Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Summary

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

Abstract

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

Introduction

Often in undergraduate general biology courses, the topics of cell cycle regulation and cancer are touched upon but not explored in detail because the breadth of content in these courses leaves little time for depth. In addition, undergraduate biology students are not typically exposed to the advanced techniques associated with animal cell culture. To help students develop a deeper understanding of these concepts, while applying and analyzing what they have learned, a laboratory activity was developed as a modification of the Walter Reed Army Institute of Research (WRAIR) extended laboratory activity¹. The lab module uses a step-wise, experimental strategy that includes growing and characterizing a cancer cell model, developing and executing cell counting methods, establishing optimal time course and dosages for treating cells with anti-proliferative agents, and identifying aberrant cell-signaling pathways. The experiment also allows for open-ended inquiry.

Most of the techniques required for this activity can be accomplished in a typical biology-teaching laboratory. The activity starts with students characterizing the morphology and growth rate of the mouse mammary tumor (MMT) cell line, a model for human breast cancer² . Breast cancer was chosen as the model cancer because of its prevalence in the population, its familiarity to college-aged students, and the widespread data available. The MMT cell line was specifically selected because it is easily obtainable, well characterized, has a short doubling time and is easy to grow. In addition, MMT cells are estrogen-dependent which is consistent with most female breast cancers. Students then identify aberrant cell-signaling pathways in the MMT cells by treating the cells with chemotherapy drugs whose mechanism of action is well established.The concentration of the drugs and length of the treatments are varied allowing students to evaluate the effect of these variables on the rate of cell division. The key assay for this activity is the determination of cell viability, which simply requires cell counting, using one of two methods. Each method depends on strong microscopy skills. Students determine cell viability by using a standard, hemocytometer method and a novel photomicroscopy method and propose. Based on their findings, they can propose and test modifications to the activity. Students then represent their data and interpret the results to refine their hypothesis and devise new experimental strategies.

This laboratory activity is suited for freshman or sophomore level students majoring in the biological sciences. It is condensed into a one-week lab module that can be completed in a first year, general biology or second year, cellular/molecular biology course. Skills needed for proper completion of the activity include basic arithmetic and algebra, familiarity with an array of core laboratory skills (e.g., pipetting, solution making, sterile technique), data analysis, basic light microscopy and time management, along with instructor knowledge of cell culture and spreadsheet software. Reagents required include an animal cell line model for cancer (e.g., mouse mammary tumor cells, MMT²), chemotherapy agents (e.g., tamoxifen, curcumin, metformin, and aspirin), trypan blue and cell culture media (e.g., Eagle's Minimum Essential Medium; EMEM) with appropriate supplements (e.g., donor horse and fetal bovine serum). Instruments needed include an inverted light microscope with digital camera attachment, computer, 100 mm and 24 well tissue culture plates, CO₂ incubator (or equivalent), biosafety cabinet (BSC; Class II), hemocytometer, and digital microscopy software.

There are good examples of specific lab activities that rely on animal cell culture to teach undergraduate students about concepts in cell biology³. However many require supplies or techniques that are not easily accessible (e.g., radioactive isotopes, live animal tissue, advanced imaging equipment^1,4,5), describe protocols that are quite advanced (e.g., suitable for a 400 level course⁶), or require multi-week or semester long projects^6,7. The lab activity described here is straightforward and can be conducted in a single week with common lab equipment.

In summary, this lab module effectively introduces or reinforces the concepts of cell cycle, cellular signaling pathways and cancer while teaching basic and advanced lab skills, experimental data analysis, the method of animal cell culture and the scientific process. The laboratory module is simple and economically accessible and provides both flexibility and opportunity for open-ended inquiry. The activity encourages student creativity by providing a template experimental strategy that acts as a guide but not a recipe. Most importantly, the activity satisfies all learning domains of Blooms Taxonomy⁸ as it requires remembering, understanding, applying, analyzing, evaluating and creating by engaging students in a process that pulls them out of the textbook and into the world of scientific research.

