Name: longitudinal in vivo imaging and quantification of human pancreatic islet grafting and contributing host cells in the anterior eye chamber
Uploaded: 2020-06-11T14:30:00
Description: Watch this Scientific Journal Video about Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber at JoVE.com

This study was supported by the Swedish Research Council, Strategic Research Area Exodiab, Dnr 2009-1039, the Swedish Foundation for Strategic Research Dnr IRC15-0067 to LUDC-IRC, the Royal Physiographic Society in Lund, Diabetesf&#246;rbundet and Barndiabetesf&#246;rbundet.

The Regional Ethics Committee in Lund, Sweden, approved the study according to the Act Concerning the Ethical Review of Research Involving Humans. Animal experiments were performed in strict accordance with the Swedish ethics of animal experiments and approved by the ethics committees of Malm&#246; and Lund. 6 to 8-week-old immunodeficient NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/- (NOD.ROSA-tomato.Rag2-/-) recipient mice were used as recipients for tra...

<ol>
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A method is presented to study the human pancreatic islet cell grafting process by observing the involvement of recipient and donor tissue. After a minimal invasive surgery implanting human islets into the anterior chamber of an immunodeficient mouse eye, the mouse recovers quickly within minutes after surgery. The procedure is performed on one eye. Generally, from 5&#8211;7 days postimplantation onwards the cornea is sufficiently healed to perform intravital imaging.
In this protocol, the qua...

Diabetes mellitus describes a group of metabolic diseases characterized by elevated levels of blood glucose as results of insufficient insulin production from loss or dysfunction of pancreatic islet beta cells, often accompanied by insulin resistance. Type 1 (T1D) and type 2 diabetes (T2D) are complex diseases in which the progressive dysfunction of the beta cells causes disease development. T1D is precipitated by an autoimmune attack on the beta cells, while T2D is considered to be driven by metabolic factors, albeit with increasing evidence of low-grade systemic inflammation1. Transplantation of donor human islets, particularly to T1D patients, offers the potential for providing physiological glycemic control. However, a shortage of tissue donors and poor islet engraftment has prevented islet transplantation to become a mainstream therapeutic option. A substantial proportion of the functional islet graft is lost in the immediate posttransplantation period (24&#8211;48 h) due to the hypoxic, in&#64258;ammatory, immunogenic host environment2,3. To evaluate the efficiency of intervention methods for the improvement of islet survival, continuous monitoring of such transplantations is necessary.
In vivo techniques to image and track the fate of transplanted human pancreatic islets after transplantation still remains a challenge for diabetes research4,5. To date, noninvasive imaging techniques, including positron emission tomography (PET), magnetic resonance imaging (MRI), or ultrasound (US) show potential for the quantification and functional evaluation of transplanted islets in experimental conditions5. However, given the small islet sizes, quantitative measurements by those modalities suffer from insufficient resolution. The anterior chamber of the eye (ACE) as a transplantation site for observation is a promising noninvasive imaging solution offering effectively higher spatial resolution and frequent monitoring over long time periods6. This method has been successfully exploited to study mouse islet biology (reviewed in Yang et al.7), autoimmune immune responses8, as well as human islet grafting9,10.
Here the ACE transplantation method is combined with a 2-photon imaging approach to investigate the dynamics of the human pancreatic islet engraftment process by continuous and repeated recordings on individual islet grafts for up to 10 months after transplantation. The multiphoton imaging properties of greater imaging depths and reduced overall photobleaching and photo damage overcome the imaging limitations of confocal microscopy11. Quantification of fluorescent imaging involves several stages, including islet sample preparation, islet transplantation, image acquisition, image filtering to remove islet noise or background, segmentation, quantification, and data analysis. The most challenging step is usually partitioning or segmenting an image into multiple parts or regions. This could involve separating signal from background noise, or clustering regions of voxels based on similarities in color or shape to detect and label voxels of a 3D volume that represents islet vasculature, for example. Once segmented, statistics such as object volume sizes are typically straightforward to extract. Provided is a method for the quantification and extraction of the imaging data, such as segmentation and data visualization. Particular attention is paid to the removal of autofluorescence in human islets and distinction between islet vasculature and islet capsule forming recipient cells.

