lecture 4a culture equipment

Fig. 3.2 Laminar flow cabinet (class II)

Biosafety Levels• Biosafety Level 1 is suitable for work involving well-

characterized agents not known to consistently cause disease in healthy adult humans, and of minimal potential hazard Precautions against the biohazardous materials in question are minimal, most likely involving gloves and some sort of facial protection.

• Biosafety Level 2 moderate potential hazard to personnel and the environment. Includes various bacteria and viruses that cause only mild disease to humans,

Biosafety levels contd.• Biosafety Level 3 is applicable to agents which may cause serious or

potentially lethal disease as a result of exposure by the inhalation route. The laboratory has special engineering and design features.

• Biosafety Level 4 is required for work with dangerous and exotic agents that pose a high individual risk of aerosol-transmitted laboratory infections, agents which cause severe to fatal disease in humans for which vaccines or other treatments are not available. Multiple airlocks are employed and are electronically secured to prevent both doors opening at the same time.

Fig. 3.6

Tissue culture flasks (T-flasks)

Fig. 3.7 Multi-well plates

Fig. 3.10 Spinner bottle with suspended paddle

Fig. 3.11a Roller bottles of various sizes

Bottles in a roller system Fig. 3.11b

Fig. 10.17

A confluent layer of BHK cells covers two microcarriers

Exchange Capacity (meq/ g)

0 1 2 3 4 5

Pro

duct

ivity

(10

6 cel

ls/

litre

/ h)

0

2

4

6

8

10

12

14

16

Cell yield on microcarriers with varying charge

Non-porous microcarriers• Cytodex

• Cells grow adherent to the surface of the bead• Used for adherent cells• Allows for the growth of adherent cells in scaled up

processes

• 3 varieties: 1, 2, and 3• 1 = positively charged throughout the bead • 2 = positively charged on the surface of the bead• 3 = collagen coated to promote attachment

1 & 3 Most commonly used Cytodex species

Porous microcarriers• Cytopore

• Dextran mesh increases surface area• Cells can lodge themselves in the dextran mesh

• Protection from shear forces• Designed for CHO cells• Positive charge throughout the bead

• 2 varieties: 1 and 2• 1 = 1.1 mEq charge• 2 = 1.8 mEq charge

Amersham Biosciences

Fig. 10.18Comparison of a unit system and a multiple system

Cell encapsulationFig. 10.19

Typical culture vessels suitable for cell growthCulture vessel

Number of culture wells/ unit

Max. culture volume (ml)

Vessel size Growth surface (cm2)

Material

Multiple well plate

96 0.37 10.8 x 6.4 mm (D x diam.)

0.32 plastic

" "

24 3.4 17.6 x 15.5 mm "

1.88 plastic

" "

12 6.9 17.6 x 22.1 mm "

3.8 plastic

" "

6 16.8 17.6 x 34.6 mm "

9.4 plastic

Medical flat bottle

10 125 ml 22 glass

" " " 15 250 ml 30 glass

Roux bottle 50 500 ml 200 glass

T-flask, 25 5.0 50 ml 25 plastic

" , 75 15-30 250 ml 75 plastic

" , 150 75 600 ml 150 plastic

" , 175 50-100 750 ml 175 plastic

Culture vessel 100-200 1250 ml 490 plastic

Culture vessel 100-250 2200 ml 850 plastic

Culture vessel 100-500 4900 ml 1750 plastic

Culture vessel 100 250 ml glass

Culture vessel 250 500 ml glass

Typical culture vessels suitable for cell growthcontinued

Culture vessel Number of culture wells/ unit

Max. culture volume (ml)

Vessel size Growth surface (cm2)

Material

Fig. 3.12a Inverted microscope

Fig. 3.12b Standard microscope

Lecture 4 Animal Cell BiotechnologyCell Culture Conditions and Media

Culture Conditions (Incubator)• cells grow best at 37oC and pH 7.4

• grow slower at slightly < 37oC, > 37oC (39-40oC) may destroy cells

• pH of liquid cultures maintained by bicarbonate-CO2

buffer system

CO2 + H2O HCO3- + H+ , pKa = 6.3

• pH further dependent on enriched CO2 atmosphere in the incubator

Double chamber CO2 incubator Fig. 3.3

Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P 34.

“Good” Buffers - Maintaining pH of a cell culture

- HEPES (pKa = 7.4)- MOPS- Tris

- Each has a specific pKa value- HEPES expensive- Allow pH to drift downwards due to lactic acid

accumulation, HEPES will buffer the system and maintain it +/- of 7.4.