Cassio, D., Hamon-Benais, C., Guerin, M. and Lecoq, O. (1991) Hybrid cell lines constitute a potential reservoir of polarized cells: isolation and study of highly differentiated hepatoma-derived hybrid cells are able to form functional bile canaliculi in vitro. J. Cell Biol. 115: 1397-1408.

Ihrke, G., Neufeld, E.B., Meads. T., Shanks, M.R., Cassio, D., Laurent, M., Schroer, T.A., Pagano, R.E., Hubbard, A.L. (1993) WIF-B cells: an in vitro model for studies of hepatocyte polarity. J. Cell Biol. 123: 1761-1775.

Shanks, M.R., Cassio, D., Lecoq, O. and Hubbard, A.L. (1994) An improved polarized rat hepatoma hybrid cell line: generation and comparison to its hepatoma relatives and hepatocytes in vivo. J. Cell Science 107: 813-825.

Meads, T. and Schroer, T.A. (1995) Polarity and Nucleation of Microtubules in Polarized Epithelial Cells. Cell Motility and the Cytoskeleton 32:00-00.

B. Culture Medium:

- Modified Ham’s F12 Medium

- This medium is supplemented with:

- 33 mM NaHCO₃ (2.7 g/l)

- 1x HAT (Hypoxanthine Aminopterin Thymidine)*

- 5% Fetal Bovine Serum

200 Unit/ml Penicillin G

0.5 m g/ml Fungizone (Amphoterin B)

* See protocol recipe II, III.

C. Culture Conditions:

• 7% CO₂ atmosphere
• 37° C temperature

• Medium is renewed (changed) three times a week (ex. For 10 cm dish: 15 ml on Mon. and Weds., 20 ml on Fri).
• Cells are grown on tissue culture dishes (e.g. Falcon #3003 10 cm tissue culture dish (TCD)), and glass cover slips.
• A confluent (1.5 x 10⁵ cells/cm²) container is split 1:4-16 (see subculture protocol).
• If cells are being grown for routine maintenance, the cells should be passed approximately every 10 days, when the cells have been sown at a density of 1-2 x 10⁵/10 cm dish.

1. Nutrient Mixture F12; 5% FCS Medium (FOR 1 LITER)

     Materials:

Protocol for 1 Liter:

1. Add 1 1L package of Nutrient Mixture F12 Medium powder to 900 ml DDH2O.
2. Add 2.7 g of Sodium Bicarbonate
3. Add 10 ml 100x SPF Stock.
4. Add 1 ml 1000x HAT Stock.
5. Bring volume up to 1 L with DDH₂O, and mix until dissolved.
6. CO₂ gas to medium to attain the pH of a 6.8 (after sterile filtration pH should be ~7.0, the color should be a salmon-pink {medium can be pH’ed with HCl also}).
7. Add 5% FBS to medium.
8. Sterile filter (0.22 m m) the medium into the appropriate labeled (sterile) bottles, and then incubate sample at 37° C overnight to check for sterility. Store at 4° C to 8° C.

Alternate Protocol for 1 Liter:

1. Add 1 1L package of Nutrient Mixture F12 Medium powder to 900 ml DDH2O.
2. Add 2.7 g of Sodium Bicarbonate.
3. Add 5 ml reconstituted Penicillin G-Streptomycin (sigma cat. No. P-3539 or P-0781)
4. Add 90 mg Penicillin G (Sigma cat. No. P-3032)
5. Add 2 ml Amphotericin B (Sigma cat. No. A-2942)
6. Add 1 ml 1000x HAT Stock
7. Bring volume up to 1 L with DDH₂O, and mix until dissolved.
8. CO₂ gas to medium to attain the pH of a 6.8 (after sterile filtration pH should be ~7.0, the color should be a salmon-pink {medium can be pH’ed with HCl also}).
9. Add 5% FBS to medium.
10. Sterile filter (0.22 m m) the medium into the appropriate labeled (sterile) bottles, and then incubate sample at 37° C overnight to check for sterility. Store at 4° C to 8° C.

II) 1000x HAT Stock

Material

1. Hypoxanthine, Anhydrous M.W. 136.1, Sigma Cat. #H-9636
2. Aminopterin, Anhydrous M.W. 440.4, Sigma Cat. #A-3411
3. Thymidine, Anhydrous M.W. 242.2, Sigma Cat. #T-1895

Protocol

1. Add to 136.12 mg Hypoxanthine 0.6 ml 1 N NaOH and dissolve.
2. Add 1.76 mg Aminopterin to a couple drops of 1 N NaOH and dissolve.
3. Combine #1, #2 and 38.76 mg Thymidine with 100 ml with DDH₂O.
4. Sterile filter (0.22 m m).
5. Make 5ml aliquots, incubate overnight at 4° C and then store at -20° C.

