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28DOI: 10.5281/zenodo.2589998 UDC: 611.36.018:57.085.2
Hepatocytes isolation from adult rats for liver recellularization
*Mariana Jian, BioD; Vitalie Cobzac, MD, PhD Applicant; Victoria Vartic, MD; Viorel Nacu, MD, PhD, Professor
Tissue Engineering and Cells Cultures Laboratory Nicolae Testemitsanu State University of Medicine and Pharmacy, Chisinau, the Republic of Moldova
^Corresponding author: [email protected] Manuscript received January 10, 2019; revised manuscript March 01, 2019
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Abstract
Background: Currently hepatocytes obtaining is prerequisite to create the necessary conditions for medical research, because it is an important tool in developing of new strategies in tissue engineering domain, which represents obtaining functional organs in laboratory conditions. Material and methods: The study was made on adult Wistar rats liver with body weight 274.66± 2.52 g (n=3) which were used for hepatocytes extraction by perfusion through the upper cave vein with combination of type II collagenase and type I dispase and Hank's 0.9 mM MgCl2, 0.5 mM EDTA and 25 mM HEPES (HiMedia, India).
Results: The cells were counted with trypan blue 0.25% in hemocytometer and cultured in William's E medium (HiMedia, India) with 2 mM L-glutamine, 5% fetal bovine serum (Lonza, Belgium), antibiotic antimycotic solution (HiMedia, India), 100 nM dexamethasone and 100 nM insulin, with 2.5 x 105 cells per well in 12-well plates. After isolation were obtained 324, 48 ± 1, 25 x 106 hepatocytes, with a viability of 94.7 ± 0.9 % which indicates a high yield of cells viability.
Conclusions: The hepatocyte isolation method by liver perfusion with the combination of collagenase-dispase is feasible for obtaining a large amount of functional hepatocytes intended for the recellularization in vitro of decellularized liver scaffolds. The yield and viability of hepatic cells could be increased by enzymatic digestion of liver tissue using combination of collagenase/dispase solution due to the less cytotoxic effect. Key words: hepatocytes, cell separation, cell survival, collagenases, dispase, in vitro techniques.
Introduction
Liver is the organ with a large functional metabolic profile, being a subject of an impressive number of biochemical investigations. Its main function in the body is the transformation of nutrients into physical and chemical forms that are used in the body, and on the other hand they are excreted. What is important is that the liver functions in the liver are only performed by parenchymal cells (hepatocytes). They constitute 90 - 95%, the majority, from the total weight of the other types of cells and 2/3 of the total cell population. This relative morphological uniformity of the liver as well as the linkage between hepatocytes with its organo-specif-ic function make it possible to use parenchymal cells as a model for investigating various biochemical, biophysical, pharmacological and physiological processes that happen in the liver [1, 2, 3, 4]. The sources of hepatocytes may be from the liver that has been rejected after transplantation, portions of liver resected from patients with cirrhosis or other hepatic diseases and portions of liver resected from healthy patients [5, 6, 7, 8, 9, 10]. The hepatocytes isolated from rats, mice or human liver are a useful technique for studying liver function in vitro as well as for the recellularization of decellularized hepatic scaffolds. Also, the hepatocytes obtained after isolation have a major utility to be transplanted, as cell therapy of hepatic diseases for correction of metabolic disorders of the liver in the absence of donors liver for transplantation [11, 12, 13, 14, 15, 16, 17, 18].
Due to multitude possibilities for using isolated hepato-cytes, there are various hepatocyte isolation protocols. What
is important, is that they should be allowed for obtaining a higher number of hepatocytes with good viability and purity, preservating in vitro all functions specific for them in vivo [19].
The aim of our study is the isolation and cultivation of viable hepatocytes in large amounts from the liver of an adult rat for the in vitro liver recellularization.
