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

Summary

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)26Sor^tm4-Rag2^-/-mouse recipient followed by repeated 2-photon imaging.

Abstract

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)26Sor^tm4-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.

Introduction

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 (24–48 h) due to the hypoxic, inflammatory, immunogenic host environment^2,3. 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 research^4,5. 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 grafting^9,10.

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.

Protocol

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 Malmö and Lund. 6 to 8-week-old immunodeficient NOD.(Cg)-Gt(ROSA)26Sor^tm4-Rag2^-/- (NOD.ROSA-tomato.Rag2^-/-) recipient mice were used as recipients for transplantation of human islets¹⁰.

1. Islet preparation for transplantation

1. Culture human islets in CMRL 1066 supplemented with 10 mM HEPES, 2 mM L-glutamine, 50 μg/mL gentamycin, 0.25 μg/mL fungizone, 20 μg/mL ciproxfloxacin, 10 mM nicotinamide (NIC), and 10% heat-inactivated human serum at 37 °C in 5% CO₂ and humidified air until transplantation, as described previously¹².
   NOTE: Islets should be free of exocrine tissue and not touch each other in culture. Exocrine tissues appear translucent.
2. On the day of transplantation, transfer culture media containing the islets to a new Petri dish using an aspirator tube assembly connected to a pulled glass capillary.
   NOTE: Alternatively, use a 200 µL pipette. Coloring the back of the Petri dish helps make the islets more easily distinguishable under the stereo microscope.
3. Using a stereo microscope, pick ~20–40 islets per transplantation and transfer to a 1.5 mL tube. Fill the tube to the top with culture media from the incubator.
4. Seal the tubes with paraffin film and store on ice until transplantation. Prepare an appropriate amount for the number of transplantations performed.
5. Alternatively, ensure a CO₂ incubator is available in the surgery room to keep islets in culture and pick them immediately prior to each transplantation.

2. Preparation of transplantation equipment and surgery table

NOTE: All surgical tools should be autoclaved, and the surgery table and instruments disinfected with 70% alcohol.

1. Connect a stereotaxic head holder to anesthesia via a nose mask and turn on the heating pad.
2. Connect a gastight Hamilton syringe to polyethylene tubing and a blunt end eye cannula.
   NOTE: It is recommended to fill all parts with PBS before assembly. Check for trapped air bubbles and remove if present.
3. Attach the Hamilton syringe tightly to the table (Figure 1a) or a movable base (Figure 1e) and attach the tubing to the stereo microscope, with cannula hanging down (i.e., waiting position).
   NOTE: Use surgical tape, because it is easy to remove and reattach.
4. Prepare a 1 mL syringe connected to a 30 G needle filled with 0.1 mg/kg buprenorphine solution.
5. Prepare a syringe with sterile PBS. Alternatively, use a pipette.
6. Set aside a clean wake-up cage with heating lamp.

3. Anesthesia and positioning of recipient mice for surgery

NOTE: All animals were bred and maintained in a pathogen-free environment at the animal facilities at Lund University.

1. Anesthetize the mouse in a chamber filled with 40% O₂/60% N₂/3% isoflurane and transfer the anesthetized mouse to the head holder platform on a warm heating pad (Figure 1a). Check for the lack of pedal reflexes.
   NOTE: Isoflurane anesthesia is the preferred method of anesthesia for fast recovery after surgery. The microscope room must be properly ventilated to use isoflurane.
2. Place the snout of the mouse into the anesthesia mask connected to 40% O₂/60% N₂/0.9%–1.5% isoflurane anesthesia machine. Use the thumb and finger to lift the head up slightly and fasten it using the metallic pieces on the sides. Ensure that the earpieces fix the head directly below the ears. Inject 0.1 mg/kg buprenorphine solution subcutaneously on the back of the mouse.
   NOTE: Buprenorphine is used as an analgesic.
3. Tilt the head so that the eye to be operated on is facing upwards and is close to the researcher.
4. Gently retract the eyelids of the eye to be transplanted using blunt forceps, pop the eye out, and loosely fix with a pair of tweezers. Ensure that the tips of the tweezers are covered with a polythene tube attached to the head holder platform (Figure 1a, insert).
5. Always keep both eyes wet by applying a droplet of sterile PBS onto the eye.
6. Transfer the human islets from the sealed 1.5 mL tube (section 1) to a Petri dish with sterile PBS and make sure that the islets are close to each other to minimize the amount of cell culture media transferred (Figure 1c).
7. Pick up ~20–30 islets in the eye cannula connected via polythene tubing to the Hamilton syringe.
   NOTE: Take up as little liquid as possible with the islets.
8. Hang the tubing upside down and attach to the stereo microscope (Figure 1d). Tape the tubing carefully to let the islets sink to the end of the tube toward the cannula.

4. Transplantation procedure

NOTE: This method has been previously described for the transplantation of mouse islets⁶. A slightly modified procedure is presented here.

