Cell Culture Laboratory

(Lec I)
Dr. Anum Gul
General principles for Cell culture Lab Design

 The rooms should be designed for easy cleaning.

 Furniture should fit tightly to the floor or be suspended from the

bench with a space left underneath for cleaning.

 Cover the floor with a vinyl, acrylic coating, or other dustproof finish,

and allow a slight fall in the level towards a floor drain located outside

the door of the room (i.e., well away from the sterile cabinets).
General principles for Cell culture Lab Design

 If possible, it is preferable for the tissue culture lab to be

separated from the preparation, washup, and sterilization areas,

while still remaining adjacent.

 If you have a large tissue culture lab with a separate washup and

sterilization facility, it will be convenient to have this on the same

floor as, and adjacent to, the laboratory, with no steps to negotiate,

so that carts or trolleys may be used.

Layout, Furnishing, and Services

 The need to maintain asepsis distinguishes the tissue culture laboratory

from most others, so it is important to be dust free and have no through

traffic.

 Layout depends on the type and scale of the operations and the number

of users.
Requirements

 Number of users: How many people will work in the facility, how long will

they work each week, and what kinds of culture will they perform?

 Space: What space is required for each facility? The largest area should

be given to the culture operation, which has to accommodate cell culture

hoods, cell counters, centrifuges, incubators, microscopes, and using stocks

of reagents, media, glassware, and plastics, and, if possible, a quarantine

area.
Requirements

The second largest is for washup, preparation and sterilization, the third is for

storage, and the fourth is for incubation. A reasonable estimate is 4:2:1:1, in

the order just presented.

 Location of preparation area

Facilities for washing up and for sterilization should be located (a) close to the

aseptic area that they service and (b) on an outside wall to allow for the

possibility of heat extraction from ovens and steam vents from autoclaves.
Requirements

 Aseptic work area:

The major requirement of a cell culture laboratory is the need to maintain

an aseptic work area that is restricted to cell culture work.

Although a separate tissue culture room is preferred, a designated cell

culture area within a larger laboratory can still be used for sterile handling,

incubation, and storage of cell cultures, reagents, and media.

Cell Culture Equipment

 The specific requirements of a cell culture laboratory depend mainly on

the type of research conducted; for example,

 The needs of mammalian cell culture laboratory specializing in cancer

research is quite different from that of an insect cell culture

laboratory that focuses on protein expression.

Cell Culture Equipment

 However, all cell culture laboratories have the common

requirement of being free from pathogenic microorganisms (i.e.,

asepsis), and share some of the same basic equipment that is

essential for culturing cells.

Basic equipment

 Cell culture hood (i.e., laminar-flow hood or biosafety cabinet)

 Incubator (humid CO2 incubator recommended)

 Water bath

 Centrifuge

 Refrigerator and freezer (–20°C)

 Hemocytometer

 Inverted microscope

 Liquid nitrogen (N2) freezer or cryostorage container

 Sterilizer (i.e., autoclave)

Requirements

 Cell culture hood

The simplest and most economical way to provide aseptic conditions is to

use a cell culture hood (i.e., biosafety cabinet).

Three kinds of cell culture hoods, designated as Class I, II, and III, have

been developed to meet varying research and clinical needs.

Requirements
Requirements
Biosafety Cabinet Class I

 A Class I cabinet is defined as a ventilated cabinet for personnel

and environmental protection.

 Class I cabinets do not offer product protection from

contamination, significantly limiting their applications.

 Class I cabinets have a similar airflow pattern to a fume hood, but

they also have a HEPA filter at the exhaust outlet.

 They may or may not be ducted outside. Class I cabinets are safe
for use with agents requiring Biosafety Level 1, 2 or 3 containment.
Biosafety Cabinet Class I
Biosafety Cabinet Class II

 A Class II cabinet is defined as a ventilated cabinet for personnel,

product and environmental protection for microbiological work or

sterile pharmacy compounding.

