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Cell culture Supervisor: Prof. Luke O’Neill

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1 Cell culture Supervisor: Prof. Luke O’Neill
Sinead Cunningham, Oluwasola Lawal, Alicia Smyth.

2 Revolutionary Technique.
Ease of growing and manipulating cultured cells in different ways. Growing excitement in this field. Evidence for the importance of the cell culturing technique was highlighted in 2012 when the Noble Prize for Physiology and Medicine went to John B. Gurdon of the University of Cambridge in England and Professor Shinya Yamanaka of Kyoto University in Japan.

3 It is an essential tool for bettering human health prospects.
The cell culture technique opens up many possibilities for the study of: abnormal differentiation and malignancy. for screening systems. for diagnosis. for drug development for known pathologies. It is an essential tool for bettering human health prospects.

4 Theoretical basis of the Technique Sinead Cunningham

5 What is cell culture? Cell culture refers to the removal of cells from an animal or plant (multicellular organism). It is the subsequent growth and maintenance of these cells in an artificial favourable environment. Cells are removed from the tissue directly and disaggregated (separated) by enzymatic or mechanical means. They can also be derived from a cell line or cell strain that has already been established.

6 Cell culture terminology
Primary culture - stage of the culture after the cells are isolated from the tissue and have proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence).  Sub-culturing- transferring primary culture cells to a new vessel with fresh growth medium to provide more room for continued growth. Senescence- limited replicative potential of primary cells in culture. Cells that can not proliferate indefinitely are described as “mortal”. An “immortal” cell line is one that can proliferate indefinitely, such as tumour cells. A cell line - After the first subculture, the primary culture becomes known as a cell line or subclone. 

7 Trypsinization - the process of using trypsin, a proteolytic enzyme which breaks down proteins.
It dissociates adherent cells from the vessel in which they are being cultured. When added to a cell culture, trypsin breaks down the proteins which enable the cells to adhere to the vessel. Trypsinization is often used to passage cells to a new vessel. Passaging - the process of sub-culturing cells in order to produce large numbers of cells from pre-existing ones. Passaging (also known as subculture or splitting cells) involves splitting the cells and transferring a small number to a new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density.

8 History of the Cell Culture Technique: Key Events!
The Beginning 1907, Ross Granville Harrison Looked at frog tissue in vitro. Frog embryo nerve fibre outgrowth in vitro. Alexis Carrel, 1912. Grew pieces of chick embryo in various media initially maintained for 85 days, and subsequently for years. Trypsinization is the process of using trypsin, a proteolytic enzyme which breaks down proteins, to dissociate adherent cells from the vessel in which they are being cultured. When added to a cell culture, trypsin breaks down the proteins which enable the cells to adhere to the vessel. Trypsinization is often used to passage cells to a new vessel. In cell culture, passaging is the process of sub-culturing cells in order to produce large number of cells from pre-existing ones. Passaging (also known as subculture or splitting cells) involves splitting the cells and transferring a small number into each new vessel. Cells can be cultured for a longer time if they are split regularly, as it avoids the senescence associated with prolonged high cell density. Suspension cultures are easily passaged with a small amount of culture containing a few cells diluted in a larger volume of fresh media. For adherent cultures, cells first need to be detached, commonly done with a mixture of trypsin-EDTA. A small number of detached cells can then be used to seed a new culture, while the rest is discarded. Also, the amount of cultured cells can easily be enlarged by distributing all cells to fresh flasks. In the average lab, adherent cells are grown in petri dishes, multi-wells plates or culture flasks, with FCS containing culture media at 37 °C with 5 % CO2. In the case of more or less fast growing cells like HeLa, a freshly seeded plate will reach confluency in two or three days. If nothing is done, the food will run out and the cells will die shortly thereafter, so passaging is required. Thus, the media is removed, the cells are washed with phosphate buffered saline (PBS), then 1 ml of trypsin is added to make the cells detach from the bottom of the plate. Trypsin works best in a warm surrounding, so the plate is incubated for five minutes in 37 °C. In order to stop the reaction, 1 ml of trypsin inhibor stock solution is added (1mg/ml in water or PBS). Then, 8 ml of PBS is added and the cells are resuspended. An appropriate number of cells in suspension is then transferred new plates, fresh media is added to each plate, and the new plates are incubated for the next growth phase. For best results, cells are kept less than 100 % (log phase of growth) but more than 10 % confluent. Cells die if they get too lonely (“apoptotic death of neglect”) or much too crowded (death by metabolic decay). Trypsin is an endopeptidase produced by the gastro-intestines of mammals, and has an optimal operating pH of about 8 and an optimal operating temperature of about 37 °C. In vivo, trypsin is produced in the pancreas in the form of inactive zymogen, trypsinogen. After secretion into the duodenum, the enzyme enteropeptidase activates a small number of the enzymes into trypsin by proteolytic cleavage, followed by autocatalysis of trypsins to activate the whole secreted mass. Then trypsin acts to hydrolyse pepsin-digested peptides by hydrolysis of peptide bonds. The aspartate residue (Asp 189) located in the catalytic pocket (S1) of trypsins is responsible for attracting and stabilizing positively-charged lysine and/or arginine. Thus, trypsin predominantly cleaves proteins C-terminally of the amino acids lysine and arginine, except when either is followed by proline. Trypsins should be stored at very cold temperatures (between −20 °C and −80 °C) or at a pH of 3 in order to prevent autolysis 1916, Rous and Jones discovered that trypsin is capable of degrading matrix proteins, thus separating cells. Rous and Jones began plating out cells.