<table>
	<tbody>
		<tr>
			<td>Anasthesia machine, e.g. Anaesthesia Unit U-400</td>
			<td>Agnthos</td>
			<td>8323001</td>
			<td>used for isofluran anasthesia during surgery and imaging</td>
		</tr>
		<tr>
			<td>-induction chamber 1.4 L</td>
			<td>Agnthos</td>
			<td>8329002</td>
			<td>connect via tubing to U-400</td>
		</tr>
		<tr>
			<td>-gas routing switch</td>
			<td>Agnthos</td>
			<td>8433005</td>
			<td>connect via tubing to U-400</td>
		</tr>
		<tr>
			<td>AngioSense 680 EX</td>
			<td>Percin Elmer</td>
			<td>NEV10054EX</td>
			<td>imaging agent for injection, used to image blood vessels in human islet grafts</td>
		</tr>
		<tr>
			<td>Aspirator tubes assemblies</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>connect with pulled capillary pipettes for manual islet picking</td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic) 0.3mg/ml</td>
			<td>Schering-Plough Europ&#233;</td>
			<td>64022</td>
			<td>fluid, for pain relief</td>
		</tr>
		<tr>
			<td>Capillary pipettes</td>
			<td>VWR</td>
			<td>321242C</td>
			<td>used together with Aspirator tubes assemblies</td>
		</tr>
		<tr>
			<td>Dextran-Texas Red (TR), 70kDa</td>
			<td>Invitrogen</td>
			<td>D1830</td>
			<td>imaging agent for injection</td>
		</tr>
		<tr>
			<td>Eye cannula, blunt end , 25 G</td>
			<td>BVI Visitec/BD</td>
			<td>BD585107</td>
			<td>custom made from Tapered Hydrode lineator [Blumenthal], dimensions: 0.5 x 22mm (25G x 7/8in) (45&#8304;), tip tapered to 30 G (0.3mm)</td>
		</tr>
		<tr>
			<td>Eye gel</td>
			<td>Novartis</td>
			<td></td>
			<td>Viscotears, contains Carbomer 2 mg/g</td>
		</tr>
		<tr>
			<td>Hamilton syringe 0.5 ml, Model 1750 TPLT</td>
			<td>Hamilton</td>
			<td>81242</td>
			<td>Plunger type gas-tight syringe for islet injection</td>
		</tr>
		<tr>
			<td>Head holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Head holding adapter</td>
			<td>Narishige</td>
			<td>SG-4N-S</td>
			<td>assemled onto metal plate</td>
		</tr>
		<tr>
			<td>-gas mask</td>
			<td>Narishige</td>
			<td>GM-4-S</td>
			<td></td>
		</tr>
		<tr>
			<td>-UST-2 Solid Universal Joint</td>
			<td>Narishige</td>
			<td>UST-2</td>
			<td>assemled onto metal plate</td>
		</tr>
		<tr>
			<td>-custom made metal plate for head-holder assembly</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Dumont #5, straight</td>
			<td>Agnthos</td>
			<td>0207-5TI-PS or 0208-5-PS</td>
			<td>attached to UST-2 (custom made)</td>
		</tr>
		<tr>
			<td>Heating pad, custom made</td>
			<td></td>
			<td></td>
			<td>taped to the stereotaxic platform</td>
		</tr>
		<tr>
			<td>Human islet culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-CMRL 1066</td>
			<td>ICN Biomedicals</td>
			<td></td>
			<td>cell culture media for human islets</td>
		</tr>
		<tr>
			<td>-HEPES</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-L-glutamin</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Gentamycin</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Fungizone</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Ciproxfloxacin</td>
			<td>Bayer healthcare AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Nicotinamide</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Bitplane</td>
			<td></td>
			<td>Imaris 9</td>
		</tr>
		<tr>
			<td>Image Aquisition software</td>
			<td>Zeiss</td>
			<td></td>
			<td>ZEN 2010</td>
		</tr>
		<tr>
			<td>Infrared lamp</td>
			<td>VWR</td>
			<td>1010364937</td>
			<td>used to keep animals warm in the wake-up cage</td>
		</tr>
		<tr>
			<td>Isoflurane Isoflo</td>
			<td>Abott Scandinavia/Apotek</td>
			<td></td>
