Cultured Autologous Oral Mucosal Epithelial Cell-Sheet for Corneal Epithelial Reconstruction (CAOMECS) One-year follow-up for 17 patients/25 included ASCRS, Boston, april 2010 C.Burillon, O.Damour Tissus et Cells HCL Bank The authors have no financial interest in the subject matter of this poster Ophthalmology department

Limbal epithelial deficiency (LSCD) Population Consequences  Thermal or chemical Burns  Stevens Johnson and Lyell Syndrom  Ocular pemphigoid  Aniridia  Severe dry eye  Limbal stem-cell deficiency after cornea transplantations  Other bilateral disorders of the ocular surface  Ulcers  pain  Dryness  Neovascularisation  Conjunctivalisation  Symblepharon Total bilateral LSCD gives rise to  blindness

What do we have to do in LSCD? To Bring an Epithelium Autologous limbus graft Cultured Autologous limbal Epithelium Oral Mucosa Graft For unilateral deficiency Autograft from controlateral safe eye Brings stem cells to the corneal epithelium 1-3 mm² Biopsy For unilateral deficiency For bilateral deficiency Oral mucosa very closed to cornea epithelium +++

Purpose Hypothesis of CAOMECS use: 1 – To restore the ocular surface 2 – To obtain sufficient Cornea transparency when stroma is healthy. 3 – To allow secondary donor cornea graft when stroma is opaque.

Materials and methods We use a specific support for the culture: an UpCell ® Insert which is a thermosensitive polymer - The temperature responsive polymer (PIPAAm) is immobilized by covalent binding on the surface. - The surface change between hydrophobic and hydrophilic is temperature-dependent and reversible. - At the temperature 20~25 ℃, cells can't keep attachment because the surface turned to hydrophilic to liberate hydrophobic interaction. - The surface antigen and extra cellular matrix (ECM) of harvested cells are intact, because trypsin or dispase is not necessary to harvest the cells. 37°C 20°C UpCellBasal membrane hydrophobichydrophilic

Biopsy and Culture using UpCell Insert ® UpCell ® -Insert Biopsy Culture on Upcell Insert at 37°C: 3 weeks Isolation NIH/3T3 in storage Temperature reduced to 20ºC PVDF doughnut ring as a white ring

After three weeks, we obtain in 100% cases: Robust, viable, multilayered epithelial cell sheets, Transparent to read a letter Arial size 10. Holoclones is garant of their functionality. Keratin 3, p63 and b1 integrin and Laminin 5 expression on the sheet Final Product Validation Cytokeratin 3 Laminin 5 Integrin beta-1 p63 Efficiency CFE > 2.1 ± 0.9% Quality keratin 3/76, p63, β1 integrin Microbiological Safety On biopsy transport medium On the culture medium 4 days before harvest the sheet Graft day: on the medium just before harvest the sheet Transport medium  Clinical Trial Hospices Civils de Lyon, France prospective, open, non comparative and monocentric study Stage I/II study by Gehan study design

Results: safety and efficacy at 12 months Grade changes   improvement in blue   worsening in red # Primary endpointSecondary endpoint 1 Corneal burn / 4 keratoplasties cataract surgery, Trabeculectomy + Mitomycine No Ulcer anymore No more vessels  Comfort (dryness or watering, pain and photophobia) 2 Neuroparalytic keratitis 1 lamellar keratoplasty  PEK (2-level improvement) No vessels anymore(3 to 0)  visual acuity,  Comfort (pain, photophobia, dryness watering) 3 Rosacea keratitis / no surgical treatment No Ulcer anymore,  vascularisation Recurrence of few localized vessels  Visual acuity  Comfort (dryness or watering and pain) 4Corneal burn / 1 amniotic membrane  Blood vessels’ activities Recurrence Localized conjunctivalisation since at 6 months, due to surgery or contact lens  Comfort (dryness or watering)  Visual acuity 5 Lyell syndrome / 1 lamellar keratoplasty SAE Corneal Perforation at 6 month, but not related with CAOMECS 6 Corneal burn / 1 lamellar keratoplasty, 1 amniotic membrane No more PEK  Corneal vascularisation (uncountable to 1), blood vessels activity  Comfort (dryness and pain)  1 level decrease of visual Acuity 7 Corneal burn / 1 keratoplasty 1 amniotic membrance No more Ulcer No more PEK  Corneal vascularisation (5 to 1)  Comfort (Photophobia, dryness, pain)

