DES115 Cell Culture - Week 8-9 Lab Session PDF

Laboratory Session Plan Course: Program: Principles of Cell Biology and Genetics Week 6 8-9 DES115 Dentistry Ad. Assoc. Prof. Kousparou C. Module: Introduction to cell culture: plate, split and freeze Year: 1 Dr Charous C....

Laboratory Session Plan Course: Program: Principles of Cell Biology and Genetics Week 6 8-9 DES115 Dentistry Ad. Assoc. Prof. Kousparou C. Module: Introduction to cell culture: plate, split and freeze Year: 1 Dr Charous C. human cells Semester: 1 Dr Tomouzou C. Lab Session Activities Culture, split, re-plate and freeze human cancer cells Lab Session Location Laboratories N34, N23 N41, N42 Lab Session Duration 2hrs x 5 GROUPS A B C D E Team- Time Cultured human cancer cells, in each laboratory Structure related reagents. Dr Harous C. Dr Tomouzou C. 2 hrs x 8 GROUPS Lab ABCDEFGH Rotations In place in each laboratory I. Learning Objectives This laboratory session aims at educating students on how human immortalized cell lines are maintained and propagated in appropriate culture conditions. The fact that these cell-based applications are an integral part of not only basic research, but also biochemical studies, vaccine development, and drug discovery is stressed. II. Laboratory Session Description Activity Culture, split, re-plate and freeze human cancer cells Description Activity 120 min Duration (in minutes) Objective Techniques understanding and analysis and link to theoretical part taught in lectures. Instructions Your group will be assigned to a specific teaching station. During this session you will: Culture, split, re-plate and freeze human cancer cells INTRODUCTION Several approaches can be used to separate a particular type of cell from the cells that surround it in the body. If the cells are part of a compact tissue, they must first be dissociated from each other. This is often accomplished using proteolytic enzymes and other agent that disrupt the adhesive bonds between cells. Next, the different types of cells in the tissue must be isolated from each other. A fluorescence-activated cell sorter allows the isolation of specific types of cells. The isolated cells can be used for biochemical analysis or for establishing cell cultures. Many animal and plant cells survive and proliferate in culture provided they have suitable medium containing nutrients and the necessary growth factor proteins. Experiments performed using cultured cells are said to be carried out in vitro (‘in glass’, ‘in culture’) to contrast them with experiments on intact organisms, which are said to be carried out in vivo (‘in the living’). Most vertebrate cells cease to proliferate after a finite number of cell divisions. Like most human somatic cells, these cells do not express the enzyme telomerase, whose renew the ends of chromosomes at each cell division. As a result the chromosomes of human somatic cells progressively shrink at each cell division, and cell division stops when critical information is lost from the ends of chromosomes. This feature ensures that somatic cells do not divide indiscriminately and develop into cancerous cells. Cells that can divide indefinitely as the result of a genetic change are said to be immortalized and can be propagated in culture as a cell line. Immortalized cell lines can be regenerated by providing the cells with the gene that encodes the catalytic subunit of telomerase. The cell lines provide a convenient source of homogeneous cells. Among the most promising cell lines to be developed are the human embryonic stem (ES) cell lines. The critical importance of these cell lines is the fact that the cells are undifferentiated; and given the appropriate treatment, they can give rise to any tissue in the body. They form and will further form the basis of the most developed treatments for diseases. Morphology of cells in culture Cells in culture can be divided into three basic categories based on their shape and appearance (i.e., morphology). Cell culture equipment The specific requirements of a cell culture laboratory depend mainly on the type of research conducted; for example, the needs of mammalian cell culture laboratory specializing in cancer research is quite different from that of an insect cell culture laboratory that focuses on protein expression. However, all cell culture laboratories have the common requirement of being free from pathogenic microorganisms (i.e., asepsis), and share some of the same basic equipment that is essential for culturing cells. This section lists the equipment and supplies common to most cell culture laboratories, as well as beneficial equipment that allows the work to be performed more efficiently or accurately or permits a wider range of assays and analyses. Note that this list is not all inclusive, the requirements for any cell culture laboratories depend on the type of work conducted. Cell culture hood (i.e., laminar flow hood or biosafety cabinet) - Biological safety cabinets (BSC) precisely control the airflow of the work area and are essential in protecting the samples as well as for the safety of lab personnel. Safety and ergonomics are factors to consider when buying a BSC. Incubator (humid CO₂ incubator recommended) - CO2 incubators provide optimal conditions for cell growth, controlling temperature, humidity and CO2 levels (O2 control optional). Uniformity and recovery of parameters and contamination control are important buying considerations. Water bath Centrifuge - Centrifuges are used in the harvesting workflow step to separate or concentrate cells or cell components. Precise temperature control and accurate centrifugal speeds are critical for this process. Refrigerator and freezer (–20°C) Cell counter (Automated Cell Counter or hemocytometer) Inverted microscope Liquid nitrogen (N₂) freezer or cryostorage container Sterilizer (i.e., autoclave) Additional supplies Cell culture–treated vessels (e.g., flasks, dishes, multiwell