Standard Techniques for Cell Culture


Cell lines
Happy Cells
Subculturing
Cryoconservation
Transfection


Cell lines

CELL LINES USED IN THE CBL

Based on hteir morphology and/or functions 3 types of cell lines can be differenciated:
Epithelial-like cells grow attached to a surface and appear flat and polygonal.
Fibroblast-like cells grow attached to a surface and appear elongated bipolar.
Lymphoblast-like cells grow suspended and appear spherical.

Cell line Description Medium Type Inoculum Subculture Freeze BSL ATCC
HeLa Human cervix (epithelial) DMEM/F12 + 10% FBS adherent 1 * 104/cm2 1:10 75% DMEM/F12, 20% FBS, 5% DMSO 2
(HPV-18) 		CCL-2
Caco-2 Human colon (epithelial-like) DMEM/F12 + 10% FBS adherent 1 * 104/cm2 1:10 75% DMEM/F12, 20% FBS, 5% DMSO 1
HTB-37
MCF7 Human breast (epithelial) EMEM + 10% FBS + 0.01 mg/ml insulin or
DMEM/F12 + 10% FBS + 0.01 mg/ml insulin ?		 adherent @ 37 °C
not adherent @ RT 		1 * 104/cm2 1:10 85% EMEM, 10% FBS, 5% DMSO 1 HTB-22
HT-29 Human colon (epithelial-like) DMEM + 10% FBS adherent 5 * 103/cm2 1:20 75% DMEM, 20% FBS, 5% DMSO 1
HTB-38
THLE-3 Human liver (epithelial) WME-L + 10% FBS adherent 104/cm2
coated flask		 1:10 75% WME-L, 20% FBS, 5% DMSO 2
(SV40) 		CRL 11233
THP-1 Human monocytes (monocyte) RPMI1640 or DMEM/F12 + 10% FBS suspension --- 1:10 75% medium, 20% FBS, 5% DMSO 1
TIB 202


Happy Cells

Some general recommendations for having happy cells :-)

Medium

- Buy and store Gln-free medium and add it as dipeptide
- Add 5 - 10 mM HEPES to medium to have a more stable pH
- Avoid exposition of medium to UV/fluorescent light (peroxide formation)

Subculture

- Subculture cells at 80 - 90 % confluent
- When subculturing, use prewarmed medium
- Use dissociating agents at low conc. and temperature; inactivate/remove them afterwards
- Centrifuge cells at low revolutions: 5 min @ 5 g to get a soft pellet
- Keep cell suspensions chilled (e.g. while counting) to avoid clumping
- Seed ~104 N/cm2 and use enough medium: 0.25 ml/cm2

Plastics

- Serum provides attachement factors like fibronectin, vitronectin
- A low cost coating material is gelatin
- Pretreated plastics accelerates cell attachemnt (e.g. CellBIND from "Corning" or Nunclon from "Nunc")


Subculturing

When adherent monolayer cells occupate all growth space the cells stop to grow due to contact inhibition. Therefore, when the cell growth is 80 - 90 % confluent the cells have to be "diluted" = subcultured.
In contrast, suspension cells can directly be diluted with fresh growth medium.

Examine cells

Observe the cells under an inverted microscope (phase contrast) or (far less good, but cheaper) under a stereomicroscope: 100x.
- pH-shift (medium not red)
- Microbial contamination (medium turbid)
- Cell morphology (dead cells are floating or round)
=> Iniciate subculturing when 80 - 90 % confluent: ~105N/cm

Preparation

- Turn on dry bath (37 °C)
- Clean Biological Safety Cabinet (BSC) with 70 % EtOH. Place tips, pipettes, (clean) waste flask into the BSC
- Turn on UV light in BSC (10 min). Afterwards turn on the blower
- Prewarm medium to 37 °C (15 ml for T-75, 5 ml for T-25), prewarm trypsin/EDTA and DPBS to RT
- Label flask or multititerplate (cell line; date; working lot; experiment)

