Name: using mouse mammary tumor cells to teach core biology concepts: a simple lab module
Uploaded: 2015-06-18T13:00:00
Description: Watch this Scientific Journal Video about Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module at JoVE.com

This work is supported by the Joseph Alexander Foundation, the ASBMB Undergraduate Research Award, 2013-2014, and a Science Award Grant, Marymount Manhattan College, 2012-2013.

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
<ol>
	<li>Grow cells in 10 cm tissue culture dishes containing 10 ml of nutrient rich media that consists of Eagles Minimum Essential...

<ol>
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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 ...

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&#160;.&#160;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&#39;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&#160;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 equipment1,4,5), describe protocols that are quite advanced (e.g., suitable for a 400 level course6), or require multi-week or semester long projects6,7. 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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>ESCO Labculture Reliant</td>
			<td></td>
			<td>Class II Type A2 Biological Safety Cabinet</td>
		</tr>
		<tr>
			<td>Waterjactor CO2 Incubator</td>
			<td>CEDCO</td>
			<td>Model 1510</td>
			<td></td>
		</tr>
		<tr>
			<td>Bright-line Hemocytometer</td>
			<td>American Optical</td>
			<td></td>
			<td>with two separate grids</td>
		</tr>
		<tr>
			<td>Motic Images Plus</td>
			<td></td>
			<td></td>
			<td>Mac OSX Verison 2.0 or higher</td>
		</tr>
		<tr>
			<td>Gilson Pipetman</td>
			<td>Rainin instrument co. inc</td>
			<td></td>
			<td>P-20D, P-200D, P-1000D</td>
		</tr>
		<tr>
			<td>CK30/CK40 Culture Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>4 objective inverted light microscope with camera</td>
		</tr>
		<tr>
			<td>200 uL Pipet tips</td>
			<td>MidSci</td>
			<td>40200C</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 uL Pipet tips</td>
			<td>MidSci</td>
			<td>AVR4</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Seriological Pipets</td>
			<td>TPP</td>
			<td>TP94010</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plates</td>
			<td>CoStar- Tissue Culture Cluster</td>
			<td align="right">3524</td>
			<td>24 wells, 16 mm well diameter, Radiation sterilized</td>
		</tr>
		<tr>
			<td>Trypan Blue Solution 0.4%</td>
			<td>Sigma</td>
			<td>T8154</td>
			<td>100 mL, cell culture tested non-haz</td>
		</tr>
		<tr>
			<td>Bright-line Hemacytometer replacement coverslip, non-haz</td>
			<td>Sigma</td>
			<td>Z375357</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Mammary Tumor(MMT) cells</td>
			<td>ATCC</td>
			<td>CCL-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Eagle Minimum Essentail Medium (EMEM)</td>
			<td>ATCC</td>
			<td>30-2003</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F0926</td>
			<td>500 mL</td>
		</tr>
		<tr>
			<td>Meformin Hydrochloride</td>
			<td>Sigma</td>
			<td>PHR1084</td>
			<td>500 mg</td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma</td>
			<td>T5648</td>
			<td>white or white-yellow powder</td>
		</tr>
		<tr>
			<td>Curmumin</td>
			<td>Sigma</td>
			<td>C1386</td>
			<td>yellow-orange powder</td>
		</tr>
		<tr>
			<td>Aspirin</td>
			<td>Sigma</td>
			<td>A2093</td>
			<td>meets USP testing specifications</td>
		</tr>
	</tbody>
</table>

using mouse mammary tumor cells to teach core biology concepts: a simple lab module

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.

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</str...

Watch this Scientific Journal Video about Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module at JoVE.com

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

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.

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

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