The authors thank the donors' families and the Organ Procurement Organizations involved in this research and Maria Martino and Irina Kusmartseva for their expert assistance. This work was funded by the Juvenile Diabetes Research Foundation (M.C-T., M.A.) in support of the Network for Pancreatic Organ Donors with Diabetes.

1. Set-up
<ol>
	<li>Label cassettes for paraffin blocks manually with a pencil, or automatically with a cassette printer. Include the donor identifier (CaseID), sample type, aliquot number, and any additional unique identification (Case worksheet, <a href="https://www.jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">Appendix 1</a>).</li>
	<li>Label OCT molds as for cassettes with a permanent marker. Cut out 5-10 cm rectangles of aluminum foil.</li>
	<li>Print or manually label with permanent marker all vials and tubes. Include enough vials for minced snap frozen samples and tubes with RPMI media for fresh shipments. For the snap frozen vials with RNAlater, add 1 mL RNAlater to each cryovial.</li>
	<li>Set-up dissection boards in biosafety hood and counter tops with sterilized dissection instruments (forceps, scalpels, blades, gauze pads (4x4)) as well as cassettes and OCT molds arranged in order of use.</li>
	<li>Prepare a freezing bath by putting dry ice in a medium sized Styrofoam box (10x10 cm) to a depth of at least 3 cm and add isopentane until the dry ice is completely covered.</li>
	<li>Optional: Fill a small Dewar container with liquid nitrogen.</li>
	<li>If pancreas is needed for electron microscopy (EM), prepare TAAB fixative by adding 1.25 mL 16% paraformaldehyde, 1.25 mL 8% glutaraldehyde, and 7.5 mL 0.1M PBS. Prepare Lowicryl fixative by adding 2.5 mL 16% paraformaldehyde to 7.5 mL 0.1M PBS.</li>
	<li>Prepare solutions used for isolating cells. To 500 ml bottle of DMEF (with glutamine), follow aseptic techniques while adding the following:
	<ol>
		<li>Fetal bovine serum (FBS): add 50mL, final 10%.</li>
		<li>Penicillin/Streptomycin (Pen/Strep, 10,000 U/mL / 10,000 ug/mL stock) - add 5 mL Pen/Strep stock to RPMI for final concentration of 100 U/mL / 100ug/mL. Also add 5 ml to a 500 ml bottle of DPBS when used for holding lymph nodes or spleen samples.</li>
	</ol>
	</li>
</ol>
2. Personnel
<ol>
	<li>Procedure is optimally accomplished with three staff members however the procedure can be conducted with one well trained individual. Two individuals can generally complete the procedure in 90 minutes. Actual duration is dependent on numbers of tissues received and ease of finding pancreatic lymph nodes. Samples for cell isolations are held at 4 &deg;C until completion of tissue dissection while serum samples are processed in parallel.</li>
	<li>Pathologist, pathology assistant, or equivalent- an experienced staff member leads the tissue handling and performs major dissections including division(s) of pancreas head and harvesting pancreatic lymph nodes. Initial efforts are directed towards pancreas followed by examination of peri-pancreatic fat for lymph nodes.</li>
	<li>Laboratory technologist or equivalent- staff member trained in tissue trimming whose primary responsibilities are to weigh pancreas and regions and trim tissues for fixed and frozen blocks and for snap frozen vials. Receives duodenum and spleen from pathology assistant and processes these organs while the pathology assistant is processing the pancreas.</li>
	<li>Laboratory assistant- staff member who assists the others including block and vial labeling, sample mincing for cryovials, and serum processing.</li>
</ol>
3. Pancreas Dissection
<ol>
	<li>Remove the duodenum and spleen and clean the pancreas of extraneous fat, vessels, and nerves using blunt and sharp dissection</li>
	<li>Place the cleaned pancreas on a dissecting board covered with paper towels. Dip a cotton-tipped applicator in blue ink and spread on the anterior surface of the pancreas. Blot off excess.</li>
	<li>Optional: Dip a new applicator in black ink and spread on the posterior surface of the pancreas. Return pancreas to normal orientation with anterior surface facing prosector.</li>
	<li>Cut the pancreas into 3 regions: head, body, and tail (Figure 1). Cut the junction between head and body then divide the remaining portion into ~equal portions for body and tail.</li>
	<li>Weigh each region of pancreas and record weights (Case workup sheet (<a href="https://www.jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">Appendix 1</a>)).</li>
	<li>Section (~0.5 cm) each region of the pancreas in a transverse "bread loaf" manner (Figure 1).</li>
	<li>Use the sections from both junctions between regions for minced samples. Depending on pancreas size, a sample from the body-tail junction region may be difficult to obtain in which case the number of paraffin blocks can be decreased to obtain minced samples or only samples from the pancreas head-body region can be obtained. Label cryovials according as head or body to denote proximal region at the junction.
	<ol>
		<li>Mince pieces and divide evenly between cryovials (&ge;1 gram per vial). Rapidly freeze vials without RNALater in liquid nitrogen or in the dry ice - isopentane slurry then transfer to a -80 &deg;C freezer.</li>
		<li>Keep vials with RNALater at room temperature for 30 minutes to allow for equilibration, remix and rapidly freeze in liquid nitrogen or in the dry ice-isopentane slurry then transfer to a -80 &deg;C freezer.</li>
	</ol>
	</li>
