5

Mouse Islet Isolation

Simona Marzorati and Miriam Ramirez-Dominguez

Contents
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Steps in Mouse Islet Isolation and Key Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Surgery and Pancreas Harvesting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pancreas Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Practical Issues: Quality, Time, and Cost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Purity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Yield . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Time and Cost . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Factors to Consider in Setting Up a New Rodent Islet Isolation Laboratory . . . . . . . . . . . . . . . . .
Infrastructure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Staff . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Budget . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cross-References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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S. Marzorati
Cell Biology Unit, S. Raffaele Scientific Institute, Diabetes Research Institute-DRI, Milan, Italy
e-mail: marzorati.simona@hsr.it
M. Ramirez-Dominguez (*)
Department of Pediatrics, Faculty of Medicine and Odontology, University of the Basque Country,
UPV/EHU, University Hospital Cruces, Leioa, Pas Vasco, Spain
e-mail: miriamrd@gmail.com
M.S. Islam (ed.), Islets of Langerhans, DOI 10.1007/978-94-007-6686-0_33,
# Springer Science+Business Media Dordrecht 2015

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Abstract

Pancreatic islets transplantation is a therapeutic option for patients affected by

type 1 diabetes. While it is always desirable to perform experiments directly with
human islets, the limited availability of this precious tissue makes mouse islets a
feasible option for experimental research in diabetes in many laboratories worldwide. This chapter summarizes from a practical perspective the main aspects of
mouse islet isolation. We will discuss the technical factors that affect its success,
as well as the practical issues (quality assessment, time, and cost) to take into
account when selecting an islet isolation protocol. Finally, we will provide some
guidelines on the necessary logistics for setting up this kind of methodology.
Keywords

Islet isolation Islet purification Animal models Quality assessment

Introduction
The success of the Edmonton Protocol (Shapiro et al. 2000) contributed to the
worldwide expansion of human transplantation programs and to the improvement
of access to human tissue in translational studies. Unfortunately, the success of
clinical islet transplantation is influenced by numerous variables, such as the
location of the implant site, and strong inflammatory and immunological reactions.
Moreover, the islet isolation process disrupts the interaction between blood vessels
and endocrine cells, which, once the islets are transplanted, can lead to metabolic
exhaustion and cell death (Piemonti et al. 2010).
In this context, rodent, and also pig and monkey islets, are useful in overcoming
the challenge of clinical islet isolation. In particular, mouse islet isolation can
provide a reliable means of studying islet isolation. New protocols are always
under investigation, even though the protocols currently used are relatively straightforward. It is essential to carefully design preclinical studies to obtain consistent
and reliable results; in this scenario it is really important to select the correct animal
model, even though this is not always simple (Cantarelli et al. 2013). Lessons
learned from animal models determine improvements in the different steps of islet
isolation, culture techniques, and the function and viability of the transplanted
islets. Animal models are important, for example, in reducing the donor-to-recipient ratio. In fact, as observed in human islet isolation, the procurement rate of
mouse islets is tremendously affected by numerous factors and is far from optimal
(Nano et al. 2005). It is usual to recover far less than 50 % of the islets present in the
rodent pancreas. It has been estimated that a mouse pancreas has an average of
2,000 islets, but a typical murine islet isolation yield is ~200 islets (~10 %) per
mouse (Carter et al. 2009). The yield from one mouse, as observed in humans
(Nano et al. 2005) depends on body weight, size of the pancreas, total islet mass,
and also on genetic background, as pointed out in an interesting paper by Bock and
colleagues in 2005 (Bock et al. 2005). In particular, a mouse strain-dependent

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85

hierarchical total islet number exists: B6, DBA/2, NOD, 129S6, C3H, CBA. The
average number engrafted in murine islet transplants is 300500, resulting in a donorto-recipient ratio of 1.52.5 (Biarnes et al. 2002). Also, just an average of 40 % of the
murine islets engraft, the rest are lost in the early post-transplant period.
Owing to these similarities, improving the yield rate in mice could become a
starting point to increase the yield of human islet transplantation. Mice are also
extremely useful in studying the etiopathogenesis of type 1 diabetes, increasing the
usefulness of this animal model in diabetes research. A recent study performed on
rodents is particularly relevant because it has not only been shown to improve the
isolation procedure, but also developed a method of establishing primary islet cell
clusters (Venkatesan et al. 2012). The researcher approaching animal studies has to
be aware of some of the limitations of this tool. In fact, differences in the cytoarchitecture of mouse and human islets have been described (Cabrera et al. 2006),
suggesting that a prototypical mammalian islet type may not exist. While in mouse
pancreatic islets there are around 77 % cells and 18 % cells, these percentages
change to 55 % and a 38 % respectively in humans. Besides, the distribution is also
different. In mice, cells are located in the core of the islet, whereas in humans
cells are scattered through the islet, intermingled with and cells. Consequently,
the differences in the structure of the islets have implications for the interactions
between cells and, therefore, for the islet response to glucose and different
metabolites, as well their ability to survive. It is clear that the use of mouse
pancreatic islets in diabetes research, specifically in the islet isolation procedure,
cannot yet be replaced by non-biological or computer-generated models, but, thanks
to all the recent improvements, it should be possible to optimize and decrease the
number of mice needed to obtain the same amount of islets.
Therefore, a successful mouse islet isolation laboratory must approach different
interrelated aspects with different challenges, which this chapter aims to do from a
wide perspective. The main aspects addressed in this chapter are: the steps and key
aspects of rodent islet isolation, practical issues regarding islet isolation, and factors
to consider in setting up a new mouse islet isolation laboratory.

Steps in Mouse Islet Isolation and Key Aspects

Modern islet research started in 1911 with the pioneering work of Bensley on the
handpicking of guinea pig islets and staining them with neutral red (Bensley 1911).
Since preliminary advances in the field, including the introduction of digestion of
the pancreas with collagenase in islet isolation by (Moskalewski 1965) and the
distention of the pancreas via the pancreatic duct followed by incubation of
chopped tissue in collagenase by Lacy in 1967 (Lacy and Kostianovsky 1967),
the fields of both islet isolation and islet transplantation in animal models have
evolved in parallel (Lacy 1967; Kemp et al. 1973; Scharp et al. 1975). This has
paved the way for the translation of these preclinical studies to human islet isolation
and clinical transplantation. A complete review of all the steps that led to the
development of more advanced protocols for pancreatic islet isolation have been

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well highlighted by Piemonti and Pileggi (Piemonti and Pileggi 2013). In this sense,
the primary goal of isolating mouse pancreatic islets (either for in vivo transplantation or for in vitro studies) is to consistently provide viable and functional islets.
Despite islet isolation being a work of craftsmanship and each laboratory having
its own recipe (ODowd 2009; Zmuda et al. 2011; Kelly et al. 2003; Salvalaggio
et al. 2002; Szot et al. 2007; Li et al. 2009), the main steps in any rodent islet
isolation procedure consist of:
1. Surgery and pancreas harvesting
2. Pancreas digestion
3. Islet purification
4. Culture of the islets
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 step, the method of islet purification from pancreatic acinar
tissue and the culture conditions following isolation. Identifying factors influencing
the efficacy of the isolation procedure of pancreatic islets in rodents mice is
mandatory for the standardization of this procedure, the reduction of variability,
and the harvesting of good quality islets for subsequent experiments. In this section,
we compare and contrast the advantages and potential disadvantages of the most
common islet isolation protocols reported in the scientific literature and provide
details of a successful setting up of the procedure (Table 1). Despite rats also
usually being used as models in islet isolation, there are substantial differences in
the isolation procedure (Table 2). Many published islet isolation protocols specific
for rat islet isolation provide the necessary details to successfully perform the
complex procedures. For further details on rat isolation see (Kelly et al. 2003).

