日本組織培養学会第85回大会
JTCA85 2012 Kyoto
シンポジウム２細胞接着と細胞機能制御の最先端形態制御が与える機能面の変化
肝細胞初代培養の経験からFunctional changes induced by morphologicalregulation in primary hepatocyte culture
絵野沢 伸
Shin Enosawa
国立成育医療研究センター 臨床研究センター 先端医療開発室
Division for Advanced Medical Sciences,
National Center for Child Health and Development
平成24年5月18日 京都

1

謝辞・利益相反・共同研究者
Acknowledgment・COI・Collaborators
本研究において肝組織・肝細胞ならびに手術摘出がん組織を提供してくださいました患者さま、ご家族にまずは深く御礼申し上げます。
Authors would like to express sincere gratitude to people who donate hepatocytes andsurgically resected tumor tissues for scientific research.
本研究は厚生労働科学研究費政策創薬マッチング官民共同研究事業「創薬研究における人由来初代細胞および幹細胞の利用円滑化に向けた研究」の補助金およびトランスパレント社の委託研究費により行いました。
Supported by Grant for Public-private sector joint research on Publicly Essential Drugs by
Ministry of Health, Labour and Welfare and contract research with Transparent.
＜共同研究者＞
高橋由里子（株式会社トランスパレント）
Yuriko Takahashi, Transparent
城村友子 （株式会社トランスパレント）
Tomoko Jomura, Transparent
池谷武志 （株式会社トランスパレント）
Takeshi Ikeya, Transparent
宮本義孝 （国立成育医療研究センター）
Yoshitaka Miyamoto, National Center for Child Health and Development2

セルエイブル概観

Outline of Cell-Able

超親水性高分子コーティングがなされタンパク質はもちろん細胞も接着しない
Interculture area coated by blockco-polymer. Neither proteins norcells can adhere.

****

細胞接着部分
Cell culture area

100μm
3

ふたつの技術要素
Two Technological factors in Cell-able System
培養表面のポリマー加工によって規格化されたスフェロイドを作ることができる．
Formation of uniform-size spheroids by
microfabrication of culture surface.
フィーダー細胞を利用すると細胞の接着がよくなり、機能維持にも優れる．
Use of feeder cells promotes hepatocytes
attachment and long-term maintenance offunctions.
4

細胞非接着面を形成する感光材料の特徴
感光材料: PEG誘導体（感光化）
ON3R
N3Rn感光性基

親水性

感光性基

光硬化後の感光材料表面の性質（水の静的接触角）
感光材料（光硬化後）
8°

Ultra-hydrophilicコントロール (TCPS)
42°

コントロール (PSt)92°

5

Production of Cell-able™
Photosensitive
polymer coating

UV exposure

Water development

Substrate

Substrate

Photo mask

Substrate

Non-adhesion area
*

*

*

*

Cell culture area

開発者

100μm

大塚英典、長崎幸夫、片岡一則、池谷武志

Developed by Hidenori Ootsuka, Yukio Nagasaki, Kazunori Kataoka,
Takeshi Ikeya

6播種細胞が接着するまでMigration of seeded cells
Day 0Day 17

Cell-ableによる肝細胞初代培養の標準プロトコール
Standard Protocol of Primary Hepatocyte Culture
on Cell-able① Feeder Cells Optional② Feeder(optional)+ Hepatocytes

フィーダー細胞との共培養の利点
Advantage of co-culture with feeder cells
CYP活性の長期維持
Long-lasting CYP activities
低接着性凍結肝細胞の培養実験使用が可能
Cryopreserved hepatocyte with low attaching capability can be cultured8

肝細胞スフェロイドが完成するまで
Formation of hepatocyte spheroids9Microstructure of Hepatocyte-feeder cell
heterospheroids formed on the Cell-able
HH

Downward viewm

Hpm

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 thatmigrate from the culture plate andenwrap spheroidal hepatocyte mass.
10材料と方法

Materials and Methods

ヒト肝細胞 [Human Hepatocytes]
Fresh; isolated from surgically resected liver in National Center for Child Health and Development (IRBpermission No.385, 396)
Cryopreserved; Xenotech, IVT
フィーダー細胞 [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)
培地 [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)
IVT; InVitroGRO HI Medium
XENOTECH; Hepatocyte culture media
BD; BD Hepatocyte Culture Medium Kit
培養方法 [Culture]
2x10^4 human hepatocytes /one well of 96-well
When feeder cells were used, 8x10^3 cells / one well were seeded two days before hepatocytes inoculation.
機能評価（CYP活性測定） [CYP activity]
Testosterone 6 beta hydroxylation, Testosterone glucronidation. CYP induction; rifampicin
トランスポーター活性 [Transporter activities]
Influx; tritiated ([6,7-3H(N)]-estrone sulfate, Efflux; carboxy-dichlorofluorescein diacetate (CDF-DA)
11

