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BIO BITS
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Cell Culture – From clone screening to protein production
Introduction
The use of incubation shakers is gaining more significance for the cultivation of animal cells. The cultivation of CHO (Chinese Hamster
Ovary) cells in a variety of shaker flasks shows that the Multitron Cell incubation shaker (Infors HT) is very well suited for the production of
seed cultures.
Multitron Cell - stackable exclusive shaker for cell culture
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Hygienic Direct Stream humidification prevents evaporation
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Antimicrobial surface eliminates microorganisms to prevent contamination
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Special box for Microtitre plates & Disposable Shaker Bag options
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Access ports for external sensors for verification / documentation
10
9
For the production of seed culture as an inoculum for later cultivation & SEAP
(secreted alkaline phosphatase) production in bioreactors, three shaker flask
cultures with the CHO XM-111 cell line were set up.
7
5
8
4
3
pH
6
5
2
4
3
1
2
1
3
4
5
6
7
8
250 ml pH
500 ml pH
1000 ml pH
250 ml Ammonium
500 ml Ammonium
1000 ml Ammonium
250 ml Glutamate
500 ml Glutamate
1000 ml Glutamate
250 ml Lactate
500 ml Lactate
1000 ml Lactate
100
1.00E+07
80
60
1.00E+06
40
20
0
1.00E+05
0
1
2
3
4
5
6
7
8
glucose in g/L
process time in d
250 mL viable cell density
500 mL viable cell density
1000 mL viable cell density
250 mL viability
500 mL viability
1000 mL viability
4
2
3
1.5
2
1
1
0.5
glutamine in mmol
The Direct Steam Humidification has an important influence on fluid loss and also
changes the osmolarity. Especially in small working volumes, the maximal
evaporation is less than 0.4% in one day.
2
process time in d
For the optimization of CHO seed culture, three different sizes of shaker flasks were
set up under comparable conditions for several days in the Multitron Cell incubation
shaker.
A comparison of the maximum cell concentration showed 500 mL shaker flask
culture produced up to 4.65 x 106 cells per mL. Glucose and glutamine
consumption, particularly in the 500 mL shaker flask, showed that substrate was
available in adequate quantities up to the fifth day. The simultaneous formation of
ammonium, lactate and glutamate is known to reduce the rate of cell growth.
1
viability in %
Analysis of results
0
viable cell density in mL
flasks were shaken in a Multitron Cell incubator (Infors HT) at a constant 122 rpm
over several days for cultivation.
0
0
Culture took place in 250 mL, 500 mL and 1000 mL shaker flask in each case, using
the serum and protein free HP-1 medium 2 g/L Pluronic F-68 and 2.5 mg/L
tetracycline were added to the medium. For optimal cultivation conditions,
temperature of 37°C, humidity of 85%* and Co2 saturation of 5% were selected. All
Glutamate, Ammonium in mmol
and Lactate in g/l
Experimental specifications
0
0
0
1
2
3
4
5
6
7
8
process time in d
250 mL glucose
500 mL glucose
1000 mL glucose
250 mL glutamine
500 mL glutamine
1000 mL glutamine
Summary
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The shaker flask cultures reach a total number of cells between 1.8 x 108 and 1.4 x 109 after 2 to 5 days.
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Early exchange of medium is preferred.
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Small inoculum volume & an optimal feeding strategy permit additional cell culture nutrients
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Low evaporation of less than 0.5% at 85% relative humidity results in a stable osmolarity in the culture and reproducibility of
processes. The shaker culture is available for further process steps, e.g. inoculation of a bioreactor or of disposable bags
*if condensation occurs humidity set can be reduced
Biochemistry Analyzer
The development of alternatives to fossil fuels as an energy source is an urgent
global priority. Cellulosic biomass has the potential to contribute to meet the
demand for liquid fuel, but land-use requirements and process inefficiencies
represent hurdles for large-scale deployment of biomass-to-biofuel technologies.
Genomic information gathered from across the biosphere, including potential
energy crops and microorganisms able to break down biomass will be vital for
improving the prospects of significant cellulosic biofuel production.
YSI's innovative biosensor technology uses the inherent specificity of enzymes for
a single target analyte. Its proprietary immobilized enzyme electrodes allow a
rapid, accurate and largely interference free measurement to be made in about a
minute. The unique fluidics and chamber design resist clogging – even at high cell
densities. YSI analyzers do not require a highly trained individual to see
meaningful results. The analyzer is self-calibrating, self-cleaning, and totally
automated.
Next generation YSI Series can monitor Glucose, Lactate, Glutamine, Glutamate,
Ammonium, Potassium, Xylose, Ethanol, Methanol, Sucrose, Galactose,
Lactose, Choline, Glycerol & Hydrogen peroxide. It has wide range of application
like Critical bioprocess monitoring and fermentation control, Biofuel production
and research, Clinical blood chemistry, Food and beverage processing
For more information visit www.ysilifesciences.com
Second Generation Bioreactor for
Biofuel Research
Production of ethanol from lignocellulose has become a popular field of research
and development due to its advantage of availability and diversity of the raw
material. The production process of second generation biofuels consists of three
major steps. The chemical pre-treatment to enhance the accessibility of cellulosic
compounds, the enzymatic hydrolysis to convert cellulose to sugar and the
microbial fermentation itself to form ethanol from the sugars. Used for
Lignocellulosic material e.g. wheat straw, wood waste, bagasse.
