection since the patient’s own cells make up the new organ.

<span style="font-family: Arial,Helvetica,sans-serif;">Mammalian Cell Culture History
<span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif;">


 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Along with Wilhelm Roux’s initial basic invention of mammalian cell culture in 1885, this technique has had quite an extensive history.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">In 1913 <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> Alexis Carrel <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> revealed that cells could be grown for long periods of time in a cell culture if fed regularly under aseptic conditions.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">In 1952 <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">George Otta Gey <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> and his colleagues establish a continuous line of cells derived from a human cervical carcinoma, which later become the well-known HeLa cell line.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">In 1955 <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Harry Eagle <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> created the first systematic investigation of the essential nutritional requirements of cells in culture and found that animal cells can propagate in a defined mixture of small molecules supplemented with a small proportion of serum proteins.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">In 1964 <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Littlefield <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> introduces HAT medium for the selective growth of somatic cell hybrids. Together with the technique of cell fusion, this makes somatic-cell genetics accessible.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">In 1965 <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Ham <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> introduces a defined, serum-free medium able to support the clonal growth of certain mammalian cells.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">In 1976 <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Sato <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> and associates publish the first of a series of papers showing that different cell lines require different mixtures of hormones and growth factors to grow in serum-free medium.
 * <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">In 1998 <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Thomson <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> and <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">Gearhart <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;"> and their associates isolate human embryonic stem cells. <span style="font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">[[file:///C:/Users/SUNY%20OSWEGO/Documents/Cell%20Phys%20Midterm%203.docx#_ftn4|[4]] <span style="background-color: #ffffff; font-family: Arial,Helvetica,sans-serif; line-height: 1.5;">]

== <span style="font-family: Arial,Helvetica,sans-serif;">Mammalian Cell Culture and its Application Today ==


 * <span style="font-family: Arial,Helvetica,sans-serif;">Research on Mammalian cell culture did not end in the 1990’s. There is still a lot of research being done today on, and involving cell culture, especially with the strong advocacy to cure cancer. One article, //<span style="font-family: Arial,Helvetica,sans-serif;">Genetic and epigenetic instability in human pluripotent stem cells //<span style="font-family: Arial,Helvetica,sans-serif;"> (2012), is a review article that was really aiming on focusing the research for stem cell research. There was increasing evidence that human pluripotent stem cells (hPSCs) were prone to epigenetic instability during in vitro culture. This inference turned out to be true after reviewed and searching through past articles involving the cell culture of the specified cells. Many epigenetic aberrations had been detected in hPSCs and recurrent genetic alterations gave a selective advantage in a culture to the altered cells which led to overgrowth of abnormal, culture adapted cells. The reason for this is not quite yet understood, but due to the high reports of epigenetic alterations and altered phenotypic characteristics of abnormal cells, researchers are now able to control for the epigenetic integrity of hSPCs before any clinical application is a necessity.[[file:///C:/Users/SUNY%20OSWEGO/Documents/Cell%20Phys%20Midterm%203.docx#_ftn5|[5]]] This paper revealed that not all is understood about cell culture, and so many cells are so specialized that specific measures should be taken for each individual type of cell culture.
 * <span style="font-family: Arial,Helvetica,sans-serif;">One issue that has been realized in cell culturing techniques is how cells respond to the rigidity of their environment. The research article, //<span style="font-family: Arial,Helvetica,sans-serif;">Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture //<span style="font-family: Arial,Helvetica,sans-serif;"> (2010), addresses that problem in terms of muscle stem cells (MuSCs). These muscle stem cells exhibited robust regenerative capability in vivo but is lost in culture. Researches then bioengineered a substrate to recapitulate the key biophysical and biochemical features using an algorithm that ultimately revealed substrate elasticity is an important regulator of MuSC fate in culture. Unlike rigid plastic dishes, MuSCs cultured much better on soft hydrogel substrates that mimicked the elasticity of a muscle.[[file:///C:/Users/SUNY%20OSWEGO/Documents/Cell%20Phys%20Midterm%203.docx#_ftn6|[6]]] This is extremely important for further studies using these cells, and this algorithm can be applied to other cell types to improve other cells cultures. This revealed that cells are highly unique and cannot survive just anywhere if they are given the proper nutrients. Just like human could not survive in the arctic with plenty of food and no coat, certain cells cannot survive in any kind of substrate even if it has the proper nutrients.
 * <span style="font-family: Arial,Helvetica,sans-serif;">As medicine and technology improve, scientists have new ways of conducting research that they never had before. Human cells clearly do not grow on a flat surface; the world they are a part of is three dimensional. In the review article, //<span style="font-family: Arial,Helvetica,sans-serif;">Organotypic 3D cell culture models: using the rotating wall vessel to study host–pathogen interactions //<span style="font-family: Arial,Helvetica,sans-serif;">, delves into three dimensional cell culture. These researchers were able to establish 3D hierarchical models that range in complexity from a single cell type to multicellular co-culture models that recapitulate the 3D architecture of tissues observedin vivo. These new findings can be applied to the study of infectious diseases and how they affect the body and in what other ways cures can be administered in the ultimate three dimensional model, humans.[[file:///C:/Users/SUNY%20OSWEGO/Documents/Cell%20Phys%20Midterm%203.docx#_ftn7|[7]]] What was important about this topic is that new steps are being taken to mimic the actual mammalian structure even more and more. This is allowing better results and applications of new discoveries to be applied to new treatments and cures.

