3. Feeding -
2-3 times/week.
4.
Measurement of growth and viability. The viability of cells can be observed
visually using an inverted phase contrast microscope. Live cells are phase
bright; suspension cells are typically rounded and somewhat symmetrical;
adherent cells will form projections when they attach to the growth surface.
Viability can also be assessed using the vital dye, trypan blue, which is
excluded by live cells but accumulates in dead cells. Cell numbers are
determined using a hemocytometer.
V. SAFETY
CONSIDERATIONS
- Assume all cultures are hazardous since they may harbor latent viruses
or other organisms that are uncharacterized. The following safety
precautions should also be observed:
- pipetting: use pipette aids to prevent ingestion and keep aerosols down
to a minimum
- no eating, drinking, or smoking
- wash hands after handling cultures and before leaving the lab
- decontaminate work surfaces with disinfectant (before and after)
- autoclave all waste
- use biological safety cabinet (laminar flow hood) when working with
hazardous organisms. The cabinet protects worker by preventing airborne
cells and viruses released during experimental activity from escaping the
cabinet; there is an air barrier at the front opening and exhaust air is
filtered with a HEPA filter make sure cabinet is not overloaded and leave
exhaust grills in the front and the back clear (helps to maintain a uniform
airflow)
- use aseptic technique
- dispose of all liquid waste after each experiment and treat with bleach
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 media with you
partner (or anyone else) because if a
culture or a bottle of media gets
contaminated, 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 sterile
solutions. 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.
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