Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
Primary and established cell line cultureKAUSHAL SAHU
Introduction
Primary Culture
Steps of Primary Culture
Isolation Of Tissue
Dissection And Disaggregation
Types Of Primary Culture
Primary Explants Culture
Enzymatic Disaggregation
Mechanical Disaggregation
Cell Line( Finite & Continuous)
Naming A Cell Line
Choosing A Cell Line
Maintenance Of Cell Line
Conclusion
Reference
Introduction
History
Scale up in suspension:Stirred culture,Continuous flow culture,Air- lift culture,Nasa bioreactor
Scale up in monolayer culture: Roller bottle culture , multisurface culture,fixed -bed culture
Other type of culture for scaling up: HARV Vessels,STLV vessels
Monitoring of scale up
Conclusion
References
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
1. INTRODUCTION TO CELL CULTURE
2. SOURCES & TYPES OF CONTAMINATION
3. MONITORING OF CONTAMINATION IN CELL CULTURE
4. CROSS CONTAMINATION
5. ANTIBIOTIC USE
Bioreactors for animal cell suspension cultureGrace Felciya
1. Types of culture
2. Techniques of cultivating animal cell
3. suspension culture/ Non anchorage dependent
4. Bioreactor consideration
5. Requirements of Bioreactor
6. Reactors used in cultivation
Cellular coning refers to generation of genetically identical cells from parent cells. This presentation teaches differences between cell coning and molecular cloning and various methods of cell cloning. Sample questions are also provided for your review of concept learned
In this modern era, many companies are focusing on the research of monoclonal antibodies to treat cancer and other autoimmune diseases. Hence, cell culture plays an important role to produce therapeutic proteins. To make process efficient we should know alpha and gamma about cell line. The growth of cells in the medium is the most important area or checkpoint. The type of cell line is also discussed here.
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
The heart of the fermentation or bioprocess technology is the Fermentor or Bioreactor. A bioreactor is basically a device in which the organisms are cultivated to form the desired products. it is a containment system designed to give right environment for optimal growth and metabolic activity of the organism.
A fermentor usually refers to the containment system for the cultivation of prokaryotic cells, while a bioreactor grows the eukaryotic cells (mammalian, insect cells, etc).
Introduction
History
Scale up in suspension:Stirred culture,Continuous flow culture,Air- lift culture,Nasa bioreactor
Scale up in monolayer culture: Roller bottle culture , multisurface culture,fixed -bed culture
Other type of culture for scaling up: HARV Vessels,STLV vessels
Monitoring of scale up
Conclusion
References
INTRODUCTION
HISTORY
NEED OF SYNCHRONIZATION
SYNCHRONOUS CULTURES CAN BE OBTAINED IN SEVERAL WAYS:
Physical fractionation .
Chemical appro ach
CENTRIFUGAL ELUTRIATION
Inhibition of DNA synthesis
Nutritional deprivation
SYNCHRONIZATION AT LOW TEMPERATURE
CELLULAR TOTIPOTENCY
SOME HIGHLIGHTS OF CELL SYNCHRONIZATION
REFERENCES
1. INTRODUCTION TO CELL CULTURE
2. SOURCES & TYPES OF CONTAMINATION
3. MONITORING OF CONTAMINATION IN CELL CULTURE
4. CROSS CONTAMINATION
5. ANTIBIOTIC USE
Bioreactors for animal cell suspension cultureGrace Felciya
1. Types of culture
2. Techniques of cultivating animal cell
3. suspension culture/ Non anchorage dependent
4. Bioreactor consideration
5. Requirements of Bioreactor
6. Reactors used in cultivation
Cellular coning refers to generation of genetically identical cells from parent cells. This presentation teaches differences between cell coning and molecular cloning and various methods of cell cloning. Sample questions are also provided for your review of concept learned
In this modern era, many companies are focusing on the research of monoclonal antibodies to treat cancer and other autoimmune diseases. Hence, cell culture plays an important role to produce therapeutic proteins. To make process efficient we should know alpha and gamma about cell line. The growth of cells in the medium is the most important area or checkpoint. The type of cell line is also discussed here.
As opposed to common belief, the measurement of growth in cell culture is fairly simple. Most of the tecchniques that are applied for measurement of microbial growth can be applied to cell culture.Of course with some modification. This presentation exactly explains growth measurement techniques with respect to cell culture. At the end you will also find sample multiple choice questions for practice.
