In the study, it was found that HPMCs could be successfully cultured from PDE of patients who underwent the PD catheterization for less than 2 weeks.
At the early growth stage, HPMC was spindle-shaped and hard to be differentiated from fibroblasts. After about 7–10 days, the cells gradually grew, contacted with adjacent cells and fused into polygons. When reaching confluency, cells presented a cobble-stone appearance, which could be clearly distinguished from fibroblasts(15). While fibroblasts were negative for cytokeratin and positive for Vimentin, endothelial cells were negative for cytokeratin antigen, the cultured cells were confirmed to be HPMC with both positive expression of cytokeratin and Vimentin by immunofluorescence staining.
So far, there are two main sources of HPMC culture(11, 14). Detaching HPMCs from the human omentum tissue by trypsin digestive methods is very classical. It is of high cell yield and reproducible(12). However, it is an invasive method and difficult to obtain human omentum samples as well as control the time of trypsin digestion. In addition, the omentum tissue needs to be clipped, which will bring about increased chance of cell damage. Above all, it is susceptible to the contamination of fibroblasts or erythrocytes(16). By contrast, the method of culturing HPMC from PDE by centrifugation is simple and non-invasive. The PDE samples were easily obtained from patients. The results showed that PDE-derived HPMCs generally showed a uniform growth pattern with good quality(5). In brief, PDE is a feasible source for the culture of HPMC. But the culture of HPMC from PDE might encounter some problems, such as insufficient cell number, long waiting time, failure of monolayer formation and wide variety in cell phenotype(14). Alternatively, the success rate of PDE-derived culture was unpredictable. And as such, we attempted to establish a practicable method of culturing PDE-derived HPMCs and explore the clinical factors associated with the success rate of culture, which provides a reference to select better PDE samples for the culture of HPMC.
In total, 36 patients were recruited in our study. PDE-derived HPMCs could grow normally and form confluent monolayer with typical cobblestone appearance in 22 patients. The growth condition showed significant interpatient variations, which indicated that the discrepancies in cell morphology and growth condition in PDE-culture could be attributed to the difference in peritoneal membrane status between patients. By comparative analysis of PD patients between two groups, we found that the average days from catheterization of the successful group were less than 9 days, while the failed group were more than 11 days. There were more patients complicated with diabetes mellitus or treated with the erythrocyte lysate in the successful group. According to the results of univariate and multivariate logistic regression analysis, the success rate of culture was found to be associated with days from catheterization and the level of CRP.
On the basis of differences between the two groups, it was speculated that days from catheterization could affect the quantity, morphology and vitality of HPMC. The peritoneal membrane of PD patients will permanently be exposed to bioincompatible peritoneal dialysate, with high glucose, high permeability and low pH value. Besides, glucose degradation products and advanced glycosylation end products generated by stimulation of thermal sterilization and reactive oxygen were included(17). Among them, glucose and glucose degradation products can stimulate the production of transforming growth factor-β (TGF-β) and vascular endothelial growth factor (VEGF), resulting in inflammatory cell infiltration and angiogenesis(18, 19). This in turn lead to the destruction of peritoneal structure, such as the loss of mesothelial cell layer, the thickening of the mesothelial dense area and the gradual occurrence of peritoneal fibrosis(20). Changes in peritoneal structure and function will occur in all patients undergoing PD. Within one to two years of PD, signals of peritoneal fibrosis can be detected in 50–80% of patients(21, 22). To investigate the treatment of peritoneal fibrosis, many scholars have devoted themselves to the induction of cell fibrosis with high glucose or TGF-β1. Results suggested that it took only 24 hours to successfully induce fibrosis (23, 24). HPMCs obtained from PDE have been exposed to high glucose and low pH environment as well as chronic inflammation status of uremia, it is expected that these cells were in a pathological status. Hence, PDE samples should be collected as soon as possible to obtain cells with greater viability.
