Lecture 3 Contamination Controls, Safety Aspect and Bioethics in Cell Culture.

1. Lecture 3 Contamination Controls, Safety Aspect and Bioethics in Cell Culture.

2. LECTURE CONTENT CELL CULTURE CONTAMINATION • Consequences of contamination • Types of contaminates • Microorganisms • Source of contaminations • Control method • How do Biological contaminants enter cultures • Contamination investigation • Sterile VS. Aseptic • Steps for reducing contaminations. BIOSAFETY • Who may be at risk • Biosafety Level • Biosafety Act 2007 • Hazard potential from mycoplasma • BIOLOGICAL WASTE MANAGEMENT • Why it is important? • Whose responsible? • Types of biological waste • Waste container requirement • BIOETHICS • What is bioethics? • Malaysian Bioethics Council • Ethical issue in bioethics

3. CELL CULTURE CONTAMINATION

4. CONSEQUENCES OF CONTAMINATIONS “Contamination can not be totally eliminated, but it can be managed to reduce both its frequency of occurrence and the seriousness of it consequences” – John Ryan, Ph.D (Corning Incorporate).

5. TYPE OF CONTAMINANTS TYPES OF CONTAMINANTS Chemical Contaminations Biological Contamination Media Easy to Detect Difficult to Detect Sera/ Serum Media Media Endotoxin Media Media Media Storage Vessel Media Media Fluorescent lights Media Incubator Water

6. MICROORGANISMS Bacteria • Gram positive & negative. • Media used for cell culture will maintain bacteria. • Bacteria will compete for nutrients in media. • Detect by microscopic observation or effects on the culture (pH shift, turbidity and cell destruction). Fungi • Mold, yeast. • Float in air and are on surfaces. • Grow prolifically.

7. Viruses • Enter into cells and destroy them in their quest to replicate. • May come from natural products used to grow cells. • Small size, difficult to detect. Mycoplasma • Can alter their host culture’s cell function, growth, metabolism. • Bacteria-like microbes, smallest self- replicating organism know, lack a cell wall. • Can grow high density in cell culture, w/o any signs of contamination- no turbidity, pH changes or even cytopathic effects.

8. SOURCE OF CONTAMINATIONS

9. CONTROL METHOD: Raw Materials. Starting materials can be a major source of contamination. To help prevent this, manufacturers must know what types and quantities of microbes are likely to be present. This is one of the reasons your company tests its starting materials. Staring materials must always be handles and stored in a manner to prevent contamination. Some important rules are: • Store in clean, dry locations • Store off the ground and under over • Always keep containers sealed when not in use • Be alert to damaged packages • Never use dirty or wet sampling or measuring equipment

10. CONTROL METHOD: Equipment Equipment can also be a source of contamination. In order to keep equipment clean, you should: • Store in a clean, dry manner • Drain and store all hoses correctly after use • Remove all product residue • Use approved sanitising procedures • Reinspect equipment before use • Maintain equipment

11. CONTROL METHOD: Process Water • Regular microbiological monitoring programmers • Sanitize the system regularly • Take samples carefully, being sure not to contaminate the system or the sample Process water is a key ingredient of many medicinal products. Because water is a key requirement for microbial growth, a lot of effort is needed to control any water where it is an ingredient of the product or is used to wash processing equipment. The key control over process water are:

12. CONTROL METHOD: Environment When products are exposed to the environment, the environment must be controlled. Some ways of achieving this include: • Air handling systems - Filter the air - Maintain positive air pressure - Control temperature, humidity and air flow patterns • Designated manufacturing zones - Keep doors and windows shut - Change outer garments • Cleaning programmers - Regularly check air filters - Clean ducts and air filters regularly - Keep area clean and dry at all times

13. CONTROL METHOD:Operator (Personal Hygiene) People are potentially the target source of microorganisms and are the hardest factor to control. To work in this industry, you must follow the strictest hygiene practices: • Always wear protective garments/ lab coats. • Never handle products with bare hands. • Wash hands after visiting the toilet. • Report any infections to supervisor. • Don’t eat in the factory.

14. HOW DO BIOLOGICAL CONTAMINANTS ENTER CULTURES?

15. CONTAMINATION INVESTIGATION • Weak spots of the process must broadly examined. • Perform investigation while the process is still active (visual inspection). • Perform sampling on the contaminated culture. • Quarantine all left over of raw materials/ supplements/ equipment components etc. perform sampling if necessary. • Review the cleaning and sterilization process. • Review or perform air bioburden test. • Perform personnel monitoring test ( glove print test). • Perform bacterial identification test of contaminated culture and other samples. • After the investigation completed, assign a most probable cause diagram (i.e Ishikawa diagram) for cause tracking.

16. STERILE VS. ASEPTIC

17. Key building blocks for successfully managing cell culture operations

18. Steps for reducing Contaminations Problems. • Apply good aseptic techniques • Use clean lab coats to protect against shedding contaminants from skin or clothes. • Work with only 1 type of cell line at a time in the hood. • Aliquot sterile solution in small volumes. • Reduce accidents - Be careful when labeling and choice of acronyms. - use standardized recordkeeping form. - Use written protocols/ formulation sheet when preparing media and solutions. 3. Keep the lab clean - Routinely wipe floor and work surface. - Periodic cleaning and disinfecting of incubator, water bath. - Autoclaving of any waste that have been in contact with cells.

