Unveiling the lamellar structure of the human cornea over its full thickness using polarizationresolved SHG microscopy Clothilde Raoux et al. 2023
Raoux et al. Light: Science & Applications (2023) 12:190 Official journal of the CIOMP 2047-7538
https://doi.org/10.1038/s41377-023-01224-0 www.nature.com/lsa
A key property of the human cornea is to maintain its curvature and consequently its refraction capability despite daily changes in intraocular pressure. This is closely related to the multiscale structure of the corneal stroma, which consists of 1–3 μm-thick stacked lamellae made of thin collagen fibrils. Nevertheless, the distribution, size, and orientation of these lamellae along the depth of the cornea are poorly characterized up to now. In this study, we use second harmonic generation (SHG) microscopy to visualize the collagen distribution over the full depth of 10 intact and unstained human corneas (500–600 μm thick). We take advantage of the small coherence length in epi-detection to axially resolve the lamellae while maintaining the corneal physiological curvature. Moreover, as raw epi-detected SHG images are spatially homogenous because of the sub-wavelength size of stromal collagen fibrils, we use a polarimetric approach to measure the collagen orientation in every voxel. After a careful validation of this approach, we show that the collagen lamellae (i) are mostly oriented along the inferior–superior axis in the anterior stroma and along the nasaltemporal axis in the posterior stroma, with a gradual shift in between and (ii) exhibit more disorder in the anterior stroma. These results represent the first quantitative characterization of the lamellar structure of the human cornea continuously along its entire thickness with micrometric resolution. It also shows the unique potential of P-SHG microscopy for imaging of collagen distribution in thick dense tissues.
Corneal storage methods: considerations and impact on surgical outcomes Gabriela Wojcik , Stefano Ferrari , Vito Romano , Diego Ponzin , Sajjad Ahmad & Mohit Parekh (2020): Corneal storage methods: considerations and impact on surgical outcomes, Expert Review of Ophthalmology, DOI: 10.1080/17469899.2021.1829476. 

ABSTRACT Introduction: With recent developments in the field of eye banking, human corneas are not only procured and preserved, but also processed and prepared for transplantation. However, one of the challenges that still persists is the long-term storage of tissues without damaging the corneal endothelial cells. Thus, the review aims at reporting the influence of tissue storage conditions on the clinical outcomes. Areas covered: Endothelial cell loss (ECL), graft survival, and contamination from the tissues stored in hypothermic storage and organ culture and; other storage options such as cryopreservation and lyophilization. Expert opinion: Hypothermic storage and organ culture have shown similar ECL. However, due to the relatively new techniques and limited long-term clinical studies, further evaluation is essential to assess the effect of storage time and conditions on the grafts deemed for endothelial keratoplasty.

Post Market Clinical Follow Up


Clinical results of KT

Influence of organoculture medium on the clinical results of keratoplasty transfixiante

Coralie Ouilhon; Under the direction of Anne-Sophie Marty, 2017LYO1M206 – 2017
(Presentation SFO 2018, 5 – 8 May)

Compare the influence of two organoculture media on the prognosis of Keratoplasty Transfixiante (KT). We conducted a Monocentric retrospective study, including patients who benefited from a transfixiante keratoplasty between 1 January 2008 and 31 December 2015 at the Edouard Herriot Hospital in Lyon. The grafts came from the cornea Bank of the Edouard Herriot Hospital. They were conserved in organoculture in a conventional conservation Medium (CorneaMax ®) or in a non-compound animal-based conservation Environment (STEMALPHA2 ®). Prior to grafting, the grafts were déturgescence in a conventional medium containing Dextran T500 (CorneaJet ®) or Poloxamer 188 (STEMLPHA3 ®). Endothelial cell densities (DCE) were compared as a function of conservation media at the beginning and end of conservation, followed by 6, 12, 24, and 36 months after intervention. The main judgement criterion was endothelial decay at 12 months. The secondary judgement criteria were the best corrected visual acuity (CAVM), corneal astigmatism, minimal corneal thickness, and complications. Five hundred and Thirty-nine KT were included, of which 247 were in the StemAlpha group. The daily endothelial loss during conservation was lower in the StemAlpha group (0.19 vs. 0.31, p < 0.001). Endothelial decay at 12 months was 42.34% in the Cornea group and 36.94% in the StemAlpha Group (p = 0,138). There was no statistically significant difference in CAVM, corneal astigmatism, pachymetry, and intraocular pressure between the two groups. The STEMALPHA2 ® conservation medium improves health safety and induces less endothelial cellular lesions during conservation. However, the contribution of Poloxamer 188 during the déturgescence phase on postoperative endothelial decay was not highlighted.