	<li>Remove alternating sections for paraffin and OCT blocks beginning with paraffin (Figure 1).</li>
	<li>Lean all transverse sections to the right.</li>
	<li>Place ~1.5 x 1.5 x 0.5 cm sections in the cassettes. If the transverse sections are too large, cut in half (Figure 2b). Label cassettes for blue anterior half as "A" and posterior half as "B".</li>
	<li>If pieces are still too large, cut each section perpendicular to the previous cut (Figure 2c). Label cassettes A-D in a clockwise manner. Trim pieces further as needed to fit within cassettes.</li>
	<li>Number blocks sequentially within region, starting with the most medial section.</li>
	<li>For paraffin blocks, position cassettes so the label is to the left and place tissue into the cassette, maintaining its orientation. Close cassette lid and transfer to a container with ~500 ml 10% neutral buffered formalin (NBF). Start timing fixation when the last cassette is placed in fixative.</li>
	<li>For OCT blocks, pour a thin layer of OCT media into the prelabeled mold then lay the tissue in the mold being sure to maintain orientation and cover tissue with OCT. Rapidly immerse the OCT molds in a dry ice - isopentane slurry for ~1 minute. Remove from slurry and place on dry ice for temporary storage or in -80 &deg;C freezer for long term storage.</li>
	<li>Obtain ~0.5 x 0.5 cm slices from the head-body junction for electron microscopy (EM). Place one slice in the TAAB fixative and the other slice in the Lowicryl fixative. Store both types of fixative with samples at 4 &deg;C for at least 48 hours and/or indefinitely until further processing for EM.</li>
</ol>
4. Spleen, Lymph Node Dissections and Duodenal Mucosa Dissections
<ol>
	<li>Isolate pancreatic lymph nodes (PLN) from fat and trim all connective tissue to the node capsule. Place in sterile cell culture dish containing DMEF. Depending on total numbers isolated, divide PLN into aliquots for fresh shipments, cryopreserved cell isolations, paraffin and/or OCT blocks, and vials. Mince large nodes into pieces no larger than 0.5 cm.</li>
	<li>Mince non-pancreatic lymph nodes and spleen into small (~0.5 cm) cubes. Place in fixative, OCT media, and or DMEM for fresh sample shipments or cryopreserved cell isolations according to amounts of starting materials.</li>
	<li>Open the duodenum and gently wipe the duodenal mucosa clean of mucous with a moistened gauze pad. Cut sections of the mucosa for paraffin and/or frozen OCT blocks and minced samples for vials, with and without RNAlater. Freeze as for other samples.</li>
</ol>
5. Wrap OCT blocks in pre-labeled aluminum foil and place OCT blocks and vials in boxes for long term storage at -80 &deg;C.
6. Submit Fixed Samples for Paraffin Processing
<ol>
	<li>Fix tissues for 16 hours (range 20 &plusmn;4 hours) using an automatic paraffin processor or using manual timing. Process to paraffin blocks using an automatic processor (<a href="https://www.jove.com/files/ftp_upload/4039/4039appendix2.doc" target="_blank">Appendix 2</a>).</li>
	<li>Embed sections in the exact orientation as placed by the prosector with the cassette label to the left. Embed the duodenal mucosa with the cut surface down to allow examination of the mucosa perpendicular to the submucosa.</li>
</ol>
7. Clean dissection areas and disinfect. Wash dissection instruments and re-sterilize. Place remnant human tissues in formalin fixative for storage as biomedical waste. Discard biomedical waste according to local regulations within one month.
8. Update the Sample Inventory System
9. Representative Results
This procedure will process an intact human pancreas with representative sampling throughout the organ including demarcation of the three major regions, namely, head, body and tail within 2 hours. The uncinate region, found in the posterior head region, is included in the head dissection. The median pancreatic weight in organ donors without diabetes was 82.4 grams (52.7 - 139.0 grams). Pancreas regional weights were approximately equal (Figure 3).
This procedure can also allow for reconstruction of the entire pancreas using stained slides from sequential paraffin and OCT blocks. Multiple formats are feasible allowing for maximum utilization for current and future technologies. The current nPOD case worksheet is provided (<a href="https://www.jove.com/files/ftp_upload/4039/4039appendix1.doc" target="_blank">Appendix 1</a>) that is used to document recovered samples and subsequent processing by aliquot types and quantities.
<img alt="Figure 1" src="/files/ftp_upload/4039/4039fig1.jpg" /> 
Figure 1. Overall scheme of the procedure. The entire pancreas is processed while maintaining anatomical orientations. The head region is longer in the superior-inferior axis due to the presence of the ventral pancreatic lobe in the posterior region that includes the uncinate region. Hatched area at junctions denote regions used for minced samples in cryovials. P, Paraffin; O, OCT.
<img alt="Figure 2" src="/files/ftp_upload/4039/4039fig2.jpg" /> 
Figure 2. Lettering scheme for pancreas sections. Transverse pancreas slices can be further divided into halves or quarters and lettered in a clockwise manner.
<img alt="Figure 3" src="/files/ftp_upload/4039/4039fig3.jpg" /> 
Figure 3. Pancreas weights from organ donors without diabetes. The intact pancreas from adult donors (&gt;17 years old) was weighed then divided into regions and regional weights obtained. Data are means &plusmn; SEM (N=15).