Surgery and Pancreas Harvesting

Although it is desirable to perform this step under sterilized conditions, inside a
laminar flow hood, it can be performed outside, with a clean technique without
greatly increasing the incidence of contamination. Nevertheless, all reagents and
surgical instruments should be sterile in order to avoid contamination. In detail, the
procedures consist of the following steps.
Animal surgery: The first aspect to take into account in order to perform an
efficient surgical procedure is the choice of a euthanasia method for the donor
animals. Euthanizing the donor rodent is a procedure that can be differently
regulated in different countries (e.g. cervical dislocation, CO2 mouse asphyxiation,
drugs). In addition, numerous authors and our personal experience suggest that after
anesthesia and just before cannulation, the best option is to exsanguinate animals.
This step is fundamental in order to avoid possible blood contamination during the

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Table 1 Summary of the steps of the rodent islet isolation procedure and key aspects
Step
Surgical
procedure

Essential Equipments
Critical step
Autoclave,
Duct cannulation
Hood (optional),
Surgical instruments:
Surgical forceps (straight
and curved), Fine forceps
and scissor, hemostatic
forceps to clamp off the
bile duct
Ice
Dissecting microscope
27-gague needle and 3 ml
syringe

Pancreas
digestion

Hood for cell culture with

vertical laminar flow
Water bath with
temperature control
Centrifuge (with
temperature control)
Filter mesh

Purification Reagent
step
15/50 ml conical tubes
Centrifuge (with
temperature control)
Ice

Culture of
the islet

Reagents (media, FBS,

Penicillin, streptomycin)
Culture dishes (10 cm
Petri Plates)
Incubator with
temperature and gas
composition controls
Optical microscope

Time of digestion
in order to not
overdigest or
underdigest

Choice of
purification
methods

Culture density

Cautions
Plan correctly the number of
the animal that you want to
use as donor based on your
surgical expertise, in order to
dedicate 1 hours and half
maximum to this step to
obtain a good quality of islets.
During the pulling of pancreas
for harvesting pay attention to
avoid contamination breaking
intestine or stomach.
Keep pancreas perfuse on
4  C before start digestion
Shaking and centrifugation
steps induce sheer stress so
centrifuge at 900 rpmi for
2/3 minutes max.
Stop digestion with FBS and
ice
Wash carefully the mesh to
avoid loosing of material
stucked on mesh
The solution for purification
are toxic so keep all the
solution at 4  C in order to
reduce the stress of the islet
once they are in contact with
the solution
Centrifuge the islet without
brake in order to avoid mix of
the solution
Mix carefully islets in the
heaviest gradient solution,
otherwise a purification will
be not good
Choose the correct media
based on your experiment, in
order to keep islet in good
shape and no in activate
metabolic state.
Culture the islet as pure as
possible, because starting
from day +1 post culture it
became difficult distinguish
endocrine tissue from
exocrine tissue

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Table 2 Summarizing the differences between mouse and rat pancreatic islet isolation
Surgery

euthaninization

needle

Pancreas
digestion

collection of
pancreas
dissecting
microscope
enzyme

digestion time
Purification number of pancreas
step
for each 50 ml tube
Culture
culture media
culture density

Mouse
cervical dislocation,
CO2 asphyxiation,
drugs
30 or 27 Ga 1/2" needle
Each pancreas is ready
for digestion
mandatory

Rats
Isoflurane (3-5 %) in a sealed
chamber or Ketamine/Xylazine ip
PE 50 Polyethylene tubing o
Blunt needles 0.6 mm  25.4 mm
Each pancreas has to be clean
from lymph node, fat
not necessary

collagenase Type XI
or V
12 min max
up to 5

Collagenase type XI or liberase

12/20 min
1-2

RPMi
250-300 IEQ/ml

CMRL 1066, 10 % FBS, PenStrep

not more than 300 IEQ per ml

subsequent steps and alterations in the efficacy of collagenase activity. Exsanguination, usually performed by cutting the vena cava, allows a good-quality batch of
islets to be obtained. Next after euthanization, the mouse should be placed in a
supine position; the abdomen cleaned with 70 % ethanol and opened with a
V-incision from the pubic region up to the diaphragm, in order to expose the
abdominal cavity.
Pancreas perfusion and harvesting instructions: Position the lobes of the liver
against the diaphragm. Find the hepatic artery, portal vein, and bile duct bundle
leading into the liver by gripping the duodenum with curved forceps. Once the liver
is exposed, grip the duodenum again with curved forceps following the bile duct
and clamp at the level of the ampulla, in order to spread the collagenase solution
only in the pancreas and not in the intestine. If the clamp is too high or too low the
enzyme will not perfuse the pancreas properly. The ampulla is a triangular white
area located at the duodenum surface, where there is a confluence between the bile
duct and the duodenum with bile draining from the gall bladder and enzymes from
the pancreas coming together before entering the intestines. Optimal needle placement is important to prevent backflow into the liver and to be sure it drains the
splenic tail of the pancreas, an islet-rich area. Particular care should be taken to
avoid penetration of the fascia tissue, which would result in perfusing only the
surrounding connective tissue. Once the common bile duct is clamped, it can be
cannulated using different techniques. The standard procedure consists of direct
swelling via injection of collagenase solution into the common bile duct (Fig. 1). In
this method, the needle must be inserted into the Y-shaped junction of the cystic
duct and the hepatic duct.
Contrary to this classic approach, a suggested method in the literature
involves catheterization without a microscope through the duodenum end of the

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Fig. 1Anatomy of the mouse upper intraperitoneal cavity. The mouse lying on its back with head
toward the surgeon. Panel A. The arrow indicates the site of ampulla. Panel B. Injection site and
common bile duct

common bile duct, after occlusion of the junction site of the hepatic and cystic
ducts. At first glance this method could seem easier, but the perfusion from the
intestine to the liver could drag bacteria into the pancreas, causing contamination.
The pancreas is then excised and digested at 37  C (Gotoh et al. 1985). It is
important to remove fat tissue because it may affect digestion and reduce the
yield (Li et al. 2009).
General Considerations The choice of the methods for cannulation depends on
the skills and expertise of the operator. However, one consideration must be
taken into account: perfusing the pancreas by common bile duct cannulation allows
the collagenase to spread inside the pancreas using the anatomical structure and
allows a 50 % higher islet yield to be obtained, because collagenase interacts
closely with connective tissue, and is more cost effective (Shapiro et al. 1996). A
pioneering approach in the scientific literature suggests avoiding clamping the duct,
directly excising the pancreas, and cutting it into 1- to 2-mm pieces, followed by
digestion of the pieces in a collagenase solution stirring or shaking directly as
detailed below (Lacy and Kostianovsky 1967; ODowd 2009). However, this
method is less efficient and can only be considered when pancreas perfusion is
not possible.

Pancreas Digestion
The basis of islet isolation is the intraductal injection of collagenase in order to
digest the tissue. Once the pancreas is harvested, the next step is its digestion with
collagenase. The digestion can be static and/or dynamic. The collagenase used in
the procedure is not a pure preparation; the ability of different batches of enzyme to
yield a successful isolation is still one of the greatest obstacles to improving this
procedure. The key points to take into consideration during this procedure are
described below.

