Functional Evaluation of 3D-culture of Human Hepatocytes on Cell-able under Newly Optimized Condition.

Shin Enosawa1, Yuriko Takahashi1,2, Tomoko Jomura2, Emiko Ozeki2, and Takeshi Ikeya1,2
1. Division for Advanced Medical Sciences, National Center for Child Health and Development, 2-10-1 Ookura,
Setagaya-ku, Tokyo 157-8535, Japan
2. Transparent Inc.
A multi-well plate microfabricated with highly hydrophilic polymer, Cell-able facilitates 3-dimensional (3D) culture ofprimary hepatocytes and induces long-term well-functioning culture, comparing to conventional monolayer. We report
here the improved outcome using novel culture medium specialized for human hepatocytes and mouse ﬁbroblasts as
feeder cells. [Methods] Primary human hepatocytes were isolated from surgically resected liver tissue (Eticalpermission No.385 and 396) or purchased as cryopreserved hepatocytes (Xenotech). Hepatocytes (2x10^4/well) wereseeded on 96-well type Cell-able that had been precultured with mouse ﬁbroblasts (JCRB9019 (same as ATCC
CCL-92) or ATCC CCL-163). Cells were cultured with RM101 (Transparent) based on Williams medium E containing1% fetal bovine serum. Hepatocyte functions were evaluated by testosterone metabolism and transportation of tritiatedtaurocholic acid and carboxy-dichloroﬂuorescein diacetate (CDF-DA). [Results and Discussion] Hepatocytes cultured
on Cell-able with RM101 showed 1.55 to 3.43 fold higher activity of testosterone 6-beta hydroxylation of other 5 media(3 commercially available and 2 previously reported media) examined on day 7 and the superiority increased thereafter
until day 21. Activity of testosterone 6-beta hydroxylation was maintained well for 54 days. Noticeably, the cells onday 54 increased the activity from 0.15 to 0.80 fmol/cell/min in response to rifampicin. Time course changes intaurocholate uptake were 24.1 fmol/cell (day of isolation), 2.84 (day 3), and 6.83 (day 7), while those of monolayer
hepatocytes were 0.49 (day 3) and not detectable on day 7. The increase of the activity from day 3 to 7 on Cell-able wasthought to be due to maturation of spheroid structure. The evidence of bilially efﬂux was examined by cofocal
microscope using CDF-DA. The clear ﬂuorescent spots were detectable intercellular area of the spheroids. Whencalcium ion was withdrawn from the culture medium, the intensity and sharpness of ﬂuorescent spots became weakerand vaguer, suggesting the formation of small bile duct-like structure between hepatocytes. The present results indicatethat Cell-able culture system suits for the examination of a sequence of reactions of DMPK research.
ADDRRESS CORRESPONDENCE senosawa@nch.go.jp
17th ISSX 2011 Atlanta
1

Outline of Cell-Able

Interculture area coated by block
co-polymer. Neither proteins norcells can adhere.

****

Cell culture area2Standard Protocol of Primary Hepatocyte
Culture with Feeder Cells
 Plate surface coatedwith Block co-polymer

FeederFeeder  Advantage of co-culture with feeder cells
Long-lasting CYP activities
Cryopreserved hepatocyte with low attaching capability canbe cultured
Optimization of culture medium enabled long-lasting CYP
activities in feeder-free culture (see below)
3

Microstructure of Hepatocyte-feeder cell
heterospheroids formed on the cell array
HH

Downward view

mHpm

Horizontal view

Magnified horizontal view

Diesse space-like structure was observed
between hepatocyte (Hp) and feeder (HH)cell with microvilli rooted from hepatocyte
was observed. m; mitochondria.

Arrow heads indicate HH cells that
migrate from the culture plate andenwrap spheroidal hepatocyte mass. 4Materials and Methods
[Hepatocytes]
Fresh; isolated from surgically resected liver in National Center for Child Health and Development (IRB permission No.
385, 396)
Cryopreserved; Xenotech (799, HC2-6, HC5-7), IVT (TSF, GHA)
[Feeder cells]
HH bovine aortic epithelial cells (JCRB0099), Mouse 3T3 fibroblasts (ATCC CCL-92, ATCC CCL-163), Rhesus monkey
retinal epithelial cells (ATCC CRL-1780)[Culture medium]
RM100; medium for rat hepatocytes (Transparent)
RM101; Medium for human hepatocytes (Transparent)
SE & YY; Williams E-base Matrigel-containing medium (reported by Enosawa and Yamada in JSSX2009, Kyoto)
IVT; InVitroGRO HI Medium
XENOTECH; Hepatocyte culture media
BD; BD Hepatocyte Culture Medium Kit[Culture]
2x10^4 human hepatocytes /one well of 96-well type Cell-able
When feeder cells were used, 8x10^3 cells / one well of 96-well type Cell-able were seeded two days before
hepatocytes inoculation. In addition, cryopreserved feeder cell-seeded plates were also used.
[CYP activity]
Hepatocytes were incubated with 100 micro-mol/L testosterone or phenacetin for 3 hrs. Formation of metabolites (6beta hydroxytestosterone, testosterone glucronide, acetaminophen) were determined by UPLC (Waters). CYP induction
was performed by 72-hr incubation with rifampicin (25 micro-mol/L) or omeprazole (5 micro-mol/L).
[Transporter activities]
Influx 1; tritiated ([3H(G)]) taurocholate (1 micro-mol/L) as a substrate and Rifamycin SV (100 micro-mol/L) as an
inhibitor.
Influx 2; tritiated ([6,7-3H(N)]-estrone sulfate (1 micro-mol/L) as a substrate and taurocholate (100 micro-mol/L) as an
inhibitor.
Efflux; carboxy-dichlorofluorescein diacetate (CDF-DA) (10 micro-mol/L)

