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 non- sterile reagents

Refrigerator CO2 incubator microscope Laminar Flow

Working area Storage and preparation Washing area

Working area Preparation area

soaking washing Deionize water supply 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

water softener cartridge 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

CO2: maintain pH of culture medium pH7.2---7.4 concentration: 5% 2. Gas phase CO2: maintain pH of culture medium pH7.2---7.4 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

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 ---

 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( 0.2-0.5% mL/cm2)

 change water frequently  avoid microbial contamination 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: 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

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