DESIGN, CONCEPTS AND LAYOUT OF CELL CULTURE LABS

Concepts
Effects of contaminants
Facilities in TC labs
Equipping the TC labs
Essential equipment
Beneficial equipment

ASEPTIC TECHNIQUE
Sources of contaminants
Type of contaminants
Minimising contamination
pipetting
sterile handling
pouring
Objectives of aseptic technique

Laminar flow hood and microbiological safety cabinet

Types and functions of cabinets
Important points regarding the use of the cabinets
Organizing the hood for routine work
Use of antibiotics
 DESIGN CONCEPTS AND LAYOUT OF CELL-CULTURE LAB
CONCEPT
 Tissue culture lab need to maintain asepsis.
 Handling of cultured cells in sterile environment.
 Provision must be made to ensure that cultures and culture media
are maintained free from contaminating micro-organism
• The lab should be only for the purpose of TC handling and propagation
Should not be used for any other experimental work involving the TC eg.
virus infection of TC, cell hybridization

Effects of Contaminant
rapid growing in culture media
kill the animal cells
release toxin
deplete the nutrient medium
depress the medium pH

FACILITIES NEEDED IN TISSUE CULTURE LAB

1. Sterile handling area
2. Incubation -incubators
3. Preparation area
4. Wash up area
5. Sterilization
6. Storage
Sterile handling area with

Minimal movement of people past through the clean area

Should have a proper separation between clean area and

dirty operations including waste disposal

Vinyl flooring with dust proof finishing

Air conditioning

Other facilities essential to cell culture work

Laminar flow cabinets

Incubators (CO2)

Inverted microscope

Instrument and equipment benches-centrifuge, pipettes

Media fridges (sterile working solutions only)

Freezers for culture reagents requiring storage at -20°C esp. serum

-80°C for cryopreservation of cells before storage in liqiud nitrogen (-196oC)

Media and solutions

Preparation area

General cold storage facility for chemicals and non-sterile reagent

General preparation bench

In a separate room :
Liquid nitrogen freezer (tanks) for frozen cell stocks

Storage area for unopened plastic ware

Sterilization
Autoclave
Drying oven
Water purification system

Wash up area with sinks, soak tanks

EQUIPPING THE TC LABORATORY

The specific needs of the tissue culture laboratory like most labs,
can be divided into three categories ;

Essential – you cannot perform a job without them

Beneficial – the work would be done better, more efficiently, quicker, or

with less labour

Useful – it would make life easier, improve working conditions, reduce

fatigue, enable more sophisticated analysis to be made, or generally make
your working environment more friendly and attractive.
 Essential equipment
Incubator – temperature, gas phase and humidity are controlled.
Consideration for purchasing incubator :
Large enough
Have forced air circulation
Temperature control ± 0.5°C
Safety thermostat
Corrosion resistant-stainless steel
Easily cleaned

Steriliser
Antibiotic
Autoclave
Filtration
Chemical sterilisation
Fumigation
Liquid disinfectants
Irradiation
Ultraviolet (260nm) – to sterilize air in cell culture cabinet
Gamma rays – has a very good penetrating properties,
items can be completely sealed and packages
Inverted microscopic
Cell freezing unit
Water purification
Centrifuge

Beneficial equipment
Cell counter-essential for precise quantitative growth kinetics
e.g Coulter counter
Aspiration pump-a vacuum pump or simple tap siphon
Balances, pH meter and osmometer
Upright microscope-attach to the camera
Dissecting microscope
Magnetic stirrer
Pipette and micropipette
Large column dispensing unit
Repetitive dispensing unit
Useful additional equipment
Low temperature freezer
Glassware washing machine
Colony counter
Controlled rate freezer
 ASEPTIC TECHNIQUE
Successful cell culture is absolutely dependent on
fastidious aseptic technique.

Source of contamination
- Bacteria
- mycoplasma-invisible (need mycoplasma test)
- yeast
- fungal spores

Type of contamination
- can be minor and confined to one or two cultures
-can spread among and several other cell lines and
infect a whole experiment
- can be widespread and wipe out the entire stock or even the whole lab
Minimising Contamination by:

• cultures are checked through microscopy

• culture maintain without antibiotics (where possible)

• reagents are checked for sterility before use

• bottles of media are not shared with other people or

used for different cell lines

• the standard of sterile technique and good microbiological practice

and techniques are kept high at all times

•Sloppy cell culture technique can be very disruptive to the lab.

Occasional isolated contamination can happen to anyone, but commonly
prove to be no more than a nuisance.

CONTAMINATION IS TROUBLESOME,
TIME CONSUMING AND EXPENSIVE.
Pipetting

Never use pipette more than once.