各種フィーダー細胞を用いた場合のCYP活性
Optimum feeder cellComparison 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

day14GHA200

day7HC5-7

day14

200

HC2-6

day7400

TSF

pmole/2x10^4 cells/h

day3TSF

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

12フィーダー細胞の有無と培地比較
Optimum Culture Medium
ASSAY:

day7day14

day21

Sph_H10Monolayer

CULTURE CONDITION:
Sph H+F;
spheroids formed with human
cryopreserved hepatocytes and feeder cells(HH) on Cell-able
Sph H;
feeder-free spheroids of human
cryopreserved hepatocytes on Cell-able
Monolayer; monolayer culture of human cryopreserved
hepatocytes on type I collagen-coated plate

RM100

RM101

SE&YY(JSSX)RECOMMENDED

IVT

XENOTECH

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_H0
Sph_H+F

pmole/2x10^4cells/hr20Testosterone 6 beta hydroxylation (CYP3A4)

BD

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

13

CYP活性および誘導能の長期維持
Long-lasting CYP Activity of Cryopreserved Human
Hepatocytes Cultured on Cell-able

pmol/2x10^4 cells/h

Testosterone → 6-beta Hydroxytestosterone (CYP3A4)
HC2-6

1500

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.
14

CDF-DAをプローブとして見た肝細胞スフェロイドが示す排出トランスポーター活性Hepatocyte spheroids show efflux transporter activities
examined by CDF-DA exclusion into intercellular bile pools
Culture day 2

day 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.

15[6,7-3H(N)]-estrone sulfateをプローブとして見た
肝細胞スフェロイドが示す取込トランスポーター活性

Spheroid culture by
Cell-able
Day 1
15000

GrossNon-Specific

10000

5000

0012min3[6,7-3H(N)]-estrone sulfate Incorporated
Dpm/10^6 hepatocytes[6,7-3H(N)]-estrone sulfate Incorporated
Dpm/10^6 hepatocytes

Hepatocyte spheroids show influx transporter activities examined by[6,7-3H(N)]-estrone sulfate exclusion into intercellular bile pools
Monolayer culture bycollagen-coated plate
Day 115000
Gross
Non-Specific

10000

5000

00

123minHepatocyte spheroids showed good influx transporter activity.
Non-specific incorporation was determined under the existence of inhibitor (taurocholate).

16

小括

Brief Summary Cell-able培養においてマウス3T3細胞はヒト肝細胞長期培養の際のフィーダー細胞として優れることがわかった。
Mouse 3T3 fibroblasts are more effective on long-term
hepatocyte culture as feeder cells than bovine endothelialof monkey epithelial cells on Cell-able.
 Cell-able培養においてヒト肝細胞スフェロイドは成熟とともに取り込み、排泄トランスポーター活性を示すことがわかった。
The human hepatocyte spheroids formed on Cell-able showed
influx and efflux transporter activities.
17

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate

Seeded human cryopreserved
hepatocytes

Day 0Day 14

96-well Cell-ablewith feeder (3T3)
96-well Collagencoat plate

Gene Expression byAgilent microarray

1. Well間差 Inter-well difference
2. Day14/Day 0発現比 Day14/Day 0 Ratio
18

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate
Cell-able

Collagen coat plate

Day7Day1419

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate

Cell-ableの方がWell間差が少ない
Lower difference in Cell-able than conventional plate

20

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate＜以後のデータ表示＞
Global normalizationののち、Day14／Day0比で表示しCell-able培養とConventional plate（コラーゲンコートプレート）培養を比較
14日間培養した細胞における発現＝14日間の培養の影響による変化

解凍時＝in vivo肝組織＜Data expression＞
After global normalization, expressed as a Day14/Day0 ratio and compare
the effect of Cell-able culture and conventional collagen plate culture.
i.e.,Expression of 14days culture= Effect of 14 day culture
Expression of liver in vivo
21

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate
主要CYP
CYP1A1CYP1A2CYP2B6CYP2C19
CYP2C9CYP2D6
CYP3A4CYP3A5Cell-able
1.44
0.76
0.59
0.31
0.37
0.26
0.44
0.32