9 1920’s- 1950’s 1923, Fischer and Carrel began to generate what was termed cell strains. 1943, Earle et. al established the first continuous cell line, the L-cell mouse fibroblast 1948, Antibiotics were beginning to be used in tissue culture which enabled long term cell line propagation, although many people were sceptical about harbouring antibiotic resistant contaminations. 1948, Sanford cloned the first cell strain called L292. It was isolated by capillary cloning from mouse L-cells. Trypsinisation then became popular in the 1950’s In Dulbecco’s work as a way of making single cell suspensions for single cell cloning

10 1950’s and 1960’s It was then in 1952 that George Gey established the first continuous immortal human cell line from cervical carcinoma in a patient named Henrietta Lacks. He called them HeLa cells. HeLa cells were subsequently extremely important in the development of vaccines for polio. 1955, Eagle developed a defined media which ultimately led to the development of serum free media. Requirement of defined media for serum growth factors. 1961, Hayflick and Moorhead defined the finite life span of normal human cells 1964 – the pluripotency of embryonic stem cells established by Kleinsmith and Pierce .

11 Late 1960’s and 1970’s , measels, mumps and rubella vaccines in WI-38 human lung fibroblasts were developed (Wiktor et. al., ; Sokol et al., 1968) 1965, Serum free cloning of Chinese Hamster Cells ( Ham, 1965) 1967, He-La cell cross contamination (Gartler, 1967.) Density Limitation of cell proliferation (Moore et al., 1967 ; Gerper et al., 1969) 1970’s- development of laminar flow cabinets. Hybridomas- monoclonal antibodies

12 1970’s – 2000’s 1977, Cross-contamination of many cell lines with HeLa cells confirmed, Nelson-Rees and Glandermeyer. 1983, Regulation of cell cycle and cycling reported by many. , Development of many specialized cell lines. 1989, Transformation, malignancy, oncogenes reviewed by Weinberg. 1990, Application of cell culture to production of biotherapeutic agents. 1998, Production of cartilage by tissue engineered cell culture by Aigner et al. 2000, Mapping of the human genome. 2007, Use of viral vectors to reprogram adult cells to embryonic state (induced pluripotent stem cells) by Yu et al. 2008 and beyond, Era of induced pluripotent stem cells – promises and challenges.

13 Ethics The use of animal models is decreased as various tests can be performed in vitro. Not sacrificing animals when not necessary. The process of implanting foetal neurons into patients with Parkinson’s disease could benefit these patients. Society must decide to what extent foetal and gamete material can be used.

14 Economic Benefit Cultures can be exposed to a reagent at a lower and defined concentration with direct access to the cell. Therefore less reagent is required in cell culture experiments than for injection of a reagent in vivo, where 90% may be lost by excretion and distribution to other tissues other than those under study. Screening tests with many variables and replicates are cheaper, and the legal, moral and ethical questions of animal experimentation are avoided. Cells grow rapidly.