<span style="background-color: #ffffff; font-family: 'Times New Roman',serif; font-size: 10pt;"> <span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">"Basic techniques for mammalian cell tissue culture." Current Protocols in Cell Biology. georgelab.eng.uci.edu/resources/Basic%20Technique%20for%20Tissue%20Culture.pdf (accessed October 6, 2013). <span style="background-color: #ffffff; font-family: Calibri,sans-serif; font-size: 10pt;"> <span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">"Basic techniques for mammalian cell tissue culture." (accessed October 6, 2013) <span style="background-color: #ffffff; font-family: 'Times New Roman',serif; font-size: 10pt;"> <span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">Alberts, Bruce. //Molecular biology of the cell//. 4th ed. New York: Garland Science, 2002. <span style="background-color: #ffffff; font-family: Calibri,sans-serif; font-size: 10pt;"> <span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">Alberts, Bruce. // Molecular biology of the cell //. 4th ed. New York: Garland Science, 2002. <span style="background-color: #ffffff; font-family: 'Times New Roman',serif; font-size: 10pt;"> <span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">Nguyen, H. T.; Geens, M.; Spits, C. (2012). "Genetic and epigenetic instability in human pluripotent stem cells".//Human Reproduction Update// **19** <span class="apple-converted-space" style="background-color: #ffffff; font-family: 'Times New Roman',serif;"> (2): 187–205. <span style="background-color: #ffffff; font-family: Calibri,sans-serif; font-size: 10pt;"> <span style="background-color: #ffffff; font-family: Arial,sans-serif; font-size: 9pt;">Gilbert, P.M. et al. //Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture//. Science 329, 1078–1081 <span style="background-color: #ffffff; font-family: 'Times New Roman',serif; font-size: 10pt;"> <span style="background-color: #ffffff; font-family: 'Times New Roman',serif;">Barrila, Jennifer, Andrea L. Radtke, Aurélie Crabbé, Shameema F. Sarker, Melissa M. Herbst-Kralovetz, C. Mark Ott, and Cheryl A. Nickerson. "Organotypic 3D Cell Culture Models: Using The Rotating Wall Vessel To Study Host–pathogen Interactions." //Nature Reviews Microbiology// <span class="apple-converted-space" style="background-color: #ffffff; font-family: 'Times New Roman',serif;"> 8, no. 11 (2010): 791-801. [] (accessed October 6, 2013).