University of California, Davis

Department of Chemical Engineering and Materials Science

ECH 161L: Bioprocess Engineering Laboratory

Spring 1999 Professor Karen McDonald

I. INTRODUCTION

Purpose:

The purpose of this experiment is to expose students to a novel technique for recombinant protein production while giving them hands on experience with aseptic techniques and bioreactor operation. Students will be using insect cells (Spodoptera frugiperda, the fall armyworm, which belongs to the Lepidoptera family consisting of moths and butterflies) and a baculovirus (double stranded DNA viruses that are pathogenic for invertebrates but nonpathogenic to mammals, birds, fish and reptiles) to produce a "model protein", E. coli -galactosidase. Students should gain a thorough understanding of the insect cell / baculovirus expression system, as well as practical experience operating a laboratory scale fermentor.

Figure 1. Sf9 Cells (from Invitrogen Web Catalog)

Background:

The insect cell/baculovirus system for protein production is becoming a popular, method for producing recombinant proteins and insecticides. Insect cells are interesting because they can 1) perform many of the post-translational modifications that higher eucaryotes utilize, 2) be subcultured continuously (i.e. capable of indefinite replication) without being transformed, 3) be grown to fairly high cell densities at higher growth rates than mammalian cells and 4) be adapted to grow either in suspension or attached to a surface. The insect cells serve as a host for the replication of a baculovirus, Autographa californica nuclear polyhedrosis virus (AcMNPV). During the late stages of infection (18 hours post infection), occlusion bodies referred to as polyhedra or nuclear polyhedrosis virus particles are observed in the nucleus of the host cell. The primary structural protein in the occlusion body is a 29kDa protein known as polyhedrin. The polyhedrin gene is transcribed at a very high level late in the infection cycle but is not essential for viral infection or replication. Thus, a foreign gene inserted under the control of the polyhedrin promoter will be transcribed at high levels late in the infection cycle and will be expelled to the media following lysis of the host insect cell.

The baculovirus used in this experiment is the AcMNPV type encoded with E. coli -galactosidase. Although the wild type baculovirus produces the occlusion bodies described above, the baculovirus containing the -galactosidase gene will not since the inserted gene replaces the polyhedrin gene eliminating the production of the polyhedrin protein. The experiment will be performed in a 5L BioFlo 3000 bioreactor (New Brunswick) equipped with pitched blade impellers, temperature, pH, DO and agitation control. Process data (agitation, T, pH and DO) can be directly recorded but samples will have to be taken manually to determine cell concentrations, glucose levels, and intracellular and extracellular levels of -galactosidase.

II. LABORATORY PREPARATION

Reading:

In order to understand the insect cell/baculovirus system and the dynamics of the baculovirus infection cycle, students should read Chapters 1 and 2 in Baculovirus Expression Systems and Biopesticides by Shuler et al. (1995). Relevant reference articles are also listed in Section IV. In addition, read all relevant equipment manuals (Bioflo 3000, YSI sugar analyzer, Coulter Counter, Microplate luminometer, hemocytometer, sonicator), MSDS and protocols (-galactosidase assay) for the experiment.

Group Organization:

Groups will be consolidated into larger groups for this experiment. This experiment runs for approximately 10 days. Each group must meet with the TA the week before the experiment to familiarize themselves with the equipment and procedure. The TA will be responsible for cleaning and sterilizing the bioreactor, preparing the inoculum, determining the cell concentration of the inoculum, preparing the virus stock, and determining the PFU (plaque forming units) of the viral stock. The entire lab group should be present for the loading of the medium, setting up the shake flasks and bioreactor, calibration of sensors, inoculation, and virus infection procedures. The group will have to schedule a timeline for taking and analyzing samples among themselves.

III. EXPERIMENTAL

Procedure:

Students will be required to meet with the TA the afternoon before their scheduled lab period to load sterile medium into the sterilized bioreactor. The TA will sterilize the bioreactor and all necessary equipment for each lab group. Students are responsible for transferring medium into the sterile 2 L dual-port vessel using aseptic techniques in the laminar flow hood, and sterile insertion of approximately 2.75 L of medium into the bioreactor. The insect cell culture medium used in this experiment is a serum-free insect cell culture medium, ESF 921 (Expression Systems Inc.). The 2 L vessel will have to be used twice to insert 2.75 L of medium, and consequently, students should carefully maintain the sterility of the 2 L vessel during medium insertion.

Once the medium has been inserted into the bioreactor, the bioreactor can be started up. Carefully read the operation section of the BioFlo 3000 manual to insure that all start-up tasks are completed. Once the cooling water is initiated and the cooling water pump has been primed, the temperature control can be put into PID mode and the temperature setpoint adjusted to 27 ^oC. This is the desired operating temperature for the entire experiment. After the motor drive has been carefully inserted onto the agitation coupling, the agitation control can be put in PID mode with a setpoint of 75 rpm. Turn on pure oxygen to the bioreactor at a flow rate of approximately 1 L/min and let the medium saturate with oxygen overnight. In the morning, calibrate the DO probe by setting the span of the DO probe to 100.0.

Once the DO probe is calibrated, set the DO setpoint to 50% saturation and allow the system to equilibrate. The DO-pH control mode on the BioFlo 3000 can be used to simultaneously control the DO and pH. Alternatively the pH can be controlled by acid/base addition two peristaltic pumps set to 1 RPM and connected to 1.0N HCl and 0.5N NaOH. The pH setpoint should be set to 6.2. In preparation for inoculation, count the cell concentration in the flasks of Sf9 cells provided by the TA. The TA will show you how to use the Coulter Counter; instructions are also given in this handout. Inoculate the bioreactor to obtain a starting cell concentration of approximately 1 10⁶ cells/mL. It would be a good idea to inoculate several shake flasks at the same time to use as an end-point comparison with the bioreactor data. Following inoculation, take several initial samples from the bioreactor to determine the starting conditions and monitor cell growth until cells reach a concentration of about 3 10⁶ cells/mL. Once samples are taken, handle the samples according to the sample analysis procedure in this handout.