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Protocol

Notes: Conduct all work with cells and cell culture reagents in a Class II biosafety cabinet (BSC)⁹. MMT cells are classified as Biosafety Level I, as they pose low to moderate biological risk. Apply proper cleaning and decontamination procedures to the BSC between uses (e.g., ultraviolet light, 70% ethanol wipe down).

1. Grow MMT cells

1. Grow cells in 10 cm tissue culture dishes containing 10 ml of nutrient rich media that consists of Eagles Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and 1% Antimycotic/Antibiotic (10 units penicillin, 100 µg streptomycin, 0.25 µg amphotericin B). Cultivate cells in a humidified 5% CO₂ chamber, at 37 °C. Plate cells at a density of 3.6 x 10⁶ cells/cm² (see Counting Cells in step 2).
2. Replace half of the cell culture media with fresh media every 48 hrs unless otherwise indicated. Check cell viability and morphology by examination with an inverted phase contrast light microscope.
   1. Note and characterize cells for size, structure, shape, organization and estimated number under at 100 - 200X magnification. At this density, the cells should appear in one plane, not clumped atop each other and in close proximity. Each cell consists of a thick, spherical core with thin, long, branch-like extensions expanding from it that come to a point (see Figure 1).
3. Subdivide cells when they reach a cell density of 7.2 x 10⁶ cells/cm². Cells exhibit a doubling rate of 1.8 x 10⁶ cells/cm² per 24 hrs. Designate cells Passage X (Px) to denote the number of times they have been split.
   1. At generation 3 (i.e., after three passages, P3), harvest, count, and plate the cells onto a 24 well tissue culture plate at a density of 3.6 x 10⁶ cells/cm².
4. View cells every day and record digital photomicrographs. Note and describe cell morphology (i.e., size, shape, light reflective properties). Determine cell doubling time by counting cells regularly and relating elapsed time to cell number.

2. Count MMT Cells

Note: Count cells to determine if cells need to be subcultured, to set up for an experiment or to determine cell viability. There are two methods presented here.

