Materials and Reagents

Paper towels
Foam or cork board
Silicon tube (Ø4.76 x 7.94 mm²) (TOKYO RIKAKIKAI, EYELA, catalog number: 125540 )
Collagen type I-coated plate
96-well plate (IWAKI, catalog number: 4860-010 )
24-well plate (IWAKI, catalog number: 4820-010 )
12-well plate (IWAKI, catalog number: 4815-010 )
6-well plate (IWAKI, catalog number: 4810-010 )
100 mm dish (IWAKI, catalog number: 4020-010 )
Non-coated tissue culture plate
96-well plate (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 167008 )
24-well plate (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 142475 )
6-well plate (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 140675 )
100 mm dish (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 172931 )
Sterilized cotton gauze (Osaki Medical Corporation, catalog number: 15095 )
100 ml centrifugation tube (IWAKI, catalog number: 2355-100 )
50 ml conical centrifuge tube (Thermo Fisher Scientific, Thermo Scientific^TM, catalog number: 339652 )
Stainless 60 μm cell strainer (Ikemoto Scientific Technology, catalog number: 802-570-01 )
Wide bored P200 tip (Thermo Fisher Scientific, catalog number: 118-96RNS )
Cannula Ø1.2 mm (plastic outer needle equipped with intravenous cannula) (Top corporation, catalog number: 01151 )
4-0 Sterilized braid silk suture (Akiyama Medical MFG, catalog number: DEWB0404 )
0.22 μm filter (Stericup-GP), 250 ml (Merck, catalog number: SCGPU02RE )
25-ml pipette (Corning, Costar^®, catalog number: 4489 )
Wistar rats aged at 5 weeks to 20 weeks (both male and female animals can be used)
CAUTION: All animal experiments must comply with national and institutional regulations.
PAS kit (Sigma-Aldrich, catalog number: 395B-1KT )
ISOFLURANE Inhalation Solution (Pfizer, catalog number: 871119 )
Ethanol (Wako Pure Chemical Industries, catalog number: 054-07225 )
E-MEM (Sigma-Aldrich, catalog number: M4655 )
Percoll (GE Healthcare, catalog number: 17089102 )
PBS(-) (Nissui, catalog number: 05913 )
TrypLE Express (Thermo Fisher Scientific, Gibco^TM, catalog number: 12604013 )
Fetal bovine serum (FBS) (Gibco, catalog number not available)
Note: FBS should be heat inactivated at 56 °C for 30 min before use.
Cryopreserved MEFs (EmbryoMax^® Primary Mouse Embryo Fibroblasts) (Merck, catalog number: PMEF-CF )
CELLBANKER^®1 Cell Freezing Media (Takara Bio, catalog number: CB011 )
Liquefied nitrogen
Sodium bicarbonate (NaHCO₃) (Wako Pure Chemical Industries, catalog number: 191-01305 )
Sodium chloride (NaCl) (Wako Pure Chemical Industries, catalog number: 192-13925 )
Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
Sodium phosphate monobasic dehydrate (NaH₂PO₄·2H₂O) (Wako Pure Chemical Industries, catalog number: 192-02815 )
Sodium phosphate dibasic dodecahydrate (Na₂HPO₄·12H₂O) (Wako Pure Chemical Industries, catalog number: 196-02835 )
Glucose (Sigma-Aldrich, catalog number: G6152 )
EGTA (DOJINDO, catalog number: 346-01312 )
EDTA (DOJINDO, catalog number: 345-01865 )
Phenol red (Sigma-Aldrich, catalog number: P3532 )
HEPES (Sigma-Aldrich, catalog number: H3375 )
Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C7902 )