Material and methods
The study was done on the liver from Wistar adult rats with body mass 274.66±2.52 g from which the hepatocytes were extracted. Before the isolation process, the substrate for hepatocytes adhesion was prepared. With 3-4 hours prior to initiating the experiment the cell culture surfaces of the culture plates or culture flasks were coated in a thin layer with Type I collagen solution at a concentration of 40 |ig / ml. They were subsequently washed with HBSS (Hanks' balanced salt solution) without calcium and Mg (HiMe-dia, India) and allowed to dry in a Nuve 090 laminar flow hood. The hepatocytes were isolated by superior cava vein perfusion with 0.05% type I collagenase (HiMedia, India), 0.1% type I dispase (HiMedia, India) and HBSS without calcium and Mg (HiMedia, India) with 0.9 mM MgCl2, 0.5 mM EDTA (Ethylenediaminetetraacetic acid) and 25 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HiMedia, India) after washing the intrahepatic blood vessels with Buffer I solution. Prior to initiating the hepato-cyte isolation process Buffer I and II solutions were heated
in the water bath at 42° C. The work surface was sterilized with 70% alcohol after which it was left under UV rays for at least 15 minutes. During the perfusion process a 75 watt incandescent light lamp was set at 25-30 cm above the perfused liver area to maintain the temperature at around 37° C, in order to avoid hepatocytes death caused by temperature lowering during liver perfusion with solutions which become colder. Also, to maintain temperature of perfused liver, to prevent drying of its surface and to remove unwanted particles that precipitate from the air, with a syringe the liver surface was wetted continuously with warm 0.9% NaCl solution.
The isolated hepatocytes were counted with triptan blue in the hemocytometer and the biochemical parameters such as ALT (Alanine aminotransferase), glucose, total protein and glucose-6-phosphatase were determined with the Eli-tech kit.
Results
Isolation of hepatocytes from the liver of adult rat is required to study the recellularization liver process in vitro. Prior to sacrificing the animals 5000 IU of heparin was injected intraperitoneally. The general anesthesia was performed with 60 mg/kg ketamine and 5 mg/kg xylazine, then was removed the fur with a trimmer and it was processed with 70% alcohol. With scissors, the toracoabdominal wall was removed and a suprahepatic portion of the inferior cave vein was channeled with 18 G plastic catheter during persisting cardiac contractions, for keeping hepatocytes alive. We used a two step perfusion by collagenase/dispasse solution for hepatocytes isolation from adult Wistar rat liver through the superior vena cava with Buffer I solution (Hank's 0.9 mM MgCl2, 0.5 mM EDTA and 25 mM HEPES) through a 0.22 mm filter for 4-5 minutes at a speed of 15 -20
ml/min. At the same time the inferior vena cava was ligated and the portal vein was sectionated and every 2 minutes it was clipped and released for decompression. This procedure is repeated 6-8 times (fig. 1). The second step was performed by Buffer II solution (collagenase/dispase) liver perfusion at a rate of 25-30 ml/min until it became flaccid, it took about 10-12 minutes, with clamping and realising of the portal vein every 2-3 minutes. Later the liver was extracted from the abdominal cavity and underwent mechanical disintegration for hepatocyte release (fig. 2). It was placed under a laminar flow hood in a Petri box with William E and 5% FBS nutrient solution placed on the ice for 5 minutes and was removed the liver capsule and the hepatocytes were released by shaking. With a pipette the released cells were collected and filtered through a 100 ^m strainer placed on a 50 ml tube. After isolation, was appreciated the cells viability, that was 92%. Some of them were used for biochemical parameters tests, and some were preserved to be used in recel-lularization of decellularized liver matrices.
The cells were counted with 0.25% blue trypan solution in haemocytometer and apreciated the cellular viability. Then the cells were cultured in William E (HiMedia, India) medium with 2 mM of L-glutamine, 5% fetal bovine serum (Lonza, Belgium), antifungal antibiotic solution (HiMedia, India), 100 nM deaxmethasone and 100 nM insulin. The cells were seeded in 12-well plates by 2.5 x 105 cells per well and incubated at 37°C with 5% CO2. Following the hepatocytes isolation were obtained a total of 324, 48±1, 25 x 106 cells with a viability of 94.7± 0.9%, indicating a high yield of viable cells (fig. 3).
