Cell-sheet technology

Autologous cell-laden hydrogel sheet

2016, 20th of May

Autologous cell-laden hydrogel sheet for prevention of post-surgical

abdominal adhesion (PAA)

PhD project of Dr. BRESSON

Director Dr. CHAI (INSERM U1008)

Co-Director Pr. TAKEUCHI (Takeuchi Lab, IIS, Tokyo, Japan)

Clinic problem

Post-surgical abdominal adhesions (PAA)

� Morbidity, infertility, chronic pain, digestive occlusions, etc.

� One of major public health issues

The most frequent post-operative complication:� 50 ~ 90% of the patients� replacement of the peritoneum

by fibrosis 2

Inhibition of inflammation (Avsar et al., 2001)

Prevention of fibrin formation and promotion

Existing strategies for reducing PAA

Prevention of fibrin formation and promotion of fibrinolysis (Topal et al., 2010)

Anti-angiogenesis (Chiang et al., 2000; Greene et al., 2005)

Tissue separation using hyaluronan-based membranes (Becker et al., 1996) (Becker et al., 1996)

No satisfaction!

Best solution � regenerate the mesothelium (Asano et al. 2006, Di Paolo 2007, Kawashini et al. 2013)

3

Function of mesothelium

Mutsaers, “The Mesothelial Cell”, 20044

Regenerative medicine:Cell therapy or Tissue engineering

• Cell isolation before the• Cell isolation before thesurgical intervention

• Cell expansion (with or withoutscaffold)

• Transplantation on the injuredorgan during the surgery

Prevention of PAA

5

Cell source :

Mesothelial cells (MCs)

Adipose stem cells

Scaffold-free (cellular therapy ) :Cell-sheet tranplant

Project outline

Adipose stem cells

(ASCs)

Transplantation in PAA rat model

Scaffold (tissue engineering ):Cell-laden hydrogel transplant 6

State of art: Cellular therapy or Tissue engineering

Cell-sheet technologyInjection of cellsCellular therapy(without Scaffold)

Free-floating MCs are incorporated into the regenerating mesothelium. Kawanishi K, et al. 2013

rASC injection suppressed peritoneal inflammation.Kimi, et al. 2014

�Temperature response culture surface

�Mono- or multilayer cell sheet

�Extra Cellular Matrix (ECM)

Okano et al, 2004

(without Scaffold)

Retention of transplanted cells

in engrafted areas remainsproblematic.

8

Cell-sheet technology

Monolayer of ASCs reversed wall thinning in the scar area and improved cardiac function in rats model of

Multilayered (MCs+ Fb ) cell sheet prevents

the formation of PAA. (Kawanishi K, et Al. 2013)

State of art: Cellular therapy or Tissue engineering

area and improved cardiac function in rats model of

myocardial infarction. (Miyahara, et al., 2006)

the formation of PAA. (Kawanishi K, et Al. 2013)

9

State of art: Cellular therapy or Tissue engineering

Scaffold

Hydrogel matrixPeritoneal graft

10

Denost et Al.Surgery 2015.

Colorectal tissue engineering: Adipose stem cells +

chitosan hydrogel patches to replace colon

Study design

Cell-sheet technologyInjection of cells

Scaffold-free

or

Scaffold

Hydrogel matrixPeritoneal graft

11

Study design

MC MCs exposed to the peritoneal cavity using a Peritoneal graft of Tunica vaginalisMC

Structure of the peritoneal graft

FB Fibroblats (FB) exposed to the peritoneal cavity using the opposite side of Tunica vaginalis graft to expose the subserosa face

MC-sheet MC+FB multilayered sheet graft

Control no graft

FB

12

•PAA model= Coagulation + Suture of parietal peritoneal right flank on 5×15 mm•N=5/group•Sacrifice after 2 weeks

• Qualitative and Quantitative PAA assessment based on macroscopic observation (Macro)

• Histological examination: standard stainning (HPS) and immunohistochemistry (IHC) to labell MCs

Peritoneal graft Subserosal graft No graft, model

Results (1)

Cell-sheet graft

Intr

a o

pe

rati

ve

Intr

a o

pe

rati

ve

vie

w

Sa

crif

ice

vie

w

13

c

h

Sa

crif

ice

vie

w

No adhesion on MC graft; PAA prevention in comparison with the

three other groups: PAA in 100 % of cases.

