Isolation of Human Hepatocytes

Materials and Reagents

1.Additives for Liver Perfusion Solutions

● 0.5 M calcium chloride dihydrate: Put 14.7 g of calcium chloride dihydrate in a 200 mL mixing container. Dissolve in ultrapure Type 1 water with 18.2 MΩ ionic purity. Top up to a volume of 200 mL with Type 1 water. Process into a sterile bottle by membrane filtration with a 0.2 μm filter. Store solution at 4 ℃.

● 100 mM EGTA: Put 7.61 g of EGTA in a 200 mL mixing container. Dissolve in120 mL of Type 1 water. Adjust pH to 7.4. Top up to a volume of 200 mL with Type 1 water. Process into a sterile bottle by membrane filtration with a 0.2 μm filter. Store solution at 4 ℃.

2.Liver Perfusion Solutions

● 10× Perfusion Solution 1: 1540 mM sodium chloride, 200 mM HEPES, 56 mM potassium chloride, 50 mM glucose, 250 mM sodium hydrogen carbonate, pH 7.4. To a 2 L mixing container with gentle stirring, add 1.5 L of Type 1 water. Add 180 g of sodium chloride, 96 g of HEPES, 8.4 g of potassium chloride, 18 g of glucose, and 42 g of sodium hydrogen carbonate. Top up mixing container with Type 1water to the 1.9 L mark. Adjust pH to 7.4. Top up with Type 1 water to reach final volume of 2 L. Process the solution immediately into sterile bottles by membrane filtration with a 0.2 μm filter. Store solution at 4 ℃.

● Perfusion Solution 1: 154 mM sodium chloride, 20 mM HEPES, 5.6 mM potassium chloride, 5 mM glucose, 25 mM sodium hydrogen carbonate, pH 7.4. To a 2 L mixing container with gentle stirring, add 0.2 L of 10× Perfusion Solution 1 and 1.7 L of Type 1 water. Adjust pH to 7.4. Top up withType 1 water to reach final volume of 2 L. Process the solution immediately into sterile bottles by membrane filtration with a 0.2 μm filter. Store solution at 4 ℃.

● Perfusion Solution 2: 152.5 mM sodium chloride, 19.8 mM HEPES, 5.5 mM potassium chloride, 5 mM glucose, 24.8 mM sodium hydrogen carbonate, 1 mM EGTA, pH 7.4. Shortly before liver perfusion, add 10 mL of 100 mM EGTA to 990 mL of Perfusion Solution 1.

● Perfusion Solution 3: 152.5 mM sodium chloride, 19.8 mM HEPES, 5.5 mM potassium chloride, 5 mM glucose, 24.8 mM sodium hydrogen carbonate, 5 mM calcium chloride dihydrate, pH 7.4. Shortly before liver perfusion, add 10 mL of 0.5 M calcium chloride dihydrate to 990 mL of Perfusion Solution 1.

● Digestion Solution: 152.5 mM sodium chloride, 19.8 mM HEPES, 5.5 mM potassium chloride, 5 mM glucose, 24.8 mM sodium hydrogen carbonate, 5 mM calcium chloride dihydrate, 250–400 U/mL collagenase, pH 7.4. Shortly before isolation, add appropriate amount of collagenase to 400 mL of Perfusion Solution 3. Process the solution immediately into a sterile bottle by membrane filtration with a 0.2 μmfilter. The appropriate collagenase activities are as follows; 250 U/mL for less than 25 g of liver 300 U/mL for 25–40 g of liver, 350 U/mL for 41–80 g of liver and 400 U/mL for more than 80 g of liver.

3.Cell Suspension Solutions

● Hepatocyte Suspension Solution: 120 mM sodium chloride, 10 mM HEPES, 0.9 mM calcium chloride dihydrate, 6.2 mM potassium chloride, 0.1% w/v albumin, pH 7.4. To a 5 L mixing container with gentle stirring, add 4.8 L of Type 1 water. Add 35.1 g of sodium chloride, 11.9 g of HEPES, 0.66 g of calcium chloride dihydrate, 2.31 g of potassium chloride, 5 g of bovine serum albumin. Adjust pH to 7.4. Top up with Type 1 water to reach final volume of 5 L. Process the solution immediately into sterile bottles by membrane filtration with a 0.2 μm filter. Store solution at 4 ℃.