			<td>fluid, for anesthesia</td>
		</tr>
		<tr>
			<td>Needle 25 G (0.5 x 16mm), orange</td>
			<td>BD</td>
			<td>10442204</td>
			<td>used as scalpel</td>
		</tr>
		<tr>
			<td>Petri dishes, 90mm</td>
			<td>VWR</td>
			<td>391-0440</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Photon/confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-LSM7 MP upright microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Ti:Sapphire laser Tsunami</td>
			<td>Spectra-Physics, Mai Tai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-long distance water-dipping lens 20x/NA1.0</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-ET710/40m (Angiosense 680)</td>
			<td>Chroma</td>
			<td>288003</td>
			<td></td>
		</tr>
		<tr>
			<td>-ET645/65m-2p (TR)</td>
			<td>Chroma</td>
			<td>NC528423</td>
			<td></td>
		</tr>
		<tr>
			<td>-ET525/50 (GFP)</td>
			<td>Chroma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-ET610/75 (tomato)</td>
			<td>Chroma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-main beam splitter T680lpxxr</td>
			<td>Chroma</td>
			<td>T680lpxxr</td>
			<td>Dichroic mirror to transmit 690 nm and above and reflect 440 to 650 nm size 25.5 x 36 x 1 mm</td>
		</tr>
		<tr>
			<td>Polythene tubing (0.38mm ID, 1.09 mm OD)</td>
			<td>Smiths Medical Danmark</td>
			<td>800/100/120</td>
			<td>to connect with Hamilton syringe and eye canula</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Model SMZ645, for islet picking</td>
		</tr>
		<tr>
			<td>Stereomicroscope (Flourescence)</td>
			<td></td>
			<td></td>
			<td>for islet graft imaging</td>
		</tr>
		<tr>
			<td>-AZ100 Multizoom</td>
			<td>Nikon</td>
			<td></td>
			<td>wide field and long distance</td>
		</tr>
		<tr>
			<td>-AZ Plan Apo 1x</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-AZ Plan Apo 4x</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-AZ-FL Epiflourescence with C-LHGFI HG lamp</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-HG Manual New Intensilight</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block TEXAS RED</td>
			<td>Nikon</td>
			<td></td>
			<td>contains EX540-580, DM595 and BA600-660</td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block G-2A</td>
			<td>Nikon</td>
			<td></td>
			<td>(EX510-560, DM575 and BA590)</td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block B-2A</td>
			<td>Nikon</td>
			<td></td>
			<td>(EX450-490, DM505 and BA520)</td>
		</tr>
		<tr>
			<td>-DS-Fi1 Colour Digital Camera (5MP)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1-ml, Omnitix</td>
			<td>Braun</td>
			<td>9161406V</td>
			<td>for Buprenorphine injection, used with 27 G needle</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal in vivo imaging and quantification of human pancreatic islet grafting and contributing host cells in the anterior eye chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

Non-labeled human islets were transplanted into the ACE of 8-week-old female NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-(NOD.ROSA-tomato.Rag2&#8722;/&#8722;) recipient mice. To prevent human tissue rejection, immunodeficient Rag2 knockout mice were chosen as recipients. In these transgenic mice, all cells and tissues expressed a membrane-targeted tomato fluorescence protein (mT) that allows clear identification of the recipient and the donor tissue. Repe...

Watch this Scientific Journal Video about Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber at JoVE.com

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

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