EFFECT OF PERCOLL CENTRIFUGATION ON CELL VIABILITY AND ENZYME ACTIVITIES IN HUMAN CRYOPRESERVED PRIMARY HEPATOCYTE

Zhihong Zhang O’Brien*, Melanie Hann, W.Perry Gordon*, Yong Hee Lee, Kevin Holme and Julie Doerr-Stevens

1 Lion Bioscience, San Diego, CA 92121

ABSTRACT

The human cryopreserved hepatocyte (HCH) has been used in short-term drug metabolism and toxicity studies. To minimize the number of dead cells and optimize the metabolic enzyme activities in HCH system, percoll centrifugation has been applied to traditional cell processing procedure (rapid thawing rate, slow dilution rate and low temperature (4 o C) during cell processing) and the effect of percoll centrifugation on cell viability and enzyme activities has been investigated. Recently, more studies have done on further investigations of the relationships of cell viability, medium, temperature and cell dilution rate. After the hepatocytes were purified by 25% Percoll centrifugation using traditional cell processing procedure, 7-ethoxycoumarin (7-EC, 75  M), a substrate for enzyme activity assessment, was incubated in the presence of HCH (100,000 cells/well) for 0, 1, 2 and 4 hours. Cell viability was determined using Trypan Blue exclusion at 0, 1, 2 and 4 hours. Enzyme activities were assessed by monitoring % remaining of 7-EC and formation rates of 7-hydroxycoumarin (7-HC), 7-hydroxycoumarin glucuronide (7-HCG) and 7-hydroxycoumarin sulfate (7-HCS). Cellular viabilities, UDP glucuronosyltransferase (UDPGT) and sulfotransferase (ST) activities were all improved significantly (P< 0.05, t-test) by Percoll centrifugation. Percentage remaining of 7-EC showed a significant decrease (P< 0.05, t-test) by Percoll purification. The investigation of the relationships of cell viability, medium, temperature and cell dilution rate has been done using HCH (single and pooled donor). The results indicated that: 1) medium temperature and Percoll centrifugation are the two key factors in obtaining high cell viability of human cryopreserved hepatocyte after thawing; and 2) different types of media used during cell processing have no effect on the initial cell viability; but do effect post incubation cell viability. In conclusion, percoll centrifugation should be performed routinely for cryopreserved cell processing to optimize enzyme activities.

INTRODUCTION

Hepatocyte has been recognized as a powerful tool for acquiring quantitative and qualitative information of metabolism and toxicity of new chemical entities at early stage of drug development. It is a valuable

in vitro

experimental model with similar performance as

in vivo

, such as synthesis and secretion of plasma proteins, intact membrane transportation, and production and storage of energy. It possesses both the simplicity of what subcellular liver fractions have and the complex architectures of the intact liver.

Therefore, it is a compromise between convenience and relevance, which a good

in vitro

system should have. Hepatocytes can be easily isolated by two-step collagenase perfusion of liver. Therefore, the primary hepatocyte has been well established and widely used in toxicological and pharmacological studies. However, the main problem in all studies is the accuracy of interspecies extrapolation, especially from laboratory animals to human. Thus, the human hepatocyte is of high interest. But the major problems of human hepatocyte are encountered by usage of large amount of hepatocyte cells generated during isolation and its limited source. Cryopreservation enhances the long term preservation of isolated hepatocytes. Several laboratories have successfully developed and optimized the cryopreservation of freshly isolated hepatocytes in the past few years. However, the low post-thaw cell viability and decline of some Phase II enzyme activities are still problems encountered after cryopreservation. To circumvent these problems, Percoll centrifugation has been widely used for the improvement of post-thaw cell viability and Phase II enzyme activities. Recently, Xenotech, LLC, has demonstrated good post-thaw cell viabilities for their cryopreserved cells using a thawing protocol that is distinct from those traditionally utilized. Our lab further investigated the factors that affect post-thaw cell viability and the effect of Percoll wash on enzyme activities using 7-ethyoxycoumarin (7-EC) as a substrate.

