CHEN 4800/5800: Bioprocess Engineering, Spring 1996
University of Colorado-Boulder
, Chemical Engineering Dept.
Dhinakar Kompala
, Mike Sportiello, Gautam Banik

Lab Exercise 4: Perfusion Cultures of CHO Cells

Objectives

In this laboratory exercise, we will determine the performance characteristics of the inclined settler in selectively recycling the larger, live CHO cells back to the bioreactor, while removing the smaller, dead cells and cell debris in the overflow stream. In addition, we will examine the production of reporter protein at two different growth rates in two different bioreactors.

Background

The recombinant CHO cell line used in this lab exercise is a suspension-adapted cell line, which has been stably transfected with the plasmid pSVgalneo. This plasmid contained the lacZ gene and the gene for resistance to neomycin under the influence of the SV40 early promoter. The lac Z gene codes for the reporter protein -galactosidase.

These recombinant CHO cells are cultivated in two types of bioreactor systems. One bioreactor is operated in chemostat mode and the second bioreactor is operated in perfusion mode. Selective viable cell retention is achieved in the perfusion mode using an inclined settler. A schematic drawing of the inclined settler is shown in Diagram I. The principle behind the operation of the inclined settler is that viable cells are bigger, settle faster than dead cells and hence get recycled back to the reactor while non-viable cells being smaller are selectively washed out in the harvest line. You will sample the reactor contents in both the reactors. In addition for the perfusion reactor you will also sample the medium underflow and overflow streams from the settler. Collect the samples in 15 ml centrifuge tubes and label the tubes appropriately. These samples will be used for viable cell density and % viability determination using a hemocytometer, cell size determination using the coulter counter, and determination of the intracellular -galactosidase content in the cells growing at two different growth rates.

Experimental Procedures

A) Determining Total and Viable Cell Concentration using a Hemacytometer

Trypan blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live cells do not take up certain dyes whereas dead cells do and appear blue under the microscope.

1) Take 0.5 ml of cell suspension. Add 50 µl of 0.25% trypsin. Incubate at 37°C for 15 minutes. Trypsin is used to dissociate the aggregates and form a single cell suspension.

2) After 15 minutes, add 0.5 ml of 0.4% trypan blue to the above mixture. Allow to stand for 5 min. The dilution factor in this case is (0.5 ml cell suspension + 0.05 ml trypsin + 0.5 ml trypan blue)/(0.5 ml cell suspension) = 1.05/0.5=2.1

3) With the cover-slip in place, using a micropipettor transfer a small amount of the trypsin-trypan blue-cell suspension mixture to both chambers of the hemacytometer by carefully touching the edge of the cover-slip with the pipette tip and allowing both chambers to fill by capillary action. Do not overfill or underfill the chambers.

4) Starting with one chamber of the hemacytometer, count all the cells (non-viable cells stain blue) in the 1 mm center square and the four 1 mm corner squares (see Diagram II). Keep a separate count of the viable and non-viable cells. Note: Count cells on top and left touching the middle line of the perimeter of each square. Do not count cells touching the middle line at the bottom and right sides (see Diagram III).

5) Repeat this procedure for chamber 2. Note: If greater than 10% of the cells appear clustered, repeat the entire procedure making sure that the cells are dispersed by longer incubation with trypsin, vortexing and vigourous pipetting of the trypsin-cell suspension mixture. If less than 200 or greater than 500 cells (20 - 50 cells per square) are observed in the 10 squares, repeat the procedure, adjusting for an appropriate dilution factor.

6) Cell Counts and % viabilty: Each square of the hemacytometer, with the cover slip in place, represents a total volume of 0.1 mm3 or 10-4 cm3 or 10-4 ml. Thus the subsequent cell concentration per ml will be determined by the following:

Cells/ml = the average count per square x dilution factor x 104 . For example, if the average viable cell count per square is 50 cells then the viable cell concentration can be determined as 50 x 2.1 x 104 =1.05 x 106 cells/ml.

% viability = total viable cells (unstained) / total cells (stained + unstained)

B) Determining CHO Cell Size Distribution Using the Coulter Counter

The Coulter Counter will be used to determine the size distribution of the CHO cells in each of the samples, according to the procedures below:

1) With the orifice tube immersed in a beaker of saline solution, flush the orifice tube by turning (in a clockwise direction) the bottom of the two knobs on the upper right of the sampling stand to the FILL position (designated by an unfilled rectangle), and then turn the top knob in a clockwise direction to RESET (unfilled rectangle). After about ten seconds...

2) Turn the top knob to COUNT, and the bottom knob to CLOSE (always in a clockwise direction).

3) Add 1 ml of your sample to a vial containing 15 ml of saline solution. Gently mix.

4) Immerse the orifice tube into your sample vial.

5) Turn the top knob to RESET.

6) When the green baseline on the monitor appears, press the Reset button on the Counter itself, and the previous data will be deleted from the graphic display.

