1. Introduction to mammalian cell culture
Lecturer: Assist. Prof. Dr. Mirza Suljagić Assistant: Jasmin Sutkovic
March, 16th 2018

2. Chapter 1 – Introduction
Textbook: „Introduction to Mammalian Cell Culture“ (D. Ler, U. Glamočlija, M. Suljagić)

3. Introduction
Cell Culture – removal of cells from an organism and their subsequent growth in a favorable, artificially controlled environment
Primary Cell Culture – removal from tissue
Can be sub-cultured
Cell line – due to proliferation
Finite – limited life span
Immortal – due to transformation (Continous cell line)

4. Primary Cell Culture Contains heterogenous cell population
Sub-culturing leads to cell line generation
Different purification methods
Tissue sample: Dissection, Rinsing, Mechanical Dissagregation (Enz. Digestion) in trypsin
Different separation techniques:
Density gradient separation
MACS
FACS
Figure – Cell line

5. Essential for viable state
Sub-culturing – cells are harvested, diluted in a fresh growth medium and replaced in a new culture flask to promote further growth
Essential for viable state
PRIMARY CUTURE  Sub-culturing  CELL LINE (SEC.CULTURE)
Cell line – transfer of cell from one to another culture vessel
Providing fresh nutrients and growing space
Figure – Primary Cell Culture

6. Types of cell lines (on the basis of life span of culture):
Finite cell lines - limited life span limited number of cell generations (40-60 doublings) slow growth rate doubling time h
Continuous cell lines - transformed under laboratory conditions grow in a monolayer or in suspension rapid growth rate doubling time h
Cell strain – positive selection of cells either from primary culture or cell line by cell cloning
Limited division potential, lose ability to proliferate (Hayflick limit)

7. CHAPTER 2 BASICS OF CELL CULTURE

8. Cell Culture Requirements and Equipment
Differ depending on a cell culture (e.g. Cancer & GMO cells)
Microbiologically free
Equipment common to most cell culture labs:
Essential Equipment - cell culture hood, humid CO2 incubator, centrifuge, refrigerator (2-8ͦ C) and freezer (-20 ͦ C)
Automated Cell Counter - inverted microscope, pipettor, micropipettes, cultureware
Outside culture environment - liquid nitrogen container, sterilizer (autoclave), waterbath

9. Work Surfaces
Bench tops, wallsm flooring – smooth and resistant to temperature and cracking (for liquid nitorgen)
Major requirement – to maintain aseptic work area
Use cell culture hood (for aseptic conditions)

10. Basic Equipment
Clean Benches – horizontal or vertical laminar flow provide product protection
- dust-free assembly of sterile equipment never used for handling of cell cultures
2. Cell Culture Hood – laminar flow cabinet or tissue culture hood HEPA filter

11. Figure – Laminar Flow Hood
Figure – Cell Culture Hood

12. Types of Cell Culture Hood
Class I – significant protection levels to lab personnel
- no culture protection from contamination
Class II – designed for work involving BSL-1,2,3 materials provide aseptic environment for handling potentially hazardous materials (viruses, toxic/carcinogenic reagents, etc.)
Class III – highest level of protection
- working with human pathogens and BSL-4 material

13. 3. Incubators – for growth conditions (temperature, CO2, humidity)
Mammalian cell cultures – at 37 ͦ C, 5-10% CO2
Large, forced air circulation, have temperature control ± 0.2 ͦ C, made of stainless steel
Mammalian cells – kept at humid CO2 incubators
Humid CO2 incubators – incubation of cells in sterile flasks
Dry incubators – water dish controles humidity

14. 4. Centrifuge – for maintanance of most cell lines and
4. Centrifuge – for maintanance of most cell lines and cyropreservation
Balance the tubes
Centrifuge rate – xg
Higher forces may promote cell damage
Tubes ml
Storage
Storage areas for liquids (media, antibiotics) and consumables ( disposable pipettes, gloves, media bottles)
Follow the instructions