plates) Pipettes and pipettors Syringes and needles Waste containers Media, sera, and reagents Cells Cell Culture Media for healthy mammalian cell cultures Cell culture medium is vital to your culture environment as it provides components (e.g., nutrients, growth factors) necessary to support cell growth and function. In addition, media can be formulated to regulate environmental conditions in cell culture, including osmotic pressure and pH. A variety of cell culture media formulations have been developed to support a wide range of cell types and experimental applications. Fetal Bovine Serum FBS Fetal Bovine Serum (FBS) is the most commonly used serum as it provides the most robust culture system for the widest range of cell types. Gibco FBS can provide the cell health, maintenance and viability critical for optimal performance and cell growth in your culture medium. Aseptic technique Successful cell culture depends heavily on keeping the cells free from contamination by microorganisms such as bacteria, fungi, and viruses. Nonsterile supplies, media, and reagents, airborne particles laden with microorganisms, unclean incubators, and dirty work surfaces are all sources of biological contamination. Aseptic technique, designed to provide a barrier between the microorganisms in the environment and the sterile cell culture, depends upon a set of procedures to reduce the probability of contamination from these sources. The elements of aseptic technique are a sterile work area, good personal hygiene, sterile reagents and media, and sterile handling. Cryogenic storage Cell lines in continuous culture are likely to suffer from genetic instability as their passage number increases; therefore, it is essential to prepare working stocks of the cells and preserve them in cryogenic storage. We do not store cells in –20°C or –80°C freezers, because their viability decreases when they are stored at these temperatures. There are two main types of liquid nitrogen storage systems, vapor-phase and liquid-phase, which come as wide-necked or narrow-necked storage containers. Vapor-phase systems minimize the risk of explosion with cryostorage tubes, and are required for storing biohazardous materials, while liquid-phase systems usually have longer static holding times and are therefore more economical. Narrow-necked containers have a slower nitrogen evaporation rate and are more economical, but wide-necked containers allow easier access and have a larger storage capacity. Instructions for Equipment and materials you will use: today’s Cell culture hood experiment Incubator (optimal temperature, humidity and C02) Water bath Centrifuge machine Refrigerator and freezer (–20°C) Additional Supplies: Cell culture vessels (Flasks, Petri dishes, multi-well plates) Pipettes and pipettors Waste containers Media and reagents Cells Sterile handling Always wipe your hands and your work area with 70% ethanol. Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood. Avoid pouring media and reagents directly from bottles or flasks. Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids and use each pipette only once to avoid cross-contamination. Do not unwrap sterile pipettes until they are to be used. Keep your pipettes at your work area. Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resealable bags to prevent microorganisms and airborne contaminants from gaining entry. Never uncover a sterile flask, bottle, dish, etc., until the instant you are ready to use it, and never leave it open to the environment. Return the cover as soon as you are finished. If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down. Use only sterile glassware and other equipment. Be careful not to talk, sing, or whistle when you are performing sterile procedures. Perform your experiments as rapidly as possible to minimize contamination. Experimental Procedure: 1.Take a flask of cells stored at the incubator (Make sure the incubator is closed properly after use). 2.Remove and discard the used cell culture media from the culture vessel (Make sure the caps or covers face down on the work area). 3.Wash cells using balanced salt solution (PBS). Gently add wash solution to the side of the vessel opposite the attached cell layer to avoid disturbing the cell layer, and rock the vessel back and forth several times. Note: Be careful not to touch the pipette tip to anything non-sterile, including the outside edge of the bottle threads. 4.Remove and discard the wash solution from the culture vessel. Repeat steps 3 and 4. 5.Add the pre-warmed dissociation reagent trypsin (1ml) to the side of the flask; use enough reagent to cover the cell layer. Gently rock the container to get complete coverage of the cell layer 6.Incubate the culture vessel at room temperature for approximately 10-15 minutes. Note: The actual incubation time varies with the cell line used. 7.Observe the cells for detachment. If cells are 90% detached, then add 5ml of pre-warmed fresh media to the flask to resuspend any cell pellets. 8.Collect the cells and transfer them into a 6-well plate, 2mL to each well. Make sure you label the plate with your group number. 9.Incubate the cells using proper temperature at the incubator for the following laboratory session. 10.Make sure you wipe the work surface with 70% ethanol before you leave.  This is what you should be seeing in the flasks of attached cells using the inverted microscope. Try to think of one actual application in real-life science for which cell culture is a useful, necessary technique. __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ __________________________________________________________________________________________ Bibliography Mack GS (2007). Nature Biotech 25:631–638. McLenachan S, Sarsero JP, and Ioannou PA (2007). Genomics 89:708–720. Nayak S and Herzog RW (2009). Gene Ther 17:295–304. Pfeifer A and Verma IM (2001). Annu Rev Genomics Hum Genet 2:177–211.