IGEM:University of Debrecen:Cell Passaging

Scientific Background
Medium reconstitution: You have to reconstitute a new bottle of medium when you don't have enough medium for the subsequent processes (cell culturing, Transfection etc.)

Cell passaging or splitting is a technique that enables an individual to keep cells alive and growing under cultured conditions for extended periods of time. Cells should be passed when they are 90%-100% confluent. You have to do the cell passage on every second to fourth day (i.e. on every Monday, Wednesday and Friday).

After reaching the confluency, the cells do not get enough nutrients and do not have enough place where they can extend. The colour of the medium switches from reddish-pink to orange or yellow which shows acidic metabolic products.

Materials
For 293T-cells, in 10cm Petri dishes:

I. Medium reconstitution:

500 ml of DMEM (Dulbecco's modified Eagle Medium)

The basic solution has to be completed with the following substances:

50 ml FBS (Foetal Bovine Serum)

10 ml L-Glutamine solution

5 ml Penicillin-Streptomicin solution

pipettor, pipette tips for the pipettor

II. Cell passaging

Ethanol squirt bottle

kimwipes

5 mL and 10 mL sterile pipets

confluent cells in Petri dishes

Media (DMEM) with 10% (50ml) serum and antibiotics

Trypsin-EDTA

1% PBS (Phosphate Buffered Saline)

Pasteur Pipettes+ vacuum for aspirating the used medium

15 mL centrifuge tube

Petri dishes

Automatic pipettors

gloves

37°C water-bath

37° thermostat

laminar box

tube holders

Procedure
I. Medium reconstitution:

NOTE: every material which goes into the box have to be sprayed down with 70% alcohol! Don't forget it, we must keep the sterility.

Storage: Medium in the cold roomat 4 degrees;

PS, L-Glutamine are stored in the freezer, at -20 degrees

FBS is in room temperature

1. Prepare solutions: put them into the 37 degrees termostator-waterbath (FBS needs at least half an hour for thawing)

2. Open the sterile laminar box (Hood), turn on the ventillator and wait for 15 minutes.

3. Put on gloves, wipe the cabinet with 70% alcohol, take the solutions and squirt the tubes with alcohole before you put them in the sterile box.

4. Put the pipettor and the tube holder into the box (after you sprayed them down), and do the same with the pipette tips(don't use alcohol because of the wrapping paper).

5. Put the PS, Glutamine, and FBS into the basic Medium solution using the pipettor and the pipette tips.

6. Screw down the cap of the Media vigorously, invert the bottle gently

7. write your name, date,and the components on the bottle

The Completed Media can be used immediately

Storing: the complete medium can be stored in the fridge (+4 Celsius)

Preparing: bring it to physiological temperture (for 10-15 minutes)

In the box everything is sterile, so don't open the flasks, Petri dishes, bottles, tubes, pipette tip boxes outside the box

To sterilize glass materials, you can use Bunsen

Whenever you pulling out your hands from the box, you have to use 70% alcohol before you reach in again

II. Cell Passaging for adherent culture (also called Splitting, subculturing):

1. Warm media, trypsin-EDTA and PBS in 37°C waterbath

2. Check cells in 10 cm Petri dish under Phase-contrast microscope to confirm that the cells are 90%-100% confluent

3. Clean hood with 70% alcohol

4. Sterilize all materials, bottles, etc. which are loaded into the hood. Spray hands with ethanol. Sterile pipets may be placed in the hood directly

5. Spray hands with ethanol. Remove Petri dishes from the incubator and quickly place them in the hood. (Do not spray flasks with ethanol)

6. Attach a Pasteur pipette to vacuum. Turn on vacuum system by opening vacuum valve in hood

7. Using the empty liquid media covering cells. Be careful to not touch the pipet to anything outside of the Petri dish

8. Add 2-3 mL of PBS to Petri dish. Lightly swirl PBS on base of Petri dish. Aspirate PBS from dishes

9. Add 2 mL trypsin-EDTA to Petri dish. Lightly swish trypsin

10. Place flask in incubator until detached (2-3 mins, epends on the cell-type)

11. Remove cells from incubator. Tap side of Petri dish on hard surface or your hand. Repeat several times. Visually check to ensure lumps of cells are dispersed

12. Check cells under microscope to confirm that cells are detached from the surface

13. Add 5 mL of media to dilute trypsin. Media contains antitrypsin. (Note: The liquid suspension now contains the cells.) Carefully re-suspend cells by using pipettor and pipettes

14. Aliquot appropriate volume of cell suspension into freshly prepared Petri dishes with media (The total volume in a Petri dish is 10 mL; 2mL Trypsin-EDTA, 5 ml DMEM for trypsin dilution, 3 further mL of DMEM)

15. Replace media and cells to mix. Place Petri dishes in incubator

16. Turn off aspiration

17. Dispose of liquid and solid biohazards wastes properly

18.Clean hood with ethanol. Spray ethanol liberally over surfaces and wipe clean with kimwipe

Notes & troubleshooting
NOTES:

DMEM (Dulbecco's modified Eagle Medium) it contains basic salt solution, glucose, essential amino acids, vitamins, buffer system, phenol red (for the indication of pH changes)

(Foetal Bovine Serum): serum provides a wide variety of macromolecular proteins, low molecular weight nutrients, carrier proteins for water – insoluble components, and other compounds necessary for in vitro growth of cells, such as hormones and attachment factors. Serum also adds buffering capacity to the medium and binds or neutralizes toxic components.

L-glutamine media: Most commercially available media are formulated with free L-glutamine which is added to liquid formulations at time of use. L-glutamine is unstable at physiological pH in liquid media. It breaks down to ammonium and pyroglutamate at rates that make it a problem in many biomanufacturing applications.

Penicillin-Streptomycin solution to prevent the media from bacterial contaminatio

Trypsin/EDTA is a combined method for detaching cells. Trypsin cuts the adhesion proteins in cell-cell and cell-matrix interactions (i don't remember the specific site), and EDTA is a calcium chelator, which integrins needs to interact with other proteins for cell adhesion-- no calcium, no cell adhesion.Also that it can decrease the clumbing of cells.

We use PBS to wash out the remained media from the cells.The buffer helps to maintain a constant pH. The osmolarity and ion concentrations of the solution usually match those of the human body (isotonic).

You should not take off the cap of the dish, only in the box. The optimal Temp. for trypsin is 37°C, it's better to bring the Trypsin- containing tube into the box right before you use it.