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The choice of the enzyme: The standard digestion protocol for mouse isolation
uses a crude collagenase type XI or V. Unfortunately, variations in the composition
of preparations that are commercially available requires each lot to be individually
tested and optimized prior to general use. The same considerations that must be
taken into account when the enzyme blend is chosen for human isolation should
also be borne in mind for rodent isolation. In fact it has already been reported that
thermolysin concentration, the ratio of collagenase I to II, and the purity of the
enzyme blends are three of the most significant parameters for achieving optimal
islet isolation (Wolters et al. 1992, 1995). For human islet isolation, collagenase I is
considered essential (Barnett et al. 2005); on the contrary, collagenase II is reported
to play a major role in murine pancreatic islet isolation (Wolters et al. 1995;
Vos-Scheperkeuter et al. 1997). With all these considerations, a highly purified
preparation eliminates the intensive process of lot testing normally required for
crude or enriched collagenase products, and ensures homogeneity. Recently,
numerous publications have reported how using a more purified collagenase mixture can be more effective. In 2009, Yesil et al. showed that an increase in enzyme
purity correlates with an increase in islet yield (Yesil et al. 2009). Stull
et al. reported in 2012 that using a purified protease mixture increased the islet
yield in different mice strains: up to 260 islet/mouse for C57Bl/6, 200300 islets/
mouse for CD1, 200300 islets/mouse for 129/B6, 120200 islets/mouse for
BLKS-db/db, 100150 islets/mouse for NOD (10 weeks old), and 100150 islets/
mouse for NOD-SCID (Stull et al. 2012). Those studies lead to more productive use
of resources and improved flexibility in experimental design. Moreover, collagenase formulations have been identified as containing endotoxins in varying
amounts. As observed in humans, the level of endotoxin should be considered
when choosing collagenase mixtures, because it usually correlates with
pro-inflammatory cytokines in models of transplantation (Jahr et al. 1999). Some
interesting experiments performed in 2001 demonstrated that using endotoxinfree reagents during islet isolation (not only the enzyme, but also the wash and
culture media) is a key factor for successful islet transplantation (Berney
et al. 2001).
Control of digestion: The digestion step is really critical and must be taken
seriously when choosing between different methods. Digestion time, digestion
temperature, and collagenase administration are key factors in obtaining a good
islet yield. Regarding the digestion time, this step varies among the different
protocols because it is strictly dependent on the choice of cannulation methods.
In fact the cannulation of the bile duct allows collagenase to spread intact pancreas
and to digest the connective tissue more closely, which could result in shortening of
the duration of digestion. Usually, the digestion time should take between 8 and
11 min. A good suggestion is to perform a static digestion for 68 min and
perform an additional mechanical digestion at 37 C for 2 min. The mechanical
digestion, by shaking (manually or automatically), is necessary because the islet
yield could be low if the tissue is not well broken. The strain and the age of the
rodent can also influence the time of digestion. This is because the connective
tissue is different between animals. Once the correct point of the digestion has

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91

been decided, the action of the enzyme must be terminated. Stopping the
reaction is usually achieved by a combination of a cooling procedure and the
removal of the enzyme. The decrease in the temperature to 4  C and the washing
could be achieved by adding Hanks solution supplemented with 10 % FBS. Some
studies suggest adding some protease inhibitor such as BSA to the stop solution to
prevent cell lysis and the release of proteolytic enzymes (Wolters et al. 1990;
Perdrizet et al. 1995). In fact, endogenous proteolytic activity released during the
dissociation process exerts a deleterious effect, causing rupture of the cells and
release of DNA.
General Considerations Incubation time with the enzyme and the termination
technique vary among different perfusion conditions and is strictly collagenase
dependent. It is important to calibrate the optimal incubation time for each new
enzyme batch and use frozen stock to mimic the real experimental conditions.
Precise guidelines that suggest how to choose and use collagenase were reported by
de Haan et al. in 2004 (De Haan et al. 2004).

Purification
The goal of the purification step is to extract the islets from the exocrine tissue
contained in the digested pancreas. The purification step defines the mass, viability,
and function of your isolated islets. The purity could affect the immunogenicity and
safety of the islet recipient (Gotoh et al. 1986); in fact, acinar cells are able to
secrete different digestive enzymes (e.g., gastrin-releasing peptide, amylase), that
are really dangerous for the survival of the islets. On the contrary, preservation of
ductal cells and fibroblast appears pivotal for the survival of pancreatic islet. Taking
all this into account, the importance of the choice of the purification method is
evident. Before purification, a filtration step through stainless steel filters is
required. The tissue is poured through a 0.419-mm mesh that allows digested tissue
to be separated from non-digested tissue, fat and lymph, and a pellet of tissue to be
prepared for purification. Despite there has been a consensus on the importance of
separating endocrine tissue from acinar tissue, there is some debate regarding the
method of purification, particularly about the use of a density gradient to obtain a
first clean-up of islet preparation (Carter et al. 2009). Specifically, the most common alternatives for purifying islets are the following.
Sedimentation: The easiest and least expensive method of purifying the filtrate
tissue is sedimentation, although it is also the most inefficient method. In fact, islets
normally show intrinsic variation in diameter (50500 m, which can overlap with
acinar cell diameters).
Density gradient purification: The standard method for islet purification is
density gradient centrifugation or isopycnic centrifugation (separation according
to differences in density). Islets are centrifuged on density gradients long enough
for them to reach the point of the gradient of equal density. Acinar cells and islets
have high differences in density and are, therefore, easily separated by this method.
However, acinar tissue density depends on the secretory status of the acinar cells

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and their density may be affected by the size of the aggregates formed (Chadwick
et al. 1993a, b; De Duve 1971), by the collagenase digestion of the pancreas and their
swelling after the isolation procedure, leading to some overlapping (Berney et al.
2002). Albumin, sucrose, iodixanol, Histopaque, and Ficoll are the most common
solutions used to separate islets from acinar tissue by this method of purification.
Among them, the solution most widely used for density gradient purification, as
reported in the literature, is Ficoll, because it leads to a higher level of purification
in comparison with other gradients, and provides a more physiologically osmotic
environment for the islets (Lindall et al. 1969). Usually, Ficoll is layered in a
discontinuous way and islets are isolated from acinar tissue, which is heavier. Ficoll
is expensive, potentially toxic to islets because of its high content of glucose and
has been associated with release of IL-1, NO, and reactive oxygen species by islets
in vitro (Jahr et al. 1995, 1999). To overcome some of these drawbacks, many
laboratories have adopted other solutions, even though each of these solutions has a
different osmolarity, which can lead to varied islet numbers, viability, purity,
and functionality. Histopaque is a hypertonic solution also used in isolating other
cell types. For this reason, some laboratories prefer this gradient (Zmuda
et al. 2011). Iodixanol is a non-ionic, iso-osmolar solution, first used for pig
isolation and since 2007 also used for rodents, with good results. Recently, McCall
et al. (McCall et al. 2011) made a comparison of different protocols of purification
(dextran, Ficoll, Histopaque, iodixanol) and concluded that Histopaque was as good
as Ficoll.
It should be noted that Histopaque and Ficoll are both gradients widely used and
accepted. As general guidelines, independently of the kind of solution used, the
tissue should be suspended in the most dense gradient. The pellet should be
resuspended very well in order to avoid a decrease in purification efficiency. The
gradient must be built with different layers of the solution going from the most
dense solution in the bottom of the tube to the lightest solution, to avoid mixing the
layers. Then the gradient should be spun at a specific temperature and speed
according to the nature of the solution, tissue volume, etc. At the end of centrifugation islets can be retrieved from the first and second gradient interfaces; the purity
of the first layer is usually higher, because the second layer can also include
embedded islets.
Other purification techniques: Some protocols also consider handpicking the
islets before culture as an option to purify the suspension. The handpicking protocol
includes a stereomicroscope with side illumination to identify islets suspended in a
Petri dish with a black background. It should be performed in a laminar flow cabinet
using a dissecting microscope to minimize the risk of islet contamination. Using a
2 objective, islets are selected from the acinar tissue and transferred to a second
culture or third dish (depending on the initial purity of the preparation) containing
culture media. An instrumental step that does not allow the widespread use of the
handpicking technique is the fact that it is very tedious and time consuming. In fact,
this step should be performed as fast as possible in order to minimize the time
outside the sterile hood and incubator to limit exposure to contamination and pH
changes. In 2002, Salvalaggio (Salvalaggio et al. 2002) compared pancreatic digest

Mouse Islet Isolation

93

purified either by Ficoll or by filtration, claiming superiority of the second method

of islet purification. Recently, our group has compared Histopaque and filtration
purification with handpicking as the gold standard method for islet purity (London
et al. 1998). Our results show the significant advantage of islet purity using
Histopaque versus the other methods, all of them with purity over 98.5 %. Once
the endocrine tissue is completely separated from acinar tissue, the islets are ready
to be cultured. In some protocols a second purification step by gradient, sedimentation or filtration is needed before culturing, to further increase islet purity and islet
yield, particularly if the volume of packed tissue is still large.
General Considerations Even though the use of density gradients is well
accepted in all the laboratories, further improvements in the purification step will
not result from the production of new density gradient media, but rather from the
continued modification of the biochemical composition of the solvents in which the
established gradient media are dissolved in order to produce a more suitable
physiological environment for the islet (London et al. 1998). The final purity of
the isolated islet depends on the type of gradient, but is also influenced by the mouse
strain. The total number of islets varies considerably depending on the strain and
age of the rodent, the expertise of the technician as well as the method of isolation
(Gotoh et al. 1985). It has been reported, for example, that lean rodents lead to a
yield of higher purity than those with more fat (De Groot et al. 2004). Certain
factors must be taken into account when choosing the purification method:
(a) The temperature: may affect the results of density gradient purification. Rodent
islet purification is more efficient at 4  C, since the characteristics of the
solution used in this procedure could damage metabolically active cells.
(b) Osmolarity of the solution: hyperosmolarity could prevent edema of acinar
tissue.
(c) Volume of tissue packed: as a general guideline the normal amount of tissue
processed in 50-ml tubes for efficient purification should be 1 ml.

