A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.
The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77±10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.
A liver cell suspension can be prepared from the liver by mechanical or enzymatic methods. Mechanical methods used to prepare whole liver cells include forcing the liver through cheesecloth 2, shaking a liver piece with glass beads in a Kahn shaker 3, using glass homogenizers with loose pestles 4,5 etc. Over the years, mechanical methods have fallen out of favor due to the damage to cell membranes and the loss of function of the isolated hepatocytes 6,7. Consequently, the use of an enzymatic method is currently the main method for isolation of hepatocytes.
Isolation of hepatocytes using an enzymatic method was greatly improved when Berry and Friend 8 perfused collagenase and hyaluronidase through the liver via the portal vein in rats. This perfusion process utilized the vasculature to allow the enzymes to come into close contact with the majority of the cells, leading to a 6-fold increase in yield of hepatocytes 8. Further, this method yielded cells that retained their structural integrity, with virtually no transformation of endoplasmic reticulum into isolated vesicles and no mitochondrial damage 8.
This method was modified by Seglen 1, who pioneered a two-step perfusion procedure for liver cell isolation. In this procedure, the rat liver is perfused with a Ca2+ free buffer followed by perfusion with a collagenase buffer containing Ca2+ 1. The removal of Ca2+ in the first step helps to disrupt desmosomes, while the addition of Ca2+ in the second step is required for optimum collagenase activity 1,9.
Given that the published work described above has been performed in rats, this article aims to demonstrate a modified procedure that can be used for isolation of hepatocytes with high viability from human livers. The use of human hepatocytes remains important for translational research and for validating experiments using animal models. The human liver pieces used in this study were acquired with consent for governance through the Human Tissue and Cell Research Foundation, a state-controlled non-profit foundation 10. After a pathologist removed what was required for diagnosis, liver pieces were collected from the remaining tissue. The tissue sectioned off by the pathologist was morphologically healthy tissue obtained from resection margins after liver resection.
1. Preparation of Perfusion and Isolation Solutions
2. Preparation of Perfusion Equipment and Solutions
3. Perfusion of the Liver
4. Isolation of Hepatocytes
Note: In this case, the trypan blue dilution factor is 10 and the hemocytometer factor is 10,000.
Perfusion Setup
The equipment required for liver perfusion should be set up according to Figure 1.
Viability and Yield of Isolated Human Hepatocytes
The average viability of isolated human hepatocytes was 77±10% and the average yield of hepatocytes was 13±11 million hepatocytes/g liver, with values expressed as means ± standard deviation. The number of hepatocyte isolations carried out to obtain these averages was 648 isolations carried out from January 1999 to December 2012.
Suitable Perfusion Parameters
In order to carry out a successful flushing and perfusion of the liver, the number of cannulae used should vary according to the weight of the liver (Figure 2A). In general, 4-8 cannulae should be used for livers ranging from below 20 g to over 80 g. A suitable rate of perfusion, which is also dependent on liver weight, should be chosen for a successful perfusion of the liver (Figures 2B and C). It has been found that an average perfusion speed of 44 ml/min cannula-1 is ideal across a range of different liver weights and therefore the perfusion speeds should be adjusted appropriately if more cannulae are used. If perfusion is successful, the liver should be pale in color and slightly plumped up.
Purity of Hepatocytes
By means of immunofluorescence, it was found that isolated hepatocytes, which stain positively for albumin, had a purity of 94±1% (N = 4 with 5 replicates each) (Figures 3A and B). Figure 3C is a representative phase contrast image showing the morphological characteristics of hepatocytes such as large cell size and polygonal shaped-cells.
Figure 1. Perfusion setup. (A) bubble trap, (B) liver piece with curved irrigation cannulae with olive tips inserted in blood vessels on a Büchner funnel, (C) glass jacketed condenser, (D) water bath, (E) oxygenation apparatus, (F) 95% O2/5% CO2 gas tank and (G) peristaltic pump.