The Labfors BioEtOH (InforsHT) can combine both the enzymatic hydrolysis and
the fermentation step in one vessel/bioreactor known as SSF (simultaneous
saccharafication and fermentation). The powerful high torque motor and the
special vessel/impeller design guarantees rapid and uniform mixing even of
extremely viscous slurry and high dry matter content and ensures ideal
fermentation conditions. Temperature is monitored and controlled throughout the
bioprocess and this prevents vessel overheating and thereby keeps enzyme activity
high.
Wide range of applications include, Research and development in the
lignocellulosic ethanol production sector, SSF process development (enzymatic
hydrolysis and fermentation) & Enzyme screening and activity studies.
For more information visit www.infors-ht.com
Email: [email protected]
Web: www.labmateasia.com
Cancer Metabolic Profile Provides New Insights To
Therapeutical Intervention
Otto Warburg described how cancer cells utilize aerobic glycolysis in preference to oxidative phosphorylation, a phenomenon
known as the Warburg effect. Warburg also observed that this high rate of aerobic glycolysis provides sufficient ATP for energy stores and the
intermediate metabolites needed to support tumor growth. Nonetheless, the reason that cancer cells demonstrate enhanced glycolysis and
reduced mitochondrial respiration remains unclear and an active area of research.
While differentiated, specialized cells typically have a large spare capacity to respond to increased ATP demand, cancer cells often
have a low spare capacity. Highly proliferating cells typically have a basal OCR (Oxygen Consumption Rate) closer to the maximal OCR
owing the highly anabolic nature of these cells. The extrinsic pathway is initiated by the extracellular binding of members of the TNF
(Tumor Necrosis Factor) ligand family, including TRAIL (TNF-related apoptosis -inducing ligand). TRAIL can be selectively toxic to tumor
cells and initiates apoptosis through the death-inducing signaling complex (DISC). ATP is needed for this extrinsic-mediated apoptotic cell
death, apoptotic cells have been shown to have elevated ATP levels.
Robinson, et al2 use Xf24 Extracellular Flux Analyzer (Seahorse Bioscience) to demonstrate that the glycolytic phenotype in Z138
cells (Mantle Cell Lymphoma cells) is more complicated than described by Warburg. Initially, Robinson inhibited glycolysis by treating the
Z138 cells with 2-deoxyglucose (2-DG) to block hexokinase, the first enzyme in the glycolytic pathway, and observed a reverse Warburg
change in metabolism as expected. He showed that 2-DG treatment of Z138 cells produced an almost complete inhibition of glycolysis
(ECAR- Extra Cellular Acidification Rate) and an approximately 30% reduction in oxidative phosphorylation (OCR) as shown in Fig.A and
showed an increased sensitivity to TRAIL.
Robinson observed a surprisingly different phenotypic change in metabolism when he inhibited glycolysis by growing the Z138
cells in glucose-free medium, as illustrated in Fig.B. Although depriving the cancer cells of glucose caused a reverse Warburg phenotype, the
Z138 cells oxidative phosphorylation levels increased by approximately 60%, in contrast to the decrease observed with 2-DG. This switch
from aerobic glycolysis to oxidative phosphorylation was most notable after 20 hours of growth in glucose-free media. Continued growth
under these conditions resulted in a slower growth rate an almost complete conversion to oxidative phosphorylation and these cells were
significantly less sensitive to TRAIL .
Fig.A
1000
800
600
- 2DG
400
200
+ 2DG
BASAL OCR
(pmol/min/106 cells)
B. Z138 cells cultured for a
minimum of 7 days in 2mM
glutamax and 1 mM pyruvate
containing media with ( ) or
without ( ) 11 mM glucose.
1000
BASAL OCR
6
(pmol/min/10 cells)
A. Z138 cells cultured for 20 h in
11mM glucose, 1mM pyruvate
and 2mM glutamax supplemented
medium with ( ) or without ( ) 5
mM 2-DG
Fig.B
800
600
- Glucose
400
+ Glucose
200
0
0
0 40 120 200 280
BASAL ECAR
(mpH/min/106 cells)
0
40
80 120 160
BASAL ECAR
(mpH/min/106 cells)
Metabolic Switch induced by 2-DG inhibition of glycolusis results in a less aerobic
phenotype than metabolic switch induced by glucose-free culture.
1.
2.
Warburg, On the origin of cancer cells. Science 123: 309-314; 1956
Robinson, GL., et al., Switching from aerobic glycolysis to oxidative phosphorylation modulates the sensitivity of mantle cell
lymphoma cells to TRAIL. Oncogene (2012)
Seahorse XF Analyzer is the world's most advanced cell metabolism analyzer
For more information visit www.seahorsebio.com
Email: [email protected]
Web: www.labmateasia.com
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Bertin Technologies introduces Precellys® Evolution next generation homogenizer.
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The unique 8 multi directional motion provides the same high level energy to all the tubes.
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Can process 4 different size tubes 0.5ml, 2ml, 7ml, 15ml simultaneously.
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Grind, homogenize or lyse any type of soft or hard sample up to 24 nos.
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A 6. Lack of Oxygen
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M 9. Controlled fermentation
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LABMATE (ASIA) PVT. LTD.
Baid Mehta Complex, # 183, Mount Road,
Chennai - 600015, India Phone: 044 2220 0066 / 0166
Email: [email protected] Web: www.labmateasia.com
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Feb 2015
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