February 2025
Volume 14, Issue 2
Open Access
Retina  |   February 2025
Effect of the Sugar Present in the Culture Medium on the Preservation of Human RPE Cell Suspensions
Author Affiliations & Notes
  • Shohei Kitahata
    Department of Ophthalmology and Micro-technology, Yokohama City University, Minami-ku, Yokohama, Japan
  • Hinako Ichikawa
    Department of Ophthalmology and Micro-technology, Yokohama City University, Minami-ku, Yokohama, Japan
  • Yuji Tanaka
    New Industry Creation Hachery Center (Niche), Tohoku University, Sendai, Miyagi, Japan
  • Shin Tanaka
    Department of Ophthalmology and Micro-technology, Yokohama City University, Minami-ku, Yokohama, Japan
  • Tatsuya Inoue
    Department of Ophthalmology and Micro-technology, Yokohama City University, Minami-ku, Yokohama, Japan
  • Maiko Maruyama-Inoue
    Department of Ophthalmology and Micro-technology, Yokohama City University, Minami-ku, Yokohama, Japan
  • Kazuaki Kadonosono
    Department of Ophthalmology and Micro-technology, Yokohama City University, Minami-ku, Yokohama, Japan
  • Correspondence: Shohei Kitahata, Department of Ophthalmology and Micro-technology, Yokohama City University, 4-57 Urafunecho, Minami-ku, Yokohama 232-0024, Japan. e-mail: [email protected] 
  • Yuji Tanaka, New Industry Creation Hachery Center (Niche), Tohoku University, 6-6-10 Aoba, Aoba-ku, Sendai, Miyagi 980-8579, Japan. e-mail: [email protected] 
Translational Vision Science & Technology February 2025, Vol.14, 1. doi:https://doi.org/10.1167/tvst.14.2.1
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      Shohei Kitahata, Hinako Ichikawa, Yuji Tanaka, Shin Tanaka, Tatsuya Inoue, Maiko Maruyama-Inoue, Kazuaki Kadonosono; Effect of the Sugar Present in the Culture Medium on the Preservation of Human RPE Cell Suspensions. Trans. Vis. Sci. Tech. 2025;14(2):1. https://doi.org/10.1167/tvst.14.2.1.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose: The retinal pigment epithelium (RPE) is crucial for photoreceptor function, and its dysfunction is associated with several retinal degenerative diseases. This study examines how different sugars in preservation media affect the viability of RPE cells, highlighting the need for effective storage solutions for cell transplantation.

Methods: Human RPE cells were cultured and suspended in modified media with various sugars. The survival rate was measured for cells cultured under adhesion for 3 weeks and for those stored at 37°C for 24 hours. Metabolism was evaluated using liquid chromatography, whereas the responses to hypoxia were assessed with specific markers.

Results: Fructose-containing media significantly enhanced RPE cell survival, even under hypoxic conditions. In adherent cultures, fructose showed higher survival rates compared to galactose, which had a notably low survival rate. Chromatography results indicated that fructose played a role in non-anaerobic metabolism, helping to explain its effectiveness. In suspension, fructose maintained higher cell viability than glucose and suppressed hypoxia markers, suggesting increased resistance to hypoxic stress.

Conclusions: The study highlights the critical role of sugar composition in preservation media on RPE cell survival, positioning fructose as a potential enhancer. Its antioxidative properties suggest fructose could be effective in suspension preservation. These findings indicate that fructose-containing media are promising for preserving RPE cells and could have broader applications in preserving various cell types and tissues.

Translational Relevance: The results of this study may allow for longer-term storage of RPE cells, potentially increasing the versatility of cell transplantation therapy.

