Biofibrillar/nanofibrillar cellulose
hydrogel as 2D/3D cell culture
material
Marjo Yliperttula
Division of Biopharmaceutics and Pharmacokinetics,
Faculty of Pharmacy, University of Helsinki Finland
Native plant cellulose nanofiber
hydrogels support 3D liver cell (Hepa RG
and HepG2) and 3D stem cell spheroid
formation without bioactive matrix
Optimized cell culture systems
have potential in basic biomedical research, drug
development, and cell-based transplantations
n
Predictive cell models for preclinical drug discovery are urgently needed (EU and FDA) to improve the current success rate of 10% in clinical drug testing
n
Improved 3D cell culture systems are needed for tissue
engineering and cell transplantation purposes due to lack of organ donors
3D cell culture: the missing link in drug discovery
S. Breslin & L. O’Driscoll, Vol 18, March 2013, Pages 240–249 Drug Discovery Today
Methods available for 3D multi-cellular spheroid formation:
a) Forced-floating of cells
b) Hanging drop methods
c) Matrices or scaffolds
d) Microfluidic systems
Biophysical microenvironment and 3D culture
physiological relevance
A. Asthana and W.S. Kisaalita, vol 18, July, 2013, 533-40 (Drug Discovery Today)
Illustrative schematic of liver tissue in vivo TEM picture of HepG2 cells cultured on 3D porous polystyrene scafolds TEM picture of pericanalicular region of two adjoining peri- portral hepatocytes of adult rat tissue
The liver
n
The major role in drug metabolism
n
Several cell types:
1.
Hepatocytes
2.
Stellate cells
3.
Sinusoid endothelial cells
4.
Kupffer cells
5.
cholangiocytes
The existing liver models
n Tissue fractions; Liver slices n Cellular models
§ Primary hepatocytes § Cell lines
§ Collagen sandwich method § Three-dimensional cell
aggregate method n Subcellular models
§ human liver microsomes
n Tissue fractions; Difficult to get
n Cell lines
• the loss of various liver-specific functions
• show low levels of drug-metabolism
n Primary human hepatocytes
• unpredictable availability
• limited growth activity and life-span
• phenotypic alterations rapidly after isolation
n Microsomes; Only metabolism
Problems of current models
Future:
- 3D liver cell culture systems
stem cell based, primary cells or cell lines
Criteria:
Morphology
Polarity
Biofibrillar cellulose
Cellulose nanofiber (CNF)
hydrogel
Classifica(on of nano-‐sized celluloses
n
Bacterial nanocellulose
- Production by biosynthesis
- Controllable structure and pure fibers
n
Plant derived nanocellulose
- Nanofiber networkn
Microcrystalline cellulose
-
Aggregated fibrils (particles)
n
Nanowhiskers and cellulose nanorods
Rheological properties of CNF
hydrogels
!" 1 10 100 0,01 0,1 1 G' o r G' ' [ P a] Frequency [Hz] G' G'' #" 0,001 0,01 0,1 1 10 100 1000 10000 100000 0,01 0,1 1 10 100 Vi sc o si ty [Pa s]Shear stress [Pa]
0.1% 0.2% 0.3% 0.5% 1 % a) Frequency dependence of storage (G') and loss modulus (G'') of a 0.5 wt-% CNF hydrogel.
b) Flow curves of 0.1-1% CNF hydrogels as function of shear stress.
Molecular diffusion in CNF
b)
Influence of molecular
radius to permeability (P)
in hydrogel , N = 6
a)
Percent release of
fluorescently labeled dextrans
(FITC-dextrans) from 0.5%
hydrogel as a function of time,
N = 6
Mechanical
adhesion
and
release of the
particles
Cell studies with biomaterials
including nanofibrillar
cellulose hydrogel
Needle
tests
!
Viability of ARPE-19 cells cultured in native CNF
hydrogel after transferring the cells with a syringe
needle of different sizes. The viability is presented
as relative fluorescence intensity.
