Difference between revisions of "20.109(S11):Start-up biomaterials engineering (Day1)"
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==Protocols== | ==Protocols== | ||
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Half the class today will start in the cell culture facility and half will start with experimental design. Midway through class, you'll switch places. In your notebooks, you should only write up Part 2 of the protocol. For Part 1, the only thing you need to write down is your cell count data, for use in a later FNT calculation. The Part 1 protocol will be posted on each tissue culture hood for your reference. | Half the class today will start in the cell culture facility and half will start with experimental design. Midway through class, you'll switch places. In your notebooks, you should only write up Part 2 of the protocol. For Part 1, the only thing you need to write down is your cell count data, for use in a later FNT calculation. The Part 1 protocol will be posted on each tissue culture hood for your reference. | ||
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====Background==== | ====Background==== | ||
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In the past century, we have learned a tremendous amount by studying the behavior of mammalian cells maintained in the laboratory. Tissue culture was originally developed about 100 years ago as a method for learning about mammalian biology. The term tissue culture was originally coined because people were doing exactly that, extracting tissue and letting it live in a dish for a short time. Today, most tissue culture experiments are done using cells rather than tissues. Much of what we know about cancer, heritable diseases, and the effects of the environment on human health has been derived from studies of cultured cells. | In the past century, we have learned a tremendous amount by studying the behavior of mammalian cells maintained in the laboratory. Tissue culture was originally developed about 100 years ago as a method for learning about mammalian biology. The term tissue culture was originally coined because people were doing exactly that, extracting tissue and letting it live in a dish for a short time. Today, most tissue culture experiments are done using cells rather than tissues. Much of what we know about cancer, heritable diseases, and the effects of the environment on human health has been derived from studies of cultured cells. | ||
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One type of familiar immortalized cell is the cancer cell. Tumor cells continuously divide allowing cancer to invade tissues and proliferate. Cancer cells behave the same way in culture, and under the right conditions, cells can be taken from a tumor and divide indefinitely in culture. Another type of immortalized cell is the embryonic stem cell. Embryonic stem cells are derived from an early stage embryo, and these cells are completely undifferentiated and pluripotent, which means that under the right conditions, they can become any mammalian cell type. Mouse embryonic stem cells have become a valuable research tool, and it is this cell type that we will be using for our practice cell culture today. | One type of familiar immortalized cell is the cancer cell. Tumor cells continuously divide allowing cancer to invade tissues and proliferate. Cancer cells behave the same way in culture, and under the right conditions, cells can be taken from a tumor and divide indefinitely in culture. Another type of immortalized cell is the embryonic stem cell. Embryonic stem cells are derived from an early stage embryo, and these cells are completely undifferentiated and pluripotent, which means that under the right conditions, they can become any mammalian cell type. Mouse embryonic stem cells have become a valuable research tool, and it is this cell type that we will be using for our practice cell culture today. | ||
+ | [[Image:Be109normalmousefibroblasts.jpg|thumb|250px|left|'''Normal Mouse Fibroblasts; Photographs courtesy of G. Steven Martin''']] | ||
+ | [[Image:Be109transformedmousefibroblasts.jpg|thumb|250px|center|'''Transformed Mouse Fibroblasts; Photographs courtesy of G. Steven Martin''']] | ||
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The art of tissue culture lies in the ability to create conditions that are similar to what a cell would experience in an animal, namely 37°C and neutral pH. Blood nourishes the cells in an animal, and blood components are used to feed cells in culture. Serum, the cell-free (and clotting-factor free) component of blood, contains many of the factors necessary to support the growth of cells outside the animal. Consequently, serum is frequently added to tissue culture medium, although serum-free media exist and support some types of cultured cells. | The art of tissue culture lies in the ability to create conditions that are similar to what a cell would experience in an animal, namely 37°C and neutral pH. Blood nourishes the cells in an animal, and blood components are used to feed cells in culture. Serum, the cell-free (and clotting-factor free) component of blood, contains many of the factors necessary to support the growth of cells outside the animal. Consequently, serum is frequently added to tissue culture medium, although serum-free media exist and support some types of cultured cells. | ||
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====Protocol==== | ====Protocol==== | ||
− | Each of you will have a 35 mm dish of mouse embryonic stem (MES) cells that you will use to seed a six-well dish. You and your partner will seed the dishes at different concentrations so you should decide who will seed at 1:10 and who will seed at 1:2. | + | Each of you will have a 35 mm dish of mouse embryonic stem (MES) cells that you will use to seed a six-well dish. You and your partner will seed the dishes at different concentrations so you should decide who will seed at 1:10 and who will seed at 1:2. As you work, try to put into practice what you learned about sterile technique in the demo last week. A member of the teaching faculty will be present to guide you. |
#Each tissue culture hood is partly set up for you. Finish preparing your hood according to the demo, first bringing in any remaining equipment you will need, then picking up the pre-warmed reagents from the water bath. Don't forget to spray everything down with 70% ethanol. | #Each tissue culture hood is partly set up for you. Finish preparing your hood according to the demo, first bringing in any remaining equipment you will need, then picking up the pre-warmed reagents from the water bath. Don't forget to spray everything down with 70% ethanol. | ||
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#To dislodge the cells from the dish, you will add trypsin, a proteolytic enzyme. Using a 2 ml pipet, add 0.7 ml of trypsin to the flask. Be careful not to pull up liquid too quickly or it will go all the way up your pipet into the pipet-aid! | #To dislodge the cells from the dish, you will add trypsin, a proteolytic enzyme. Using a 2 ml pipet, add 0.7 ml of trypsin to the flask. Be careful not to pull up liquid too quickly or it will go all the way up your pipet into the pipet-aid! | ||
#Tip the flask in each direction to distribute the liquid evenly. Incubate the cells at 37°C for 3-5 minutes, until the cells round up and are easily dislodged from the plate by tapping. | #Tip the flask in each direction to distribute the liquid evenly. Incubate the cells at 37°C for 3-5 minutes, until the cells round up and are easily dislodged from the plate by tapping. | ||
− | #While you are waiting, you can | + | #While you are waiting, you can add 1 ml of sterile 0.1% gelatin to the two leftmost wells of a six-well dish (one dish per pair) and also label the dish. |
− | #*The | + | #*The gelatin will be removed before you seed your MES cells but it is important to pre-treat the dish this way. MES must grow on either a "feeder layer" of fibroblasts, or on a gelatin-coated surface. '''The pre-treatment must be done for at least 10 minutes.''' |
− | #After retrieving your cells, add 1.3 ml of media to the trypsinized MES cells and pipet the liquid up and down ( | + | #*Label the plate lid with your group colour, today s date, and the cell line (called [http://www.atcc.org/ATCCAdvancedCatalogSearch/ProductDetails/tabid/452/Default.aspx?ATCCNum=SCRC-1010&Template=cellBiology J1 ]). Label the well you used with your initials and the cell dilution you did, and make sure your partner does the same. |
− | #According to the procedures below, either begin counting your cells or begin plating them (no matter what count you get, you will plate a 1:2 and a 1:10 dilution), depending on microscope availability. | + | #After retrieving your cells, add 1.3 ml of media to the trypsinized MES cells and pipet the liquid up and down ( |