Introduction
The retinal pigment epithelium (RPE) plays a crucial role in supporting and maintaining the functions of the photoreceptors in the eye, including supporting the visual cycle, regulating the transport of nutrients to the photoreceptors, phagocytosis, and absorbing stray light. Dysfunction of the RPE is known to be associated with numerous retinal diseases, including age-related macular degeneration (AMD) and Stargardt disease.1 Moreover, recent studies have shown that the preservation of RPE thickness helps to minimize complications following autologous retinal transplantation.2 Recent advancements in stem cell technology have made embryonic stem cells/ induced pluripotent stem cell-RPE (iPSC-RPE) cells viable for use in regenerative medicine. Several preliminary clinical trials have been carried out, focusing on the treatment of both atrophic and neovascular AMD.36 Various forms of grafting have been tried, including use of cell sheets, cell suspensions, and cell strips.4,7,8 Although single-cell suspension presents challenges, such as cell death, due to anoikis and an increased likelihood of cell reflux, it remains the most basic and least invasive approach, particularly in the preliminary stages of preparing both cell sheets and cell strips.810 Therefore, the development of effective methods to inhibit cell death and senescence in the state of single cell suspension is a key to improve all transplantation methods by preventing cell death during their production processes, storage, sipping, and transplantation. 
Various methods of inhibiting cell death in single cell suspensions have been studied in conjunction with the elucidation of the mechanism of cell death. Recently, we found an optimal temperature to inhibit cell death related to both hypoxia and anoikis, a form of apoptosis that occurs when cells are unable to adhere to the extracellular matrix (ECM), in the single-cell suspension of human iPSC-RPE cells.9 More recently, the use of Rho-related protein kinase inhibitors has shown the potential to effectively suppress cell death in single cell suspension of human iPSC-RPE cells, and improve their viability in transplantation.11,12 However, there is limited research focused on the basic composition of the preservation media itself.9 Although the impact of high glucose levels caused by diabetes on the RPE has been extensively studied, there is limited research on the effects of different sugars.13 Based on this, further investigation is warranted to study the effects of sugars on single cell suspension of human RPE cells and their cell death. The primary objective of this study was to assess the impact of different sugars in the preservation media on the cell viability of human RPE cells and to identify the most suitable sugars for cell suspension preservation by substituting the current storage media sugar with various alternatives. 
Methods
Human RPE Cell Culture and Sugar-Modified Media
Human primary RPE cells (Lonza Biologics, Basel, Switzerland) were cultured in maintenance media (DMEM/F12 [7:3] supplemented with B27 [Life Technologies, Carlsbad, CA, USA] and 2 mM-glutamine [Sigma-Aldrich, St. Louis, MO, USA]) containing 10 ng/mL bFGF and 0.5 µM SB431542. This media was the previously established RPE culture media, a self-adjusting media, and served as the control.9,14 The culture media was changed every 2 to 3 days. After 2 to 3 weeks of culture, the RPE cells became near-confluent. When the cells reached confluence, they were incubated with 0.25% trypsin-EDTA (Life Technologies, Carlsbad, CA, USA) at 37°C for 10 minutes. The trypsin was quickly neutralized using fetal bovine serum-containing media. Cell suspensions were prepared in each sugar-modified media in Nunc tubes (Life Technologies, Carlsbad, CA, USA) containing 5.0 × 105 cells/300 µL. For the preparation of the sugar-modified media, an equal weight of sugar was added to and dissolved in the sugar-free maintenance media. All the sugars compared had the same molecular weight and therefore all had the same molar concentration. Sodium chloride was then added to achieve an osmotic pressure equivalent to that of the maintenance media, ensuring that all media were within a range of ± 2 milliosmolar (mOsm)/L. Considering that the maintenance media contains glucose, we also prepared a glucose-containing media to verify the accuracy of the preparation of sugar-modified media, ensuring that it was equivalent to the maintenance media. 
RPE Cell Viability Assessment
Viability of the cells in the attached condition was evaluated after culture for up to 3 weeks. The RPE cells were seeded onto CELLstart-coated 24-well plates and photographed under a microscope (Fig. 1). The cells were detached with 0.25% trypsin-EDTA and the cell viability was examined by trypan blue (0.4%) staining (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The percentage of viable cells was calculated using a hemocytometer (WakenBtech Co., Kyoto, Japan) in a drop of the trypan blue/cell mixture. We then separately counted the cells that showed negative staining (viable) and positive staining (dead) with trypan blue. In the suspension cell viability assessment, RPE cells that had reached confluence in adhesion culture were trypsinized and neutralized to produce a cell suspension. The suspension was adjusted to the same concentration in sugar-modified media and stored in a Nunc tube (5.0 × 105 cells/300 µL). The tubes were placed in a 37°C incubator with 5% CO2 supply for 24 hours, and then cell viability was assessed. 
Figure 1.
 
