Lab No.

8: Mammalian Cell Culture & Transfection

Tasks to complete: Pre-lab discussion-strategic flowchart
Subculture and splitting for transfection
Cell counting with Hemocytometer
Lipid mediated transfection
Mammalian Cell lysis

Educational objectives:

By the completion of this lab you should be able to:

a) Explain the steps involved in the culturing of mammalian cells; including using sterile
techniques, and subculturing cells.
b) Explain the advantages and limitations that using mammalian cell culture can present to
researchers,
c) Carry out transient DNA transfection of mammalian cells in culture.
d) Lyse mammalian cells to prepare whole cellular lysate.

Background:

Tissue culture was initially developed at the beginning of the 1900’s as a method for
examining the behavior of animal cells outside their host. This allows researchers to test a specific
hypothesis by treating cell lines under different conditions without the systemic variation that occur
during animal testing.

There are two major advantages for utilizing mammalian cell culture. The first is the control
of the physiochemical environment of the cells which includes pH, temperature, osmotic pressure,
02 , C02, and surface tension. The second is the ability to modulate the intra and extracellular
physiological conditions of cells. The majority of cell lines require that the media be supplemented
with a combination of serum or other compounds. One must be aware that these “supplements”
can vary by batch and contain undefined elements such as hormones and other regulatory
substances, all factors which can influence cellular activities.
 Mammalian cell culture techniques must be carried out using strict aseptic techniques
because animal cells grow much more slowly compared to bacteria, mold, and yeast and are
therefore susceptible to infection. Furthermore, mammalian cell culture requires a large amount of
skill and time to grow only a few milligrams of cells that can be used in an experiment. Another
property to be aware of is alteration of cellular properties over time. Every time the mammalian cell
culture flask is confluent, i.e. the cells have no more room to grow, the mammalian cell culture
needs to be diluted or “split”. This process involves subjecting the surface of the cells to enzymatic
digestion, followed by aliquoting a fraction of the cells into a new flask containing fresh media. This
process is referred to as a passage. With each passage, artificial selection causes small alterations in
the phenotype of the cells of interest. After numerous passages, the cells which you are using in your
research may not resemble the cells which were used at the beginning of the project. Therefore,
researchers must be extremely diligent in their record keeping and characterization of cell types to
ensure that the cells remain true to their primordial line.

As you can imagine, there are numerous types of cultures that are available, given the
numerous organisms and tissue types available to utilize as a research model. However, they can be
grouped into three separate types: (1) organ culture – derived from a specific organ in which the
characteristics of the tissue in vivo are retained (e.g. embryonic organs and adult tissue fragments); (2)
primary explant culture – a fragment of tissue is placed between a glass and liquid interface where it
attaches; and (3) mammalian cell culture – is an outgrowth of the primary explant culture and is
dispersed into a cell suspension (either as a monolayer or free floating in the media suspension). In
our lab today we will be utilizing a characterized human mammalian cell culture line that is derived
from a primary explant of embryonic kidney epithelial tissue (Hek 293).

Contamination by microorganisms is a large problem in tissue culture. This can include, but
not limited to, bacteria, mycoplasma, yeast, and fungal spores all of which are typically introduced by
the researcher. Proper aseptic techniques will greatly reduce these contaminants. Therefore, to
minimize contamination in your culture and other groups cells it is important to carryout the
following: (1) check the culture for the presence of bacteria, fungus, or any other “abnormal”
substance by using an inverted microscope every time you handle a sample; (2) the cultures should
contain an antibiotic to remove any cryptic contamination; (3) the reagents are sterile; (4) the bottles
used should not be used for the maintenance of any other cell lines; and (5) sterile techniques are
used every time & at all times.
Biological containment devices have been developed to reduce the risks associated with
performing cell culture, maintenance of sterile cell lines, and for the reduction of cross
contamination. The use of proper microbiological procedures, such as aseptic techniques, and
equipment is the primary method for providing personnel and environmental protection – NOT the
biological safety cabinet. In some laboratories, the biological safety cabinet is referred to as a laminar
flow hood. This alternative name is derived from the laminar flow of air within the cabinet. Filtered
air moves at a steady velocity, is unidirectional, and moves in a parallel line to the worker. A laminar
flow hood consists of a front opening, sash, exhaust high efficiency particulate air (HEPA) filter,
rear plenum, supply HEPA filter, and a mechanical blower.

