Solutions to eradicate stem cell culture

obstacles & barriers using diverse

surface coating technology

Hyunju Lee

Regional Product manager

2

Problem of current cell culture..

•Cell’s viability: Attachment & detachment?

•Limitations in clinical applications: Animal component free?

•Reproducibility of cell culture?

•Effective scale-up for Bioproduction?

We can overcome it using surface coating technology of

NUNC!!

Obstacles & barriers of cell culture

3

Understanding diverse cell culture surface

• Dry coating Energy treatment

-Nunclon delta

-Nunclon Vita

• Wet coating ECM components or Special Polymer coating

-ECM coating

-Special polymer coating- Upcell / Hydrocell

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Understanding diverse cell culture surface

• Dry coating Energy treatment

-Nunclon delta

-Nunclon Vita

• Wet coating ECM components or Special Polymer coating

-ECM coating

-Special polymer coating- Upcell / Hydrocell

5

NUNCLON Delta

(Energy treatment)

For normal adherent cell

6

Nunclon delta surface

(%)100

0

No.

of

att

ached c

ells

Hydrophilic Hydrophobic

Silicon coating

Polystyrene

Optim

um

zone

NUNCLON DELTA

Corona Treatment

Nunclon Delta culture treated surface

•Hydrophilic surface

•Perfect for adherent cell cultures

•A proven cell culture surface for 25 years

Untreated Polystyrene surface

•Hydrophobic surface

•Suit for suspension cell’s proliferation and

growth

7

ECM Coating

(Wet)

For fastidious adherent cell

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What is ECM?

• Cells are connected to Basal Lamina or ECM through Cell Junction

Epitherial tissue

Muscle tissue

Connective tissue

Small Intestine

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ECM Coating

Media and FBS can

construct ECM

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ECM coating

Poly-D-Lysine/ Collagen/CC2 coating

•Uniform coating creates a positive

charge on the surface

•Cells with Low adherence or growth

•CC2 surface : Mimics Poly-D-Lysine

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Special energy treatment

Nunclon Vita

For ADCF stem cell culture

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INNOVATION

Surfaces for stem cell culture

Surfaces: Most stem cells require extra help from the culture surface

to promote their attachment and growth. Traditionally, to maintain and

expand undifferentiated ESCs, one has to rely on feeder cells or ECM

coating.

• Feeder Layers

• Matrigel coating

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INNOVATION

Normal Surfaces for stem cell culture <Feeder layer>

Feeder Layers –a monolayer of fibroblast cells (e.g.

MEF, hFF) to promote fastidious cell culture.

Often the cells of the feeder layer are irradiated or

otherwise treated so that they will not proliferate. In

most cases the feeder cells produce growth factors or

cytokines to facilitate stem cell growth. ESCs

Feeder layer

New Feeder layer

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INNOVATION

Normal Surfaces for stem cell culture <Matrigel>

Matrigel – is the trade name by BD Biosciences for a gelatinous protein mixture

secreted by mouse sarcoma cells. This mixture resembles the complex

extracellular environment found in many tissues and is used as a substrate for

cell culture. Culture dishes can be purchased from BD with Matrigel-coating, or

they can be coated in labs by end users.

Matrigel contains animal components that are not suitable for clinical

applications.

Human ESCs and

iPSCs cultured on

Matrigel surface.

(phase contract, 20X)

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Problem: Existing methods for cultivating human pluripotent stem cells

rely heavily on components such as ECM (Matrigel) coatings and feeder

cells.

Such methods have a number of drawbacks:

- add variability / less reproducible

- are time & labor-intensive; pricey

- have limitations in clinical applications

- not well adaptable to scalable cell expansion.

Solution: NunclonTM VitaTM is a unique energy-treated polystyrene

surface that enables growth of most cell lines directly on the surface

without using matrix coatings or feeder cells.

Which problem does Nunclon Vita solve ?

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The Nunclon Vita Surface treatment

Polystyrene Polystyrene with functional

binding groups

Plasma Treatment

Hydrophobic Hydrophilic

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hESC Culture on Nunclon Vita Surface

• No matrix coatings or feeder layers saving time and labor

• hESC can be maintained for over 10 passages in culture

• hiPS cells can be expand for at least 3 passages

• Need conditioned media & ROCK inhibitor

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INNOVATION

Media and Supplements

Conditioned Media – Media that have been

enriched by feeder cells. These cells

"condition" the media by releasing growth

factors, cytokines, chemicals, etc. into the

supernatants. CM are obtained by sterile

filtration of the supernatants.

There are many kinds of CM depending on

the cells that “condition” them. The actual

components of CM are usually unknown.

Labs make their own CM and freeze them

down for future use. This is a labor-intensive

and time-consuming process. CM can also

be purchased with a premium price.

Basic

fibroblast

growth factor

Irradiated

feeder

MEF

Media

Basic

fibroblast

growth factorES cell

colony

Matrigel-

coated

surface

Conditioned

Media (CM)

Filter and

store CM

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INNOVATION

Media and Supplements

Defined Media – also called “chemically defined medium” in which the

chemical nature of the major ingredients and their amounts are known.

They are sometimes referred to as synthetic media.

The most commonly used defined media for human ESC and iPSC are:

TesR by StemCell Technologies; Nutristem by Biological Industries; and

StemPro by Invitrogen. All of them are very expensive. These defined

media are usually used with Matrigel surface or feeder cells to support

human ESC and iPSC growth.

