Effective Isolation of Functional Islets from Neonatal Mouse Pancreas

Summary

We describe a protocol herein for isolating intact islets from neonatal mice. Pancreata were partially digested with collagenase, followed by washing and hand picking. 20 – 80 islet clusters can be obtained per pancreas from newly born mice, which are suitable for several islet studies.

Abstract

Perfusion-based islet-isolation protocols from large mammalian pancreata are well established. Such protocols are readily conducted in many laboratories due to the large size of the pancreatic duct that allows for ready collagenase injection and subsequent tissue perfusion. In contrast, islet isolation from small pancreata, like that of neonatal mice, is challenging because perfusion is not readily achievable in the small pancreata. Here we describe a detailed simple procedure that recovers substantial numbers of islets from newly born mice with visual assistance. Freshly dissected whole pancreata were digested with 0.5 mg/mL collagenase IV dissolved in Hanks' Balanced Salt Solution (HBSS) at 37 °C, in microcentrifuge tubes. Tubes were tapped regularly to aid tissue dispersal. When most of the tissue was dispersed to small clusters around 1 mm, lysates were washed three to four times with culture media with 10% fetal bovine serum (FBS). Islet clusters, devoid of recognizable acinar tissues, can then be recovered under dissecting stereoscope. This method recovers 20 – 80 small- to large-sized islets per pancreas of newly born mouse. These islets are suitable for most conceivable downstream assays, including insulin secretion, gene expression, and culture. An example of insulin secretion assay is presented to validate the isolation process. The genetic background and degree of digestion are the largest factors determining the yield. Freshly made collagenase solution with high activity is preferred, as it aids in endocrine-exocrine isolation. The presence of cations [calcium (Ca²⁺) and magnesium (Mg²⁺)] in all solutions and fetal bovine serum in the wash/picking media are necessary for good yield of islets with proper integrity. A dissecting scope with good contrast and magnification will also help.

Introduction

Isolating pure pancreatic islets is essential for assaying glucose stimulated insulin secretion (GSIS) of beta cells and for islet transplantation from cadaveric donors ^1-3. It is also necessary to establish endocrine gene expression in islet cells ^{4, 5}. For this purpose, detailed protocols have been established to allow for isolation of pancreatic islets from large pancreata (⁶ and references therein). These methods are based on enzymatic perfusion to dissociate acinar from islet tissues, coupled with gradient separation and hand picking. Thus, islet isolation from large pancreas can be performed readily in most laboratories. On the other hand, no detailed step-by-step protocol exists to allow for the isolation of islets from pancreata that are too small to perfuse.

Studying gene expression and function of neonatal islets is important. Neonate islets have different properties from adults in insulin secretion and proliferation capability ^{7, 8}. However, isolating islets from newly born animals, especially mice is challenging due to the small size of the newly born pancreas. The size prevents the usual perfusion process when collagenase is injected though the pancreatic duct. Indeed, several papers have presented studies along these lines, with enzyme or non-enzyme aided isolation procedures ^{7, 9, 10}. However, detailed description of the islet isolation process with visual aid is lacking ^{7, 9}, making it a challenge for most researchers to perform similar studies.

We have explored several different conditions that yield high quality islets from neonatal mice. Here we present a protocol that is expected to help researchers learn the key details in the islet isolation process. This protocol is applicable to mouse pancreas up to two weeks of age, after which perfusion can be performed for routine islet isolation. Islets can be directly used for insulin secretion and gene expression assays.

Protocol

Animal usage follows the procedures specified in protocol M/11/181 approved by the Vanderbilt Institutional Animal Care and Use Committee for Gu. CD1 or CBA/Bl6 mice were purchased from commercial vendors and crossed in the Vanderbilt animal facility to obtain neonatal mice. 1. Preparation of Mice, Stock Solutions, and Equipment For the mouse cross: set up a mouse cross and record the plugging dates to aid experimental planning. CD1 mice usually give birth around day 19 after matin…

Representative Results

Under optimal conditions, the presented method can yield 20 – 80 islets from each small mouse pancreas. This number depends on the genetic background, age of mice, and the size of islets to be recovered. Among the commonly used, CD1 out-bred and C57BL/6J pure-bred mice produce less islets with smaller size than hybrids between CD1 and C57BL/6 or commercial B6CBAF1/J mice do. Direct hand picking generally gave a smaller number of islets, likely due to the exclusion of small islets that cou…

Discussion

Here, we provide a step-by-step protocol on islet isolation from pancreata that are too small for conventional perfusion. It is expected to yield islets ready for all islet-based studies such as beta-cell purification, gene expression analysis, islet beta-cell maturation, proliferation, cell stress responses, cell survival, metabolism, and functional GSIS maintenance, etc. This will be, to the best of our knowledge, the first detailed visual protocol that guides new researchers to perform islet isolation from ne…

Materials

Collagenase  Type IV	Sigma Aldrich, St Louis, MO	C5138
1X HBSS with Ca2+/Mg2+	Mediatech/Cellgro, Manassas, VA	MT21020CV	If HBSS wihout Ca2+/Mg2+ is obtained, CaCl2 and MgSO4 can be added to 1.26 and 0.5 mM, respectively.
RPMI 1640 w/o glucose	Thermo Fisher Scientific/Life Technologies, Waltham, MA	11879-020	Glucose needs to be added to specific levels to not interfere with seusequent islet usage.
Glucose	Sigma Aldrich, St Louis, MO	G-7021
polysucrose and sodium diatrizoate solution	Sigma Aldrich, St Louis, MO	Histopaque-10771
Stereoscope	Carl-Zeiss, Oberkochen, Germany	Stemi2000
Stereoscope	Leica, Wetzlar, Germany	Leica M165
Microcentrifuge	Eppendorf, Hauppauge, NY	Centrifuge 5417C
Centrfuge	Eppendorf, Hauppauge, NY	Centrifuge 5810R
15-ml centrfuge tubes	VWR, Radnor, PA	89039-666
50-ml centrfuge tubes	VWR, Radnor, PA	89039-658
Precision balance	VWR, Radnor, PA	VWR-225AC
Microfuge tubes	VWR, Radnor, PA	87003-294
Pipetman P1000	Fisher Scientific,  Waltham, MA	F123602
Pipetman P20	Fisher Scientific,  Waltham, MA	F123600
100X15 millimeter dish	VWR, Radnor, PA	25384-088
60X15 millimeter dish	VWR, Radnor, PA	25384-168
12-well plates	VWR, Radnor, PA	665-180
Scissor	Fine Scientific Tools, Foster City, CA	14080-11
Tweezers	Fine Scientific Tools, Foster City, CA	5708-5
CD1 mice	Charles River Laboratories, Wilminton, MA	CD-1
C57BL/6J	The Jackson laboratory, Farmington, CT	C57BL/6J
B6CBAF1/J	The Jackson laboratory, Farmington, CT	B6CBAF1/J