Figure 1: Biological safety cabinet – (A) front opening; (B) sash; (C) exhaust HEPA filter; (D) rear
plenum; (E) supply HEPA filter; and (F). blower. Photo courtesy of Office of Health and Safety, Centers for
Disease Control and Prevention.

The primary purpose of this laboratory is to ectopically express PTEN in mammalian cells
(HEK 293). There are numerous methods available to deliver genes into eukaryotic cells, a process
called transfection, including biochemical, physical, and virus-mediated transfection. In our case, we
will be using a biochemical method of transfection known as lipid-mediated transfection.

Table 1. Various methods for Transient Transfection

Method Cell Cell Types Comments

Toxicity
Lipid-mediated Varies Adherent cells, Cationic lips are used to create artificial
primary cell lines, membrane vesicles (liposomes) that bind
suspended cultures to DNA
Calcium- No Adherent cells and Calcium phosphate forms an insoluble
phosphate suspension cultures co-precipitate with DNA, which attaches
mediated to the cell surface and is absorbed by
endocytosis
DEAE-dextran Yes Cell line specific Positively charged DEAE-dextran binds
mediated to negatively charged phosphate groups
of DNA, forming an aggregate that
binds to the negatively charged plasmid
membrane
Electroporation No Numerous High voltage current is applied to cells
that lead to the formation of nanometer-
sized pores in the plasma membrane.
DNA is taken directly into the cell
cytoplasm.
Biolistics No Primary cell lines Small particles of tungsten or gold are
used to bind DNA and inserted by a
particle accelerator system – identical to
gene therapy techniques.

Lipofection is a technique that introduces DNA into cultured mammalian cells. Every type
of lipofection reagent currently on the market adheres to the same principle. Cationic lipids are used
to create artificial membrane vesicles (liposomes) that have the ability to directly interact with the
plasmid membrane of a cell (Bangham 1992) or can be taken up by non-receptor mediated
endocytosis.
There are two types of liposome mediated transfection reagents, cationic and anionic.
However, in this lab we will only be focusing on cationic liposomal transfection. This technique was
initially developed by Peter Felgner, when he discovered that cationic lipids react spontaneously with
DNA to form a unilamellar shell which can fuse to cell membranes (Felgner et al. 1987). As with all
new technologies the types of cationic lipids has evolved over time from a monocationic lipid, which
was toxic to mammalian and insect cells, to polycationic which are less toxic. In the majority of
lipofection reagents, they consist of a mixture of synthetic cationic lipid and a fusogenic lipid
(phosphatidylethanolamine).

The first step into characterizing PTEN in vivo is to successfully be able to express PTEN in
cultured cells. This is done by constructing a plasmid that contains the sequence of interest and
inserting it into cells by transfection. In our lab we will be performing a transient cationic lipid-
mediated DNA transfection into Hek 293 cultured cells. The plasmid DNA, which has been
isolated for you, forms a complex with unilamellar liposomes that are formed by cationic lipids in
water. The main benefits of using this type of transfection compared to other methods include
higher efficiency, the ability to transfect numerous different cell lines, and lower cell toxicity (Felgner
P.L. et al. 1987). The largest draw back is the cost associated with the transfection reagents.
Pre-lab protocols:
Solutions & Consumables Required:

Reagent Section

70% Ethanol; 1.47 L of 95% EtOH plus 0.53L ddH20; store in a 2L flask labeled “70% EtOH” A

DMEM w. FBS and Pen/Strep (500 ml); (1) 450 ml of Dulbecco's Modified Eagle's Medium/Nutrient
Mixture w 4500 mg/L glucose, L-glutamine and sodium bicarbonate. Substitutes pyridoxine hydrochloride
for pyridoxal hydrochloride (Sigma Cat. No. D5796); (2) add 50 ml of Fetal Bovine Serum (Sigma Cat No.
F0926); and (3) add 5 ml of Penicillin-Streptomycin solution (100x; Sigma Cat No. P-0781).