The hepatocyte isolation is a very delicate process. During the hepatocyte isolation, it is necessary to obtain a maximum number of cells and to preserve their morphology and functionality. The biochemical parameters of the hepa-
Fig. 1. The perfusion of the liver through upper cave vein.
A - with Hank's solution with 0.9 mM MgCl2, 0.5 mM EDTA and 25 mM HEPES at a rate of 15-20 ml/min, B - with Hank's solution and type II collagenase / type I dispase displacement with speed of 25 ml/min.
Fig. 3. Liver cells visualized with phase-contrast inverted microscope KZD: A - x10, B - x40.
tocytes that were analyzed show gluconeogenesis process, glucose-6-phosphatase, which is found only in hepatocytes and alanine aminotransferase, and as well as the content of glucose and total protein (tab. 1).
Table 1
Functional - metabolic properties of isolated hepatocytes in culture
Biochemical parameters Catalytic activity
ALT, u / L 17.83 ±1.06
Glucose-6-phosphatase, u / L 3.41±0.46
Biochemical parameters Concentration
Glucose, mM / L 4.16±0.10
Total protein, g / L 61.86±2.09
Discussion
Since hepatic tissue for liver isolation is limited, improvements in hepatocyte production and cryopreserva-tion protocols are necessary to maintain cell viability during cultivation over a longer period of time and to prevent the hepatocyte number reduction after thawing [20, 21, 22].
According to the literature, there are many protocols for the isolation of hepatocytes with high viability and purity,
but they depend on the type of collagenase used which determines significant differences in hepatocyte viability after digestion [23].
In the majority of the methods, collagenase is used to isolate hepatocytes from liver tissue. But Ricky H. Bhogal et al. used a combination of collagenase / protease / hyaluroni-dase and deoxyribonuclease [24].
Another study by MN. Berry shows that after buffered perfusion containing 0.05% collagenase and 0.10% hyal-uronidase the isolated liver cells are viable and the form and function correspond to liver cells in situ with the presence of cytoplasmic vacuolization in a low number of cells and loss of potassium that are the only signs of cell lesion [25]. In another study in which the hepatocytes were isolated only by collagenase they showed a viability of 53% [26].
An effective change in our method was the insignificant reduction of collagenase concentration in 100 ml of buffer with approximately 400U to diminish, even insignificantly, the cytotoxic effect of collagenase on hepatocytes and the addition of solution of Dispase I enzyme which is not so cytotoxic at a concentration of 1U/ml.
In the hepatocytes grown medium were added supplements necessary for effective cellular respiration, since he-patocytes are highly specialized cells with intense biochemi-
Ш I
cal activity requiring high energy consumption for protein, lipid and carbohydrate metabolism. These are: Na, selenium, transferin, bovine albumin, dexamethasone, aminoplasmol, hepatic amino acid complex, growth factors for hepatocytes and fibroblasts. It is important to note that the nutrient medium used for hepatocyte cultivation was changed every 2 days. During the medium changing within the first 4 hours, it should be taken into account that the hepatocytes are fragile and can easily be damaged by direct contact, so they are only pipetted from the side of the well.
Also is very important the supply of oxygen solutions, for which the oxygenator hose was introduced during the infusion first in Buffer I, and then in Buffer II solutions. It was programmed at 0.5 L/min. After strict following the listed steps, the viability of the hepatocytes is 94.7 ± 0.9%, compared to cell viability of 10% to 85%, which was obtained for use only for the detachment of hepatocytes from collagenase.