<ol>
	<li>Brissova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+human+pancreatic+islet+architecture+and+composition+by+laser+scanning+confocal+microscopy.">Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy.</a> J. Histochem. Cytochem. 53, 1087-1097 (2005).</li><li>Rahier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pancreatic+polypeptide+cells+in+the+human+pancreas:+the+effects+of+age+and+diabetes.">The pancreatic polypeptide cells in the human pancreas: the effects of age and diabetes.</a> J. Clin. Endocrinol. Metab. 56, 441-444 (1983).</li><li>Uchida, T., Takada, T., Ammori, B. J., Suda, K., Takahashi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+reconstruction+of+the+ventral+and+dorsal+pancreas:+a+new+insight+into+anatomy+and+embryonic+development.">Three-dimensional reconstruction of the ventral and dorsal pancreas: a new insight into anatomy and embryonic development.</a> J. Hepatobiliary Pancreat. Surg. 6, 176-180 (1999).</li><li>Wittingen, J., Frey, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Islet+concentration+in+the+head,+body,+tail+and+uncinate+process+of+the+pancreas.">Islet concentration in the head, body, tail and uncinate process of the pancreas.</a> Ann. Surg. 179, 412-414 (1974).</li><li>Rahier, J., Goebbels, R., Henquin, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+composition+of+the+human+diabetic+pancreas.">Cellular composition of the human diabetic pancreas.</a> Diabetologia. 24, 366-371 (1983).</li><li>Vaught, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+NCI+perspective+on+creating+sustainable+biospecimen+resources.">An NCI perspective on creating sustainable biospecimen resources.</a> J. Natl. Cancer Inst. Monogr. 42, 1-7 (2011).</li><li>Gianani, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+results+of+screening+of+nondiabetic+organ+donors+for+expression+of+islet+autoantibodies.">Initial results of screening of nondiabetic organ donors for expression of islet autoantibodies.</a> J. Clin. Endocrinol. Metab. 91, 1855-1861 (2006).</li><li>Tauriainen, S., Salmela, K., Rantala, I., Knip, M., Hy&#246;ty, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collecting+high-quality+pancreatic+tissue+for+experimental+study+from+organ+donors+with+signs+of+&#946;-cell+autoimmunity.">Collecting high-quality pancreatic tissue for experimental study from organ donors with signs of &#946;-cell autoimmunity.</a> Diabetes Metab. Res. Rev. 26, 585-592 (2010).</li><li>In't Veld, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+insulitis+in+adult+autoantibody-positive+organ+donors.">Screening for insulitis in adult autoantibody-positive organ donors.</a> Diabetes. 56, 2400-2404 (2007).</li><li>T&#246;ns, H. A., Terpstra, O. T., Bouwman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+human+pancreata+in+perspective+of+the+isolation+of+the+islets+of+langerhans.">Heterogeneity of human pancreata in perspective of the isolation of the islets of langerhans.</a> Transplant Proc. 40, 367-369 (2008).</li></ol>