19. Routinely monitor for contamination. - Perform Bioburden and Environment monitoring. • Use frozen cell repository strategically - To reduce the need to carry large number of cultures. - stop biological time for them, preventing them from acquiring the altered characteristic that can normally occur in actively growing cells as a result of environmental/ age related changes. 6. Use antibiotics sparingly if at all.

20. BIOSAFETY

21. Biosafety Level

22. HAZARD POTENTIAL FROM MYCOPLASMA Mycoplasma: • Extremely small bacteria. • Cultivating of most mycoplasmas in artificial media requires several supplements, i.e: cholesterol. • Difficult in detection and elimination. • Able to persist in permanent cell- cultures undetected for year. How do they enter cells??? • Contamination with human commensalmycoplasma from lab personnel. • Important to the lab by establishing a primary cell culture of an infected animal. • Use of contaminated supplements (serum, cytokines, s/ natant of infected cells). • Splitting adherent cells with contaminated trypsine.

23. HAZARD POTENTIAL FROM MYCOPLASMA Why should we care about them??? • Can change several properties: growth, metabolism, morphology, and genome structure. • Compete with host nutrients. • Inhibit the synthesis of histone and nucleic acid, followed by chromosomes aberration of the host cells. • Produce hydrogen peroxide, which is directly toxic to the cells. How can we detect mycoplasms??? • Cultured detection is very sensitive, but it takes 2 weeks and several strain cannot be cultivated. • DNA staining with intercalating fluorescent substances.

24. BIOLOGICAL WASTE MANAGEMENT Why it is important??? To ensure the generation and disposal of scheduled waste is properly managed as well as complies with Environment Quality (Scheduled Waste) Regulation 2005. Whose responsible is it and what should they do??? All personnel who generate and handle the waste: • Arranging the pickup of the waste contractor (e.gKualitiAlam). • Manage and dispose the waste from their work station to the Temporary Waste Room and record it in the Waste Log Book Provided. • Identify the waste category with the labeling.

25. BIOLOGICAL WASTE MANAGEMENT TYPES OF BIOLOGICAL WASTE: • Solid non contaminated waste: all non-contaminated materials such as facemask, bouffant cap, papers, etc are disposed in waste bin that is lined with black plastic bag. • Solid contaminated/ biohazard: All solid contaminated/ biohazard waste will be collected in a waste bag and is line with biohazard waste container (Red Biohazard Bag). It must be label accordingly. • Liquid contaminated/ biohazard waste: After decontamination, liquid biohazard waste is collected is a liquid waste container and label the liquid waste container accordingly before being send to disposal. • Biohazardous Sharps: Place all sharps in a puncture- proof container either a commercially available sharps container or a study cardboard. Seal the container or box and attach a strip of autoclave tape.

26. BIOLOGICAL WASTE MANAGEMENT Solid and Liquid Waste Container Requirement • The waste containers and bags used must be compatible with the type of waste contained. • Biohazard liquid waste containers and waste bags cannot be reuse and should be completely disposed. • Do not fill the containers and bags completely. Each container must have at least 1 inch of headspace above the waste to avoid spillage. • Tie or seal the waste bags to prevent leakage or expulsion of content during storage, handling and transport. • Bio hazardous liquid waste container shall be rigid, heat resistance (suitable for autoclaving), leak proof and has tight- fitting cover. Do not use metal container for nay liquid waste. • Place sharps in a dedicated puncture resistance container before dispose it in the waste bag.

27. BIOETHICS What is bioethic??? The study of controversial ethics brought about by advances in biology and medicine. Bioethicists are concerned with the ethical questions that arise in the relationships among life sciences, biotechnology, medicine, politics, law and philosophy. Malaysian Bioethics council • Launched on 22 May 2012 by the Minister of Science, Technology and Innovation YP Datuk Seri Panglima Dr. MaximusJohnityOngkili. • Made up of representatives from noted academic experts, and representatives from government and non- government offices. • Aims to provide advice, as well as resolve and manage bioethical issues in the country. • Focusing on the impact of science and technology on the environment, society, public health, culture, laws and religion.

28. ETHICAL ISSUE IN CELL CULTURE • The use of fetal bovine serum: ethical or scientific problem??? • Fetal bovine serum (FBS) is a common component of animal cell culture media. • FBS is harvested from bovine fetuses taken from pregnant cows during slaughter. FBS is commonly harvested by means of a cardiac puncture without any form of anaesthesia. Fetuses are likely exposed to pain and/ or discomfort and therefore current practice of fetal blood harvest is inhumane. • Apart from moral concerns, several scientific and technical problems exist regarding the application of FBS in cell culture. Efforts should be made to reduce and preferably replace FBS by synthesis alternatives. On moral and scientific grounds the most promising alternative to FBS is the use of defined media.

29. ETHICAL ISSUE IN CELL CULTURE 2) Stem Cells Controversy • Not all stem cell research involves the creation, use or destruction of human embryos. For example, adult stem cells, amniotic stem cells and induced pluripotent stem cells do not involve human embryos at all. 3) Human Biopsy Materials • These aspects extend from the initial contact with the donor or their next- of- kin, through isolation and supply procedures, to use in the research facility. • It should be sup plied through officially recognized human tissue banks, and collected according to acceptable ethical standards and in compliance with national laws.