Equipments & Safety in Cell Culture Lab PDF

Assist. lecturer: Salma Zaher Equipments & Safety in Cell Culture Lab Section 3 10/14/2024 1 Assist. lecturer: Salma Zaher...

Assist. lecturer: Salma Zaher Equipments & Safety in Cell Culture Lab Section 3 10/14/2024 1 Assist. lecturer: Salma Zaher Basic Cell Culture Lab Equipment & Supplies 10/14/2024 2 Assist. lecturer: Salma Zaher Incubators ▪ The purpose of the incubator is to provide the appropriate environment for cell growth. ▪ Stainless steel incubators allow easy cleaning and provide corrosion protection, especially if humid air is required for incubation. ▪ Frequent cleaning of the incubator is essential to avoid contamination of cell cultures. 10/14/2024 3 Assist. lecturer: Salma Zaher 10/14/2024 4 Assist. lecturer: Salma Zaher Types of Incubators ▪ Dry incubators ▪ Humid CO2 incubators ▪ are more economical but require the cell ▪ are more expensive but allow superior cultures to be incubated in sealed flasks control of culture conditions. to prevent evaporation. ▪ They can be used to incubate cells ▪ Placing a water dish in a dry incubator cultured in Petri dishes or multi-well can provide some humidity, but they do plates, which require a controlled not allow precise control of atmospheric atmosphere of high humidity and conditions in the incubator. increased CO 2 tension. ▪ 10/14/2024 5 Assist. lecturer: Salma Zaher Storage ▪ A cell culture laboratory should have storage areas for liquids such as media and reagents, for chemicals such as drugs and antibiotics, for consumables such as disposable pipettes, and gloves, for glassware such as media bottles and glass pipettes, for specialized equipment, and for tissues and cells. 10/14/2024 6  Assist. lecturer: Salma Zaher Types of Storage ▪ Refrigerators ▪ Freezers ▪ For small cell culture laboratories, a ▪ Most cell culture reagents can be stored at –5°C to domestic refrigerator is an adequate and –20°C; therefore an ultradeep freezer (i.e., a – inexpensive piece of equipment for storing 80°C freezer) is optional for storing most reagents and media at 2–8°C. reagents. ▪ For larger laboratories, a cold room restricted ▪ While most reagents can withstand temperature to cell culture is more appropriate. oscillations in an autodefrost freezer, ▪ A domestic freezer is a cheaper alternative to ▪ some reagents such as antibiotics and enzymes a laboratory freezer. should be stored in a freezer that does not autodefrost. 10/14/2024 7 Assist. lecturer: Salma Zaher 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. ▪ Do not store cells in –20°C or –80°C freezers, because their viability quickly decreases when they are stored at these temperatures. 10/14/2024 8 Assist. lecturer: Salma Zaher 10/14/2024 9 Assist. lecturer: Salma Zaher liquid-nitrogen ▪ 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. ▪ liquid phase systems usually have longer static holding times and are therefore more economical. 10/14/2024 10 Assist. lecturer: Salma Zaher 10/14/2024 11 Assist. lecturer: Salma Zaher Cell Counter ▪ A cell counter is essential for quantitative growth kinetics, Cell counters not only count cells, but also perform the following: ▪ Calculate cell viability ▪ Present data visually ▪ Store data securely ▪ Allow for data export 10/14/2024 12 Assist. lecturer: Salma Zaher What is the hemocytometer? The hemocytometer is a glass slide with a centralized chamber (also called the counting chamber) that contains your cell sample. Etched onto this chamber is a grid, used to count cells when viewed under a microscope. The cells are identified in the hemocytometer by using brightfield microscopy. Viability is determined via the principle of trypan blue exclusion; whereby, trypan blue stains non-viable, or dead, cells, yet excludes viable cells. Both viable and non-viable cells are then counted under a microscope. 10/14/2024 13 Assist. lecturer: Salma Zaher 10/14/2024 14 Assist. lecturer: Salma Zaher Cell Culture Flask & Plate 10/14/2024 15 Assist. lecturer: Salma Zaher Multichannel Pipette 10/14/2024 16 Assist. lecturer: Salma Zaher Buffer 10/14/2024 17  Assist. lecturer: Salma Zaher 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. 10/14/2024 18 Assist. lecturer: Salma Zaher Laminar Flow ▪ Laminar flow hoods protect the working environment from dust and other airborne contaminants by maintaining a constant, unidirectional flow of HEPA-filtered air over the work area. The flow can be horizontal, blowing parallel to the work surface, or it can be vertical, blowing from the top of the cabinet onto the work surface. ▪ Depending on its design, ▪ Horizontal flow hood provides protection to the culture (if the air flowing towards the user) or to the user (if the air is drawn in through the front of the cabinet). ▪ Vertical flow hoods, on the other hand, provide significant protection to the user and the cell culture. 10/14/2024 19 10/14/2024 Assist. lecturer: Salma Zaher 20 Assist. lecturer: Salma Zaher Cell Culture Lab Safety 10/14/2024 21 Assist. lecturer: Salma Zaher Safety 01 02 03 04 05 Proper handwashing Decontaminating Handling Quarantining new Regularly screening and wearing work surfaces before carcinogenic cells and treating existing cell culture personal protective and after substances with care. them as potentially stocks. equipment.(gloves, experiments. infected. closed shoes, lab coat). 10/14/2024 22 Assist. lecturer: Salma Zaher Biohazards in Cell Culture ▪ Viruses pathogenic for humans are one of the most likely biohazards presented by cell cultures. Where infection with an agent pathogenic for humans is known or suspected. ▪ In addition, the generation and use of modified cells–for example; transformed cells and cells containing recombinant DNA, can be hazardous. ▪ These procedures could potentially result in the appearance of modified or reactivated viruses. ▪ Laboratory workers should never culture cells derived from their own body or tissues. 10/14/2024 23 Assist. lecturer: Salma Zaher 10/14/2024 24 Assist. lecturer: Salma Zaher Genetically modified organisms (GMOS) The generation and use of genetically modified organisms (GMOs) should be strictly controlled and regulated. Most countries have regulatory organizations to ensure the risks posed by GMOs are minimized. For example, in the UK all institutions that carry out work using and/or generating GMOs are required by law to have a Genetic Modification Safety Committee (GMSC). 10/14/2024 25 Assist. lecturer: Salma Zaher 10/14/2024 26 Assist. lecturer: Salma Zaher Cell Culture Waste Disposal Tissue culture waste (culture medium) – inactivate for at least 2 hours in a solution of hypochlorite (10,000ppm) prior to disposal to drain with an excess of water Contaminated pipettes should be placed in hypochlorite solution (2500ppm) overnight before disposal by incineration Solid waste such as flasks, centrifuge tubes, contaminated gloves, tissues, etc., should be placed inside heavy-duty sacks for contaminated waste and incinerated If at all possible waste should be incinerated rather than autoclaved Waste from specially licensed laboratories e.g. those handling genetically modified level 2 (GMO2) organisms requires specific treatment and tracking 10/14/2024 27