download - Lorentz Center

Lorentz workshop September 26th, 2012 | The Netherlands

Technische Universität Berlin | Gerd Lindner

Human skin equivalents in vitro: Increasing the level of complexity to match human biology

The diverse skin types Scalp

3) Scalp Skin - many terminal hairs organised in FUEs - large SG - strengthened APM

2) Abdominal Skin - thinner epidermis: - “fewer” non-pigmented hair follicles, - thick dermis/subcutis, - adipose tissue

1) Palm / Foot Pad -

thick epidermis: sensory/mechanical nerv no hair follicles no sebaceous glands more sweat glands

The ideal skin model Exsiting Skin Equivalents; H& E staining

MatTek „full thickness“ skin equivalent

day15 embryonic skin

The ideal skin model Exsiting Skin Equivalents

MelanDermFT, MatTek

Langerhans cells Fransson et el 1998 Br.J Derm 139 EpidermFT MatTek

Melanoma, MatTek

Psoriasis Model, MatTek

The ideal skin model Desired components

Tissue engineering of replacement skin: Metcalfe and Ferguson, J.R.Soc.Interface 4, 413-37 (2007)

Pre-existing skin engineering skills The Hair Follicle Source and target mini organ

HaCat + HF biopsy fibroblasts on fibrin gel in perfused 7d submers + 14d airlift cuture DP cell exp. + ( 3 weeks)

2 days

+

14 days

pan-Cytokeratin

Trychohyalin

SEM

in vitro culture

in humans

hair follicle

Lindner G et al.; J. Biotech (2011) 152:108 - 112

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The Hair Follicle Growth & Regression

1.) Controls of hair follicle cycling. Stenn KS, Paus R. Physiol Rev 2001: 449–494. 2.) The hair cycle. Alonso,L. & Fuchs,E. J Cell Sci 119 3.) The human hair follicle, a bistable organ? Bernard, B. ExpDerm 2012

Hair related Disorders Manipulating the hair cycle Hyperthrichosis Hirsutism Androgenetic Alopecia

Alopecia Areata Chemotherapy inducedcAlopecia

The vascular network Skin and Hair Follicle

The Vasculature In vitro and in situ

Formation of human capillaries in vitro: Montagno, I. Tissue Eng. 2010:16, 269 Vascularised human tissue models: Schanz J. J Biotech. 2010:148,56

The Organ-on-a-Chip System On-Chip pump, channels and tissue segments

Fluidic inlet Channel plate Pump membrane Glas slide

Valve inlet

Pumping inlet Pneumatic chamber Flow chamber

The aimed modular organized Skin equivalents From technically perfused to dynamic vascularized

DONOR AREA HARVESTING

Dynamic technically perfused

Dynamic vascularized

Modular based Skin equivalents Dermal, epidermal and hair follicle components Dermal Matrix preparation

Cell seeding Dermal Eq

HF equivalent Epidermal Stratification EpiDex®

Collagen Gel HDKC Matriderm®

DONOR AREA HARVESTING Fibroblast

HDMEC EpiFlex® Adipocyte

Chip insertion HDMEC covered

Skin and Hair Follicle equivalents Histology and Immunostainings of equivalents

EpiFlex® + Fib + KC diff | H&E

Mattek® + MF + EpiDex®

EpiFlex ® + Microfollicle +EpiDex®

Neopapilla GFP + HDMEC

EpiFlex ® + HDMEC + Fib

Microfollicles in collagen gel

Skin Organ Culture Foreskin biopsies in perfused MOC-bioreactor, H& E

Biopsy day 0 control

|

Biopsy 14 days in static culture

|

Biopsy 14 days in Chip

Diseased Skin in Organ Culture | Future Perspective MOC-bioreactor, H& E 1) Androgenetic Alopecia:

2) Wounded skin

3) Skin Cancer: Melanoma

4) Skin Cancer: Basal Cell Carcinoma

5) Psoriasis: skin

6) Prurigo nodularis:

disorder with excessive cell division leads to increased thickness of epidermis

Painfull itching noduls all over the body. No cure. Good access.

