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(1)

Biofibrillar/nanofibrillar cellulose

hydrogel as 2D/3D cell culture

material

Marjo Yliperttula

Division of Biopharmaceutics and Pharmacokinetics,

Faculty of Pharmacy, University of Helsinki Finland

(2)

Native plant cellulose nanofiber

hydrogels support 3D liver cell (Hepa RG

and HepG2) and 3D stem cell spheroid

formation without bioactive matrix

(3)

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

(4)

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

(5)

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

(6)
(7)

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

(8)

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

(9)

Future:

- 3D liver cell culture systems

stem cell based, primary cells or cell lines

Criteria:

Morphology

Polarity

(10)
(11)

Biofibrillar cellulose

Cellulose nanofiber (CNF)

hydrogel

(12)

Classifica(on  of  nano-­‐sized  celluloses  

n 

Bacterial nanocellulose

- Production by biosynthesis

- Controllable structure and pure fibers

n 

Plant derived nanocellulose

- Nanofiber network

n 

Microcrystalline cellulose

-

Aggregated fibrils (particles)

n 

Nanowhiskers and cellulose nanorods

(13)

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.

(14)

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

(15)

Mechanical

adhesion

and

release of the

particles

(16)

Cell studies with biomaterials

including nanofibrillar

cellulose hydrogel

(17)

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.

(18)

Reference materials

Biomaterial

Chemistry / classification Methods of scaffold formation

ExtracelTM 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

(19)

Mitochondrial

metabolic activity

no

problem

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 culture

correct

Albumin secretion

of HepG2 and

HepaRG 3D cells in hydrogels

MaxGelTM (MG), ExtraCelTM (EC),

HydroMatrixTM (HM), PuraMatrixTM

(20)

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.

(21)

"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

(22)

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

(23)

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%

(24)

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 cellulose

H9-GFP

Cell viability after cellulase treatment

To take the 3D stem cell speroids out form the

NFC hydrogel

(25)

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

(26)

AFP

nuclei

Beta tubulin-III

nuclei

Muscle actin

nuclei

iPS(IMR90)-4 cells after 3D culture

form embryoid body

(27)
(28)

Cells have normal karyotype after being

cultured in hydrogel

WA07

iPS(IMR90)-4

(29)

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.

References

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