● Trypan blue solution.

4.Cell Culture Media

● Hepatocyte Medium: DMEM media with 800 μg/L hydrocortisone, 125 U/Linsulin, 100 U/mL penicillin, 100 μg/mL streptomycin(Gibco, Life Technologies), 2 mM L-glutamine, 7.4 μg/L glucagon, and 10% FCS (Sigma-Aldrich). To a 1 L mixing container with gentle stirring, add 800 mL of DMEM media. Add 8 mL of 1,00,000 μg/L hydrocortisone, 10 mL of 12,500 U/L insulin, 10 mL of 10,000 U/mL penicillin/10,000 μg/mL streptomycin, 10 mL of 200 mM L-glutamine, 7.4 mL of 1000 μg/L glucagon and 100 mL of FCS.Adjust pH to 7.4. Top up with Type 1 water to reach final volume of 1 L. Process the medium immediately into sterile bottles by membrane filtration with a 0.2 μm filter. Store solution at 4 ℃.

Equipment

1.Gas cylinder containing a mixture of 95% oxygen and 5% carbon dioxide.

2.Bubble trap.

3.Olive-tipped irrigation cannulas.

4.Glass coil heat exchanger.

5.Gas-permeable tubings.

6.Thermostated circulating water bath.

7.Peristaltic pump.

8.Refrigerated centrifuge(Beckman Coulter).

9.Büchner funnel.

10.Scalpel.

11.Tubing clamps.

12.Crystallizing dish.

13.Forceps.

14.Nylon mesh (210 μm).

15.Nylon mesh (70 μm).

16.Centrifuge tubes (50 and 200 mL).

17.Counting chamber.

18.Serological pipette(Ucallm).

Procedure

A.Preparation Before Liver Perfusion

1.Set up the perfusion equipment as shown in Fig. 1.

2.The various solutions listed in the Materials section should be prepared in advance.

3.Shortly before the arrival of the liver, turn on the water bath and set it at an appropriate temperature (see Note 1).

4.Next, open the valves on the regulator on the gas cylinder(Fig. 1A) to start the flow of 95% oxygen and 5% carbon dioxide (see Note 2).

B. Perfusion of the Piece of Liver

1.A piece of liver with only one surface cut with a scalpel should be obtained.

2.Place the piece of liver on the porous plate of a Büchner funnel (Fig. 1C).

3.Open the tubing clamps on each of the eight outflow tubings(Fig. 1C) and prime the perfusion system with Perfusion Solution 1.

4.Pause the peristaltic pump and close the tubing clamps on the eight outflow tubings.

5.Choose a cannula with an appropriate gauge that can fit snugly into the largest available open blood vessel on the cut surface of the liver and attach it to one of the tubings.

6.Open the tubing clamp on the tubing and ensure that Perfusion Solution 1 is flowing through the irrigation cannula at a low rate (see Note 3).

7.Push the tip of the irrigation cannula into the blood vessel. The olive tip should fit tightly into the blood vessel and hold the cannula in place. A part of the liver perfused by the vessels branching from the cannulated vessel will lighten as blood is flushed out.

8.Repeat steps 5–7 until the largest blood vessels are cannulated and the piece of liver is lightened throughout (see Note 4). The smallest blood vessels should not be cannulated so that perfused solutions can drain out of the liver.

9.Once all the cannulas are in place, increase the flow rate from the peristaltic pump gradually until there is a slight plumping of the piece of liver.

10.Cover the piece of liver with a piece of gauze soaked in saline to keep it moist.

11.Perfuse with 1 L of Perfusion Solution 1 to flush out any remaining blood.

12. Perfuse with Perfusion Solution 2 for 10 min.

13.Perfuse with 0.5 L of Perfusion Solution 3.

14.Carry out recirculating perfusion with Digestion Solution 9–12 min or until the liver is sufficiently digested (see Note 5).