METHODS

Experimental Design: (1) Effect of Percoll on cell viability and enzyme activities using traditional Protocol:

Human Cryopreserved Hepatocyte • Pooled donor: six donor pool (In Vitro technology, Baltimore, MD) Media • William E Medium (WME) Plate Format • 96-well plate Thawing Protocols • A: post-thaw cell processing at 4 o C, without Percoll wash • B: post-thaw cell processing at 4 o C, with 25% Percoll wash Cell Viability Determination • Trypan blue exclusion method • Cell viabilities at 0 min (pre-incubation), 30 min, 60 min, 120 min & 240 min (post-incubation) Enzyme Activity Determination • Using 7-Ethyoxycoumarin (7-EC) as a substrate at 75  M • Metabolic formation rates of 7-Hydroxycoumarin (7-HC), 7-Hydroxycoumarin Glucuronide (7-HCG) & 7-Hydroxycoumarin Sulfate (7-HCS) at 240 min post-incubation

(2) Effects of cell processing temperature, medium, and Percoll wash on improvement of post-thaw cell viability

Human Cryopreserved Hepatocytes • Single donor: Lot#63, Lot#51 (In Vitro technology, Baltimore, MD) • Cell viability reported from In Vitro Technology website:

Lot#63 (75%), Lot#51 (53%)

Media • Krebs-Hansleit Buffer (KHB), William E Medium (WME) & Xenotecch Cell Processing Medium (supplemented DMEM) Plate Format • 96-well plate Thawing Protocols • A: post-thaw cell processing at 4 o C, without Percoll wash • B: post-thaw cell processing at 4 o C, with Percoll wash • C: post-thaw cell processing at 37 o C, without Percoll wash • D: post-thaw cell processing at 37 o C, with Percoll wash Cell Viability Determination • Trypan blue exclusion method • Cell viabilities at 0 min (pre-incubation), 30 min, 60 min, 120 min & 240 min (post-incubation)

Table 1. Cell thawing procedures using Protocol-A, -B, -C & -D

Procedure Steps Pre-cell processing prep A B Procedure Steps Adding insulin, gentamycin & glutamine in WME or use KHB buffer Adding insulin, gentamycin & glutamine in WME Pre-cell processing prep C Two tubes of WME or KHB D One tube of WME (or KHB)+Percoll & one tube of WME (or KHB) only, or Xenotech Media Kit Prepare 90% isotonic Percoll solution in 10XKHB Cell thaw Suspension media Cryopreservant & dead cell clean-out pH supplemented WME, or KHB buffer pH supplemented WME, 90% Percoll & 1XKHB Keep all the solutions chilled (4 o C) Quick thaw (1.5min) at 37 o C Keep all the solutions chilled (4 o C) Quick thaw (1.5min) at 37 o C Cell thaw Keep them at 4 o C Keep them at 4 o C Suspension media Quickly transfer thawed cells into chilled 50ml conical Quickly transfer thawed cells into chilled 50ml conical tubes Add WME or KHB dropwisely into the cells at a speed of 12ml/4-5min tubes Add WME dropwisely into the cells at a speed of 12ml/4 5min Cryopreservant & dead cell clean-out Centrifuge the cells at 50xg for 5min to obtain cell pellet Centrifuge the cells at 50xg for 5min to obtain cell pellet Warm all the media up to 37 o C Warm all the media up to 37 o C Quick thaw (1.5min) at 37 o C Keep them at 37 o C Quick thaw (1.5min) at 37 o C Keep them at 37 o C Pour the cells into pre warmed media Pour the cells into pre warmed media Centrifuge the cells at 40-60xg for 5min Centrifuge the cells at 60-90xg for 5min Dead cell clean-out Percoll clean-out Resuspension in fresh incubation media Resuspend the cells in WME or KHB for cell count Suspend the cells in 25% Percoll-KHB solution and then centrifuge it at 100xg for 6min Supend the cells in fresh WME and then centrifuge it at 50xg for 5min Percoll clean-out or Suspend the cells in further cryopreservant fresh WME or KHB and clean-out then centrifuge it at 40 Resuspend the cells in WME for cell count Resuspension in fresh incubation media 60xg for 3min Resuspend the cells in WMEor KHB for cell count Suspend the cells in fresh WME or KHB and then centrifuge it at 40 60xg for 3min Resuspend the cells in WME or KHB for cell count