Introduction
History
Cell culture techniques
Species cloned
Approaches of cell cloning
Monolayer culture- Dilution cloning
Microtitration plate
Suspension culture- Cloning in agar
Cloning in methocel
Isolation of clone
By clonal rings
By suspension clone
Application of cell cloning
Conclusion
Reference
Scale up means increasing the quantity or volume of cell culture. For animal cells, the scale up strategies are dependent upon cell types or i.e. whether the cells requires matrix for attachment and growth ( adherent cell culture) or grows freely in suspended form in aqueous media. The scaling up principle for adherent cells are just to increase surface area for attachment while for suspension culture is to increase culture volume. This presentation enlightens the reader about different methods of scaling up of cells culture. Readers are also provided with sample questions for better understanding
The heart of the fermentation or bioprocess technology is the Fermentor or Bioreactor. A bioreactor is basically a device in which the organisms are cultivated to form the desired products. it is a containment system designed to give right environment for optimal growth and metabolic activity of the organism.
A fermentor usually refers to the containment system for the cultivation of prokaryotic cells, while a bioreactor grows the eukaryotic cells (mammalian, insect cells, etc).
The following presentation is only for quick reference. I would advise you to read the theoretical aspects of the respective topic and then use this presentation for your last minute revision. I hope it helps you..!!
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Lecture 9b scaling up
1. Lecture 9 Animal Cell Biotechnology
Scaling up the production process
pH
• set point pH of 7.4 ± 0.1 common
• without buffering the pH could fluctuate
• for small scale operation, can maintain pH by using
gaseous CO2 to control culture pH
2. Lecture 9 Animal Cell Biotechnology
Scaling up the production process:
Controlling the pH with CO2
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P161.
3. Lecture 9 Animal Cell Biotechnology
Scaling up the production process
• for larger scale cultures, can directly add acid or base to
maintain pH
• insert probe into culture to detect changes in pH
• acid or base pumped in accordingly, under automatic
control
→ pH ↓, add base (concentrated sodium bicarbonate)
→ pH ↑, add acid (concentrated HCl)
→ not normally a problem due to lactic acid production
4. Lecture 9 Animal Cell Biotechnology
Scaling up the production process
Oxygen requirements
• supply of oxygen to satisfy cell metabolism is one of the
major problems associated with culture scale-up
• O2 consumption rate: 0.06-0.6 mM/hour for 106
cells/ml
• for small volumes (< 1 litre) O2 diffusion from the
headspace through the culture surface is sufficient to
meet the oxygen demand
• as the volume increases, the surface-volume ratio
decreases
5. Dissolved oxygen
polarographic electrode
Dissolved oxygen polarographic electrode (InPro 6050, Metler-
Toledo, 2006). The cathode, where the half-reaction with O2
(O2+2H2O+4e-→4OH-) takes place, is in contact with a
membrane, that allows the transport of the dissolved oxygen
from the external medium. At the anode, the silver oxidation
(Ag++Cl-→ AgCl + e-) takes place.
6. Fig. 9.8 Membrane-covered oxygen electrode
Pt cathode: O2 + H2O + 4e → 4OH-
Ag anode: 4Ag + 4Cl- → 4AgCl + 4e
7. The limitation of O2 supply by
diffusion through the head space
Culture Head space O2 supply O2 demand
volume (L) area (cm2) (mmol/h) (mmol/h)
1 100 0.063 0.063
10 500 0.313 0.625
100 2500 1.56 6.25
8. Lecture 9 Animal Cell Biotechnology
Scaling up the production process
OTR = oxygen transfer rate
OUR = oxygen uptake rate
To supply sufficient O2 to cells and to avoid O2 depletion:
OTR > OUR
9. Lecture 9 Animal Cell Biotechnology
Scaling up the production process
• at > 1 L, the surface-volume ratio is too low to satisfy
overall O2 demand
• surface-volume ratio of a fermentor defined by aspect
ratio:
aspect ratio = diameter of culture/height of culture
10. Fig. 9.7
Aspect ratio = width/ height of culture
11. Lecture 9 Animal Cell Biotechnology
Scaling up the production process:
Bubble death !
12. Lecture 9 Animal Cell Biotechnology
Scaling up the production process
Strategies to prevent cell damage
• use of chemical agents to reduce cell damage, such as
0.1% Pluronic F68 →
synthetic copolymer of ethylene and propylene
oxide, reduces cell:bubble interaction by preventing
attachment of cells to bubbles
• cover gas sparger with fine wire mesh to reduce the
number of bubbles reaching cells
• use of alternate fermentors (i.e. air lift fermentor)
• sparge media in a secondary vessel
• use gas permeable tubing (i.e. thin-walled silicone
tubing) within bioreactor
13. Strategies for controlling dissolved oxygen
Fig. 9.9
(b) Intermittent oxygen sparging
(a) Change in air flow rate
(c) Control of gas composition (d) Spin filter isolates cells
from sparged gass
14. Dissolved oxygen control
O2 flow rate
controller
Q1
QT = Q1 + Q2
PC
N2 flow rate
controller Q2
C (%)
DO is controlled by the adjustment of the oxygen fraction in the sparged gas.
Flow rate is kept constant and corresponds to the sum of the two controlled gases Q1 and Q2.
