1.3: Use of Trypsin for splitting a culture of NIH3T3 cells
As you proceed through this set of protocols and experiments, be sure to enter all your work into your
notebook!
Objectives
Improve dexterity with sterile pipetting in a laminar flow hood
Become familiar with feeding and splitting procedures for adherent cell cultures
Learn and adhere to safety considerations
Vocabulary
trypsinization, PBS
Outline
Aspirate old medium; wash cell monolayer with PBS; add trypsin to cells; uniformly disperse cells by
repeated pipetting; inactivate trypsin by addition of serum-containing medium; distribute cells to
multiple plates containing fresh medium.
Materials
sterile
trypsin solution
phosphate buffered saline (PBS), at room temp
3T3 growth medium mix (from previous session), prewarmed to 37C
two to four 10 cm plates/team of 4
serological pipettes
plastic pipette tips
non-sterile
37C water bath, maintained with antimicrobial additive
rack in water bath, as necessary
fully-stocked biosafety cabinet
kimwipes for swabbing
70% ethanol
DAY 1 (Friday)
Protocol
PREPARATION
1. Prepare the microscope and assess confluency
a. Remove the protective plastic cover from the microscope
b. Sterilize the microscope stage area by spraying 70% ethanol on a kimwipe and wiping
the stage area and knobs.
i. Do not spray the microscope as this may soil the objective lenses and filter sets
c. Turn on the inverted microscope light source
d. Remove a cell culture plate from the incubator and place it on the microscope stage
e. Use the microscope to assess the confluency of the cells.
i. 100% confluency means that all surface area is occupied by cells
ii. 50% confluency means half of the surface area is occupied by cells, and so on.
f. Depending on the cell type and application, determine whether the cells need to be fed
or split.
i. Allowing cultures to reach high confluence (80-100%) might permanently alter
some cell behaviors and, therefore, is to be avoided. Always seek specific
guidance when working with a new cell line/type.
2.
3.
4.
5.
ii. For NIH3T3 cells, at 70% confluency, they should be split to new plates.
g. Return the plate of cells to the incubator while preparing the hood.
h. If done with the microscope, turn off the light and replace the protective covering.
Prepare the hood
a. Turn off UV, raise the sash and ensure airflow is on at an acceptable flow rate.
b. Clutter within the hood should be kept to a minimum and any materials should be
positioned within the hood to minimize disruption of the laminar airflow.
i. Never block the front or rear vents.
c. Swab down the work surfaces with 70% ethanol and wipe clean.
Collect all materials
a. Place only the required sterile items within the hood necessary for the task at hand.
b. All materials entering the hood should be sprayed and/or wiped down with 70% ethanol
to sterilize their outer surfaces.
i. Remember, your hands are on the list of items that must be sterilized every time
they exit the hood before reentry.
c. For today, items to be sterilized include:
i. Trypsin bottle
ii. PBS bottle
iii. 3T3 growth medium bottle, prewarmed
iv. Serological pipettes and pipette aid
v. Aspirator and corresponding pipettes
vi. Plastic pipette tip boxes and pipettors (as necessary based on your calculations)
vii. 10 cm plates
Label the appropriate number of new culture plates.
a. Remember to label in small printing near the edge of the cover. The label should include
the name of the cell line (NIH3T3), the date on which the cells are thawed, today’s date,
your initials, and your group number.
i. Example: NIH3T3, 9/29/10-10/1/10, RMN, Group 42
ii. The number of plates you split into depends on the starting confluency,
doubling time for the cell line, and number of days before the next split.
Loosen the screw caps for the various (thawed) components
WASHING ADHERENT CELLS WITH PBS
6. Retrieve the cell culture plates from the incubator
7. Aspirate the old medium from the plates
a. Aspirate medium at one edge of the plate, moving the tip from the surface of the liquid
down the inner wall of the plate until almost all liquid is removed.
i. Tipping the plate slightly will facilitate aspiration of the liquid medium.
ii. Do not touch the aspirator tip to the floor of your plates. This is where your cells
are and doing so can pull them off the plate and into the aspirator.
iii. Do not allow the cells on your plates to sit without any aqueous solution on
them for more than a couple minutes.
