Visualization of Normal & Transformed Cells - Kit 1. Introduction. Your kit includes the following materials:
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1 Visualization of Normal & Transformed Cells - Kit 1 CellServ@FAES/NIH Introduction Cells in culture may assume a variety of morphologies, most often quite different from their shape in their native tissue. Nevertheless, the range of morphologies is predictable and can be correlated with the origin of the cell type, its developmental status and the in vitro environments (e.g. substratum, serum source, crowding). This exercise will provide students with an opportunity to examine cells with different shapes and patterns of growth in culture stemming from their different origins, human lung fibroblasts (IMR-90) vs. a mouse subcutaneous cell derived from an areolar and adipose tissue biopsy (clone L-929), variations in cell density, and stage in the cell cycle (interphase vs. mitosis). This exercise also shows that the "social behavior" of cells from tumor (transformed) cells is different from that of non-transformed cells. Finally, by having students stain the cells, an opportunity is presented to introduce basic concepts of an important cytological/histological technique. We have grown selected cell types in 24-well plates which contained round coverslips which served as attachment surfaces for the cells. The coverslips with attached cells were retrieved and fixed with methanol in order to kill the cells without markedly modifying their morphology. (See Appendix 1 for details regarding subculturing methodology). These fixed coverslip cultures are ready for staining by the students. Your kit includes the following materials: Coverslips with IMR-90, a normal, diploid human cell line which shows the typical fibroblastic morphology. Coverslips with L929, a transformed mouse cell which shows cells of varying morphology and giant cells. STAIN #1 and STAIN #2. (Be careful! Avoid contact with skin and clothes.) Permount (mounting medium). (Keep in closed container. Avoid prolonged contact with air.) Printed background and procedural information as well as a Glossary of Terms, References, & Further Reading. If your kit is not complete, contact us immediately.
2 Teacher Preparation The following materials will need to be made available to each student or group of students: 1. Monocular or binocular microscope (10-40X objectives) 2. Forceps, pipettes, microscope slides. 3. Distilled or deionized water aliquotted in small tubes or in squeeze bottles (a few mls per student/group is all that is needed). 4. Stains #1 & #2. These stains can be reused a number of times provided that caution is taken against excessive carry over between stains. NOTE: Those terms which are highlighted throughout this manual are defined in more detail in the Glossary of Terms, References, and Further Reading section supplied with this kit. Objectives Technique to be learned 1. To stain and mount cells which have been grown attached to coverslips. Time requirement: minutes, including casual observations. Observations to be made and skills to be developed 1. To examine the stained, mounted cells at 100X and 400X magnification in order to discern the most common cell shapes. 2. To recognize the nucleus, its position, and the variety of shapes it may assume. 3. To recognize the nucleoli and the variety of shapes they may assume. 4. To be able to distinguish typical interphase cells from cells in mitosis. 5. To recognize typical fibroblastic morphology of cultured cells. 6. To recognize monolayer growth. Time requirement: 40 to 60 minutes, depending upon detail requested of student. Kit 1 Visualization of Normal & Transformed Cells 2
3 Background Information Normal and Transformed Cells When subjected to various factors, whether biological or environmental, normal cells can undergo neoplastic transformation and become cancerous. Whether this process occurs in vivo or in vitro the behavior of these transformed (tumorigenic) cells will be quite different from the ancestral normal cell. These observable and measurable differences between normal and transformed cells provides the researcher with a valuable tool in determining the various factors which control cell division, cell-to-cell interactions, the influence of growth factors on cell growth, the cell cycle, and much more. Although the growth environment of an in vitro system does not directly match that of an in vivo system, there exists extensive evidence that indicates a direct correlation between cell behavior in vitro and cell behavior in vivo. What causes a normal cell to undergo transformation? Although the picture is not complete, researchers know that several factors can cause a cell to become transformed thereby resulting in behavior quite different from that of a normal cell. Some transforming factors are as follows: Environmental carcinogens or mutagens - When cells are exposed to certain chemicals some genes of these cells may undergo mutations. These mutations can result in the formatio and proliferation of tumorigenic cells. UV radiation - Exposure to harmful UV radiation can result in mutations in a normal cell's DNA. As a result, the cells can become tumorigenic or cancerous. Retroviruses (RNA tumor viruses)- Upon infection of a cell with a retrovirus the genome of the virus (RNA) is reverse transcribed into DNA which becomes inserted into the DNA of the host. The location of this DNA insertion can have profound effects on the normal genome and growth behavior of the host cell. Once inserted, the viruses genome is replicated along with the host DNA and this results in continued multiplication of the virus. The location of insertion of the viral genome may result in the overexpression of a host gene which can result in the cancerous phenotype or the viral genome may itself contain genes, which when expressed, can result in the cancerous phenotype (these viral cancer causing genes are among a group of regulatory genes termed oncogenes). DNA Tumor Viruses - The genome of these viruses is composed of DNA. In some DNA viruses the viral DNA will become inserted into the host's genome. This insertion can result in the transformation of infected cells. Examples of these tumorigenic viruses are some types of human papillomavirus (HPV) and Epstein-Barr virus (EBV). Kit 1 Visualization of Normal & Transformed Cells 3