Detach cells

- Remove and discard medium (pipette)
- Wash the cells once with DPBS in order to remove traces of FBS which inhibits trypsin: 8 ml for T-75; 3 ml for T-25
- Add 0.05 % trypsin/EDTA and incubate about 10 min at RT: 1.5 ml for T-75 or 0.5 ml for T-25
  - DO NOT hit or shake flask in order to avoid clumping of the cells
  - Observe the cells under inverted microscope or stereomicroscope: detached cells becomes round
Add medium to inactivate trypsin/EDTA: 7.5 ml for T-75 or 2.5 ml for T-25
  - pipette up and down to wash the cells from bottom and to isolate them

Cell count

Place a drop of homogeneous cell suspension onto a improved Neubauer-Kammer - due to capillar force the liquid enters equally to the chamber.
Count the cell number (N) of all the 25 small (yellow) squares and multipicate this number with 10,000 to get the N/ml.
Improved Neubauer-Kammer

red square: 1 mm2 = 100 nl
green square: 0.0625 mm2 = 6.25 nl
yellow square: 0.04 mm2 = 4 nl
blue square: 0.0025 mm2 = 0.25 nl
depth: 0.1 mm
 Neubauer

Inoculum

Inoculate monolayer cells with ~1 * 104N/cm2.
Subculturing is only done with flasks, not with MTP; the numbers given here are an orientation to inoculate experiments.
  T-75 T-25 6-well MTP 12-well MTP 96-well MTP
working vol. (ml) 15 5 2.5 1 0.1
working surface (cm2) 75 25 9.5 4 0.32
Total cells (N) at 100% confluence 8 * 106 3 * 106 1 * 106 4 * 105 3.5 * 104
Wash DPBS (ml) 8 3 1.5 0.5 0.1
Trypsin/EDTA (ml) 1.5 0.5 -- -- --
Add new medium (ml) 7.5 2.5 -- -- --
Expected cells (N/ml) 1 * 106 1 * 106 -- -- --
Needed cells (N): 4d subculturing
                    in 2 days 70% confluent
                    next day 70% confluent		 5.0 * 105
--
-- 		2.0 * 105
--
-- 		--
1.75 * 105
3.5 * 105 		--
7.0 * 104
1.4 * 105 		--
6.0 * 103
1.2 * 104
Expected inoculum: 4d subculturing
                    in 2 days 70% confluent
                    next day 70% confluent		 500 µl
--
-- 		200 µl
--
-- 		--
175 µl
350 µl 		--
 70 µl
140 µl 		--
  6 µl
 12 µl
These values are rough estimations. For fast growing cells, e.g. HeLa, a lower inoculum while for slow growing cells, e.g. Caco-2, a higher inoculum should be used.

Troubleshooting

Cell clumps:
- Medium is viscous (DNA has been released from lysed cells)
  -> Dissociating process is too harsh: less pipetting, lower trypsin concentration
  => Add 1 drop of sterile DNase (1 mg/ml)
- Cells are reaggregating
  => Chill cells when not used directly
  => Centrifuge cells less strong

No reattachment:
- Cells lack attachment factors
  => Use serum and/or coated plates
- Dissociation enzymes (trypsin) not inactivated
  => Centrifuge cells (5 min, 125 g) and/or add an inhibitor (e.g. serum)
  serum does not inactivate collagenase!