1. Pinch the pads on the hind legs to make sure that the mouse is asleep.
2. Tighten the forceps restraining the eye without disrupting the blood flow and apply a droplet of sterile PBS onto the eye.
3. Using a 25 G needle as a scalpel, bevel upwards, carefully penetrate only half of the tip in the cornea and make a single lateral incision. Make the hole in an upward angle; the hole will seal more easily after the transplant (Figure 1f).
4. Carefully lift up the cornea with the cannula preloaded with islets and slowly apply islets in the eye. Avoid insertion of the cannula into the anterior chamber to prevent damage of the iris, but rather push carefully against the corneal opening (Figure 1g).  Slowly retract the cannula from the ACE.
   NOTE: Aim for an injection volume of 3–8 µL. If the volume is too large, it will expose the eye to unnecessarily high intraocular pressure and may result in reflux of the injected islets out of the anterior chamber.
5. When facing difficulties with insertion of the islets due to increased pressure in the eye chamber, enlarge the incision site by reinforcing the lateral incision site and reapply islets.
   NOTE: Occasionally, introduced air bubbles can be used as space holders.
6. Apply eye gel to the eye, loosen the eye-restraining forceps and leave the mouse on isoflurane in the same position for 8–10 min to let the islets set.
7. Remove the forceps holding the eyelid and put the eyelid back to its normal position.
8. Remove the mouse from the head holder and transfer it to a wake up cage.
9. When the mouse is awake and moving, transfer it back to the original cage and keep in the animal housing until scanning (at least 5 days are recommended).

5. Imaging of implanted human islets by 2-photon microscopy

NOTE: Taking overview images of the eye using a fluorescence stereoscopic microscope (Figure 2a–c) 4–5 days after transplantation prior to 2-photon imaging is recommended to localize the islets of interest. Avoid restraining the eye too tightly this early after transplantation. Use 2-photon imaging 6–7 days posttransplantation.

1. Start the image acquisition software (see Table of Materials). In the “Laser” menu activate the Mai Tai laser (Power “ON”) and in the “Light Path” menu set the wavelength to 900 nm and apply a minimal transmission laser power starting with 5%–10% laser power (use sliders).
   NOTE: While scanning, adjust the laser power as needed.
2. Set green, orange, and red channels. Collect emission light simultaneously onto three nondescanned detectors (NDD) using a dichronic mirror (LBF 760) and emission filter information as follows: Red/Angiosense 680, 690–730 nm; Green/Autofluorescence, 500–550 nm; and Orange/Tomato, 565–610nm (Figure 2d).
3. Place the head holder stage onto the motorized microscope stage and connect the gasmask to the tubing of the anesthesia machine and tubing connected to ventilation system. Turn on the heating pad.
4. Anesthetize the recipient mouse, transfer to the head holder platform, restrain the eye for imaging, and administer buprenorphine solution as described above (steps 3.1–3.5).
5. Adjust isoflurane vapors as needed. A breath rate of ~55–65 breaths per minute (bpm) indicates optimal anesthesia. If anesthesia is too deep, the rate will be <50 bpm with heavy breathing or gasping; if too light, the rate will be >70 bpm with superficial breathing. Carefully monitor mice during anesthesia by visual inspection every 15 min.
   NOTE: Anesthetization varies from mouse to mouse, between mouse strains, and as time under anesthesia progresses¹³.
6. Administer enough eye gel onto the eye as an immersion liquid between the cornea and the lens, allowing it to slowly accumulate (Figure 2f, insert). Avoid air bubbles.
   NOTE: Side illumination with a flexible metal hose lamp is recommended to adjust the focus and localize islet grafts.
7. To visualize blood vessels, administer 100 µL of the imaging agent (e.g., Angiosense 680) intravenously into the tail vein using a disposable insulin 30 G syringe.
8. In “Acquisition mode” adjust the frame size to 512 x 512 and the scan speed.
   NOTE: Slower scans (i.e., increasing dwell time) will improve the signal-to-noise ratio.
9. In the “Channels” menu adjust Master Gain for each PMT in Volts to amplify the signal until an image is seen on screen in the Live scanning mode. The higher this value, the more sensitive the detector becomes to signal and noise.
   NOTE: Preferably, keep values between 500–800 V.
10. In the “Z-stack” menu define the beginning and the end of the z-stack by manually moving the focus to the top of the islet graft. Save position by selecting “Set First”. Move to the last bottom plane that can be focused in the islet graft and save position by selecting “Set Last”. Use a z-step size of 2 µm.
11. Collect the final image stack by clicking the “Start Experiment” tab and save as 8-bit CZI (i.e., Carl Zeiss format) file.

6. Imaging of implanted human islets by confocal microscopy

NOTE: The total volume, morphology, and plasticity of transplanted islets can be assessed by monitoring the in vivo scattering signal in a separate scan (i.e., separate track) by detection of laser backscatter light¹⁰.

1. Take out the main beam splitter (i.e., LBF Filter) and in the “Light Path” dialog set up a separate track for confocal imaging. Choose the Argon laser with wavelength of 633 nm and detection at the same wavelength as the laser light. Z-stacks are acquired with a step size of 2–3 µm for backscatter light signal.
2. Readjust the z-stack settings to make sure to record the whole islet (see step 5.10).
3. Acquire the image stack and save as 8 bit CZI file.