1. Amphotericin B (Fungizone), Sigma Cat. #A-2942.

IV) 100x SPF Stock

Material

1. "Penicillin G, 10,000 Unit/ml – Streptomycin, 10 mg/ml" 20 ml vial, Sigma Cat. #P-3539 or P-0781 can substitute.
2. Penicillin G, 1,670 Units/mg, Sigma Cat. #P-3032
3. 500x Amphotericin B (Fungizone) Stock (Sigma Cat. No. A-2942)

Protocol

1. Add 8 ml of 500x Amphotericin B (Fungizone) stock and 12 ml of DDH₂O to the 20 ml vial of "Penicillin G, 10,000 Unit/ml – Streptomycin, 10 mg/ml" (P-3539).
2. Add 360 mg of Penicillin G, 1,670 Units/mg, to the 20 ml dilution.
3. Bring the volume up to 40 ml with DDH₂O and sterile filter (0.22 m m).
4. Make 5 ml aliquots, and store at -20° C.

Alternate Protocol: Prepare desired amount of 100x SPF for use and add directly to medium without filtration, so the SPF is only filtered once with the medium.

V) ATV (0.5% Trypsin, 0.2g/L ETDA)*

Material

1. Sodium Chloride, NaCl, M.W. 58.44
2. Potassium Chloride, KCl, M.W. 74.56
3. Sodium Bicarbonate, NaHCO₃, M.W. 84.01
4. Dextrose, HOCH₂CH(CHOH)₄O, M.W. 180.16
5. EDTA, M.W. 372.24
6. Trypsin, Sigma Cat. #T-7409
7. DDH₂O

Protocol for 1 Liter

1. Add to 500 ml of DDH₂O:

8 g NaCl

0.4 g KCl

0.58 g NaHCO₃

1 g Dextrose

0.2 g EDTA

0.5 g Trypsin

2. Bring volume up to 1 L with DDH₂O, and pH to 7.4 with HCl.
3. Incubate at 37° C for at least 1 hour to activate the trypsin.
4. Sterile filter (0.22 m m) and make 10 ml aliquots.
5. Refrigerate at 4° C overnight, and then store at -20° C.

* Commercially available products may be substituted.

• Aliquots (vials are thawed rapidly and reseeded within recommended sowing range.

*When thawing, most cell death occurs between –50° C and 0° C.

• Always check cells the following day for healthy, viable cells and freedom from contamination.

Protocol for 1x10⁶ cells/vial:

1. Remove vial from liquid nitrogen and immerse immediately into 37° C water bath until the medium is liquid, (approximately 2 minutes), and constantly agitate.
2. Tighten vial cap, ethanol vial thoroughly on outside. Add 0.5 ml of medium into the vial, drop by drop. Add cell suspension into 8 ml of fresh medium in a 15 ml conical tube (9 ml total).
3. Plate cells in two 10 cm dishes, 3 ml and 6 ml, respectively.
4. Place in the incubator, and once the cells have attached, renew with fresh medium (do this no later than 24 hours). Observe cells daily for growth and freedom from contamination.

F. Cell Characteristics:

• Generation time is 1 generation/~72 hours.
• Cloning efficiency is 80-90%.
• Cell pellet should be light brown in color.
• The cells have 1 to 2 nucleoli.
• During mitosis the cells are attached.
• The cells form bile canaliculi (BC), phase lucent structures.
• When cells are confluent, they are viable in this stage for 1 week.
• Cells are very similar to WIF-12-1 cells, except they do not go into the crisis stage.
• The cells grow better and produce more BC when cells are in high density, >2.8 x 10⁴ cell/cm² (1.5 x 10⁶ cell/10cm dish, for experimental set up). When the cells are confluent they exhibit many BC.
• At confluency 90% of the cells exhibit BC.
• For additional characteristics see the publications,
• Cells do begin to show signs of apoptosis after 7 to 10 days in culture. This can increase with further time in the culture during a given passage. Cellular debris is clearly visible, cells can be rinsed (F12 without serum or a HANKs buffer are fine), then fed to remove debris

G. Subculture (Passing):

Materials:
               ATV* (TRYPSIN) solution:

               0.05% Trypsin-0.5 mM EDTA-137 mM NaCl-5.4 mM KCl (0.058% NaHCO₃)

               Medium (5% FBS)

               PBS or Hanks

              *See protocol recipe.

