1. 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. 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 non-
sterile reagents

3. Refrigerator
CO2 incubator
microscope
Laminar Flow

4. Working area
Storage and preparation
Washing area

7. Working area
Preparation area

8. soaking
washing
Deionize water
supply
Pipette
washer

11. 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

12. 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

13. 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

14. water softener cartridge
tapwater 
water softener cartridge RO
ultrapurification system
ultrapure water
Direct-Q™ 5 Ultrapure Water Systems

15. III. Equipping the laboratory
A. Laminar flow hoods

16. 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

17. Vertical Hood
Horizontal Hood

18.  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

19. B. CO2 Incubator

23. CO2 Incubator
1. Temperature
Mammalian cells : 37 oC
Avian cells : > 37 oC
Mammalian skin cells : <37 oC
Cold blooded animals: their normal
expose range

24. CO2: maintain pH of culture medium pH7.2---7.4 concentration: 5%
2. Gas phase
CO2: maintain pH of culture medium pH
concentration: 5%
10% for serum free medium
CO2+H2O  H2CO3  H++ HCO3-
NaHCO3  Na+ + HCO3-
(add NaHCO3 additionally in the medium)
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

25. Incorporate into medium to stabilize pH
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
Earl’s salt
DMEM
NaHCO3
4mM
10mM
26mM
44mM
CO2
Atmospheric and evolved from culture 2% 5%
10%
HEPES
20mM
50mM
---

26.  Rely mostly on glycolysis in cell culture
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( % mL/cm2)

27.  change water frequently  avoid microbial contamination
3. Humidity
 change water frequently
 avoid microbial contamination
Water plate

28. C. Microscope
 Inverted microscope
for observation of :
cell morphology, degree of spreading,
membrane blebbing, proportion of
multinucleate, vaculation, microbial
contamination
 Fluorescent microscope

29. Inverted Microscope

30. Fluorescent microscope

31. D. Centrifuge  bench top centrifuge
 cell collection

33. E. Fridges and freezer( -20oC, -70oC, liquid
nitrogen)
 media storage
F. Miscellaneous small items of equipment
 Water bath 37oC
 Submersible magnetic stirrer
 Aspirator jar

34. Culture plastic ware and associate small consumable items
A. The most commonly used items of plastic ware:
For growing cells( electric treatment on
surface of culture plate
 flat bottomed flask
 petri-dish
 multi-well dish
 conical flask roller bottle
 Tubes

35.  culture flask
25 cm cm cm2

36.  culture dish petridish: 35mm, 60mm, 90mm,….
Multiwell dish 6 wells,12 wells,24 wells,…

37. Chamber Slides

38. 2. For handling solutions and cell suspensions
Volumetric pipettes
Plastic Pasteur pipette
Micropipette tips
 Centrifuge tube

40. 15 ml centrifuge
50 ml centrifuge

41. 3. For storage  Sample tube  Eppendorf tube  Cryotube
 Large volume screw capped bottle

43. 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

44. Spinner flask
conical flask on a rotating platform

45. B. Filters for sterilizing tissue culture solutions
pore size: 0.2um um
 How to choose the filters
 Low protein binding
 Minimum dead space
 Large volume

47. C. Pipettes
 micropipettes with disposable autoclaveable
tips
volume: ul ul ul
5 ml
 Disposable Pasteur pipettes
 Glass volumetric pipette
volume ml

48. D. Pipette aid or multichannel micropipette
E. Glassware for tissue culture use
F. Miscellaneous small items

51. 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

52. 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