Conclusions
1. The high hepatocyte viability in dynamics is a priority and the sustained hepatocyte growth is determined by adding supplements necessary for effective cellular metabolism in the cell culture medium used.
2. The hepatocyte isolation method by liver perfusion with the combination of collagenase-dispase is feasible for obtaining a large amount of functional hepatocytes intended for the recellularization in vitro of decellularized liver scaffolds.
3. The yield and viability of hepatic cells could be increased by enzymatic digestion of liver tissue using combination of collagenase/dispase solution due to the less cyto-toxic effect.
References
1. Hang H, Shi X, Gu G, Wu Y, Ding Y. A simple isolation and cryopreser-vation method for adult human hepatocytes. Int J Artif Organs. 2009 Oct;32(10):720-7.
2. Li WC, Ralphs KL, Tosh D. Isolation and culture of adult mouse hepatocytes. Methods Mol Biol. 2010;633:185-96. doi: 10.1007/978-1-59745-019-5_13.
3. Sasaki K, Kon J, Mizuguchi T, Chen Q, Ooe H, Oshima H, Hirata K, Mitaka T. Proliferation of hepatocyte progenitor cells isolated from adult human livers in serum-free medium. Cell Transplant. 2008;17(10-11):1221-30.
4. Wallace K, Fairhall EA, Charlton KA, Wright MC. AR42J-B-13 cell: an expandable progenitor to generate an unlimited supply of functional hepatocytes. Toxicology. 2010 Dec 30;278(3):277-87. doi: 10.1016/j.tox.2010.05.008. Epub 2010 Jun 1.
5. Akhter J, Johnson LA, Gunasegaram A, Riordan SM, Morris DL. Hepatocyte transplantation: a review of laboratory techniques and clinical experiences. Surgeon. 2007 Jun;5(3):155-64.
6. Alexandrova K, Griesel C, Barthold M, Heuft HG, Ott M, Winkler M, Schrem H, Manns MP, Bredehorn T, Net M, Vidal MM, Kafert-Kasting S, Arseniev L. Large-scale isolation of human hepatocytes for therapeutic application. Cell Transplant. 2005;14(10):845-53.
7. Baccarani U, Sanna A, Cariani A, Sainz-Barriga M, Adani GL, Zam-bito AM, Piccolo G, Risaliti A, Nanni-Costa A, Ridolfi L, Scalamogna M, Bresadola F, Donini A. Isolation of human hepatocytes from livers rejected for liver transplantation on a national basis: results of a 2-year experience. Liver Transpl. 2003 May;9(5):506-12.
I U6
8. Hsu HC, Matsuno N, Machida N, Enosawa S. Improved recovery of hepatocytes isolated from warm ischemic rat liver by citrate phosphate dextrose (CPD)-supplemented Euro-Collins solution. Cell Med. 2013 May 14;5(2-3):97-101. doi: 10.3727/215517913X666521. eCollection 2013 Nov 10.
9. Sahi J, Grepper S, Smith C. Hepatocytes as a tool in drug metabolism, transport and safety evaluations in drug discovery. Curr Drug Discov Technol. 2010 Sep;7(3):188-98.
10. Tanaka K, Soto-Gutierrez A, Navarro-Alvarez N, Rivas-Carrillo JD, Jun HS, Kobayashi N. Functional hepatocyte culture and its application to cell therapies. Cell Transplant. 2006;15(10):855-64.
11. Baccarani U, Adani GL, Sainz M, Donini A, Risaliti A, Bresadola
F. Human hepatocyte transplantation for acute liver failure: state of the art and analysis of cell sources. Transplant Proc. 2005 Jul-Aug;37(6):2702-4.
12. Bartlett DC, Newsome PN. Hepatocyte cell therapy in liver disease. Expert Rev Gastroenterol Hepatol. 2015;9(10):1261-72. doi: 10.1586/17474124.2015.1073106. Epub 2015 Jul 27.
