Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Summary

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Abstract

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma ^1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis ^2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA ⁴, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation.

Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies ⁵. Small pieces of pancreas of about 0.5-1 cm³ were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 °C until sectioning. Forty serial sections of 10 μm thickness were cut on a cryostat under a -20 °C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 °C.

Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol ⁶. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 μl lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Protocol

1. Freezing of Human Pancreatic Tissue

1. Remove adipose tissue, blood vessels, nerves and non-parenchymal tissue with a scalpel and tweezers, and cut the pancreatic tissue into pieces (~0.5-1 cm cubes).
2. Place one pancreatic tissue piece in the center of a Cryomold, and cover it completely with cold Tissue-Tek O.C.T. Compound. Put the Cryomold into a jar whose bottom is covered with pre-cooled 2-Methylbutane and snap-freeze in liquid nitrogen. Store the frozen sample at -80 °C.

2. Preparation of Frozen Sections

1. Wear gloves throughout all steps to avoid denaturation of RNA.
2. Cle....

Results

As shown in Figure 1, the modified dehydrating protocol led to an improvement of beta cells autofluorescence compared to the previous published protocol ⁶. Applying the described protocol, each of 39 surgical pancreatic specimens was used to generate 40 serial cryosections for an average of 31'544'704 μm³ tissue/ pancreatic specimen (range: 8'742'390 - 81'522'153 μm³) as shown in Table 1. This represents the volume of about 18 pancreatic islets of.......

Discussion

We describe a reliable approach for the laser microdissection (LMD) of human islets from surgical pancreatic specimen. Provided that a LMD microscope is available, this procedure could be implemented at any research institution performing partial pancreatectomies, thereby increasing access to human islet material from both non-diabetic and type 2 diabetic subjects. This is especially relevant given the paucity of pancreata offered for islet isolation. Favourable aspects of LMD compared to islet isolation by collagenase d.......

Materials

Name of the reagent	Company	Catalogue number	Comments
2-Methylbutane (isopentane)	ROTH	3927.1
AdhesiveCap (opaque, 500 μl)	ZEISS	415190-9201-000
Cellstar Tubes (50 ml)	greiner bio-one	210 261	with support skirt
Cellstar Tubes (50 ml)	greiner bio-one	227.261
Diethyl pyrocarbonate (DEPC)	SIGMA	D 5758
Dry ice
Ethanol absolute	VWR	20821.310
Liquid nitrogen
Paint brush
PALM MicroBeam	ZEISS
Peel-a-Way embedding moulds (truncated), 12 x 12 mm	ProSciTech	RR12	Top internal 22x22 mm, depth 21 mm
Arcturus PicoPure Frozen RNA Isolation Kit	Applied Biosystems	KIT 0204
Plastic clamp
Razor
RNase-Free DNase Set	Qiagen	79254
RNaseZAP	SIGMA	R 2020
Scalpel / surgical blade	Techno Cut	2800111
SuperFrost Plus adhesion microscope slides	Thermo Scientific	J1800AMNZ	25x75x1.0 mm
Tissue-Tek O.C.T Compound	Sakura	4583 or 0807000022
Tweezers	BRAUN	BD168R
Xylene	VWR	28975.291