G. Ho Wang Yin_J Fr Ophtalmol_2016

G. Ho Wang Yin_J Fr Ophtalmol_2016
Impact des caractéristiques du greffon cornéen sur les résultats cliniques après Descemet stripping automated endothelial keratoplasty (DASEK).

Effect of donor graft characteristics on clinical outcomes in Descemet stripping automated endothelial keratoplasty (DASEK).

R Hristova et al, 2016


ЛИМБАЛНА БИОПСИЯ След получаване на информирано съгласие бе проведена минимално инвазивна лимбална биопсия. Процедурата включва внимателна дисекция на лимбален епител с размер 2х2 мм в зона с демонстративни палисади на Vogt на 12 ч. при стриктно спазване на асептични условия. В два от случаите за донор бе използвано контралатералното здраво око. При другите двама пациенти биопсията бе получена от зона със запазени палисади на засегнатото око. Полученият материал бе транспортиран до тъканна банка Биорегенерация в среда Stem α 2 (STEM ALPHA, Rhône-Alpes, France).

R. Hristova, Y. Zdravkov, M. Hristova, I. Tanev

The aim of the current study is to investigate the application of autologous ex vivo expanded limbal stem cell transplantation in the management of ocular surface disease. This is the fi rst study in Bulagria in which autologous ex vivo expanded stem cells were transplanted to four patients with mean age 56.5 years from the Department of Ophthalmology, University Hospital Alexandrovska. All patients presented signs of unilateral limbal stem cell defi ciency, associated with trophic disease of the ocular surface. After informed consent was obtained a minimally invasive limbal biopsy was performed. The limbal materials were cultured using a novel protocol without additional xenobiotic products. The grafts were transplanted after fourteen days. Anterior segment optical coherence tomography before and after the intervention was used for assessment of the condition. Success of the procedure was defi ned as 1 . complete epithelialization of the corneal surface, 2. partial or complete reduction of neovascularization, 3. achieving normal corneal transparency, thickness and refl ex, 4. visual acuity improvement. Clinical recovery was observed in all patients, as well as improvement of subjective ocular comfort. In three of the cases all four success criteria were met. Visual acuity of one patient did not change, due to neoplastic process, pervading the optic nerve. No graft rejection reactions were observed. Limbal biopsy did not induce limbal stem cell defi ciency in the donor eye. Transplantation of autologous ex vivo expanded limbal stem cells is an effective and safe method for ocular surface reconstruction, which can be applied in different conditions, associated with limbal stem cell defi ciency.

Contact address: Rozaliya Hristova, 1 G. Sofi iyski str. Sofi a 1431 е-mail: alleta@abv.bg

