Comparative Study On The Biological Characteristics Of Human Amnion Epithelial Cells After Cryopreservetion And The Potential Effect On LPS-induced Acute Lung Injury

AIM:1. Whether cryopreserved amniotic epithelial cells (human Amniotic EpithelialCells, hAECs) in liquid nitrogen would impact its stem cell’s activity, rarely reported.The research would observe the biological activity and stem cell characteristics ofhAECs after cryopreservation from the five aspects, as cell morphology, cell growth,expression of cell surface markers and stem cell genes, osteogenic and adipogenicdifferentiation in comparison with freshly isolated hAECs，to study the reliability ofcryopreserved hAECs in liquid nitrogen.2. Sepsis remains the most common cause of acute lung injury, there is still alack of effective treatment. Our study would detect the lung histopathology andcytokine in lung tissue and serum, to oberve biological effects and the possiblemechanisms of hAECs on endotoxin acute lung injury, in order to explore thefeasibility of the amniotic epithelial cells on treating acute lung injury.METHODS:1. HAECs were obtained by subsequent trypsin digestion after elective cesareansection of healthy pregnancy women at term. Digested hAECs with trypsin for severaltimes and got purification hAECs after several differences digestion in culture. Thenadjusted the purified P2cells to the density of5×106/mL,and added DMSO cryopreservation solution to cells slowly.The frozen pipes would be rapidly placedinto program freezing equipment, and moved to liquid nitrogen tank for preservationuntil the temperature dropped to-100℃. After a month, hAECs were thawed toculture. Detected the cell viability, growth curve, cell surface markers（CD29、CD73、CD166、CD34、CD44、CD90、HLA-ABC）by flow cytometry, and the capacity todifferentiate into osteogenic, adipogenic lineages with certain inducing factors in vitro.Data were analyzed using SPSS19.0statistical software, measurement data wereexpressed as mean±SD, analyzed the differences between the two groups usingindependent sample t–test.2. The newbornal SD rats was inoculated intraperitoneally with20μL/g(5mg/kg)endotoxin (LPS) prepared from Escherichia coli to be lipopolysaccharide-inducedacute lung injury. CM-Dil fluorescence-labeled P2generation amniotic epithelial cellswould be administrated intratracheally after2h of endotoxin. Subgroups of animalswere killed at24and72h. There were five rats at each time point of groups.Newbornal SD rats were randomly divided into four groups:(A) normal group (n=5);(B) model group (n=5): intraperitoneal injection of LPS5mg/kg;(C) cell group (n=5):intraperitoneal injection of LPS5mg/kg+intratracheal instillation hAECs (5×105/40μL);(D) control group (n=5): intraperitoneal injection of LPS5mg/kg+intratracheal instillation40μLPBS. CM-Dil labeled P2generation amniotic epithelialcells were infused through trachea after two hours of modeling, cells, and thensacrificed animals after24h,72h to collect specimens. There are five rats in eachgroup at each time point. Measured lung wet-dry ratio, dected lung injury score andthe thickness of the alveolar septa after HE staining, using ELISA immunosorbentassay to detect lung tissue malondialdehyde (MDA) concentration and superoxidedismutase (SOD) activity, IL-10and IL-6concentrations in serum. Data wereanalyzed using SPSS19.0statistical software, measurement data were expressed asmean±SD, analyzed the differences between the two groups using ANOVA.RESULTS:1. HAECs morphology was not changed after cryopreservation, showed a cobblestone-like growth. Compared with the cells before cryopreservation, the timeof digesting the frozened cells was shortened（P<0.05）.There were no significantstatistical differences in cell viability, time of generation and passages compared withhAECs before cryopreservation.The growth curve of fresh and frozen cells had thesame retention period, logarithmic and plateau phase, which showed S-shaped.2. HAECs after resuscitation can still express stem cell surface markers. Theexpression of CD73, CD29and CD166was more than90%, moderate expression ofCD90, CD44, low expression of HLA class I (HLA-ABC), and negative expression ofhematopoietic stem cell marker CD34. Oct-4and Nanog gene expression was positiveby RT-PCR. hAECs were induced to osteogenic differentiation and adipogenicdifferentiation by appropriate culture medium.3. After intraperitoneal injection of LPS, the lung tissues were congestion insome extents, vascular endothelial cells injuried, some alveolar collapse atelectasis,some alveolar septal thickening and destruction. Compared with the normal group,lung injury score and the thickness of alveolar septum in model group weresignificantly increased (P<0.01), while lung wet-dry ratio reduced (P=0.04).Detection of biochemical markers in lung tissue and serum, we found concentration ofMDA increased gradually while SOD activity reduced with no statistical significance(P>0.05) in the model group compared with the normal group. Concentration ofproinflammatory cytokines IL-6in serum gradually increased and anti-inflammatorycytokine IL-10gradually reduced in model group, compared with the normal group,the level of IL-6at24h is1.25±0.01（P=0.762）, at72h is1.61±0.29(P=0.006); thelevel of IL-10at24h is13.68±1.14(P=0.219),at72h is12.49±0.40(P=0.005),concentration of two cytokines has significant statistical difference at72h.4. After hAECs administration, injury scores and the thickness of alveolarseptum were significantly decreased in comparsion with the control group at24h and72h,the difference was statistically significant (P<0.01). Lung wet and dry ratio (D/Wvalue) in cell group was increased than the control group, the difference wasstatistically significant (P=0.046) at24h, while at72h P=0.366showed no statisticallysignificant.In comparison with the control group, the concentration of the oxidation factor MDA in lung tissue gradually reduced, and anti-oxidants SOD activityincreased gradually in cell group. Levels of proinflammatory cytokines IL-6reducedand levels of anti-inflammatory cytokine IL-10elevated in cell group, compared withthe control group at72h P<0.05, had a statistically significant difference.CONCLUTION:1、Excepted the decreasing of cell adherence, cell morphology、cell cycle、cellproliferation curve、stem cell surface markers and multi-differentiation potential ofcryopreserved hAECs were not changed. Cryopreserved in liquid nitrogen can be areliable method for long-term preservation of hAECs.2、Administrated hAECs from trachea, reduced lung injury score, decreased thethickness of the alveolar space, alleviated pulmonary edema. It demonstrated thathAECs can protect injuried lung tissue from endotoxin by reducing the infiltration ofinflammatory cells and the effusion of alveolar.3、HAECs can reduce the oxidative stress response, alleviated the injury ofoxygen radicals, modulate the balance of pro-inflammatory cytokines andanti-inflammatory factors, to protect the injury tissue and promote the recovery ofdamaged tissue.