Tissue Culture Methods-3

REFERENCES:

R. Ian Freshney, Culture of Animal cells: A manual of basic techniques, Wiley-Liss, 1987.

VI. TISSUE CULTURE PROCEDURES

Each student should maintain his own cells throughout the course of the experiment. These cells should be monitored daily for morphology and growth characteristics, fed every 2 to 3 days, and subcultured when necessary. A minimum of two 25 cm² flasks should be carried for each cell line; these cells should be expanded as necessary for the transfection experiments. Each time the cells are subcultured, a viable cell count should be done, the subculture dilutions should be noted, and, after several passages, a doubling time determined. As soon as you have enough cells, several vials should be frozen away and stored in liquid N₂. One vial from each freeze down should be thawed 1-2 weeks after freezing to check for viability. These frozen stocks will prove to be vital if any of your cultures become contaminated.

Procedures:1. Media preparation. Each student will be responsible for maintaining his own stock of cell culture media; the particular type of media, the sera type and concentration, and other supplements will depend on the cell line. Do not share mediawith you partner (or anyone else) because if a culture or a bottle of media getscontaminated, you have no back-up. Most of the media components will be purchased prepared and sterile. In general, all you need to do is sterily combine several sterilesolutions. To test for sterility after adding all components, pipet several mls from each media bottle into a small sterile petri dish or culture tube and incubate at 37EC for several days. Use only media that has been sterility tested. For this reason, you must anticipate your culture needs in advance so you can prepare the reagents necessary. But, please try not to waste media. Anticipate your needs but don't make more than you need. Tissue culture reagents are very expensive; for example, bovine fetal calf serum cost ~ $200/500 ml. Some cell culture additives will be provided in a powdered form. These should be reconstituted to the appropriate concentration with double-distilled water (or medium, as appropriate) and filtered (in a sterile hood) through a 0-22 μm filter.

All media preparation and other cell culture work must be performed in a laminar flow hood. Before beginning your work, turn on blower for several minutes, wipe down all surfaces with 70% ethanol, and ethanol wash your clean hands. Use only sterile pipets, disposable test tubes and autoclaved pipet tips for cell culture. All culture vessels, test tubes, pipet tip boxes, stocks of sterile eppendorfs, etc. should be opened only in the laminar flow hood. If something is opened elsewhere in the lab by accident, you can probably assume its contaminated. If something does become contaminated, immediately discard the contaminated materials into the biohazard container and notify the instructor.

2. Growth and morphology. Visually inspect cells frequently. Cell culture is sometimes more an art than a science. Get to know what makes your cells happy. Frequent feeding is important for maintaining the pH balance of the medium and for eliminating waste products. Cells do not typically like to be too confluent so they should be subcultured when they are in a semi-confluent state. In general, mammalian cells should be handled gently. They should not be vortexed, vigorously pipetted or centrifuged at greater than 1500 g.

3. Cell feeding. Use prewarmed media and have cells out of the incubator for as little time as possible. Use 10-15 ml for T-25's, 25-35 ml for T-75's and 50-60 ml for T-150's. a. Suspension cultures. Feeding and subculturing suspension cultures are done simultaneously. About every 2-3 days, dilute the cells into fresh media. The dilution you use will depend on the density of the cells and how quickly they divide, which only you can determine. Typically 1:4 to 1:20 dilutions are appropriate for most cell lines. b. Adherent cells. About every 2-3 days, pour off old media from culture flasks and replace with fresh media. Subculture cells as described below before confluency is reached.

4. Subculturing adherent cells. When adherent cells become semi-confluent, subculture using 2 mM EDTA or trypsin/EDTA.

Trypsin-EDTA :

	a. Remove medium from culture dish and wash cells in a balanced salt solution without Ca++ or Mg++. Remove the wash solution.
	b. Add enough trypsin-EDTA solution to cover the bottom of the culture vessel and then pour off the excess.
	c. Place culture in the 37°C incubator for 2 minutes.
	d. Monitor cells under microscope. Cells are beginning to detach when they appear rounded.
	e. As soon as cells are in suspension, immediately add culture medium containing serum. Wash cells once with serum containing medium and dilute as appropriate (generally 4-20 fold).