Reference materials
Biomaterial
Chemistry / classification Methods of scaffold formationExtracelTM Hydro gel Mixture of HA+gelatin+PEG
Chemical cross-linking
Problems, X-linking components toxcicty
hydrogels formed at
physiological pH and ambient temperature
(chemical cross linking)
MaxGelTM ECM Matrix mix of human ECM
components
gel formation at ambient temperature
HydroMatrixTMPeptide cell
culture scaffold
self-assembling peptide nanofiber gel
gel formation with increase in temperature or ionic strength
Matrigel™ mix of animal ECM
components
gelation at elevated temperature
PuraMatrix™ self-assembling peptide
nanofiber gel
gelation initiated by salt concentration of ≥ 1mM
•
Mitochondrial
metabolic activity
noproblem
•
Viability
of 30 days HepaRG culture and 4 days HepG2 culture in 0.7% CNF hydrogel just fine•
Morphology
of HepG2 and HepaRG 3D cells in CNF and PM hydrogel culturecorrect
•
Albumin secretion
of HepG2 and
HepaRG 3D cells in hydrogels
MaxGelTM (MG), ExtraCelTM (EC),
HydroMatrixTM (HM), PuraMatrixTM
Conclusions
1.
Plant derived cellulose nanofiber (CNF) hydrogel
(GrowDex
TM) can be used as a 3D cell culture scaffold for
hepatocyte cell models.
2.
The CNF hydrogels possess ultrastructure and mechanical
properties that may be tuned to fulfill the requirements of
different cell types.
3.
The CNF hydrogel was biocompatible and supported cell
growth and differentiation to 3D spheroids.
4.
Beneficial cell culture properties are based on the unique
extracellular matrix mimicking structural properties of
CNF hydrogels.
5.
The CNF based cell culture scaffolds may be further
optimized for cell culture by adding bioactive components
to the scaffold.
"The use of nanofibrillar cellulose hydrogel as a
flexible 3D model to culture and differentiate
human pluripotent stem cells"
Yan-Ru Lou, Liisa Kanninen, Tytti Kuisma, Johanna Niklander, Luke Noon, Deborah Broks, Arto Urtti, Marjo Yliperttula
CONFIDENTAL
1. Xeno-free, chemically defined culture systems are needed to maintain and propagate human pluripotent stem cells
(hPSCs) for different biomedical applications.
2. Current culture systems have several drawbacks:
n
containing human-origin or animal-origin products;
n
two-dimensinal surfaces that are not mimicing the natural stem cell niche;
n
not scalable to the quantity required for therapy and research
•
Stem cell viability in NFC
hydrogel - no problem
3D hPSC spheroids in 0.5 %
NFC hydrogel – just fine
Pluripotent stemcells cultured in 3D 0.5 % NFC
hydrogel for 21 days (
world record ;-) as fas as we are
aware
)
WA07, day3, 5x
1%
0.5%
0 5 0 200 300 400 500 R el ati ve in cr ea se Cellulase (µg/mg cellulose)
Relative cell growth before and after cellulase
treatment iPS(IMR90)-4 WA07
H9-GFP
No enzyme NFC/GFP 200 µg enzyme/mg cellulose NFC/GFP NFC/GFP 50 µg enzyme/mg cellulose NFC/GFP 500 µg enzyme/mg celluloseH9-GFP
Cell viability after cellulase treatment
To take the 3D stem cell speroids out form the
NFC hydrogel
Cells transferred from 3D to 2D
LN511
LN521
Matrigel
Vitronectin
LN511
LN521
Matrigel
vitronectin
iPS(IMR90)-4
WA07
Stem cell morphology
LN511
H9-GFP
Matrigel
AFP
nuclei
Beta tubulin-III
nuclei
Muscle actin
nuclei
iPS(IMR90)-4 cells after 3D culture
form embryoid body
Cells have normal karyotype after being
cultured in hydrogel
WA07
iPS(IMR90)-4
Ackowledgements
University of Helsinki, Finland
Yan-Ru Lou, Liisa Kanninen, Madhushree Bhattacharya, Melina Malinen, Patrick Lauren, Tytti Kuisma, Johanna Niklander, Covadonna
Parras-Cicuendez, Saara Kuisma, Arto Urtti
Principe Phelippe, Valencia, Spain: Deborah Brook, Luke Noon
Université de Rennes , France: Anne Corlu, Christiane GuGuen-Guillouzo
VTT Technical Research Centre of Finland: Martina Lille
UPM-Kymmene Corporation, Finland: Timo Koskinen, Kari Luukko, Antti
Laukkanen, Esa Laurinsilta
Aalto University, Finland: Olli Ikkala
Funding: BioCenter Finland, Tekes-The Finnish Funding Agency for Technology and
Innovation, EU-FP7 (LIV-ES project, HEALTH-F5-2008-223317), Graduate School of Pharmaceutical Sciences, Academy of Finland (grant 118650), and EU-Erasmus Exchange Student Exchange Programme, UPM-GrowDex -project.