Lab 1: Mammalian Cell Culture

Micropipetting

• P20

  • used for measuring 1-20 µl

• P200

  • used for measuring 20-200 µl

• P1000

  • used for measuring 200-1000 µl (1 mL)

Cell Culture Reagents

• Phenol Red

• DMSO

• Trypan Blue

• Trypsin

Phenol Red

• pH indicator dye that…

  • turns orange if pH decreases

  • turns purple if pH increases

DMSO

• cryopreservation agent which reduces the formation of ice crystals for long term storage

PBS

• washes adherent cells in a flask primarily to remove culture media, serum, and other debris

Trypan Blue

• determines cell viability:

  • blue: dead/dying cells

  • uncoloured: living cells

Trypsin

• enzyme used to detach adherent cells from culture dishes

Biosafety

Primary vs. Immortal Cell Lines

• primary cells are taken directly from a tissue, and thus have a finite lifespan

  • useful for some applications, as they resemble in vivo physiology

  • drawback is that they are hard to grow, and finite

• immortal cell lines divide indefinitely

  • this makes them useful for lab environments, as they are easy to grow

  • however, they are less representative of in vivo systems, and acquire increased levels of genetic modification over time

  • may be adherent (attach to a substrate, and eventually get to a point where they form a solid monolayer with minimal space between the cells, known as confluency) or grown in suspension

Brightfield vs. Phase Contrast

• Brightfield:

  • setting “O”

  • useful for viewing specimens that have their own natural colour, or those stained

• Phase contrast:

  • “Ph 1”

  • makes highly transparent objects more visible

  • ideal for thin, living speciments

Tissue Culture Equipment

• BSC: biological safety cabinet

  • hood

  • protects from infectious material/toxins and protects specimens from contamination

  • differs from fume hoods, which suck air out without filtering

• CO₂ incubator: provides constant 37^oC temperature and 5% CO₂

  • assists in the short term storage of growing cells

  • maintains pH and temperature

How to Determine Cell Count/Concentration

• use of hemocytometer

  • etched grid chamber which cells are pipetted into

• cells in the 4 corners are counted as follows:

cells per ml = (# cells counted/ # large squares containing counted cells) x (volume sample + volume diluent/ volume sample) x 10⁴

Step 1

• 24 hours prior to lab, CHO cells were seeded into flasks, thereby allowing sufficient time for the cells to adhere to the bottom surface and grow to desired confluency.

Step 2

• Draw up and dispose of the media in the flask, leaving only the CHO cells adhered to the bottom

Step 3

• Add PBS to the dish, swirl over cells, and draw up/dispose of PBS

Step 4

• Add trypsin to flask, and rotate cells back and forth to ensure contact between Trypsin and all cells. Wait 3 min, then observe under the inverted scope to ensure that the cells are floating, and no longer attached

Step 5

• Add media back to the flask

Step 6

• Split cells into 2 separate tubes, and microcentrifuge to pellet the cells

Step 7

• Remove the supernatant and dispose, but do not disturb the pellet!

Step 8

• Add either:

  1. Regular media

  2. Regular media + DMSO

Step 9

• Freeze for 40 minutes

Step 10

• Mix cells with trypan blue

Step 11

• Load mixture into the hemocytometer chamber with a glass coverslip on top

Step 12

• Place hemocytometer on microscope stage and find the grid, using Phase Contrast

Step 13

• Switch between Phase Contrast (which allows you to see the cells), and Brightfield (which allows you to see the potential blue or transparent colouring of the cells) to estimate the % living vs. dead cells in your sample