MOJ MOJBB

Bioequivalence & Bioavailability
Mini Review
Volume 3 Issue 1 - 2017
Pharmacokinetics and Isolated Hepatocyte
Javad Khalili Fard*
Pharmacology and Toxicology Department, Tabriz University of Medical Sciences, Iran
Received: August 20, 2016 | Published: January 31, 2017
*Corresponding author: Javad Khalili Fard, Faculty of Pharmacy, Pharmacology and Toxicology Department, Tabriz University of Medical Sciences, Daneshgah Street, Tabriz, Iran, Tel: +98-41-33372250-1; Email:
Citation: Fard JK (2017) Pharmacokinetics and Isolated Hepatocyte. MOJ Bioequiv Availab 3(1): 00023. DOI: 10.15406/mojbb.2017.03.00023

Abstract

Hepatic metabolism is an important contributor affecting the bioavailability of chemicals. Today isolated hepatocytes are widely used to study the biological assessment of xenobiotics and drug metabolisms. In this regard, methods have been developed to implement systems to evaluate in vitro biological properties of natural and synthetic compounds. This mini review will provide a brief introduction of two isolated hepatocyte preparation methods.

Keywords: Hepatocytes; Drug metabolism; Bioavailability; Xenobiotics

Introduction

Two main groups of collagen in the body which is present in an extracellular matrix have been identified: fibrillar (Type I, II, III, V, XI) and non-fibrillar collagen [1]. A mixture of collagens is embedded in the collagenous fibrillar network of the liver. Approximately 80% of the total hepatic collagen is made up of type I and type III collagens [2]. Moreover, these types of collagen are present in the renal interstitium and blood vessels [3]. By means of this structural protein the hepatocytes are attached to other neighboring hepatocytes. Knowledge about this key component has shaped the methods of isolation of not only hepatocytes but also other mammalian cells such as kidney glomeruli.

Clostridium species excrete collagenases which break the peptide bonds in collagen and contribute to its pathogenesis. This enzyme is widely used to isolate both parenchymal and non-parenchymal cells. Non-parenchymal cells and Kupffer cells could be isolated from liver by pronase and collagenase perfusion.

Hepatocyte isolation methods

Method 1: In this method liver is perfused continuously with SC-1 solution at 37°C for 5 min followed by 15 min perfusion with 0.03% collagenase which is prepared by dissolving in SC-2 solution (Table 1). After these perfusion procedures, the liver is cut, and the cells are suspended in Geys balance salt solution (GBSS). The cell suspension is filtered using a steel mesh and centrifuged at 50 g for 1 min. The cell pellet was resuspended in GBSS solution, and the washing procedure was repeated three times [4].

Ph 7.25 (All in Mg/L)

SC-1 Solution

NaCl

KCl

NaH2PO4•H2O

Na2HPO4

HEPES

NaHCO3

EGTA

Glucose

8,000

400

88.7

120.45

2,380

350

190

900

SC-2 Solution

NaCl

KCl

NaH2PO4•H2O

Na2HPO4

HEPES

CaCl2·2H2O

8,000

KCl

88.7

120.45

2,380

560

Geys Balance Salt Solution

NaCl

KCl

MgCl2·6H2O

MgSO4·7H2O

NaH2PO4

KH2PO4

Glucose

NaHCO3

CaCl2·2H2O

8,000

370

210

70

120

30

991

227

225

Table 1: List of Chemicals Used in Method 1.

Method 2: Calcium-free Hanks balanced salt solution with HEPES (HBSSH) is prepared by combining 100 ml of each stock solution (A,B,C,D) with 600 ml deionized, distilled water and pH is adjusted to 7.5 by adding NaOH. Deionized, distilled water is added to each stock up to 1L (Table 2).

All in G/L

Stock A

Stock B

Stock C

Stock D

Glucose

MgCl2·6H2O

MgSO4·7H2O

NaCl

KCl

KH2PO4

Na2HPO4

Phenol Red

HEPES

10 g

1 g

1 g

80 g

4 g

0.6 g

0.49 g

0.1 g

48 g

Collagenase Stock Buffer (pH 7.4)

NaCl

KCl

CaCl2.2H2O

HEPES

NaOH (1N)

3.9 g

0.5 g

0.7 g

24 g

66 ml

Table 2: List of Chemicals Used in Method 2.

To prepare 1% collagenase buffer solution dissolve 50 mg collagenase type I in 50 ml collagenase stock buffer. To obtain non parenchymal cells pronase which effectively digest parenchymal cells could be used. After these procedures the liver is embedded in a dish containing HBSSH. The cell suspension is centrifuged at 50 g for 4 min at 4°C. Then supernatant is discarded and cells are resuspended in fresh medium [5].

Conclusion

Isolated hepatocytes provide a suiTable  tool which facilitates evaluating not only chemical metabolisms but also the effects of drugs on organelles in both human and animal cells. In various studies cells viability was between 85% and 97% [6]. Since in method 1 and 2 the weight/volume percentage concentration of the collagenase solution is respectively 0.03 and 1 and the washing time period differ in both methods, it is suggested that the efficacy of different methods should be simultaneously assessed in a similar experiment.

References

  1. Fratzl P (2008) Collagen: Structure and Mechanics: Springer, USA.
  2. Aycock RS, Seyer JM (1989) Collagens of normal and cirrhotic human liver. Connect Tissue Res 23(1): 19-31.
  3. Yoshioka K, Tohda M, Takemura T, Akano N, Matsubara K, et al. (1990) Distribution of type I collagen in human kidney diseases in comparison with type III collagen. J Pathol 162(2): 141-148.
  4. Tamaki N, Hatano E, Taura K, Tada M, Kodama Y, et al. (2008) CHOP deficiency attenuates cholestasis-induced liver fibrosis by reduction of hepatocyte injury. Am J Physiol Gastrointest Liver Physiol 294(2): G498-G505.
  5. Charles AT, John MF (2016) In Vitro Biological Systems: Methods in Toxicology. Elsevier, pp. 592.
  6. Carvalho M, Milhazes N, Remião F, Borges F, Fernandes E, et al. (2004) Hepatotoxicity of 3, 4-methylenedioxyamphetamine and α-methyldopamine in isolated rat hepatocytes: formation of glutathione conjugates. Arch Toxicol 78(1): 16-24.
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