Abstract
The aim of any pancreatic islet isolation is obtaining pure, viable and functional pancreatic islets, either for in vitro or in vivo purposes. The islets of Langerhans are complex microorgans with the important role of regulating glucose homeostasis. Imbalances in glucose homeostasis lead to diabetes, which is defined by the American Diabetes Association as a “group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both” (American Diabetes Association 2011). Currently, the rising demand of human islets is provoking a shortage of this tissue, limiting research and clinical practice on this field. In this scenario, it is essential to investigate and improve islet isolation procedures in animal models, while keeping in mind the anatomical and functional differences between species. This chapter discusses the main aspects of mouse islet isolation research, highlighting the critical factors and shortcomings to take into account for the selection and/or optimization of a mouse islet isolation protocol.
Keywords
3.1 Introduction
Modern islet research started with Bensley and his pioneering scientific contribution in 1911 [2]. One hundred years later, the field has evolved tremendously, especially since the success of the Edmonton protocol. It was developed by the islet transplantation team at the University of Alberta, in Canada, which first introduced a steroid-free immunosuppressive regimen, resulting in 100 % insulin independence at 1 year in seven individuals [3]. This advance contributed to the worldwide expansion of human transplantation program and the access to human tissue for translational studies.
The islet community has recently appealed for a higher investment in human islet isolation and distribution to the NIH, JDRF and American Diabetes Association [4, 5]. The current rising number of researchers working with human islets [from 35 active users in the Integrated Islet Distribution Program (IIDP) in 2010 to 104 in 2014] and the consequent rising demand for this tissue has resulted in a bottleneck in the research islet supply. Thus, the use of mice as animal models for islet isolation and other in vitro and in vivo purposes has emerged as an alternative to study the pathophysiology of diabetes as well as to conduct islet isolation and transplantation .
The aim of pancreatic islet isolation is obtaining viable, pure and functional islets, either for in vitro or in vivo studies (Fig. 3.1). However, to obtain a successful yield and good quality islets, different key aspects must be taken into account: the type and concentration of the digestive enzyme, the method of enzyme administration, the temperature and duration of the pancreas digestion, the method for islet purification and the culture conditions following isolation [6]. It is instrumental to identify the factors influencing the efficacy of the isolation procedure of mouse pancreatic islets in order to standardize the procedure, reduce the variability, and harvest good quality islets.
The main steps in any mouse islet isolation are the following: pancreas distention and dissection; pancreas digestion; islet purification and islet culture. In this chapter, we describe the main methodological aspects of mouse islet isolation as well as the key aspects and challenges.
3.2 Pancreas Distention and Dissection
3.2.1 General Considerations About the Procedure
First of all, the animal should be euthanized by cervical dislocation, CO2 asphyxiation, etc., depending on the regulations in that country and/or the laboratory choice. While it is desirable to perform all the manipulations of the mouse inside the laminar flow hood in sterile conditions, it is also acceptable to perform them outside, with a “clean technique”, with sterile reagents and surgical instruments in order to avoid contamination.
The mouse is then placed under a stereomicroscope in supine position, with the abdomen cleaned up with 70 % ethanol. A laparotomy is performed, cutting the skin and the muscular tissue of the thorax with a V-incision from the pubic region up to the diaphragm, in order to expose the abdominal cavity. The skin must be well separated from the organs exposed, in order to avoid contamination with the mouse’s hair.
The lobes of the liver are then positioned against the diaphragm in order to expose the gall bladder and the proximal segment of the common bile duct. Then, the duodenum is gripped with forceps following the common bile duct and clamped at the level of the Vater ampulla, in order to distend only the pancreas with the collagenase solution. The Vater ampulla is a triangle-shaped white area located at the confluence between the common bile duct and the duodenum .
Once the common bile duct is clamped, it can be cannulated. The standard procedure [7–10] consists in inserting the needle in the Y-shaped junction of the cystic duct and the hepatic duct and injecting the collagenase solution into the common bile duct, distending directly the pancreas. It is important to cannulate well with an optimal needle placement in order to prevent backflow into the liver and drain the splenic tail of the pancreas, an islet rich area. The pancreas is then excised and digested at 37 °C [6, 11]. It is important to remove fat tissue because it may affect digestion and reduce the yield [9].
3.2.2 Enzyme Administration
The key element of this step is the method of the enzyme administration. Originally, the standard procedure set by Lacy et al. [12] was based on the administration of cold saline buffer by the common bile duct to distend the pancreas taking advantage of the anatomical structures, so the enzyme would penetrate and distend better the pancreas, improving the release of the islets from the exocrine tissue. The pancreas was then dissected and the tissue minced in small pieces (1–2 mm), increasing the surface area for the digestion [13]. However, some years later, some modifications to this method were introduced. Gotoh et al. [11] suggested to inject the collagenase in the common bile duct and do a stationary digestion avoiding the mincing step. This is the most common method used nowadays [6–10, 14, 15], since it has a better access with a better digestion of the connective tissue, it produces a yield approximately 50 % higher and it is more cost effective [16].