 Class II BSCs are designed with an open front with inward airflow

(personnel protection), downward HEPA-filtered laminar airflow

(product protection) and HEPA-filtered exhaust air (environmental

protection).
Biosafety Cabinet Class II

 These cabinets are further differentiated by types based on

construction, airflow and exhaust systems.

 The types include A1, A2, B1, B2 and C1.

Biosafety Cabinet Class II
Biosafety Cabinet Class II

Type B1 Type B2
Biosafety Cabinet Class II
Biosafety Cabinet Class III

 A Class III cabinet is defined as a totally enclosed, ventilated cabinet

with leak-tight construction and attached rubber gloves for performing
operations in the cabinet.

 Class III biosafety cabinets are also called glove boxes.

 The cabinet has a transfer chamber that allows for sterilizing materials
before they leave the glove box.
Biosafety Cabinet Class III

 The cabinet is maintained under negative pressure and supply air is

drawn in through HEPA filters.

 The exhaust air is treated with either double HEPA filtration or HEPA

filtration and incineration.

 Class III cabinets are safe for work requiring Biosafety Level 1, 2, 3 or

4 containment.
Biosafety Cabinet Class III
Air-flow characteristics of cell culture hoods

 Cell culture hoods protect the working environment from dust and

other airborne contaminants by maintaining a constant, unidirectional

flow of HEPA-filtered air over the work area.

 The flow can be horizontal, blowing parallel to the work surface, or it

can be vertical, blowing from the top of the cabinet onto the work

surface.
Clean benches

 Horizontal laminar flow or vertical laminar flow “clean benches” are

not biosafety cabinets; these pieces of equipment discharge HEPA-

filtered air from the back of the cabinet across the work surface

toward the user, and they may expose the user to potentially

hazardous materials.
Clean benches
Clean benches

 These devices only provide product protection. Clean benches can be

used for certain clean activities, such as the dust-free assembly of

sterile equipment or electronic devices, and they should never be

used when handling cell culture materials, or when manipulating

potentially infectious materials.

Cell culture hood layout

 A cell culture hood should be large enough to be used by one person at a

time, be easily cleanable inside and outside, have adequate lighting, and

be comfortable to use without requiring awkward positions.

 Keep the work space in the cell culture hood clean and uncluttered, and

keep everything in direct line of sight.

 Disinfect each item placed in the cell culture hood by spraying them with

70% ethanol and wiping clean.

Cell culture hood layout
Incubator

 There are two basic types of incubators, dry incubators and humid CO2

incubators.

 Dry incubators are more economical, but require the cell cultures to be

incubated in sealed flasks to prevent evaporation.

 Placing a water dish in a dry incubator can provide some humidity, but

they do not allow precise control of atmospheric conditions in the

incubator.
Incubator

 Humid CO2 incubators are more expensive but allow superior control

of culture conditions.

 They can be used to incubate cells cultured in Petri dishes or

multiwell plates, which require a controlled atmosphere of high

humidity and increased CO2 tension.

Aseptic work area

 The major requirement of a cell culture laboratory is the need to maintain

an aseptic work area that is restricted to cell culture work.

 Although a separate tissue culture room is preferred, a designated cell

culture area within a larger laboratory can still be used for sterile

handling, incubation, and storage of cell cultures, reagents, and media.

 The simplest and most economical way to provide aseptic conditions is to

use a cell culture hood (i.e., biosafety cabinet).

Ventilation Considerations for Biosafety Level 2
Laboratories

 Potentially harmful aerosols can escape from the containment of the

laboratory room unless the room air pressure is negative to adjacent

non-laboratory areas.

 As a general rule, air should flow from low hazard to high hazard

areas.
Ventilation Considerations for Biosafety Level 2
Laboratories

 Ideally a tissue culture laboratory should be at positive pressure

relative to surrounding work areas, to avoid any influx of contaminated

air from outside.

 Tissue culture rooms that involve the use of biohazardous agents shall

be negative.
Thank You