Trypsin inhibitor (Sigma-Aldrich, catalog number: T9128 )
Collagenase (Wako Pure Chemical Industries, catalog number: 032-22364 )
L-proline (Sigma-Aldrich, catalog number: P0380 )
Sodium hydroxide (NaOH) (Wako Pure Chemical Industries, catalog number: 197-02125 )
Bovine serum albumin (BSA) (Sigma-Aldrich, catalog number: A9647 )
Human epidermal growth factor (EGF) (Sigma-Aldrich, catalog number: E9644 )
Dexamethasone (100 mg) (Wako Pure Chemical Industries, catalog number: 047-18863 )
Ascorbic acid-2 phosphate (Asc2P) (Wako Pure Chemical Industries, catalog number: 013-12061 )
Y-27632 (25 mg) (RHO/ROCK pathway inhibitor; Inhibits ROCK1 and ROCK2) (Wako Pure Chemical Industries, catalog number: 251-00514 )
A-83-01 (10 mg) (Activin/NODAL/TGF-β pathway inhibitor; Inhibits ALK5, ALK4, and ALK7) (Wako Pure Chemical Industries, catalog number: 035-24113 )
Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855 )
CHIR99021 (25 mg) (WNT pathway activator; Inhibits GSK3) (Axon Medchem, catalog number: 1386 )
DMEM/F12 containing 2.4 g/L NaHCO₃and L-glutamine (Thermo Fisher Scientific, Gibco^TM, catalog number: 11320082 )
DMEM, high glucose, pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11995065 )
Antibiotic/antimycotic solution (Thermo Fisher Scientific, Gibco^TM, catalog number: 15240062 )
Insulin-transferrin-serine (ITS)-X (Thermo Fisher Scientific, Gibco^TM, catalog number: 51500-056 )
Nicotinamide (Sigma-Aldrich, catalog number: N0636 )
Recombinant Mouse Oncostatin M (OsM) (25 μg) (R&D Systems, catalog number: 495-MO-025 )
Matrigel (Corning, catalog number: 354230 )
0.5% (10x) trypsin-EDTA (Thermo Fisher Scientific, Gibco^TM, catalog number: 15400054 )
mTeSR^TM 1 (STEMCELL Technologies, catalog number: 85850 )
Hydrochloric acid (HCl) (35-37%) (Wako Pure Chemical Industries, catalog number: 081-03475 )
Leibovitz’s L-15 medium (with additives: sodium pyruvate; L-glutamine; phenol red) (Thermo Fisher Scientific, Gibco^TM, catalog number: 11415064 )
HBSS (10x), no calcium, no magnesium, no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 14185052 )
7.5% NaHCO₃ solution (see Recipes)
Pre-perfusion buffer (see Recipes)
0.05% phenol red solution (see Recipes)
0.05% collagenase solution (see Recipes)
2 M HEPES solution (see Recipes)
L-proline stock solution (see Recipes)
5 N NaOH solution (see Recipes)
5% BSA stock solution (see Recipes)
10 μg/ml EGF stock solution (see Recipes)
10^-4 M dexamethasone (see Recipes)
1 M nicotinamide (see Recipes)
100 mM Asc2P stock solution (see Recipes)
Y-27632 (5 mM) (see Recipes)
A-83-01 (2.5 mM) (see Recipes)
CHIR99021 (15 mM) (see Recipes)
Small hepatocyte medium (SHM) (see Recipes)
10 μg/ml mouse OsM stock solution (see Recipes)
Matrigel stock (see Recipes)
Matrigel-coated plate (see Recipes)
0.05% (1x) trypsin-EDTA (see Recipes)
Hepatic induction medium for Step 1 (HIM-1) (see Recipes)
Hepatic induction medium for Step 2 (HIM-2) (see Recipes)
BEC induction medium for Step 1 (BIM-1) (see Recipes)
BEC induction medium for Step 2 (BIM-2) (see Recipes)
L-15-based medium (for complete Percoll solution) (see Recipes)
Complete Perocll solution (see Recipes)