No conflicts of interest declared.

The goal of this procedure is to define a standard pancreas processing that can be harmonized across multiple collection sites and thus allow comparisons of results obtained by different groups. The dissection method is based on standard surgical pathology practices with additional steps for annotation of major pancreas regions followed by intra-regional subdivisions. Standardization of biospecimen processing methods is required to facilitate collection of high quality samples exemplified by the publication of best pract...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments </td>
		</tr>
		<tr>
			<td>Dissection instruments including scalpels with 22 blades</td>
			<td>Various</td>
			<td>Various</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 and 50 ml)</td>
			<td>Fisher Scientific</td>
			<td>05-539-5, 430828</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Cryotubes(1.8 ml)</td>
			<td>VWR North America</td>
			<td>89004-300</td>
			<td>Sterile, bar codes optional</td>
		</tr>
		<tr>
			<td>RNAlater</td>
			<td>Ambion</td>
			<td>AM7021</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Surgical Pathology Ink</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cassettes</td>
			<td>Mercedes Medical</td>
			<td>CAS ECO109</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Fisher Scientific</td>
			<td>23-245-685</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>OCT Freezing Media</td>
			<td>TissueTek</td>
			<td>4583</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Molds</td>
			<td>ThermoScientific</td>
			<td>58952</td>
			<td>Various sizes</td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Fisher Scientific</td>
			<td>O3551-4</td>
			<td>OCT chilling bath</td>
		</tr>
		<tr>
			<td>Dry ice</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>OCT chilling bath</td>
		</tr>
		<tr>
			<td>Liquid nitrogen in Dewar flask</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Snap freezing cryovials</td>
		</tr>
		<tr>
			<td>Cell culture dish (100mm)</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>Hyclone DMEF</td>
			<td>ThermoScientific</td>
			<td>SH30023.01</td>
			<td>Sterile, store refrigerated, glutamine included</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Cellgro</td>
			<td>35-010-CV</td>
			<td>Sterile, store frozen aliquots</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin/Antifungal</td>
			<td>GIBCO</td>
			<td>15240-062</td>
			<td>Sterile, store frozen aliquots</td>
		</tr>
		<tr>
			<td>Type 9 Paraffin</td>
			<td>Richard-Allan Scientific</td>
			<td>8337</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1X D-PBS</td>
			<td>GIBCO</td>
			<td>14190-144</td>
			<td>Sterile</td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EM Sciences</td>
			<td>15710</td>
			<td>Make fresh, store refrigerated</td>
		</tr>
		<tr>
			<td>8% Glutaraldehyde</td>
			<td>EM Sciences</td>
			<td>16019</td>
			<td>Make fresh, store refrigerated</td>
		</tr>
		<tr>
			<td>Biosafety Level 2 hood</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Ultra-low freezer</td>
			<td>Various</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sakura Cassette Printer</td>
			<td>Sakura</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sakura VIP300</td>
			<td>Sakura</td>
			<td>&nbsp;</td>
			<td>Automatic paraffin processor</td>
		</tr>
		<tr>
			<td>Paraffin Embedding Station</td>
			<td>ThermoFisher</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
Table 1. Specific reagents and equipment.

collection protocol for human pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

Watch this Scientific Journal Video about Collection Protocol for Human Pancreas at JoVE.com

Collection Protocol for Human Pancreas

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Research

JoVE Journal

Medicine

14.7K 次观看.  University of Florida. 该视频演示了加工成多种存储格式的人类胰腺的解剖过程。解剖方向，保持整个胰腺地区允许区域胰岛成分和密度的定义。

视频: Collection Protocol for Human Pancreas

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

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 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;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 &mu;l 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.