15. Fig. 9.11
Modulated feedback control
+
Set point
_
16. A typical control loop
controller actuator process
+ error electric
PID bioreactor
set-point resistance
-
sensor
measured
value thermometer
A set-point is compared to the measured value by the sensor.
An error measurement based on a signal to the electric resistance (actuator)
is generated by the controller, that will heat up the bioreactor (process).
17. d error
actuation
• P.error I. error.dt D.
On-off controller, in which the action can only assume two states (on or
dt
off).
• Controller modulated by pulse width (Pulse-Width Modulation or
PWM). In this control type, the action is also on or off but the time that
the actuator stays on within a certain cycle can be adjusted
continuously. , allowing a final operation of different intensities.
• Cascade controller, composed of one master and one slave loop. This is
used when a more rigid control of a process variable is required, for
instance, the temperature of the culture medium.
• P-I-D controller, or Proportional-Integral-Derivative. It's based on the
principle that the action is taken not only on how large is the error
(difference between desired and measured values), but also on the sum
of past errors (integral of the error) and to the rate that the error is
changing (derivative of the error). where actuation is the controller
output, error is the difference between the desired value (set-point)
and the one measured by the sensor, t is time and P, I and D are
constants that need to be adjusted for each system. The adjustment of
the constants for a process, is called P-I-D controller tuning.
18. Proportional control.
• The output of the controller is proportional to
the error signal.
• = 0 + k.E
• where = output signal of the controller
• where 0= output signal when the error is zero
• where k = controller gain or proportional band
• where E = error or deviation from the set point
19. Integral and derivative control.
• Integral control. The output of the controller is a function of
the integral of error and time. Here, the control action
increases with time as long as the error is registered.
• = 0 + tI E dt
• where tI = integral time constant
• Derivative time. The output of the controller is a function of
the rate of change of the error.
d E
• = 0 + td .
dt
• where td = derivative time constant
20. Feeding flow rate control system based on glucose concentration measured in real time
(adapted from Ozturk et al., 1997)
21. Other bioreactor types
• Airlift fermenter
• Packed bed bioreactor
• Hollow fiber bioreactor
• Single use bioreactor
22. Airlift fermenter
• Gas mixture sparged
into the reactor at the
base.
• The gas flow cause the
culture medium to rise.
• No mechanical
agitators.
23. The fermenter is 200' high and 25 ft diam. (Chem. Eng. News, 10-Apr-78)
24. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Air lift bioreactor
Waites et al. 2001. Industrial Microbiology: An Introduction. Oxford: Blackwell Science. P 98
27. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Air lift bioreactor
• sparged gas agitates and aerates column
• no mechanical parts, no shear stress
• gas flow through inner tube lifts cells and medium,
• cells and medium spill out over draft tube, circulate
down side
• 2-2000 litre reactors available
28. Hollow-fiber bioreactor
• bioreactor consists of a cartridge containing bundles of
synthetic, semi permeable hollow fibers which are
similar to the matrix of the vascular system
• good for anchorage-dependent or independent cells
Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p84
29. Hollow fiber bioreactor
• In fibrous-bed bioreactor, the cells
are immobilized on the fibers in
the bioreactor.
• Following is scanning electron
microscope photos of human
osteosarcoma cells in an artificial
growth medium, a fibrous-bed
bioreactor.
• The cells cling to Dacron fibers
34. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Packed-bed/fixed-bed bioreactor – glass bead column
• good for anchorage-dependent cells
• 1-100 litre volumes
• cells attach to surface of beads (3-5 mm)
• aerated medium is pumped in from a secondary vessel
• inoculation could be a problem, uneven growth
• heterogenous bioreactor – environment may not be the
same throughout the column
36. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Packed-bed/fixed-bed bioreactor – ceramic bioreactor
• ceramic cartridge (30 cm long, 4 cm wide) containing a
series of parallel channels (1 mm2 square channels)
→ cells attach and grow on the walls of the channels
• fresh medium is pumped in through the
chambers, spent medium is returned to main reservoir
• secreted products can be isolated from the spent
medium
38. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Packed-bed/fixed-bed bioreactor – the cell cube
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P170-171.
39. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Packed-bed/fixed-bed bioreactor – the cell cube
• stack of 20 cm2 polystyrene plates separated by 1 mm
spacers
• cells attach to either side of plate
• culture medium is “sprayed” over the surface of the
plates by multiple inlet ports
40. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Fluidized-bed bioreactor
• similar to packed
bed, but particles/ micro-
carriers are separated by
liquid media
• immobilized cells are
held in suspension by an
upward flow of liquid
medium
M.Butler and M.Dawson. 1992. Cell Culture Labfax. Oxford:BIOS Scientific Publishers. p205
41. Lecture 10 Animal Cell Biotechnology
Other fermentor Systems:
Fluidized-bed bioreactor
Butler, M. 2004. Animal cell culture and technology 2nd ed. London and New York:Garland Science/BIOS Scientific Publishers. P172.