3.3 Pancreas Digestion
3.3.1 General Considerations About the Procedure
This step entails the digestion of the pancreatic tissue with collagenase once the pancreas is harvested. Usually the tissue is incubated in a water bath at 37 °C, but the duration of the digestion depends on the strength, concentration and formulation of the collagenase. It is also dependent on the strain and age of the mouse [14]. The incubation can be static [11, 12] and/or dynamic [17]; the tissue can be hand-shaken manually (to improve mechanically the dissociation) in the middle of the incubation and/or afterwards, and the tissue can be minced [14, 17] or not [11], depending on the protocol chosen .
In some protocols, the digested tissue is passed through a 14G needle and/or a 450 μm mesh filter, to improve mechanically the dissociation of the tissue. Next, the tissue is washed once or several times before proceeding to the purification phase.
3.3.2 Selection of the Enzyme
The choice of the enzyme is critical. Without a good digestion of the tissue, the purification cannot be effective. Therefore, the knowledge of the collagen composition in the extracellular matrix is crucial for an adequate selection and formulation of the most appropriate enzyme according to the donor’s characteristics.
Traditionally, the enzyme used in islet isolation is the bacterial collagenase Clostridium histolyticum. The rationale for the use of this enzyme is that collagen is an important component of the pancreatic extracellular matrix (ECM). The use of this collagenase was introduced for the first time by Moskalewski in 1965 [18], and it allows the enzymatic degradation of the ECM and release of islets during the isolation procedure [19].
Traditionally, crude collagenase blends such Collagenase V (Sigma), were used for rodent as well as for human islet isolation [20, 21]. Original crude collagenase preparations from Clostridium histolyticum are mixtures of six collagenases, a neutral protease and several enzymes with tryptic-like activity, which also influence the dissociation process [22]. In fact, it has been reported that tryptic-like activity is a key component that optimizes the efficiency of islet isolation , reducing dissociation time and minimizing lot-to-lot variability. The six collagenases are divided in two subtypes: G (or class I) and H (or class II) collagenases. However, there are contradictory studies about their role and importance in human and rodent isolation, probably due to the difference in the extracellular matrix composition between species or the different blends used. Fujio et al. [23] suggested that it is possible that some components of the rat extracellular matrix could only be digested by class II collagenases. Wolters et al. did pioneering work in this regard [24] and reported the predominant role of class II collagenase in rat islet dissociation versus incomplete dissociation obtained with class I collagenase alone. However, Brandhorst et al. reported that the highest yield of rat islet isolation was obtained using the same proportion of class I and class II collagenase (C-ratio of 1.0) [25, 26]. In human islet isolation , collagenase class I is considered essential [27].
With the evolution of this research field, it was observed that crude collagenases, which are derived from bacterial cultures, contained impurities. Key active components often had an imbalanced formulation, there was significant batch-to-batch and vial-to-vial enzyme variability, and high endotoxin levels and pigment contamination were detected [20, 27, 28]. Specifically, endotoxin contamination correlates positively with increased cytokine production and apoptosis and negatively with engraftment in rat islet transplantation models as well as in clinical outcomes [29]. Therefore, the current enzymes used in human islet isolation are purified, despite the suggestion by some authors to use filtrated crude collagenases in human islet isolation to decrease costs [21]. In 2009, Yesil et al. showed a correlation between enzyme purity and yield [30]. The current combinations used for islet isolation consist, mainly, of class I and class II collagenases from Clostridium histolyticum and a neutral protease. The neutral protease can be from Clostridium histolyticum as well, although the gold standard neutral protease in use is Thermolysin, which is derived from Bacillus thermoproteolyticus. The reasons of its success are its low cost, stable production and strong digestion efficacy. However, a recent publication suggests that clostripain (a protease from Clostridium histolyticum with tryptic-like activity) could have a synergistic effect with neutral protease and collagenases derived from the same bacteria in rat islet isolation , increasing the efficiency and outcomes of the procedure [31].