Culture
After performing islet isolation, proper culture conditions are imperative to ensuring that the islets can recover from the insult of collagenase digestion and isolation
procedure. Appropriate islet culture conditions must be considered:
Culture medium: The common medium used for rodent islet culture is RPMI
1640, even though other media are used equally as frequently in different laboratories; some authors suggest using a medium commonly used in human islet
culture, CMRL 1066. It is reported, for example, that this medium induces a
decrease in alloreactivity; however, it seems that some immune cells, such as
dendritic cells and endothelial cells, do not survive for a long period of time in
CMRL 1066 (Benhamou et al. 1995). Another key factor to take into account in the
choice of the medium is glucose concentration. Media with glucose concentrations
below 11 mM can reduce islet insulin content and down regulate key genes related
to glucose metabolism.

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S. Marzorati and M. Ramirez-Dominguez

Culture density: Another important consideration is islet density in culture: it is

common not to culture too many islets in the same dish, because this can induce
necrosis as a consequence of cell damage and competition for nutrients (Dionne
et al. 1993). Usually, 250300 islets equivalent/ml for a 60-  15-mm dish does not
appear to stress the islets. Suspension culture dishes are preferred in order to
decrease islet attachment.
Culture temperature: The standard protocol for rodent islet culture foresees
placing them at 37 C with 5 % CO2 infusion and humidified air. Some authors
incubate islets at 24  C for the first 4872 h following isolation, with the aim of
dampening their metabolic activity, preserving their viability, and smoothing their
transition to in vitro culture (Ricordi et al. 1987). Moreover, it seems that culturing
islets below 37  C helps to almost completely remove the intra-islet lymphoid cells
(Moore et al. 1967).
Effects of culture on future transplantation: Recently, thanks to new advances in
technologies, new methods of culturing islets have been explored in the literature.
Islet culture prior to transplantation is controversial for many reasons: death of
endothelial cells with the impossibility of re-establishing islet vasculature in vitro;
proliferation of fibroblasts with effects on gene expression; development of a
necrotic core in the islets, etc. On the other hand, in vitro culture of pancreatic
islets reduces their immunogenicity and prolongs their availability for transplantation. In fact, after a few hours of culture, the majority of acinar cells die, increasing
the purity of the preparation (e.g., because endothelial cells die and these intra-islet
blood vessels may be crucial if the islets are to be transplanted). Fibroblasts in
culture can grow in a few days, totally changing the expression of genes such as
vimentin. A necrotic core islet could also appear during culture. Recently, biomaterials (e.g., hydrogel, polyglycolic acid scaffold [PGA]) have been explored
(Vaithilingam et al. 2014; Jun et al. 2013) in order to maintain islet 3D culture, and
simulated microgravity (sMG) is believed to confer benefits to cell culture (Song
et al. 2013).
General Considerations It is usually good practice to change media 2448 h
post-isolation to better preserve islet function and to remove dead islets and debris.
Many laboratories, in order to increase islet yield for the experiment, perform more
than one isolation and pull together the preparations at the end. It is important not to
mix islets from different isolations, because the cells could be influenced by many
factors, including the time spent in culture (Zmuda et al. 2011). Therefore, in order
to reduce variability, the best option is to perform experiments on islets isolated at
the same time. In addition, some cryogenic methods for long-term preservation of
the islets have been explored. Warnock et al. (Warnock et al. 1987) reported a
successful protocol for cryopreservation, with -cell responsiveness in vitro and
in vivo. Others suggest that is not possible to preserve a good endocrine function of
frozenthawed islets (Piemonti et al. 1999). Finally, selection of appropriate media
has been shown to depend again on the animal source of the islets (Holmes
et al. 1995).

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95

Practical Issues: Quality, Time, and Cost

Islets are the primary source of either in vitro or in vivo experiments in the diabetes
research field. Therefore, it is of great importance that the isolation procedure yields
islets of sufficient quality. In that sense, it is also necessary that the assessment of
that quality is performed according to standardized criteria. However, the establishment of standardized assessment tests has proved to be a challenge and it is
under development in several laboratories. Islet preparations are difficult to characterize for many reasons (Colton et al. 2007):
1. Islets are cellular aggregates. Therefore, they have 3D structure, with an asymmetric shape. This implies some technical inconveniences, since many techniques suitable for single cells or cells in monolayers are unsuitable for islets.
Besides, the wide range of sizes makes it difficult for them to be accurately
quantified or proper visual estimations to be made, which are operator dependent
and highly variable.
2. It is not easy to obtain a preparation that is 100 % pure. Different variables could
affect the purity of the preparations obtained, including some aspects of the
isolation procedure, as explained in the previous section (e.g., type of collagenase, methods of purification), or characteristics of the donors (animal age, sex
or strain)
3. Islets are dynamic. Islet volume decreases with time after isolation because they
are under stress from the moment the pancreas is harvested, during the islet
isolation process, in culture, etc. Therefore, when the experiment starts islets are
not completely representative of their original status.
In this section we will discuss some practical issues regarding quality, time, and cost of
the islet preparations to be taken into account before starting any experiment (Table 3).

Morphology
When islets are inspected under a light microscope, islets appear spherical and
golden-brown, and range between 50 to over 400 m, most of them between
50 and 250 m in diameter (Carter et al. 2009; Bertera et al. 2012). Bertera and
colleagues reported that, in general, 20 % of the islets measure less than 50 m, 30 %
between 50 and 100 m, 35 % between 100 and 250 m, and 15 % greater than
250 m (Bertera et al. 2012). Mouse islets are easily identified by their semi-opaque
color in comparison to the relatively transparent exocrine tissue. Healthy isolated
islets present a defined smooth rounded surface. Usually, islets show a well-preserved
capsule after an overnight recovery. However, darker hypoxic areas in the center
of larger islets or cells disrupting the defined surface of the islets can appear over time
as a sign of reduced health. As explained in the previous section, an optimized ratio
between the number of islets and the volume of culture media should be established
to maintain islets with good morphology and good viability (Zmuda et al. 2011).

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Table 3 Summary of the parameters, assays, and information to be considered in order to select a
rodent islet isolation protocol
Parameter
Assay
Morphology Observation by light microscopy

Purity

Dithizone staining

Yield

Stimulation of the number in a

sample. Observation under the
stereomicroscope
Membrane integrity tests.
Observation by fluorescence
microscopy.
Glucose Static Insulin Secretion
Dynamic Perifusion
Timing of the isolation/purification
procedure
Summing up the cost of the reagents
taking into consideration staff wages

Validity

Function
Time
Cost

Information Obtained
Shape and aspect of the islets (under
stereomicroscope), presence/absence of
dark hypoxic areas (under inverted
microscope)
What fraction of the preparation is
endocrine and exocrine tissue
What number of islets have been obtained
What is the number of IEQ
Percentage of viable tissue in each islet and
in the preparation
Insulin secretory capacity under glucose
challenge
if the protocol is the most suitable according
to the volume of work of the laboratory
if the protocol is the most suitable according
to the economy of the laboratory

Purity
Purity is a key factor in islet preparation, since it is intimately linked to quality.
As explained in the previous section, contamination of the preparation by acinar
cells can affect the immunogenicity of the graft for in vivo experiments and also
its functionality, both in vitro and in vivo, owing to the interaction with the
secretory response of the islets (Piemonti et al. 1999) The traditional method of
assessing islet purity is by the zinc-specific binding dye dithizone (DTZ) staining
(Latif et al. 1988), since pancreatic islets contain a high concentration of zinc
because of its role in the synthesis, storage, and secretion of insulin. This
dye stains the islets red, so that they can be visually distinguished from
exocrine tissue under a light microscope. It is also helpful to quantify the islet
yield both in human and mouse islets, although mouse islets can be more
easily distinguished from exocrine cells at a glance. However, dithizone has a
potentially toxic effect on islet function; thus, it is considered a non-vital stain
(Conget et al. 1994).
Usually islets are examined with a phase contrast microscope. Islet mass can be
quantified by islet counting and determination of islet diameter. A calibrated grid in
the eyepiece could be useful for measuring the diameter of the islets (Ricordi
et al. 1988). The purity of the islets could be determined by estimating the volume
of the islets relative to the non-islet tissue. The purity is calculated as the percentage
of the intersections that overlie islets out of the number of the intersections that
overlie islet and non-islet tissues (Ricordi et al. 1990).

