Laboratory Session Plan

Lab Session Activities Wet lab & CAL Week 1 1-3

Lab Session Location Lab N23 & Lab N34 CAL M2 & DEN PC Rm

Lab Session Duration 3 hrs Time

Courses: Module: Topic: Session: Team- Time Wet Lab N23 Wet Lab N34

Medical Genetics Structure function Cells 1 Structure 12:30-14:20 A1-A3 A4-A6

cultivation
Lab Rotations 15.00-17:30 B1-B3 B4-B6
& Gene
Transfer CAL- CAL-

12:30-14:20 B1-B3 B4-B6

15.00-17.30 D1-D3 D4-D6

Professor(s) Stephanou Program(s) Structure & Function: Body Systems in Health I

I. Learning Objectives

1. Be able to identify different types of cells grown in culture and provide examples of specific stable cell lines commonly used in culture.
2. Be able to explain the cell culture work environment and identify the significance of specific cell culture equipment (laminar flow hoods, CO2 incubators, Microscopes,
Preservation equipment, storage vessels).
3. Be able to explain how and where cells are appropriately preserved and stored.
4. Recognize important safety considerations when conducting cell culture experiments and be sure to observe all safety precautions.
5. Describe aseptic techniques for working with mammalian cell cultures and the materials needed to practice and ensure aseptic technique
6. Be able to use an inverted microscope and analyse and visualize different cell types and growth rates (cancer cells vs normal cells).
7. To be able to know how we can manipulate cells by gene transfer using the DNA transfection protocol (Green fluorescent protein cDNA plasmid)

II. Laboratory Session Description

Activity / Station # 1
Activity Description Procedure for cell cultivation

Activity Duration (in 180mins

minutes)
Cell culture refers to the removal of cells from an animal or plant and their subsequent growth in a favorable artificial environment.
Instruction 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 already 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 subcultured (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 (i.e., they
are finite; see below), and as they are passaged, cells with the highest growth capacity predominate, resulting in a degree of
genotypic and phenotypic uniformity in the population. Cell Strain If a subpopulation of a cell line is positively selected from the
culture by cloning or some other method, this cell line becomes a cell strain. A cell strain often acquires additional genetic changes
subsequent to the initiation of the parent line

Basic Equipment:
Cell culture hood (i.e., laminar-flow hood or biosafety cabinet) , Incubator (humid CO2 incubator recommended), Water bath ,
Centrifuge , hemacytometer, Inverted microscope, freezer or cryostorage container, Sterilizer (i.e., autoclave)

Aseptic technique
Successful cell culture depends heavily on keeping the cells free from contamination by microorganisms such as bacterial, fungi, and
viruses. Non-sterile supplies, media, and reagents, airborne particles laden with microorganisms, unclean incubators, and dirty work
surfaces are all sources of biological contamination.
Aseptic technique, designed to provide a barrier between the microrganisms in the environment and the sterile cell culture, depends
upon a set of procedures to reduce the probability of contamination from these sources. The elements of aseptic technique are a
sterile work area, good personal hygiene, sterile reagents and media, and sterile handling.

• Always wipe your hands and your work area with 70% ethanol.
• Wipe the outside of the containers, flasks, plates, and dishes with 70% ethanol before placing them in the cell culture hood.
• Avoid pouring media and reagents directly from bottles or flasks.
• Use sterile glass or disposable plastic pipettes and a pipettor to work with liquids, and use each pipette only once to avoid
cross contamination. Do not unwrap sterile pipettes until they are to be used. Keep your pipettes at your work area.
• Always cap the bottles and flasks after use and seal multi-well plates with tape or place them in resealable bags to prevent
microorganisms and airborn contaminants from gaining entry.
• Never uncover a sterile flask, bottle, petri dish, etc. until the instant you are ready to use it and never leave it open to the
environment. Return the cover as soon as you are finished.
• If you remove a cap or cover, and have to put it down on the work surface, place the cap with opening facing down. •
Use only sterile glassware and other equipment.
• Be careful not to talk, sing, or whistle when you are performing sterile procedures.
• Perform your experiments as rapidly as possible to minimize contamination.

Procedure
1. Remove and discard the spent cell culture media from the culture vessel.
2. Wash cells using a balanced salt solution without calcium and magnesium (approximately 2 mL per 10 cm2 culture surface area).
Gently add wash solution to the side of the vessel opposite the attached cell layer to avoid disturbing the cell layer, and rock the
vessel back and forth several times.
Note: The wash step removes any traces of serum, calcium, and magnesium that would inhibit the action of the dissociation reagent.
3. Remove and discard the wash solution from the culture vessel
4. Add the pre-warmed dissociation reagent such as trypsin or TrypLE™ to the side of the flask; use enough reagent to cover the cell
layer (approximately 0.5 mL per 10 cm2). Gently rock the container to get complete coverage of the cell layer.
5. Incubate the culture vessel at room temperature for approximately 2 minutes. Note that the actual incubation time varies with the
cell line used.
6. Observe the cells under the microscope for detachment. If cells are less than 90% detached, increase the incubation time a few
more minutes, checking for dissociation every 30 seconds. You may also tap the vessel to expedite cell detachment.
 7. When ≥ 90% of the cells have detached, tilt the vessel for a minimal length of time to allow the cells to drain. Add the equivalent of
2 volumes (twice the volume used for the dissociation reagent) of pre-warmed complete growth medium. Disperse the medium by
pipetting over the cell layer surface several times.
8. Transfer the cells to a 15-mL conical tube and centrifuge then at 200 × g for 5 to 10 minutes. Note that the centrifuge speed and
time vary based on the cell type.
9. Resuspend the cell pellet in a minimal volume of pre-warmed complete growth medium and remove a sample for counting.
10. Determine the total number of cells and percent viability using a hemacytometer, cell counter and Trypan Blue exclusion, or the
Countess® Automated Cell Counter. If necessary, add growth media to the cells to achieve the desired cell concentration and recount
the cells. Note: We recommend using the Countess® Automated Cell Counter to determine the total number of cells and percent
viability. Using the same amount of sample that you currently use with the hemacytometer, the Countess® Automated Cell Counter
takes less than a minute per sample for a typical cell count and is compatible with a wide variety of eukaryotic cells. For more
information on using a traditional hemacytometer, see Support Protocols,.
11. Dilute cell suspension to the seeding density recommended for the cell line, and pipet the appropriate volume into new cell
culture vessels, and return the cells to the incubator.

Resources: Wet Lab

Bibliography https://www.thermofisher.com/cy/en/home/references/gibco-cell-culture-basics/introduction-to-cell-culture.html