7. Image analysis

NOTE: Commercial software (see Table of Materials) was used for this step.

1. Removing islet autofluorescence (Figure 3b)
   1. In the “Image processing” tab choose “Channel arithmetic´s” and type “ch1-ch2”. This creates a new channel 4 (ch 4); rename as “Vasculature”.
      NOTE: The Green/Autofluorescence channel is subtracted from Red/Angiosense channel.
   2. Repeat the previous step and type “ch3-ch2” to create a new channel (ch 5); rename as “Tomato (all)”.
      NOTE: The Green/Autofluorescence channel is subtracted from the Orange/Tomato channel.
2. Defining islet mask by manual drawing (Figure 3c)
   1. Create a new surface (blue symbol) and in the wizard choose “Edit manually”. Keep the pointer in “Select” mode and in 3D view unclick “Volume” (under Scene) to visualize sections.
   2. For easier islet border discrimination, activate all channels, including ch 1–ch 3.
      NOTE: The Orange/Tomato channel is useful to define islet borders by the Tomato capsule signal. Alternatively, increase channel intensities to use multichannel islet autofluorescence and detector background signal as guidance.
   3. In the “Drawing” tab choose “Contour” and click “Draw” to start drawing contours around the islet border starting in slice position 1.
   4. Move to a new slice position and repeat drawing contours. Finish with the last slice on the top of the islet and end by clicking the “Create surface” tab. Usually it is enough to draw contours every 10^th slice.
3. Segmentation of “Islet vasculature” and “Islet tomato” fluorescence using islet mask (Figure 3d).
   1. Choose the previously defined “Islet mask” object, go to the editing tab (pencil symbol), and click the “Mask all” tab, which opens a new window.
   2. Choose the previously named channel “Vasculature” (ch 4) in the channel selection dropdown menu and activate options “Duplicate channel before applying mask”, “Constant inside/outside”, and set voxels outside surface to “0.000”, which creates a new channel; rename as “Islet vasculature” (ch 6).
   3. Repeat steps 7.3.1 and 7.3.2 and choose the previously created channel “Tomato (all)” (ch 5) in the channel selection drop down menu to create the new channel; rename as “Islet tomato” (ch 7).
4. Surface rendering of islet vasculature (Figure 3e)
   1. Create a new surface in the “Scene” menu and in the wizard choose “Automatic creation”.
   2. Set the source channel to previously created “Islet vasculature” (ch 6) and choose background subtraction. Automatic threshold estimation can be adjusted if needed. Compare to the responding fluorescence channel (e.g., by blending in/out the newly created surface tab). Proceed in the wizard.
   3. Optionally, use filters. For example, choose “Volume” and adjust the filter (yellow) in the window, which can remove selected surface objects. Finish the wizard and name the new surface object “Islet vasculature”.
5. Segmentation of “Islet tomato vasculature” fluorescence signal (Figure 3f)
   1. In the previously created “Islet vasculature” surface object, go to editing tab and click the “Mask all” tab, which opens a new window.
   2. Choose the previously named channel “Islet tomato” (ch 7) in the channel selection drop down menu and set voxels outside surface to “10.000”, which creates the new channel; rename as “Islet tomato vasculature” (ch 8).
6. Segmentation of “Tomato capsule” fluorescence signal (Figure 3g)
   1. Choose “Channel Arithmetic’s” in the “Image processing” tab and type “ch7-ch8”, creating the new channel; rename as “Tomato capsule” (ch 9).
      NOTE: The “Islet tomato vasculature” fluorescence signal is subtracted from the total “Islet tomato” fluorescence signal.
7. Surface rendering of “Islet tomato vasculature” and “Tomato capsule” (Figure 3h)
   1. Follow step 7.4, and in the wizard choose source channels “Islet tomato vasculature” (ch 8) or “Tomato capsule” (ch 9) to create new surface objects accordingly.
8. Surface rendering of total islet surface (Figure 3i)
   1. Open the islet backscatter file and create a new surface.
   2. In the wizard, choose “Automatic creation” and define “Region of interest”.
      NOTE: "Region of interest" is used to separate the signals of multiple islets and to define the depth of the islet to be analyzed (e.g., top 75 µm).
   3. In “Absolute intensity” adjust threshold if needed. The surface object can be clicked on or off to cross-check with corresponding channel intensity. Close the wizard.
9. Quantification (Figure 3j)
   1. Select a created surface object in the “Scene” menu and go to the “Statistics” tab.
   2. To retrieve detailed volume data in the selected surface object choose the “Detailed” tab and select “Specific values” and “Volume” from dropdown menu. To retrieve a total volume value of the selected surface object, go to the “Detailed” tab and choose “Average values”.

Results

Non-labeled human islets were transplanted into the ACE of 8-week-old female NOD.(Cg)-Gt(ROSA)26Sor^tm4-Rag2^-/-(NOD.ROSA-tomato.Rag2^−/−) 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. Repeated imaging...

Discussion

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 5–7 days postimplantation onwards the cornea is sufficiently healed to perform intravital imaging.

In this protocol, the qua...

Materials

List of materials used in this article

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