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97

Yield
The yield obtained after islet isolation is an important parameter, taking into account
that the number of mice that will be sacrificed for the experiment usually depends on
the average yield obtained per mouse. In fact, the average yield per mouse is an
important parameter to consider in setting up islet isolation. This yield, as explained
in the previous section, also depends on different variables, such as the experience of
the operator (e.g. surgery ability), and characteristic of the donor (age, weight, and
strain) (De Haan et al. 2004; De Groot et al. 2004). On average, rats yield between
300 and 800 islets per animal, while the yield from a mouse pancreas is lower, around
100150 increasing to 200400 islets with an experienced technician, which means
between 10 and 30 % of the islets in the typical rodent pancreas (Carter et al. 2009;
Zmuda et al. 2011; Kelly et al. 2003; Gotoh et al. 1985).
One method of directly quantifying the yield in mouse islet preparations is
handpicking. This is a time-consuming procedure, making this tedious process
unfeasible for large-scale islet isolations. Thus, the standard practice is to take a
small sample of the preparation (e.g., 100 l), stain it with dithizone, and proceed to
counting under the light microscope with an ocular micrometer.
According to the consensus report of 1990 (Ricordi et al. 1990), developed after
the workshop of the 2nd Congress on Pancreas and Islet Transplantation (Minneapolis, 1989), the standard practice for expressing the total volume of islets is Islet
Equivalents (IEQ). One IEQ is the number of standard islets of 150 m in
diameter that would have the same total volume (Buchwald et al. 2009). Therefore,
when counting, islets are classified according to their diameter in ranges of 50 m
and their number of IEQs calculated by multiplying the number of islets with a
conversion factor that is derived from the mean volume of islets in that range group
divided by the volume of an islet with a diameter of 150 m. In this regard, there is
some controversy over the quantification of islets between laboratories. Although
the use of IEQ could be more statistically representative of the total volume of the
preparation, others consider using the absolute number of islets a more accurate
measure of preparations with a small number of islets.

Viability
The viability of an islet preparation is an important parameter in quality assessment,
since it is expected that a high degree of viability will correlate with a successful
transplantation outcome. Pancreatic islets are cell aggregates, and it is always
controversial and time-consuming to choose the correct test to check viability.
A good suggestion is to perform more than one test in order to obtain a more
precise picture. The most common tests are reported below.
Fluorescein diacetate and propidium iodide (FDA/PI) assay: The standard
international method of determining viability is double staining with fluorescein
diacetate and propidium iodide (FDA/PI) assay. This is an assay based on the
simultaneous discrimination of live versus dead cells by membrane integrity stains.

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FDA functions as an inclusion dye, while PI is an exclusion dye. FDA is a colorless,

non-polar ester that passes through the plasma membrane and becomes hydrolyzed
by non-specific cellular esterases to produce fluorescein, resulting in a strong green
fluorescence. On the other hand, PI is polar and only enters dead cells, as intact
membranes remain impermeable to it. Once inside the cell, it fluoresces orange/red
when bound to nucleic acids. After the islet sample is stained with both dyes, it is
examined under a fluorescence microscope and the approximate volume fraction of
cells stained of each color is visually assessed (Colton et al. 2007).
The advantages of using FDA/PI are that it is a quick, easy, and cheap assay and
therefore very convenient, apparently. However, it has some limitations (Colton
et al. 2007; Barnett et al. 2004; Boyd et al. 2008):
It does not distinguish between islets and non-islets.
It is a subjective method, operator-dependent.
It is not accurate. Islets are tridimensional structures and with fluorescence
microscopy only the surface of the islets can be seen; therefore, information
from the inside of the aggregate is lacking.
It only differentiates between dead and non-dead cells; it does not distinguish
apoptotic cells.
The assay has to be technically optimized. The final concentration, incubation
times, type of solvent, and storage conditions influence the final scoring of
viability.
There is a lack of correlation between membrane integrity stains in general and
other viability assays, including mitochondrial function assays, such as MTT
and ATP.
It does not correlate with the transplantation outcome, neither in animals nor in
humans.
It overrates the percentage of live cells and the FDA can also cause background
fluorescence.
Regarding the last point, a comparison study between different dyes (Barnett
et al. 2004) reported that FDA/PI produced intense staining that obscured the PI
signal in the core of the islets. The viability was higher than that obtained with
SYTO/EB (Syto-13 ethidium bromide), and there was also a higher degree of
background fluorescence, owing to extracellular, nonspecific esterase activity.
However, in a more recent study (Boyd et al. 2008) a wider variety of stains were
compared, and the results were found to be questionable for all dyes.
Alternative methods: Effort is are therefore being invested in the exploration of
alternative methods that are currently under investigation, such as stains that are
responsive to metabolic activity (e.g. tetrazolium salts, ATP, ADP/ATP, oxygen
consumption rate) and/or mitochondrial membrane potential (Janjic and Wollheim
1992; Barbu and Welsh 2004; Goto et al. 2006; Papas et al. 2007). In fact,
Colton et al. (2007) suggest mitochondrial function assays as an interesting
alternative to membrane integrity tests, since they are able to identify cells that
are damaged, but do not reach the membrane permeabilization step. Therefore, this
kind of assay would provide a more accurate measurement of the viability of an
islet preparation.

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99

Functionality
In the assessment of the mouse islet preparations it is fundamental to characterize
islet function. Pancreatic cells are very sensitive to changes in extracellular
glucose and the ability of the islets to regulate insulin release defines its functionality (Carter et al. 2009). Islets show a dose-dependent pattern of insulin release
when incubated with different glucose concentrations. The time course of insulin
secretion in response to glucose stimulation is biphasic. There is a first phase with a
rapid spike in insulin secretion followed by a decrease to a prolonged second phase
plateau of insulin that continues with the duration of the stimulus. Islet functionality
can be assessed in vitro or in vivo.
In vitro functional test: The standard methods used to measure islet function are
glucose standard insulin secretion (GSIS) and dynamic perifusion. While the first
method can be useful for establishing doseresponse curves in response to insulin
secretagogues, questions about the kinetics of insulin secretion in response to
glucose are addressed with regard to islet perifusion experiments (Nolan and
ODowd 2009).
The GSIS assays consist of cultivating the islets first at a low, basal glucose
concentration in order to synchronize the cells before starting the real assay. Next,
the islets are incubated at a low glucose concentration, then at a high glucose
concentration, and then again with low glucose. When islets are exposed to low
glucose (usually 2.8 mM) they are under basal or unstimulated conditions. On
the other hand, when islets are stimulated at the high glucose concentrations, the
concentration of insulin can be raised to different concentrations, depending on the
laboratory, to 11.1 mM, which is half maximal, or over 28 mM, which is maximal.
The purpose of alternating basal or unstimulated conditions with stimulated
ones is to check that the islets in the preparation are able to respond properly,
increasing and also decreasing insulin secretion according to the changes in glucose
concentration. The decrease in the insulin secretion with the second basal also
discards the possibility of sustained high values of insulin secretion after stimulating conditions due to cell death processes. The stimulation index (SI) can be
calculated as the ratio of stimulated-to-basal insulin secretion. Healthy islets can
have an SI of 220 depending on different factors such as strain, age, and body
weight (Carter et al. 2009). A more physiological assessment of the detection of
insulin release in vitro is dynamic perifusion. This technique is an adaptation of the
principle of the flow respirometer developed by Carlson and colleagues (Olsson and
Carlsson 2005) for the measurement of the uptake of oxygen by amphibian nerve.
Even though this method is time-consuming and expensive, the technique
provides accurate quantitative data on the dynamic responses to biologically active
compounds and adds information on the dynamics of the hormone in terms of
secretion and clearance. This technique allows the detection of insulin release in
response to elevated glucose concentration and secretagogues. Islets in a perifusion
media (RPMI-1640, or more often a specific medium, depending on the experimental aim) are sandwiched between two layers of sterilized material in a
microchamber. Gassing, flow rates, the addition of secretagogues and fraction