In rodent islet isolation , collagenase V (Sigma, Ayrshire, UK), collagenase XI (Sigma, Ayrshire, UK) and collagenase P (Roche, Mannheim, Germany) are routinely used. However, these enzymes are not the only responsible of the tissue dissociation of the pancreas. The pancreas itself is a source of proteolytic endogenous enzymes that are continuously released by the exocrine tissue during the digestion [32]. In fact, Wolters et al. suggested that proteolytic activity caused cell lysis and release of DNA, making the separation of islets from the exocrine tissue difficult. Therefore, they reported that adding inhibitors to the digestion medium to suppress the proteolytic activity, like bovine serum albumin and trypsin inhibitors (purified clostripain, egg white trypsin inhibitor, soybean trypsin inhibitor, etc.) increased the islet yield . However, in a recent study of the effects of some endogenous protease inhibitors (specifically serine protease inhibitors such as Pefabloc, Trasylol and Urinary Trypsin Inhibitor) it was shown that some of them have detrimental effect on the action of bacterial neutral proteases [33]. Pefabloc, in particular (which is widely used in human islet isolation ) affects insulin response. In contrast, Urinary Trypsin Inhibitor, which is not yet approved by the FDA, enhances bacterial neutral protease action in addition to inhibiting endogenous proteases. Therefore, the quality and formulation of the digestion enzyme is key for the islet isolation outcomes, both for experimental and clinical islet isolations.
3.3.3 Duration of Digestion
The duration of digestion is dependent, on one hand, on the strength, concentration and formulation of the enzyme; and, on the other, on the strain and age of the animal, according to the guidelines of factors influencing islet isolation published by the Haan [34], since differences in the connective tissue have been observed. Then, the quality of the enzyme and the duration of the digestion constitute a tandem that is critical for the islet isolation outcomes. A prolonged digestion not only causes overdigestion and degradation of the islets but also affects morphology, yield, viability and even functionality [27]. Therefore, due to the batch-to-batch variability in the enzymes used for mouse islet isolation , the duration of the digestion should be carefully optimized prior to any experiment. The typical range of digestion time in mouse pancreas for collagenase P (Roche, Mannheim, Germany) (3 mg/ml) is around 6–7 min and for collagenase V (Sigma, Ayshire, UK) (1 mg/ml) is up to 10 min. This variability is due to the formulation, the concentration, the activity of the enzyme for that batch and the procedure, as well .
3.4 Islet Purification
Once the pancreatic tissue is digested and chemically and mechanically dissociated, the next step is to separate the endocrine of the exocrine tissue and purify the islets. Purification is a key step for islet isolation outcomes as well as for clinical applications, since highly pure preparations lead to engraftment, reduced graft immunogenicity in transplants and suitability for immunomodulation procedures (Fig. 3.2) [6, 7, 35]. Furthermore, an impure preparation can cause important postsurgical complications after intraportal transplantation, such as thrombosis and embolism [36]. While contamination with acinar cells may impair the integrity, viability and functionality of the islets [37], some extra-insular tissue potentially containing progenitor cells could potentially increase islet viability and functionality and improve clinical outcomes as well.
3.4.1 General Considerations About the Procedure
The traditional method used for mouse islet purification is discontinuous density gradient centrifugation (or isopycnic centrifugation) with Ficoll [38]. With this method, the cells in a suspension are separated according to their intrinsic differences in cell density (≈1.059 g/ml for islet tissue and 1.059–1.074 g/ml for exocrine tissue) [39]. The researcher should build a gradient of different layers of Ficoll solutions of different densities, from the densest layer at the bottom of the tube (which is mixed with the cells) to the less dense at the top. The tube is spun at a certain speed and temperature, depending on the density of the cells that we want to purify. Then, each cell type migrates through the gradient to the interface of the layers with the same density. After the centrifugation, the cells are retrieved and washed several times to eliminate the rests of Ficoll .
3.4.2 Density Gradient Purification
The first islet purification was done by Bensley in 1911 [2]. It consisted in hand-picking of islets to separate them manually from the exocrine tissue. However, it was a long and tedious procedure, and not feasible for large-scale experiments. In 1967, Lacy introduced the islet purification by differential density elutriation using the discontinuous sucrose density gradient [12]. However, the osmolarity was very high, due to the very high molar concentration of sucrose in the gradient, and it damaged the islet integrity. Ficoll, a high molecular weight polymer of sucrose (40 kD) was introduced in 1969 by Arnold Lindall [40]. However, since islets obtained by Ficoll purification exhibited impaired insulin secretion, Lacy’s team started dialyzing Ficoll with positive results [38].
While bovine serum albumin, percoll or metrizamide have been used in the past [41], the most common discontinuous density gradients used today are Ficoll, Histopaque (a Ficoll-based solution), dextran and iodixanol [42, 43]. Ficoll is the most widely used, as it reduces cell swelling and increases the density differences between islets and exocrine tissue [38]. Although several publications have reported toxic effects of Ficoll on islets, affecting its quality [44–46], more recent studies suggest that neither Ficoll nor Ficoll-based gradients [43, 47] exhibit deleterious effects. In fact, McCall et al. [43] compared different density gradient solutions in terms of islet quality (Ficoll, Histopaque, Dextran and Iodixanol) and showed that the best in terms of islet quality and cost-effectiveness was Histopaque, which is Ficoll-based. Iodixanol is a non-ionic, iso-osmolar solution that was initially used for porcine islet isolation [48] and later applied for rodent [46] and human islet isolation [49]. Iodixanol is currently displacing the use of Ficoll in human islet isolation with the advantage of a lower cost [50, 51].
