PhytoCellTec™ Malus Domestica

PhytoCellTecTM Malus Domestica
Plant stem cells for skin stem cell protection

© Mibelle Group Biochemistry, Switzerland 2012

PhytoCellTecTM Malus Domestica
3 Stories

1. PhytoCellTecTM technologie
2. Swiss apple story
3. Stem cell cosmetics

© Mibelle Group Biochemistry, Switzerland 2012

PhytoCellTecTM Malus Domestica
Innovation Prize

• BSB Innovation Prize for
most innovative natural
active

• Received during in-cosmetics
2008

• Patent pending in CH, EU,
USA and Korea

© Mibelle Group Biochemistry, Switzerland 2012

1. Plant Cell Cultures

• New cell culture technology makes it now possible to cultivate
cells of endangered and rare species

• Selected plant material (leave, fruit) is wounded to induce the
formation of a callus tissue

• The callus of a plant consists of dedifferentiated, totipotent
cells (plant stem cells)

© Mibelle Group Biochemistry, Switzerland 2012

The 3 Steps of PhytoCellTec

1. Preparing a sterile plant preparation
2. Wounding the plant to induce callus tissue
3. Growing callus cells in liquid cultures

© Mibelle Group Biochemistry, Switzerland 2012

Plant Cellculture Technology
(PhytoCellTecTM =PCT)

• Suspension cultures from plant stem cells are grown in bioreactors
• Medium consists of sucrose, trace elements and plant hormones

(cytokinins and auxins)
• Cultures grow with oxygen and without light (chlorophyll free)
• Batch is harvested after all sugar has been metabolized
• The cells are washed and homogenized to release the secondary

metabolites
• Oil- and water-soluble ingredients are extracted by liposomal structures

© Mibelle Group Biochemistry, Switzerland 2012

2. Swiss Apple Story

© Mibelle Group Biochemistry, Switzerland 2012

Storage Ability of Apples and Skin Aging

• Apples with very good storage
abilities stay fresh over months

• This was an important factor for
cultivar selection

• Modern apple cultivars are selected
for intensive cultivation and for a
sweet flavour since storage is no
longer a problem

• Therefore cultivars with excellent
storage abilities disappeared from
the market

© Mibelle Group Biochemistry, Switzerland 2012

Uttwiler Spätlauber

• An old cultivar is still alive in
Switzerland but no longer cultivated

• The Spätlauber variety derives from
the middle of the 18th century

• It was famous for its tremendous
storability without shrinking

• Today this is a very rare and
endangered apple variety with only a
few trees left in the world

© Mibelle Group Biochemistry, Switzerland 2012

Uttwiler Spätlauber

The importance of the Uttwiler
Spätlauber apple was honored by
the release of a stamp.

© Mibelle Group Biochemistry, Switzerland 2012

Young and Old

© Mibelle Group Biochemistry, Switzerland 2012

The Secret of Storage Ability

• High acid and tannin concentrations
• Longevity of cells
• Undiscovered factors?

This apple can only be used by
PhytoCellTecTM technology

© Mibelle Group Biochemistry, Switzerland 2012

PhytoCellTecTM Malus Domestica

Composition 9.0 %
Malus Domestica Stem Cells 0.14%
Phospholipids 0.4 %
Glycerin 1.4 %
Phenoxyethanol 1.0 %
Keltrol T ad.100 %
Deionized water

CTFA/INCI
Malus Domestica Fruit Cell Culture Extract (and)
Xanthan Gum (and) Glycerin (and) Lecithin (and)
Phenoxyethanol (and) Aqua/ Water

© Mibelle Group Biochemistry, Switzerland 2012

3. Stem Cell Cosmetics

© Mibelle Group Biochemistry, Switzerland 2012

What are Skin Stem Cells?

Properties of skin stem cells:

• Self-renewal - the ability to go through
numerous cycles and cell divisions while
maintaining the undifferentiated state

• Differentiation - the capacity to
differentiate into other cell types of the
same tissue

• Primary roles - replenish dying cells
and regenerate damaged tissues

© Mibelle Group Biochemistry, Switzerland 2012

Adult Stem Cells in Skin

In skin, different types of stem cells have been found:

Type Place
Epidermal skin stem cells Basal layer of the epidermis
Hair bulge stem cells Hair follicle

© Mibelle Group Biochemistry, Switzerland 2012

Epidermal Stem Cells

Stratum corneum
Granular layer

Spinous layer Differentiating progeny of Epidermal
Basal layer one epidermal stem cell stem cell
Dermis
Transient
amplifying
cells

Basement
membrane

Stem cells are located in the basal layer where 2-7% of the cells are stem cells

© Mibelle Group Biochemistry, Switzerland 2012

Hair Bulge Stem Cells

Epidermal layer Infundibulum
Basal layer Sebaceous gland

Outer root sheath Hair bulge
Inner root sheath stem cells
Hair shaft

Hair follicle matrix Dermal papilla

Lineages derived from bulge stem cells
Differentiation pathways supplied by bulge stem cells

© Mibelle Group Biochemistry, Switzerland 2012

Stem Cells and Aging

• Stem cells have a limited life
expectation

• Environmental and intrinsic stress
factors effect stem cells

• In aged skin the number and
activity of stem cells is reduced

• Skin stem cells have to be
protected and supported to delay
preliminary aging

© Mibelle Group Biochemistry, Switzerland 2012

Epigenetic Factors
Composition of Plant Stem Cells

• Plant stem cells contain similar epigenetic factors as human
stem cells

• Plant stem cell extracts provide important nutrients to human
stem cells

 Plant stem cell nutrients improve the viability and healthiness of
human stem cells