DMEM: Dulbecco's Modified Eagle's Medium/Nutrient Mixture With 4500 mg/L glucose,L-glutamine and A
sodium bicarbonate. Substitutes pyridoxine hydrochloride for pyridoxal hydrochloride (Sigma Cat. No.
D5796)
Fetal bovine serum; Canadian Qualified (Endotoxin level: <=50 EU/ml (levels routinely <= 10 EU/ml) A
Hemoglobin level: <=25 mg/dl.) Sigma Cat. No. F-0926)
Penicillin-Streptomycin Solution (100X): Contains 10,000 units of penicillin (base) and 10,000 µg of A
streptomycin (base)/ml utilizing penicillin G (sodium salt) and streptomycin sulfate in 0.85% saline. (Sigma
Cat. No. P-0781)

Trypsin: 0.5 g/L porcine trypsin and 0.2 g/L EDTA•4Na in Hank's Balanced Salt Solution with phenol red, B
1X, cell culture tested

Phosphate-buffered Saline (PBS -1X); (1) 137 mM NaCl – dissolve 8 g of NaCl (Fisher Cat No. BP358-1, E
F.W. = 58.43) in 800 ml of ddH20; (2) 2.7 mM KCl – add 0.2 g of KCl (Fisher Cat. No. BP366-500, F.W.
74.56); (3) 10 mM Na2HPO4 – add 1.44 g of Na2HPO4 (EMD CHEMICALS INC. CA Sodium Phosphate,
Dibasic, Anhydrous, GR F.W.= 141.96); and (4) 2 mM KH2PO4 – add 0.24 g of KH2PO4 (Fisher Scientific
Cat. No. BP-352; Phosphoric Acid, Monopotassium Salt KH2PO4,F.W. 136.09).

Adjust the pH to 7.4 with HCl. Add H2O to 1 liter. Dispense the solution into aliquots and sterilize them by
autoclaving for 20 minutes at 15 psi (1.05 kg/cm 2) on liquid cycle or by filter sterilization. Store the buffer at
room temperature.
GenCarrier-1: (Epoch Biolabs) E
pCMV5.nFLAG.PTEN DNA (glycerol stock in -80) F
Lysis buffer (100ml): (1) 50 mM Tris-HCl (buffering agent prevents protein denaturation): in 80 ml of F
ddH20 add 0.79 g Tris; (2) 150 mM NaCl (salt prevents non-specific protein aggregation): add 0.90g NaCl
adjust the pH to 7.4 with HCl; (3) 1 % NP-40 (non-ionic detergent to extract proteins; 10% stock solution in
H20) add 10 ml of 10% NP-40; (4) Protease Inhibitors: Phenylmethylsulfonyl fluoride (PMSF) (200 mM
stock solution in isopropanol; store at room temperature), Leupeptin (store frozen in aliquots, 1 mg/ml in
H2O), Aprotinin (store frozen in aliquots, 1 mg/ml in H2O), Pepstatin (store frozen in aliquots, 1 mg/ml in
methanol), Add 1 ml of 100 mM EDTA (final concentration 1mM) to the solution. Adjust the volume of the
solution to 100 ml using a graduated cylinder. Store RIPA buffer at 2-8°C until ready to use.
Safety Requirements & Warnings:

Good laboratory practices require that you wear a lab coat, disposable gloves, and safety glasses at all
times.

All Material Safety Data Sheets (MSDS) are located within the laboratory, pre-
loaded on all lab computers, or on-line @ www.uwindsor.ca/biotech

Equipment Required:

Laminar flow hood

CO2 incubator
Inverted microscope
Pipet aid
Serological pipette (10 ml, 5ml, and 1 ml)
Biohazard bag
10 cm mammalian cell culture plates
Kim wipes
Microcentrifuge
Benchtop centrifuge (4°C)
Ice bucket
Sonicator
Marker
Freezer box
Benchtop centrifuge
Cell counter (Neubauser hemocytometer)
Cleaned P1000, P200, and P20 pipettes – cleaned with 70% ethanol
P1000, P200, and P20 tips (sterile)
15/50 ml centrifuge tube rack
1.5 ml centrifuge tube rack
Procedure

Overview:

A. Split pre-initiated cell line (provided)

B. Quantification of cell numbers
C. Transfect pCV5.FLAG.PTEN into Hek 293 cells
D. Cell lysis

Mammalian cell culture:

A. Subculturing and splitting of Hek 293 mammalian cells

1. Warm prepared media, containing fetal bovine serum, and antibiotics in 37°C water
bath.

2. Wipe down the work surface and all other inside surfaces of laminar-flow hood,
including the front screen with 70% ethanol and a paper towel.

