EFFECT OF PERCOLL CENTRIFUGATION ON CELL VIABILITY AND ENZYME ACTIVITIES IN HUMAN CRYOPRESERVED PRIMARY HEPATOCYTEZhihong Zhang O’Brien*, Melanie Hann, W.Perry Gordon*, Yong Hee Lee, Kevin Holme and Julie Doerr-Stevens1

Lion Bioscience, San Diego, CA 92121

EFFECT OF PERCOLL CENTRIFUGATION ON CELL VIABILITY AND ENZYME ACTIVITIES IN HUMAN CRYOPRESERVED PRIMARY HEPATOCYTEZhihong Zhang O’Brien*, Melanie Hann, W.Perry Gordon*, Yong Hee Lee, Kevin Holme and Julie Doerr-Stevens1

Lion Bioscience, San Diego, CA 92121

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 (4oC) 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, 75M), 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.

ABSTRACTABSTRACT

INTRODUCTIONINTRODUCTION

METHODSMETHODS RESULTSRESULTS

CONCLUSIONCONCLUSION

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.

• 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.

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

Procedure Steps A B Procedure Steps C D

Pre-cell processing prep

Adding insulin, gentamycin & glutamine in WME or use

KHB buffer

Adding insulin, gentamycin & glutamine in WME

Pre-cell processing prep

Two tubes of WME or KHB

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

pH supplemented WME, or KHB buffer

pH supplemented WME, 90% Percoll & 1XKHB

Keep all the solutions chilled

(4oC)

Keep all the solutions chilled

(4oC)

Warm all the media up

to 37oC

Warm all the media up

to 37oCCell thaw Quick thaw (1.5min) at 37oC Quick thaw (1.5min) at 37oC Cell thaw Quick thaw (1.5min) at

37oC

Quick thaw (1.5min) at

37oCSuspension media Keep them at 4oC Keep them at 4oC Suspension media Keep them at 37oC Keep them at 37oC

Cryopreservant & dead cell clean-out

Quickly transfer thawed cells into chilled 50ml conical

tubes

Quickly transfer thawed cells into chilled 50ml conical

tubes

Cryopreservant & dead cell clean-out

Pour the cells into pre-warmed media

Pour the cells into pre-warmed media

Add WME or KHB dropwisely into the cells at a speed of

12ml/4-5min

Add WME dropwisely into the cells at a speed of 12ml/4-

5min

Centrifuge the cells at 40-60xg for 5min

Centrifuge the cells at 60-90xg for 5min

Centrifuge the cells at 50xg for 5min to obtain cell pellet

Centrifuge the cells at 50xg for 5min to obtain cell pellet

Dead cell clean-out Suspend the cells in 25% Percoll-KHB solution and then centrifuge it at 100xg for 6min

Percoll clean-out Supend the cells in fresh WME and then centrifuge it at 50xg for 5min

Percoll clean-out or further cryopreservant clean-out

Suspend the cells in fresh WME or KHB and then centrifuge it at 40-60xg for 3min

Suspend the cells in fresh WME or KHB and then centrifuge it at 40-60xg for 3min

Resuspension in fresh incubation media

Resuspend the cells in WME or KHB for cell count

Resuspend the cells in WME for cell count

Resuspension in fresh incubation media

Resuspend the cells in WMEor KHB for cell count

Resuspend the cells in WME or KHB for cell count

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 4oC, without Percoll wash• B: post-thaw cell processing at 4oC, 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 4oC, without Percoll wash• B: post-thaw cell processing at 4oC, with Percoll wash• C: post-thaw cell processing at 37oC, without Percoll wash• D: post-thaw cell processing at 37oC, 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)

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

**

* *

0

10

20

30

40

50

60

70

80

90

0 min 60 min 120 min 240 min

Time

Cel

l V

iab

ilit

y (%

)

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)

*

*

0

5

10

15

20

25

30

35

40

7-HC 7-HCG 7-HCS

Fo

rma

tio

n R

ate

(p

mo

le/m

in/m

illi

on

ce

lls

)

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 Medium Protocol Treatment Yield per vial(million cells) 0 min 60 min 120 min 240 min

Six donor pool WME A 4oC, no Percoll 5.4 +/- 1.02 66 +/- 5.21 57 +/- 7.69 54 +/- 5.90 48 +/- 5.31

WME B 4oC, 25% Percoll 4.5 +/- 0.74 74 +/- 3.58 71 +/- 4.24 68 +/- 3.54 58 +/- 6.04

WME C 37oC, no Percoll 5.3 71 62 64 60

WME D 37oC, 25% Percoll 4.2 +/- 0.77 90 +/- 1.30 74 +/- 3.21 73 +/- 2.49 70 +/- 1.52

Cell Viability (%)

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

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

0

10

20

30

40

50

60

70

80

90

100

0 min 30 min 60 min 120 min 240 min

Time

Ce

ll V

iab

ilit

y (

%)

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 4oC without Percoll wash (), 37oC without Percoll wash () and 37oC with Percoll wash ().

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

Hepatocyte Medium Protocol Treatment Yield per vial(million cells) 0 min 60 min 120 min 240 min

Six donor pool WME A 4oC, no Percoll 5.4 +/- 1.02 66 +/- 5.21 57 +/- 7.69 54 +/- 5.90 48 +/- 5.31

WME B 4oC, 25% Percoll 4.5 +/- 0.74 74 +/- 3.58 71 +/- 4.24 68 +/- 3.54 58 +/- 6.04

WME C 37oC, no Percoll 5.3 71 62 64 60

WME D 37oC, 25% Percoll 4.2 +/- 0.77 90 +/- 1.30 74 +/- 3.21 73 +/- 2.49 70 +/- 1.52

Cell Viability (%)