© Mibelle Group Biochemistry, Switzerland 2012

PhytoCellTecTM Malus Domestica
Provides two Different Modes of Activity

1. Use of plant stem cells to protect skin stem cells
The components of plant stem cells such as Malus domestica are rich in
specific factors relevant to protect and maintain the function of skin stem
cells

 epigenetic factors

2. Use of longevity cell extract to delay skin aging
The apple Uttwiler Spätlauber is known to contain ingredients which
insure longevity of cells

 metabolites

© Mibelle Group Biochemistry, Switzerland 2012

Novel Progenitor Cell Targeting
Technology

• Biopsy: human epidermal cells  ca. 6% of cells are stem cells (optically not
different from normal basal cells)

• Medium: specifically designed, fully defined medium that mimics the
physiological environment of the in vivo stem cell niche
 enrichment of so called progenitor cells (stem cells)

3x

© Mibelle Group Biochemistry, Switzerland 2012

Activity Assay for Epidermal Stem Cells

 Stimulation of growth or over-expression of markers are not good
parameters for the activity of stem cells

• Colony forming efficiency (CFE)
Cell cultures are seeded at low density. The number of colonies formed is a
value for the concentration and activity of progenitor/stem cells

+ apple stem cell extract Determination
Incubation of CFE

© Mibelle Group Biochemistry, Switzerland 2012

Increase of CFE by
PhytoCellTecTM Malus Domestica

+ 92%

Result
Number of active and
healthy stem cells is
increased compared to
untreated cultures

S-234

© Mibelle Group Biochemistry, Switzerland 2012

Result

PhytoCellTecTM apple stem cell extract enhanced CFE up to 92%
compared to the control.
Maintenance of the stem cell vitality of epidermal stem cells was improved.

© Mibelle Group Biochemistry, Switzerland 2012

Growth of Hair Follicle

Hair follicles From human skin fragments
(from micro dissection)
Test substance
Design PhytoCellTecTM Malus Domestica 0.2%

Length of hair follicle increases until day 14
→ after day 14 induction of senescence
→ length of hair follicle decreases

S-157

Day 0 Day 4 Day 7

© Mibelle Group Biochemistry, Switzerland 2012

Length of Hair Follicle

Control 0.2% Malus Domestica stem cell extract

Change in length compared to day 14 in % 20 Senescence of hair
10 (stem cells) can be delayed
S-157
0 © Mibelle Group Biochemistry, Switzerland 2012
-10 Day 18 Day 20
-20
-30
-40
-50
-60
-70

Day 16

PhytoCellTecTM Malus Domestica
Effect on Gene Expression in Senescent
Dermal Fibroblasts

Cell line Normal Human Dermal Fibroblasts
Design
 H2O2 induced senescence (2h)
Parameters followed by a treatment with
2% PhytoCellTecTM Malus
Domestica or not (144 h)

 Gene profile (microarray)

© Mibelle Group Biochemistry, Switzerland 2012

PhytoCellTecTM Malus Domestica
Gene Profile (Microarray)

↓ Gen, that is down-regulated by senescence induction (see control)
↑ Gen, that is up-regulated or normalized in senescent cells by PhytoCellTec Malus Domestica

Gen After H2O2 After H2O2
Control + 2% Malus Domestica
cyclin B1: induces proliferation
cyclin E1: cell cycle regulator 73 ↓ stem cell extract
p53: tumor suppressor gene 78 ↓
insulin-like growth factor II: cell proliferation enhancer 63 ↓ 130 ↑
heme oxigenase 1: antioxidans enzyme 71 ↓
89 ↓ 135 ↑

137 ↑

117 ↑

211 ↑ S-194

© Mibelle Group Biochemistry, Switzerland 2012

Capacity to Form 3D Epidermis

Study design
Comparison of 3D epidermis formed by early
passage (young) and by late passage (old)
epidermal progenitor cells.
Test substance
• without test substance (control)
• 0.01% PhytoCellTecTM Malus Domestica

S-244

© Mibelle Group Biochemistry, Switzerland 2012

Day 14 Day 21 Day 28

Young cells (P5)
(control)

No cell growth No cell growth Old cells (P14)
(control)

Old cells (P14)
+ PhytoCellTec MD

S-244

© Mibelle Group Biochemistry, Switzerland 2012

Result

Epidermal progenitor (stem) cells of late passage (P14) loose the capacity to
form a 3D epidermis. But when these cells are cultured with PhytoCellTecTM
Malus Domestica, they retain the capacity to form a 3D epidermis similar to
early passage progenitor cells.

PhytoCellTecTM Malus Domestica clearly helps to conserve the progenitor
cell characteristics. Also cells of late passage are able to proliferate and to
undergo the complex process of stratification and differentiation.

© Mibelle Group Biochemistry, Switzerland 2012

In-Vivo Study: Anti-Wrinkle Effect of
PhytoCellTecTM Malus Domestica

• Analysis of wrinkle depth in the crow‘s feet area
• 20 volunteers
• 28 days
• Application twice daily
• Against inital conditions

© Mibelle Group Biochemistry, Switzerland 2012

In-Vivo Study: Anti-Wrinkle Effect of
PhytoCellTecTM Malus Domestica

2% PhytoCellTec Malus Domestica

16

Decrease of wrinkle depth in % 12

8

4 S-186

0 Day 28
Day 14

© Mibelle Group Biochemistry, Switzerland 2012

In-Vivo Study: Anti-Wrinkle Effect of
PhytoCellTecTM Malus Domestica

S-186

© Mibelle Group Biochemistry, Switzerland 2012

PhytoCellTecTM Malus Domestica
Applications

• Stem cell cosmetics
• Rejuvenation products for face and

body
• Fresh cell treatments
• Anti-Aging formulations
• Rejuvenation of hair follicles
• Delay of hair aging

© Mibelle Group Biochemistry, Switzerland 2012