Background

According to the August 2021–August 2023 cycle of the National Health and Nutrition Examination Survey (NHANES) published by the Centers for Disease Control and Prevention, 15.8% of US adults are estimated to have diabetes. Type 1 diabetes (T1D) is caused by the autoimmune destruction of pancreatic islets, the cells that produce insulin in response to elevated glucose levels [1]. In contrast, type 2 diabetes (T2D) generally begins with reduced insulin sensitivity in insulin-responsive tissues, such as the liver, muscles, and adipose tissue. The body often compensates for such insulin resistance by islet proliferation. However, failure in compensatory islet proliferation and islet dysfunction leads to T2D onset [2]. Therefore, strategies that promote islet regeneration or function hold great potential in treating diabetes.

Among the medications used in diabetes treatment, only DPP4 and GLP-1 receptor agonists can potentially improve islet function [3]. No medications have been proven to promote islet regeneration, apart from possibly harmine [4], which is still under clinical trial. One obstacle in the development of islet-targeting drugs is the difficulty in culturing islets in vitro for the long term. Pancreatic islets are spherical clusters of cells with diameters ranging from 50 to 500 μm [5]. A human adult pancreas contains 1–2 million islets, composing approximately 2% of the pancreatic mass. Compared to the acinar and ductal tissues of the pancreas, islets are highly vascular and receive 20% of the blood supply to the pancreas [6]. The most commonly used protocol for isolating human islets uses the Ricordi automated method [7], which utilizes physical forces and enzymatic reactions to separate islets from exocrine tissues and purifies islets by gradient centrifugation. To then culture the isolated islets, the two most commonly used media are RPMI 1640 supplemented with 10% serum or serum-free Connaught’s Medical Research Lab (CMRL) medium, neither of which can support normal islet function for more than a week [8]. Commercial media with undisclosed recipes exist, such as the PIM(S)^® from Prodo Lab, which may permit long-term islet culture for up to 21 days, as reported by the company’s documents. It therefore remains a challenge to sustain islet function long-term in vitro.

Part of the difficulty in long-term culturing of islets is due to the fact that islet isolation protocols disrupt the islets’ rich vascular connections. Most endothelial cells within the islet are believed to deteriorate soon after isolation, leaving cells within the center of the avascular islet with limited nutrients and oxygen [9]. Various strategies to vascularize in vitro cultured islets include prefabricated chips [10] or bioprinting [11]. These strategies require highly specialized techniques and do not achieve an intra-islet vascular density comparable to that of native islet capillaries. In this protocol, we convert generic endothelial cells (ECs) into adaptable ECs by overexpression of the endothelial pioneer transcription factor ETV-2. Adaptable ECs functionally vascularize islets in vitro and sustain islet function for more than 20 weeks. This approach provides an ideal platform for screening therapeutics to promote islet functional improvement or regeneration.

Beyond drugs to augment islet functional mass, islet β-cell replacement therapy is considered another promising treatment for diabetes, which provides finely tuned insulin secretion at physiological levels by transplanting insulin-secreting islet cells derived from either human deceased donors or from stem cells [12]. Clinically, islet transplantation is used to treat T1D patients with severe hypoglycemic complications. There are several obstacles preventing cellular therapy from expanding to a wider T1D population or even to T2D patients, including the use of immunosuppressants, scarcity of donor cells, and high costs. Furthermore, clinical transplantation protocols involve the infusion of either deceased donor-derived or stem cell–derived islets into the portal vein of the liver [13]. Portal infusion of transplanted cells carries with it the small but very serious risk of thrombosis, also limiting the infusion of anything larger than naked islets. Therefore, attempts to encapsulate islets for immune shielding have involved alternative transplantation sites such as the subcutaneous space [14] and the omentum [15]. Among the various alternative transplantation sites, the subcutaneous space is the most promising due to its large size, ease of access and monitoring, and ability to retrieve transplanted cells. Compared to most other sites, such as the kidney capsule, omentum, and anterior chamber of the eye, the subcutaneous space is enormous, making it ideal to construct sophisticated structures not only to protect grafts from immune attack but to establish a tissue-specific vascular niche to sustain islet functional mass. Transplanting islets in the subcutaneous space, however, has long been hampered by the difficulty in establishing a sufficient blood supply to the grafts [16,17]. In this protocol, we present a method of co-transplanting islets with ETV-2-expressing adaptable ECs to achieve functional engraftment of human islets within the subcutaneous space.