 |
|||||||||||||||||||
|
Newsletter March 2010 |
|||||||||||||||||||
|
|
|||||||||||||||||||
| Content | |||||||||||||||||||
|
|||||||||||||||||||
| Publication | |||||||||||||||||||
|
Biotechnology and Bioengineering (2010), Vol. 105, No. 2, pp 260-275 Microscale experiments offer a potential platform to obtain key process data early and cost effectively.
Barrett and co-workers demonstrate that considering the corresponding engineering parameters, cell culture experiments performed in shaken microwell plates provide data that is both reproducible and comparable to currently used shake flask systems. They describe a detailed engineering characterization of liquid mixing and gas-liquid mass transfer in microwell systems and its impact on suspension adapted mammalian cell cultures.
In contrast with stirred systems, oxygen transfer in shaken cultures occurs solely through surface aeration. In order to investigate whether oxygen transfer in a 24-well plate covered with a breathe-easy membrane is sufficient for the cells, the dissolved oxygen tension (DOT) was measured at different shaking speeds and liquid fill volumes using fluorescent oxygen sensor spots. Similar experiments were also performed in shake flasks. The DOT in microwell cultures varied between 65 and 90% air saturation at fill volumes of 800µL and shaking speeds between 120 and 300 rpm which is sufficient for cell culture requirements.
Mixing in shaken wells is also an important factor when considering the use of microtiter plates. By injecting a tracer dye the distinct mixing patterns in 24-well plates were demonstrated with impressive images on high-speed video. At a shaking speed of 120 rpm the dye flows to the base of the well and then rises through the liquid as a horizontal front. Above 250 rpm heterogeneities exist for less than a second before the dye is dispersed evenly. Mixing times in 24-well plates increase with fill volume at lower shaking speeds.
Via computational fluid dynamics (CFD) simulation the mean energy dissipation rate (P/V) was predicted in shaken microwells, suggesting that the hydrodynamic environment is unlikely to have a detrimental effect on the cultured cells.
Since the shaking speed in microwell cultures can have a distinct impact on engineering parameters such as mixing time or kLa, the influence of shaking speed was investigated for hybridoma cells in 24-well plates in a range of 120 - 250 rpm (800µL fill volume). Hydrodynamics and gas-liquid mass transfer were not found to be limited over the range of shaking speeds tested.
To investigate the influence of fill volume, the growth and antibody production of hybridoma cultures were determined in a 24-well plate filled with either 800 or 2000 µL at speeds between 120 – 250 rpm. In contrast to shaking speed, variations in fill volume had a significant impact on culture performance. At 120 rpm and with 800µL of medium, the peak viable cell concentration was almost double that obtained at 2000µL.
An engineering comparison was performed at matched energy dissipation rates (P/V) with microwell and shake flask cultures to test the utility of microplates for high throughput process development. The average P/V in the microwell plate was predicted to be 40 Wm-3 (120 rpm, 800 µL fill volume). Operating conditions for a 250mL shake flask were adjusted to an equal magnitude of P/V (120 rpm, 100mL fill volume). Cell growth kinetics, antibody production rate and metabolite profiles were approximately the same in the different vessels.
These findings indicate that shaken microplates provide quantitative and reproducible bioprocess data when compared to shake flask methods and offer advantages such as a reduction in operation scale, reduced costs through lower material requirements and increased throughput.
In our free publication database you will find abstracts and links for this paper and most of the articles cited in it.
|
|||||||||||||||||||
| Dr. Shaker´s helpful hint no.2 |
|||||||||||||||||||
|
Plug and Shake The mass transfer resistance of a sterile plug is mainly determined by the neck geometry of the shake flask and to a lesser extent by the plug material and density.
When cultivating micro organisms with high respiration activity (e.g. Escherichia coli), wide neck flasks with cotton plugs are recommended.
For organisms requiring less oxygen such as plant cells, narrow neck flasks with cotton plugs are suitable.
In the autoclave, aluminium foil protects the cotton plugs from dripping water and should be removed shortly afterwards.
If aluminium caps are used they should be fitted firmly onto the flask. When the caps move during the shaking operation, the gas exchange is not reproducible and there may be a greater risk of contamination because of the convective gas flow.
You can find more information about the mass transfer resistance of different sterile plugs in shaking bioreactors in the following publication:
Mrotzek, C., Anderlei, T., Henzler, H-J. und Büchs, J.:
"Mass transfer resistance of sterile plugs in shaking bioreactors"
Biochemical Engineering Journal (2001), Vol. 7 (2), pp 107 - 112
Search our free publication database for more interesting papers dealing with sterile plugs!
|
|||||||||||||||||||
| Current news |
|||||||||||||||||||
|
|||||||||||||||||||
| ------------------------------------------------------------------------------------------------------------------- | |||||||||||||||||||
|
|||||||||||||||||||
| ------------------------------------------------------------------------------------------------------------------- | |||||||||||||||||||
|
|||||||||||||||||||
Please klick here if you no longer wish to receive this newsletter.