Time-course of preservation of human RPE cells in the attached condition in each sugar-containing media. Human RPE cells were cultured for 5 weeks at 37°C.
Figure 1.
 
Time-course of preservation of human RPE cells in the attached condition in each sugar-containing media. Human RPE cells were cultured for 5 weeks at 37°C.
We utilized another assay to evaluate the impact of sugar on cell viability to ensure the reliability of our results. Specifically, in the WST-8 assay, we quantified the production of formazan dye, which occurs in the presence of an electron carrier when the highly water-soluble tetrazolium salt, WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt), is reduced by dehydrogenases.15 Each tube containing a cell suspension of 5 × 105 cells in 300 µL of various sugar-modified media was incubated at 37°C with 5% CO₂ for 22 hours. After incubation, 100 µL of the supernatant was transferred to a 96-well plate, followed by the addition of 10 µL of WST-8 (Dojindo, Kumamoto, Japan), and incubated for 2 hours. The absorbance was measured at 450 nm using a microplate reader, with a reference wavelength of 600 nm to account for background absorbance (Tecan, Männedorf, Switzerland). 
Liquid Chromatography/Mass Spectrometry
For the chromatographic assay of pyruvic acid, lactic acid, and LDH, the RPE cells were cultured under adherent conditions in each sugar-modified media for 7 days, after which the supernatant was collected. The RPE cell suspensions were stored separately at 37°C for 3 and 6 hours and the supernatants were collected after centrifugation. We confirmed the absence of any significant change in the cell viability for up to 6 hours of storage at 37°C. Therefore, the results of the chromatographic assay conducted after 6 hours of storage were considered as not being affected by the cell viability. The analyses were performed by liquid chromatography/mass spectrometry (MS; Triple Quadrupole LCMS-8060, Shimadzu, Japan) using a C18 column (3 µm, 2.1 mm × 15 mm). Mobile phase A consisted of 0.1% formic acid in water, whereas mobile phase B consisted of 0.1% formic acid in acetonitrile. In all the experiments, the elution gradient was started at 0% mobile phase B for 1 minute, then linearly increased to 95% mobile phase B in 9 minutes, kept constant for 4 minutes, and then taken back to the initial conditions in 1 minute. The gradient was followed by an equilibration period of 2 minutes before the next injection. During these steps, the column temperature was maintained at 40°C, the flow rate was set as 0.35 mL/min, and the sample injection volume was 1 µL. In the MS analysis, the results were shown as the area ratio between the 2-isopropylmalic acid reference standard and the target amino acids. 
Determination of the Oxygen Tension
To detect hypoxia in the RPE cell suspensions, we used the hypoxia-detecting probe (mono azo rhodamine [MAR]; Goryo Chemical, Hokkaido, Japan), in accordance with the manufacturer's instructions.16 This fluorescent probe consists of a rhodamine moiety with an azo group directly attached to the fluorophore. Due to ultrafast structural changes around the N = N bond, this compound does not emit fluorescence under normoxic conditions. However, in hypoxic conditions, the azo group is reduced, releasing a strongly fluorescent rhodamine derivative, which serves as an indicator of hypoxia response. RPE cell suspensions under each condition were incubated with 1 µM MAR containing 0.1% dimethyl sulfoxide for 6 hours. The fluorescence intensity was then analyzed by fluorescence microscopy (BZ-710; KEYENCE, Osaka, Japan). In order to determine the MAR fluorescence in individual cells, we used the Image J software to circle each cell boundary under phase contrast and superimposed the same image field under the MAR-stained condition to measure the MAR fluorescence intensity within each cell boundary (Supplementary Fig. S1). 
Results
Viability of the RPE Cells in the Attached Condition in Each of the Sugar-Containing Media
To examine the impact of different sugars contained in the media on the viability of the RPE cells after 5 weeks of culture, we used trypan blue staining. The RPE cells were cultured in media containing glucose, galactose, mannose, or fructose and preserved at 37°C for up to 5 weeks (see Fig. 1). The number of live and dead cells was counted by the standard trypan blue exclusion assay. After 3 weeks, the viable cell count was 1.02 ± 0.13 × 105 cells in the control media, 1.05 ± 0.08 × 105 cells in the glucose-containing media, 0.83 ± 0.03 × 105 cells in the galactose-containing media, 0.96 ± 0.06 × 105 cells in the mannose-containing media, and 1.14 ± 0.12 × 105 cells in the fructose-containing media (Fig. 2). Additionally, to assess cell death in a different system, lactate dehydrogenase (LDH) levels were measured using chromatography.17 Consistent with the trypan blue results, galactose showed the highest levels. The control, maintenance media, and glucose showed similar levels, while fructose demonstrated significantly lower LDH levels (Supplementary Fig. S2). 
Figure 2.
 