EDTA alone:

	a. Prepare a 2 mM EDTA solution in a balanced salt solution (i.e., PBS without Ca++or Mg++).
	b. Remove medium from culture vessel by aspiration and wash the monolayer to remove all traces of serum. Remove salt solution by aspiration.
	c. Dispense enough EDTA solution into culture vessels to completely cover the monolayer of cells.
	d. The coated cells are allowed to incubate until cells detach from the surface. Progress can be checked by examination with an inverted microscope. Cells can be gently nudged by banging the side of the flask against the palm of the hand.
	e. Dilute cells with fresh medium and transfer to a sterile centrifuge tube.
	f. Spin cells down, remove supernatant, and resuspend in culture medium (or freezing medium if cells are to be frozen). Dilute as appropriate into culture flasks.

5. Thawing frozen cells.

	a. Remove cells from frozen storage and quickly thaw in a 37°C waterbath by gently agitating vial.
	b. As soon as the ice crystals melt, pipet gently into a culture flask containing prewarmed growth medium.
	c. Log out cells in the "Liquid Nitrogen Freezer Log" Book.

6. Freezing cells.

	a. Harvest cells as usual and wash once with complete medium.
	b. Resuspend cells in complete medium and determine cell count/viability.
	c. Centrifuge and resuspend in ice-cold freezing medium: 90% calf serum/10% DMSO, at 106 - 107 cells/ml. Keep cells on ice.
	d. Transfer 1 ml aliquots to freezer vials on ice.
	e. Place in a Mr. Frosty container that is at room temperature and that has sufficient isopropanol.
	f. Place the Mr. Frosty in the -70°C freezer overnight. Note: Cells should be exposed to freezing medium for as little time as possible prior to freezing
	g Next day, transfer to liquid nitrogen (DON'T FORGET) and log in the "Liquid Nitrogen Freezer Log" Book.

7. Viable cell counts. USING A HEMOCYTOMETER TO DETERMINE TOTAL CELL COUNTS AND VIABLE CELL NUMBERS (Reference: Sigma catalogue)Trypan blue is one of several stains recommended for use in dye exclusion procedures for viable cell counting. This method is based on the principle that live cells do not take up certain dyes, whereas dead cells do.

1. Prepare a cell suspension, either directly from a cell culture or from a concentrated or diluted suspension (depending on the cell density) and combine 20 μl of cells with 20 μl of trypan blue suspension (0.4%). Mix thoroughly and allow to stand for 5-15 minutes.

2. With the cover slip in place, transfer a small amount of trypan blue-cell suspension to both chambers of the hemocytometer by carefully touching the edge of the cover slip with the pipette tip and allowing each chamber to fill by capillary action. Do not overfill or underfill the chambers.3. Starting with 1 chamber of the hemocytometer, count all the cells in the 1 mm center square and four 1 mm corner square.  Keep a separate count of viable and non-viable cells.4. If there are too many or too few cells to count, repeat the procedure either concentrating or diluting the original suspension as appropriate.5. The circle indicates the approximate area covered at 100X microscope magnification (10X ocular and 10X objective). Include cells on top and left touching middle line. Do not count cells touching middle line at bottom and right. Count 4 corner squares and middle square in both chambers and calculate the average.6. Each large square of the hemocytometer, with cover-slip in place, represents a total volume of 0.1 mm³ or 10^-4 cm³. Since 1 cm³ is equivalent to approximately 1 ml, the total number of cells per ml will be determined using the following calculations:Cells/ml = average cell count per square x dilution factor x 10⁴;

Total cells = cells/ml x the original volume of fluid from which the cell sample was removed; % Cell viability = total viable cells (unstained)/total cells x 100.