Cryopreservation of corneal endothelial cells in vitro, ex vivo, and on a tissue engineered endothelial graft

IF 3 3区医学 Q1 OPHTHALMOLOGY

Acta Ophthalmologica Pub Date : 2025-01-19 DOI:10.1111/aos.16953

Tomy Sagnial, Melis Coban, Inès Aouimeur, Louise Coulomb, Gain Philippe, Zhiguo He

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引用次数: 0

Abstract

Aims/Purpose: In response to the global shortage of corneas, alternatives such as cell injection therapy and tissue-engineered endothelial keratoplasty (TEEK) have emerged, both relying on the mass production of primary cultured corneal endothelial cells (CECs). Cryopreservation of corneas (the primary cell culture source), cultured CECs (in vitro), and TEEK grafts (final products) is crucial to facilitate their industrialization and clinical application. While corneal cryopreservation has long been a challenge, advancements in cryoprotectants warrant a renewed investigation. This study aims to assess various cryopreservation methods for CECs n the three different states.

Methods: Ten cryopreservation media and varying cooling rates (-2°C/min, -1°C/min, or -0.5°C/min) were first tested on cultured CECs. After thawing, cell survival rates were assessed using Trypan blue staining and an automated cell counter. Subsequently, the best conditions identified for cultured CECs were applied to native CECs adhered to their Descemet's membrane and CECs on TEEKs. Post-thawing viability was assessed using triple labeling with Hoechst, Ethidium, and Calcein-AM (Pipparelli et al. IOVS 2011).

Results: A gradual cooling rate of -1°C per minute and the cryopreservation medium CryoStor CS10 provided the best conditions for in vitro CECs, ensuring an average viability of 91 ± 1% post-cryopreservation. However, applying the same conditions to native CECs on corneas or CECs on TEEK grafts resulted in a viability of less than 80%. Upon thawing, CECs tended to detach easily from the DM or bio-engineered grafts, leading to significant areas without cells.

Conclusions: Cryopreservation is effective for in vitro cultured CECs but remains very challenging for CECs attached to the DM or TEEKs. The next step will be to address cell attachment issues during cryopreservation.

References

Aurélien Pipparelli, Gilles Thuret, David Toubeau, Zhiguo He, Simone Piselli, Sabine Lefèvre, Philippe Gain, Marc Muraine; Pan-Corneal Endothelial Viability Assessment: Application to Endothelial Grafts Predissected by Eye Banks. Invest. Ophthalmol. Vis. Sci. 2011;52(8):6018-6025. https://doi.org/10.1167/iovs.10-6641.

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角膜内皮细胞在体外、离体和组织工程内皮移植上的低温保存

目的/目的：为了应对全球角膜短缺，出现了细胞注射治疗和组织工程内皮角膜移植术（TEEK）等替代方案，这两种方法都依赖于原代培养角膜内皮细胞（CECs）的大量生产。角膜（原代细胞培养源）、体外培养的CECs和TEEK移植物（最终产品）的低温保存对于促进其产业化和临床应用至关重要。虽然角膜冷冻保存长期以来一直是一个挑战，但冷冻保护剂的进步值得重新研究。本研究旨在评估三种不同状态下cec的冷冻保存方法。方法：首先对10种低温保存介质和不同的冷却速率（-2°C/min, -1°C/min，或-0.5°C/min）在培养的CECs上进行测试。解冻后，使用台盼蓝染色和自动细胞计数仪评估细胞存活率。随后，将确定的最佳培养条件应用于粘附在Descemet膜上的天然CECs和TEEKs上的CECs。使用Hoechst、乙锭和钙素- am （Pipparelli等）三重标记评估解冻后的生存能力。IOVS 2011)。结果：冷却速度为-1℃/ min，低温保存介质CryoStor CS10为体外cec提供了最佳条件，冷冻后平均存活率为91±1%。然而，将相同的条件应用于角膜上的天然CECs或TEEK移植物上的CECs，其存活率低于80%。解冻后，CECs容易从DM或生物工程移植物上脱离，导致大面积无细胞。结论：冷冻保存对于体外培养的CECs是有效的，但对于附着在DM或TEEKs上的CECs仍然是非常具有挑战性的。下一步将是解决冷冻保存过程中的细胞附着问题。参考文献aur lien Pipparelli、Gilles Thuret、David Toubeau、何志国、Simone Piselli、Sabine lef vre、Philippe Gain、Marc Muraine；泛角膜内皮细胞活力评估：在眼库预解剖内皮移植中的应用。投资。角膜切削。生物学报，2011;52(8):6018-6025。https://doi.org/10.1167/iovs.10 - 6641。

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来源期刊

Acta Ophthalmologica 医学-眼科学

CiteScore

7.60

自引率

5.90%

发文量

433

审稿时长

6 months

期刊介绍： Acta Ophthalmologica is published on behalf of the Acta Ophthalmologica Scandinavica Foundation and is the official scientific publication of the following societies: The Danish Ophthalmological Society, The Finnish Ophthalmological Society, The Icelandic Ophthalmological Society, The Norwegian Ophthalmological Society and The Swedish Ophthalmological Society, and also the European Association for Vision and Eye Research (EVER). Acta Ophthalmologica publishes clinical and experimental original articles, reviews, editorials, educational photo essays (Diagnosis and Therapy in Ophthalmology), case reports and case series, letters to the editor and doctoral theses.