Basic Requirement For Laboratory Design
I. The Laboratory Design
1. Design Concepts and Layout
a. To allow sterile handling of culture cells
b. Minimum movement of people past or through
the clean area
c. Adequate separation of the clean area for sterile
handling of cells from dirty operation
2. Facilities Essential to Cell Culture
a. Laminar flow cabinet
b. CO2 Incubator
c. Storage space for sterile equipment and
solution
d. Iinstrument and equipment bench
e. Media fridge ( sterile working solution only)
f. Freezer for culture reagents requiring storage at
- 20oC or below
g. Preparation area for media and other solution
h. General cold storage facility for chemicals and nonsterile reagents
Laminar Flow
Refrigerator
CO2 incubator
microscope
Working area
Storage and
preparation
Washing
area
Working area
Preparation area
Deionize
water
supply
soaking
washing
Pipette
washer
If possible In a separate room:
a. Liquid nitrogen freezers for cell stock
b. General preparation area
c. Storage area for unopened plastic ware
d. Sterilization oven and autoclave
e. Drying oven
f. Water purification system
g. Washing-up area
Others:
1. Warm room at 37oC for large scale culture
2. Laminar flow for tissue dissection for primary culture
3. Electronic cell counter ( for large number in cell
growth kinetic research)
4. Air conditioning
5. Cold room at 4oC for media storage or liquid nitrogen
freezer
II. Services
1. Water supply to sink
2. Gas tap for Bunsen burner
3. CO2
4. Ultra pure water
water may be used for:
a. Solvent for culture media
b. Solvent for supplement
c. For rinsing of glassware
tapwater

water softener cartridge

RO

ultrapurification system

ultrapure water
Direct-Q™ 5 Ultrapure Water Systems
III. Equipping the laboratory
A. Laminar flow hoods
class II cabinet offers:
 Front screen which minimize the turbulence
drown in front side
 Exhausting air flow is mostly recycled
 Equipped air flow monitor alarm system
Vertical Hood
Horizontal Hood
 How to choose a laminar flow?
1 . Does the hood require an internal power point
or gas tap?
2. Is the inside of the hood easy to clean?
3. Will it be necessary to open the front of the hood
regularly to introduce large vessel?
4. Would it be useful for the height of the
working surface of the hood to be adjustable
B. CO2 Incubator
CO2 Incubator
1. Temperature
Mammalian cells : 37 oC
Avian cells : > 37 oC
Mammalian skin cells : <37 oC
Cold blooded animals: their normal
expose range
2. Gas phase
CO2: maintain pH of culture medium
pH7.2---7.4
concentration: 5%
10% for serum free medium
CO2+H2O  H2CO3  H++ HCO3NaHCO3  Na+ + HCO3(add NaHCO additionally in the medium)
3
NaOH+H2CO3  NaHCO3+H2O  Na++HCO3-+H2O
(similar effect to neutralize pH if add alkali)
 Inclusion of pyruvate increase production of CO2
 LeibovitzL15 has high sodium pyruvate, lack NaHCO3 
does not require CO2
Buffer used for medium
Incorporate into medium to stabilize pH
Relationship Between NaHCO3, CO2 and HEPES
compound
Eagle’s MEM Hank’s salt
Low HCO3- buffer
Eagle’s MEM
DMEM
Earl’s salt
NaHCO3
CO2
HEPES
4mM
Atmospheric and evolved
from culture
10mM
10mM
26mM
44mM
2%
5%
10%
20mM
50mM
---
Oxygen
 Rely mostly on glycolysis in cell culture
 Dissolved O2 is toxic by producing free radical ---------may be reduced by free radical scavager, i.e.
- mecaptoethanol or dithiothreitol
 Higher O2 percentage for organ culture 95%
 Keep depth of medium within 2-5 mm( 0.2-0.5% mL/cm2)
3. Humidity
 change water frequently
 avoid microbial contamination
Water plate
C. Microscope
 Inverted microscope
for observation of :
cell morphology, degree of spreading,
membrane blebbing, proportion of
multinucleate, vaculation, microbial
contamination
 Fluorescent microscope
Inverted Microscope
Fluorescent microscope
D. Centrifuge
 bench top centrifuge
 cell collection
E. Fridges and freezer( -20oC, -70oC, liquid
nitrogen)
 media storage
F. Miscellaneous small items of equipment
 Water bath 37oC
 Submersible magnetic stirrer
 Aspirator jar
Culture plastic ware and associate small consumable items
A. The most commonly used items of plastic ware:
1. For growing cells( electric treatment on
surface of culture plate
 flat bottomed flask
 petri-dish
 multi-well dish
 conical flask roller bottle
 Tubes
 culture flask
25 cm2 75cm2 175 cm2
 culture dish
petridish: 35mm, 60mm, 90mm,….
Multiwell dish 6 wells,12 wells,24 wells,…
Chamber Slides
2. For handling solutions and cell suspensions
 Volumetric pipettes
 Plastic Pasteur pipette
 Micropipette tips
 Centrifuge tube
15 ml centrifuge
50 ml centrifuge
3. For storage
 Sample tube
 Eppendorf tube
 Cryotube
 Large volume screw capped bottle
For large scale adherent cell culture:
 anchorage dependent culture:
▪roller bottle
▪ micro carrier beads
▪ meshes
 Suspension culture:
▪ spinner flask
▪ conical flask on a rotating platform
▪ static culture on nonadherent plastic ware
Spinner flask
conical flask on a rotating
platform
B. Filters for sterilizing tissue culture solutions
pore size: 0.2um---0.45um
 How to choose the filters
 Low protein binding
 Minimum dead space
 Large volume
C. Pipettes
 micropipettes with disposable autoclaveable
tips
volume: 1---20 ul
20---200 ul
100---1000 ul
5 ml
 Disposable Pasteur pipettes
 Glass volumetric pipette
volume 5---25 ml
D. Pipette aid or multichannel micropipette
E. Glassware for tissue culture use
F. Miscellaneous small items
IV. Washing re-usable tissue culture equipment
1. Soaking
 residual medium must be removed
 dried on medium will be difficult to removed
 Inadequate wash may introduce toxic products into
the new the new solution
 after soaking, glass ware must be
a. rinse with cold tap water
b. remove all tap water
c. remove all markers
2. washing
 Use detergent or acid
 Machine or hand wash
 Rinse with R.O. water after wash
3.washing pipette
 Soak in the plastic cylinder
 Wash with sonicator or pipette washer
http://www.youtube.com/watch?v=_fjZ-MHV22w