Low viability:
- Cells dissociated too strong
  => use less concentrated dissociation soln.
- Cells kept for longer time in tube
  => use (plate) cells immediately


Cryoconservation

During prolonged (sub)cultivation the cell lines can change their phenotypic characteristics and expression pattern (genetic drift). This means, that the behaviour of the cells changes during subculturing: thereby frequently subcultured cell lines can not be considered as the true (original) cell model. For pharmaceutical investigation only 5 (five!) passages (subculturing) are accepted for a cell line. This effect is less pronounced in immortal cancer lines (the majority), primary cell lines are still mortal and they just die. To avoid these genetic changes the cells have to be congelated in liquid nitrogen for their preservation.
During congelation you can cause celular damage:
slow cooling fast cooling
external water freezes first internal water freezes first
-> water migrates out of the cells (osmosis) -> water remains in the cells
-> avoids formation of intracelular ice crystals -> avoids osmotic imbalance
=> but high osmotic imbalance => but formation of ice crystals

Cryoprotection

In order to archieve both conditions you have to
a) add a cryoprotectant like DMSO or glycerol (which are toxic to the cells!) and
b) congelate the cells with about 1 °C/min.

Examine cells

Observe the cells under an inverted microscope (phase contrast) or (far less good, but cheaper) under a stereomicroscope: 100-200x.
- pH-shift (medium not red)
- Microbial contamination (medium turbid)
- Cell morphology (dead cells are floating or round)
- Cells should be grown to ~90 % confluent: ~7 * 106N (T-75)

Preparation

- Clean Biological Safety Cabinet (BSC) with 70 % EtOH. Place tips, pipettes, (clean) waste flask into the BSC
- Turn on UV light in BSC (10 min). Afterwards turn on the blower
- Prewarm DMSO, trypsin/EDTA and DPBS to RT. Keep the medium at 4 °C
- Prepare "Mr. Frosty": add 250 ml isopropanol and place it in the refrigerator
- Label cryovials (cell line; date; seeding lot)

Detach cells

- Remove and discard medium (pipette)
- Wash the cells once with 8 ml DPBS in order to remove traces of FBS which inhibits trypsin
- Add 1.5 ml 0.05 % trypsin/EDTA and incubate about 10 min at RT
  - DO NOT hit or shake flask in order to avoid clumping of the cells
  - Observe the cells under inverted microscope or stereomicroscope: detached cells becomes round
Add 5 ml Resuspending-medium to inactivate trypsin/EDTA
  - pipette up and down to wash the cells from bottom and to isolate them
- Chill the cells on ice

Freezing cells

- Prepare 5 ml of 2x Freezing medium: Normal medium with double concentrated DMSO and FBS.
- Transfer 500 µl 2x Freezing medium into 1.2 ml cryovials (10)
- Add 500 µl cells, mix briefly by pipetting, close tap and place tube into the cold "Mr. Frosty"
- When finished with all 10 cryovials, place "Mr. Frosty" for ~4 h into freezer (-20 °C)
Afterwards transfer "Mr. Frosty" overnight (~12 h) into ultrafreezer (-80 °C)
- The next day transfer the 10 cryovials onto 2 cryocanes and place them into (the vapor phase) of the liquid nitrogen
It is important to be fast at this step, that the cells do not thawn up meanwhile!

Thawing up cells

When the cells are stored submerged in the LN2 then place it for 24 h to the highest position in a cryocane (i.e. in the vapor phase)

Preparation

- Turn on dry bath (37 °C)
- Clean Biological Safety Cabinet (BSC) with 70 % EtOH. Place tips, pipettes, (clean) waste flask into the BSC
- Turn on UV light in BSC (10 min). Afterwards turn on the blower
- Prewarm medium to 37 °C (15 ml for T-75, 5 ml for T-25)
- Label flask (cell line; date; working lot; experiment)

Thaw up cells

Use adequate personnal protection: cryogloves and faceshield
- Remove cryovial from cryocane. Be aware that the cap can blow off violently
- Place the cryovial into dry bath for to thaw the cells rapidly
- Pipette complete content into T-25 flask and add immediately 5 ml of complete medium
- Incubate cells at 37 °C for 6 - 8 h (or overnight)
- Remove medium (cells should be attached) and add 5 ml fresh complete medium (removing cryoprotection)