Follow up of safety and efficacy at 12 months Grade changes   improvement in blue   worsening in red # Primary endpointSecondary endpoint 8 Trachoma / Several corneal grafts No Ulcer anymore neither PEK No vascularisation anymore(20 to 0)  Visual acuity  Comfort (no dryness Photophobia,) 9 Lyell syndrome / 1 lamellar keratoplasty No more ulcer  PEK  Corneal vascularisation (20 to 2)  Visual acuity  Comfort (no dryness, no photophobia) 11 Corneal burn / 1 keratoplasty No Ulcer anymore  PEK No vascularisation anymore(12 to 0)  Visual acuity But  Corneal opacification  Comfort (no photophobia, no dryness) 12 Corneal burn / 1 amniotic membrane, 1 lamellar keratoplasty No Ulcer anymore Recurrence Vessel (firstly decrease 20 to 0, but shown recurrence to 5 at 2-month and 7 at 12 month post- graft Recurrence PEK (firstly decreased grade to 0, but shown recurrence to + since 3 –month post-graft)  Comfort (no photophobia and no pain) but  Comfort (dryness was appeared after 1 month.) Patient No 13 could not come for the follow-up assessments since 2-month time.

Follow up of safety and efficacy at 12 months Grade changes   improvement in blue   worsening in red # Primary endpointSecondary endpoint 10 Rosacea keratitis/ 3 keratoplasties, Cataract surgery No Ulcer anymore  PEK  Corneal vascularisation (15 to 3), vessels activity  visual acuity,  Comfort (no photophobia, no dryness) 14 Corneal burn/ 1 Palpebral reconstruction 1 amniotic membrance KPS de ++ to 0 Recurrence of neovasculatisation at 8months  visual acuity,  Comfort (no pain) 15 Aphakia post-congenital cataract + corneal anoxia induce by contact lens/ no surgical treatment  PEK  Corneal vascularisation(40 to 2), Blood vessels activity  visual acuity,  Comfort (no pain, photophobia, no dryness) 16 Corneal burn/ 1 keratoplasty, 1 amniotic membrane No more Ulcer  PEK  Corneal vascularisation (30 to 3  visual acuity,  Comfort (no pain, no dryness, photophobia) 17 Lyell syndrome/ 1 amniotic membrane  PEK  Corneal vascularisation (30 to 8), vessels activity  Comfort (dryness +++ to +)

Without fluoresceinwith fluorescein InclusionOne year follow upInclusionOne year follow up Some examples: Primary criteria : Ocular Surface observed by Slit Lamp Examination After corneal graft

Conclusions Significant regenerated epithelium on the ocular surface: –new good functional epithelium –less vascularisation and vessels activities When stroma and endothelium are healthy, visual acuity can be improved When stroma and endothelium are damaged, visual acuity is difficult to be improved with only CAOMECS treatment but it allows a conventional donor graft without rejections and visual acuity is improved (5 cases up to day) Histology of cornea After CAOMECS + corneal graft

Aknowlegments GCS/CTC tissus and cells bank Odile Damour Chantal Héloire Eric Venet Pascale Pascal Céline Auxenfans Laboratoire Immunocytology Serge Nataf CellSeed Yukio Hasegawa Nao Nakamura Ayumi Murayama Virginie Justin Ophthalmology Carole Burillon Preliminary Human Study Nishida et al, N. Engl.J.Med, 2004, 351,