Protocol:

1. Withdraw medium, rinse with PBS or HANKS, aspirate and add ATV (37° C, e.g. 2 ml/T25 flask, 3 ml/10 cm tissue culture dish), roll back and forth over cells for 1 to 2 minutes.
2. If cells are not coming off rounding up for transfer within 5 minutes, place dish (or flask) at 37° C. Replace trypsin stock if cells do not quickly respond to trypsin.
3. Tap on both sides of flask or dish to release cells. Using a small pipette, gently spray off the cells from the plastic. (It is important to attain as many single cells as possible at this point).
4. Add appropriate amount of medium (bring total volume to 10 ml) and pipette cell suspension up and down a few times.
5. Centrifuge at 1000 rpm (1600 g) for 5 minutes in a 15 ml conical tube. Aspirate off supernatant, and immediately resuspend in 10 ml of fresh medium.
6. Count the cells with a hemacytometer and sow cells for propagation dishes: plate 1x10⁵ cells and 2x10⁵ per 10 cm TCD. It is very important to have the majority of the cells as single cells, to have the cells spread evenly in sowing, and for the sowing density to be within the correct range.
7. Cells used for either biochemical or morphological experiments are plated at 5x10⁵ cells per 10 cm TCD. For morphology 6 glass 1x1 cm cover slips are placed in the bottom of a 10 cm TCD. The next day, after cells are attached, cover slips are removed and placed in individual 6 cm Falcon dishes. Use 4 ml of medium for 6 cm dishes.

Materials:

               Medium (with 15% FBS)

               DMSO (final concentration is 10% v/v)

               1.8 ml sterile cryovial (Nunc #3-66656)

               Cryo 1 freezing container, Nalgene (VWR Cat. No. 55710-200)

Protocol:

1. Renew (change medium) cells 24 hours prior to freezing. Cells should be healthy and at the edge of confluence at time of freezing. They should also have many mitoses evident by phase microscopy (~ day 8 of propagation).
2. Trypsinize cells as described in the subculture methods. Count cells and determine the amount per vial to be frozen (a good cell number per vial is between 1 and 5 x 10⁶).
3. After cells have been harvested, as described in the subculture methods and counted, the cells should then be centrifuged again, the supernatant aspirated and the cells resuspend in the predetermined volume of medium with 15% FBS (the volume of the medium in which the cells are resuspended is determined as follows: 0.45 ml medium x vials to be frozen). This should be performed in a stock Gently pipette cell suspension up and down a few times.
4. Slowly (dropwise) add predetermined volume of DMSO to the stock tube (50 m l DMSO x vials to be frozen = 10% v/v of DMSO), while gently mixing the suspension.
5. Place 0.5 ml aliquots into cryovials labeled with cell type, passage number, date, number of cells and initials.
6. Transfer vials into a cryo 1° freezing container and place into a -80° C freezer overnight. Freeze rate down to approximately 1° /min.
7. Transfer vial to liquid nitrogen and record location and all other information on vial.

Alternate Protocol:

1. Renew (change medium) cells 24 hours prior to freezing. Cells should be healthy and at the edge of confluence at time of freezing. They should also have many mitoses evident by phase microscopy.
2. Trypsinize cells as described in the subculture methods. Count cells and determine the amount per vial to be frozen (a good cell number per vial is between 1 and 5 x 10⁶).
3. Resuspend cells in the appropriate volume of Freezing Medium (90% Medium with 15% FBS; 10% DMSO). Please work quickly once cells are resuspended in Freezing Medium, DMSO is toxic to cells.
4. Place 0.5 à 1 ml/cryo vial (10⁶ cells) labeled with cell type, passage number, date, number of cells and initials.
5. Transfer vials into a Cryo 1° freezing container and immediately place into a -80° C freezer overnight (freeze rate = ~ 1° /min.).
6. Transfer vial to liquid nitrogen and record location and all other information on vial.

I. Recombinant adenovirus:

The following protocol is used for WIF-B cells grown on 1x1 cm coverslips and housed in 6 cm Falcon dishes.

1. Remove medium. Rinse with 2-5 ml of sterile Hanks balanced salt solution or PBS++.

2. Virus sup application:

1. for 200 ml of viral sup (try this vol first, a 1:10 dilution may also be appropirate to test) suction dish and around coverslip edges with a sterile pasteur pipet to reduce surface tension. Process a max of 3 coverslips at a time, the      cells will dry out if left too long before addition of medium.  Apply 200ml of virus sup to coverslip.
2. for 1-2 ml of virus sup ( use this vol if 200 ml does not give expression). Apply 1-2 ml of virus sup to dish.
3. for control uninfected, apply appropriate vol of 10% FCS DMEM.

3. Alternatively, purified virus may be used for infecting WIF-B cells on coverslips (estimate is ~2 x 10⁶ cells/ coverslip)

                 b. Apply 200ml of diluted virus (=1-5 x 10⁹ virus particles).

4. Incubate at 37 °C for

5. Remove virus, wash once as in step 2, then replenish cells with medium and incubate at 37 °C for 24 to 48 hrs. Test for expression by IMF or western blotting.

II. Selection of Clones stably expressing YFG. 

Contact Gudrin Ihkre at gi200@cus.cam.ac.uk