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. Clean the cryostat knife, specimen holder and paintbrush with cold 100% ethanol and RNase Away. Place the frozen tissue inside the cryostat.
3. Wait until the tissue, knife and brush reach -20 °C. Label the SuperFrost Plus slides and pre-cool them inside the cryostat chamber while waiting for the tissue to reach -20 °C.
4. Apply the Tissue-Tek O.C.T. Compound on the specimen holder and place the frozen tissue on top.
5. Once the Tissue-Tek O.C.T. Compound is frozen trim the cryoblock and remove any excess of Tissue-Tek O.C.T. Compound with a razor blade.
6. Cut a total of 40 consecutive 10 μm slices from the pancreatic tissue block. Additionally we recommend cutting a first and middle slice to be saved for overview drawings of the pancreatic section that can facilitate the identification of islets within the slices during the microdissection process.
7. Transfer the sections from the knife blade to the center of a SuperFrost Plus slide by touching the backside of the slide with your finger (covered with a glove) to warm up only the region to which the section shall adhere.
8. Dry the sections 1-2 min inside the cryostat at -20 °C, after which you put them into a slide box placed on dry ice. Store the sections at -80 °C.
9. If necessary, clean the knife blade with paper towels and cold 100% ethanol.

3. Dehydration of Frozen Sections

1. Prepare the Reagents: pour 30 ml freshly prepared 70% ethanol, 30 ml DEPC-treated water, 2x30 ml 100% ethanol and 30 ml xylene into 50 ml Cellstar tubes (Falcons); chill and keep all reagents (except xylene) on ice.
2. Clean the working space under the hood with 100% ethanol and RNaseZAP.
3. Process two cryosections as follows: fix in ice cold 70% ethanol for 30 sec, wash with 5-6 quick dips in ice cold DEPC-treated water, dehydrate twice for 1 min in ice cold 100% ethanol, incubate for 4 min in xylene at room temperature (25 °C), and air dry for 3-5 min. Repeat this step with another pair of cryosections.
4. Insert two dried cryosections back to back into a 50 ml Cellstar tube wrapped with aluminium foil to protect them from light and moisture.

4. Laser Microdissection of Beta-cells with PALM MicroBeam, Zeiss

1. Clean the microscope with RNaseZAP and mount the Adhesive Cap in the RoboMover.
2. Set the following settings: Filter set 09 (excitation: 450-490 nm, emission > 515 nm), AutoLPC mode, 50% laser energy, 65% laser focus, 100% laser speed, 5 μm distance between AutoLPC shots, 6 μm distance to line, working height: -10,100.
3. Insert one slide into the stage.
4. Locate the autofluorescent beta cells under microscopic visualisation (5x or 10x lens).
5. Switch to the 40x lens, select the beta cells with the "Freehand" selection tool and microdissect them using the laser.
6. Capture the tissue of four dehydrated cryosections into one AdhesiveCap.
   Comment 1: the time to dissect the beta cells of one dehydrated cryosection should not be longer than 10-20 min to avoid rehydration of the tissue sections which would result in RNA degradation.
   Comment 2: verify successful microdissection process by visual inspection after moving the stage to the checkpoint.

5. Lysis of Microdissected Beta Cell Enriched Tissue

1. Remove the AdhesiveCap from the RoboMover.
2. Pipet 10 μl extraction buffer (XB, PicoPure RNA Isolation Kit, Arcturus) into the lid of the AdhesiveCap and incubate it upside down at 42 °C for 30 min.
3. Spin down at 10,000 x g and put the lysate on dry ice. Store it at -80 °C until the RNA extraction is performed.
4. Repeat steps 3.) to 5.) with all other sections.

6. RNA Extraction

1. Combine all ten 10 μl lysates
2. Proceed with the RNA isolation by working at room temperature according to the protocol of the PicoPure RNA Isolation Kit, Arcturus, using a 1:1 ratio Extraction Buffer (XB) to 70% Ethanol (including DNase Treatment).

7. RNA Analysis

1. Use 1 μl RNA for analysis of quality and quantity using an Agilent 2100 Bioanalyser (PicoChip). Quantitative evaluation is done in reference to a standard RNA loaded in parallel on the chip.

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	Company	Catalog 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