预汇合视网膜细胞的制备提高了体外和体内移植的存活率：一个小鼠模型。

Preparation of pre-confluent retinal cells increases graft viability in vitro and in vivo: a mouse model.

机构信息

Catherine McAuley Clinical Research Centre, University College Dublin, Dublin, Ireland.

出版信息

PLoS One. 2011;6(6):e21365. doi: 10.1371/journal.pone.0021365. Epub 2011 Jun 29.

DOI:10.1371/journal.pone.0021365

PMID:21738643

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3126823/

Abstract

PURPOSE

Graft failure remains an obstacle to experimental subretinal cell transplantation. A key step is preparing a viable graft, as high levels of necrosis and apoptosis increase the risk of graft failure. Retinal grafts are commonly harvested from cell cultures. We termed the graft preparation procedure "transplant conditions" (TC). We hypothesized that culture conditions influenced graft viability, and investigated whether viability decreased following TC using a mouse retinal pigment epithelial (RPE) cell line, DH01.

METHODS

Cell viability was assessed by trypan blue exclusion. Levels of apoptosis and necrosis in vitro were determined by flow cytometry for annexin V and propidium iodide and Western blot analysis for the pro- and cleaved forms of caspases 3 and 7. Graft viability in vivo was established by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and cleaved caspase 3 immunolabeling of subretinal allografts.

RESULTS

Pre-confluent cultures had significantly less nonviable cells than post-confluent cultures (6.6%±0.8% vs. 13.1%±0.9%, p<0.01). Cell viability in either group was not altered significantly following TC. Caspases 3 and 7 were not altered by levels of confluence or following TC. Pre-confluent cultures had low levels of apoptosis/necrosis (5.6%±1.1%) that did not increase following TC (4.8%±0.5%). However, culturing beyond confluence led to progressively increasing levels of apoptosis and necrosis (up to 16.5%±0.9%). Allografts prepared from post-confluent cultures had significantly more TUNEL-positive cells 3 hours post-operatively than grafts of pre-confluent cells (12.7%±3.1% vs. 4.5%±1.4%, p<0.001). Subretinal grafts of post-confluent cells also had significantly higher rates of cleaved caspase 3 than pre-confluent grafts (20.2%±4.3% vs. 7.8%±1.8%, p<0.001).

CONCLUSION

Pre-confluent cells should be used to maximize graft cell viability.

摘要

目的

移植物失功仍然是实验性视网膜下细胞移植的障碍。关键步骤是制备有活力的移植物，因为高水平的坏死和凋亡会增加移植物失功的风险。视网膜移植物通常从细胞培养中获取。我们将移植物制备过程称为“移植条件”（TC）。我们假设培养条件会影响移植物的活力，并研究使用小鼠视网膜色素上皮（RPE）细胞系 DH01 进行 TC 后，活力是否会降低。

方法

通过台盼蓝排斥试验评估细胞活力。通过流式细胞术检测 Annexin V 和碘化丙啶检测凋亡和坏死水平，通过 Western blot 分析检测 caspase 3 和 7 的前体和裂解形式。通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）和视网膜下同种异体移植物中裂解 caspase 3 的免疫标记评估体内移植物的活力。

结果

未达到汇合的培养物中的非存活细胞明显少于达到汇合的培养物（6.6%±0.8%比 13.1%±0.9%，p<0.01）。在任何一组中，TC 后细胞活力均无明显变化。汇合水平或 TC 后 caspase 3 和 7 均无改变。未达到汇合的培养物中凋亡/坏死水平较低（5.6%±1.1%），TC 后无明显增加（4.8%±0.5%）。然而，培养超过汇合会导致凋亡和坏死水平逐渐增加（高达 16.5%±0.9%）。与来自未达到汇合的培养物的移植物相比，来自达到汇合的培养物的同种异体移植物在术后 3 小时的 TUNEL 阳性细胞明显更多（12.7%±3.1%比 4.5%±1.4%，p<0.001）。来自达到汇合的细胞的视网膜下移植物的裂解 caspase 3 率也明显高于来自未达到汇合的移植物（20.2%±4.3%比 7.8%±1.8%，p<0.001）。