Although the development of the chronic complications of T1DM is multifactorial, there is clear evidence that they are closely linked to the tight glycaemic control. In addition to observations made in studies involving identical twins, studies such as the Diabetes Control and Complications Trial (DCCT) have elegantly demonstrated that intensive insulin regimens resulting in tight glycemic control can significantly reduce the incidence of secondary complications of diabetes compared with conventional insulin treatment [1]. However, the intensive regimens are also associated with life-threatening hypoglycemia [2]. The challenge, therefore, is to develop treatments that are well tolerated, and that ensure restoration of normal glucose homeostasis at an early stage of the disease (i.e. treatments that truly reverse T1DM), in order to prevent the secondary complications from developing, rather than trying to treat these severe conditions once the “horse has already bolted”. In addition, as T1DM is principally diagnosed in the first 2 decades of life, any candidate treatment needs to be applicable to children and young adults.

The first attempt to cure diabetes using β-cell replacement was performed in Bristol in 1883, when minced pancreas procured from a sheep was injected into a patient with T1DM [6]. This attempt failed, and with our current knowledge of transplant immunology, this is not surprising! In 1916 the first pancreatic tissue allograft was performed in Newcastle-upon-Tyne by Dr Pybus. In his report, published in The Lancet in September 1924, he reported a small reduction in urinary excretion of glucose in one of two diabetic patients whom he transplanted with fragments of pancreatic tissue obtained from human cadaveric donor [7]. Once the association between islets of Langerhans and insulin secretion had been established in the 1920s, scientists started isolating islets from animals in order to study islet physiology, with the hope of treating and curing diabetes. Initially, islet isolation was performed using mechanical disruption, microsurgical dissection, and hand-picking. However, in 1965 Moskalewski developed a new technique of tissue dissociation using a crude preparation of enzymes obtained from culture supernatants from the bacterium Clostridium histolyticum and successfully isolated pancreatic islets from a guinea pig [8]. This technique was quickly adopted by Paul Lacy and Mary Kostianovsky in order to isolate islets from rats [9]. It was not until 1974, however, that the first clinical islet allotransplant using isolated islets of Langerhans was performed [10].

Improvements in both islet isolation and islet transplantation occurred over the next few decades, including the development of a semiautomated method for pancreas digestion by Ricordi and the introduction of a large-scale method for islet purification by Lake [11]. However, despite the fact that 494 islet transplants were reported to the International Islet Transplant Registry between 1974 and the end of the 1990s, and the fact that in animal models reversal of diabetes following an islet transplant was almost routine, the results of clinical islet allotransplantation were disappointing, with overall 9% of patients achieving insulin-independence and about 40% achieving partial graft function in the form of C-peptide production (C-peptide levels >0.3 ng/ml) [12].

However, all this changed in 2000 when Shapiro published the results of a small series of islet transplants performed in Edmonton, Canada [13]. Using a steroid-free immunosuppression regime, together with a protocol that involved each patient receiving at least two consecutive islet transplants utilizing large islet numbers, he reported that all seven of his patients had achieved insulin-independence. In addition, the main indication for transplantation in this group was the severe complication of hypoglycemic unawareness, which was also universally reversed by the islet transplants. This landmark publication stimulated the reactivation of other islet-transplant centers across the world, as well as the creation of many new centers. Soon leading groups were reporting insulin-independence rates of 80–85% at 1 year, success rates that were comparable with whole-pancreas transplantation [14].

Islet transplantation involves two broad components, namely islet isolation and the islet transplant itself. One of the major challenges of clinical islet transplantation has been to consistently obtain sufficient numbers of high-quality islets to routinely achieve insulin independence after transplantation. There are a number of factors which affect islet-isolation outcome, including donor selection, the variability of pancreas digestion, and the recovery of islet function after isolation and transplantation [15–17].

Pancreases for clinical islet isolation should be retrieved in an identical manner to that used for vascularized pancreas transplantation, with the exception of the vessel dissection [26]. The pancreas is therefore removed en bloc with the duodenum and spleen using a no-touch technique. During pancreas dissection, a meticulous technique is required, with avoidance of excessive use of diathermy. Adequate perfusion of the pancreas is of paramount importance and in situ perfusion is often complemented by additional ex vivo perfusion on the back table. During multiorgan retrieval, intravascular cooling is achieved by either aortic or aorto-portal flush. The latter has negative effects on the pancreas and should be avoided. Reduction of the pancreatic core temperature is considered one of the most important steps during pancreas retrieval [27]. Intravascular flush with cold preservation fluid is usually not sufficient and needs to be complemented by external pancreas cooling using copious amounts of ice slush placed in the lesser sac.

Standard preservation techniques involve simple cold storage at 4 °C in University of Wisconsin solution (UW), although some centers use HTK, Celsior, EuroCollins, and Kyoto solutions [28,29]. The pancreas is immersed in at least 300 ml of preservation solution and double-bagged. These conditions allow for preservation of the pancreas for up to 8 hours, with minimal loss of organ quality.