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collection are controlled by a programmable perfusion/perifusion apparatus. Fractions are collected every 10/30 min over 3 h. Recently, new tools (e.g., glucose
nanosensor) reported in the literature have allowed the widespread use of this
quality assessment technique.
Besides, it is important to take into account the purity of the preparation
when any of these functional studies are performed, since the presence of
exocrine tissue can interact with the secretory response of the islets. It is also
advisable to culture the islets between 12 and 18 h prior to functional experiments
to allow the replacement of receptors, which may have been proteolyzed during
collagenase treatment (Nolan and ODowd 2009). However, some reports have
shown improved islet function in rodents with fresh islets (King et al. 2005; Olsson
and Carlsson 2005). Deciding to perform functional testing on the day of isolation
or after recovery correlates with the aim of the experiments, whether the goal is to
perform in vitro experiments or preclinical studies. In the latter case, it is better to
perform the functional test on the day of the transplant in order to better mimic the
situation of the islets on the day of engraftment.
Insulin and C-peptide content could be another measure of islet functionality.
Briefly, both peptides can be extracted from the isolated islets by acidified ethanol
and sonication, and can be quantified on supernatants through immunoassay tests
(Rabinovitch et al. 1982).
In vivo functional test: Moreover, islet cell potency can be assessed in vivo
by diabetes reversal after islet transplantation into chemically diabetic mice.
Diabetes is induced in mice by streptozotocin or alloxan injection. The differences
between the two methods are well explained by Cantarelli et al. (2013). Nonfasting
glycemia and metabolic testing, such as the intraperitoneal glucose tolerance
test or the intravenous glucose tolerance test can be performed in transplanted
mice, in order to test the functionality of the islets in the graft (Juang
et al. 2008; Morini et al. 2007). Histological examination of the graft can
provide useful information on the morphology and cellular composition of the
transplanted islets.

Time and Cost

Although obtaining good quality islets is instrumental in any experimental study,
time and cost are also side issues that are gaining relevance in the daily routine of
mouse islet laboratories.
As McCall and collaborators suggest (McCall et al. 2011), a mouse islet isolation protocol must be chosen when the time and costs invested are worth the
benefits in terms of quality, and vice versa. In this sense, we have recently
compared the quality, time, and cost of a standard rodent islet purification protocol
using Histopaque with a filtration-based method, an alternative that is not mouse so
well established in laboratories, taking handpicking as the gold standard method of
islet purification (Ramirez-Dominguez and Castano 2014).

Mouse Islet Isolation

101

Our study suggests that, while both protocols yield good-quality islets, purification of islets by filtration with 100-m cell strainers saves almost 90 % of the time
spent in purification using Histopaque density gradient. If staff time and wages are
also taken into account, an important economic saving for the laboratory could be
made. Therefore, it would be particularly convenient for large-scale islet isolations
and/or laboratories with limited staff. The drawback of this filtration protocol is that
one third of the islets are lost compared with the yield obtained by handpicking, and
therefore, more animals are needed.
Thus, this study points out the importance of time and cost investments in
addition to the quality of the islets in the selection of the mouse islet isolation
protocol, and their impact on the management of the laboratory routine.

Factors to Consider in Setting Up a New Rodent Islet Isolation

Laboratory
Performing studies on isolated human islets are unquestionably crucial for the
improvement and widespread use of this clinical procedure. Unfortunately, the
availability of human donors can be a hindrance, and for this reason mouse islets
serve as a useful surrogate for human islets. Animal use in scientific studies is
regulated by OLAW (Office of Laboratory Animal Welfare) guidelines and IACUC
(Institutional Animal Care and Use Committees) committees when planning the
protocol for any experiment, although the use of animals in research is always
controversial.
Basic elements to consider when setting up a new laboratory for mouse islet
isolation include adequate space to grow, well-trained staff, and sufficient funding
to support the research.

Infrastructure
Facilities: Provision of adequate space is usually one of the general problems in any
institution. In order to have an optimal supply of mouse pancreas it is mandatory
that the institution where the laboratory is going to be set up has a fully
working animal facility that facilitates the provision of mice of the required strain,
sex, and age. Taking into account the fact that the surgery can be performed
either under sterile conditions or using an aseptic technique it is not compulsory
to carry it out at the animal facility; it can be done in the laboratory.
The laboratory should have an area for performing the purification and quality
assessment assays. In addition, there should be a separate cell culture unit where
the islets will be cultured after isolation and also where the in vitro experiments will
be carried out.
Equipment: A mouse islet isolation laboratory is composed mainly of the general
equipment of a cell biology laboratory plus some specific materials for performing

102
Table 4 Essential
Facility
equipment for the setting
Equipment
up of a mouse islet
isolation program

S. Marzorati and M. Ramirez-Dominguez

Laboratory
Microcentrifuge
Magnetic stirrer
with heating
Scale
Vortex
Water Bath
pHmeter
Fridge with a
20 C freezer
80 C freezer
Planetary Shaker
Plate Reader

Cell culture unit

Laminar Flow Hood
Fluorescence Inverted
Microscope
Stereomicroscope
Centrifuge
Incubator
Water Bath

surgery (Table 4). It is important to establish a routine for the cleaning and correct
maintenance of the equipment since the isolations must be carried out using an
aseptic technique and the islets will be used in other experiments (either in vitro or
in vivo) requiring sterility.

Staff
The most important factor in the success of a new laboratory is the members of staff
who will be part of the team. The workers recruited must meet the necessary skills,
knowledge, and motivation requirements to set up the new laboratory efficiently.
Since mouse islet isolation requires a significant degree of expertise, it is highly
desirable to hire personnel with previous expertise in the field. However, if the
candidates lack the necessary scientific background, they must be adaptable and
smart and capable of learning new techniques. The number and composition of
the members of the team is dependent on size of the laboratory, the dynamics of
the institution, and the budget. In order to have a constant source of islets it is
desirable to have at least two technicians devoted to the isolation procedure
who master all the techniques in the laboratory, and who can help and/or
complement each other.

Budget
The final goal of the establishment of a mouse islet isolation laboratory is the
translation of the studies performed with those islets to the clinic with pre-clinical
assays. Therefore, the institution can receive funding from government sources at
different levels, institutions, industry or private research sources. These organizations can provide either funding or the development of different research lines.
Therefore, the initial investment in setting up the laboratory from the recipient
institution is recovered once the laboratory is fully working.

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103

Conclusions
Mouse islets are a constant and reliable raw material for any in vitro or in vivo study in
the diabetes field. Thus, despite differences between human tissue and animal models,
improvements in the mouse isolation procedure could be somehow translated to the
optimization of human isolation and therefore, to better transplantation strategies.
Mouse islet isolation is a work of craftsmanship, since many variables, as well
as operator expertise, influence the outcome, therefore requiring the acquisition
of specific technical skills and know-how. Once the protocol is defined and
islets are isolated, their quality can be assessed by different parameters using
standard methods. However, time and costs invested in the selected protocol
are also issues to be considered, with a direct impact on the management of the
laboratory.
The set-up of a mouse islet isolation laboratory is not only an economic
investment, but also a human and institutional one. It is the foundation stone for
pre-clinical studies. Therefore, it brings not only scientific benefits by translating
the knowledge gained from the studies of mouse islets to the clinics, but also a very
valuable logistical experience when undertaking to establish a human islet isolation
laboratory.
Acknowledgements This work was supported by a grant of the Basque Government for Groups
of Excellence (IT-472-07) and a grant of the Department of Industry of the Basque Government
(SAIO09-PE09BF01).