Results (2)Peritoneal graft Subserosal graft Cell-sheet graft No graft, model

g: coloration

standard rat

HPS

•*** ***•

Peritoneum Adominal

muscle

Intestin

c

standard rat

30

k: HBME rat

30

h

IHC

•

•***

***

***

Adominal muscle

Adominal

muscle

Intestin

Non

specific

Liver

Muscle

14

Cytokeratin AE1/AE3 IHC stained a thin top layer of MCs ( ) on the peritoneal graft. No stainning

on the injured surface in the other groups. Non specific staining on others epitheliomas like

intestine mucosa. Fibrotic tissue (•); leucocytic infiltrate (***); granuloma formation and foreign

body reaction (surrounded by black circle).

• specific

staining

Liver Adominal

muscle

Results (3)

Qualitative score of PAA

15

Extent of PAA

Conclusion on cellular therapy and tissue engineering

Cell-sheet technologyInjection of cells

Disadvantages

1. No mechanical

resistance

Scaffold-free

or

Scaffold

resistance

2. Delicate to

transplant

Hydrogel matrix

Peritoneal graft prevented

PAA occurrence.

But no clinical application!!(invasif removal, limited

quantity, tumoral tissue)

Needs

• Scaffold

• MCs

16

MCs ASCsA

dv

an

tag

es • Well-differenciated:

multi-function

• Ease of harvesting tissue

• Ease of isolating stem cells

• Self renewal: long-term proliferation

State of art: choice of cellA

dv

an

tag

es

multi-function • Self renewal: long-term proliferation

capacity

• Multipotent differenciation

Dis

ad

va

nta

ge

s • Limited expansion capacity

• Invasive method of tissue harvest

with limited quantity

• Potential problems with source

tissue

• Trans-differentiation to mesothelial

cell has yet to be proven

Comparative study of MCs/ASCs

in Rat (r) and Human (h) (prospective clinical study)

Dis

ad

va

nta

ge

s

tissue

17

Tissue source :

•Testis vaginalis

Results obtained on «choice of cell»

rMCsIsolation,

Culture

Enzymatic

digestion

Culture

Phenotype characterization

rMCs, P0, D7VimentinCytokeratin 8+18 18

Enzymatic

rASCs from subcutaneous fat tissue

Results obtained on «choice of cell»

digestion

Multilineage differentiation

Self renewal:Colony forming efficiency (28%)

Expansion in vitro

Adipogenic differenciation, D14

Oil red O lipid staining

Osteogenic differenciation, D31. Alizarin red calcium staining

rASCs,, P0, D6

19

Features of required biomaterial

State of art: Scaffold

1. Injectable for clinical application 1. Injectable for clinical application by open or mini invasive surgery

2. Auto-adherent3. Biocompatible4. Degradable5. 3D environment for Cell Growth

and Cell Differentiation

20

and Cell Differentiation

State of art: Scaffold

BD PuraStat® Peptide Hydrogel

1. Synthetic matrix2. Standard amino acids (2.5 % w/v)3. Nanometer scale fibrous structure (pore size of 50-200 nm)4. Biocompatible5. Resorbable

21

5. Resorbable6. No animal-derived material and pathogens7. Injectable8. Gelation induced by saline buffer pH 79. pH=2.3

1%

State of art: BD PuraStat® PeptideBlood loss (g)

Markers of

(Dental pulpstem cells)

Purastat permits stem cell

Con

trol

Pur

asta

t2%

Pur

asta

t1%

Gel

atin

Markers of odontoblast

22Cavalcanti BN, et al (2013) Dental Materials 29: 97-102.Song H, et al (2010) Macromol. Bioscience, 10, 33–39

Purastat can reduce hemorrhage, with only minimal tissue responses.

Purastat permits stem cell adhesion, growth and differentiation.Usefull for tissue engineering based on stem cells.

Results obtained with BD PuraStat® Peptide

Cells encapsulation in Purastat, in vitro & in vivo

Factors tested:• Purastat concentrations: 1.25%, 0.75%, and 0.5%• Type of cells: continuous cell line, primary MCs• Cell concentration: 1 M cell/mL and 500 Kcell/mL

Results:

23

Results:• Cell survival has been shown: 79-89%.• Improvement of the technic of manipulation of the gel.• Waiting for results of MC encapsulated cells

transplantation on PAA model.

PerspectivesMCS

� In vitro MCs function assessment with and without scaffold.

� Characterization of MCs by transmission electron microscopy (TEM) and

scanning electron microscopy (SEM) 2015

ASCs

� ASCs cells sorting by FACS techniques: CD34+CD31-CD45-CD146-.

� ASCs labeling strategies for in vivo cell-tracking experiments,

Hydrogel choice

� Improve Purastat

2015

2016

� Other scaffold to find

Cell laden scaffold construction

� Improve MC proliferation into the scaffold

� Test ASC proliferation but also differentiation in the scaffold

24

2017

Merci pour votre attention!

25