1. Use of a hemocytometer, a standard technique to count cells.
   1. Obtain a bright-line hemocytometer, a 0.2% solution of trypan blue dye, a compound light microscope, sterile test tubes, pipettes and a manual cell counter.
   2. Under the BSC, use a 10 ml pipette to remove cells from a 10 cm tissue culture dish. If cells are grown on a 24 well plate use a 1 ml pipette or 1,000 µl micropipette to remove the media.
   3. Draw cell media into the pipette, place the tip of the pipette against the bottom of the dish and expel the media while sliding the pipette tip across the plate. Repeat 4 - 5 times to help dislodge cells, dissociate clumps and lead to a more accurate cell count.
   4. Place the resulting cell suspension in a 15 ml sterile tube (or 1 ml micro centrifuge or other small volume tube). Otherwise, leave the cells in the original tissue culture plate/well.
   5. Under the BSC, combine 10 µl of the cell suspension with 10 µl of the trypan blue solution (1:1 ratio) in a non-sterile micro centrifuge or other small volume tube.
      Note: Trypan blue is a vital stain that is not absorbed by healthy viable cells. When cells are damaged or dead, trypan blue can enter the cell allowing dead cells to be counted (aka dye exclusion method). Therefore under a microscope, dead cells appear dark purple while viable cells are a bright and light in color.
   6. Incubate at RT for 5 min.
   7. Place the cover slip on the hemocytometer. Apply 10 µl of the trypan blue-cell suspension mixture into the groove. View the hemocytometer at 100 - 200X magnification. A four-corner grid is apparent (see Figure 2).
   8. Count the number of viable cells (unstained) versus total cells in each gridded area. Average the four grids and multiply by 1 x 10⁴ to obtain cells/ml.
      1. Extrapolate the total number of cells per dish (or cells/cm²). Use this number to either determine the correct volume of the cell suspension to use to seed a new tissue culture plate at the desired density of 3.6 x 10⁶ cells/cm² or determine total number of viable cells on the plate or in the well.
         Note: For additional guidance on the use of a hemocytometer, see¹⁰.
2. Use software and a digital camera to count viable cells.
   1. Obtain a digital camera, its accompanying software, a 0.4% solution of trypan blue dye, a compound light microscope, sterile test tubes, pipettes and a computer.
      Note: A Moticam 2,000 digital camera with accompanying Motic Software is employed here. Any comparable camera and software should be sufficient. Ensure that the digital camera software has been calibrated in advance (according to manufacturer’s protocol).
   2. Under the BSC, use a 1 ml pipet or 1,000 µl micropipette to remove the media from the MMT cells growing in a 24 well tissue culture dish. Wash the adherent cells by gently applying a small amount (approximately 500 µl) of un-supplemented (i.e., EMEM without any supplements) fresh media to the plate then removing it. Apply 10 µl of 0.2% trypan blue (made by diluting 1:1 with un-supplemented EMEM) directly to the well.
   3. Incubate the plate at RT for 5 min.
   4. View the plate under the microscope at 100 - 200X magnification with a digital camera attached. Plate may be washed with unsupplemented media if staining with 2% trypan blue is too dark.
   5. Open the software on the computer and verify that the software is calibrated to the objective used via the settings tab. An image of the FOV should appear.
   6. Select the Grid from the Measure tab. A grid of squares approximately 0.005 cm in width will appear. Select Grid Info to confirm the area of each square.
   7. Choose a specific area of the grid to count cells (i.e., 5 squares x 9 squares). Select Rectangle from the Measure tab. Click the corner of a square and drag your cursor to encompass the squares of choice.
      Note: A shade of green will cover the squares of choice and a white box will appear in the corner that provides the width, height, area, and perimeter of the section of the grid chosen (see Figure 3).
   8. Count the number of viable cells within the determined area. Do the same for two other locations in the same well or plate by moving the plate under the microscope.
   9. Be sure that the measured area is the same and the magnification has not changed. Calculate the average number of viable cells within the area. Using the area of the well (for a 24 well plate, one well has an area of 2 cm², for a 100 mM dish the area is 78.6 cm²), extrapolate the number of viable cells from the area delineated in the grid to the total number of cells within the well (cells/cm²).