15. 5. Refrigerators and Freezers - for small cell culture lab
5. Refrigerators and Freezers - for small cell culture lab (domestic refrigerators)
Storing at -5 to -20 ͦ C
Ultra-deep freezer ͦ C storing
6. Plastic-ware for Cell Culture
Includes Petri-dishes, multi-well plates (6, 12, 24, 48, 96 wells per plate)
Screwcap flasks – classified acc. to surface areas (T-25, T-75, T-175, T-225 cm2)
Provide hydrophilic surface to facilitate attachment of anchorage dependent cells

17. 7. Inverted Microscope – used to visualize cells in culture
Easy to operate
Useful for observing living cells or organisms at the bottom of a dish
Provide information about cell morphology and cell state
Cell culture can be viewed in flasks, dishes, multi-well plates
Liquid Nitrogen – cellular maintenance for prolonged period

19. Biosafety
Main aim – prevent injury, protect property, avoid harm to individuals and environment
Common hazards: accidental punctures with syringe needles, spills, splashes onto skin and mucous membrane, inhalation, ingestion
Strict adherence to standard microbiological practices and techniques
Type of risks: a) Low risk – non-human cont. cell lines (well characterized)
b) Medium risk – poorly characterized cell lines (mammalian) c) High risk – primary cells derived from human/primate tissue

20. Biohazards
Biological substances that represent a threat to human health
Medical waste, toxins, viruses, m.o.
Most common biohazards presented by cell culture – human pathogenic viruses
Cell products, biomolecules with modulations
Figure – Symbol of biohazard materials

21. Biosafety levels
„Biosafety in Microbiological and Biomedical Laboratories“ by NIH
Describe safety equipment, microbiological practices
1. BSL-1
Basic level of protection
Common to most labs
For agents that are not known to cause disease to humans (Bacillus subtilis, Canine hepatitis, E.coli)

22. 2. BSL-2
For moderate-risk agents
Cause mild human disease (by ingestion)
Work with lentiviral vectors (Zika, Hepatitis A, B, C, HIV, Salmonella)
3. BSL-3
For agents with known potential for aerosol transmission
Agents may cause lethal infection (for which vaccines exist)
West Nile Virus, Typhus, TBC, Plasmodium falciparum

23. 4. BSL-4
For agents that pose high individual risk of life threatening disease
No treatment
Involves special equipment
Contains UV light, showers, vacuum room, showers, autonomous detection system

24. Disinfection and Waste Disposal
To minimize risk of contamination of cell culture
Personal protective equipment should be used
Main disinfectant:
Hypochlorite (Sodium hypochlorite) - for general purpose - cannot be used on metal surfaces - should be made fresh daily - active against viruses
b) Alcohol (Ethanol, Isopropanol)
effective against bacteria, viruses (only ethanol)

25. c) Aldehyde (Formaldehyde)
irritants, use should be limited due to sensitization problems
Used only in well ventilated areas
Different waste forms (different treatments)
Tissue culture waste
Contaminated pipettes
Solid waste

26. Aseptic Manipulations
Keep the cells free from m.o. contamination
Wear basic protective equipment
Aseptic manipulation – barrier between m.o and environment
Elements of aseptic technique
Sterile Work Area - use gloves, lab coat, cell culture hood
- disinfect surrounding areas before and after use Bunsen burner – not recomended

27. b) Personal Hygiene
Wash hands to remove adherent m.o that would contaminate cell cultures
Keep places clean
Avoid talking, sneezing, coughing
One pipette – cannot be used between two different bottles of media

28. Cell Culture Contamination
Contaminants:
Chemical – media, sera, detergents, water, endotoxin impurities b) Biological – bacteria, yeast, viruses, etc.
Bacteria – contamination is easily detected (pH drop, cloudly appearance, thin films on a surface)
Yeast – little pH change until the contamination is heavy
- appear as individual ovoid or spherical particles
Mycoplasmas – Difficult to detect until they achieve extremely high densities decrease proliferation rate, not cell death fluorescent staining

29. Molds – grow as multicellular filaments
- appear as thin filaments or dense clumps of spores
Viruses – take over cell machinery to reproduce hard to detect detected by ELISA, PCR, electron microscopy