用于人类角膜器官培养和消肿的无动物成分培养基和泊洛沙姆

Animal compound-free medium and poloxamer for human corneal organ culture and deswelling.

作者信息

Thuret Gilles, Manissolle Chloe, Campos-Guyotat Lydia, Guyotat Denis, Gain Philippe

机构信息

Ophthalmology Department, University Hospital of Saint-Etienne, 25 Boulevard Pasteur, F 42055 Saint-Etienne Cedex 2, France.

出版信息

Invest Ophthalmol Vis Sci. 2005 Mar;46(3):816-22. doi: 10.1167/iovs.04-1078.

DOI:10.1167/iovs.04-1078

PMID:15728536

Abstract

PURPOSE

Eliminating fetal calf serum (FCS) from corneal organ culture (OC) media has long been a challenge. This study was an assessment of a new animal compound-free (ACF) medium for corneal storage and of its combination with poloxamer for end-of-storage corneal deswelling.

METHODS

A randomized controlled study with masked assessment compared the ACF medium to standard commercialized media containing 2% FCS and their combination with dextran for deswelling. Paired human corneas were randomly allocated at procurement, one to the ACF medium and the other to the FCS media, and then assessed at day (D)2 and D30 of OC storage and after 48 hours of deswelling. Comparison criteria were endothelial cell density (ECD) and morphometry by a corneal analyser, quality of endothelial visualization (using saline), EC mortality (trypan blue), corneal thickness, corneal transparency, and folding. Fifty-six corneas (28 pairs) with ECD of 2000 cells/mm(2) or more were enrolled. Data were compared using paired tests with P < 0.01 deemed significant.

RESULTS

Parameters were similar at baseline (D2) between groups. Daily EC loss during the 30 days of storage was reduced with the ACF compared with standard (-0.31% +/- 0.30% vs. -0.88% +/- 0.38%, P < 0.001). With poloxamer 188 (Lutrol F68; BASF, Ludwigshafen, Germany), EC loss was substantially reduced (-1.43% +/- 3.60 vs. -15.41% +/- 10.13%, P < 0.001) and morphometry better preserved, despite thickness reduction, transparency improvement and folding reduction comparable to dextran. After 30 days of storage in ACF medium and deswelling in poloxamer 188, ECD was 30% higher (2466 +/- 447 cells/mm(2) vs. 1729 +/- 281 cells/mm(2), P < 0.001). ACF medium alone and combined with poloxamer 188 considerably facilitated EC visualization at D30 and after deswelling.

CONCLUSIONS

The ACF medium combined with poloxamer 188 for deswelling showed superiority over standard FCS medium in its ability to preserve EC viability and facilitate endothelial visualization. This innovative use of poloxamer for deswelling appears far less toxic than does dextran.

摘要

目的

长期以来，从角膜器官培养（OC）培养基中去除胎牛血清（FCS）一直是一项挑战。本研究评估了一种用于角膜储存的新型无动物成分（ACF）培养基及其与泊洛沙姆联合用于储存末期角膜消肿的效果。

方法

一项采用盲法评估的随机对照研究，将ACF培养基与含2% FCS的标准商业化培养基及其与右旋糖酐联合用于消肿的情况进行了比较。配对的人角膜在获取时随机分配，一个置于ACF培养基中，另一个置于含FCS的培养基中，然后在OC储存的第2天（D）和第30天以及消肿48小时后进行评估。比较标准包括内皮细胞密度（ECD）、角膜分析仪测量的形态学指标、内皮可视化质量（使用生理盐水）、内皮细胞死亡率（台盼蓝）、角膜厚度、角膜透明度和褶皱情况。纳入了56只角膜（28对），ECD为2000个细胞/mm²或更高。数据采用配对检验进行比较，P < 0.01被认为具有显著性。

结果

各组在基线（D2）时参数相似。与标准培养基相比，ACF培养基在30天储存期间每日内皮细胞损失减少（-0.31% ± 0.30% 对 -0.88% ± 0.38%，P < 0.001）。使用泊洛沙姆188（Lutrol F68；德国路德维希港巴斯夫公司）时，内皮细胞损失大幅减少（-1.43% ± 3.60对 -15.41% ± 10.13%，P < 0.001），尽管厚度降低、透明度提高和褶皱减少与右旋糖酐相当，但形态学指标得到更好的保留。在ACF培养基中储存30天并在泊洛沙姆188中消肿后，ECD高出30%（2466 ± 447个细胞/mm²对1729 ± 281个细胞/mm²，P < 0.001）。单独的ACF培养基以及与泊洛沙姆188联合使用在D30和消肿后显著促进了内皮可视化。