Trypan blue exclusion assay for cell viability examination in the attached condition. Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 2.
 
Trypan blue exclusion assay for cell viability examination in the attached condition. Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Viability of the RPE Cell Suspension in Each of the Sugar-Containing Media
Next, the viability of the cells was measured in cell suspensions in media containing sugars other than those that reduced the viability of the cells in the attached state (shown in Fig. 2). Because suspension culture presents more challenging conditions for RPE cell survival compared to adherent culture, observations were made after 24 hours of storage.9 After 24 hours of preservation, the viable cell counts were examined by trypan blue staining. The viable cell counts in the suspensions were 0.51 ± 0.09 × 105 cells in the control media, 0.56 ± 0.07 × 105 cells in the glucose-containing media, 1.08 ± 0.13 × 105 cells in the fructose-containing media, and 0.84 ± 0.09 × 105 cells in the mannose-containing media (Figs. 3a, 3b). The cell suspension in fructose-containing media had the highest viable cell count, followed by that in the mannose-containing and then the control media. The results suggested reliability, as there were no significant differences between the viability in the adjusted media and the control media. We conducted additional evaluations using the WST-8 assay. The assessment showed that the highest cell viability was observed with fructose, followed by mannose, glucose, and the maintenance media as the control. The sugar-free media exhibited the lowest viability. This finding was consistent with the results obtained using trypan blue (Supplementary Fig. S3). 
Figure 3.
 
Trypan blue exclusion assay for cell viability examination in cell suspensions. Human RPE cell suspension tubes were stored for 24 hours at 37°C. (a) Trypan blue exclusion assay images of samples from each sugar condition. Scale bars = 50 µm. (b) Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 as compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 3.
 
Trypan blue exclusion assay for cell viability examination in cell suspensions. Human RPE cell suspension tubes were stored for 24 hours at 37°C. (a) Trypan blue exclusion assay images of samples from each sugar condition. Scale bars = 50 µm. (b) Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 as compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Evaluation of the Glycolytic Intermediates by Chromatography
RPE cell suspensions are known to be affected by hypoxia due to cell deposition, causing cell death.9 We used chromatography to measure the contents of pyruvate and lactate involved in anaerobic metabolism. At weeks 3 and 5 of incubation, the contents of lactic acid and pyruvic acid were measured by chromatography. In the third week, LDH levels were also measured. The results of chromatography after 5 weeks showed clearly low levels of pyruvic acid, fructose, and galactose. In regard to the levels of lactic acid, the values were lower in the fructose-containing media than in the mannose- and glucose-containing media, but not as low as the level in the galactose-containing media (Fig. 4). The levels of lactic acid and pyruvic acid were then checked in the cell suspensions in the glucose-containing, fructose-containing, and sugar-free media by chromatography. 
Figure 4.
 
Changes in the lactate and pyruvate levels over time in adherent cultures of RPE cells in each sugar-containing media assessed by chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 4.
 