8. Add 5ml of PBS to each aspirated cell culture plate
a. Take special care to not touch the pipette tip to any unintended surfaces. Likewise, do
not touch insides of caps, edges or rims of containers with your hands.
b. If dexterity allows, do not set vessel lids down on any surface at any time. Hold the cap
until the desired volume has been removed and then immediately replace the cap on
the vessel.
c. Hold the plate lid by the edges above the plate just enough for the pipette to dispense
media into the plate.
d. Be sure to dispense the solution from the pipette gently so as not to create any splash
or dislodge the cells.
e. Disperse the medium evenly by gently rocking the plate(s).
f. Take special care not to spill the liquid or to get the lids of the plates wet.
g. 20 seconds of gentle washing is sufficient
TRYPSINIZATION OF ADHERENT CELLS
9. Aspirate the PBS from the plates
10. Add 1ml of prewarmed Trypsin solution to each 10cm plate. Scale the volume downward
accordingly for plates with smaller amounts of surface area.
a. With the lid replaced on the dish, gently rock the plate(s) to ensure complete coating of
all cells on the plate with the trypsin solution.
b. The length of time needed to release cells from the plate varies for different types of
cells. For NIH3T3 cells, 2 to 3 minutes should be sufficient. Check by periodically tilting
the plate and looking for the cell monolayer to “slip” from the plate surface. If you are
unsure, you can check on the microscope. The cells will become spherical as they
detach from the plate and ECM.
c. Gently tapping the upright plate against an object, such as the side of the tissue culture
hood wall or the floor of the hood, can facilitate the “slipping” of the cell monolayer
from the plate surface.
11. While waiting for the trypsinization to occur, pipette 10 ml prewarmed 3T3 growth medium into
the desired number of fresh 10 cm plates.
12. Once the cell monolayer is mostly released from the plate surface, add 4mL of growth medium
to the plate.
a. The serum in the growth medium contains protease inhibitors that will block further
activity of the trypsin enzyme.
b. If splitting more than one plate of cells, the cells and medium from one plate can be
combined with that from additional plates.
13. Once all the desired cells are collected, suck them up into the pipette, place the pipette opening
firmly against the inside growth surface of the old plate, and completely dispense the liquid back
into it to mechanically disperse the cells. Repeat this process 3-4 times.
REPLATING TRYPSINIZED CELLS
14. Pipette the desired volume of cells into each fresh plate containing prewarmed 3T3 growth
medium to seed the cells at the desired initial confluency.
a. It is common to not replate all cells if there are more cells than needed for a particular
downstream application.
b. If not all the cells are to be replated, once the desired amount are replated, dispose of
the remainder by aspiration.
15. Gently rock the fresh plates to evenly distribute the cells.
a. The inverted microscope can be used to assess the effectiveness of the redistribution of
the cells. A common problem is for cells to clump into the center or outer edges of the
plate if you “swirl” the plates.
16. Place the fresh plate(s) in the 37C incubator.
DAY 2 (Monday)
Protocol
1. Confirm attachment. Observe your cells under the microscope and estimate the % confluence.
2. Refeed or split if the cells are likely to reach >70% confluence before the next lab session.
Questions
1. Starting with a 10cm plate of cells at 80% confluency on a friday, you split the cells from that
plate to four fresh 10cm plates.
a. What is the predicted initial confluency of the cells on the fresh plates?
b. Assuming a doubling time of 24 hours, when will the cells again require splitting?
c. Do you look forward to tending your cells on Sundays?
2. Starting with a 10cm plate of cells at 75% confluency, propose a splitting scheme that will yield
plates approaching 70% (+/- 5%) confluency between weekdays and a separate splitting plan for
across the weekend.
3. Why might spilling media out of a plate increase the chance of a contaminating microorganism
entering the plate?