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.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

A Detailed Protocol for Perspiration Monitoring Using a Novel, Small, Wireless Device

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the patients are unaware of such disorders or are having difficulty expressing their symptoms. Until now, several devices for perspiration monitoring have been developed; however, such devices tend to have a relatively large exterior, considerable power consumption, and/or less sensitivity.
Recently, we developed a small, wireless device for perspiration monitoring. The device consists of a temperature/relative humidity (T/RH) sensor, battery-driven small data logger, and silica gel as a desiccant in a small cylindrical exterior. The T/RH sensor is placed between the detection windows (through which the water vapor from the skin enters) and the silica gel. The underlying principle of the perspiration monitoring device is based on Fick&#39;s law of diffusion, which means that water vapor flux from the skin to the silica gel (i.e.&#160;transepidermal water loss and perspiration) can be captured by change in humidity at the T/RH sensor. In addition, a baseline subtraction method was adopted to distinguish perspiration and transepidermal water loss.
As shown in the previous report, the developed device can monitor the perspiration at any sites of the body in an easy, wireless manner. However, detailed methods of how to use the device have not been disclosed yet. In this article, therefore, we would like to show the point-by-point tutorials of how to use the device for perspiration monitoring, by showing the sympathetic activity test with the sympathetic skin response monitoring as an example.

Recently, we developed a small wireless device for perspiration monitoring. In this article, we present detailed protocols on how to use the device for perspiration monitoring with an example of the sympathetic activity test.

Perspiration monitoring can be utilized for the detection of certain diseases, such as thermoregulation and mental disorders, particularly when the ...

Mouse Model for Pancreas Transplantation Using a Modified Cuff Technique

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the availability of the widest range of molecular probes and reagents to perform in vivo as well as in vitro studies. Based on our experience with various murine transplantation models, we developed a heterotopic pancreas transplantation model in mice with the intent to analyze mechanisms underlying severe ischemia reperfusion injury-associated early graft damage. In contrast to previously described techniques using suture techniques, herein we describe a new procedure using a non-suture cuff technique. 
In recent years, we have performed more than 300 pancreas transplantations in mice with an overall success rate of &gt;90%, a success rate never described before in mouse pancreas transplantation. The backbone of this non-suture cuff technique for graft revascularization consists of two major steps: (I) pulling the recipient vessel over a polyethylene/polyamide cuff and fixing it with a circumferential ligature, and (II) placing the donor vessel over the everted recipient vessel and fixing it with a second circumferential ligature. The resultant continuity of the endothelial layer results in less thrombogenic lesions with high patency rates and, finally, high success rates.
In this model, arterial anastomosis is achieved by pulling the abdominal aorta of the donor graft over the everted common carotid artery of the recipient animal. Venous drainage of the graft is achieved by pulling the portal vein of the graft over the everted external jugular vein of the recipient. This manuscript provides details and crucial steps of the organ recovery and organ implantation procedures, which will allow researchers with microsurgical skills to perform the transplantation successfully in their laboratories.

Among abdominal solid organ transplantation, pancreatic grafts are prone to develop severe ischemia reperfusion injury-associated graft damage, leading eventually to early graft loss. This protocol describes a model of murine pancreas transplantation using a non-suture cuff technique, ideally suited for analyzing these early, deleterious damages.

Mouse models have several advantages in transplantation research, including easy handling, a variety of genetically well-defined strains, and the ...

Dissection of the Mouse Pancreas for Histological Analysis and Metabolic Profiling

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at the 12 codon (Ptf1acre/+;LSL-KrasG12D/+) mouse strain as a model of human pancreatic cancer. The goal of our current studies is to identify novel metabolic biomarkers of pancreatic cancer progression. We have performed metabolic profiling of urine, feces, blood, and pancreas tissue extracts, as well as histological analyses of the pancreas to stage the cancer progression. The mouse pancreas is not a well-defined solid organ like in humans, but rather is a diffusely distributed soft tissue that is not easily identified by individuals unfamiliar with mouse internal anatomy or by individuals that have little or no experience performing mouse organ dissections. The purpose of this article is to provide a detailed step-wise visual demonstration to guide novices in the removal of the mouse pancreas by dissection. This article should be especially valuable to students and investigators new to research that requires harvesting of the mouse pancreas by dissection for metabolic profiling or histological analyses.