Equipment

Surgical scissors (any company’s product should be fine)
Surgical tweezer (any company’s product should be fine)
Peristaltic pump (TOKYO RIKAKIKAI, EYELA, catalog number: RP-1000 )
Small animal anesthetizer (Muromachi Kikai, catalog number: MK-A110 )
Air tight chamber (Muromachi Kikai, catalog number: MK-TB1 )
Bubble trap (prepared in-house)
CO₂ incubator (Panasonic Healthcare, catalog number: MCO-170AICUVH-PJ )
Compact Tabletop Refrigerated Centrifuge (KUBOTA, model: Model 2800 )
Hemocytometers (Improved Neubauer) (Sunlead Glass, catalog number: A126 )
Micropipette (Gilson, catalog numbers: F123600 for P20; F123601 for P200; F123602 for P1000)
Unit water bath Thermominder SDminiN (TEITEC, model: SDminiN, catalog number: 0068750-000 )
Magnetic stirrer (any company’s product should be fine)
150 ml plastic storage bottle (Corning, catalog number: 431175 )
Refrigerator (any company’s product should be fine)

Procedure

Timing
Steps 1-18, preparing rat primary MHs: 2-3 h
Step 19, induction of CLiPs from primary MHs: 2 weeks
Steps 20-24, harvesting CLiPs for various applications: 0.5-1 h
Steps 25-29, hepatic induction of CLiPs: 11 d
Steps 30-34, biliary induction of CLiPs: 12 d
Steps 35-36, long-term culture of CLiPs through continuous subculturing (until reaching P10): 2.5-4 months
Steps 37-39, freeze CLiP stocks: 0.5-1 h

Isolation of primary mature hepatocytes (MHs), TIMING 2.5-3.5 h
Notes:
1. Before starting, place 20 ml E-MEM at room temperature, which will be used for digesting the extracted liver.
2. Before starting, warm pre-perfusion buffer and collagenase solution to 38 °C.
3. Before starting, set the perfusion rate approximately at 25-30 ml/min.
1. Set up the perfusion apparatus as shown in Figure 8.
  
  Figure 8. Equipment setup for liver perfusion. The upper panel presents the complete view of the apparatus for liver perfusion. The bottom panels present detailed images from the upper panel. Pre-perfusion buffer and collagenase solution should be warmed to 37 °C. Paper cushion can be altered with any materials, such as aluminum foil.
2. Anaesthetize a rat by inhalation of isoflurane vapor. Place a paper towel cushion on a foam board or cork board, and lay the rat on the cushion. Sterilize the animal by wiping its fur with 70% EtOH. Open the abdomen using surgical scissors to expose the liver.
3. Place a knot with sterilized suture surrounding the portal vein at the location approximately 0.5 cm from the bifurcation (Figure 9). Start drip of pre-perfusion buffer (see Recipes) by turning on the pump at 25-30 ml/min. Half-cut the portal vein at the location approximately 1.5 cm from the bifurcation of the portal vein, and insert the cannula. Tie the suture to fix the cannula.
  
  Figure 9. Cannulation of the portal vein. A. Schematic representation of the location of the cannula. B-C. Penetrate the tissue beneath the portal vein with a surgical tweezer, and make a knot with a suture surrounding the portal vein at the location approximately 0.5 cm from the bifurcation. D-E. Half-cut the portal vein with surgical scissors at the location approximately 1.5 cm from the bifurcation. F. Insert the cannula into the incision of the portal vein. G. Tie the suture to fix the cannula. H-I. Expose the vena cava, and cut it with surgical scissors to let the blood go out.
4. Immediately after confirming that the color of the liver changes from reddish brown to yellowish, cut the inferior vena cava (Figures 9 and 10). Perfuse approximately 450 ml pre-perfusion buffer (approximately 15 min).
  