Changes in the lactate and pyruvate levels over time in adherent cultures of RPE cells in each sugar-containing media assessed by chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Lactic acid and pyruvic acid were then tested for glucose, fructose, and sugar-free in suspension. No significant change in the viability was observed in the cell suspensions up to 6 hours of storage, indicating that cell death was not affected. Although no significant changes in either the lactic acid or pyruvic acid levels were observed after 3 hours of storage, after 6 hours of storage, both the levels of lactic acid and pyruvic acid were significantly lower in the fructose-containing media than in the glucose-containing media (Fig. 5). 
Figure 5.
 
Changes in the lactate and pyruvate levels over time in RPE cell suspensions in each sugar-containing media assessed using chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 5.
 
Changes in the lactate and pyruvate levels over time in RPE cell suspensions in each sugar-containing media assessed using chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Preservation Conditions Affect Oxygen Tension
Based on the chromatography results, we then analyzed the suspension conditions at 37°C preservation with the hypoxia detection marker, MAR (Figs. 6a, 6b). In the glucose-containing media used as a control, as well as in the galactose and mannose media, the fluorescence intensity of MAR was similar; however, it was significantly lower in the fructose media compared with those in the other conditions. 
Figure 6.
 
Hypoxia detection in the cells under the control (glucose), galactose, fructose, and mannose conditions. (a) Imaging of the cell hypoxia marker mono azo rhodamine (green, MAR) in human RPE cells under in each preservation media after 6 hours of incubation at 37°C in a 5% CO2 atmosphere. Scale bar = 50 mm. (b) MAR fluorescence intensity in the same samples as those referred to in (a) (n = 4, means ± SD are presented, P values were calculated by Student t-test, *P < 0.05, **P < 0.01).
Figure 6.
 