3. Clean your pipettes by using 70% ethanol and a Kim Wipe – your T.A. will
demonstrate the technique.

4. Remove mammalian cell culture reagents from the water bath and wipe down seal
with 70% ethanol. Ensure that all the aliquots are dry. Bring them into the hood.

5. Using the inverted microscope examine the culture carefully for signs of
contamination or deterioration.

6. Take the culture back to the sterile work area (flow hood)

7. Loosen, but do not remove, the caps of all bottles and aliquots that are to be used.

8. Remove the cap of your bottle into which you are about to pipette and the bottles
that you wish to pipette from, and place the caps open side uppermost on the work
surface, at the back of the hoods and behind the bottle, so that your hand does not
pass over them - 1X PBS, Tryspin, DMEM+FBS/+P/S

9. Insert 5mm of the larger end of a sterile Pasteur pipette into the vacuum line in the
hood then aspirate the media from the culture.

10. Select the appropriate pipettes; open the pack at the top, peel the ends back, turning
them outside in, and withdraw a pipette from the wrapping without touching any
part of the outside wrapping; discard the wrapping into the waste bin.

11. Insert pipette in a pipette aid, pointing the pipette away from you and hold it well
above the graduations, so that the part of the pipette entering the bottle or flask will
not become contaminated.
12. The pipette aid will now be at a right angle to your arm. Take care that the tip of
the pipette does not touch the outside of a bottle or the inner surface of the
hood.

13. Add 10 ml of PBS slowly to the side of the plate opposite to the cells in attempt to
avoid dislodging the cells. Tilt the plate gently from back to front to rinse the cells,
then aspirate the PBS off the plate. This step is designed to remove traces of serum
that would inhibit the action of the trypsin.

14. Add 1 ml of trypsin to the side of the plate opposite of the cells. Ensure that the
monolayer is completely covered. Put the plate into the incubator for 1 minute then
bring it back into the hood. Gently tap the side of the plate with your hands to help
dislodge the cells.

15. Add 10 ml of medium to the plate and resuspend the cells by tilting the plate slightly
and repeatedly (GENTLY) pipe ting up-and-down over the surface bearing the
monolayer three times to wash down all the cells. With the plate still tilted, pipette
the suspension up and down sufficiently to disperse the cells into a single cell
suspension (6-8 times).

16. Pipette the suspension into a 15 ml centrifuge tube (to determine cell count). Label
the tube with your name.
B. Quantification of cell numbers (Hemocytometer)

Proper knowledge of total cell numbers is required for reproducible cell maintenance
and for reducing experimental variability. The concentration of a cell suspension can be
determined by placing the cells in an optically flat chamber, called a hemocytometer, and
viewing it under a microscope. The cell number within a defined area of known depth is
counted and the cell concentration is derived from the count.

Grid printed on a hemocytometer – arrow pointing to area in which counts are performed.

Steps:

1. Take the hemocytometer and cover slip and clean with 70% ethanol, taking care not
to scratch the semi-silvered surface.

2. Take the cover slip and gently place it down over the grooves. Bring the
hemocytometer into the hood.

Haemocytometer chamber; Left (top view) – is the two chambers which cell samples are loaded;
and Right (cross section) – figure shows where cell sample is contained within the haemocytometer.
3. Mix the cell sample by pipe ting with a 10 ml serological pipette vigorously to disrupt
any clumps, collect 10 µl into the tip of Pasteur pipette

4. Transfer the cell suspension to the edge of the hemocytometer change and let the
suspension run out of the pipette and be drawn under the coverslip by capillary
action. Do not overfill or under fill the chamber, or else the dimensions of the
chamber will change.

5. Reload the pipette and fill the second chamber.

6. Transfer the slide to the microscope slide.

7. Select a 10 x objective and focus the grid lines in the chamber.

8. Move the slide so that the field that you see is one of the corner 4 x 4 square areas of
the grid. With a standard 10 X objectives this area should approximately fill your
field of view.

9. Count the cells lying within this area. If more than half of the cells in the field are
clumped to other cells, repeat steps 1-8, making sure to fully mix the cell suspension
this time.

