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Cell and Tissue Culture

Setting Up a Cell/ Tissue Culture

• Consider budget
• Number of the Personnel
• Available area assesment
• Special requirements
• Reduce the act of entering and exiting
• Maintain positive pressure in the room
Ece ÖZYAĞCI
(HEPA filtered air)
Selay TORNACI
Ebru TOKSOY ÖNER • Smooth and untexture floors (ensure easy
cleaning)
Marmara University, Department of Bioengineering

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Medium Size Cell Culture Laboratory Large Size Cell Culture Laboratory

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Basic Equipment Cell Culture Hood
• Cell culture hood (laminar-flow hood or biosafety cabinet)
• Incubator (humid CO2 incubator recommended)
• Inverted microscope
• Water bath
• Centrifuge
• Cell counter (Automated Cell Counter or hemacytometer)
• Refrigerator and freezer (+4 and –20° C)
• Cryostorage container
• Sterilizer (autoclave)
Personnel protection ✓
Product protection ✓
Environmental protection ✓

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Incubator (CO2 or N2) Microscopes
• Create the right environment to promote optimal cell growth

HEPA Filter

Inverted Fluorescence
Inverted

Dissecting
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Water bath and Centrifuge Cell counter

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Refrigerator and freezer Stor
e
Cryostorage Container Liquid Nitrogen Tank
acco all me
rdin d
g to ia, reag
the e
instr nts, an -196 °C
uctio d
ns o chemic
n th a
e la ls
bel!
+4 °C !! -80 °C

-20 °C

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Sterilizer Other Equipments
• maintaining steam at 121 ° C
• 100 kPa (15 PSI)
• 15 or 20 minutes

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Cell and Tissue Culture
Safety in CTC Labs (Personal and Environmental Safety)
• All work places have some common safety risks. CCT
labs have unique dangers tied to working with human
or animal cells, tissues, and harmful chemicals.
• Specific hazards
• Handling and manipulating human or animal cells and
tissues, corrosive or mutagenic solvents and reagents.
• Accidental punctures with syringe needles or other
contaminated sharps
• Spills and splashes onto skin and mucous membranes
• Ingestion through mouth pipetting or inhalation exposures
to infectious aerosols.
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Cell and Tissue Culture
Safety in CTC Labs (Personal and Environmental Safety)
• For animal cell culture the level of risk is dependent
upon the cell line to be used and is based on whether
the cell line is likely to cause harm to humans. (Cells
collected form HIV patients)
• The key to safety in a cell culture lab is following
standard microbiological practices and techniques
closely
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Personal and Environmental Safety Safety Equipment
• Primary barriers: Biosafety cabinets, enclosed containers, and
additional safety measures to reduce contact with dangerous
substances
• Personal protective equipment (PPE): Create a quick
barrier between people and the hazardous agent

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General Rules Disinfection (Decontamination)
• Always wear appropriate personal protective equipment. Change gloves • of culture waste, work surfaces and equipment is
when contaminated, and dispose them with other contaminated lab waste. crucial for reducing risks
• Wash your hands before leaving the laboratory.
• Do not eat, drink, smoke, handle contact lenses or apply cosmetics Hypochlorites Alcohols
• Follow the institutional policies regarding safe handling of sharps Good for general purpose disinfectant Good for daily purpose disinfectant
• Take care to minimize the creation of aerosols and/or splashes.
Active against bacteria, viruses, Active against bacteria and most
• Before and after experiments, decontaminate work surfaces and promptly mycobacteria and fungal spores viruses
disinfect in case of spills. Regularly clean lab equipment, even when not
Should be made fresh daily (inactivated Mode of action is dehydration and
visibly contaminated. by organic matter) and highly corrosive fixation
• Decontaminate all potentially infectious materials before disposal.
Effective concentration: Effective concentrations: 70%
• Report any incidents that may result in exposure to infectious materials to • Surface (1000ppm)
appropriate personnel (e.g., laboratory supervisor, safety officer) • Tissue culture waste and spillages
(10,000 ppm)

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Waste Disposal Safety in CTC Labs
• To dispose of tissue culture waste (culture medium), soak (Sample Safety with Aseptic Technique)
it in a 10,000 ppm hypochlorite solution for at least 2
• Maintaining a successful cell
hours, then rinse it with excess water before draining. culture relies on preventing
• Contaminated pipettes should be left in a 2,500 ppm
contamination by
hypochlorite solution overnight and then disposed of by
microorganisms like bacteria,
autoclaving and incineration.
fungi, and viruses.
• Solid waste like flasks, centrifuge tubes, contaminated
• Aseptic technique aims to create
gloves, and tissues should be placed in heavy-duty sacks a barrier between these
for contaminated waste and then incinerated. microorganisms in the
• Whenever possible, incineration is preferred over
environment and the sterile cell
autoclaving for waste disposal. culture.

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Aseptic Technique ✓ For routine cleaning: 70% Ethanol
• Sterile Work Area: ✓ UV light to sterilize air and surface
✓ Only required items for experiment
Aseptic Technique
• Sterile Reagents and Media
✓ Always sterilize any reagents, media, or solutions using the
appropriate sterilization procedure (autoclave or sterile filter)

• Good Personal Hygiene

✓ Wash hands before and after
working with cell cultures
✓ Use PPE, confined long hair,
avoid loose and hanging clothes
and jewelry

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Sterile Handling Aseptic Technique Checklist
• Always wipe hands and the work area outside of the containers, flasks,
plates, and dishes with 70% ethanol.
• Avoid pouring media and reagents directly from bottles or flasks.
• Use sterile glass or disposable plastic pipettes to work with liquids, and
use each pipette only once to avoid cross-contamination.
• Always cap the bottles, flasks, and multi-well plates after use.
• Never uncover a sterile flask, bottle, petri dish, etc. until needed, and never
leave it open to the environment. Return the cover as soon as possible.
• After removing a cap or cover, place it down with the opening facing down.
• Use only sterile glassware and other equipment.
• Be careful not to talk, sing, or whistle while performing sterile procedures.
• Perform experiments as rapidly as possible to minimize contamination.

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Biological Contamination Biological Contamination
• Contaminating cell cultures is the most common issue in cell culture
labs if the aseptic technique is not applied correctly.

Bacteria Contamination

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Biological Contamination Biological Contamination

Yeast Contamination Mold Contamination

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Biological Contamination Non-Biological Contamination

Mycoplasma Contamination

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Cell and Tissue Culture

How to enter and exit to the cell culture labotatory

(BSL2)?

BSL2 laboratory - YouTube

BSL2 rules in practice

Lab Safety Training (BSL-2) - YouTube
Ece ÖZYAĞCI
Selay TORNACI
Ebru TOKSOY ÖNER
Marmara University, Department of Bioengineering

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