BENG0011-lecture 3.pdf

Introduction to Introduction to Cell Culture and Aseptic Processing Lecture 3 BENG0011-Manufacturing Regenerative Medicines: From Lab Bench to Industry Dr Rana Khalife 1 Introduction Cell culture refers to a culture derived from...

Introduction to Introduction to Cell Culture and Aseptic Processing Lecture 3 BENG0011-Manufacturing Regenerative Medicines: From Lab Bench to Industry Dr Rana Khalife 1 Introduction Cell culture refers to a culture derived from dispersed cells taken from original tissue, from a primary culture, or from a cell line or cell strain by enzymatic, mechanical, or chemical disaggregation. What is biosafety? Biosafety is the application of safety precautions that reduce a laboratory risk of a potential infection and limit contamination of the work environment What is meant by aseptic technique? Aseptic technique is a process or procedure used to achieve asepsis to prevent the transfer of potentially pathogenic micro-organisms to a susceptible site that may result in the development of infection (Wilson, 2019).  What are the principles of aseptic techniques Creating a microorganism-free environment (sterile field) Use of sterilized instruments Maintaining sterility of sterile field and instruments by preventing microbial contamination by contact with non-sterile objects How aseptic environment is achieved: Safety Equipment Personal Protective Equipment biosafety cabinets (PPE) enclosed containers gloves laboratory coats shoe covers Respirators face shields safety glasses Safety -PPE PPE is equipment that will protect the user against health or safety risks at work. It can include items such as safety helmets, gloves, eye protection, high-visibility clothing, safety footwear and safety harnesses. It also includes respiratory protective equipment (RPE) Safety - PPE Lab Requirements depends on: Number of users Space Location of preparation area Storage Access Containment and sterility Cell culture facilities Lab Equipment Hood Laminar flow hood It is used to provide sterile environment and protect the researcher from exposure to aerosols from cells culture Air is filtered through a high efficiency particulate air filter before leaving the cabinet Cabinet’s surface should be wiped with ethanol before and after use The hoods are equipped with UV light to sterilize the surface Laminar flow hood Laminar flow hoods are classified at : Class I- cabinets are the simplest and easiest to maintain but offer least sterile protection to cell culture Class II- most widely used, offer good protection to researcher and cell culture since air is passing over the working area is a high efficiency particulate air filtered Class III- completely sealed units and are used for more hazardous types of work Laminar flow hood https://www.labconco.com/articles/laminar-flow-in-the-laboratory Contaminations Source of contamination: Technique Manipulations, pipetting, dispensing Work surface Operator hair, hands, breath, clothing Materials and reagents Glassware and screw caps Instruments, pipettes Equipment and facilities Room air Laminar-flow hoods Dry incubators Incoming cell lines Contaminations: source and prevention Contaminations Methods of sterilization How aseptic environment is achieved: Gibco Lab Equipment Hood Microscope Incubator Microscope A simple compound microscope invented by British microscopist Robert Hooke in the 1660s The microscope must accomplish three tasks: produce a magnified image of the specimen, separate the details in the image, and render the details visible to the human eye or camera Microscope Incubator Provide a controlled humid atmosphere by adding water to the tray Air is circulated around the incubator to keep Co2 and temperature uniform The levels of gas like oxygen, Co2 and temperature can be controlled Cryopreservation The cell suspension is frozen in the presence of a cryoprotectant such as glycerol or dimethyl sulfoxide (DMSO). Optimal freezing of cells for maximum viable recovery on thawing depends on minimizing intracellular ice crystal formation and reducing the damage of high concentration solutes formed when intracellular water freezes. This is achieved : by freezing slowly to allow water to leave the cell but not so slowly that ice crystal growth is encouraged by using a hydrophilic cryoprotectant to sequester water by storing the cells at the lowest possible temperature by thawing rapidly to minimize ice crystal growth and generation of solute gradients formed as the residual intracellular ice melts. Part 2 Cell Culture Aim and objectives Understand the importance of cell culture and its application Be able to differentiate between cell line and primary cell culture and its derivation Discuss the difference between serum and serum free media and its impact on cell culture Be able to explain how to passage cells Growth of Cell Culture Number of hits in PubMed for ‘‘cell culture’’ from 1965. The pre-1960 figure is derived from the bibliography of Fischer.(Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition) Cell Culture Applications  Advantages of Cell culture Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, (6th Edition)  Limitations of Cell culture Expertise Quantity Reprogramming and Selection Origin of Cells Instability Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, (6th Edition) Primary cultures Vs Cell line  Types of cell culture Primary Continuous On the basis of the life span of culture, the Normal cells cell lines are categorized into two types cultured without any changes in their division rate Finite Indefinite Are formed It is nearly the after the first same as finite subculture of but the cells here can divide primary cells indefinitely by Will proliferate immortalization for a limited Cell cultures number of cell derived from division after human tumors which they will are often senesce immortal Cell line  Primary cultures Cell Line Cells are disaggregated from an A cell line is a permanently organism enzymatically, chemically or established cell culture that will mechanically and placed in suitable proliferate indefinitely(if culture environment. transformed or isolated from tumor) given appropriate fresh medium and space Primary cells are morphologically similar to the parent tissue. A cell culture developed from a single cell and therefore Primary cells have a finite life span, consisting of cells with a after which they enter a non- uniform genetic make-up proliferative state called senescence. Primary Culture contains a heterogeneous population of cells. Subculturing of primary cells leads to the generation of cell lines How can we isolate cells from tissues? Before attempting to work with human or animal tissue, make sure that your work fits within medical ethical rules or current legislation on experimentation with animals Experiments with human biopsies or foetal material usually requires the consent of the local ethical committee and the patient and/or his or her relatives Do not dissect animals in the tissue culture laboratory, as the animals may carry microbial contamination  How can we obtain a cell line? Normal- Taken from a tissue as single cell Transformed- Normal cells underwent a genetic change Stem cell- generating other cell lineages Types of Cell Culture Suspension Cells Adherent cells Proliferate without being Propagate as a monolayer, attached to attached to a substratum the cell culture vessel Culture Vessels The choice is determined by The number of cells required If the cells grow in monolayer or in suspension The sampling regime Cell Passaging-Adherent cells Cell Passaging-Suspension cells Cell Passaging Name of cell line Passage number Date of passaging Density Your name The passage number is the number of times that the culture has been subcultured, whereas the generation number is the number of doublings that the cell population has undergone, given that the number of doublings in the primary culture is very approximate.  Media Initial attempts to culture cells were performed in natural media based on tissue extracts and body fluids, such as chick embryo extract, plasma, serum, and lymph. With the propagation of cell lines, the demand for larger amounts of a medium of more consistent quality led to the introduction of chemically defined media based on analyses of body fluids and nutritional biochemistry: Eagle’s Basal Medium (1955) Eagle’s Minimal Essential Medium (MEM) (1959) Supplemented with calf, human, or horse serum, protein hydrolysates, and embryo extract. More optimized media for different cell types were made (e.g., RPMI 1640 for lymphoblastoid cell lines)… However, many industrial scale production techniques now use serum-free media to facilitate downstream processing and reduce the risk of Culture of Animal Cells and adventitious infectious agents. Conventional Medium for certain cell lines COMPLETE MEDIA The term complete medium implies a medium that has had all its constituents and supplements added and is sufficient for the use specified. Examples: Amino Acids Vitamins Salts Glucose Organic Supplements Hormones and Growth Factors Antibiotics Media Composition Media Composition Media Composition Replacement of Medium Four factors indicate the need for the replacement of culture medium: A drop in pH Cell concentration. Cell type Morphological deterioration SERUM Serum is composed of: Protein Growth Factors Nutrients and Metabolites Lipids Minerals Hormones Inhibitors Disadvantages: Batch to Batch variation Can not use it in clinical trials May carry some contaminants ADVANTAGES OF SERUM-FREE MEDIA Improved reproducibility between cultures and avoid batch to batch variation of serum by creating a Standard Medium : pure constituents are used a given medium formulation can be standardized regardless of where it is used and by whom. allow easier validation of industrial processes, and research labs can replicate conditions to repeat and confirm experimental data. Selective media for a particular cell type. For example fibroblastic overgrowth can be inhibited in breast and skin cultures by using one type of serum free media (MCDB 170) by choosing the correct growth factor or group of growth factors. Regulation of proliferation and differentiation: ability to select for a specific cell type the possibility of switching from a growth-enhancing medium for propagation to a differentiation-inducing medium by altering the concentration and types of growth factors and other inducers. Less protein interference in bioassay DISADVANTAGES OF SERUM-FREE MEDIA Multiplicity of media : Each cell type appears to require a different recipe Selectivity: the transition to serum free conditions is not always as straightforward as it seems. Some media may select a sub-lineage that is not typical of the whole population, and even in continuous cell lines, some degree of selection may still be required. Reagent purity: The removal of serum also requires that the degree of purity of reagents and water and the degree of cleanliness of all apparatus be extremely high Cell proliferation: Growth is often slower in serum-free media