Comparative study

Hung J, et al. Stem Alpha versus Eurobio : comparaison à un an des résultats de 49 kératoplasties transfixiantes. SFO Mai 2016.
Résumé : Contexte : Afin d’étudier l’efficacité et la sécurité clinique d’un milieu de conservation en organoculture intégralement synthétique, nous avons mené une étude post-greffe comparative entre des patients receveurs de greffons conservés dans les milieux standards contenant des substances d’origine animale et ceux receveurs de greffons des milieux Stem Alpha. Méthode : Une étude rétrospective a été réalisée par inclusion consécutive de 49 patients ayant bénéficié d’une kératoplastie transfixiante (KTF) à but optique du 1er janvier 2013 au 30 juillet 2014 au CHRU de Lille opérés par deux chirurgiens, et suivis pendant un an. Une analyse descriptive et comparative a été menée. Nous avons recueilli 26 greffons conservés dans les milieux Eurobio et 23 greffons des milieux Stem Alpha. Résultats : Nous n’avons pas retrouvé de différence significative concernant les résultats visuels (acuité visuelle à un an 3,50 +/- 2,7 vs 3,84 +/-2, p=0,66), ni en terme de complications (échecs primaires, infection, hypertonie oculaire, décompensation endothéliale, rejet…), ni d’astigmatisme (4,9 +/- 2,4 D vs 4,5 +/- 2,0 D, p=0,66). Nous avons cependant noté une différence significative quant à la densité cellulaire endothéliale plus importante dans le groupe Stem Alpha, révélée dès le comptage préopératoire (2522 vs 2246 p<0,001) et cette différence se maintient significativement à 6 mois (1770 vs 1142 p=0,009) et à un an (1525 vs 1020 p=0,031) après la KTF.
Conclusion : Les greffons conservés dans les milieux Stem Alpha donnent des résultats visuels identiques au milieu standard à un an de suivi. En outre, ils permettent une meilleure préservation des cellules endothéliales.

A-S Marty, C Burillon, A Desanlis, O Damour, V Kocaba, C Auxenfans. Validation of an endothelial roll pr,eparation for Descemet Membrane Endothelial Keratoplasty by a cornea bank using ‘no touch’ dissection technique. Cell and Tissue Banking, pp 1-8, 2016. Spinger link : http://link.springer.com/article/10.1007/s10561-016-9544-y

In vivo structural imaging of the cornea by polarization-resolved second harmonic microscopy
Gaël Latour,1 Ivan Gusachenko,1 Laura Kowalczuk,2 Isabelle Lamarre,1 and Marie‑Claire Schanne-Klein 1,*
1Laboratory for Optics and Biosciences, École Polytechnique—CNRS—INSERM, 91128 Palaiseau, France
2Laboratory for Applied Optics, ENSTA ParisTech—École Polytechnique—CNRS, 91761 Palaiseau, France
*Corresponding author: marie-claire.schanne-klein@polytechnique.edu
Received 7 Nov 2011; revised 29 Nov 2011; accepted 29 Nov 2011; published 1 Dec 2011
(C) 2011 OSA 1 January 2012 / Vol. 3, No. 1 / BIOMEDICAL OPTICS EXPRESS 1

Delessalle N. et al, 2007. Contrôle de qualité externe des milieux utilisés dans la greffe de cornée : résultats des tours de contrôles de 2005 à 2006. AFSSAPS – DLC, unité Produits sanguins et thérapie cellulaire St denis et Microbiologie.

Thuret G., Manissolle C., Campos-Guyotat L, Guyotat D and Gain P. (2005) Animal compound free medium and poloxamer for human corneal organ culture and deswelling, Investigative Ophthalmology & Visual Science, 46, 816-822.

G. Thuret, C. Manissolle, O. Garraud, S. Acquart, L. Campos, D. Guyotat, J. Maugery and P. Gain. New animal compound free medium for human corneal organ culture, Invest Ophthalmol Vis Sci 2004 ; 45 : E-Abstract 4868.

EEBA 2014

ACF cornea storage media STEM ALPHA

STEM ALPHA is a medium without animal component that allows optimal preservation of endothelial cells with good tolerance and effectiveness of transplanted corneas.

Communication SFO 2019

Validation d’un injecteur prêt à l’emploi pour les greffes de cornée type Descemet Membrane Endothelial Keratoplasty : étude expérimentale.