  Figure 10. Colour change of the liver before and under perfusion
5. Pause the pump, transfer the inlet tube to the collagenase bottle, and resume perfusion. Perfuse approximately 300-350 ml collagenase (approximately 10 min).
6. Stop perfusion, and remove the cannula from the portal vein. Cut the liver from the abdominal cavity, and transfer it to a 100-mm sterile plastic dish. Remove the attaching surrounding tissue, such as the diaphragm and pancreas.
  Note: From this step forward, perform all procedures on a clean bench.
7. Add 20 ml E-MEM kept at room temperature and 20 ml of the remaining collagenase solution (see Recipes). Mince the liver with surgical scissors into pieces that are less than 3 mm in size.
8. Incubate the minced tissue at 37 °C in a CO₂ incubator for 15 min. After taking out the dish from the incubator, further dissociate the remaining tissue by gently pipetting the digested solution with a wide-bore pipette, such as a 25-ml pipette, several times.
  Note: If large undigested fragments remain, remove them with sterilized tweezers.
9. Filter the undigested tissue fragments by transferring the digested solution into a 100-ml tube equipped with single-layered gauze on the top. Discard the single-layered gauze from the tube top, and transfer the filtered solution into a 100-ml tube with the double-layered gauze.
  Notes:
  1. When the gauze is clogged with undigested tissue fragments, remove the clogging materials from the filter using a pipette.
  2. From this step forward, all the procedures should be performed on ice, and all the reagents should be kept at 4 °C or on ice.
10. Aliquot the filtered solution into two 50-ml tubes, add E-MEM into each tube up to 50 ml by decantation, and mix by gentle inversion. Centrifuge the tubes at 57 x g for 1 min at 4 °C.
11. Aspirate the supernatant, and loosen the cell pellet by gently tapping the tubes. Add 10 ml E-MEM to each tube, and resuspend the cell pellet by gently pipetting several times. Decant E-MEM into each tube to 50 ml, and mix by gentle inversion. Filter the cell suspension through a double-meshed stainless cell strainer into new tubes. Centrifuge the tubes at 57 x g for 1 min at 4 °C.
  Note: When pipetting the MH suspension, a wide-bore pipette should be used to avoid damaging MHs by extensive shear stress.
12. Aspirate the supernatant, and loosen the cell pellet by gently tapping the tubes. Add 10 ml complete Percoll solution to each tube, and resuspend the cell pellet by gently pipetting several times. Aliquot the remaining complete Percoll solution to each tube (approximately 14 ml/tube), and mix by gentle inversion. Centrifuge the tubes at 57 x g for 10 min at 4 °C.
13. Aspirate the supernatant, and loosen the cell pellet by gently tapping the tubes. Add 10 ml E-MEM to each tube, and resuspend the cell pellet by gently pipetting several times. Decant E-MEM into each tube to 50 ml, and mix by gentle inversion. Centrifuge the tubes at 57 x g for 2 min at 4 °C.
14. Repeat the above wash process (Step 13) once more.
15. Resuspend the cells in 15 ml SHM(-), combine the 2 tubes, and measure the cell concentration.
16. (Optional) If extensive aggregation disturbs cell counting, filter the cell suspension with a 40-μm cell strainer before cell counting. There is no difference in reprogramming efficiency under microscopic observation between cells isolated with and without this filtration step.
17. Mix 30 μl of cell suspension with 120 μl SHM(-) to prepare a 5 fold-diluted cell suspension. Remove 30 μl from the diluted cell suspension, and mix it with 30 μl of trypan blue. Count the viable and dead cell numbers using a hemocytometer. Determine the suspension volume to be used for culture.
18. Seed the isolated MHs at approximately 1 x 10⁴ cells/cm² in SHM(+) (if desired, SHM(-) can be also used as control) (for example, we seed cells at 2.5 x 10⁴ cells/well, 5 x 10⁴ cells/well and 1 x 10⁵ cells/well in a 24-well plate, 12-well plate and 6-well plate, respectively). To obtain homogeneous seeding of MHs, shake the plates vigorously back and forth 10 times and then right and left 10 times. Let the plates stand still for 15 min so that MHs fall down to the bottom of the plate, and then, transfer the plates into a CO₂ incubator.