Skin and Hair In ml and µl scale..

Beren Ataç - Organs on Chips 2012

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Hair Production of Microfollicles

KC

MC

+

ECM

+

DP


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Hair Production of Microfollicles


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Morphogenesis of Microfollicles Developmental stages – Scanning electron microscopy

Neopapilla K

Microfollicle M

20µm

Lindner et al.; J. Biotech (2011) 152:108


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Morphogenesis of Microfollicles Developmental stages - Light microscopy B

A

150µm

150µm

C

F

D

150µm

150µm

Lindner et al.; J. Biotech (2011) 152:108


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Microfollicle characterization

C-kit K15 Vimentin

Col IV

Trychohyalin

Chondroitin-4suphate 150µm

150µm


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Skin and Hair Integration of Microfollicles to skin models BioVaSc

Groeber et al, Biotechnol. J. 2012, In Press


Skin and Hair Integration of Microfollicles to skin models BioVaSc in static vs. dynamic perfusion

1 week

3h DF + HDMECs

1 day NP/MF added

Dynamic perfusion

1 week

Air liquid interphase

12 days

Keratinocytes added


Fixation Analysis

Skin and Hair Integration of Microfollicles to skin models Static trials

EpiFlex® + MF + Keratinocytes


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Skin and Hair Integration of Microfollicles to skin models Micromanipulation

EpiFlex® + MF + Keratinocytes


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Skin and Hair Integration of Microfollicles to skin models Live cell imaging EpiFlex® + MF + Keratinocytes D30 Green: DP –GFP tagged Red: Martix – SHG Blue: Cell nucleus


32

Skin and Hair Integration of Microfollicles to skin models Static trials EpiFlex® + MF + Keratinocytes ColIV

PanCK


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Skin and Hair Multi-organ-chip in vitro vs. ex vivo D14 D25Preputium EpiFlex® + Static MF + EpiDex®

D14 Preputium Dynamic

ColI

ColI- Tenascin

PanCK

Ki67 -TUNEL

ColI- Tenascin

Ki67 -TUNEL


ColIV Vimentin

CK10- CK15

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Skin and Hair ColI - PCK

Multi-organ-chip ex vivo D25 FUEs

Rhodamin


TUNEL - Ki67

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Summary I

• A proprietary procedure to generate human Neopapillae and Microfollicles for testing and transplantation has been established • A dynamic micro-bioreactor platform for organo-typic tissue culture is in place vitro: • Static and dynamically perfused skin models in ml and µl scale are in progress for Microfollicle integration • Bioreactor Organ culture of Hair Follicle Units, normal and diseased skin is ongoing


Summary II and Outlook

•

We could succesfully recellularize Epiflex® decellularized dermal matrix with dynamic perfusion

•

Modular organized and technically perfused full-thickness skin equivalents with integration of microfollicles are under investigation

•

Microfollicle with Endothelial cells is in progress for skin integration studies

•

Vascularized blood perfused skin models with hair microfollicles is envisioned


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Acknowledgement

Roland Lauster Director

Uwe Marx CEO TissUse & Head of GO-BIO

Technische Universität Berlin Institute of Biotechnology Department of Medical Biotechnology Reyk Horland Ilka Wagner Eva Materne Martina Fischer Katharina Schimek Silke Hoffmann Sven Brincker Alexandra Lorenz Matthias Gruchow Ulrike Menzel Alexander Thomas

Annika Jaenicke Agnes Schumacher Luzie Reiners-Schramm Desiree Geßner Karolina Tykwinska Devasena Kanthi Jennifer Binder Shirin Fatehi Mark Rosowski Chris Drewell Lutz Kloke

Funding German BMBF: GO-BIO

IGB, Stuttgart Heike Walles Florian Groeber Jan Hansmann

IWS, Dresden Frank Sonntag Niels Schilling Udo Klotzbach