RESULTS (1) Effect of Percoll on cell viability and enzyme activities using traditional cell processing protocol:

90 80 70 60 50 40 30 20 10 0

*

0 min

* * *

60 min

Time

120 min 240 min Fig. 1. Post-thaw cell viabilities of human cryopreserved hepatocyte using traditional cell processing protocol, with (  ) and without (  ) Percoll wash. Data are expressed as the mean ± standard deviation of thirty-five experiments without Percoll wash and eight experiments with Percoll wash. *, significantly different from the corresponding value of without Percoll wash cell process at the given time point (p < 0.05, student t-test) 25 20 15 10 5 0 40 35 30 7-HC

*

7-HCG

*

7-HCS Fig. 2. Phase I and Phase II metabolism of 7-EC by human cryopreserved hepatocyte using traditional cell processing protocol, with (  ) and without (  ) Percoll wash. Data are expressed as the mean ± standard deviation of twenty-eight experiments without Percoll wash and eight experiments with Percoll wash. *, significantly different from the corresponding value of without Percoll wash cell process at the given time point (p < 0.05, student t-test)

(2) Effects of cell processing temperature, medium, and Percoll wash on cell viability improvement

Table 3. Effect of Temperature and Percoll wash on cell viability in cryopreserved primary human hepatocytes* Hepatocyte

Six donor pool

Medium

WME WME WME WME

Protocol

A B C D

Treatment

4 o C, no Percoll 4 o C, 25% Percoll 37 o C, no Percoll 37 o C, 25% Percoll

Yield per vial (million cells)

5.4 +/- 1.02

4.5 +/- 0.74

5.3

4.2 +/- 0.77

0 min

66 +/- 5.21

74 +/- 3.58

71 90 +/- 1.30

Cell Viability (%) 60 min

57 +/- 7.69

71 +/- 4.24

62 74 +/- 3.21

120 min

54 +/- 5.90

68 +/- 3.54

64 73 +/- 2.49

* Data are expressed as mean  SD of twenty-nine independent experiments (4 o C, no Percoll), thirty-five independent experiments (4 o C, 25% Percoll), one experiment (37 o C, no Percoll) and five independent experiments (37 o C, 25% Percoll).

Table 4. Effect of medium on cell viability in cryopreserved primary human hepatocytes Hepatocyte

Six donor pool

Medium

WME WME WME WME

Protocol

A B C D

Treatment

4 o C, no Percoll 4 o C, 25% Percoll 37 o C, no Percoll 37 o C, 25% Percoll

Yield per vial (million cells)

5.4 +/- 1.02

4.5 +/- 0.74

5.3

4.2 +/- 0.77

0 min

66 +/- 5.21

74 +/- 3.58

71 90 +/- 1.30

Cell Viability (%) 60 min 120 min

57 +/- 7.69

71 +/- 4.24

62 74 +/- 3.21

54 +/- 5.90

68 +/- 3.54

64 73 +/- 2.49

240 min

48 +/- 5.31

58 +/- 6.04

60 70 +/- 1.52

240 min

48 +/- 5.31

58 +/- 6.04

60 70 +/- 1.52

100 90 80 70 60 50 40 30 20 10 0 0 min 30 min 60 min

Time

120 min 240 min

CONCLUSION

Figure 3. Demonstration of improvement of cell viability in human cryoprserved hepatocyte lot# 51 from 53% (reported value from IVT web-site) to 94% by utilizing new protocol. Hepatocytes of Lot#51 were processed under 4 o C without Percoll wash (  ), 37 o C without Percoll wash (  ) and 37 o C with Percoll wash (  ).

• • • Medium temperature and Percoll centrifugation are the two key factors in obtaining high cell viability of human cryopreserved hepatocyte after thawing.

Different types of medium used during cell processing have no effects on initial cell viability, however they do have effects on cell viability post-incubation.

Percoll centrifugation should be performed routinely for cryopreserved cell processing to optimize enzyme activities.

1 Pre-clinical/Development, Neurocrine Biosciences, San Diego, CA 92121