4 Once a cell has become transformed it will behave quite differently, both in vivo and in vitro, from the normal cell. Some characteristics of transformed cells grown in vitro are as follows: Transformed cells continue to divide indefinitely. Normal cells, on the other hand, will undergo a limited number of cell divisions (population doublings) after which time they will stop dividing. Transformed cells usually contain abnormal numbers of chromosomes. The cell is said to be aneuploid and the chromosome number is usually greater than the normal (diploid) number of chromosomes. Transformed cells behave differently when grown in culture. Whereas, normal cells are anchorage dependent, that is to say, they require a substratum and cannot grow in suspension. In addition, when normal cells grow in vitro they will divide until they cover the entire surface after which time they will stop dividing (this is termed contact inhibition). Tumor cells, on the other hand, will continue to divide and "pile" up on each other, even after the growth surface is covered with cells. Transformed cells undergo morphological changes. Many normal cells have a distinct shape common to that particular cell type. Tumor cells, on the other hand, may loose this constraint on morphology and will exhibit various shapes, or become rounded up and grow while loosely attached to the substratum. Transformed cells grow in vitro under less stringent conditions than those required for normal cells. The quantity and types of growth factors normally required to effectively grow normal cells in culture may not be required for many tumor cells. This may be a result of the tumor cells producing these necessary growth factors. Kit 1 Visualization of Normal & Transformed Cells 4
5 N L Student Procedures 1. Each student or pair of students should receive one coverslip with each cell type attached. The coverslips contain markings which when oriented correctly indicates the side of the coverslip to which the cells are attached. It is important to remember that the cells are affixed to this side. L N L929 (transformed) IMR-90 (normal) Cells are on surface facing you, when coverslips are in this orientation. CAUTION: Coverslips are very fragile. Handle Gently. 2. Gently grasp an edge of the coverslip with forceps and dip the coverslip into STAIN #1 for 1 SECOND ONLY. Repeat once more. Allow excess stain to drain by touching the edge of the coverslip to the stain container. Transfer the coverslip immediately to STAIN #2 and dip for 1 SECOND ONLY. Repeat once more. Caution should be taken to avoid overstaining of the coverslips. 3. Rinse coverslip in distilled (deionized) water for a few seconds to wash off any residual stain. AIR DRY THE COVERSLIP COMPLETELY. A stream of warm air or blowing may help speed up the process. Incomplete drying will result in very poor resolution when the mounting medium (Permount) is added. 4. To mount the coverslip, add 1-2 drops of Permount to a microscope slide in the area where the coverslip is to be mounted. Place the coverslip - CELL SIDE DOWN - onto the Permount. Apply gentle pressure to the coverslip to evenly distribute the Permount between the coverslip and slide. Eliminate/avoid air bubbles. Allow Permount to dry. Avoid getting Permount on the top of the coverslip. 5. To observe the cells, place the slide on a microscope stage, scan the slide and locate a population of cells. Switch to a higher power to observe cytological details. Low power (100X total magnification) should reveal shapes, growth patterns, and cell distribution. High power (400X) should reveal nuclear and nucleolar shapes, chromosomes, and cytological aberrations such as giantism, multinuclearity, cytoplasmic vacuolization, and nuclear blebbing, if present. Kit 1 Visualization of Normal & Transformed Cells 5
6 Results The photographs below show some of the various shapes that can be observed on the stained coverslips. The intensity of the stain is dependent upon the length of time that the coverslip is immersed in the stain; caution should be used to avoid overstaining. Figure 1. L929 Figure 1. L929 "Ruffling" membrane Giant cell showing multiple nuclei Typical morphology of L929 cells Note the appearance of the various cell shapes characteristic of L929 cells. Due to migrations across the coverslip during growth, the cell membranes will extend in various directions. This membrane "ruffling" is characteristic of migratory cells and cells internalizing (drinking) droplets of medium by pinocytosis. Cells in culture sometimes form "giant" cells. Note the multinucleated giant cell in the center of the photograph. This abnormality may result from the fact that the cells are grown on an artificial environment (plastic). The presence of these giant cells is by no means an indication of problems in the cell culture environment unless the frequency of these rise above normal levels. The multiple nuclei result from a loss of coordination between karyokinesis and cytokinesis, thereby resulting in an increased number of nuclei per cell. Figure 2. L929 (CONT) Figure 2. L929 (CONT) This photograph again exhibits the common variations in cellular morphology characteristic of this cell line. The cell in the center of the photograph which has the long membrane extension was probably undergoing migration when it was fixed. Note the variety of cell shapes throughout the photograph. Also, note the binucleated cell. Again, this is not rare for this particular cell type. Figure 3. IMR 90 L929 cell showing long membranous