Addition of human platelet lysate to islet culture medium suppresses islet loss and improves transplantation outcomes

Dear Editor,

The world’s first islet transplantation was performed in 1974 ¹ . Since the report of the Edmonton Protocol in 2000 ² , a large number of clinical islet transplantations have been conducted worldwide^2,3. In a worldwide survey of activities and practices in clinical islet of Langerhans transplantation (2000–2020), 4365 islet allotransplants (2608 in Europe, 1475 in North America, 135 in Asia, 119 in Oceania, 28 in South America) were reported in 2170 patients ³ . Culturing isolated islets before transplantation has become a common practice, leading to progress in the development of islet culture techniques. Culturing islets after isolation reduces contamination with exocrine tissue in the transplant graft. Furthermore, this provides time for undertaking quality control tests during the culture period and allows pre-transplant administration of immunosuppressive agents to the recipient. The culturing also offers logistical benefits for recipients living far from the transplant center by securing the necessary travel time. However, as isolated islets tend to deteriorate rapidly during culture, a method that can prevent islet loss during culturing is needed.

For research purposes, human islets are generally cultured using Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 5%–10% fetal bovine serum (FBS). Since FBS contains a large amount of various growth factors, adhesion molecules, and hormones, it is widely used in research islet culture. However, in clinical islet culture intended for transplantation, the use of culture media containing xenogeneic proteins should be avoided as far as possible. In a report by Martin et al. ⁴ , on culturing human cells in FBS-containing media, non-human animal-derived components, such as the sialic acid N-glycolylneuraminic acid (Neu5Gc), were detected on the cell surface. Thus, the clinical use of cells cultured under conditions containing animal-derived components raises safety concerns. Hence, culture media free of xenogeneic proteins are used for culturing clinical islet intended for transplantation.

At the University of Alberta in Edmonton, which has conducted the highest number of islet transplants worldwide, a serum-free medium based on Connaught Medical Research Laboratory (CMRL) 1066 medium supplemented with human serum albumin (HSA) and several growth factors is used ⁵ . In the Clinical Islet Transplantation (CIT) Consortium Phase 3 Trial sponsored by the National Institutes of Health (NIH), a medium composed of CMRL 1066 Supplemented medium with HSA, heparin, and insulin-like growth factor (IGF) was used ⁶ . However, this media contains fewer growth factors and is considered to provide a less favorable culture environment compared with FBS-containing media. Moreover, addition of recombinant growth factors to the culture medium is expensive.

In recent years, culture media supplemented with human platelet lysate (hPL) has been reported to be effective for the in vitro expansion of various cell types^{7 –9}. hPL contains a wide variety of growth factors and bioactive molecules released from platelet granules during lysis ⁹ . hPL has already been introduced for ex vivo expansion of various types of mesenchymal stromal cells (MSCs) for both research and clinical applications ⁷ , and has also been used for research-grade expansion of endothelial colony-forming cells ⁸ .

We investigated whether the addition of hPL to culture medium could suppress the deterioration of isolated islets and improve islet function. Porcine islet isolation was performed (n = 6; Supplemental Tables 1 and 2), and the islets were cultured in each medium listed in Supplemental Table 3 at 37°C for 24 and 48 h. Student’s t-test was used for comparisons between two independent groups using Microsoft Excel. In the Alb group, islet morphology was disrupted, and the number of islets was significantly decreased compared with that in the hPL group (Fig. 1a). To investigate whether hPL suppressed the decrease in islet number during culture, islet equivalents (IE) were compared between the two groups after 24 and 48 h of culture. Islets cultured with hPL (n = 6) had significantly higher IE values than those cultured with albumin (n = 6) (Fig. 1b), suggesting that hPL suppressed the reduction in the number of porcine islets during culture. The stimulation index of the cultured islets tended to be higher in the hPL group (n = 6, from six separate islet isolations), at 1.69 ± 0.14, compared with 1.49 ± 0.19 in the Alb group (also n = 6); the difference was not statistically significant (Fig. 1c). The ATP content of porcine islets cultured with hPL (n = 6) was 0.93 ± 0.04 pmol/IE, which was significantly higher than that of the Alb group (n = 6; 0.74 ± 0.06 pmol/IE) (P < 0.05; Fig. 1d).

Figure 1.

Culture of isolated islets in each solution. (a) Culture of isolated islets in albumin-supplemented solution or in hPL-supplemented solution. Porcine islets were incubated in each culture medium at 37°C for 24–48 h. Scale bar = 200 μm. (b) Number of porcine islets after culture. A total of 2000 IE of porcine islets were cultured in each medium for 72 h. Islets were counted and IE calculated at 24 and 48 h (n = 6 per group). **P < 0.01. (c) After 24 h of culture in each solution, islet function was evaluated based on insulin secretion in response to glucose stimulation. A total of 1200 IE were incubated at 37°C for 2 h in RPMI 1640 medium containing either 2.8 mM or 25 mM glucose. The supernatant was collected, and insulin concentrations were measured using a commercially available ELISA kit. The stimulation index was calculated by dividing the amount of insulin secreted at high glucose by that at low glucose. n = 6 for both the Alb and hPL groups. (d) ATP content in cell lysates after islet culture was measured using an ATP assay. ATP content was normalized to IE. n = 6 for both the Alb and hPL groups; *P < 0.05. Data are presented as mean ± SE.

