Upload
others
View
1
Download
0
Embed Size (px)
Citation preview
Human Hepatocytes HepG2
BIOMIMESYS® Liver has been tested on HepG2 and on cryopreserved Human Hepatocytes. It makes 3D cell culture easy and provides a robust in vitro and reliable model for metabolism & toxicological studies. To know more about BIOMIMESYS®Liver visit our website: www.biomimesys.com +33(0) 769 999 137; [email protected]
BIOMIMESYS®Liver is ready to use and compatible with all analytical technologies
• Available in a ready-to-use format (96 well plates) it enables the culture of hepatocytes under physiological conditions that are representative of the microenvironment found in liver tissue(2).
• Hepatocytes are simply seeded on top of the hydroscaffold and placed in the incubator.
• The media can be refreshed easily by pipetting. Being transparent makes it suitable for microscopy (immunofluorescence, bright field) and use in plate readers (OD, fluorescence & luminescence).
• Thanks to its mechanical properties, the hydroscaffold can be easily handled with fine forceps and proteins, nucleic acid can be extracted by directly adding the lysis buffer to the hydrogel, due to its high porosity.
BIOMIMESYS®Liver is physiological
BIOMIMESYS® range are hyaluronan based hydroscaffold developed to overcome the 2D flat culture limitations by recreating an in vivo-like physiology within the in vitro environment.
In comparison to in vivo decellularized liver tissue
SEM observation of a human decellularized liver tissue (from Mazza et al. 2015(1) )
SEM observation of a BIOMIMESYS®Liver section, highlighting the collagen chain, (artificially coloured)
Biomimetic structure
References:
(1) Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation. Mazza G. et al. Sci Rep.7; 5:13079, 2015.
(2) Decellularization and cell seeding of whole liver biologic scaffolds composed of extracellular matrix. Faulk D. et al. J Clin Exp Hepatol. 5; 1:69-80, 2015.
BIOMIMESYS®Liver hydroscaffold is made of RGDS and galactosamine-grafted Hyaluronic acid, Adipic acid dihydrazide crosslinker and extracellular matrix (ECM) proteins (collagen type I and collagen type IV) to mimic liver-ECM composition.
0
50
100
150
Day 7 Day 14 Day 21 Day 28
Dia
met
er (
µm
)
HepG2 spheroids sizes
100µm 400µm
100µm
Alive Dead
Day 28
Viability and Growth
Albumin Secretion
• HepG2 form spheroids with an average diameter of 80 to 100 µm after one month of cultivation.
• The amount of albumin secreted by HepG2 is 20 to 30 times higher in BIOMIMESYS®Liver compared to 2D.
• Cryopreserved human hepatocytes form small aggregates, 40 to 80 µm, which have a very good viability until day14.
Key genes expression levels
Toxicity study
• Cryopreserved human hepatocytes grown in BIOMIMESYS®Liver represent model for routine drug testing in 96-well format.
n=3 0
20
40
60
80
100
120
Day 7 Day 14
Dia
me
ter
(µm
)
Human Hepatocytes spheroids sizes
donor 1 donor 2
3D
Day 7
2D
100µm
25µm 25µm
Day 21
100µm MRP-2
Biliary Canaliculi Formation
• MRP2 expression, its colocalization with actin and CFDA fluorescence confirms the presence of active biliary canaliculi in HepG2 during 3 weeks.
CYP activities
• Basal and induced CYP1A1/A2, CYP2B6 and CYP3A4 activities are higher in BIOMIMESYS®Liver compared to 2D collagen sandwich.
Basal activity 2D vs 3D Basal and induced activities in 3D
0
50
100
150
200
CYP1A1/A2
2D 3D
Ace
tam
ino
ph
en/1
00
ng
of
DN
A
0
500
1000
1500
2000
CYP3A4
6β
-OH
-Te
sto
ster
on
e (n
M)/
10
0n
g o
f D
NA
0
20
40
60
80
CYP2B6
OH
-Bu
pro
pio
n
(nM
)/1
00
ng
of
DN
A
0
1000
2000
3000
CYP1A1/A2 Basal Induced
***
Ace
tam
ino
ph
en/1
00
ng
of
DN
A
0
1000
2000
3000
4000
5000
CYP3A4
***
6β
-OH
-Te
sto
ster
on
e (n
M)/
10
0n
g o
f D
NA
0
100
200
300
400
500
600
CYP2B6
***
OH
-Bu
pro
pio
n
(nM
)/1
00
ng
of
DN
A
Day 7
Viability and Growth
100µm
Day 7
100µm
Day 14
400µm 100µm 100µm
• HepG2 grown in BIOMIMESYS®Liver allow acute and chronic dose (3 doses) drug exposure. -10%
10% 30% 50% 70% 90%
110% 130%
0,01 0,2 4 80
Via
bili
ty (
ATP
co
nte
nt)
Chlorpromazine (µM)
BIOMIMESYS®Hepatocyte
-10% 10% 30% 50% 70% 90%
110% 130%
0,1 2 40 800
Amiodarone (µM)
BIOMIMESYS®Liver, a 3D cell culture model for maintaining and promoting hepatocytes functions for metabolism and toxicity studies
Day 14
400µm
25µm
Day 21
Actin MRP2
Day 14
CFDA 100µm
Toxicity study
Day 7 Day 8 Day 9 Day 10
1 dose
1st dose 2nd dose 3rd dose
Viability
Viability
Treatment 1 dose : Day 7 - Viability analysis : Day 8
• Better expression of phase I & II enzymes and transporters genes in 3D Day 7
0
2
4
6
mR
NA
no
rmal
ized
by
RP
LPO
CYP1A1
0
1
2
3 UGT1A1
0
2
4
6
NTCP
0
2
4
6
8 BSEP
Patented process
0,0
0,5
1,0
1,5
2,0
2,5
Day 7 Day 14 Day 21
µg a
lbu
min
/10
0n
g o
f D
NA
2D BIOMIMESYS®Hepatocyte
*** ***
***
BIOMIMESYS®Liver
-5% 15% 35% 55% 75% 95%
115% 135%
0,01 0,1 1 10 100
Via
bili
ty
Chlorpromazine (µM)
BIOMIMESYS®Hepatocyte - Chronic BIOMIMESYS®Hepatocyte - Acute
BIOMIMESYS®Liver - Chronic
BIOMIMESYS®Liver - Acute
BIOMIMESYS®Liver