Never enter a culture vessel or reagent bottle with a used pipette

Never draw so much fluid into the pipette as to wet the plug

Do not allow fluid to drip from the pipette tip

Never blow bubbles in media or cell suspensions

When using pipette to disperse cells, keep the tip below fluid level and
a deep, narrow-mouthed bottle or flask

Clean up any spillage immediately with 70% ethanol

Sterile handling
Swabbing

Swab down working surface before and during work particularly

following spillage

Swab down after finished

Swab bottles, particularly from the cold room, before using for the first
time each day

Capping

Deep screw caps should be used in preference to stoppers

Flaming

The necks of the bottles and screw caps should be flamed before and
after opening a bottle and after closing
If working outside the Class II cabinet, Work close to the flame
where there is an up-current due to convection,
and do not leave bottles open

IT IS NOT ENCOURAGE TO USE FLAMES IN CLASS II CABINETS

AS THEY CAUSE TURBULENCE AND DISTURBED THE AIR FLOW

Screw caps should be place open-side down on a clean surface and flamed
before replacing on the bottle

Screw caps may be held in the hand during pipetting,

avoiding the need to flame or lay down
Handling bottles and flasks

Pouring

The major risk in pouring lies in the generation of a bridge of

liquid between the outside of the bottle and the inside,
which may infection to enter the bottle.

Bottles or flasks that are stored or incubated after pouring are at

a significantly higher risk
 Thus it is essential that the cell culture specialist adopt the
best possible working habits (good microbiological practice and techniques)
and continually be on the outlook for problems and their causes

Objectives of aseptic technique

To provide a barrier between micro-organism in the environment
outside the culture and the pure uncontaminated culture
within its flask and dish.

Personal hygiene
Hand washing
Gloves
Caps, gowns and face mask
Flu-wear face mask
Talking is permissible-if working in the Class II microbiological
safety cabinet
LAMINAR FLOW HOOD AND MICROBILOGICAL SAFETY CABINET

Working in the laminar flow hood

Routine handling of cultured cell is recommended to be carried out in a
class II microbiological safety cabinet.

Functions and limitation of laminar flow

Laminar flow hoods are engineered to eliminate particulates,
to reduce the chance that a particle will enter an open culture dish or flask.

Two basic types of laminar flow hood are used :

vertical flow-air flow is straight down

horizontal flow – force the air out of the front cabinet, no glass screen
 Laminar flow clean air cabinet
Horizontal air-flow cabinet

This protects the work against microbial contamination by a laminar flow

of sterile air.

This type of cabinet is suitable for making media and handling

non-infected tissue cultures.

It is not suitable for infectious work as personnel are exposed to

the exhaust air.

1 Open front and exhaust air

2 Working space
3 Hepa filters and laminar flow
4 Fan
 Class I microbiological safety cabinet

.
This protects the worker. Air is drawn from the room,
with the air-stream away from the worker thus protecting
him against infection.
The incoming air is not sterile and may contaminate the work.
The exhaust air is filtered to protect the environment.

1 Open front with inward airflow

2 Working space
3 Exhaust fan
4 Exhaust hepafilter
5 Sterile exhaust air
This protects both the work and the worker.
Incoming and exhaust air is filtered.
The open front is protected by an air current,
which acts as a curtain.
Highly contagious material must be handled in
a Class ll cabinet as air and liquid spilling
through the front are not absolutely preventable.

1 Open front with air curtain

2 Window
3 Working space
4 Inlet with hepafilter
5 Inlet fan
6 Air inlet with prefilter
7 Air vents in the bottom
8 Exhaust fan
9 Exhaust hepafilter

Class II microbiological safety cabinet

(vertical flow)
Important points regarding the use of laminar flow hoods
Laminar airflow creates a clean work environment, not a sterile one

The laminar flow hood is the most critical work area in the lab,
despite the filtered air, it is the most likely site where cultures can
become contaminated

Horizontal-flow hoods must never be used in work with pathogens or

with human and primate cell lines

Perform all activities in the hood with the understanding that

airflow can carry contaminants to the culture system

Hands and arms (or gloves and sleeves) can be a serious source items
for of contaminant – laden particulate (eg. Dry skin, talc, and lint).
Organizing the hood for routine work
Minimize the clutter

Position items for efficiency of movement and to minimize traffic over the
centre of the field where handling of cultures will be performed

Leave the front and rear vents clear for the best airflow

The flasks or dishes of cultured cells or tissue for primary culture should be
the last items transferred to the hood

Minimize the introduction of dust and particles to the hood by first wiping
with 70% ethanol

Incubators are a prime source of contamination.

Flasks and trays of cultures from the incubator be wiped with 70% ethanol
before they are placed in the hood
 USE OF ANTIBIOTICS

Antibiotics introduced into culture media – to reduce contamination

Use of laminar flow hoods and strict/good microbiological practice

and techniques makes antibiotic unnecessary

Disadvantages of using antibiotics

i. They encourage the development of antibiotic resistant organisms

ii. They hide the presence of low level cryptic contaminants esp.
Mycoplasma that will flare-up once the antibiotic is removed or
changes made eg change of culture conditions
iii. They have antimetabolite effects that can cross react
with mammalian cells
vii. They encourage poor aseptic techniques. Fungal and yeast contaminants
that cannot be controlled by antibiotics will set in.
Use of antibiotics is restricted
to primary culture
Large scale labor intensive experiments with high consumable costs