3. Treat MMT Cells with anti-proliferative Agents

1. Prepare solutions of the selected anti-proliferative therapeutic agents (tamoxifen, curcumin and metformin) and optional drug, aspirin under the BSC.
   1. Dissolve curcumin and tamoxifen in 100% ethanol to generate a stock concentration of 27 mM. Dissolve metformin and aspirin in unsupplemented EMEM to generate a stock concentration of 500 mM and 15 mM, respectively.
2. Establish a Dose Response.
   1. Treat MMT cells with the three anti-proliferative therapeutic agents (tamoxifen, curcumin and metformin) and optional drug (aspirin) at varying concentrations for 96 hrs to generate a dose response curve. Initially administer all drugs at a range of concentrations based on published reports^1,11-16 and then at concentrations larger or smaller than those published.
      Note: A dose response determines the minimum concentration of a drug necessary to produce the desired results. Here the desired result is a reduction in cell proliferation as compared to the control.
      1. For tamoxifen and curcumin, use concentrations (and corresponding volumes) of 0.054 mM (1 µl), 0.108 mM (2 µl), 0.162 mM (3 µl) and 0.216 mM (4 µl).
      2. For metformin, use concentrations (and corresponding volumes) of 2 mM (2 µl), 4 mM (4 µl), 6 mM (6 µl), 8 mM (8 µl) and 10 mM (10 µl).
      3. For aspirin, use concentrations (and corresponding volumes) of 0.030 mM (1 µl), 0.060 mM (2 µl), 0.099 mM (3.3 µl), 0.150 mM (5 µl), and 0.216 mM (6.7 µl).
   2. Split MMT cells from the 10 cm dish onto a 24 well plate at a concentration of 3.6 x 10⁶ cells/cm². Determine initial cell concentration by both cell-counting methods (Step 2). Call this new 24 well plate of cells “Day Split”.
   3. 24 hrs after cell plating, treat the MMT cells with each of the anti-proliferative therapeutic agents, tamoxifen, curcumin, metformin and aspirin, at the concentrations described in Step 3.2.1. Refer to this as “Day 0”.
      1. As each well can hold a maximum of 500 µl of media, use micropipettes with sterile micropipette tips and a new tip each time a new well is treated to avoid cross contamination. Use two wells as controls: cells grown in the absence of any drug treatment (negative control) and cells grown in the presence 100% ethanol (solvent control).
   4. Grow cells and re-administer drugs at the described concentrations when the cells are fed every other day (see Step 1.2) for 96 hrs (until Day 4). On Days 1 - 4 of treatment, observe the cells under the microscope and count using the method in Step 2.2 (see Figure 4).
   5. Repeat the experiment at least three times.
   6. Determine optimal concentration of each drug by graphing the relationship between cell viability and drug dosage over the length of the experiment (see Figure 5).
3. Establish a Time Course.
   1. Treat MMT cells with the three anti-proliferative therapeutic agents (tamoxifen, curcumin and metformin) at a fixed concentration for varying time periods. Use the optimal concentration identified through the Dose Response experiments (see Step 3.2).
      1. Use the following concentrations: 0.216 mM tamoxifen, 0.216 mM curcumin and 10 mM metformin.
         Note: A time course determines the amount of time necessary for a drug to produce its optimal desired result. Here, the desired result is a reduction in cell proliferation as compared to the control.
   2. Split MMT cells from the 10 cm dish onto a 24 well plate at a concentration of 3.6 x 10⁶ cells/cm². Determine initial cell concentration by both cell-counting methods (Step 2). Call this new 24 well plate of cells “Day Split”.
   3. 24 hrs after cell plating, treat the MMT cells with each of the anti-proliferative therapeutic agents, tamoxifen, curcumin, metformin and aspirin, at the optimal concentrations identified in Step 3.2. Refer to this as “Day 0”.
      1. As each well can hold a maximum of 500 µl of media, use micropipettes with sterile micropipette tips and a new tip each time a new well is treated to avoid cross contamination.
      2. Use two wells as controls: cells grown in the absence of any drug treatment (negative control) and cells grown in the presence 100% ethanol (solvent control).
4. Grow cells and re-administer drugs at the selected concentration when the cells are fed every other day (see Step 1.2) for 96 hrs (until Day 4). On Days 1 - 4 of treatment, observe the cells under the microscope and count using the method in Step 2.2 (see Figure 4).
5. Repeat the experiment at least three times.
6. Determine optimal exposure time of MMT cells to each drug by graphing the relationship between cell viability and length (time) of drug treatment at the selected concentration (see Figure 6).

4. The Lab Module

Note: The following is a 5-day lab schedule for the lab module. Figure 7 is a flow chart of what the schedule would entail within the 5 days. For this activity to be completed in five days either a time course or a dose response curve is generated. There is not sufficient time to generate both curves. A dose response experiment is described below. A time course experiment can be easily interchanged