Figure 2. (A) The number of cannulae used, (B) perfusion rate (ml/min), or (C) perfusion rate (ml/min.cannula) for various sizes of liver (g). Values represent means ± standard deviation with N = 25, 41, 18, 14 and 9 for 0-20 g, 20-40 g, 40-60 g, 60-80 g and >80 g liver respectively. aSignificantly different from the 0-20 g condition, P<0.05. abSignificantly different from the 20-40 g, 40-60 g and 60-80 g condition, P<0.05.
Figure 3. Immunofluorescent images of isolated cells positive for (A) albumin (stained in green) and the corresponding (B) negative control (200X magnification). Nuclei are stained in blue using DAPI. (C) Phase contrast image of isolated cells (100X magnification).
Solution | Constituent | Final Concentration |
Solution 1 | Sodium chloride | 154 mM |
HEPES | 20 mM | |
Potassium chloride | 5.6 mM | |
Glucose | 5 mM | |
Sodium hydrogen carbonate | 25 mM | |
Solution 2 | Sodium chloride | 152.5 mM |
HEPES | 19.8 mM | |
Potassium chloride | 5.5 mM | |
Glucose | 5.0 mM | |
Sodium hydrogen carbonate | 24.8 mM | |
EGTA | 1 mM | |
To prepare Solution 2, add 10 ml of 100 mM EGTA to 990 ml of Solution 1. | ||
Solution 3 | Sodium chloride | 152.5 mM |
HEPES | 19.8 mM | |
Potassium chloride | 5.5 mM | |
Glucose | 5.0 mM | |
Sodium hydrogen carbonate | 24.8 mM | |
Calcium chloride dihydrate | 5 mM | |
To prepare Solution 3, add 10 ml of 0.5 M calcium chloride dihydrate to 990 ml of Solution 1. | ||
Solution 4 | Sodium chloride | 152.5 mM |
HEPES | 19.8 mM | |
Potassium chloride | 5.5 mM | |
Glucose | 5.0 mM | |
Sodium hydrogen carbonate | 24.8 mM | |
Calcium chloride dihydrate | 5 mM | |
Collagenase | See Table 2 | |
To prepare Solution 4, add appropriate amount of collagenase to Solution 3. | ||
Solution 5 | Sodium chloride | 120 mM |
HEPES | 10 mM | |
Calcium chloride dihydrate | 0.9 mM | |
Potassium chloride | 6.2 mM | |
Albumin | 0.1% w/v |
Table 1. Perfusion and isolation solutions.
Size of liver piece (g) | Collagenase concentration (%) | Collagenase activity (U/ml) |
<25 | 0.10 | 250 |
25 – 40 | 0.11 | 300 |
41 – 80 | 0.13 | 350 |
>80 | 0.15 | 400 |
Table 2. Collagenase concentrations (%) and activities (U/ml) to be used for various sizes of liver pieces (g).
This protocol results in the isolation of human hepatocytes with high viability and purity. In order to achieve these results, it is important to start with a suitable piece of liver. The piece of liver should have intact Glisson’s capsule on all surfaces except for 1 cut surface. Another important factor is the particular batch of collagenase used, as different batches can result in marked differences in viabilities of hepatocytes after digestion 11. Therefore, different batches of collagenase should be tested and the batch that produces hepatocytes with the best viability should be obtained in large quantities. Finally, a suitable digestion time has to be chosen based on the yield and viability of the cells obtained. For example, a high viability with low yield could indicate an insufficient digestion time, and a high yield with low viability could indicate that the digestion time is too long.
This method can be adapted to isolate non-parenchymal cells and hepatocytes from the same liver piece. One way of doing this is to use the supernatant from the first centrifugation step (step 4.6) directly for non-parenchymal cell isolation 12. A second way is to remove the required aliquot of cell suspension after filtration through the nylon mesh for hepatocyte isolation (step 4.5) and subject the remaining cell suspension to an additional pronase digestion step before non-parenchymal cell isolation in order to increase the yield of non-parenchymal cells 13. For a higher yield of non-parenchymal cells at the expense of the hepatocytes, this method can be modified by substituting collagenase alone (step 3.10) for pronase and collagenase and using the resultant cell suspension for non-parenchymal cell isolation 14.