15 Media for culturing cells
When cells are growing in vitro there is an absence of homeostatic regulation which is found in vivo, such as a nervous and an endocrine system. This means that the conditions in which the cells find themselves may not be truly representative of the tissue from which the cells were derived. This is why additions must be made. Initial attempts to culture cells were performed in natural media based on tissue extracts and body fluids, such as chick embryo, plasma, serum and lymph. With the propagation of cell lines (L929 cells, HeLa cells etc.) the demand for larger amounts of medium of a more consistent quality. This led to the introduction of chemically defined media based on analysis of body fluids and nutritional biochemistry. Eagle’s Basal Medium (in 1955) and subsequently Eagle’s Minimal Essential Medium (MEM) became widely adopted. Supplemented with bovine calf (CS), foetal bovine(FBS), human (usually only with human cell lines) or adult horse serum, protein hydrolysates, and embryo extract. Serum contains growth factors which promote cell proliferation, and adhesion factors which promotes cell attachment. Serum also contains lipids, minerals and hormones many of which are bound to protein. As more continuous cell lines became available it became clear that this media was adequate for the majority of them.

16 Minimum Essential Medium
Minimum Essential Medium (MEM) is one of the most widely used of all synthetic cell culture media.  Early attempts to cultivate normal mammalian fibroblasts and certain subtypes of HeLa cells revealed that they had specific nutritional requirements that could not be met by Eagle's Basal Medium (BME).  Additions to BME could be made to aid growth of a wider variety of fastidious cells.  MEM, which incorporates these modifications, includes higher concentrations of amino acids so that the medium more closely approximates the protein composition of mammalian cells. MEM has been used for cultivation of a wide variety of cells grown in mono-layers. Succeeding developments were aimed at replacing serum Optimizing media for different cell types.

17 Examples of Media RPMI 1640 – for lymphoblastoid cell lines. RPMI-1640 was developed by Moore et. al. at Roswell Park Memorial Institute, hence the acronym RPMI. RPMI utilizes a bicarbonate buffering system and alterations in the amounts of amino acids and vitamins. DMEM (Dulbecco’s modification of Eagles medium) –cells grown for the formation of products, as hosts for viral propagation, or for non-cell-specific molecular studies rely on this medium supplemented with serum. contains a four-fold higher concentration of amino acids and vitamins than MEM, as well as additional supplementary components. The original DMEM formula contains 1000 mg/L of glucose and was first reported for culturing embryonic mouse cells. Serum-free-media –facilitates downstream processing and reduce risk of infectious agents. Antibiotics such as penicillin and streptomycin can be added to media to manage contamination

18 Cell Types and Media Cells or cell line Medium Serum
Chick embryo fibroblasts Eagles MEM calf serum CS Chinese hamster ovary (CHO) Eagles MEM, HAMs F12 Endothelium DMEM, M199, MEM Glioma MEM, DMEM/F12 Foetal bovine FB HeLa cells L cells L929 Mouse leukemia Fisher’s, RPMI 1640 Foetal Bovine FB, Horse serum HoS

19 Adherent cells and suspension cells
There are two basic systems for growing cells in culture as monolayers on an artificial substrate (i.e., adherent culture) or free-floating in the culture medium (suspension culture). Cell adhesion Cell adhesion- cells from solid tissues grow as adherent monolayers. Cells need to spread out on the substrate before they start to proliferate. Cell adhesion is mediated by specific cell surface receptors for molecules in the extracellular matrix.

20 So, having made the right choice of Media for the particular cell type that is to be grown and considering the benefits of this technique the next question is – How is it performed?

21 How to perform the technique
Oluwasola Lawal

22 Method Cells can be isolated from different sources i.e tissue, blood etc Then placed in a flask containing medium

23 Method Medium contains nutrients for cells
Flask is then placed in an incubator with appropriate temperature and gaseous atmosphere

24 Method Change medium from time to time to minimize contamination

25 Cell Counting Cells are counted using a device known as hemocytometer
Cells stained with trypan blue dye

26 Cell Counting Trypan blue dye used to distinguish between viable and nonviable cells Viable cells = bright unviable = blue Number of cells per ml = no of viable cells*dilution factor*104

