cell & molecular biology

Experiment 5

Introduction:

Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favourable artificial environment. The cells may be removed from the tissue directly and disaggregated by enzymatic or mechanical means before cultivation, or they may be derived from a cell line or cell strain that has been already established.

Primary Culture: Primary culture refers to the stage of the culture after the cells are isolated from the tissue and proliferated under the appropriate conditions until they occupy all of the available substrate (i.e., reach confluence). At this stage, the cells have to be sub cultured (i.e., passaged) by transferring them to a new vessel with fresh growth medium to provide more room for continued growth.

Cell Line: After the first subculture, the primary culture becomes known as a cell line or subclone. Cell lines derived from primary cultures have a limited life span and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of genotypic and phenotypic uniformity in the population. A cell strain often acquires additional genetic changes subsequent to the initiation of the parent line.

Culture Conditions: Culture conditions vary widely for each cell type, but the artificial environment in which the cells are cultured invariably consists of a suitable vessel containing a substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones, and gases (O2, CO2), and regulates the physicochemical environment (pH, osmotic pressure, temperature). Most cells are anchorage-dependent and must be cultured while attached to a solid or semi-solid substrate (adherent or monolayer culture), while others can be grown floating in the culture medium (suspension culture).

Preservation Steps:

Cryopreservation: If a surplus of cells is available from sub culturing, they should be treated with the appropriate protective agent (e.g., DMSO or glycerol) and stored at temperatures below –130°C (cryopreservation) until they are needed.

Cell lines in continuous culture are prone to genetic drift, finite cell lines are fated for senescence, all cell cultures are susceptible to microbial contamination, and even the best-run laboratories can experience equipment failure. Because an established cell line is a valuable resource and its replacement is expensive and time consuming, it is vitally important that they are frozen down and preserved for long-term storage. As soon as a small surplus of cells becomes available from sub culturing, they should be frozen as a seed stock, protected, and not be made available for general laboratory use.

Working stocks can be prepared and replenished from frozen seed stocks. If the seed stocks become depleted, cryopreserved working stocks can then serve as a source for preparing a fresh seed stock with a minimum increase in generation number from the initial freezing. The best method for cryopreserving cultured cells is storing them in liquid nitrogen in complete medium in the presence of a cryoprotective agent such as dimethyl sulfoxide (DMSO).Cryoprotective agents reduce the freezing point of the medium and also allow a slower cooling rate, greatly reducing the risk of ice crystal formation, which can damage cells and cause cell death.

Note: DMSO is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials. Dispose of the reagents in compliance with local regulations

Materials Needed

1. Culture vessels containing cultured cells in log-phase of growth

2. Complete growth medium

3. Cryoprotective agent such as DMSO (use a bottle set aside for cell culture; open only in a laminar flow hood) or a freezing medium such as Synth-a-Freeze® Cryopreservation Medium or Recovery™ Cell Culture Freezing Medium

4. Disposable, sterile 15-mL or 50-mL conical tubes

5. Reagents and equipment to determine viable and total cell counts (e.g., Countess® Automated Cell Counter, or hemacytometer, cell counter and Trypan Blue)

6. Sterile cryogenic storage vials (i.e., cryovials)

7. Controlled rate freezing apparatus or isopropanol chamber

8. Liquid nitrogen storage container for freezing adherent cells, in addition to the above materials, you need:

a) Balanced salt solution such as Dulbecco’s Phosphate Buffered Saline (D-PBS), containing no calcium, magnesium, or phenol red

b) Dissociation reagent such as trypsin or TrypLE™ Express, without phenol red.

Revival Process:

The thawing procedure is stressful to frozen cells, and using good technique and working quickly ensures that a high proportion of the cells survive the procedure. As with other cell culture procedures, we recommend that you closely follow the instructions provided with your cells and other reagents for best results.

• Thaw frozen cells rapidly (< 1 minute) in a 37°C water bath.

• Dilute the thawed cells slowly, using pre-warmed growth medium.

• Plate thawed cells at high density to optimize recovery.

• Always use proper aseptic technique and work in a laminar flow hood.

• Always wear personal protective equipment, including a face mask or goggles. Cryovials stored in liquid-phase present a risk of explosion when thawed.

• Some freezing media contain DMSO, which is known to facilitate the entry of organic molecules into tissues. Handle reagents containing DMSO using equipment and practices appropriate for the hazards posed by such materials.

Materials Needed:

• Cryovial containing frozen cells

• Complete growth medium, pre-warmed to 37°C

• Disposable, sterile centrifuge tubes

• Water bath at 37°C

• 70% ethanol

• Tissue-culture treated flasks, plates, or dishes

Protocol for Thawing Frozen Cells:

The following protocol describes a general procedure for thawing cryopreserved cells. For detailed protocols, always refer to the cell-specific product insert.

1. Remove the cryovial containing the frozen cells from liquid nitrogen storage and immediately place it into a 37°C water bath.

2. Quickly thaw the cells (< 1 minute) by gently swirling the vial in the 37°C water bath until there is just a small bit of ice left in the vial.

3. Transfer the vial into a laminar flow hood. Before opening, wipe the outside of the vial with 70% ethanol.

4. Transfer the thawed cells dropwise into the centrifuge tube containing the desired amount of pre-warmed complete growth medium appropriate for your cell line.

5. Centrifuge the cell suspension at approximately 200 × g for 5–10 minutes. The actual centrifugation speed and duration varies depending on the cell type.

6. After the centrifugation, check the clarity of supernatant and visibility of a complete pellet. Aseptically decant the supernatant without disturbing the cell pellet.

7. Gently resuspend the cells in complete growth medium, and transfer them into the appropriate culture vessel and into the recommended culture environment. Note: The appropriate flask size depends on the number of cells frozen in the cryovial, and the culture environment varies based on the cell and media type.

Reference Material

Cell thawing: www.youtube.com/watch?v=9zPK-rg608Y

Cell culture:https://www.youtube.com/watch?v=jA9DKdx04Xg&feature=emb_title

Cell cryopreservation:https://www.youtube.com/watch?v=ee4F51IXuME&feature=emb_title

Revival of cells from frozen:https://www.youtube.com/watch?v=Azad5OQJp9c&feature=emb_logo

Questions

1. Give examples of synthetic media those can be used for animal cell culture.

2. What is HEPES? write about its function.

3. What is the function of Trypsin during primary cell culture process?

4. What is the full form of EMEM and DMEM?