  Note: Due to the relatively heavy weight of MHs, be careful to keep the cell suspension homogeneous. Loading a large amount of the cell suspension in a pipette and releasing it to multiple plates/wells may result in inconsistent seeding density among plates/wells. We typically apply well-mixed undiluted cell suspension (generally approximately 3-8 x 10⁶ cells/ml) using a micropipette directly to plates that are already filled with SHM(+). During the cell seeding step, ensure that the cell suspension is kept homogeneous by periodically inverting the tube to mix it.
Induction of CLiPs from primary MHs using the small molecule cocktail YAC, TIMING 2 weeks
1. One day after plating primary MHs, replace the culture medium with fresh SHM(+) (if desired, SHM(-) also as control). Thereafter, renew the medium every 2-3 days. Given that proliferative YAC(+) cells are bipotential (see Steps 24-33), we designate these cells as chemically induced liver progenitors (CLiPs).
Harvesting CLiPs for various application. Steps 20-24. TIMING 0.5-1 h.
When CLiPs reach 70-100% confluency, harvest CLiPs to perform hepatic induction (Steps 25-29) or biliary induction (Steps 30-34), establish stable CLiPs (Steps 35-37), or prepare frozen stocks for future use (Steps 38-40).
1. Wash plates with PBS(-) twice.
2. Add 0.05% trypsin-EDTA (see Recipes) or TrypLE Express (these two reagents can be used equivalently for harvesting CLiPs), and incubate at 37 °C in a CO₂incubator until the cells are detached from the plate.
  Notes:
  1. Make sure that the dissociating reagent completely covers the culture areas. We typically apply 2 ml of dissociation reagent to a 100-mm dish and use a similar ratio for other culture scales.
  2. CLiPs are relatively resistant to trypsinization, and it typically takes 10-15 min to detach them from plates.
3. Further dissociate the cells physically by pipetting with a P1000 tip to harvest as completely as possible, and transfer them to a 15-ml or 50-ml collection tube.
  Note: Despite its relatively damaging effect on the cells, this procedure is indispensable. Unlike general cell lines, even if the cells exhibit round-shaped morphology following trypsinization, many of them still attach to the plate tightly and can rarely be harvested by directly applying medium onto the plates.
4. Collect the cells remaining in a plate with 5 ml SHM + YAC supplemented with 5%-FBS (heat inactivated) or SHM + YAC for trypsin-EDTA or TrypLE Express (TrypLE Express does not require neutralization of the enzymatic activity).
5. Count the cell number using a hemocytometer, determine the suspension volume to be used for passage, transfer the required volume to a new tube, and centrifuge the tube at 200 x g at 4 °C for 5 min. Aspirate the supernatant, and loosen the cell pellet by tapping the tubes. Resuspend the cells with medium to be used for each application.
Hepatic induction of CLiPs (at P1). Steps 25-29. TIMING 0.5-1 h for passage, 2 d for culturing in SHM(+), and 8 d for hepatic induction
For primary CLiPs, we typically use cells cultured for 2 weeks (14-16 days).
1. Harvest the cells following Steps 20-24, and obtain the required volume of the cell suspension for hepatic induction. For early passage CLiPs, we seed the cells at approximately 5 x 10⁴ cells/cm² (1 x 10⁵ cells/well and 2 x 10⁵ cells/well for 24- and 12-well plates, respectively). Centrifuge the tube at 200 x g at 4 °C for 5 min.
  Note: The cell density can be reduced to 1-2.5 x 10⁴ cells/cm² when stable CLiPs are used for hepatic induction.
2. Aspirate the supernatant, resuspend the cells in 0.5 or 1 ml SHM + YAC supplemented with 5% FBS, and seed to a well of collagen-coated 24- or 12-well plates, respectively.
  Note: To increase the attachability of primary CLiPs on culture plates, we recommended using 5% FBS-supplemented SHM + YAC.
3. One day after seeding, replace the medium with fresh SHM + YAC, and culture the cells for 2 days.
4. Replace the medium with HIM-1 (see Recipes), and continue culturing for another 6 days. Replace the medium with fresh HIM-1 every other day.