27 Aseptic Technique Cell culture work should be done in the laminar flow-hood – clean air 70% alcohol should be used as a disinfectant – for gloves and materials

28 Equipment Cell culture hood Incubator Centrifuge Water bath
Cell counter (hemocytometer) Autoclave Refrigerator and freezer

29 Applications of Cell culture
Alicia Smyth

30 Basic Research Cell cultures provide excellent model systems for the study of the basic physiology and biochemistry of eukaryotic cells. It allows a particular cell type to be grown in carefully controlled and easily observable conditions. This provides us with the ability to manipulate that environment in order to observe particular features of the cells or to expose them to particular stimuli in order to identify its effect on the cell It provides the means to study; morphology, intercellular signalling, the effects of disease on cells, the cell cycle, host-pathogen interactions on a cellular level and gene expression as well as many other cellular processes.

31 Virology and Vaccines Virology benefits from advances in cell culture as many animal viruses cannot survive independently and must be grown in animal cells in order to be studied. Poliovirus, measles, mumps, rubella and varicella vaccines are currently being manufactured using cell culture based methods. Live attenuated vaccines generally rely on human cell culture and involve cultivating the virus in successive human cell cultures and selecting the strains that grow poorly in human cells leading to attenuated strains which replicate poorly in humans but can still induce a strong immune response.

32 Genetic Engineering The ability to transfect cultured cells with new genetic material has led to massive advances in both the study of genetics and in the production of biological compounds E.coli is generally used in the production of recombinant proteins but has many disadvantages including the inability to make posttranslational modifications. CHO cells are the primary mammalian cell line used for the production of therapeutic recombinant therapeutics. Including erythropoietin, Herceptin, clotting factors, interferon-β Biological products produced by recombinant DNA technology in animal cell cultures include monoclonal antibodies, enzymes, synthetic hormones, and anticancer agents.

33 Antibodies and hybridoma
Monoclonal antibodies are vitally important in diagnostics, targeting drug therapy and identifying different proteins. A hybridoma cell consists of a myeloma cell (an immortal cancerous cell) fused to a normal antibody producing cells from a rat or mouse spleen. The antibodies produced can be used in diagnostics and treatment of disease. They are also used in research to identify and locate proteins. They are part of a number of cell sorting technologies including flow cytometry, western blot, immunoprecipitation and ELISA.

34 Drug screening and toxicity testing
Live cell based assays are becoming an increasing element of the drug discovery environment. Cultures of healthy cells are increasingly required for research and screening. It is a cost-effective, ethical and efficient alternative to animal screening. Toxicity screenings for drugs and cosmetics take advantage of cell culture techniques to assess basal functions of the cell or specialised cell functions. Cytotoxicity can also be assessed in specialised cell lines, when in vivo models have already proven toxic, in order to establish the mechanism of the toxic action of the drug.

35 Measuring toxicity General toxic effects Organ specific toxic effects
Vital staining Membrane Integrity Cytosolic enzyme release Metabolic alterations Cell growth Synthesis or release of specific molecules/enzymes Cloning efficiency Interactions of compounds with cells e.g. phagocytosis

36 Cancer Research Cell culture techniques are the vital basis for a large majority of experimental cancer research. Almost all malignant tumour entities were established as immortal cell lines, and many of them are available commercially. The reproducibility and reliability of these cellular systems, however, is highly influenced by cell and tissue culture conditions, and many cellular functions can be modulated purely by altering the conditions of the culture.

37 Tissue Engineering Tissue culture is an extension of cell culture where entire tissues are grown and sustained as opposed to individual cells. The conditions required to maintain the tissue and allow it to survive must be carefully mediated, including; oxygen, pH, humidity, temperature, nutrients and osmotic pressure maintenance. Cells are grown on a structure or matrix which supports the three dimensional element of tissue formation. These are scaffolds. Tissues produced this way include artificial bladders, pancreas, cartilage, skin, bone and bone marrow and cardiac tissue.

38 Conclusion Theory Cell culture- is growing and maintaining cells outside of the biological system of an organism. Supplements and additives must be used to create a favourable environment for the cells to grow in. Long history of research and discovery building on the cell culture technique. Method Isolation Plating Cell counting Aseptic technique Equipment Applications Basic research Virology Genetic Engineering Toxicity Cancer research Tissue engineering

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