Design and Equipment

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Dulcie Richardson
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1 Cell Culture

2 Design and Equipment Laboratory Design Microbiological Safety Cabinets Centrifuges Incubators Work Surfaces and Flooring Plasticware and Consumables Care and Maintenance of Laboratory Areas

3 Safety Aspects Risk Assessment Disinfection Hypochlorites (e.g. Chloros, Presept) Alcohol (e.g. ethanol, isopropanol) Waste Disposal Tissue culture waste (culture medium) Contaminated pipettes Solid waste

4 Main types of Cell Culture Primary Cultures Continuous Cultures

5 The Cell Environment Basic Constituents of media Inorganic Salts Buffering Systems Carbohydrates Vitamins Proteins and Peptides, Fatty Acids and Lipids Trace Elements Serum Guidelines for serum use Origin of Serum

6 Storage of Cell Lines Cryopreservation of Cell Lines Method Advantages Disadvantages Electric (-135ºC) Freezer Liquid Phase Nitrogen Vapor Phase Nitrogen Ease of maintenance Steady temperature Low running costs Steady ultra-low (-196ºC) temperature Simplicity and mechanical reliability No risk of crosscontamination from liquid nitrogen Low temperatures achieved Simplicity and reliability Requires liquid nitrogen back-up Mechanically complex Storage temperatures high relative to liquid nitrogen Requires regular supply of liquid nitrogen High running costs Risk of cross-contamination via the liquid nitrogen Requires regular supply of liquid nitrogen High running costs Temperature fluctuations between - 135ºC and - 190ºC

7 Good practices: The Do's Use personal protective equipment at all times. Wear dedicated PPE for tissue culture facility and keep separate from PPE worn in the general laboratory environment. Keep all work surfaces free of clutter. Correctly label reagents including flasks, medium and dishes with contents and date of preparation. Only handle one cell line at a time. Clean the work surfaces with a suitable disinfectant between operations and allow a minimum of 15 minutes between handling different cell lines.

8 Good practices: The Do's Maintain separate bottles of media for each cell line in cultivation. Examine cultures and media daily for evidence of gross bacterial or fungal contamination. Quality Control all media and reagents prior to use. Keep cardboard packaging to a minimum in all cell culture areas. Ensure that incubators, cabinet, centrifuges and microscopes are cleaned and serviced at regular intervals. Test cells for mycoplasma on a regular basis.

9 Good practices: The Don'ts Do not continuously use antibiotics in culture medium Don t allow waste to accumulate particularly within the microbiological safety cabinet or in the incubators. Don't have too many people in the lab at any one time. Don't handle cells from unauthenticated sources in the main cell culture suite. Avoid keeping cell lines continually in culture without returning to frozen stock.

10 Good practices: The Don'ts Avoid cell culture becoming fully confluent. Always sub-culture at 70-80% confluency or as advised on ECACC's cell culture data sheet. Do not allow media to go out of date. Shelf life is only 6 weeks at +4ºC once glutamine and serum is added. Avoid water baths from becoming dirty Don t allow essential equipment to become out of calibration. Ensure microbiological safety cabinets are tested regularly.

11 Sterile Technique Aim: To ensure all cell culture procedures are performed to a standard that will prevent contamination from bacteria, fungi and mycoplasma and cross contamination with other cell lines. Materials Chloros / Presept solution (2.5g/l) 1% formaldehyde based disinfectant e.g.virkon,tegador 70% ethanol in water (Prod. No. R8382) Equipment Personal protective equipment (sterile gloves, laboratory coat) Microbiological safety cabinet

12 Sterile Technique Procedure Sanitize the cabinet using 70% ethanol before commencing work. Sanitize gloves by washing them in 70% ethanol and allowing to air dry for 30 seconds before commencing work. Sanitize all materials and equipment (media bottles, pipette tip boxes, pipette aids) into the cabinet prior to starting work after sanitizing the exterior surfaces with 70% ethanol.

13 Sterile Technique Procedure: do not contaminate gloves whilst working by touching anything outside the cabinet (especially face and hair). If gloves become contaminated re-sanitize with 70% ethanol as above before proceeding. Movement within and immediately outside the cabinet must not be rapid. Slow movement will allow the air within the cabinet to circulate properly. Speech, sneezing and coughing must be directed away from the cabinet so as not to disrupt the airflow.

14 Sterile Technique Procedure: After completing work disinfect all equipment and material before removing from the cabinet. Spray the work surfaces inside the cabinet with 70% ethanol and wipe dry with tissue. Dispose of tissue by autoclaving. Cell culture discard must be kept in the cabinet for a minimum of two hours (preferably overnight) prior to discarding down the sink with copious amounts of water.

Yeo Soh Bee / Zhou Lei A/Prof Dan Yock Young 29/9/2017 Prepared by Approved By Issue Date

Page: 1 of 6 Yeo Soh Bee / Zhou Lei A/Prof Dan Yock Young 29/9/2017 Prepared by Approved By Issue Date 1. OBJECTIVE The purpose of this document is to outline operating procedures for the safe working