Introduction : La Descemet Membrane Endothelial Keratoplasty a démontré tout son intérêt clinique dans le traitement des pathologies endothéliales. Malgré ses avantages anatomiques et fonctionnels, de nombreux chirurgiens hésitent à pratiquer cette chirurgie difficile notamment à cause de la première étape de dissection du greffon endothéliaux-descemétique. L’objectif de notre travail est de développer et valider un injecteur prêt à l’emploi pour améliorer l’accessibilité à La Descemet Membrane Endothelial Keratoplasty.

Matériels et Méthodes : Nous avons inclus 56 cornées non validées pour un usage clinique mais ayant un consentement pour la recherche. Tous les greffons étaient disséqués selon la méthode “No Touch”. Un groupe test de greffons colorés 3 minutes au bleu trypan puis conservés 72 heures à 22°C (N=30) dans un injecteur en verre Geuder rempli d’un milieu d’organoculture (Stem Alpha ou Eurobio) était comparé à un groupe contrôle (N=15) de greffons prédécoupés et conservés 72 heures flottants. Après amendement, 11 greffons dans le groupe test étaient conservés à 4°C en Stem Alpha. À 72 heures, nous avons évalué le maintien de la coloration dans le groupe test, la densité cellulaire endothéliale par comptage manuel, la viabilité cellulaire par triple coloration Hoechst/ Ethidium Homodimer/ Calcein AM HEC et l’expression de ZO-1 dans les 2 groupes. Les données étaient validées si la coloration au bleu trypan était visible et si nous obtenions moins de 15 % de perte cellulaire entre la DCE avant découpe et la DCE après découpe, coloration et conservation d’au moins 72 heures dans les deux groupes. Résultats : À 72 heures 73,3% des greffons conservés à 22°C maintenaient une bonne coloration (100% Stem Alpha, et 46,6% Eurobio). Le pourcentage de perte cellulaire endothéliale était de 13,5% ± 12,43 dans le groupe test versus 7,8 % ± 11,9 dans le groupe contrôle (p=0,4). Il n’y avait pas de différences statistiquement significatives sur la densité cellulaire endothéliale, le pourcentage de mortalité et de viabilité entre les deux groupes avec la coloration HEC. À 4°C, 100% des greffons maintenaient une bonne coloration, la perte cellulaire était de 8% ± 3,26.

Discussion : Nos résultats concordent avec ceux retrouvés dans la littérature pour d’autres systèmes d’injecteur.

Conclusion : Notre travail montre que la conservation des greffons endothéliaux colorés dans un injecteur en verre pendant 72 heures entraine une perte cellulaire non significativement supérieure aux greffons seulement prédécoupés et conservés flottants, acceptable (<15%), et comparable aux autres études.

Marie Regnier et al. Medicine 2017

Marie Regnier et al. Medicine 2017

Eye bank prepared versus surgeon cut endothelial graft tissue for Descemet membrane endothelial keratoplasty: An observational study.
Regnier, Marie MD a; Auxenfans, Celine PhD b; Maucort-Boulch, Delphine MD, PhD c,d,e; Marty, Anne-Sophie MD a; Damour, Odile PhD b; Burillon, Carole MD a,b,c; Kocaba, Viridiana MD a,b,c,*
[Article] Medicine. 96(19):e6885, May 2017.

