Protocol for Batch Animal Cell Cultivation
Growth & maintenance conditions in T flasks –
Cultivation of hybridoma cell line in suspension culture features utilization of Iscove’s modification of Dulbeeco’s medium (ICN flow, Flow laboratories Ltd, Irvine U.K.) with supplementation of Glutamine (5 mM/L) and Glucose (25 mM/L). This medium is to be supplemented with 5 % Fetal clone Bovine Serum (Hyclone Laboratories Inc. Utah, U.S.A) 1 % Gentamycin and 3.2 g/L sodium bicarbonate. The medium (with-out serum and gentamycin) is to be sterilized with 0.22 µ Hydrophillic hollow fiber membrane filter and stored at 4ºC. The serum and gentamycin is to be added prior to use.
Precultures –
Precultures are to be grown as 50 mL stationary culture in 600 mL T flasks with filter caps (Greiner Laboratories, Frickenhausen, Germany) in a humidified CO2 incubator (CO25 New Brunswick Scientific Co. Edison, U.S.A.) at 37+ 1ºC and 5 % CO2. Sub culturing of the T flask cultures is to be done three times a week by diluting cells in the late exponential growth phase (48 hours) by fresh ICN flow medium. Inoculum in the new T flask is to be adjusted in such a way that the final living count is 3X105 cells/mL. After 48 hours growth the right amount of living cells of spinner flask has to be inoculated to make the final living cell count of 3 X 105 cells/mL in the fermenter. Inoculum is to be transferred to sterile fermenter in the laminar hood and fresh medium has to be added immediately with the help of sterile flask and peristaltic pump assembly.
Batch culture studies –
A 3 liter fermenter (Applikon Schidam, The Netherlands) with working volume of 1.5 liters and equipped with 7cm axial flow turbine (Setric Impeller) operating at 150 rpm is to be used in the study. Temperature pH and dissolved oxygen is to be controlled by ADI 1030 biocontroller The temperature is to be maintained at 37 + 1ºC by heating blanket wrapped around the fermenter. The pH is to be controlled at 7.2 + 0.2 with CO2 and sodium bicarbonate solution. The cultures are to be aerated by passing O2 through the hollow fiber cartridge submerged in the liquid medium which should maintain the dissolved oxygen constant right at the set point. The dissolved oxygen tension is to be maintained at 50 + 1 % of air saturation by passing O2and N2 gases through the hollow fiber cartiridge. The exit gases leaving the fermenter, has to be allowed to pass through a condenser kept at 4ºC by a cryostat to reduce evaporation of the fermentation broth. The fermenter is to be initially filled with 0.9 % NaCl (Physiological saline solution) for calibration of probes. The pH probe is to be first calibrated by 4 and 7 pH buffers. All other probes and inoculation assembly have to be then assembled and autoclaved. Dissolved oxygen probe has to be then removed. Inoculum and fresh medium was then added aseptically in the laminar hood. Later on the probes are connected to respective controllers. Take intermittent samples to follow the culture dynamics.
Analytical methods –
Viable / dead cell concentrations and viability has to be determined by Haemocytometer using trypan blue dye exclusion procedure. Glucose and lactate is to be measured by YSI analyzer. Monclonal antibodies can be measured to establish product concentration.
Protocol for operation of model simulator
Bioreactor –
The Bioreactor portion illustrates the different components which are adequately described in the theory link. It also describes the purging of the sterile air (please see the air filter in the air inlet) from the sparger and rotation of the impeller. The impeller is bottom driven. This kind of arrangement is possible with BioEngineering AG Switzerland Bioreactor. This kind of agitation gives more space on the lid so that there is no overcrowding of different inlet ports & sensors and it is particularly useful when flame is used to connect acid /alkali pipes and when transfer of inoculum is done to the reactor. The constant temperature water flows in the jacket of the reactor (marked green) to maintain the temperature of the reactor.
Select parameters –
Beneath Select parameters label there are spaces for Simulation Start time, Time increment step and Simulation end time wherein the User have the option to enter different relevant numbers to start the simulations using different time intervals till the end of the simulation. The maximum limits are also indicated User has to ensure that the values do not exceed the maximum values.
Beneath above table there is another table which allows the Users to input the values of different model parameters (the minimum and maximum range is indicated in the brackets and the users has to ensure the input with in the range).
Underneath above table there is yet another table which allows the Users to input the initial conditions for the model simulation at time t=0.
Run Simulation –
After entering the above entries the User can enter the RUN Simulation button and the graph and table for X, S, P vs time is printer on the screen.
More graphs are available for Batch Microbial Cultivation wherein the user also sees the rate specific rates and yields of the biomass substrate and product vs time. This data along with the kinetic profile can be used to understand the culture behavior.
Out put Graph –
This section describes the kinetic profile of different fermentations in graphs.
Save Results –
By pressing this button the User goes to the Excel Sheet where all the values of kinetic profile is tabulated which can used for the detailed analysis of the system and /or making more desired trends in the Excel Sheet.