In addition to isolating human hepatocytes, this method presented can also be adapted to isolate hepatocytes from liver pieces collected from other species with comparable sizes or comparable vasculature sizes. This may be important for researchers who use alternative models, such as porcine, canine or primate models.
Isolations of human hepatocytes are generally done using livers from two sources: whole livers deemed unsuitable for transplantation or morphologically normal liver tissue from resection margins. The advantage of the latter source used in this method is that the liver pieces are made available to the laboratory sooner. Immediately after resection, the resected liver is brought to a pathologist who removes what is required for diagnosis. The pathologist will then remove a suitable piece of morphologically normal liver for hepatocyte isolation from what is slated for discarding. In general, a liver piece arrives in the laboratory ready for perfusion in an average time of 56±29 min (N = 103). In comparison, whole livers that are not suitable for transplantation will only be released to the laboratory between 13±2 hr and 16±12 hr 15. In addition, due to ethical considerations and the shortage of donor livers, isolation of hepatocytes from whole livers that do not meet all of the criteria for transplant is avoided in the Eurotransplant region. This is because these livers could still be used for transplantation with extended donor criteria. Some studies have shown that maximizing patient access to transplantation results in decreased wait list mortality and satisfactory outcomes to selected recipients 16,17. Another advantage is that the cells obtained from liver resection pieces are isolated from morphologically healthy liver, while livers unsuitable for transplant can be steatotic, fibrotic or cirrhotic. As such, the method here results in a high yield of 13 ± 11 million hepatocytes per gram liver compared to the 0.7±0.3 million or 3±2 million hepatocytes per gram liver obtained by Baccarani, et al. 15 using cirrhotic or steatotic livers respectively. However, liver resection pieces result in a lower total yield of hepatocytes as the size of the piece available is generally small with a range from 2-250 g and an average of 37±29 g (N = 648).
In comparison to other groups, this protocol avoids the use of cyanoacrylate adhesives. Alexandre, et al. 18 found that the use of ethyl cyanoacrylate, a commonly used all-purpose adhesive, to cover the cut surfaces of the liver, results in an increased yield of hepatocytes from 3.5±0.7 to 6.0±1.6 million hepatocytes per gram liver with values expressed in means ± standard error of mean. In comparison, this protocol is able to achieve a yield of 13.5±0.4 million hepatocytes per g liver (expressed in means ± standard error of mean) without the use of cyanoacrylate adhesives. While cyanoacrylate adhesives have been used for wound closure 19,20, medical grade cyanoacrylate adhesives such as octyl cyanoacrylate and butyl cyanoacrylate can be expensive compared to ethyl cyanoacrylate. However, shorter chain derivatives such as ethyl cyanoacrylate have been found to be more histotoxic than longer chain derivatives 21,22.
This method is simpler and more time efficient due to the use of curved irrigation cannulae with olive tips. The use of cannulae of an appropriate size will result in a tight fit that holds the cannulae in place without the use of glue 18 or sutures 23. The cannulae used have diameters ranging from 1-2 mm with tip diameters sized from 1.25-4.5 mm. An assortment of various cannula sizes should be made available when a liver piece is ready for cannulation, so that the appropriate sized cannula for a particular blood vessel can be chosen as shown in the accompanying video. This method also utilizes more cannulae in general (Figure 2A) compared to other studies where 2 23 or 2-4 18 cannulae are used. This may help to achieve a better perfusion throughout the liver piece leading to high viability and yield in a shorter digestion period.
In conclusion, this protocol isolates human hepatocytes, which are an important model for studying cellular metabolism, pharmacokinetics and toxicology of xenobiotics, liver regeneration and translational research. A previous survey on 150 drugs that cause human toxicity showed that the concordance between toxicity found in animal studies and that observed in clinical practice is 70% 24. Thus, human hepatocytes remain an important model for validating research done in animal models and for testing drug leads for adverse reactions before going to clinical trials. Further, the use of human hepatocytes isolated from remnant liver samples or hepatocytes isolated from slaughterhouse animals 25 as a model is in line with the 3R ethical framework 26 to replace the use of research animals when possible.