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Medium Requirement

hESCs in chemically defined

media with ROCKi (10 mM)

hESCs in hFF-conditioned

media with ROCKi (10 mM)

Conditioned medium is required for expansion of human

ES cells and iPS cells on Nunclon Vita surface

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INNOVATION

Media and Supplements

ROCK inhibitors – inhibit Rho kinase (ROCK) activity. A selective ROCK inhibitor,

Y-27632, is known to enhance human ESC recovery upon single cell dissociation

and following cryopreservation. Recently, it has been reported that Y-27632

improves survival of human ESC after cell sorting by flow cytometry.

Y-27632 is widely available through chemical and media companies (e.g. Sigma,

EMD, Stemgent, etc).

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ROCK Inhibitor Requirement

0 mM 1 mM 2 mM

10 mM4 mM

Y27632 is required for seeding and maintaining

human ES cells and iPS cells on Nunclon Vita surface

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Non-Enzymatic Harvest of hESC – Removal of ROCKi

Non-Enzymatic Release and Dissociation

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Special Wet coating

Polymer coating -Upcell

For temperature responsive cell culture

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Special polymer coating- UpCell

Extremely hydrophilic

(cell detachment)Optimal for cell

attachment

(cell adhesion)

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Special polymer coating- UpCell

“Do not need Trypsin, Scrapping”

of

Trypsin EDTA/scraping UPCELL

•Good for harvest

Macrophage, Dendritic cell &

Osteoclast culture

•Do not destroy membrane

and secreted protein.

Membrane protein & cell

junction analysis

Flow cytometry

•Better recovery of harvested

cell Stem cell culture

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Special polymer coating- UpCell

“Do not need Trypsin, Scrapping”

•Good for harvest

Macrophage, Dendritic cell &

Osteoclast culture

•Do not destroy membrane

and secreted protein.

Membrane protein & cell

junction analysis

Flow cytometry

•Better recovery of harvested

cell Stem cell culture

Comparison of E-cadherin blot by

difference in harvest

S: Physical scraping

T: Temperature harvest

D: Dispase treatment

*E-cadherin : Junction protein

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Special polymer coating- UpCell

“Do not need Trypsin, Scrapping”

•Good for harvest

Macrophage, Dendritic cell &

Osteoclast culture

•Do not destroy membrane

and secreted protein.

Membrane protein & cell

junction analysis

Flow cytometry

•Better recovery of harvested

cell Stem cell culture

Trypsin

upcell

Re-attachment kinetics of A-549 cells

10 min 60 min 120 min 300 min

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Creating stemcell sheet for clinical application

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Creating stemcell sheet for clinical application

■LSCD - Persistent epithelial breakdown, superficial corneal vasularization, chronic

discomfort and impaired vision cased by the migration of conjunctival epithelial cells and

blood vessels onto the corneal surface.

■ Issues: Donor shortage, rejection

LCSD caused by Stevens-Johnson syndrome

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Creating stem cell sheet for clinical application

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Creating stem cell tissue model

5 layers of cell-sheets

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Application of cell-sheet – multiple homo cell-sheets (2)

Pile it up!

Cardiac patch

Skeletal myoblast/MSC sheet harvested from UpCell

Stick it on!

Impaired myocardium

Dilated cardiomyopathy

100um

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Application of cell-sheet – multiple homo cell-sheets (2)-Neonatal rat Cardiomyocyte sheet

Shimizu et al., 2002

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Clinical trial

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Special Wet coating

Polymer coating -Hydrocell

For spheroid culture & Embryonic body formation

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Special coating- Hydrocell

Hydrocell

(%)

100

0

No. o

f a

tta

ch

ed

ce

lls

Hydrophilic Hydrophobic

Silicon coating

-Hyd

roC

ell

Lo

w c

ell

bin

din

g d

ish

• Cultivation of cells that are sensitive to unwanted activation and differentiation signals arising from cell adhesion

> Macrophage culture

> Basophilic leukocytes

• Single cell suspension and spheroid culture

> Neurosphere formation

> Embryoid body formation of ES cells

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Special coating- Hydrocell

50um

Neurosphere generation from dental pulp of adult rat incisor

Primary sphere culture

under +EGF, +bFGF

A: Nestin positive progenitor cells (red)

B: Tuj 1 positive neuronal cells (red)

C: S100 positive glial cells (green)

Bar: 10um

Sasaki et al., 2008

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Special coating- Hydrocell

Differentiation of neurosphere

HydroCell sphere culture 7-9 days,

followed by culture on poly-L-ornithine and

fibronectin coated chamber slides 7-14 days

50um 10um 10um

Sasaki et al., 2008

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Special coating- Hydrocell

Focus Focus

HydroCellTM

（Non adherent・round・uniform size）

Non-treated

polystyrene

（Adherent・Non-uniform size）

HydroCellTM Non-treated

polystyrene

※Cultured under 20%FBS

Embryoid body formation of ES cells

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•Cell’s viability: Attachment & detachment?

•Limitations in clinical applications: Animal component free?

•Reproducibility of stem cell culture?

•Effective scale-up for Bioproduction?

Overcome Obstacles & barriers

•Nunclon Vita

•Upcell

•Hydrocell

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Thank you very much!