This document provides guidelines for maintaining proper biosafety and aseptic technique in a cell culture laboratory. It outlines considerations for laboratory layout and design such as maintaining asepsis, dust control, and limited traffic. Major areas of a cell culture lab include media preparation, wash-up, and sterilization areas. Key points for planning the layout include the number of users, available space, and location of preparation and storage areas. Guidelines are provided for handling cell lines and cultures, maintaining equipment, using proper labeling and protective equipment, and avoiding contamination.

1. ©Sharmista, 2018

2.  to maintain asepsis
 to keep the lab dust free
 to maintain minimal traffic
LAYOUT DEPENDS ON:
 type and scale of operations
 number of users
 strict rules needed to be
followed
SOME COMMON CONSIDERATIONS:
 rooms should be designed for easy
cleaning
 décor and furniture needs to be tightly
fit to the floor
 floor covered with coved vinyl, acrylic
coating or other dust proof finishing's
 slight fall in the level toward a floor drain
located outside the door of the room away
from sterile cabinets
MAJOR AREAS:
• media preparation
• wash-up area
• sterilization area
KEY POINTS THAT SHOULD BE KEPT IN MIND DURING
PLANNING LAYOUT:
 number of users
 space
 location of preparation area
 storage
 access
 containment and sterility

3. Biosafety cabinets:
Laminar air flow
cabinets attached
with HEPA and UV
CO2 incubators:
Temperature = 37
degree Celsius
CO2 % = 2-5%
Air = 95%
Humidity = 99%

4. Centrifuge Culture vessels
Milli q water
system
Water bath
Washer Dispenser Autoclave

5. Inverted microscope
Cryo-chamber of
liquid Nitrogen
Shaking chamber
Pipettes
Membrane filters
Staked vessel system

15. Cell Lines, Cell Culture Media and Cell Culture Reagents
Only handle one cell line at a time. This common-sense approach will reduce the possibility of cross
contamination. It will also reduce the spread of bacteria and mycoplasma due to the generation of aerosols
across opened media bottles and flasks in the cabinet.
Test cells for the presence of mycoplasma on a regular basis.
Examine cultures and media daily for evidence of gross bacterial or fungal contamination. This includes media
that has been purchased commercially.
Maintain separate bottles of media for each cell line in culture.
Quality control all media and reagents prior to use.
Laboratory Practices
Ensure that incubators,
cabinets, centrifuges and microscopes are cleaned and
serviced at regular intervals.
Ensure that equipment requiring calibration is
calibrated on a regular schedule, or if its performance is
suspect.
Correctly label reagents including flasks, media
and ampoules with contents and date of preparation.
Use personal protective equipment (PPE), (laboratory
coat/gown, gloves and eye protection) at all times. In
addition, thermally insulated gloves, a full-face visor and
a splash-proof apron should be worn when handling
liquid nitrogen.
Wear dedicated PPE for the tissue culture facility
and keep separate from PPE worn in the general
laboratory environment. The use of different
colored gowns or laboratory coats makes this
easier to enforce.
Use disposable head caps to cover hair.
Clean the work surfaces with a suitable
disinfectant, e.g. 70% isopropanol, between
operations and allow a minimum of 15 minutes
between handling different cell lines.
Keep all work surfaces free of clutter.
Keep cardboard packaging to a minimum in all
cell culture areas.

16. Do not continuously use antibiotics in culture medium as this will inevitably lead to the appearance of
antibiotic resistant strains and may render a cell line useless for commercial purposes.
Don’t allow waste to accumulate, particularly within the microbiological safety cabinet or in the incubators.
Don't have too many people in the lab at any one time.
Don't handle cells from unauthenticated sources in the main cell culture suite. They should be handled in
quarantine until quality control checks are complete.
Avoid keeping cell lines continually in culture without returning to frozen stock.
Avoid cell culture becoming fully confluent. Always sub-culture at 70 to 80 percent confluency, or as
advised on ECACC's cell culture data sheet.
Do not allow media to go out of date. Shelf life is only 6 weeks at +4ºC once glutamine and serum is added.
Avoid water baths from becoming dirty by using Sigma Clean (Prod. No. S5525).
Don’t allow essential equipment to become out of calibration. Ensure microbiological safety cabinets are
tested regularly.

17. 1. Culture of animal cells by R Ian Freshney
2. "Fundamental Techniques in Cell Culture
Laboratory Handbook" published by Sigma®
Life Science and European Collection of Cell
Cultures (ECACC) 2nd Edition, Cook Book
Volume 12
3. Animal Biotechnology by M M Ranga
4. Google images