Effects of different cryopreservation methods on the ultrastructure and viability of amniotic membrane

doi:10.3969/j.issn.2095-4344.2015.15.016

Abstract

Abstract:

BACKGROUND: There are currently many cryopreservation methods for the aminotic membrane, which have varying effects on the ultrastructure and biological activity of amniotic membrane, but on no one is effective.
OBJECTIVE: To compare the effects of different cryopreservation methods on the ultrastructure and viability of aminotic membrane and to seek the ideal cryopreservation method.
METHODS: Aminotic membrane separated from the fresh placenta was preserved respectively with deep-frozen cryopreservation and vitrification, and everyway was run for 3 and 6 months. Fresh aminotic membrane was used as control. The ultrastructure of aminotic membrane was observed by transmission electron microscopy, and the viability of aminotic membrane was assessed by microcomputer analysis system for biological oxygen consumption, and immunohistochemical staining combined with image analysis system was used for lactate dehydrogenase activity.
RESULTS AND CONCLUSION: After 3 and 6 months of crypreservation, the damage to the ultrastructure of aminotic membrane by vitreous cryopreservation was slighter than that of amniotic membrane cryopreserved at -80 ℃. Compared with the fresh aminotic membrane, the gray value of lactate dehydrogenase and partial pressure of oxygen were significantly decreased in the cryopreserved aminotic membrane by deep-frozen cryopreservation at 3 and 6 months (P < 0.05) and by vitreous cryopreservation at 6 months (P < 0.05), but there was no statistically significant difference in the change rate of oxygen partial pressure and the gray value of lactate dehydrogenase between the fresh aminotic membrane and the cryopreserved aminotic membrane by vitreous cryopreservation at 3 months. The present study led to the conclusion that vitreous cryopreservation protocol allows to not only maintain the integrity of AM, but also to preserve the viability of the cells. So the vitreous cryopreservation is superior to the deep-frozen cryopreservation for cryopreservation of aminotic membrane.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

全文链接：

Key words: Amnion, Cellular Structures, Lactate Dehydrogenases, Immunohistochemistry, Cryopreservation

CLC Number:

R318

Liu Dai, Jin Jie, Xie Fang, Zhang Chao, Lu Jian-jian, Xu Jia-jie, Xu Jun, Teng Li. Effects of different cryopreservation methods on the ultrastructure and viability of amniotic membrane[J]. Chinese Journal of Tissue Engineering Research, 2015, 19(15): 2376-2381.

Figures/Tables 2

Effect of different cryopreservation methods on ultrastructure changes of amniotic membranes As observed with transmission electron microscopy, fresh amniotic membranes showed intact epithelium, with surface microvillus and junctional complexes between the cells and the basal membrane. Very few cytoplasmic extrusions (blebbing) were observed on the fresh membranes (Figure 1A). Whereas no intact intracellular and extracellular structures were identified in the amnion specimens preserved in the group of deep-frozen cryopreservation, especially after 6 months, presenting no microvillus, loose cytoplasm and nucleus, and absent junctional complexes (Figures 1B, C). No matter after 3 months and 6 months, the amniotic membranes cryopreserved by vitrification showed intact epithelium with thick chromatin and dense microvillus, junctional complexes and a few blebbings (Figures 1D, E). Effect of different cryopreservation methods on oxygen partial pressure of amniotic membranes Oxygen partial pressure of amniotic membranes is referred to the pressure of the soluble oxygen in the amniotic epithelium. The pressure variety in 60 seconds examined by microcomputer analysis system for biological oxygen consumption is positive correlated with viability of the cells. Figure 2 represents the change rate of oxygen partial pressure of amniotic membranes in different conditions. The change rate of oxygen partial pressure of fresh amniotic membranes was (13.9±1.62)%, and it decreased in preserved amniotic membranes in the group of deep-frozen cryopreservation after 3 months [(2.12±0.74)%, P < 0.05] and 6 months [(0.93±0.65)%, P < 0.05], and also in the group of vitrification after 6 months [(6.85±0.99)%, P < 0.05]. But there was no statistical significance between the rate of fresh amniotic membranes and ones cryopreservated by vitrification after 3 months [(12.34±1.90)%, P > 0.05]. Effect of cryopreservations on the lactate dehydrogenase viability of amniotic membranes As shown by the immunohistochemical staining, the amniotic epithelium and extracellular matrix of fresh amniotic membranes and cryopreserved amniotic membranes by vitrification after 3 months and 6 months expressed lactate dehydrogenase with dark brown, but there was not much expression of lactate dehydrogenase in the amniotic membranes in the group of deep-frozen cryopreservation (Figure 3). Twenty positive epithelial cells of each sample were collected randomly by using Leica QWin image analysis system, and the average gray value of lactate dehydrogenase at unit area was measured (Figure 4). The average gray value of the lactate dehydrogenase in fresh amniotic membranes was 0.106±0.011, and it decreased in cryopreserved amniotic membranes: the group of deep-frozen cryopreservation after 3 months was 0.071±0.014 (P < 0.05) and 6 months was 0.047±0.015 (P < 0.05); in the group of vitrification after 3 months was 0.097±0.088 (P > 0.05) and 6 months was 0.072±0.019 (P < 0.05). There was no statistical significance between the fresh amniotic membranes and amniotic membranes in the group of vitrification after 3 months. "

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