In Vitro Methods

Cell Culture Assays
Introduction
Cell cultures of various sorts are under wide consideration and development in toxicology to replace the use of animals, supplement the use of animals, and perhaps provide information that can be obtained by no other convenient means. An Institute of Alternatives to Animal Testing operates at Johns Hopkins University on funding from a variety of private and public sources to further the application of in vitro methods in toxicology testing and research.
A variety of in vitro systems based on cell cultures or bacterial cultures are available. Perhaps the best known, and the oldest, is the Ames Assay, developed by Bruce Ames to screen chemicals for mutagenic potential. This bacterial system, based on specially designed strains of S. typhimurium, is supplemented with rat microsomes, and the back mutation rate is scored as a function of dose of chemical. Because chemicals are identified by mutagenic action, and the system uses mammalian microsomes, the screen is also popularly considered to screen for carcinogenic potential.

Simple cell cytotoxicity assays are entering the market. One system is based on the release of a cytoplasmic enzyme into the culture medium. The enzyme, lactate dehydrogenase, is measured by a simple calorimetric assay. Another test monitors dead cells by a failure to exclude Trypan blue. These tests are scored manually by counting cells on a microscope slide, and are therefore less popular.

In this experiment, we will examine the effect of heat shock on the cell’s ability to resist exposure to cadmium, a known lung toxin. We will use a simple test for cell activity (not cell viability) to determine a dose response curve for cadmium, in the presence and absence of heat shock. The assay is the uptake of a vital stain, Neutral red. Most cells will accumulate NR in lysosomes. The take up NR therefore indicates that the cells have suffered damage. The dye taken up by the cells is subsequently extracted and measured. Since the amount of dye taken up by the cells is a function of cell number, some indication of cell mass is also necessary to interpret the results. In this experiment, cell mass will be determined by a sensitive assay for protein.

Procedure
The response of the cultures to cadmium chloride solutions will be examined. Two plates are set up, so divide yourselves into teams, one team per plate. The cultures were exposed prior to the laboratory (about 9 AM today) to a range of CdCl₂ concentrations: 0.0, 5, 10, 20, and 40 µM. At the beginning of the class period, the medium will be replaced with medium containing neutral red, to a final concentration of 50 µg/mL. After an additional 1-hour exposure, you will remove the medium, rinse the cells, and determine the Neutral red uptake. Follow the protocol. The amount of NR will be determined spectrophotometrically


Cell Culture
A culture of rat lung epithelial cells are grown on solid substrate in sterile flasks. The cells are released from the substrate and suspended in medium. About 50,000 cells are plated per well in 24 well plates. The cells are plated and grown at 37°C in a medium of 5% newborn calf serum/defined medium (F12/DMEM) under 5% CO₂. Typically, the cultures reach confluence after 24 hrs. Only cultures at, or near, confluence are used in this experiment.

Heat Shock
Twelve hours before the experiment, 1 plate of cells will be transferred to a 44°C incubator for 1 hr. The plate is then returned to the regular 37°C incubator.

Exposure to Cadmuim Chloride
Replicates of 4 wells were exposed to the following concentrations of CdCl2 ₂.
Column 1: Blank, no cells
Column 2: 5 µM Cd
Column 3: 10 µM Cd
Column 4: 20 µM Cd
Column 5: 40 µM Cd
Column 6: Control, no exposure.

Neutral Red Assay
Stock Solutions

• Neutral red (0.5% solution in F12/DMEM medium, prepared for student)
• Destaining solution (1% glacial acetic acid, 49% dH₂O and 50% ethanol, V/V)
• Dulbecco’s phosphate buffered saline (PBS)

Preparation of the NR Medium

1. Dissolve NR stock solution in ethanol to final concentration of 5 mg/mL. Incubate at 37°C for 24 hrs.
2. Dilute NR stock solution 1 to 100 in cell medium (F12/DMEM) 2 hrs prior to application to the medium, and hold at 37°C.
3. Filter the NR/medium through a 0.45-u filter to remove filtrate and ensure sterility.

Assay

1. Remove the plates from the incubator (normally, this step would be done in a laminar flow safety hood; however, the short period of incubation required for this step allows us to do this on the bench top). Remove the existing medium by aspiration. Replace with 1 mL of the NR medium. Use a sterile pipette or sterile pipette tips.
2. Return the plates to the incubator for 1 hr at 37°C.
3. Observe the plates in the inverted microscope. Describe the cultures by reference to the control plate.
4. Remove medium from cells by gentle aspiration. Add 1.0 mL of PBS to each well gently. Avoid blasting cells free from the bottom of plate. Rotate the plates several times over a 5-min period.
5. Aspirate the PBS. Repeat with another identical volume of PBS.
6. Carefully add 1.0 mL of destaining solution. Gently rotate the plates several times every 5 minutes for 15 min. A plate shaker is available.
7. Read the plates on the Bio-Tech microplate reader (instructions attached).

Protein Assay
Carefully aspirate the extraction medium. Add 0.1 mL of 1 N NaOH to each well. Swirl the plates to dissolve the protein. Add 1.5 mL Biuret reagent. Read in the plate reader after 15 min. (same settings as the NR assay).

Interpretation of Results
The absorbance value at 540 nm is the amount of NR taken up by the cells. As cells lose viability, they lose the ability to take up NR.

Obtain and attach copies of both 24 well plate reports as tables in your report.

Plot your values of NR uptake, average of 4 wells versus concentration, and construct a dose response curve. You may wish to convert the NR uptake values to a percentage response for purposes of plotting. Plot results of both plates on the figure. As a separate figure, plot the Biuret absorbance versus concentration to determine if the toxin caused a lose of cell protein from the well. Comment specifically on whether heat shock altered the dose response of the cell to cadmium exposure.