Hypoxia detection in the cells under the control (glucose), galactose, fructose, and mannose conditions. (a) Imaging of the cell hypoxia marker mono azo rhodamine (green, MAR) in human RPE cells under in each preservation media after 6 hours of incubation at 37°C in a 5% CO2 atmosphere. Scale bar = 50 mm. (b) MAR fluorescence intensity in the same samples as those referred to in (a) (n = 4, means ± SD are presented, P values were calculated by Student t-test, *P < 0.05, **P < 0.01).
Discussion
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.1820 
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.21 
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 H2O2. 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.1012 
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. 
Acknowledgments
The authors thank all the members of the Department of Ophthalmology and Micro-technology, Yokohama City University and Shimadzu Diagnostics Corporation for their valuable support. We also thank Ken Matsumoto for his essential guidance and technical support in optimizing our chromatography methods, which significantly contributed to this study. 
Supported by a Grant-in-Aid for Early-Career Scientists (21K16902). 
Disclosure: S. Kitahata, None; H. Ichikawa, None; Y. Tanaka, None; S. Tanaka, None; T. Inoue, None; M. Maruyama-Inoue, None; K. Kadonosono, None 
References
Boulton M, Dayhaw-Barker P. The role of the retinal pigment epithelium: topographical variation and ageing changes. Eye (Lond). 2001; 15(Pt. 3): 384–389. [PubMed]
Kitahata S, Inoue T, Tanaka S, et al. Temporal changes in the retinal pigment epithelium–Bruch's membrane complex thickness after autologous retinal transplantation in myopic eyes. Invest Ophthalmol Vis Sci. 2024; 65(12): 25. [CrossRef] [PubMed]
Qiu TG. Transplantation of human embryonic stem cell-derived retinal pigment epithelial cells (MA09-hRPE) in macular degeneration. NPJ Regen Med. 2019; 4(1): 19. [CrossRef] [PubMed]
Mandai M, Kurimoto Y, Takahashi M. Autologous induced stem-cell-derived retinal cells for macular degeneration. N Engl J Med. 2017; 377(8): 792–793. [PubMed]
Sachdeva MM, Eliott D. Stem cell-based therapy for diseases of the retinal pigment epithelium: from bench to bedside. Semin Ophthalmol. 2016; 31(1-2): 25–29. [CrossRef] [PubMed]
Schwartz SD, Regillo CD, Lam BL, et al. Human embryonic stem cell-derived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt's macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet. 2015; 385(9967): 509–516. [CrossRef] [PubMed]
Hirami Y, Mandai M, Sugita S, et al. Safety and stable survival of stem-cell-derived retinal organoid for 2 years in patients with retinitis pigmentosa. Cell Stem Cell. 2023; 30(12): 1585–1596.e6. [CrossRef] [PubMed]
Nishida M, Tanaka Y, Tanaka Y, et al. Human iPS cell derived RPE strips for secure delivery of graft cells at a target place with minimal surgical invasion. Sci Rep. 2021; 11(1): 21421. [CrossRef] [PubMed]
Kitahata S, Tanaka Y, Hori K, et al. Critical functionality effects from storage temperature on human induced pluripotent stem cell-derived retinal pigment epithelium cell suspensions. Sci Rep. 2019; 9(1): 2891. [CrossRef] [PubMed]
Kitahata S, Mandai M, Ichikawa H, et al. Investigation of the effectiveness of gelatin hydrolysate in human iPS-RPE cell suspension transplantation. Regen Ther. 2024; 25: 238–249. [CrossRef] [PubMed]
Kitahata S, Ichikawa H, Tanaka Y, Inoue T, Kadonosono K. Transient rho-associated coiled-coil containing kinase (ROCK) inhibition on human retinal pigment epithelium results in persistent Rho/ROCK downregulation. Biochem Biophys Rep. 2020; 24: 100841. [PubMed]
Ishida M, Sugita S, Makabe K, et al. A ROCK inhibitor promotes graft survival during transplantation of iPS-cell-derived retinal cells. Int J Mol Sci. 2021; 22(6): 3237. [CrossRef] [PubMed]
Zhang Y, Xi X, Mei Y, et al. High-glucose induces retinal pigment epithelium mitochondrial pathways of apoptosis and inhibits mitophagy by regulating ROS/PINK1/Parkin signal pathway. Biomed Pharmacother. 2019; 111: 1315–1325. [CrossRef] [PubMed]
Kamao H, Mandai M, Okamoto S, et al. Characterization of human induced pluripotent stem cell-derived retinal pigment epithelium cell sheets aiming for clinical application. Stem Cell Reports. 2014; 2(2): 205–218. [CrossRef] [PubMed]
Kanemura Y, Mori H, Kobayashi S, et al. Evaluation of in vitro proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells based on cellular metabolic activity. J Neurosci Res. 2002; 69(6): 869–879. [CrossRef] [PubMed]
Piao W, Tsuda S, Tanaka Y, et al. Development of azo-based fluorescent probes to detect different levels of hypoxia. Angew Chem Int Ed Engl. 2013; 52(49): 13028–13032. [CrossRef] [PubMed]
Kopperschläger G, Kirchberger J. Methods for the separation of lactate dehydrogenases and clinical significance of the enzyme. J Chromatogr B Biomed Appl. 1996; 684(1): 25–49. [PubMed]
Kawasaki T, Akanuma H, Yamanouchi T. Increased fructose concentrations in blood and urine in patients with diabetes. Diabetes Care. 2002; 25(2): 353–357. [CrossRef] [PubMed]
The Online Metabolic and Molecular Bases of Inherited Disease | OMMBID | McGraw Hill Medical. Accessed February 20, 2024. Available at: https://ommbid.mhmedical.com/book.aspx?bookID=2709.
Topping DL, Mayes PA. The concentration of fructose, glucose and lactate in the splanchnic blood vessels of rats absorbing fructose. Nutr Metab. 1971; 13(6): 331–338. [CrossRef] [PubMed]
Frenzel J, Richter J, Eschrich K. Fructose inhibits apoptosis induced by reoxygenation in rat hepatocytes by decreasing reactive oxygen species via stabilization of the glutathione pool. Biochim Biophys Acta. 2002; 1542(1): 82–94. [PubMed]
Semchyshyn HM, Lozinska LM, Miedzobrodzki J, Lushchak VI. Fructose and glucose differentially affect aging and carbonyl/oxidative stress parameters in Saccharomyces cerevisiae cells. Carbohydr Res. 2011; 346(7): 933–938. [CrossRef] [PubMed]
Johnson RJ, Segal MS, Sautin Y, et al. Potential role of sugar (fructose) in the epidemic of hypertension, obesity and the metabolic syndrome, diabetes, kidney disease, and cardiovascular disease. Am J Clin Nutr. 2007; 86(4): 899–906. [CrossRef] [PubMed]
Spasojević I, Mojović M, Blagojević D, et al. Relevance of the capacity of phosphorylated fructose to scavenge the hydroxyl radical. Carbohydr Res. 2009; 344(1): 80–84. [CrossRef] [PubMed]
Spasojević I, Bajić A, Jovanović K, Spasić M, Andjus P. Protective role of fructose in the metabolism of astroglial C6 cells exposed to hydrogen peroxide. Carbohydr Res. 2009; 344(13): 1676–1681. [CrossRef] [PubMed]
Ben M'Barek K, Habeler W, Plancheron A, et al. Human ESC–derived retinal epithelial cell sheets potentiate rescue of photoreceptor cell loss in rats with retinal degeneration. Sci Transl Med. 2017; 9(421): eaai7471. [CrossRef] [PubMed]
Figure 1.
 