Figure 1. Perfusion setup. (A) gas cylinder containing a mixture of 95% oxygen and 5% carbon dioxide, (B) bubble trap, (C) a piece of liver perfused using olive-tipped irrigation cannulas, (D) glass coil heat exchanger, (E) gas-permeable tubings for the oxygenation of solutions, (F) thermostated circulating water bath and (G) peristaltic pump

C. Hepatocyte Isolation

1.Turn off the peristaltic pump and remove the cannulas from the piece of liver.

2.Put the piece of liver in a crystallizing dish containing Hepatocyte Suspension Solution.

3. Carefully remove the Glisson’s capsule.

4.With a pair of forceps, gently shake the digested piece to release isolated cells into the Hepatocyte Suspension Solution. It may be necessary to cut through regions that are not well perfused to release more cells trapped within. More Hepatocyte Suspension Solution may be required during the process for ease of suspension. However, try not to use more than 400 mL of Hepatocyte Suspension Solution in total.

5.Filter the cell suspension through a 210 μmnylon mesh.

6.Next, filter the cell suspension through a70 μmnylon mesh.

7.Divide the filtered cell suspension into two200 mL centrifuge tubes.

8.Centrifuge the cell suspension at 72×g for 5 min at 4 ℃.

9.Without disturbing the loose hepatocyte pellet, transfer as much of thesupernatant as possible into 50 mL centrifuge tubes.

10.Resuspend the hepatocyte pellet in 200 mL of Hepatocyte Suspension Solution.

11.Spin the hepatocyte suspension at 72×g for 5 min at 4 ℃.

12.Aspirate off the supernatant.

13. Repeat washing steps 10–12 three times.

14.On the last wash, resuspend cells in Hepatocyte Medium.

15.Count cells and determine viability using trypan blue exclusion.

Notes

1.The temperature of solutions exiting the irrigation cannulas in Fig. 1Cshould be 37 ℃. Thus, depending on the length of the tubings, the water bath may need to be set at a higher temperature. In our setup, our water bath is set to 41 ℃ to warm all the Perfusion Solutions, but not the Digestion Solution. If the temperature of the water bath has to be set much higher than 37 ℃, consider shortening the connecting tubings. Our Digestion Solution containing collagenase is warmed in a separate water bath set at 37 ℃ to reduce the loss of collagenase activity before the perfusion of a piece of liver.

2.Make sure that the gas cylinder is properly secured before use. We recommend the use ofa two-stage regulator. The instructions for the use of the particular regulator chosen should be read before use.

3.Perfusion Solution 1 should be flowing through the cannula during insertion to prevent air embolism, which can reduce or block the perfusion of the liver through the blood vasculature.

4.The extent of the perfusion can be observed in this step. A good perfusion is critical for achieving a high yield of isolated cells. Try to cannulate in such a way that as much of the piece of liver is flushed with Perfusion Solution 1 as possible. In some cases, depending on the vasculature available, it may be challenging to get a complete perfusion of the piece of liver.

5.The liver is sufficiently digested when the tissue under the Glisson’s capsule starts to visually break apart. In addition, the liver should feel softened when probed with the blunt edge of a scalpel. A good digestion would increase the yield of isolated cells.

References

1.Lee, S.M.L., Schiergens, T.S., Demmel, M., Thasler, R.M.K., Thasler, W.E. (2017). Isolation of Hepatocytes and Stellate Cells from a Single Piece of Human Liver. In: Stock, P., Christ, B. (eds) Hepatocyte Transplantation. Methods in Molecular Biology, vol 1506. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6506-9_17

2.Lee SM, Schelcher C, Demmel M, Hauner M, Thasler WE. Isolation of human hepatocytes by a two-step collagenase perfusion procedure. JVis Exp. 2013 Sep 3;(79):50615. doi: 10.3791/50615. PMID: 24056912; PMCID: PMC3857361

3.Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol. 1976;13:29-83. doi: 10.1016/s0091-679x(08)61797-5. PMID: 177845

Manufacturer’s Link

Thermo Fisher Scientific: https://www.thermofisher.com › brands › gibco

Sigma-Aldrich: https://www.sigmaaldrich.com

Beckman Coulter: https://www.beckmancoulter.com

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