In total, 1500 IE of the cultured porcine islets were transplanted under the kidney capsule of diabetic SCID mice (n = 12; two mice per islet isolated from six independent isolations). Diabetes was induced by a single intraperitoneal injection of 220 mg/kg streptozotocin (STZ). Mice were considered hyperglycemic when their non-fasting blood glucose levels exceeded 350 mg/dl for two consecutive days. Differences in graft survival between the groups were assessed using the log-rank test based on the Kaplan–Meier method, with analyses performed using StatView software. The number of mice that achieved normoglycemia (blood glucose level < 200 mg/dl) was significantly higher in the hPL group than in the Alb group (8.3% vs. 58.3%, P < 0.01; Supplemental Table 4). These results demonstrate that culturing islets with hPL improved graft function after transplantation. All the animal experiments were approved by the Institutional Animal Care and Use Committee of the University of the Ryukyus (A2022095/March 31, 2023).

Currently, many research institutions have adopted pre-transplant culture of human islets ⁵ ; however, rapid deterioration of isolated islets during culture is well documented ⁵ . A major factor contributing to this loss is apoptosis/necrosis and activation of intracellular death signaling pathways ¹⁰ . In clinical islet transplantation, many centers perform pre-transplant culture at 22°C–24°C instead of 37°C^5,6. This is based on reports showing that culturing islets at 22°C–24°C results in higher islet viability compared with 37°C ¹¹ and is more suitable for long-term culture ¹² . Lowering the temperature is believed to reduce islet cell metabolism and prevent necrosis. In other words, the culture media currently used in clinical settings may not provide an optimal environment for the islets. Our group previously reported that for up to 48 h after islet isolation, cold storage in UW solution at 4°C results in better transplantation outcomes than culturing islets at either 22°C or 37°C ¹¹ .

hPL has been reported to contain various growth factors such as epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2/bFGF), soluble CD40 ligand (sCD40L), interleukin (IL)-1α, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF)-A, platelet-derived growth factor (PDGF)-AA and PDGF-AB/BB, transforming growth factor (TGF)-β1, 2, 3, brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), and hepatocyte growth factor (HGF)^13,14. Mohamed et al. ¹⁵ reported that the level of IGF-1 in hPL (125±2.6 ng/ml) was approximately threefold higher than that in FBS (46 ± 1.6 ng/ml). In this study, the effects of the various growth factors contained in hPL contributed to superior results compared with standard islet culture media. Delabie et al. ¹³ attempted to standardize hPL production at three European blood centers and evaluated its feasibility and batch-to-batch variability. When hPL was produced using a standard operating procedure (SOP), all hPL batches demonstrated equivalent levels of total protein concentration, pH, ionic strength, and lactate content. Furthermore, analysis of the 12 growth factors showed only minimal differences between the batches. These results indicate that the standardization of hPL production is feasible and that international standardization can reduce variability.

The development of islet culture media has been explored extensively. There have been reports that the addition of various factors ⁵ —such as ascorbic acid, vitamin E, vitamin D3, HGF, arachidonic acid, linolenic acid, ghrelin, cysteine, a combination of nerve growth factor (NGF) and VEGF, and a combination of gastrin and EGF—can help maintain or improve islet yield and function. However, simultaneous addition of multiple factors to the culture medium is labor-intensive and expensive. hPL can be prepared relatively easily by freezing and thawing expired platelet products or platelets trapped in leukocyte reduction filters used for whole blood, thereby causing platelet lysis.

In conclusion, the addition of hPL to the culture medium suppressed the deterioration of isolated islets and improved their function of porcine islets. These findings contribute to the advancement of β-cell replacement therapy for patients with type 1 diabetes and suggest that islet culture using hPL may serve as a promising therapeutic strategy.

Supplemental Material

sj-docx-1-cll-10.1177_09636897251410290 – Supplemental material for Addition of human platelet lysate to islet culture medium suppresses islet loss and improves transplantation outcomes

Supplemental material, sj-docx-1-cll-10.1177_09636897251410290 for Addition of human platelet lysate to islet culture medium suppresses islet loss and improves transplantation outcomes by Hirofumi Noguchi, Chika Miyagi-Shiohira, Takuya Sadahira, Masami Watanabe and Issei Saitoh in Cell Transplantation

Hirofumi NoguchiDepartment of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Chika Miyagi-ShiohiraDepartment of Regenerative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan Takuya SadahiraDepartment of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Masami WatanabeDepartment of Urology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan Issei SaitohDepartment of Pediatric Dentistry, Asahi University School of Dentistry, Hozumi, Japan