Several methods for the cryopreservation of cultured cells have been developed and tested, with emphasis placed on using cryoprotectants that are highly water-soluble, allowing for a high degree of concentration during freezing, and have no cytotoxicity ^[3][4][12,13]. DMSO is commonly used as a low-molecular-weight cryoprotectant. It easily penetrates cell membranes, and is exemplified by ethylene glycol, trimethylene glycol, methanol, dimethylacetoamide, and glycerol, which each demonstrate cryoprotective action ^[5][14]. By contrast, high-molecular-weight cryoprotectants, such as polyethylene glycol, polyvinylpyrolidon, hydroxylethyl starch, dextran, and albumin, show cryoprotective action that stops outside of cells ^[5][6][14,15]. Of these cryoprotectants, glycerol is frequently used as a substitute for DMSO to preserve red blood cells at a concentration of 10%, although DMSO is clearly superior with respect to cell survival rate ^[7][8][16,17]. However, DMSO also shows strong cytotoxicity, and in addition to being used as a cryoprotectant, it is also used as a differentiation inducing agent for cultured cells. Therefore, the use of DMSO is not recommended when aiming for stability during cell storage ^[9][10][11][18,19,20]. In addition, serum is often used during cryopreservation with DMSO, which poses another problem. When animal serum is used, there is a concern of zootic infection such as bovine spongiform encephalopathy ^[12][21]. In addition to its high cost, these disadvantages have motivated the development of a serum-free cryopreservation liquid. Fructan as a natural plant compound shows good potential in this regard. It is assumed that fructan in plants may serve as an anti-freezing agent (cold protectant), as well as an osmotic protectant (protection against dryness or acid) or other protectant against drought. Furthermore, fructan is known for its cell membrane strengthening, stabilization action, anti-allergy effects, and anti-tumor activity ^[11][13][20,22]. In particular, Graminan-type fructan is a polysaccharide stored in plants such as rakkyo (Japanese leek) and wheat, and various activities are expected since it has a highly branched structure ^[14][23]. The rakkyo fructan used in this experiment was graminan-type, which links at a 3:1 ratio of beta-2,1 linking to beta-2,6 linking. It is characterized by the fact that it is easily dissolved in cold water (30% or more solubility in 4 °C water) and is capable of high-pressure steam sterilization; Ogawa et al. ^[15][7] demonstrated its efficacy as a mammalian cell cryoprotectant. The key factors contributing to this effect are that it is highly water-soluble and can be highly concentrated during freezing, and that it prevents cell membrane damage from ice crystal formation on the outside of cells by enveloping the membranes like a net ^[15][7].

In our study, rakkyo fructan was used to determine its potential application as a pancreatic islet preservation liquid using rat pancreatic islets. First, the optimum concentration was determined by comparing survival rates in order to apply fructan to the cryopreservation of pancreatic islets. Fructan concentration-dependent improvement in survival rate was observed, with significantly higher survival at concentrations of 30% or less (p < 0.05).

The survival rate immediately after thawing in the 30% fructan group was 97.2 ± 3.6%, and survival decreased gradually but was maintained above 93% after 3 days. Between fructan concentrations of 30% and 40%, the 30% group showed signs of a higher pancreatic islet survival rate, but no significant difference was found. Moreover, 30% is thought to be the optimum concentration for cryopreservation of pancreatic islets with fructan, i.e., the result of ruling out toxicity to pancreatic islets even at high concentrations of fructan.

Next, we compared the effects of 30% fructan (considered to be the optimum concentration) with those of 10% DMSO solution (the commonly used cryopreservation liquid). Although there was no difference in the survival rate between the two groups immediately after thawing, there was a sharp decrease in survival rate for the 10% DMSO group, reaching 64.8 ± 14.6 by the third day. Morphological observations of cultured pancreatic islets after thawing showed that islet cell clusters in the fructan groups were well-maintained even after 3 days, whereas the cell cluster margins had collapsed and vacuolation was found in the islets in the DMSO groups. As mentioned above, fructan is considered to demonstrate a protective effect against the structural collapse of cells due to cold stress by stabilizing the cell membranes, but its action on pancreatic islets (which are cell clusters) is assumed to be due to protection from external stimulation (for islets, not individual cells). On the other hand, although DMSO is commonly used as a cryoprotectant to protect cells from ice crystal formation due to freezing on the inside and outside of cells, it ultimately decreased the survival rate, which is likely a result of penetrating the cell clusters in pancreatic islets leading to their collapse. There was no synergistic effect of the fructan-only group. In addition, addition of the low (2.5%) DMSO concentration to fructan resulted in better survival of pancreatic islets compared to the high (10%) DMSO concentration. This suggests that the cytotoxic effects of DMSO outweigh its benefits as a freezing agent for rat pancreatic islets.

The insulin release assay of pancreatic islet function showed that the stimulation index exceeded 2 in both the 30% fructan group and the DMSO groups, suggesting that the beta cells that escaped apoptosis from the freezing and thawing process were still capable of glucose concentration-dependent insulin secretion. Although there was no difference in the stimulation index between the DMSO and fructan groups, the insulin secretion volume at each glucose concentration was significantly higher in the fructan groups, which might reflect the difference in pancreatic islet survival rates (beta cell survival rate).

After pancreatic islets cryopreserved with 30% fructan were transplanted to diabetic rats, individual glucose levels were normalized in all cases and a stable blood glucose level was achieved. Furthermore, the survival of pancreatic islets transplanted to renal capsules was confirmed microscopically. No notable adverse events were found in rats, even when the low-concentration fructan (0.1%) + 3 mL PBS solution was intravenously injected into the rat tail veins. Although there is potential that fructan might be harmful to the living body, this has not yet been completely ruled out, as data on survival rates suggests that it could be considered safe.