The authors have nothing to disclose.
This work was made possible by the Human Tissue and Cell Research Foundation, which makes human tissues available for research. Financial support for this work was received from the Federal Ministry of Education and Research (grant name: Virtual Liver Network, grant number: 0315759) and Hepacult GmbH. Our thanks also go to the technical assistants from the Grosshadern Hospital Tissue bank for the collection of the liver samples and the technical assistants from the Cell Isolation Core Facility for carrying out the liver perfusion and hepatocyte isolation. In particular, we would like to thank Natalja Löwen for demonstrating this procedure in the video. Finally, we would like to thank Natalja Löwen and Edeltraud Hanesch for creating the illustrations for Figure 1 and the figures in the schematic overview of the video.
Name of Reagent/Material | Company | Catalog Number | Comments |
Bubble trap | Gaßner Glastechnik | ||
Glass jacketed condenser | Gaßner Glastechnik | ||
41 °C Water bath | Julabo | 35723-H24/EG | |
37 °C Water bath | GFL | 1083 | |
Compressed gas cylinder (95 % O2/5 % CO2) | Linde | ||
Gas permeable tubing | Neolab | 2-4440 | |
Peristaltic pump | Ismatec | IP65 | |
Scalpel | Feather | 320010 | |
Forceps | Omnilab | 5171014 | |
Conical flasks 1 L | Schott Duran | 2121654 | |
Conical flasks 5 L | Schott Duran | 2121673 | |
Beakers | Schott Duran | 2110654 | |
200 ml centrifuge tubes | Becton Dickinson | 352075 | |
Crystallizing dish | Omnilab | 5144063 | |
Curved irrigation cannulae with ball tips | Ernst Kratz GmbH | 1464LL/ 1465LL A+B/ 1472LL | |
Micro vascular clamps | Ernst Kratz GmbH | ||
Büchner funnel | Carl Roth | HT38.1 | |
Nylon mesh 210 μm | Neolab | 4-1413 | |
Nylon mesh 70 μm | Neolab | 4-1419 | |
0.22 μm sterile filters | Peske | 99505 | |
500 ml bottles | Schott Duran | 2180144 | |
1 L bottles | Schott Duran | 2180154 | |
Hemocytometer | Peske | 06-0001 | |
1.5 ml tubes | Eppendorf | 0030 120,086 | |
50 ml conical tubes | BD Biosciences | 352070 | |
Ice bucket | Neolab | 1508454 | |
Sterile Pasteur pipettes | Brand | 747715 | |
Motorised pipette filler (Pipette boy acu) | Integra | 155017 | |
Refridgerated centrifuge | Eppendorf | 5810R | |
Laminar flow | Kendro | Hera safe-KS9 | |
Aspirator (Low-flow surgical suction pump) | Atmos | C361 | |
Laboratory Gas Burner | Integra | Fire Boy eco | |
Disposable laboratory coat | Paperlynen GmbH | MD0202414 | |
Surgical mask with visor | Kimberly-Clark | 48247 | |
Surgical hood | Barrier | 42072 | |
Latex gloves | Semper Care | CE0321 | |
Collagenase (Batch number NB 4G) | Serva | 17465 | |
Calcium chloride dihydrate | Merck | 2382 | |
EGTA | Sigma | E4378 | |
Sodium chloride | Roth | 9265.2 | |
Hepes | Roth | 9105.3 | |
Potassium chloride | Serva | 26868 | |
Albumin | Biomol | 01400-2 | |
Glucose | Serva | 22700 | |
Sodium hydrogen carbonate | Serva | 30180 | |
0.4% Trypan blue solution | Lonza | 17-942E | |
Cold storage solution | Hepacult GmbH |