Our study demonstrated that the sugars contained in the culture media had profound effects on the metabolism of RPE cells in suspension. The composition of the preservation media is very important in the transport/storage of cell preparations, however, the effects of the sugars contained in the media on the viability of RPE cells had not yet been examined. In this study, we found that fructose contained in the preservation media had the effect of promoting anaerobic metabolism in the RPE cells and of thereby improving the cell survival under hypoxic conditions.
It is known that under suspension conditions, that is, in the state of being detached from the scaffold, can induce cell death in RPE cells by anoikis, a type of apoptosis. Therefore, we first investigated the effects of the sugars contained in the media under adherent culture conditions, an environment in which RPE cells survive more easily. Differences were observed in the survival rates for various sugars contained in the media, and galactose exhibited a low survival rate. By the third week of galactose cultivation, the concentration of pyruvate had decreased to nearly undetectable levels. We believe that these are caused by cell death. Among glucose, mannose, and fructose, in which cell survival was observed, pyruvic acid decreased over time, lactic acid increased, and the metabolism of glucose and mannose was glycolytic. On the other hand, as for fructose, pyruvate was significantly lower than the other two, and lactic acid was increased, suggesting that anaerobic metabolism may not be involved. Generally, in the absence of oxygen, cells switch to glycolytic metabolism, synthesize pyruvate, and after synthesizing a small amount of ATP, turn it into lactic acid. This suggests that fructose may have supported cell survival through alternative metabolic pathways rather than solely through anaerobic metabolism. Differences were also seen in the suspension state, with glucose increasing both pyruvate and lactic acid more than fructose. Lactate production is also decreased in the sugar-free condition, indicating that sugar is important for cells to generate energy, as is the poor survival rate. Compared with the adherent culture conditions, the lactic acid value exceeded 40 after 6 hours, suggesting that the suspension state is highly stressful for the cells. Thus, in both the adhesion and suspension states, fructose was less biased toward anaerobic metabolism than glucose, and cells survived.
In regard to the systemic metabolic pathway, the metabolic properties of fructose differ from those of glucose, with fructose being rapidly taken up and metabolized mainly by the liver. Fructose is converted to fructose 1-phosphate by fructokinase, the enzymatic activity of which is much higher than that of hexokinase involved in glucose metabolism, and then split into glyceraldehyde and dihydroxyacetone phosphate by fructose 1-phosphate aldolase, immediately flows into the glycolytic system via triose. Furthermore, fructose uptake into the liver is not regulated by insulin; it follows a metabolic pathway distinct from that of glucose.
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In the fructose-containing preservation media of the RPE cells, suppression of the hypoxic response, with suppressed production of lactate and pyruvate suggestive of anaerobic metabolism, was also observed. These results suggest that the cells would remain resistant to hypoxic stress that may occur during cell deposition in suspension storage. In fact, it has been reported that fructose has the ability to protect tissues against oxidative stress related to anoxia and hypoxia.
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Fructose has been shown to have a stronger reducing capacity as compared with glucose and to be a faster initiator of the glycation reaction.
22 It is noteworthy in this context that there has been significant consideration given to the role of chronic fructose consumption in various health issues like hypertension, obesity, metabolic syndrome, diabetes, kidney, and heart diseases.
23 Hence, it is important to emphasize that consistent consumption of high levels of fructose could disrupt the cellular redox status under normal conditions, thereby impacting their usual functioning.
24 As previous reports and also this study have shown, while constant intake is not desirable, fructose does have a positive effect in short-term, high-stress situations. Short-term use of fructose is associated with protective effects under conditions of oxidative stress, as demonstrated in astroglial cells exposed to excessive external H
2O
2. Therefore, brief supplementation of fructose through diet or infusion could be beneficial in the cytoprotective treatment of neurodegenerative conditions associated with uncontrolled oxidation. Because fructose can penetrate the blood-brain barrier, it has the potential to offer antioxidative protection to the nervous tissue in living organisms. Therefore, contrary to the widely recognized adverse effects of regular fructose intake in normal bodily conditions, immediate administration or consumption of fructose in the presence of oxidative stress could be advantageous in the cytoprotective treatment of neurodegenerative disorders associated with oxidative stress.
25 Numerous studies have explored the impact of fructose on cell preservation.
A previous report has also indicated that administration of fructose acutely could potentially counteract the body’s response to external antioxidants during therapy for specific pathophysiological conditions linked to oxidative stress, such as sepsis, neurodegenerative diseases, atherosclerosis, cancer, and certain pregnancy complications.
24 RPE cells in suspension is a high-stress situation that is associated with anoikis, and it is precisely in such a critical situation that fructose can have a positive impact.
Regenerative medicine utilizing RPE cells is presently under investigation to address issues highlighted in clinical studies regarding transplantation methods. Whereas cell suspension transplantation represents the simplest and least invasive approach, it suffers from a low cell survival rate. In contrast, cell sheet transplantation offers a high cell survival rate, but managing cell sheets poses challenges. Therefore, various methodologies are being explored, including the utilization of biomaterials, such as the amniotic membrane, which allows for the flexible control of thickness and transparency, as cell carriers, and transplantation methods using a stripe structure.
26 The fructose-containing media investigated in this study not only enhances the viability of cell suspensions but also holds potential for culturing and forming any of the transplantation forms currently under consideration, as described above. Furthermore, it may contribute significantly to improving cell protection, potentially in combination with ROCK inhibitors and gelatin hydrolysate, as well as facilitating the long-term preservation and transport of cells, which are essential for practical applications.
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In conclusion, it was found that RPE cells in storage are greatly affected by the sugars contained in the media. Out of all the sugars examined, fructose appeared to favor cell survival when the cells were under critical condition. The conditions and mechanisms explored in this study are expected to be relevant for various types of cells and tissues, potentially offering valuable insights for cell therapy applications beyond just RPE.