Other preservation protocols have been developed, including the TLM, in which the pancreas is preserved in hypothermia on the interface between oxygen-charged perfluorocarbon and preservation fluid.

Although recent papers have questioned the validity of the TLM, showing very limited tissue penetration of oxygen and of the perfluorocarbon itself [30–32], the 2009 Collaborative Islet Transplant Registry (CITR) report revealed that up to 33% of pancreases retrieved for islet isolation were preserved using this method. Examples of experimental preservation techniques include persufflation of pancreases with gaseous oxygen [33,34] and continuous and pulsatile hypothermic pump perfusion [35]. Thus far, none of these techniques has been used clinically, although recently presented results are encouraging.

Although groups have been undertaking islet isolation for many years [36–39], the human islet isolation procedure is still far from optimized, providing transplantable yields of islets in less than 50% of pancreas preparations, even in centers with significant experience. The main goal of islet isolation is to extract viable islets that are free from surrounding acinar tissue.

Most centers worldwide use a modified semiautomated technique for human islet isolation described by Ricordi [37]. This involves a two-stage procedure. First, the pancreas digestion stage involves the pancreas being dissociated by a combination of mechanical and enzymatic breakdown, resulting in the islets being liberated from the surrounding exocrine tissue. During this stage, the pancreas is transferred to the isolation facility, where it is dissected and the duodenum, spleen, and superficial fat are removed. The pancreatic duct is identified and cannulated with a venous catheter, and commercially available bacterial collagenase dissolved in Hanks’ solution at 4 °C is delivered to the pancreatic parenchyma by intraductal injection, either by a hand-held syringe or by recirculating pumps. The distended pancreas is then divided into a number of pieces and transferred into a metallic or polycarbonate chamber containing marbles or ball bearings. This chamber is connected to a fluid-filled system, in which a medium (usually minimal essential medium, MEM) is circulated at 37 °C. This system allows for accurate control of temperature and fluid flow, which can both be adjusted depending on the progress of digestion. When free islets appear in the digest, the process is stopped by addition of cold buffer into the circuit, and islets are collected into an albumin-enriched medium, commonly containing a cocktail of protease inhibitors.

In the second stage of islet isolation, islet purification, the liberated islets are separated from the exocrine tissue using centrifugation on a density gradient [39–41]. Following incubation of the pancreatic digest for 30–60 minutes in UW, resuspended tissue is loaded on the top of a Ficoll-based continuous gradient within a spinning COBE 2991 cell separator. During centrifugation, tissue fragments and cellular debris accumulate in the top layer (lowest density), big fragments of tissue move towards the bottom of the gradient (highest density), and liberated islets stay in between the two extremes (1.08–1.09 g/l). Purified islets can be transplanted immediately or following a short-term culture.

In order to comply with recent European Union (EU) and US Food and Drug Administration (FDA) regulations, human islet isolation for allotransplantation must now be performed in purpose-built good manufacturing practice (GMP)-grade facilities. As a result, an increasing number of islet transplant networks is developing, in which several centers transplant islets isolated from one core facility. This “hub and spoke” model not only means that the costs can be rationalized, but enables islet-isolation expertise to be centralized and maintained [42].

Islet culture

Although the original Edmonton protocol emphasized the importance of transplanting freshly isolated islets, most groups now recommend a period of 24–48 hours of culture prior to transplantation. Pretransplant culture of clinical preparations has two main purposes. First, it allows for a more thorough assessment of graft quality before the final decision to transplant [43]. Often an islet preparation can look excellent immediately after islet isolation, but a period of culture can show that in fact cell death was commencing, and after 24 hours of optimal culture the islets may become fragmented and have reduced function. This is particularly the case with large volume islets, in which central necrosis may not be evident immediately [44,45]. It is clearly beneficial to discover this before rather than after the islet transplant. On the other hand, there is clear evidence that a period in culture can also enable islet function to improve, as islets recover from the islet isolation procedure [46]. Second, the logistics of transplanting fresh islets used to mean that patients often had to be admitted in the middle of the night, and the procedure itself became an emergency. A period of islet culture enables the procedure to become semielective and carefully planned.

The specifics of different culture protocols are beyond the scope of this book. However, multiple measures are undertaken in order to limit the damage caused by hypoxic conditions, including low islet seeding density, low depth of medium to facilitate oxygen diffusion, and a wide range of supplements to reduce production and release of pro-inflammatory cytokines and free radicals.

In many centers, gas-permeable bags (similar to the bags used for platelet storage) have been used to facilitate oxygen diffusion to islets in culture. The purification process ends with several fractions containing different degrees of contamination with exocrine tissue. It is a common practice to culture high- and low-purity fractions separately. Purified tissue is placed in large culture flasks (175 cm² cultivation area), usually in cell-culture medium, such as CMRL-1066. The seeding density is about 20 000–30 000 IEq in 25–30 ml of culture medium. This seeding density, combined with a low depth of culture medium, helps prevent hypoxia in cultured islets. At the end of the culture period, the preparation is reassessed (islet number/loss, viability, microbiological status) and its suitability for transplantation is reevaluated. Although technically possible, prolonged culture is avoided in clinical transplantation and limited to a maximum of 48 hours.