3.4.3 Other Purification Methods
Although density gradient/isopycnic centrifugation has been the gold standard purification method since the 1960s, other purification methods have also been reported in the literature to purify pancreatic islets: hand-picking [47], phototermolysis [52], radiation [53], differences in osmolality [54], cryo-isolation [55] and cell sorting [56], among others. Specifically, in the case of rodent islet isolation , the main alternative methods that have been reported are: magnetic microspheres coated with islet or cytotoxic anti-acinar monoclonal antibodies [57], osmotic shock [39] and filtration [9, 45, 47]. Ramírez-Domínguez and Castaño [47] reported for the first time a comparison study of Histopaque and filtration purification in terms of quality, time and costs. In the McCall’s study [43] Histopaque was recommended over other gradient solutions, but whether islets obtained by filtration had a good quality in comparison with Histopaque-purified islets had not been reported before. According to the above study, islets purified by Histopaque and filtration were of comparable quality, but filtration was more cost-effective because it saved 90 % of the time devoted to purification with Histopaque. Despite anatomical interspecies differences in islets [58–61] the range of islet sizes is similar in mice, humans and other species, with an average diameter of 140 μm and an upper limit of size at around 500–700 μm of diameter [62]. Owing to this, filtration could be considered a method with high translational potential .
3.5 Islet Culture
During the isolation process, islets are affected by many stressing stimuli: “Anoikis” due to the detachment of islets from the surrounding extracellular matrix [63, 64], nutrient and oxygen deprivation, presence of endotoxins, the release of proteolytic enzymes form the acinar tissue, etc. [65]. Therefore, a step of islet culture can be used to restore viability and functionality, improving the islet isolation outcome.
3.5.1 General Considerations About the Procedure
This step could be interpreted as an extension of the purification step, since exocrine tissue does not survive well in culture, enriching therefore the islet fraction (Fig. 3.3). In the culture procedure, aspects such as the choice of culture media and the cell density have to be taken into account.
The most common islet culture medium for murine islets is RPMI 1640 (Rosewell Park Memorial Institute Medium). However, some laboratories prefer to use CMRL 1066 (Connaught Medical Research Laboratories), which is also widely used to culture human pancreatic islets. Preference for CMRL 1066 is based on the observation that some immune cells, such as dendritic and endothelial cells, cannot grow in that medium, which may therefore induce a decrease in alloreactivity.
One component of the medium that is key for the physiology of the islets is glucose. A concentration of 11 mM glucose resulted in islets with lower apoptosis rates and increased viability than those grown with a different concentration. With glucose concentrations below 11 mM, insulin content was reduced and downregulation of key genes for glucose metabolism was observed. Similarly, glucose concentrations over 11 mM resulted in toxicity [66].
Islet culture media are commonly supplemented with 10 % fetal bovine serum to keep the cell viability and antibiotics/antimycotics to avoid the possibility of contamination.
With reference to other practical issues, murine islets are cultured in suspension dishes, avoiding the growth of acinar tissue and preventing islet attachment. It is also recommended to let islets recover overnight in culture and change the media after 24–48 h post-isolation, to remove debris and prevent cell competition for nutrients. Specifically, it is suggested to seed a maximum of 300 islets in 60 × 15 mm dishes to avoid cell stress and keep optimum culture conditions.
Since the publication of the Edmonton protocol [3], there is some controversy on whether islets should be transplanted fresh or previously cultured. Although some authors have reported the superiority of fresh islets in in vitro and in vivo performance [67], murine islets are generally used after a short culture period.
3.6 Concluding Remarks
This chapter reviews critical aspects of murine islet isolation and highlights the importance of mice as providers of raw material to conduct biological studies on diabetes, with an additional translational potential. This emphasizes the need of implementing an efficient isolation protocol, not only to obtain high quality islets, but also to justify a rational use of animals and laboratory resources. While working with mice is economical and simple, factors such as strain, age, etc. highly influence the isolation outcome. Function, purity and yield are also donor strain-dependent.
Finally, a full translation of the mouse isolation method has yet to be achieved. Research efforts are focused in developing new purification methods that maximize the yield and the islet quality. Another challenge is to investigate and develop other factors discussed in this chapter (enzyme formulation, collagen composition in the extracellular matrix, etc.) with the goal of improving the efficiency and addressing the shortcomings of current protocols .
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Ramírez-Domínguez, M. (2016). Isolation of Mouse Pancreatic Islets of Langerhans. In: Ramírez-Domínguez, M. (eds) Pancreatic Islet Isolation. Advances in Experimental Medicine and Biology, vol 938. Springer, Cham. https://doi.org/10.1007/978-3-319-39824-2_3
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