extension Binucleated L929 cell Figure 3. IMR 90 Note the growth pattern of these cells. This diploid human lung fibroblast cell, IMR-90, is much larger than the L929 cell and also shows an oriented growth pattern (in contrast to the random growth pattern of L929 cells). Note the darkly stained nucleus in each cell. Typically, the nucleus is elliptical. The differences in size probably represent differences in cell cycle positions. When a confluent monolayer of cells is formed in the culture flask they will cease growing and migrating. On the other hand, most (but not all) transformed cells are not contact inhibited and will continue to "pile" up on each other when confluency is reached. (Note - L929 usually does not continue to grow once confluency is reached). Kit 1 Visualization of Normal & Transformed Cells **Photos were taken with a Zeiss AxioSkop 2 equipped with an Optronics Microfire camera using PictureFrame software. 6
7 Student Assessment 1. What is meant by contact inhibition? 2. Give an example of an agent (biological or environmental) which can result in the formation of a transformed cell. 3. Describe 2 distinct morphological differences between the IMR-90 cells and the L-929 cells used in this exercise What causes the formation of "giant" cells and why do they form? 5. Describe the purpose and action of trypsin during the detachment phase of cell harvesting. 6. What is aneuploidy and in what type of cells would you expect this condition? 7. Explain the differences between the terms in vivo and in vitro. 8. When one first examines a specimen with a microscope should low power or high power be used first? Why? 9. Why is it important to do cell culture work using aseptic technique? 10. If a researcher isolated a population of cells many years ago and has continued to maintain that cell line through subculturing, would you expect this cell line to be normal or transformed? Why? Kit 1 Visualization of Normal & Transformed Cells 7
8 Further Study 1. Both biological and environmental factors can result in the formation of tumorigenic cells. Identify and research an example of each type of factor and describe the nature of their actions. 2. Anchorage dependent cells have special biochemical and physical requirements in order for them to grow in tissue culture flasks. Describe the nature by which cells will attach to the plastic growth chamber incorporating both the biochemical nature of the attachment as well as the ways in which the plastic flasks can be treated. Appendix 1 Methods used to prepare cultures for CellServ Kit #1 The cells provided have been prepared using the procedures outlined below. The coverslip cultures provided are in a stable condition and can be stored for an indefinite period of time. Please see STUDENT PROCEDURES (page 5) for information outlining the steps to be completed in the lab by the students. 1. Cells are grown in a T 25 tissue culture flask to a confluency approaching 80-90% in the presence of serum-containing EMEM (Eagle's Minimal Essential Medium; see Glossary of Terms and References). 2. The "old" (conditioned) medium is poured off and the cells are rinsed two times with 5 ml of CMF-PBS. This insures that any residual serum from the medium is removed ml of cold trypsin is then added to the cells attached to the bottom of the flask. The trypsinization reaction is monitored with a microscope and is allowed to proceed until all the cells have detached from the bottom of the flask. A gentle tapping of the culture flask enhances cell detachment ml of serum-containing EMEM is then added to the flask to neutralize the enzymatic effects of the trypsin. This step is critical as overtrypsinization can result in a drastic reduction in cell viability. 5. The cell suspension is then aseptically transferred to a sterile 15 ml conical centrifuge tube and spun down in a centrifuge at 200g for 3-5 minutes. The supernatant is removed and 1 ml of CMF-PBS is then added to the cell pellet and titurated to achieve homogeneity. 6. A viable cell count is then determined with a hemocytometer using the trypan blue dye exclusion method. 7. Approximately 100,000 cells in 1 ml of EMEM are then added to each coverslip-containing well of a 24-well plate. The 24-well plate is placed in a 37 C incubator. The cells will reach the desired confluency in approximately 3 days. 8. The coverslip is removed and rinsed with CMF-PBS. This is followed by its immersion in methanol for 3-5 seconds. The cells on the coverslip are now fixed and ready for shipment. Kit 1 Visualization of Normal & Transformed Cells 8
9 Teacher Evaluation Kit In order to provide the best instructional material possible, we would greatly appreciate your comments. Please answer the following questions, and do not hesitate to include comments and suggestions that may not be covered. In order for us to improve and expand our program, please be frank in your comments. Please use back of sheet if necessary. 1. Was the information provided you adequate enough to cover this material with your class? 2. Was the material and information provided to the students easily understood and straightforward? 3. Did this material meet your expectations? Please indicate other cell culture materials that you may be interested in introducing to your students. (Keep in mind the stability of materials as well as the transportability of materials). 4. Were the student assessment questions appropriate for the level at which you would like to teach this section. (Were they too difficult/easy)? Were the further study problems adequately stated and useful for future study by your students? If you feel inclined please provide sample questions which you feel would be appropriate for students using these kits. THANK YOU Please return this sheet to: CellServ FAES/NIH Bldg. 60; Room Cloister Court Bethesda, MD Tel. (301) ; Fax (301) Kit 1 Visualization of Normal & Transformed Cells 9
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