5. Replace the medium with HIM-2 (see Recipes), and continue culturing for another 2 days.
  Note: Prepare HIM-2 immediately prior to use. Use P1000 or wide-bore 200 μl tips chilled at 4 °C or on ice.
Biliary induction of CLiPs. Steps 30-34. TIMING 0.5 h for thawing MEFs, 1 d for culturing MEFs, 0.5-1 h for passage of CLiPs, 13 d for culturing CLiPs for BEC induction
Passage primary CLiPs for differentiation into BECs.
1. One day before harvesting CLiPs, seed cell cycle-arrested MEFs on 12-well collagen plates at 5 x 10⁴cells/well. Briefly, thaw cryopreserved MEF cells in a 37 °C water bath, and dilute the suspension in 10 ml 10%FBS-DMEM. Following centrifugation at 200 x g at 4 °C for 5 min, resuspend in 10%FBS-DMEM at 5 x 10⁴ cells/ml, and seed 1 ml/well to a 12-well collagen plate.
2. Harvest the primary CLiPs following Steps 20-24, and obtain the desired volume of the cell suspension for hepatic induction. We seed the cells at 5 x 10⁵ cells/well in 12-well plates. Centrifuge the tube at 200 x g at 4 °C for 5 min.
3. Aspirate the supernatant, resuspend the cells in 1 ml SHM + YAC supplemented with 5% FBS, and seed onto the pre-inoculated MEFs.
  Note: To increase the attachability of primary CLiPs on culture plates, we recommend using 5% FBS-supplemented SHM + YAC.
4. (Step 1) On the following day, start biliary induction by replacing the medium with BIM-1 (see Recipes). Culture the cells for 6 days while replacing the medium with fresh BIM-1 every other day.
5. (Step 2) Replace the medium with BIM-2 (see Recipes). Culture the cells for another 6 days while replacing the medium with fresh BIM-2 every other day.
  Note: Prepare BIM-2 immediately prior to use. Use P1000 or wide-bore 200 ml tips chilled at 4 °C or on ice.
Long-term culture of CLiPs through continuous subculturing. Steps 35-36. TIMING 2.5-4 months (until reaching P10)
CLiPs can be cultured stably through continuous passages; however, the proliferation rates transiently decrease from approximately P2 to P7. During long-term culture, the cells can be frozen for backup or future use.
Notes:
1. CLiPs undergo a transient decrease in their proliferative capacity between P2 and P7 (if not they enter complete senescence). Thus, during this period, it takes 15-40 days until the cells obtain 30-60% confluence, depending on experimental batches (Figure 5).
2. Prepare a Matrigel-coated plate before starting passage (we typically use 35-mm, 60-mm, and 10-cm dishes for cell maintenance).
Primary CLiPs are typically passaged on days 14-16. Subcultured CLiPs are further passaged when they reach 50-90% confluence between P1 and P2, 30-60% confluence between P3 and P7, and 50-90% confluence thereafter. We typically seed cells at 2 x 10⁶ viable cells/10 cm-dish in earlier passages (P1 to P7). However, the actual seeded cell number is much lower than 2 x 10⁶ viable cells/10 cm-dish because the cells in the earlier passage cannot attach efficiently to the plates. Once cell proliferation rates increase (typically after approximately P6), we reduce the seeding density to 1-10 x 10⁵cells/10 cm-dish so that they become subconfluent after 3-7 days of culture.
1. Harvest the cells following Steps 20-24, and obtain the desired volume of the cell suspension for passage. Centrifuge the tube at 200 x g at 4 °C for 5 min.
2. Resuspend the cells in an appropriate volume of SHM + YAC, seed on a desired plate coated with Matrigel, and culture the cells in a CO₂ incubator. Medium should be replaced every 1-3 days.
Freeze stocking of CLiPs. Steps 37-39. TIMING 0.5-1 h
1. Harvest the cells following Steps 20-24, and obtain a desired volume of the cell suspension for preparation of frozen stocks (typically 0.5-10 x 10⁶ cells/vial). Centrifuge the tube at 200 x g at 4 °C for 5 min.
2. Aspirate the supernatant, and resuspend the cells in a desired volume of CELLBANKER^®1. The volume of CELLBANKER^®1 used is 0.5 ml/vial.
3. Aliquot to serum tubes equipped with screw caps. Seal the caps tightly, and store at -80 °C for at least one day. Then, transfer samples to liquefied nitrogen.