10. Record your results in your book.

11. Count the three additional squares.

12. Calculate the average of the 4 counts and derive the concentration of your sample by
multiplying by 1 x 104 to give you the concentration of cells/ml.

13. Return to the flow hood and wait your turn before plating your cells.

14. Pipette 9 ml of media into three 10 cm cell culture plates.

15. To the plate labeled Subculture, pipette 1 ml of cell suspension.

16. To the other two plates, pipette an amount of cell suspension to give 1 million cells
per plate. Place the cells back into the CO2 incubator. Ensure that your name, date,
sample plate name, and cell type are indicated on the lid of the 10 cm mammalian
cell culture plate.
C. Transfection of pCV5.FLAG.PTEN into Hek 293 cells:

1. Label two 1.5 ml microcentrifuge tubes: (1) pCMV.FLAG.PTEN & (2) negative
control

2. Add 12 µl of the provided pCMV.FLAG.PTEN DNA to your tube labeled

“flagPTENdna”.

3. Add 12 µl of the provided pCMV.FLAG. DNA (empty vector) to your tube labeled
“negative control”.

4. Add 750 µl of media (with serum and antibiotics) to each microcentrifuge tube.

5. Add 25 µl of the lipofection reagent (PEI) to each microcentrifuge tube. Note: try
not to touch the walls of the plastic microcentrifuge tube with the pipette tip.

6. Vortex each tube for 3 seconds and then flick the tube to bring all of the liquid to the
bottom.

7. Incubate for 5 minutes at room temperature.

8. Your TA will provide you with two – 10 cm plates containing Hek 293 cells pre-
labeled “pCMV.FLAG.PTEN” & “Negative control”

9. Using a P1000 pipette gently add the transfection reagent: DNA complexes to the
appropriate plate of cells in a drop-wise manner. Swirl the plates in a T-style motion
to ensure distribution over the entire plate surface.

10. Repeat the process for the negative control.

11. Return the cells to the incubator to allow for DNA incorporation and protein
expression.

D. Cell Lysis:

1. Acquire two plates from your TA that have been transfected for you. These plates
contain Hek 293 cells that have had negative control plasmid, pCMVflag, or
pCV5.nFLAG.PTEN transfected into them by the same mechanism that you just
completed.

2. Remove the medium by aspiration.

3. Gently wash the monolayer culture of cells with 10 ml of room temperature PBS
(sterile 1X).

4. Remove the PBS and add 1.0 ml of lysis buffer containing protease inhibitors.
 5. Detach cells from the plate by tilting the plate and scraping the cells into a pool using
a cell scraper. Once you have scraped the cells off half of the plate, rotate the plate
180° and scrape again into a pool. Using your P1000, slowly pipette the cell lysate up
and down 5 times.

6. Transfer the cells to the appropriate 1.7 ml microcentrifuge tube labeled control or
pCV5.FLAGPTEN.

7. Sonicate your cell lysates with 3 – 10 second bursts. Remember to place the tip of
the sonicator approximately ½ inch (4mm) below the surface of the liquid in the
tube to avoid spraying your sample out of the tube.

8. Spin tube in a refrigerated centrifuge for 15 minutes at 4°C on maximum speed.

9. Transfer the supernatant to a new 1.7 ml tube, be careful not to pick up the pellet,
pipette slowly. Indicate using a marker your group number, lab section, sample
name, and date. Place these tubes in your freezer box at -20°C.

References

Duzgunes N. and Felgner P.L. 1993. Intracellular delivery of nucleic acids and transcription
factors by cationic liposomes. Methods Enzymol. 221:303-306.

Felgner P.L. and Ringold G. 1989. Cationic liposome-mediated transfection. Nature

337:387-388.

Felgner P.L., Gadek T.R., Holm M., Roman R., Chan H.W., Wenz M., Northrop J.P.,
Ringold G.M., and Danielsen M. 1987. Lipofection: A highly efficient, lipid-mediated DNA-
transfection procedure. Proc. Natl. Acad. Sci. 84:7413-7417.

Kruse, R.H, Puckett, W .H., Richardson, J.H. 1991. Biological Safety Cabinetry. Clinical
Microbiology Reviews 4:207-241.