5

Optimum Culture Medium
Medium
ASSAY:CULTURE CONDITION:
SphH+F; spheroids formed with human cryopreserved
hepatocytes (Xenotech 799) and feeder cells(HH) on Cell-able
feeder-free spheroids of human cryopreservedSphH;
hepatocytes (Xenotech 799) on Cell-able
Monolayer; monolayer culture of human cryopreserved
hepatocytes on type I collagen-coated plate

20

day14

day21

Sph_H

day7

Monolayer

10

RM100

RM101

SE&YY(JSSX)RECOMMENDEDIVTXENOTECH

Monolayer

Sph_H

Sph_H+F

Monolayer

Sph_H+F

Sph_H

Sph_H+F

Monolayer

Sph_H

Sph_H+F

Monolayer

Sph_H

Sph_H+F

Monolayer

Sph_H

0
Sph_H+F

pmole/2x10^4cells/hr

Testosterone 6 beta hydroxylationBD

RM101 (Transparent) showed Excellent CYP activities evenwith feeder-free culture6Optimum feeder cell
Hepatocytes; TSF, HC2-6, HC5-7, GHAComparison of feeder cells (basal activity)
Testosterone→6βHydroxytestosterone

Comparison of feeder cells (basal activity)
Testosterone→Testosterone glucronide  

RECOMMENDED    

           

   

          

  pmole/2x10^4 cells/h pmole/2x10^4 cells/h





RECOMMENDED
Cell lines examined

Designation

Code No.

OriginHH

JCRB0099

Bovine aortic epithelium

3T3-Swiss albino

ATCC CCL-92(JCRB9019*)

Mouse fibroblast

BALB/3T3 clone A31ATCC CCL-163

Mouse fibroblast RF/6A

ATCC CRL-1780

Rhesus monkey retinal epithelium

*ATCC CCL-92 is also distributed by JCRB as JCRB9019 in Japan

7Optimum feeder cell (Induction study)
Hepatocytes; Xenotech HC2-6
Induction study (Omeprazole)
Phenacetin→Acetaminophen  induced

RECOMMENDED      3

0.151

Chromatography 

1; 6-beta hydroxytestosterone

0.10

2; Testosterone glucronide
3; Testosterone

0.05

-0.05

 

RECOMMENDED

0.20

0.00

  

              

induced  



  
        

pmole/2x10^4cells/h    

pmol/2x10^4cells/h

Induction study (Rifampicin)
testosterone →6βhydroxy testosterone

20246810min

8Long-lasting CYP Activity of Cryopreserved
Human Hepatocytes Cultured on Cell-able
Hepatocytes; Xenotech HC2-6 and HC5-7Feeder cells; ATCC CCL-92

pmol/2x10^4 cells/h

Testosterone → 6-beta Hydroxytestosterone
HC2-6

1500

HC5-7

1000

500

0Control induction Control induction Control induction
day7day14

day54

The initial activity of each lot was 549.6 and 214.8
pmol/2x10^4/h, respectively.

9Influx Transporter Activity (1)Hepatocytes; freshly isolated human hepatocytesFeeder; ATCC CCL-163
Tracer; [3H(G)]-taurocholate
inhibitor; Rifamycin SV 6000

Gross
Non-Specific

4000

2000

005min

10Monolayer culture bycollagen-coated plateDay 7[3H(G)]) taurocholate Incorporated
dpm/4x10^4 hepatocytes

[3H(G)]) taurocholate Incorporated
dpm/4x10^4 hepatocytes

Spheroid culture byCell-able
Day 7
6000

Gross
Non-Specific

4000

2000

00510min

Hepatocyte spheroids showed good influx transporter activity.
Non-specific incorporation was determined under the existence of inhibitor (Rifamycin SV ).

10Influx Transporter Activity (2)Spheroid culture byCell-able
Day 1
15000[6,7-3H(N)]-estrone sulfate Incorporated
Dpm/10^6 hepatocytes

[6,7-3H(N)]-estrone sulfate Incorporated
Dpm/10^6 hepatocytes

Hepatocytes; cryopreserved human hepatocytes from surgically resected liverFeeder; ATCC CCL-163
Tracer; [6,7-3H(N)]-estrone sulfateinhibitor; taurocholateGross
Non-Specific

10000

5000

00123Monolayer culture bycollagen-coated plate
Day 1
15000
Gross
Non-Specific

10000

5000001min

23miniHepatocyte spheroids showed good influx transporter activity.
Non-specific incorporation was determined under the existence of inhibitor (taurocholate).

11

Hepatocyte spheroid showing efflux transporter activities examinedby CDF-DA exclusion into intercellular bile pools
hepatocytes; freshly isolated human hepatocytes
feeder cells; ATCC CCL-92 Culture day 2day 4

day 7

Bile pool formation and CDF exclusion were becoming marked with the increase ofculture days or maturation of spheroid. (Above)
Ca2+ removed

Bile pools almost disappearedby removal of Ca2+ ions. (Left)

12

Conclusion
The best medium for human hepatocyte spheroid culture on Cellable is RM101 by Transparent. [Slide No.6]



The RM101 medium can maintain CYP activities without feeder cells
on Cell-able. [Slide No.6]

Mouse 3T3 fibroblasts are more effective on hepatocytes culture as
feeder cells than bovine endothelial of monkey epithelial cells onCell-able. [Slide No.7, 8]

The human hepatocyte spheroids formed on Cell-able showed influxand efflux transporter activities. [Slide No.10, 11, 12]

Acknowledgment
Authors would like to express sincere gratitude to people who donate
hepatocytes for scientific research. This work was supported by grantin aide by Japan Health Sciences Foundation (KHD1023).
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