Notes

Trouble shooting

Recipes

7.5% NaHCO₃ solution
Dissolve 7.5 g of NaHCO₃ in 100 ml dH₂O
Filter with a 0.22-μm filter, and store at 4 °C until use. Use within 1 month
Pre-perfusion buffer
1. Prepare 10x pre-perfusion buffer stock by dissolving the following reagents in 500 ml distilled water: 40 g NaCl, 2.0 g KCl, 0.39 g NaH₂PO₄·2H₂O, 0.76 g Na₂HPO₄·12H₂O, 4.5 g glucose, 1.9 g EGTA, 3.7 g EDTA, 0.0030 g phenol red, and 11.9 g HEPES
2. This stock solution can be stored at room temperature. Before use, make a 1x solution by diluting 50 ml of 10x stock solution with 450 ml dH₂O
3. After autoclaving, add 2.35 ml of 7.5% NaHCO₃ solution and 2 ml of 1 N NaOH solution
0.05% phenol red solution
Dissolve 0.05 g of phenol red in 100 ml dH₂O
Store at room temperature
Note: If the solution contains precipitates, mix the solution with a magnetic stirrer prior to each use.
0.05% collagenase solution
Prepare 1.2 L of collagenase basal solution as below:
1. Dissolve the following reagents in 1.2 L of dH₂O supplemented with 14.4 ml of 0.05% phenol red solution: 9.6 g NaCl, 0.48 g KCl, 0.094 g NaH₂PO₄·2H₂O, 0.18 g Na₂HPO₄·12H₂O, 2.86 g HEPES, 0.888 g CaCl₂·2H₂O and 0.42 g NaHCO₃
2. Store this basal solution at 4 °C until use
3. For perfusion, pour 400 ml into a beaker and adjust pH to 7-7.4 with 5 N NaOH and/or 6 N HCl
4. Then, dissolve 0.02 g trypsin inhibitor and 0.2 g collagenase
5. After adjusting the pH to 7.6 with 5 N NaOH and/or 6 N HCl, filter with a 0.22-μm filter, and store at 4 °C until use
Note: The prepared collagenase solution should be used within one week.
2 M HEPES solution
Dissolve 47.7 g HEPES in 100 ml dH₂O
Filter with a 0.22-μm filter, and store at 4 °C until use
L-proline stock solution
Dissolve 0.3 g of L-proline in 10 ml of PBS(-)
Filter with a 0.22-μm filter, and store at 4 °C until use
5 N NaOH solution
1. Weigh approximately 20 g of NaOH and immediately record the precise weight
2. Calculate the required volume of sterilized dH₂O (for 20 g of NaOH, 100 ml is required)
3. Add NaOH to approximately 80 ml of sterilized dH₂O while stirring the solution
4. After completely dissolving NaOH, increase the volume to the required amount with sterilized dH₂O, transfer to a plastic storage bottle, and store at room temperature
Notes:
1. Because NaOH readily becomes moist, measure the weight as soon as possible.
2. Given that dense NaOH solution dissolves glass, use a plastic bottle for storage
5% BSA stock solution
Dissolve 5 g BSA in 100 ml of PBS(-)
Filter with a 0.22-μm filter. Protect from light and store at 4 °C until use
10 μg/ml EGF stock solution
1. Prepare 20 ml of 0.1% BSA solution by diluting 0.4 ml of 5% BSA with 19.6 ml PBS(-), and dissolve 0.2 mg EGF
2. Store 500-μl aliquots at -20 °C until use
10^-4 M dexamethasone
1. Prepare 10^-2 M primary stock solution by dissolving 100 mg dexamethasone in 25.4 ml ethanol, and store 500-μl aliquots at -20 °C until use
2. To prepare 50 ml of working stock solution (10^-4 M), dilute 500 μl of primary stock with 49.5 ml dH₂O
3. Store the working stock solution at 4 °C in the dark
1 M nicotinamide
Dissolve 12.21 g NA in 100 ml PBS(-)
Filter with a 0.22-μm filter, and store at 4 °C until use
100 mM Asc2P stock solution
Dissolve 2.9 g of Asc2P in 100 ml PBS(-)
Filter with a 0.22-μm filter
Protect from light and store at 4 °C until use
Y-27632 (5 mM)
Dissolve 25 mg of Y-27632 in 14.8 ml sterilized dH₂O
Store 1-ml aliquots at -20 °C until use
A-83-01 (2.5 mM)
1. Prepare a primary stock solution (25 mM) by dissolving 10 mg of A-83-01 in 949 μl DMSO, and store 100-μl aliquots at -20 °C
2. To prepare a working stock solution (2.5 mM), add 900 μl DMSO to one vial of primary stock, and store 100-μl aliquots at -20 °C
CHIR99021 (15 mM)
Add 3.58 ml DMSO into the vial containing 25 mg CHIR99021, and store 100-μl aliquots at -20 °C
