Maintenance and Frozen stock preparation of HEK-293 Cell lines.

HEK 293 Cell Line
Protocol-at-a-Glance
(PT3494-2)
CLONTECH’s HEK 293 Cell Line is offered for use with the Adeno-XTM Expression Systems. The
general maintenance of HEK 293 cells is described below. Instructions on how to use HEK 293 Cells
to produce infectious recombinant adenovirus (1) are given in all Adeno-XTM Expression System
User Manuals.
Note: Upon receipt, frozen cells should be promptly stored in liquid nitrogen or cultured immediately.
Starting HEK 293 Cell Cultures From Frozen Stocks
Growth Medium: DMEM (or Minimum Essential Medium, a Modification [a-MEM]) supplemented
with 100 units/ml penicillin G sodium, 100 mg/ml streptomycin, 4 mM L-glutamine, and 10% fetal
bovine serum. (Tet System Approved Fetal Bovine Serum is available from CLONTECH, #8630-1.)
1. Thaw 293 cells rapidly by briefly immersing the vial in a 37°C water bath (e.g., 2–3 min with
constant agitation). Upon thawing, immediately wipe the outside of the vial with 70% EtOH,
then transfer the contents of the vial to a 10-cm culture plate or a T25 flask.
2. Add an additional 4 ml of medium to the flask/plate. Gently rock or swirl the flask/plate to
distribute cells evenly over the growth surface. Place the culture in a 37°C, 5% CO2,
humidified incubator.
3. The next day, examine the cells under a microscope. Healthy cells display a flat
morphology and adhere well to the plate. Aspirate the medium and replace with fresh,
prewarmed growth medium.
4. Expand the culture as needed. Cell cultures should be split every 2–4 days, when they
reach 70–80% confluency. As a general rule, HEK 293 cells should not be allowed to
become confluent nor should they be seeded too sparsely. (We suggest plating cells at a
density of 100 cells/mm2.)
Split the cells as follows. Remove the medium and wash the cells once with prewarmed
sterile PBS (containing no Ca2+ or Mg2+). Add 1–2 ml of trypsin-EDTA solution and treat for
1–2 min, or longer, until cells detach. To stop trypsinization, add 5–10 ml of growth medium,
then resuspend the cells gently but thoroughly. Count cells (2), then transfer the desired
number of viable cells to a new culture flask or plate containing an appropriate volume of
growth medium. Gently rock or swirl the plate or flask to evenly distribute the cells.
Preparing Frozen Cultures of HEK 293 Cells
Once you have established HEK 293 cells in culture, we recommend that you prepare frozen stock
from an early passage to ensure a renewable source of cells.
Freezing Medium: Can be purchased from Sigma or prepared (0–20%, a-MEM, 70–90% FBS, and
10% DMSO).
1. Trypsinize cells from the desired number of flasks. (See Step 4 above.)
2. Pool cell suspensions together, count cells, and calculate total viable cell number (2).
3. Centrifuge cells at 125 x g for 10 min. Aspirate the supernatant.
4. Resuspend the pellet at a density of at least 1–2 x 106 cells/ml in freezing medium.
5. Dispense 1-ml aliquots into sterile cryovials.
6. Freeze slowly (1°C per min). If a specialized freezer is not available, you may use
Nalgene’s cryo-containers (#5100) for this purpose (freeze at –70°C overnight). Alternatively,
place vials in a thick-walled styrofoam container at –20°C for 1–2 hr. Transfer to
–70°C overnight. Remove vials from styrofoam container or cryo-containers the following
day and place in liquid nitrogen for storage.
7. Two or more weeks later, confirm the viability of the frozen stocks by starting a fresh culture
from frozen cells as described above.