Lab 8 Cell Culture Lab & Transfection

8/8/2019 Lab 8 Cell Culture Lab & Transfection

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Lab No. 8: Mammalian Cell Culture & Transfection

Tasks to complete: Pre-lab discussion-strategic flowchartSubculture and splitting for transfectionCell counting with HemocytometerLipid mediated transfectionMammalian 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 1900s 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.


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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 toensure 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, tominimize 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


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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.

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

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.


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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 CellToxicity

Cell Types Comments

Lipid-mediated Varies Adherent cells,primary cell lines,suspended cultures

Cationic lips are used to create artificialmembrane vesicles (liposomes) that bindto DNA

Calcium-phosphatemediated

No Adherent cells andsuspension cultures

Calcium phosphate forms an insolubleco-precipitate with DNA, which attachesto the cell surface and is absorbed byendocytosis

DEAE-dextran

mediated

Yes Cell line specific Positively charged DEAE-dextran binds

to negatively charged phosphate groupsof DNA, forming an aggregate thatbinds to the negatively charged plasmidmembrane

Electroporation No Numerous High voltage current is applied to cellsthat lead to the formation of nanometer-sized pores in the plasma membrane.DNA is taken directly into the cellcytoplasm.

Biolistics No Primary cell lines Small particles of tungsten or gold areused to bind DNA and inserted by a

particle accelerator system identical togene 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


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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.


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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/NutrientMixture w 4500 mg/L glucose, L-glutamine and sodium bicarbonate. Substitutes pyridoxine hydrochloridefor 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 andsodium bicarbonate. Substitutes pyridoxine hydrochloride for pyridoxal hydrochloride (Sigma Cat. No.D5796)

A

Fetal bovine serum; Canadian Qualified (Endotoxin level:


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Safety Requirements & Warnings:

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

Equipment Required:

Laminar flow hoodCO2 incubatorInverted microscopePipet aidSerological pipette (10 ml, 5ml, and 1 ml)Biohazard bag10 cm mammalian cell culture platesKim wipesMicrocentrifugeBenchtop centrifuge (4C)Ice bucketSonicatorMarkerFreezer boxBenchtop centrifugeCell counter (Neubauser hemocytometer)Cleaned P1000, P200, and P20 pipettes cleaned with 70% ethanolP1000, P200, and P20 tips (sterile)15/50 ml centrifuge tube rack1.5 ml centrifuge tube rack

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


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Procedure

Overview:

A.

Split pre-initiated cell line (provided)B. Quantification of cell numbersC. Transfect pCV5.FLAG.PTEN into Hek 293 cellsD. 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 37C waterbath.

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. willdemonstrate the technique.

4. Remove mammalian cell culture reagents from the water bath and wipe down sealwith 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 ofcontamination 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 worksurface, at the back of the hoods and behind the bottle, so that your hand does notpass 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 thehood then aspirate the media from the culture.

10.Select the appropriate pipettes; open the pack at the top, peel the ends back, turningthem outside in, and withdraw a pipette from the wrapping without touching anypart 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 wellabove the graduations, so that the part of the pipette entering the bottle or flask willnot become contaminated.


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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 thehood.

13.Add 10 ml of PBS slowly to the side of the plate opposite to the cells in attempt toavoid 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 serumthat 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 themonolayer is completely covered. Put the plate into the incubator for 1 minute thenbring it back into the hood. Gently tap the side of the plate with your hands to helpdislodge the cells.

15.Add 10 ml of medium to the plate and resuspend the cells by tilting the plate slightlyand 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, pipettethe suspension up and down sufficiently to disperse the cells into a single cellsuspension (6-8 times).

16.Pipette the suspension into a 15 ml centrifuge tube (to determine cell count). Labelthe tube with your name.


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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, andviewing 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.

Steps:

1. Take the hemocytometer and cover slip and clean with 70% ethanol, taking care notto scratch the semi-silvered surface.

2. Take the cover slip and gently place it down over the grooves. Bring thehemocytometer 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.

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


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3. Mix the cell sample by pipe ting with a 10 ml serological pipette vigorously to disruptany clumps, collect 10 l into the tip of Pasteur pipette

4.

Transfer the cell suspension to the edge of the hemocytometer change and let thesuspension run out of the pipette and be drawn under the coverslip by capillaryaction. Do not overfill or under fill the chamber, or else the dimensions of thechamber 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 yourfield of view.

9. Count the cells lying within this area. If more than half of the cells in the field areclumped to other cells, repeat steps 1-8, making sure to fully mix the cell suspensionthis 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 bymultiplying 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 mammaliancell culture plate.


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C. Transfection of pCV5.FLAG.PTEN into Hek 293 cells:

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

2. Add 12 l of the provided pCMV.FLAG.PTEN DNA to your tube labeledflagPTENdna.

3. Add 12 l of the provided pCMV.FLAG. DNA (empty vector) to your tube labelednegative control.

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

5. Add 25 l of the lipofection reagent (PEI) to each microcentrifuge tube. Note: trynot 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 theappropriate plate of cells in a drop-wise manner. Swirl the plates in a T-style motionto 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 proteinexpression.

D. Cell Lysis:

1. Acquire two plates from your TA that have been transfected for you. These platescontain Hek 293 cells that have had negative control plasmid, pCMVflag, orpCV5.nFLAG.PTEN transfected into them by the same mechanism that you justcompleted.

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.


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5. Detach cells from the plate by tilting the plate and scraping the cells into a pool usinga cell scraper. Once you have scraped the cells off half of the plate, rotate the plate180 and scrape again into a pool. Using your P1000, slowly pipette the cell lysate upand down 5 times.

6.

Transfer the cells to the appropriate 1.7 ml microcentrifuge tube labeled control orpCV5.FLAGPTEN.

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

8. Spin tube in a refrigerated centrifuge for 15 minutes at 4C 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, samplename, and date. Place these tubes in your freezer box at -20C.

References

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

Felgner P.L. and Ringold G. 1989. Cationic liposome-mediated transfection. Nature337:387-388.

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.

Documents

Lab 8 Cell Culture Lab & Transfection