Small hepatocyte medium (SHM)
1. Remove 27.3 ml from a 500-ml bottle of DMEM/F12
2. The removed DMEM/F12 can be used to prepare Matrigel-coated plates (see below)
3. To the remaining DMEM/F12, add 1.25 ml of 2 M HEPES, 500 μl L-proline, and 5 ml antibiotic/antimycotic
4. Then, add 250 μl of 5 N NaOH to adjust pH to approximately 7.5. Then, add 5 ml of 5% BSA, 500 μl of 10 μg/ml of EGF, 5 ml of ITS-X, 500 μl of 10^-4 M dexamethasone, 5 ml of 1 M nicotinamide and 5 ml of 100 mM Asc2P
5. Store at 4 °C
10 μg/ml mouse OsM stock solution
1. Prepare 2.5 ml of 0.1% BSA/PBS(-) by diluting 50 µl of 5% BSA with 2.45 ml PBS(-)
2. Dissolve 25 μg mouse OsM in 2.5 ml of 0.1% BSA solution
3. Store 100-μl aliquots at -20 °C until use
Matrigel stock
1. Thaw a bottle of Matrigel in a refrigerator overnight. Prepare 0.5-1 ml aliquots on ice
2. Store aliquots at 4 °C for short-term use (within 2 weeks) or at -20 °C for later use
Note: Chill tips and pipettes for handling Matrigel at 4 °C or -20 °C before use because highly concentrated Matrigel readily solidifies at room temperature.
Matrigel-coated plate
1. Dilute Matrigel at a ratio of 16 µl per ml of DMEM/F12
2. Add approximately 0.1-0.2 ml/cm² of the diluted Matrigel solution (we typically add 0.5 ml, 1 ml, 2.5 ml and 5 ml to a well of 12- and 24-well plates, a 35-mm dish, a 60-mm dish and a 100-mm dish, respectively)
3. Incubate at 37 °C for 20 min
4. Wash once with an equal volume of SHM(-) before seeding cells (we typically add 0.5 ml, 1 ml, 2.5 ml and 5 ml to a well of 12- and 24-well plates, a 35-mm dish, a 60-mm dish and a 100-mm dish, respectively)
0.05% (1x) trypsin-EDTA
Dilute 5 ml of 0.5% trypsin-EDTA with 45 ml PBS(-), and store at 4 °C until use
Hepatic induction medium for Step 1 (HIM-1)
Add 100 μl of 10 μg/ml OsM and 500 μl of 10^-4 M dexamethasone into 50 ml of SHM + YAC. Final concentrations of OsM and dexamethasone are 20 ng/ml and 10^-6 M, respectively
Hepatic induction medium for Step 2 (HIM-2)
Notes:
1. Prepare this medium on ice immediately before use.
2. Use P1000 tips or wide-bore P200 tips chilled at 4 °C or on ice when working with Matrigel.
Mix HIM-1 and Matrigel at 7:1 volume ratio. For example, mix 62.5 μl Matrigel and 437.5 μl HIM-1 (500 μl in total) and 125 μl Matrigel and 875 μl HIM-1 (1 ml in total) for one well of a 24- or 12-well plate, respectively
BEC induction medium for Step 1 (BIM-1)
BIM-1 is mTeSR^TM 1 medium supplemented with YAC. Prepare complete mTeSR^TM 1 medium by adding the 100 ml of supplement into 400 ml basal medium, and add each vial of YAC stock to the solution
BEC induction medium for Step 2 (BIM-2)
BIM-2 is BIM-1 supplemented with 2% Matrigel. Prepare this medium immediately before use. Add 1 volume of Matrigel to 49 volumes of BIM-1
L-15-based medium (for complete Percoll solution)
Dissolve the following reagents in 99 ml of Leibovitz’s L-15 medium (Gibco, with additives: sodium pyruvate; L-glutamine; phenol red): 0.2 g bovine serum albumin (BSA) (Sigma-Aldrich) and 0.0429 g HEPES. Filter with a 0.22-μm filter, add 1 ml of antibiotic/antimycotic (Gibco) and store at 4 °C until use
Complete Perocll solution
Prepare complete Perocoll solution in a 50 ml conical tube just before use by mixing 25 ml of L-15-based medium, 2.4 ml of HBSS (10x) and 21.6 ml of Percoll solution

Acknowledgments

We thank Ayako Inoue for her technical assistance, and Yusuke Sakai and Takanobu Hara for helping us prepare photos used in this manuscript. This research was partly supported by Grant-in-Aid for Young Scientists B (16K16643) and the Research Program on Hepatitis (16fk0310505h0005) from the Japan Agency for Medical Research and Development (AMED). This protocol has been used in Cell Stem Cell (Katsuda et al., 2017). The authors have declared no conflict of interest.

References

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