Additionals

Although DMSO is more toxic to the cells, glycerol causes more osmotic problems. After thawing up the cells,
- the DMSO has to be diluted rapidly (adding fresh medium) or removed (centrifugation: 5 min @ 100 g)
- the glycerol has to be diluted stepwise: adding every 10 min 1 ml of prewarmed medium in order to avoid an osmotic shock
It is recommended to store the vials in the gas phase of the liquid nitrogen (-130 °C)
and not submerged in the liquid nitrogen (-196 °C) itself.
- Through the LN2 mycoplasma can enter into the cryovials
- LN2 can enter into the cryovial and when removing the vial due to the expandation of the nitrogen the cap can violently blow off
When the delivery of LN2 is complicate and slow (as here) then a submerged storage has the advantage to prolong storage time w/o necessity of refilling the storage Dewar
=> therefore: always use special cryogloves (water resistant) and a face protection
=> Remove the submerged cryovial for 24 h into the vapor phase (usually highest position at the cryocane)
=> use specially designed cryovials with external thread, silicone washer and lip seal (e.g. VWR #87003-404)


Transfection

Transfection means introducing DNA into a cell line and expressing the respective gene. In microbiology this process is called transformation. Transformation in Cell Biology refers usually to the change of a normal (mortal) cell into a immortal (cancer) cell.
In the CBL we use 2 different approaches (Lipofection and Magnetofection), though, existing several more (e.g. Electroporation, Ca-phosphate, Gene gun). First transient transfection is achieved, means the incorporated DNA will not be replicated hence will be lost during the next replications. Therefore these kind of cells should be harvested 24-48 h post-transfection. In order to achieve stable transfections, the DNA has to be integrated into the genomic DNA; this can be enforeced by applying a selection stress, e.g. by an antibiotic resistance gene (apply 24 h post-transfection) and to facilitate the recombination, the (vector)DNA should be linearilized.
Ca-phosphate: DNA is mixed with CaCl2 in HEPES-buffered saline forming Ca-phosphate crystals with the DNA bound on it (some phosphates are from DNA?). This precipitate is added to the cells (4-12 h) and those internalize these crystals. This method is cheap but usually not very effective.
Electroporation: In contrast to bacteria, cell lines are much more susceptible to an electric shock as they should be in medium with low salt concentration. Usually electroporation cuvettes with big gaps (40 mm) are used and low pulses are applied (300-1500 V/cm: 120-600 V). After the shock cells may be incubated 10 min on ice, then prewarmed growth medium is added and cells are incubated for 48 h.
Controls: Besides the sample the following controls should be considered:
- Sample: Vector with gene of interest.
- Positive control: Same vector expressing only GFP (expression control, transfection efficiency).
- Mock/negative control: Cells with same treatment but w/o vector (treatment control).
- Growth control: Cells w/o any treatment.

Lipofection

Here the DNA is mixed with liposome forming substances which then are endocytosed by the cells. The lipids are usually positively charged to a) aggregate with the negatively charged DNA and b) adsorb on the negatively charged cell surface. Typical lipids are DOPE (Di-Oleoyl-PtdEtN).
Other points to consider:
- 40-80% confluency of cells
- low passage number (< 50)
- pure, endotoxin-free DNA (~1 µg/µl)
- DNA-lipofection ratio (1:2 - may vary between 1:0.5 - 1:5): High Turbofect concentrations are toxic for the cells.
- transfection medium (generally recommended w/o serum, but some work also w/)
- absence of general inhibitor (e.g. EDTA, citrate, phosphate, RPMI, antibiotics)
- transfection time (0,5-4 h)
- Recommended promotor: pCMV (Cytomegalovirus), SV40 (Siam virus) or RSV (Rous sarcoma virus).
While ATCC recommends "FuGene"-lipofection system, we use the cheaper "Turbofect" (Fermentas, R501 [Storage: 4 °C]).