PDE was collected from patients underwent PD catheterization within 2 weeks. Although peritoneal irrigation was performed after surgery, sometimes doping erythrocytes could be seen in the cell precipitation after centrifugation. If left untreated, cells often struggled to grow and fuse. Therefore, the erythrocyte lysate was used in the culture of HPMC. In fact, the concept of erythrocyte lysate is not new(25), because it has been successfully used in the culture of many other cells (such as liver cells, monocytes, etc.)(26, 27). It is able to protect HPMCs from the contamination and toxicity of erythrocytes, which could improve the purity of HPMCs(28, 29). However, it has not been mentioned in the culture of HPMC. In our study, the comparison between the groups showed that more PD patients in the successful group used the erythrocyte lysate. This indicated that using erythrocyte lysate to eliminate erythrocytes is of value to the normal growth of HPMC and improve the success rate of cell culture by univariate logistic regression analysis. Regrettably, this was not confirmed in multivariate logistic regression analysis, which may be related to the relatively small number of PDE specimens. Although this study did not find sufficient evidence that use of erythrocyte lysate improved the success rate of HPMC culture, it can be used as experience for future culture of HPMC.
Meanwhile, we also found that the level of CRP was associated with the success rate of HPMC culture by the multivariate logistic regression analysis. Differences between groups told us that the failed group had a higher CRP level than that of the successful group, and higher than the upper limit of the normal range. CRP is a well-known marker of inflammation. It is mainly produced by hepatocytes in response to a variety of inflammatory cytokines including interleukin (IL)-6 during infection, tissue injury and inflammation, and subsequently released into circulation(30). Inflammation is a common feature in PD patients. It has been increasingly recognized as a key component in peritoneal fibrosis. The connection between inflammation and fibrosis is usually bi-directional, with one inducing the other(31). Inflammatory stimulation can enhance the production of fibrogenic and angiogenic factors, thus leading to mesothelial injury, apoptosis, and epithelial-to-mesenchymal transition (EMT) (32). The structure and function of peritoneal membrane will be damaged, including increase in the transport rate of small solutes and restriction of water transport. As a result, peritoneal ultrafiltration ability will decrease, even turn into ultrafiltration failure, then PD has to be terminated(33, 34). CRP has been identified as the best systematic marker of inflammation for clinical diagnosis by The American Heart Association and the Centers for Disease Control and Prevention(35). It was elucidated that the PDE samples from patients in the inflammatory state was not conducive to the culture of HPMC.
Beyond that, we also found that there were fewer diabetics in the successful group. Hyperglycemia is cytotoxic, and the degradation products of glucose can induce the apoptosis of PMC, renal tubular epithelial cells as well as endothelial cells, and promote the EMT of PMC(36). Furthermore, hyperglycemia can induce chronic inflammatory response, including CRP, IL-1, IL-6 and tumor necrosis factor-α (TNF-α) are thought to alter vascular permeability, affect vascular dilation and contraction, proliferation of mesangial, endothelium and vascular smooth muscle cells(37, 38). If ESRD patients are complicated with diabetes, their hyperglycemia can aggravate the destruction of peritoneal structure and function, which has been confirmed by some scholars in diabetic rat model(39, 40). But in the univariate or multivariate logistic regression analysis, it was not confirmed that diabetes was associated with the success rate, which may also be related to the small number of samples collected in this study. The evidence that ESRD patients combined with diabetes is not beneficial to primary HPMC culture is not sufficient, more PDE samples and clinical data need to be collected for further study.
It is used to believed that HPMCs could gradually become senescent as patients age, with the subsequent deterioration of cell vitality. However, it was revealed that there was no statistical difference in age between the two groups. The multivariate logistic regression analysis also demonstrated that age had no effect on the success rate of cell culture. Age is no longer a major concern when selecting PDE specimens.
The number of patients included in our study is relatively more than that of the previous study(14), but still insufficient. More PDE samples should be collected to explore the clinical factors associated with the success rate of HPMC culture, so as to establish a solid material base for subsequent experimental studies.