techniques for ex vivo expansion of epithelial...
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Techniques for ex vivo expansion of epithelial cells
Ricardo P. Casaroli-Marano
Instituto Clínic de Oftalmología – Hospital Clínic de BarcelonaDepartamento de Cirugía y Especialidades Quirúrgicas – UB
Transplant Services Foundation (TSF) – Corporación Sanitaria ClínicInstituto de Investigaciones Biomédicas August Pi i Sunyer - IDIBAPS
European Eye Bank Association – Annual Meeting 2010
Introduction
epithelium
stroma
endothelium
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Introduction
sclerocorneal limbus
limbic region
limbus
limbic region
Vogt palisades
**C
SC
BM / BO
stroma
epithelium
Ocular Surface: clonal characteristics and growth potential
conjunctiva
cornea
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limbic deficiencyperilimbic ischemia (alkali burns)loss perilimbic cells
“conjunctivalization”new vessels formationloss of transparency
Ex vivo expansion cell therapy: Indications
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Methods for cell culture and expansion
Methods for cell and tissue cultures1. Cell cultures
primary
stables
2. Explant cultures
for tissues
3. Organotypic cultures
for complex structures
conjunctivaconjunctiva
3 days 5 days
7 daysconjunctiva monolayer 15 days
Human conjuntival epithelium (explant methods)
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Techniques for ex vivo expansion
minimally invasive biopsy (2 x 1 mm)
one-step enzymatic process
two-step enzymatic process
appropriate substrates (scaffonding for biograft)
1. Preparation 2. Aminiotic membrane 3. Surgical steel mesh
Explant culture method
4. Fixation on mesh 5. Explant 6. Culture growth
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Human limbic sclerocorneal epithelial cells
Human conjunctival epithelial cells
4 days 7 days 21 days
0,7 cm2
21 días
limbo esclero-corneal conjunctiva
1 cm2
21 days
limbic cells
0,7 cm2
21 days21 días y21 days
limbic epithelial cellslimbic epithelial cells
AMAM
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Explant culture method
Transwell device system
Techniques for ex vivo expansion
minimally invasive biopsy (2 x 1 mm)
one-step enzymatic process
two-step enzymatic process
appropriate substrates (scaffonding for biograft)
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Techniques for ex vivo expansion: enzymatic cell culture methods
2 x 1 mm
+ dispase
epithelial sheets+
2 x 1 mm
+ trypsin (± EDTA) one-step me
two-step me
growth on feeder layer method (3T3-J9/SA mitomycin-C inactivated)
+trypsin
ethod
ethod
Techniques for ex vivo expansion: enzymatic cell culture method
Embryoid like bodiesEmbryoid-like bodiesClonal capabilityProgenitor characteristic cells
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Techniques for cell culture and ex vivo expansion
ADVANTAGES DRAWBACKS
Explant culture method
Simple methodologyOptimal cost‐effectiveness correlationSimple infrastructure
Culture “contamination” (fibroblasts, antigen presenting cells, etc.)
ADVANTAGES DRAWBACKS
Enzymatic cell culture method
Selected cell populationOptimal cost‐effectiveness correlationHigh efficiency
Several stepsAdditional tissue handlingMethodologically complexComplex infrastructureLearning curve process
Appropiate conditions to handling cell and tissues for human transplant purpose
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Techniques for cell culture and ex vivo expansion
Requirements 1. Culture media
adequate nutricional intake
2. Culture conditions
proper handling
intake
optimize cultures
3. Culture protection
productoperator
environment
Laminar flow
Sterile handling conditiuons
Culture media and supplements
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Appropriate infrastructure
laminar flow under sterile conditions
Biosafety Level-2 (BSL-2)
Clean Rooms – GMP Regulations
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Techniques for ex vivo expansion
minimally invasive biopsy (2 x 1 mm)
one-step enzymatic process
two-step enzymatic process
appropriate substrates (scaffonding for biograft)
Techniques for ex vivo expansion: substrates
appropriate substrates (scaffonding for biograft)
Aminiotic membrane Fibrin gel Contact lenses PRP / PRGF
• Collagen shields
• Paraffin gauze
• Polymer membranes (biocompatibles)