1. Split the class into groups: have one group establish a dose response and the other a time course choosing a concentration in the middle of the proposed dose response concentrations.
   1. Maintain sufficient cultures and drug stocks at appropriate concentrations. Unless otherwise noted, perform all work under the BSC.
2. Day 1
   1. Direct students to make media and grow cells (as in step 1).
   2. As students obtain a stock of MMT cells on a 10 cm plate, direct the students to prepare cell culture media and give a tutorial in the use of the hemocytometer, digital microscopy and the digital camera microscopy software (as in step 2).
   3. Calibrate the software to the objectives used on the microscope (e.g., 4X, 10X, 20X) now using manufacturer’s protocol.
3. Day 2
   1. Have students confirm total cell number and cell viability (as in step 2).
   2. Have students count cells on the 100 mm dish using both the microscopy software and the hemocytometer methods, to confirm similar results are obtained.
   3. Direct students to obtain a 24 well plate and split cells from the 100 mm dish to the 24 well plates to the desired starting cell plating density (as in step 1). This is considered Day Split. Place the 24 well plate in a cell culture incubator for 24 hrs.
4. Day 3
   1. Grow MMT cells for 24 hrs on a 24 well plate. Have students observe cells under microscope and count using microscopy software (as in step 2).
   2. Give the student/team samples of cancer treatment drug stocks. Have them prepare and dilute the original stock solution (as in step 3). Add the various concentrations of the drugs to their cells to establish a dose response curve. This is called Day 0.
   3. Incubate cells (step 1.2) with the drugs for maximum of 48 hrs.
5. Day 4
   1. Have students observe treated cells after 24 hrs. This is Day 1.
   2. Count each well using microscopy software. Record photographs of each well so that counting can be conducted outside the lab (as in step 2).
   3. Incubate cells (step 1.2) with the drugs for another 24 hrs.
      Note: Instructor provides additional tutorials and reviews of data analysis and graphical presentation. Students learn to create figures, plots, and statistical analysis for the following day of data collection.
6. Day 5
   1. Have students treated cells after 48 hrs, Day 2.
   2. Count each well using microscopy software. Record photographs of each well so that counting can be conducted outside the lab (as in step 2).
   3. Harvest and collect cells for counting by the traditional hemocytometer method as a means of verifying student ability to effectively use the microscopy software method (as in step 2).
      Note: Collect data and draw conclusions or revise hypotheses, accordingly.

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Results

Growing MMT cells and comparing counting methods.

Mouse mammary tumor cells were successfully grown and characterized (Figure 1) and a novel cell counting method developed using Motic Software, a digital camera-associated software program for a microscope. This new cell counting method was compared to a traditional counting method employing a hemocytometer (Figure 2) and was shown to be equally accurate in determining cell number (Table 1

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Discussion

A lab module is presented that aims to teach a variety of topics in cell biology through the advanced techniques of animal cell culture. The module achieves this by analyzing the effects of a number of anti-proliferative chemicals on the replication of cells that model human breast cancer. The primary assay relies on the fundamental technique of cell counting and introduces a novel way to count cells using microscopy software. The activities comprising the module can be conducted with instruments and equipment available ...

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Materials

Name	Company	Catalog Number	Comments
Tissue Culture Hood	ESCO Labculture Reliant		Class II Type A2 Biological Safety Cabinet
Waterjactor CO2 Incubator	CEDCO	Model 1510
Bright-line Hemocytometer	American Optical		with two separate grids
Motic Images Plus			Mac OSX Verison 2.0 or higher
Gilson Pipetman	Rainin instrument co. inc		P-20D, P-200D, P-1000D
CK30/CK40 Culture Microscope	Olympus		4 objective inverted light microscope with camera
200 μl Pipet tips	MidSci	40200C
1,000 μl Pipet tips	MidSci	AVR4
10 ml Seriological Pipets	TPP	TP94010
24 well plates	CoStar- Tissue Culture Cluster	3524	24 wells, 16 mm well diameter, radiation sterilized
Trypan Blue Solution 0.4%	Sigma	T8154	100 ml, cell culture tested non-haz
Bright-line Hemacytometer replacement coverslip, non-haz	Sigma	Z375357
Mouse Mammary Tumor(MMT) cells	ATCC	CCL-51
Eagle Minimum Essentail Medium (EMEM)	ATCC	30-2003	500 ml
Fetal Bovine Serum	Sigma	F0926	500 ml
Metformin Hydrochloride	Sigma	PHR1084	500 mg
Tamoxifen	Sigma	T5648	white or white-yellow powder
Curmumin	Sigma	C1386	yellow-orange powder
Aspirin	Sigma	A2093	meets USP testing specifications