Public-private cooperative study supported by grant in aid fromJapan Health Sciences Foundation (KHD1023).

HUMAN HEPATOCYTE 3D CULTURE ON
CELL-ABLE USING NEWLY OPTIMIZEDMEDIUM AND ITS FUNCTIONALEVALUATIONShin Enosawa1, Yuriko Takahashi1,2, Tomoko Jomura2,
Emiko Ozeki2, Takeshi Ikeya1,2
1. National Center for Child Health and Development
2. Transparent Inc.
26th JSSX Annual Meeting, Nov 16 to 18, 2011, Hiroshima

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 Cells① Plate surface coatedwith Block co-polymer② Feeder Cells - Optional③ Feeder (optional)+ Hepatocytes

Advantage of co-culture with feeder cells
Long-lasting CYP activities
Cryopreserved hepatocyte with low attaching capability can becultured
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-able
HHDownward viewmHpm

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 (6 betahydroxytestosterone, testosterone glucronide, acetaminophen) were determined by UPLC (Waters). CYP induction wasperformed 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
ASSAY:CULTURE CONDITION:
Sph H+F;
spheroids formed with human
cryopreserved hepatocytes (Xenotech 799)
and feeder cells (HH) on Cell-able
Sph H;
feeder-free spheroids of human
cryopreserved hepatocytes (Xenotech 799) onCell-able
Monolayer; monolayer culture of human cryopreserved
hepatocyteson type I collagen-coated plateday7day14
day21

10

RM100

RM101

SE&YY(JSSX)RECOMMENDED

IVT

XENOTECH

Monolayer

Sph_H

Sph_H+F

Monolayer

Sph_H

Sph_H+F

Monolayer

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

20

Testosterone 6 beta hydroxylationBD

RM101 (Transparent) showed Excellent CYP activities evenwith feeder-free culture

6Optimum feeder cell
Hepatocytes; TSF, HC2-6, HC5-7, GHA
Comparison of feeder cells (basal activity)
TestosteroneTestosterone glucronide

Comparison of feeder cells (basal activity)
Testosterone6Hydroxytestosterone

day3

HH

CCL92

CCL163

100

CRL1780

RECOMMENDED

HH

CCL92

CCL163

GHA

HC5-7

HC2-6

TSF

GHA

HC5-7

HC2-6

TSF

GHA

HC5-7

HC2-60
TSF

GHA

HC5-7

HC2-6

TSF

GHA

HC5-7

HC2-6

TSF

GHA

HC5-7

HC2-6

TSF

GHA

HC5-7

HC2-60

day14

GHA

200

day7

HC5-7

day14

200

HC2-6

day7400

TSF

pmole/2x10^4 cells/hday3TSF

pmole/2x10^4 cells/h

600

CRL1780

RECOMMENDED
Cell lines examined

Designation

Code No.

OriginHH

JCRB0099

Bovine aortic epithelium

3T3-Swiss albino

ATCC CCL-92(JCRB9019*)

Mouse fibroblast

BALB/3T3 clone A31

ATCC 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

7

Optimum feeder cell (Induction study)
Hepatocytes; Xenotech HC2-6
Induction study (Omeprazole)
PhenacetinAcetaminophen
day7800
pmole/2x10^4cells/h

day71500
day14day71000

day14500

induced

HH

CCL92

CCL163

day14600

day7day14400

induced200

HH

RECOMMENDED

CCL163

HC5-7

HC2-6

HC5-7

HC2-6

HC5-7

CCL92

CRL1780

RECOMMENDED

0.20
0.15

HC2-6

HC2-6

CRL1780

HC5-70

HC5-7

HC2-6

HC5-7

HC2-6

HC5-7

HC2-6

HC5-7

0
HC2-6

pmol/2x10^4cells/hInduction study (Rifampicin)
testosterone 6hydroxy testosterone

31

Chromatography

1; 6-beta hydroxytestosterone
2; Testosterone glucronide

0.10
0.05

3; Testosterone

20.00
0-0.05246810min8Long-lasting CYP Activity of Cryopreserved
Human Hepatocytes Cultured on Cell-able
Hepatocytes; Xenotech HC2-6 and HC5-7
Feeder cells; ATCC CCL-92

pmol/2x10^4 cells/hTestosterone → 6-beta Hydroxytestosterone
HC2-61500

HC5-71000

500

0Control induction Control induction Control induction
day7

day14

day54

The initial activity of each lot was 549.6 and 214.8
pmol/2x10^4/h, respectively.
9Influx Transporter Activity (1)Spheroid culture byCell-able
Day 76000

GrossNon-Specific4000

2000005min10[3H(G)]) taurocholate Incorporated
dpm/4x10^4 hepatocytes[3H(G)]) taurocholate Incorporated
dpm/4x10^4 hepatocytes

Hepatocytes; freshly isolated human hepatocytes
Feeder; ATCC CCL-163
Tracer; [3H(G)]-taurocholate
inhibitor; Rifamycin SV

Monolayer culture bycollagen-coated plate
Day 76000
Gross
Non-Specific

4000

2000

00510minHepatocyte 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

Gross
Non-Specific

10000

5000

0012min

3[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 sulfate
inhibitor; taurocholate

Monolayer culture bycollagen-coated plate
Day 115000
Gross
Non-Specific

10000

5000

00123minHepatocyte 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

Acknowledgment
Authors would like to express sincere gratitude to people who donate
hepatocytes for scientific research.

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, 9]

The human hepatocyte spheroids formed on Cell-able showed influxand efflux transporter activities. [Slide No.10, 11, 12]13