Conventional plate
3.51
0.68
0.36
0.110.150.180.420.25

核内受容体
NR1H3LXRa
NR1H4FXR
NR1I2PXR
NR1I3CAR
NR1I3CAR
NR3C1GR
NR3C1GRRXRA
AHR
AHR

Cell-able
0.57
0.76
0.60
0.30
0.34
0.91
1.26
1.02
0.48
0.51

Conventional plate
0.46
0.99
0.50
0.08
0.12
0.84
0.87
1.00
0.41
0.57

トランスポーター
ABCB1
MDR1ABCB11BSEP
ABCC2MRP2SLC10A1NTCPSLC10A2ASBTSLC22A1 OTC1SLC22A2 OTC2SLC22A4 OCTN1SLC22A5 OCTN2SLC22A6 OAT1SLC22A7 OAT2SLCO1A2 OATP1A2SLCO1A2 OATP1A2SLCO1B1 OATP1B1
SLCO2A1 OATP2A1
SLCO2B1 OATP2B1

Cell-able
0.89
0.56
1.03
0.40
1.12
0.47
2.35
1.24
1.26
0.48
0.61
0.17
0.67
0.46
0.29
0.73

Conventional plate
1.19
0.26
0.61
0.10
0.67
0.18
0.24
2.35
2.07
0.44
0.37
0.24
0.56
0.45
0.26
0.43

肝細胞タンパクAlbumin
HNF4 alpha
HNF4 gamma
Tyrosine amino transf
Transferrin

Cell-able
0.62
0.48
1.29
0.33
0.68

Conventional plate
0.48
0.53
1.33
0.17
0.25

22

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional PlateCYPs
Cell-ablevs day0

相関係数
決定係数

0.873852
0.763618

Collagen coat plate vs day0

相関係数
決定係数

0.84370.711829

23

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate

Nuclear Receptors
Cell-ablevs day0

相関係数
決定係数

0.924545
0.854783

Collagen coat plate vs day0

相関係数
決定係数

0.868742
0.754712

24

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate

Conjugation Enzymes
Cell-ablevs day0

相関係数
決定係数

0.954233
0.91056

Collagen coat plate vs day0相関係数
決定係数0.926838
0.859029

25

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate

Transporters
Cell-ablevs day0

相関係数
決定係数

0.976576
0.953701

Collagen coat plate vs day0

相関係数
決定係数

0.671470.450871

26

Cell-able培養と通常プレート培養における遺伝子発現状態の相違
Difference in Gene Expression between Cell-able and Conventional Plate

遺伝子発現のDay14対Day0比の相互関係 決定係数の比較
R^2 of Day14/Day0 Ratio
Cell-able

Conventional plateCYPs

0.764

0.712

Nuclear receptors

0.855

0.755

Conjugation enzymes

0.911

0.859

Transporters

0.953

0.451

Liver related genes

0.806

0.733

Cell-able培養の方が初期値（Day0）値を維持している．
Cell-able culture maintains in vivo gene expression
better than conventional plate culture.
27

Cell-able Oncology™
初代がん細胞培養
American Association of Cancer Research 2012

発表より

28

Cell-able Oncology™
患者由来初代卵巣がん細胞の形態

2D培養との比較

摘出卵巣がんovarian tumor: endometrioid
Cell-able

2D plateAACR 2012

共同発表抜粋

29

Cell-able Oncology™
卵巣がん・子宮体がんのスフェロイド培養とEdU取込

卵巣がん(serous, low grade)

卵巣がん(clear cell)

二種類のがん細胞がCell-able Oncology™上で増殖
EdU反応時間；3hr

子宮体がん(grade 2)
AACR 2012

共同発表抜粋
30Cell-able Oncology™
初代子宮体がんの増殖に関する化学療法
抗がん薬挙動の比較
Cell-Able
2D plate

Cell-Able: vehicle

PTX 10

PTX 100 (nM)

A450-630 (% of Control)

150
10050001

PTX 10

PTX 100 (nM)

34Log [nM]50-

2D-plate: vehicle

2IC50

PTX
Cell-Able

PTX
2D plate

>1000

16.5

Cell-able Oncology™培養の方が臨床上の反応性をよく再現していた．
EdU反応時間；3hrAACR 2012

共同発表抜粋
31

ふたつの技術要素がもたらす培養上の効果
Cell-able System and its Impact in Cell Culture 規格化されたスフェロイド → 初代肝細胞長期機能維持培養、少ないWell間差、in vivo発現の維持、in vivoを再現するがん細胞初代培養Uniform-size spheroids → Long-term functioning hepatocyte
primary culture, low inter-well difference, maintenance of in vivo
gene expression, tumor cell culture mimicking in vivo
 フィーダー細胞の利用 → 初代肝細胞長期機能維持培養、人工肝小葉の構築、低接着性凍結ヒト肝細胞の利用
Use of feeder cells → Long-term functioning hepatocyte
primary culture, reconstruction of artificial hepatic lobules, useof cryopreserved hepatocytes with low-attaching capability
32