Cross-References
For major details on Clinical Islet Isolation/transplantation refer to the chapters:
Advances in Clinical Islet Isolation
Islet Isolation from Pancreatitis Pancreas for Islet Autotransplantation

References
Barbu A, Welsh N (2004) The use of tetrazolium salt-based methods for determination of islet cell
viability in response to cytokines: a cautionary note. Diabetologia 47:20422043
Barnett MJ, McGhee-Wilson D, Shapiro AM, Lakey JR (2004) Variation in human islet viability
based on different membrane integrity stains. Cell Transplant 13:481488
Barnett MJ, Zhai X, LeGatt DF, Cheng SB, Shapiro AM, Lakey JR (2005) Quantitative assessment
of collagenase blends for human islet isolation. Transplantation 80:723728
Benhamou PY, Stein E, Hober C, Miyamoto M, Watanabe Y, Nomura Y, Watt PC, Kenmochi T,
Brunicardi FC, Mullen Y (1995) Ultraviolet light irradiation reduces human islet immunogenicity without altering islet function. Horm Metab Res 27:113120
Bensley RR (1911) Studies of the pancreas with the guinea pig. Am J Anat 12:297388
Berney T, Molano RD, Cattan P, Pileggi A, Vizzardelli C, Oliver R, Ricordi C, Inverardi L (2001)
Endotoxin-mediated delayed islet graft function is associated with increased intra-islet cytokine production and islet cell apoptosis. Transplantation 71:125132

104

S. Marzorati and M. Ramirez-Dominguez

Berney T, Pileggi A, Molano RD, Ricordi C (2002) Epithelial cell culture: pancreatic islets. In:
Atala A, Lanza RP (eds) Methods of tissue engineering. Academic, San Diego
Bertera S, Balamurugan AN, Bottino R, He J, Trucco M (2012) Increased yield and improved
transplantation outcome of mouse islets with bovine serum albumin. J Transplant 2012:856386
Bhonde RR, Parab PB, Sheorin VS (1999) An in vitro model for screening oral hypoglycemics. In
Vitro Cell Dev Biol Anim 35:366368
Biarnes M, Montolio M, Nacher V, Raurell M, Soler J, Montanya E (2002) -cell death and mass
in syngeneically transplanted islets exposed to short- and long-term hyperglycemia. Diabetes
51:6672
Bock T, Pakkenberg B, Buschard K (2005) Genetic background determines the size and structure
of the endocrine pancreas. Diabetes 54:133137
Boyd V, Cholewa OM, Papas KK (2008) Limitations in the use of Fluorescein Diacetate/
Propidium Iodide (FDA/PI) and cell permeable nucleic acid stains for viability measurements
of isolated islets of Langerhans. Curr Trends Biotechnol Pharm 2:6684
Buchwald P, Wang X, Khan A, Bernal A, Fraker C, Inverardi L, Ricordi C (2009) Quantitative
assessment of islet cell products: estimating the accuracy of the existing protocol and accounting for islet size distribution. Cell Transplant 18:12231235
Cabrera O, Berman DM, Kenyon NS, Ricordi C, Berggren PO, Caicedo A (2006) The unique
cytoarchitecture of human pancreatic islets has implications for islet cell function. Proc Natl
Acad Sci U S A 103:23342339
Cantarelli E, Citro A, Marzorati S, Melzi R, Scavini M, Piemonti L (2013) Murine
animal models for preclinical islet transplantation: no model fits all (research purposes). Islets
5:7986
Carlson FD, Brink F Jr, Bronk DW (1950) A continuous flow respirometer utilizing the oxygen
cathode. Rev Sci Instrum 21:923932
Carter JD, Dula SB, Corbin KL, Wu R, Nunemaker CS (2009) A practical guide to rodent islet
isolation and assessment. Biol Proced Online 11:331
Chadwick DR, Robertson GS, Rose S, Contractor H, James RF, Bell PR, London NJ (1993a)
Storage of porcine pancreatic digest prior to islet purification. The benefits of UW solution and
the roles of its individual components. Transplantation 56:288293
Chadwick DR, Robertson GS, Toomey P, Contractor H, Rose S, James RF, Bell PR, London NJ
(1993b) Pancreatic islet purification using bovine serum albumin: the importance of density
gradient temperature and osmolality. Cell Transplant 2:355361
Colton CK, Papas KK, Pisania A, Rappel MJ, Powers DE, ONeil JJ, Omer A, Weir G, BonnerWeir S (2007) Characterization of islet preparations. In: Halberstadt CR (ed) Cellular transplantation: from laboratory to clinic. Elsevier, Oxford, UK, pp 85133
Conget JI, Sarri Y, Gonzalez-Clemente JM, Casamitjana R, Vives M, Gomis R (1994) Deleterious
effect of dithizone-DMSO staining on insulin secretion in rat and human pancreatic islets.
Pancreas 9:157160
De Duve C (1971) Tissue fraction-past and present. J Cell Biol 50:20
De Groot M, de Haan BJ, Keizer PP, Schuurs TA, van Schilfgaarde R, Leuvenink HG (2004) Rat
islet isolation yield and function are donor strain dependent. Lab Anim 38:200206
De Haan BJ, Faas MM, Spijker H, van Willigen JW, de Haan A, de Vos P (2004) Factors
influencing isolation of functional pancreatic rat islets. Pancreas 29:e15e22
Dionne KE, Colton CK, Yarmush ML (1993) Effect of hypoxia on insulin secretion by isolated rat
and canine islets of Langerhans. Diabetes 42:1221
Goto M, Holgersson J, Kumagai-Braesch M, Korsgren O (2006) The ADP/ATP ratio: a novel
predictive assay for quality assessment of isolated pancreatic islets. Am J Transplant
6:24832487
Gotoh M, Maki T, Kiyoizumi T, Satomi S, Monaco AP (1985) An improved method for isolation
of mouse pancreatic islets. Transplantation 40:437438
Gotoh M, Maki T, Satomi S, Porter J, Monaco AP (1986) Immunological characteristics of
purified pancreatic islet grafts. Transplantation 42:387390