Pretransplant graft assessment

Pretransplant culture of isolated islets allows for assessment of graft quality in terms of morphology, functional status, and sterility. This reduces the chance of poor-quality or contaminated grafts from being transplanted and makes this treatment modality potentially safer than solid organ transplantation. Several parameters are assessed during this process [47–49].

Islet number, size distribution, and islet morphology

Although automated counting is an option, most centers count islets manually [49–51]. This process, while fairly robust, is operator-dependent and the results can differ significantly among centers. In order to identify islets in a suspension, a zinc chelator—dimethylthiocarbazone (Dithizone, DTZ)—is used, which stains islets crimson red [52]. The size distribution of an islet preparation is usually skewed towards small islets; therefore, islet number is not necessarily representative of β-cell volume. In order to adjust for this discrepancy, IEq is used, where 1 IEq represents an islet 150 µm in diameter [53]. The whole population of islets is stratified into size-related groups, with corresponding conversion factors. For instance, for an islet 50–100 µm in diameter, this conversion factor is 0.167, and for an islet 200–250 µm in diameter, it is 3.5. In addition to the number of IEqs, an isolation index (II) is calculated by dividing the number of IEqs by the number of islets. II provides an indication of whether the distribution is skewed towards small or large islets. Analysis of islet morphology determines the degree of fragmentation and cleavage of peri-islet exocrine tissue.

Purity of the preparation

The purity of the preparation is estimated as the percentage of islets compared to acinar tissue. It is subjective, with considerable interassessor variability, but a purity of >50% is required for an islet preparation to be signed off for transplantation in the UK. Indeed, it could be argued that a preparation of <50% is by definition an exocrine transplant!

Islet viability and function

For the purpose of clinical isolation, a fluorescent viability assay is performed [54,55]. This assesses the integrity of the cellular membrane, which in turn is a function of the cell’s metabolic state. A principle of this viability test is that dyes such as ethidium bromide or propidium iodide can diffuse into cells and bind to DNA only if cellular-membrane permeability is compromised, as in dead cells. Others, such as fluorescein diacetate or acridine orange, are actively transported inside an intact, living cell and can be either metabolized, resulting in release of fluorescent compound, or bound to DNA. The assessment of islets is performed manually using a fluorescent microscope and is again subject to operator bias.

A glucose-stimulated insulin secretion (GSIS) assay tests the ability of isolated islets to response to glucose stimulation by insulin secretion. Several modifications of this assay exist, but for clinical purposes a static incubation is usually performed, whereby islets are exposed to a baseline (2.5 mM) and/or a stimulatory (25.0 mM) glucose concentration. Following a period of incubation, the insulin concentration before and after stimulation is measured, and the stimulation index (SI) is calculated using the formula:

where insulin (stimulated) is the amount of insulin secreted during 1-hour incubation in 25.0 mM glucose medium and insulin (basal) is the amount of insulin secreted during 1-hour incubation in 2.5 mM glucose medium.

An SI in excess of 2 is indicative of a functional preparation. Although this test is used to assess the islet graft, it is usually performed after transplantation. Therefore, its results are not part of the product release criteria listed later.

Microbiological contamination and the presence of endotoxin

Culture of isolated islet allows for a thorough microbiological assessment of the graft prior to transplantation [56,57]. Routine microbiological tests prior to graft release include microscopy and Gram staining, endotoxin levels, and bacterial and fungal cultures. As part of microbiological monitoring, the donor’s viral status should be included with product release documentation (cytomegalovirus (CMV) and Epstein–Barr virus (EBV) status in particular) so that the transplanting team can consider appropriate prophylaxis [58,59].

Product release criteria

The current regulations for tissue banks in Europe dictate that clear product release criteria have to be determined and stuck to. In the UK, the following release criteria are used:

• Islet yield >200 000 IEq (although the graft is allocated based on the islet dose in IEq/kg).
  
• Purity >50% (>30% in the USA).
  
• Viability >70%.
  
• Packed cell volume (PCV) <10 ml (recent research from Edmonton suggests that PCV should be limited to 5 ml in order to eliminate the risk of portal-vein thrombosis [60]).
  
• Microscopy and Gram stain negative.
  
• Endotoxin level <3-5 EU/ml (values differ between manufacturers and isolation centers).

As islet transplantation develops as a successful treatment, the indications expand and more patients become eligible for its benefits [61]. Overall, however, there are two main groups that are included in the selection criteria worldwide. First and foremost is the small subpopulation of patients suffering from brittle type-1 diabetes, with severe life-threatening hypoglycemic unawareness. These patients benefit enormously from the minimally invasive islet transplants, but are only offered an islet transplant provided that all optimal insulin treatments have been exhausted. The second group is patients with unstable type-1 diabetes in whom a renal graft must be preserved [62]. These transplants can either be performed as a simultaneous islet and kidney transplant (SIK) or as an islet-after-kidney graft (IAK).