The purpose of this article is to examine outcomes of Descemet membrane endothelial keratoplasty (DMEK) performed with cornea bank (CB) prestripped tissue and surgeon stripped tissue (SST).
This retrospective study examined subjects who underwent DMEK with CB or surgeon prepared tissue for Fuchs endothelial corneal dystrophy. Best-corrected visual acuity (BCVA), corneal thickness, endothelial cell count (ECC), and complications were examined before and throughout a 6-month postoperative period.
Eleven CB and 22 SST subjects were included. Six months after surgery, BCVA was 20/20 or better in 36.4% of CB and 22.7% of SST subjects (P = .43). Median logMAR BCVA was 0.10 (0.00-0.20, 20/25) in group CB and 0.10 (0.10-0.30, 20/25) in group SST. Median preoperative corneal thickness was 614.0 [mu]m (577.5-662.0 [mu]m) and 658.0 [mu]m (606.0-689.0 [mu]m) in CB and SST subjects, respectively (P = .37). Six months after surgery, median corneal thickness was lower in the CB group (571.0 [mu]m [478.0-592.0 [mu]m]), than in the SST group (576.0 [mu]m [531.0-607.0 [mu]m], P = .02). At 6 months, median ECC was 1500.0 cell/mm2 (1321.5-2049.0 cell/mm2, 41% decrease) in group CB and 1403.0 cell/mm2 (972.5-2010.7 cell/mm2, 46% decrease) in group SST (P = .70). Rebubbling was required in 5 CB (45.5%) and 15 SST (68.2%) subjects (P = .39).
Fuchs’ dystrophy patients have good anatomic and functional DMEK results. Similar outcomes and complication rates occurred with eye bank and surgeon prepared donor tissue.

An-Katrien De Roo et al, ivos 2017

An-Katrien De Roo et al, ivos 2017

Identification of Circulating Fibrocytes and Dendritic Derivatives in Corneal Endothelium of Patients With Fuchs’ Dystrophy

Bogdan Spiru et al, Cornea 2017

Bogdan Spiru et al, Cornea 2017

Biomechanical Differences Between Femtosecond Lenticule Extraction (FLEx) and Small Incision Lenticule Extraction (SmILE) Tested by 2D-Extensometry in Ex Vivo Porcine Eyes Bogdan Spiru; Sabine Kling; Farhad Hafezi; Walter Sekundo  
Investigative Ophthalmology & Visual Science May 2017, Vol.58, 2591-2595. doi:10.1167/iovs.16-20211

Purpose: To evaluate the biomechanical stability of ex vivo porcine corneas after femtosecond lenticule extraction (FLEx) and small incision lenticule extraction (SmILE) refractive surgeries.
Methods: Forty-five porcine eyes were equally divided into three groups: Groups 1 and 2 were treated with FLEx and SmILE procedure, respectively. Group 3 served as control. A refractive correction of −14 diopters (D) with a 7-mm zone using either a 160-μm flap (FLEx) or a 160-μm cap (SmILE) was performed. For two-dimensional (2D) elastic and viscoelastic biomechanical characterization, two testing cycles (preconditioning stress–strain curve from 1.27 to 12.5 N, stress–relaxation at 12.5 N during 120 seconds) were conducted. Young’s modulus and Prony constants were calculated.
Results: At 0.8% of strain, FLEx (370 ± 36 kPa) could resist a significantly lower stress than SmILE (392 ± 19 kPa, P = 0.046) and the control group (402 ± 30 kPa, P = 0.013). Also, FLEx (46.1 ± 4.5 MPa) had a significantly lower Young’s modulus than the control group (50.2 ± 3.4 MPa, P = 0.008). The Young’s modulus of SmILE (48.6 ± 2.5 MPa) had values situated between untreated corneas and FLEx-treated corneas. When compared to untreated controls, the stress resistance decreased by 8.0% with FLEx and 2.5% with SmILE; Young’s modulus decreased by 5.1% with FLEx and 1.04% with SmILE. With a cap-based procedure, both anterior cap and stromal bed carry the intraocular pressure, while in a flap-based procedure, only the stromal bed does.
Conclusions: Compared to flap-based procedures like FLEx, the cap-based technique SmILE can be considered superior in terms of biomechanical stability, when measured experimentally in ex vivo porcine corneas.



Simultaneous microstructural and mechanical characterization of human corneas at increasing pressure. JMBBM 2016


Patent US 2021/0023273 A1
BEN M’BAREK et al .  Pub . Date : Jan. 28 , 2021 – PCT / FR2019 / 050529