Mouse Islet Isolation

105

Holmes MA, Clayton HA, Chadwick DR, Bell PR, London NJ, James RF (1995) Functional
studies of rat, porcine, and human pancreatic islets cultured in ten commercially available
media. Transplantation 60:854860
Jahr H, Bretzel RG, Wacker T, Weinand S, Brandhorst H, Brandhorst D, Lau D, Hering BJ,
Federlin K (1995) Toxic effects of superoxide, hydrogen peroxide, and nitric oxide on human
and pig islets. Transplant Proc 27:32203221
Jahr H, Pfeiffer G, Hering BJ, Federlin K, Bretzel RG (1999) Endotoxin-mediated activation of
cytokine production in human PBMCs by collagenase and Ficoll. J Mol Med (Berl)
77:118120
Janjic D, Wollheim CB (1992) Islet cell metabolism is reflected by the MTT (tetrazolium)
colorimetric assay. Diabetologia 35:482485
Juang JH, Kuo CH, Wu CH, Juang C (2008) Exendin-4 treatment expands graft -cell mass in
diabetic mice transplanted with a marginal number of fresh islets. Cell Transplant 17:641647
Jun Y, Kim MJ, Hwang YH, Jeon EA, Kang AR, Lee SH, Lee DY (2013) Microfluidics-generated
pancreatic islet microfibers for enhanced immunoprotection. Biomaterials 34:81228130
Kelly CB, Blair LA, Corbett JA, Scarim AL (2003) Isolation of islets of Langerhans from rodent
pancreas. Methods Mol Med 83:314
Kemp CB, Knight MJ, Scharp DW, Lacy PE, Ballinger WF (1973) Transplantation of isolated
pancreatic islets into the portal vein of diabetic rats. Nature 244:447
King A, Lock J, Xu G, Bonner-Weir S, Weir GC (2005) Islet transplantation outcomes in mice are
better with fresh islets and exendin-4 treatment. Diabetologia 48:20742079
Lacy PE (1967) The pancreatic cell. Structure and function. N Engl J Med 276:187195
Lacy PE, Kostianovsky M (1967) Method for the isolation of intact islets of Langerhans from the
rat pancreas. Diabetes 16:3539
Latif ZA, Noel J, Alejandro R (1988) A simple method of staining fresh and cultured islets.
Transplantation 45:827830
Li DS, Yuan YH, Tu HJ, Liang QL, Dai LJ (2009) A protocol for islet isolation from mouse
pancreas. Nat Protoc 4:16491652
Lindall A, Steffes M, Sorenson R (1969) Immunoassayable insulin content of subcellular fractions
of rat islets. Endocrinology 85:218223
London NJ, Swift SM, Clayton HA (1998) Isolation, culture and functional evaluation of islets of
Langerhans. Diabetes Metab 24:200207
McCall MD, Maciver AH, Pawlick R, Edgar R, Shapiro AM (2011) Histopaque provides optimal
mouse islet purification kinetics: comparison study with Ficoll, iodixanol and dextran. Islets
3:144149
Melzi R, Battaglia M, Draghici E, Bonifacio E, Piemonti L (2007) Relevance of hyperglycemia on
the timing of functional loss of allogeneic islet transplants: implication for mouse model.
Transplantation 83:167173
Moore GE, Gerner RE, Franklin HA (1967) Culture of normal human leukocytes. JAMA
199:519524
Morini S, Brown ML, Cicalese L, Elias G, Carotti S, Gaudio E, Rastellini C (2007) Revascularization and remodelling of pancreatic islets grafted under the kidney capsule. J Anat
210:565577
Moskalewski S (1965) Isolation and culture of the islets of Langerhans of the guinea pig. Gen
Comp Endocrinol 44:342353
Nano R, Clissi B, Melzi R, Calori G, Maffi P, Antonioli B, Marzorati S, Aldrighetti L, Freschi M,
Grochowiecki T, Socci C, Secchi A, Di Carlo V, Bonifacio E, Bertuzzi F (2005) Islet isolation
for allotransplantation: variables associated with successful islet yield and graft function.
Diabetologia 48:906912
Nolan AL, ODowd JF (2009) The measurement of insulin secretion from isolated rodent islets of
Langerhans. Methods Mol Biol 560:4351
ODowd JF (2009) The isolation and purification of rodent pancreatic islets of Langerhans.
Methods Mol Biol 560:3742

106

S. Marzorati and M. Ramirez-Dominguez

Olsson R, Carlsson PO (2005) Better vascular engraftment and function in pancreatic islets
transplanted without prior culture. Diabetologia 48:469476
Papas KK, Colton CK, Nelson RA, Rozak PR, Avgoustiniatos ES, Scott WE, Wildey GM,
Pisania A, Weir GC, Hering BJ (2007) Human islet oxygen consumption rate and DNA
measurements predict diabetes reversal in nude mice. Am J Transplant 7:707713
Perdrizet GA, Rewinski MJ, Bartus SA, Hull D, Schweizer RT, Scharp DW (1995) Albumin
improves islet isolation: specific versus nonspecific effects. Transplant Proc 27:34003402
Piemonti L, Pileggi A (2013) 25 years of the Ricordi automated method for islet isolation. Cell R4
1:822
Piemonti L, Bertuzzi F, Nano R, Leone BE, Socci C, Pozza G, Di Carlo V (1999) Effects of
cryopreservation on in vitro and in vivo long-term function of human islets. Transplantation
68:655662
Piemonti L, Guidotti LG, Battaglia M (2010) Modulation of early inflammatory reactions to
promote engraftment and function of transplanted pancreatic islets in autoimmune diabetes.
Adv Exp Med Biol 654:725747
Rabinovitch A, Quigley C, Russell T, Patel Y, Mintz DH (1982) Insulin and multiplication
stimulating activity (an insulin-like growth factor) stimulate islet (-cell replication in neonatal
rat pancreatic monolayer cultures. Diabetes 31:160164
Ramirez-Dominguez M, Castano L (2014) Filtration is a time-efficient option to Histopaque
providing good quality islets in mouse islet isolation. Cytotechnology. doi:10.1007/s10616014-9690-7
Ricordi C, Lacy PE, Sterbenz K, Davie JM (1987) Low-temperature culture of human islets or
in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-tomouse xenograft survival. Proc Natl Acad Sci U S A 84:80808084
Ricordi C, Lacy PE, Finke EH, Olack BJ, Scharp DW (1988) Automated method for isolation of
human pancreatic islets. Diabetes 37:413420
Ricordi C, Gray DW, Hering BJ, Kaufman DB, Warnock GL, Kneteman NM, Lake SP, London
NJ, Socci C, Alejandro R et al (1990) Islet isolation assessment in man and large animals. Acta
Diabetol Lat 27:185195
Salvalaggio PR, Deng S, Ariyan CE, Millet I, Zawalich WS, Basadonna GP, Rothstein DM (2002)
Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves
islet mass and function. Transplantation 74:877879
Scharp DW, Murphy JJ, Newton WT, Ballinger WF, Lacy PE (1975) Transplantation of islets of
Langerhans in diabetic rhesus monkeys. Surgery 77:100105
Shapiro AM, Hao E, Rajotte RV, Kneteman NM (1996) High yield of rodent islets with intraductal
collagenase and stationary digestiona comparison with standard technique. Cell Transplant
5:631638
Shapiro AM, Lakey JR, Ryan EA, Korbutt GS, Toth E, Warnock GL, Kneteman NM, Rajotte RV
(2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a
glucocorticoid-free immunosuppressive regimen. N Engl J Med 343:230238
Song Y, Wei Z, Song C, Xie S, Feng J, Fan J, Zhang Z, Shi Y (2013) Simulated microgravity
combined with polyglycolic acid scaffold culture conditions improves the function of pancreatic islets. Biomed Res Int 2013:150739
Stull ND, Breite A, McCarthy R, Tersey SA, Mirmira RG (2012) Mouse islet of Langerhans
isolation using a combination of purified collagenase and neutral protease. J Vis Exp.
doi:10.3791/4137
Szot GL, Koudria P, Bluestone JA (2007) Murine pancreatic islet isolation. J Vis Exp (7)255
Vaithilingam V, Kollarikova G, Qi M, Larsson R, Lacik I, Formo K, Marchese E, Oberholzer J,
Guillemin GJ, Tuch BE (2014) Beneficial effects of coating alginate microcapsules with
macromolecular heparin conjugates-in vitro and in vivo study. Tissue Eng Part A 20:324334
Venkatesan V, Chalsani M, Nawaz SS, Bhonde RR, Challa SS, Nappanveettil G (2012) Optimization of condition(s) towards establishment of primary islet cell cultures from WNIN/Ob
mutant rat. Cytotechnology 64:139144

Mouse Islet Isolation

107

Vos-Scheperkeuter GH, van Suylichem PT, Vonk MW, Wolters GH, van Schilfgaarde R (1997)
Histochemical analysis of the role of class I and class II Clostridium histolyticum collagenase
in the degradation of rat pancreatic extracellular matrix for islet isolation. Cell Transplant
6:403412
Warnock GL, Rajotte RV, Evans MG, Ellis D, DeGroot T, Dawidson I (1987) Isolation of islets of
Langerhans following cold storage of human pancreas. Transplant Proc 19:34663468
Wolters GH, van Suylichem PT, van Deijnen JH, van Schilfgaarde R (1990) Factors influencing
the isolation process of islets of Langerhans. Horm Metab Res Suppl 25:2026
Wolters GH, Vos-Scheperkeuter GH, van Deijnen JH, van Schilfgaarde R (1992) An analysis of
the role of collagenase and protease in the enzymatic dissociation of the rat pancreas for islet
isolation. Diabetologia 35:735742
Wolters GH, Vos-Scheperkeuter GH, Lin HC, van Schilfgaarde R (1995) Different roles of class I
and class II Clostridium histolyticum collagenase in rat pancreatic islet isolation. Diabetes
44:227233
Yesil P, Michel M, Chwalek K, Pedack S, Jany C, Ludwig B, Bornstein SR, Lammert E (2009)
A new collagenase blend increases the number of islets isolated from mouse pancreas. Islets
1:185190
Zmuda EJ, Powell CA, Hai T (2011) A method for murine islet isolation and subcapsular kidney
transplantation. J Vis Exp. doi:10.3791/2096