13. Iansante V, Mitry RR, Filippi C, Fitzpatrick E, Dhawan A. Human hepatocyte transplantation for liver disease: current status and future perspectives. Pediatr Res. 2018 Jan;83(1-2):232-240. doi: 10.1038/ pr.2017.284. Epub 2017 Dec 6.
14. Fox IJ. Hepatocyte transplantation. Gastroenterol Hepatol (NY). 2014 Sep;10(9):594-6.
15. Maruyama M, Totsugawa T, Kunieda T, Okitsu T, Shibata N, Takesue M, Kurabayashi Y, Oshita M, Nakaji S, Kodama M, Tanaka N, Kobayashi N. Hepatocyte isolation and transplantation in the pig. Cell Transplant. 2003;12(6):593-8.
16. Hamooda M. Hepatocyte transplantation in children with liver cell failure. Electron Physician. 2016 0ct;8(10):3096-101. doi: 10.19082/3096.
17. Terry C, Dhawan A, Mitry RR, Hughes RD. Cryopreservation of isolated human hepatocytes for transplantation: State of the art. Cryo-biology. 2006 0ct;53(2):149-59. Epub 2006 Jun 21.
18. Vidal I, Blanchard N, Alexandre E, Gandillet A, Chenard-Neu MP, Staedtler F, Schumacher M, Bachellier P, Jaeck D, Firat H, Heyd B, Richert L. Improved xenogenic hepatocyte implantation into nude mouse liver parenchyma with acute liver failure when followed by repeated anti-Fas antibody (Jo2) treatment. Cell Transplant. 2008;17(5):507-24.
19. Severgnini M, Sherman J, Sehgal A, Jayaprakash NK, Aubin J, Wang
G, Zhang, Chang L, Peng CG, Yucius K, Butler J, Fitzgerald K. A rapid two-step method for isolation of functional primary mouse hepato-cytes: cell characterization and asialoglycoprotein receptor based assay development. Cytotechnology. 2012 Mar;64(2):187-95.
20. Liu XL, Li LJ, Chen Z. Isolation and primary culture of rat hepatocytes. Hepatobiliary Pancreat Dis Int. 2002 Feb;1(1):77-9.
21. Mitry RR, Hughes RD, Dhawan A. Progress in human hepatocytes: isolation, culture & cryopreservation. Semin Cell Dev Biol. 2002 Dec;13(6):463-7.
22. Takesue M, Maruyama M, Shibata N, Kunieda T, Okitsu T, Sakaguchi M, Totsugawa T, Kosaka Y, Arata A, Ikeda H, Matsuoka J, Oyama T, Kodama M, Ohmoto K, Yamamoto S, Kurabayashi Y, Yamamoto I, Tanaka N, Kobayashi N. Maintenance of cold-preserved porcine hepatocyte function with UW solution and ascorbic acid-2 glucoside. Cell Transplant. 2003;12(6):599-606.
23. Lee SM, Schelcher C, Demmel M, Hauner M, Thasler WE. Isolation of human hepatocytes by a two-step collagenase perfusion procedure. J Vis Exp. 2013;(79). doi: 10.3791/50615.
24. Bhogal RH, Hodson J, Bartlett DC, Weston CJ, Curbishley SM, Haugh-ton E, Williams KT, Reynolds GM, Newsome PN, Adams DH, Afford SC. Isolation of primary human hepatocytes from normal and diseased liver tissue: a one hundred liver experience. PLoS One. 2011;6(3):e18222.
25. Berry MN, Friend DS. High-yield preparation of isolated rat liver parenchymal cells: a biochemical and fine structural study. J Cell Biol. 1969 Dec 1;43(3):506-20.
26. Mitry RR, Hughes RD, Aw MM, Terry C, Mieli-Vergani G, Girlanda R, Muiesan P, Rela M, Heaton ND, Dhawan A. Human hepatocyte isolation and relationship of cell viability to early graft function. Cell Transplant. 2003;12(1):69-74.