This video article provides a detailed demonstration of the procedures required to successfully remove the pancreas from a mouse by dissection for histological analysis and metabolic profiling.

We have been investigating the pancreas specific transcription factor, 1a cre-recombinase; lox-stop-lox- Kristen rat sarcoma, glycine to aspartic acid at ...

Robotic Lateral Pancreaticojejunostomy for Chronic Pancreatitis

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The recent rise of robotic and laparoscopic pancreatic surgery has found benefits such as reduced time to functional recovery. Few studies have reported on the feasibility, technique and outcome of robotic LPJ, especially including transection of the gastroduodenal artery. The present study describes the main steps for robotic LPJ in a patient with painful chronic pancreatitis with a dilated main pancreatic duct. The patient underwent robotic LPJ. The LPJ anastomosis is performed using a running suture technique in a longitudinal side-to-side manner. Routinely, the gastroduodenal artery is transected to drain the entire length of the main pancreatic duct. The patient is in French position; 7 trocars are placed (4 robotic, 2 laparoscopic assistants, 1 liver-retractor). After docking of the robot system, the omental bursa is opened, and the right gastroepiploic artery and vein are ligated at their base at the lower border of the pancreas. Intraoperative ultrasonography is performed in order to determine trajectory of the dilated main pancreatic duct which is opened for its entire length after the gastroduodenal artery has been suture ligated both cranially and caudally from the main pancreatic duct. A Roux limb is created, and a latero-lateral PJ is fashioned using several 3-0 barbed sutures. A stapled jejuno-jejunostomy is created at sufficient distance from the pancreatic anastomosis, aided by a 50 cm suture. The described technique for robotic LPJ is a complex but feasible operation for patients with treatment refractory CP and a dilated main pancreatic duct. Due to its complexity, implementation in high volume centers with extensive experience with CP surgery may improve outcomes.

Robotic lateral pancreaticojejunostomy (RLPJ) may be used in patients with painful, morphine dependent, chronic pancreatitis and a dilated main pancreatic duct. We describe a standardized and reproducible technique for RLPJ, which includes transection of the gastroduodenal artery.

Lateral pancreaticojejunostomy (LPJ) has shown good postoperative outcomes in patients with painful, morphine dependent, chronic pancreatitis (CP). The ...

Laparoscopic Radical Left Pancreatectomy for Pancreatic Cancer: Surgical Strategy and Technique Video

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological resection of left-sided pancreatic cancer. Several surgical techniques have been described in recent years, but few were specifically designed for minimally invasive approaches. This study describes and demonstrates a standardized and reproducible technique for an adequate oncological resection of pancreatic cancer: laparoscopic radical left pancreatectomy (LRLP).
A 61-year-old woman presented with an incidental finding of a 3 cm mass in the left pancreas suspect for malignancy. Imaging did not reveal distant metastases, central vascular involvement, or morbid obesity, hence the patient was suitable for LRLP. This study describes the main steps of LRLP for pancreatic cancer. First, the lesser sac is opened by transecting the gastrocolic ligament. The splenic flexure of the colon is mobilized and the inferior border of the pancreas including Gerota&#39;s fascia is dissected down to the inferior border of the spleen. The pancreas is tunneled and hung, including Gerota&#8217;s fascia with a vessel loop. At the pancreatic neck, a tunnel is created between the pancreas and the portal vein, likewise a vessel loop is passed. The pancreas is then transected using the graded compression technique with an endostapler. Both the splenic vein and artery are transected before completing the resection. The entire specimen is extracted in a retrieval bag via a small Pfannenstiel incision.
Duration of the surgery was 210 min with 250 mL blood loss. Pathology revealed a R0-resection (&#62;1 mm) of a well-to-moderately differentiated adenocarcinoma originating from an intraductal papillary mucinous neoplasm. A total of 15 tumor-negative lymph nodes were resected. This is a detailed description of LRLP for left-sided pancreatic cancer as is currently being used within the international, multicenter randomized DIPLOMA (Distal Pancreatectomy Minimally Invasive or Open for PDAC) trial.