Time-course of preservation of human RPE cells in the attached condition in each sugar-containing media. Human RPE cells were cultured for 5 weeks at 37°C.
Figure 1.
 
Time-course of preservation of human RPE cells in the attached condition in each sugar-containing media. Human RPE cells were cultured for 5 weeks at 37°C.
Figure 2.
 
Trypan blue exclusion assay for cell viability examination in the attached condition. Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 2.
 
Trypan blue exclusion assay for cell viability examination in the attached condition. Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 3.
 
Trypan blue exclusion assay for cell viability examination in cell suspensions. Human RPE cell suspension tubes were stored for 24 hours at 37°C. (a) Trypan blue exclusion assay images of samples from each sugar condition. Scale bars = 50 µm. (b) Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 as compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 3.
 
Trypan blue exclusion assay for cell viability examination in cell suspensions. Human RPE cell suspension tubes were stored for 24 hours at 37°C. (a) Trypan blue exclusion assay images of samples from each sugar condition. Scale bars = 50 µm. (b) Quantitative analysis of viable cells in each sugar condition (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 as compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 4.
 
Changes in the lactate and pyruvate levels over time in adherent cultures of RPE cells in each sugar-containing media assessed by chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 4.
 
Changes in the lactate and pyruvate levels over time in adherent cultures of RPE cells in each sugar-containing media assessed by chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 5.
 
Changes in the lactate and pyruvate levels over time in RPE cell suspensions in each sugar-containing media assessed using chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 5.
 
Changes in the lactate and pyruvate levels over time in RPE cell suspensions in each sugar-containing media assessed using chromatography (n = 6, mean ± SD, *P < 0.05 and **P < 0.01 compared with all other sugar conditions, 1-way ANOVA with Tukey's pairwise post hoc comparison).
Figure 6.
 
Hypoxia detection in the cells under the control (glucose), galactose, fructose, and mannose conditions. (a) Imaging of the cell hypoxia marker mono azo rhodamine (green, MAR) in human RPE cells under in each preservation media after 6 hours of incubation at 37°C in a 5% CO2 atmosphere. Scale bar = 50 mm. (b) MAR fluorescence intensity in the same samples as those referred to in (a) (n = 4, means ± SD are presented, P values were calculated by Student t-test, *P < 0.05, **P < 0.01).
Figure 6.
 
Hypoxia detection in the cells under the control (glucose), galactose, fructose, and mannose conditions. (a) Imaging of the cell hypoxia marker mono azo rhodamine (green, MAR) in human RPE cells under in each preservation media after 6 hours of incubation at 37°C in a 5% CO2 atmosphere. Scale bar = 50 mm. (b) MAR fluorescence intensity in the same samples as those referred to in (a) (n = 4, means ± SD are presented, P values were calculated by Student t-test, *P < 0.05, **P < 0.01).
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×