Cell-sheet technology
Transcript of 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)
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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
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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.
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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)
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State of art: Cellular therapy or Tissue engineering
Scaffold
Hydrogel matrixPeritoneal graft
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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
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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
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•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
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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
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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
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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
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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
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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
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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
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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
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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:
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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
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2017
Merci pour votre attention!
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