When the cell concentration in the bioreactor and shake flasks reaches 3 10⁶ cells/mL, the cells should be infected with the baculovirus. The baculovirus high titer stock and a PFU count will be provided by the TA. For infection of the bioreactor the group should determine how much of the viral stock should be added to 1L of fresh medium so that when the fresh medium containing the virus is added to the insect cell culture the resulting multiplicity of infection (MOI) is 0.5 PFU/cell. The TA will check your calculations prior to adding the virus to the fresh medium. Infect the shake flasks at a MOI of 0.5 PFU/cell as well. Once the cells are infected with the virus, continue to monitor and sample the bioreactor culture until the cell viability falls below 20%. At the end of the run sample the shake flasks as well to determine cell concentration, viability, glucose level and intracellular and extracellular -galactosidase. The TA will be responsible for sterilizing and cleaning the bioreactor vessel.

Sample Procedure:

To take a sample, squeeze the rubber bulb and then open the sample valve to draw fluid into the sterile sample vial. Allow approximately 20 mL of the culture to flow into the sample vial and shut the sample valve. Unscrew the sampling vial from the sampling hood and quickly replace it with another sterile sampling vial. Transfer 15 mL of the sample to a 15 mL centrifuge tube and centrifuge at 1000g for 30 min. Draw off supernatant and freeze approximately 1 mL at -80 ^oC for -gal analysis. Label all samples with ECH161L, Group #, date the sample was taken and description of sample. Sonicate the pellet and freeze the sonicated sample at -80 ^oC for -gal analysis, after carefully labeling the sample container. With the remaining 5 mL of sample, perform a cell count and cell viability count according to the protocol in this handout. Filter 2 mL of the sample through a 0.22 m syringe filter and determine the glucose concentration of the medium. Glucose samples may be frozen at -4 ^oC and analyzed later.

Coulter Counter Procedure:

Check the Coulter Counter settings and make sure that they match the insect cell culture settings indicated on the top of the Coulter Counter. Repipet 20 mL of Isoton II solution (filtered through a 0.22 m filter) into an accuvette. Vortex the insect cell sample gently and immediately pipette 100 L of sample into the accuvette. Cap the accuvette and mix by inverting the accuvette. Place the accuvette on the sample stand to submerge the aperture tube in the sample. Turn the count/reset knob ¼ turn clockwise to "reset" the instrument. When the light illuminates, turn the dial 1/4 turn clockwise to count. Once the light illuminates, make sure that the aperture is not blocked. The Coulter Counter will then count the cells in the sample drawn through the aperture. Multiply the average raw count (from 3 separate measurements of the sample) by 400 to determine the cell concentration in cells/ mL. Give the TA all samples containing cells and/or virus to dispose of them. Make sure that the orifice tube is left in a solution of the blue Coulter cleaning solution and is not allowed to dry out.

Cell Viability Procedure:

Add 50 L of 0.2% Trypan Blue solution to a 1.5 mL Eppendorf tube. Add 100 L of the sample containing the cells to the Eppendorf and mix gently. Place a drop of the cell/trypan blue solution onto a hemocytometer and count the total number of cells and number of viable cells (not blue) using the microscope according to the instructions for the hemocytometer. Give all used samples and slides to the TA for disposal.

Glucose Analysis:

Check the buffer and waste containers to make sure that the buffer supply is sufficient and the waste container is not full. Turn the stand by/run switch to RUN. Press CLEAR. When a number appears on the display, turn the zero knob until the display reads 0. Inject a standard glucose solution and press the black button just above the injection port. When the display reads a number, press the calibrate button and adjust the calibration knob until the display reads the appropriate value. Repeat the process until the instrument is properly calibrated. Filter the sample and rinse the injector syringe with the filtered sample. Inject the sample, press the black button near the injection port and take a reading. Give the TA any unused samples for disposal.

Luminometer Procedure:

Turn on luminometer. Once the luminometer software has booted up, open the file c:\ml3000\bgal.ems. Follow the procedure described by Tropix for chemiluminescent quantification of -galactosidase detection.

IV. REFERENCES

V. SUGGESTED COMPONENTS OF THE REPORT

Describe each of the stages in the insect cell culture/baculovirus system (cell growth, primary infection, secondary infection, protein expression and lysis). Present, analyze and discuss your experimental data for cell concentration, cell viability, glucose concentration, intracellular -galactosidase concentrations, and extracellular -galactosidase. Present data for the bioreactor operating variables during the culture including temperature, pH, DO and agitation. Determine growth rates and yields. All plots should include error bars to indicate the level of experimental error in the data. Compare your results with those presented in the literature and comment on possible extensions for future experiments.

VI. SAFETY PROCEDURES

Standard safe laboratory procedures apply to this laboratory. Read all MSDS sheets carefully before beginning the experiment and make notes of any hazardous materials and/or steps in your lab notebook. Closed toe shoes, lab safety glasses and long pants must be worn in the lab at all times and food/drinks are not allowed in the lab. Use examination gloves when handling samples. Be sure that ANY container that you use is labeled with the contents, ECH161L, Group # and date. After the experiment is completed be sure that all sample containers are properly cleaned and/or disposed of. Any samples containing cells and/or recombinant virus must be given to the TA for autoclaving and proper disposal. Use approved ear plugs when using the sonicator and insure that all others in the laboratory are using them as well.