Oncologically safe left pancreatectomy requires radical resection (R0), Gerota&#8217;s (perirenal) fascia resection, and adequate lymph node dissection. This study describes the technical details of laparoscopic radical left pancreatectomy (LRLP), used in the first international multicenter randomized trial comparing minimally invasive with open left pancreatectomy for pancreatic cancer, the DIPLOMA trial.

Radical resection margins, resection of Gerota&#8217;s (perirenal) fascia, and adequate lymph node dissection are crucial for an adequate oncological ...

Robot-Assisted Kidney Transplantation

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed in the supine Trendelenburg position. Kidney allografts are provided by a living donor. Before vascular anastomosis, the kidney allograft is prepared by inserting a double-J stent in the ureter, and the temperature for the anastomosis is lowered by wrapping it in an ice-packed gauze. A 12 mm or 8 mm port for the robotic camera and three 8 mm ports for robotic arms are placed. A peritoneal pouch is created for the kidney allograft by raising the peritoneal flaps on both sides over the psoas muscle before dissecting the iliac vessels and bladder. A 6 cm Pfannenstiel incision is made to insert the kidney into the peritoneal pouch, lateral to the right iliac vessels. 
After clamping the external iliac vein with Bulldogs clamps, a venotomy is performed, and the graft renal vein is anastomosed to the external iliac vein in an end-to-side continuous manner with a 6/0 polytetrafluoroethylene suture. After clamping the graft renal vein, the iliac vein is declamped. This is followed by clamping of the external iliac artery, arteriotomy, arterial anastomosis with a 6/0 polytetrafluoroethylene suture, clamping of the graft renal artery, and declamping of the external iliac artery. Reperfusion is then carried out, and ureteroneocystostomy is performed using the Lich-Gregoir technique. The peritoneum is closed at a few locations with polymer locking clips, and a closed-suction drain is placed through one of the working ports. After deflating the pneumoperitoneum, all incisions are closed.

This paper provides technical details for robot-assisted kidney transplantation from a living donor.

This paper describes robot-assisted kidney transplantation (RAKT) from a living donor. The robot is docked between the parted legs of the patient, placed ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

Collection, Expansion, and Differentiation of Primary Human Nasal Epithelial Cell Models for Quantification of Cilia Beat Frequency

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary dyskinesia. However, the wider application of these techniques is limited by the extreme susceptibility of ciliary function to changes in environmental factors e.g., temperature, humidity, and pH. In the airway of patients with Cystic Fibrosis (CF), mucus accumulation impedes cilia beating. Cilia function has been investigated in primary airway cell models as an indicator of CF Transmembrane conductance Regulator (CFTR) channel activity. However, considerable patient-to-patient variability in cilia beating frequency has been found in response to CFTR-modulating drugs, even for patients with the same CFTR mutations. Furthermore, the impact of dysfunctional CFTR-regulated chloride secretion on ciliary function is poorly understood. There is currently no comprehensive protocol demonstrating sample preparation of in vitro airway models, image acquisition, and analysis of Cilia Beat Frequency (CBF). Standardized culture conditions and image acquisition performed in an environmentally controlled condition would enable consistent, reproducible quantification of CBF between individuals and in response to CFTR-modulating drugs. This protocol describes the quantification of CBF in three different airway epithelial cell model systems: 1) native epithelial sheets, 2) air-liquid interface models imaged on permeable support inserts, and 3) extracellular matrix-embedded three-dimensional organoids. The latter two replicate in vivo lung physiology, with beating cilia and production of mucus. The ciliary function is captured using a high-speed video camera in an environment-controlled chamber. Custom-built scripts are used for the analysis of CBF. Translation of CBF measurements to the clinic is envisioned to be an important clinical tool for predicting response to CFTR-modulating drugs on a per-patient basis.

This protocol describes nasal epithelial cell collection, expansion, and differentiation to organotypic airway epithelial cell models and quantification of cilia beat frequency via live-cell imaging and custom-built scripts.

Measurements of cilia function (beat frequency, pattern) have been established as diagnostic tools for respiratory diseases such as primary ciliary ...