Laboratory set up for

animal cell culture

ASHIKA M
D3 BIOTECH
 The major requirement that distinguishes tissue culture
from other laboratory is the need to maintain asepsis
It is not economically viable to create large sterile
areas
It should be maintained dust free and have no through
traffic
Introduction of laminar flow hoods has greatly
simplified the problem
While designing an animal cell culture lab the following
items should be considered:
Ventilation
Accommodation
Renovations
Access
Quarantine
1. Ventilation
a. Pressure balance
Ideally , a tissue culture laboratory should be at
positive pressure relative to surrounding working area
It is done to prevent influx of contaminated air from
outside
Human material negative pressure
So a +ve pressure buffer zone outside the lab ie.
Preparation area , microscopic area or the corridor
b. Laminar flow hoods
To improve air circulation and remove excess heat
[300-500 w per hood ] from the room
Provide most of the air extraction required for the
room ,and it remains only to ensure that the incoming
air , from an air conditioner, does not interfere with
the integrity of the air flow in the hood
It is better to make it work continuosly
But if they are switched off, an alternative air
extract must be provided
 2. Accommodation
a. Staff numbers
Determines how many LAF will be required
12 LAF 50 people
b. Space
Largest area should be given to accommodate LAF, cell
counter, centrifuges, incubators,& some stock of
reagents, media , glass wares & plastics
2nd wash up, preparation, & sterilization
3rd storage
4th- incubation
c. Aseptic area
Windows disadvantage in tissue culture lab
Leads to heat gain, uv denaturation of medium &
entry of micro organisms if they are not properly
sealed
d.Hoods
space b/n hoods should be ~500mm(2ft), for
maintenance & to minimize interference
That space can be filled with a removable cart or
trolley which allows space for bottles, flasks ,
reagents & a note book
e. Incubation
large no. of flasks or large volume flasks that are
sealed are best incubated in a hot room
Open plates & dishes will requires a humid co 2
incubator
f. Preparation area
Close to the aseptic area
Outside wall should be provided for protection
heat(ovens, steam from autoclave)
3.Renovations
If a conversion of existing facilities is contemplated, then
there will be significant structural limitations
Choose location carefully , to avoid space constraints and
awkward projections

4.Access
Door ways should be both wide and high enough
Ceilings- sufficient clearance to install equipments

5.Quarantine
Newly introduced cell lines and biopsies need to screened for
mycoplasma before handled in the same room as general
stocks
Some human and primate biopsies and cell lines may carry a
biohazard risk
Minimum requirements
Sterile area, clean, quiet, and with no
through traffic
Separate from animal house and
microbiological labs
Preparation area
Wash up area (not necessarily within
tissue culture laboratory, but at least adjacent to it)
Space for incubator(s)
Storage areas:
Liquids: ambient, 4C, 20C
Glassware (shelving)
Plastics (shelving)
Small items (drawers)
Specialized equipment
Chemicals: ambient, 4C, 20C; share
with liquids, but keep chemicals in
sealed container over desiccant
CO2 cylinders
Space for liquid N2 freezer(s)
Sink
LAYOUT OF ASEPTIC ROOM OR
SUITE
Six main functions need to be accommodated in the
lab:
1. Sterile handling
2. Incubation
3. Preparation
4. Wash up
5. Sterilization
6. Storage
 If a single room is used, create a sterility
gradient i.e. The clean area for sterile handling
should be located at one end of the room, farthest
from the door, and wash up area and sterilization
facilities should be placed at the other end, with
preparation, storage and incubation in between
The preparation area should be adjacent to the
wash up and sterilization areas, and storage and
incubators accessible to sterile working area
 STERILE HANDLING
Sterile work should be located in a quiet part of the
tissue culture lab & should be restricted to tissue
culture
No traffic & disturbance
Use separate room or cubicle if laminar flow hoods
are available
 laminar flow
Greater control of aseptic conditions at lower cost
Can be moved around
Some can be operated with batteries
With individual hoods only operators arm enter the
sterile area , whereas laminar flow wall or ceiling
units , the operator is a part of working area
Necessary to wear sterile caps and gowns to avoid
contamination
QUARANTINE AND CONTAINMENT

If sufficient space is available, it is worth designating

a separate room as a quarantine and or containment
room
This is a separate aseptic room with its own LAF ,
incubators, freezer, refrigerator, etc.
Newly imported cell lines or biopsies can be handled
until they are shown to be free of contamination,
particularly mycoplasma ,HIV, hepatitis B
 Service bench
It is for keeping cell counter, microscope etc. close
to the sterile handling area and either dividing the
area or separating it from other end of the lab
Provide storage for sterile glass wares, plastics ,
screw caps, syringes etc. in drawer units below and
open shelf above
Small centrifuge
 INCUBATION
Incubators
Incubation Carried out in separate incubators
i.e. humid co2 incubators
Lose more heat when they are opened &
require more time to recover from it
In order to avoid this hot room can be used
Co2 incubator
Hot room
insulated to prevent heat loss
Wooden furnishings should be avoided as they warp
in the heat
Heaters to supply heat via fan
Air circulation - A second fan, positioned on the
opposite side of the room and with the airflow
opposing that of the fan heater, will ensure maximum
circulation
Thermostats - supply heat at a rate proportional to
the difference between the room temperature and
the set point. When the door opens and the room
temperature falls, recovery will be rapid
 PREPARATION AREA
MEDIA PREPARATION
o Large enterprise prepare its own media
o Small lab purchase ready made media, need only to
prepare reagents such as salt solutions & ethylene
diamine tetra acetic acid (EDTA), bottle these & water, &
package screw caps & other small items for sterilization.
Heat stable solutions & equipment can be autoclaved or
dry heat sterlized
WASHUP
Outside tissue culture lab
Should have plenty of space for soaking glass wares
Plenty of bench space for handling baskets of glass
wares, sorting pipettes & packaging & sealing packs for
sterilization
STORAGE

Storage must be provided for the following items

ensuring sterile and non sterile are kept separate and
clearly labeled:
(1) Sterile liquids, at room temperature (salt solutions,
water, etc.), at 4C (media), and at 20C or 70C
(serum, trypsin, glutamine, etc.)
(2) Sterile and non sterile glassware, including media bottles
and pipettes
(3) Sterile disposable plastics (e.g., culture flasks and Petri
dishes, centrifuge tubes and vials, and syringes)
(4) Screw caps, stoppers, etc., sterile and non sterile
(5) Apparatus such as filters, sterile and non sterile
(6) Gloves, disposal bags, etc.
(7) Liquid nitrogen to replenish freezers; the liquid
nitrogen should be stored in two ways:
a) In Dewars (2550 L) under the bench, or
b) In a large storage vessel (100150 L) on a trolley or
in storage tanks (5001000 L)