Turbofect

Seed to ~40% confluent 1 d before transfection
i.e. 2.5 * 105 N for 30 mm culture dish (= 250 µl) or 8 * 105 N for T-25 flask (= 800 µl)
Incubate cells 24 h
Mix 2 / 5 µg DNA (~1 µg/µl) with 200 / 500 µl serum-free DMEM (vortex).
- add 4 / 10 µl Turbofect, vortex 10 s strongly.
- incubate 20 min @ RT (not longer!)
- add mixture to the cells (30 mm culture dish or T-25 flask)
- gently rock for even distribution
Incubate cells 24-48 h.

Troubleshooting:
- EDTA, citrate, phosphate, RPMI, chondroitin sulfate, hyaluronic acid, dextran sulfate, or other sulfated proteoglycans in the growth medium or in the medium for preparing DNA:lipofection reagent-complex can interfere with the liposome formation.
- Antibiotics should not be added to the medium, becasue after lipofection cells are more sensitive (co-transport with liposomes).
- After adding the transfection complex into medium, mix thoroughly to prevent local acculumations.
- If transfection performance suddenly declines, it may be because of the cells: cells were seeded too sparse or too dense, also excessive passaging can decrease transfection performance.

Magnetofection

The principle is to adsorb the DNA onto magnetic beads, adding those to the cells, which internalize the beads (with the DNA) by endocytosis. To speed up this process, after addition of the beads to the cells they should be incubated for a short time on a magnetic plate.
In the CBL we use the system from Chemicell called PolyMAG, which are superparamagnetic magnetite particles with a matrix of polyethyleneimine.

PolyMAG

Seed to ~40% confluent 1 d before transfection
i.e. 2.5 * 105 N for 30 mm culture dish (= 250 µl) or 8 * 105 N for T-25 flask (= 800 µl)
Incubate cells 24 h
Mix 2 / 5 µg DNA (~1 µg/µl) with 200 / 500 µl serum-free DMEM (vortex).
- add 2 / 5 µl (vortexed) PolyMAG, vortex 10 s strongly. Incubate 20 min at RT.
- add mixture to the cells (30 mm culture dish or T-25 flask)
- gently rock for even distribution
- incubate 20 min on Magnetofector-plate in the incubator (not longer!)
Incubate cells 24-48 h.
 Magnetofection

Antibiotics

About 24 h post-transfection a selection pressure, usually an antibiotic, can be added (stable at 37 °C for usually 3-5 d). Which antibiotic, depends on the resistance gene of the vector. In the CBL we use the following vectors:
Basic vector Derivates Promotor Features Resistance gene Antibiotic Conc: selection / maintenance
pcDNA3.1
    (Invitrogen)		 pDHH-...
p...-HH
pDGM-...
p...-GFP 		pCMV His/Xpress-MCS-GENE
GENE-MCS-His/V5
GFP-MCS-GENE
GENE-MCS-GFP 		NEOR (APHR, KANR) G418 = Genticin
Stock: 50 mg/ml (HEPES)
store: -20 °C		       500 µg/ml / 100 µg/ml
add: 10 µl/ml / 2 µl/ml
pcDNA3.1/Hygro
    (Invitrogen)		 p..._Hyg pCMV MCS-GENE HPHR (HYGR) Hygromycin B
Stock: 40 mg/ml (HEPES)
store: 4 °C		       200 µg/ml / 100 µg/ml
add: 5 µl/ml / 2.5 µl/ml
pTet-On1
    (Clonetech)		 pPEx, pHAPEx
pPSelGFP 		pCMV GENE-MCS NEOR (APHR, KANR) G418 = Genticin
Stock: 50 mg/ml (HEPES)
store: -20 °C		       500 µg/ml / 100 µg/ml
add: 10 µl/ml / 2 µl/ml
1) The rtTA doxycyclin-regulating squence is excised
Hygromycin B (aminoglycoside): CAS 31282-04-9
Genticin = G418 (aminoglycoside): CAS 1108321-42-2 (disulfate)
      Neomycin B , Kanamycin A , and Gentamicin have similar structure to G418, but can not be used for selection in cell culture.




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Last modified: 04.12.2015