7) Press the Start button on the Counter, and your sample will be taken by the Counter and the size distribution graphed as the counts accumulate. After 15 seconds the sample will cease to be taken and you will have finished recording data for that sample. Turn the top knob to COUNT and carefully remove your sample from the sample stand.

Data Analysis

8) Set the left cursor and right cursor to bracket the populations of cells for which you want a count. For example, if you have a bimodal distribution of cell sizes, move the cursors to bracket one of the two populations of cells. The graph will tell you how many cells are within the size range you have chosen, and the lower and upper size thresholds of the range. Moving the cursors to bracket the other population will give you this information for that population also.

Saving Data (Bring a diskette to class)

9) Once you have completed analysis of the sample, save the data to the hard drive by going to the computer and accessing the IMP directory. Once you have done this, type imp and hit return. This brings you into the data import program.

10) Hit ALT F, and when the new screen comes up, go to the line that reads “Capture file into:” and type a file name into which you can import the data. Hit return.

11) Hit Alt M to get into the main menu, and then hit Alt C to start the data capture process. To actually initiate the transfer, hit the Print button on the Coulter Counter and the data will be transmitted to and stored on the hard drive. You can then copy the raw data to your diskette.

To run another sample, repeat all the above steps, beginning with step 1.

C) Determining -galactosidase production using a spectrophotometric assay

1) Take 1 ml of your undiluted sample and transfer it to a plastic eppendorf tube. Label the tube with the reactor type, sample port type and sample time. Centrifuge the tube for 5 minutes in an eppendorf centrifuge at 2000 rpm. Pipet off and discard the supernatant.

2) Resuspend the pellet in 1 ml of Phospate Buffered Saline (PBS). Put the sample on ice and sonicate it for 120 seconds with the sonicator output setting at 4, the duty cycle set at 50%, and the pulses set for one second each. Always wear protective covering for your ears when using a sonicator!

3) After sonication, again spin the tube in the centrifuge for 5 minutes at 4,000 rpm. Save the supernatant!

4) Add 1.3 ml Assay Buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1.0 mM MgSO4, 5 mM dithiothreitol, pH adjusted to 7.0; buffer already prepared) to a cuvette. Cover the cuvette with parafilm.

5) Add 500 µl of sonicated supernatant (from "c" above) to the cuvette.

6) Add 0.2 ml of a solution of 0.4% (w/v) ONPG (o-nitrophenyl--D-galactoside) to each cuvette; pipet up and down several times to mix.

7) Running the spectrophotometer. Press "Prog" and Program 0: HSHI will appear. Press "R/S". You do want to calibrate, so next press "0" and then "Enter". Insert the "blank" cuvette into the cuvette chamber in position 1 and press "R/S". After a short time for calibration, the spec will "ask" you to insert the samples and then press "R/S" to read.

8) Place the cuvettes in the sample chamber of the temperature-controlled Beckman DU-50 spectrophotometer (temperature will be controlled at 37°C) in positions 2-3. Leave the "blank" cuvette in position 1.

9) Record absorbance at 420 nm over a period of 8 min by pressing the "R/S" button.

10) The -galactosidase activity is calculated from the rate of change of the optical density for each sample and is expressed as U/ml (1 unit (U) hydrolyzes 1 µmole ONPG to o-nitrophenol and D-galactose per min at pH 7.3 and 37°C) by comparing the slope (rate of change of absorbance) for the sample to the slope for a -galactosidase standard. The spec is programmed to calculate the slope of your sample; you need to follow the directions below to get this value:

a. After your samples have run for 8 minutes, the spec will "ask" for a cell number. Press "2" and "Enter".

b. The display will read "blank"; press "Enter" for the default setting.

c. The display will read "Time 1"; press "3" and then press the "Enter" button. We will only take the data from 3 minutes to 8 minutes for our assay; the first 3 minutes are to allow the cuvettes to warm up to 37ûC in the sample chamber.

d. The display will read "Time 2"; press "Enter" for the default setting (8 minutes).

e. The display will read "Factor"; press "Enter" for the default setting.

In a few moments the data will appear at the bottom of your printout. Repeat steps a-e for the sample number 3. After this is done press "0" and "Enter" in order to exit the program. Remove your 2 sample cuvettes and the blank from the spec.

The last column of data for each cell (chamber) is "Result"; this is the slope of the absorbance readings for your sample from 3 to 8 minutes. You can determine the U/ml for your sample by comparing your slope to a b-galactosidase standard. The slope of a 1 U/ml standard under these assay conditions is 0.0487. A more exact expression of the enzyme activity is U/cell number. You can determine this quantity for your samples using the cell concentration data you have obtained using the hemacytometer.

Report - Due April 30, 1996

Write a brief report (under 5 pages, plus figures/graphs) discussing the effectiveness of the inclined settler in removing the dead cells and recycling the live cells based on their size distributions, and the production of -galactosidase at different growth rates.

Diagram I

 Diagram II

Diagram III


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Prof. Dhinakar S. Kompala / Department of Chemical Engineering
College of Engineering / University of Colorado