New Islet Model for 3D Study of Endothelial and β Cell Interactions: Relevance for the Screening of Cytoprotective Agents

Summary

This protocol provides a detailed procedure to generate stable spheroids of murine pancreatic islets, and detailed methods for 3D staining, clearing, and mounting, adapted to their small size.

Abstract

This protocol details an optimized method for the production of small stable spheroids, their culture, and 3D imaging, for the study of the endothelial and insulin-producing β cells interactions in a 3D model of pancreatic islets. The 150-200 µm spheroids, mirroring the lowest range of islet sizes, were prepared from a selected ratio combining 1 intra-islet endothelial cells (MS-1 cells) to 20 insulin-secreting cells (β-TC-6). Staining, clearing, and mounting challenges of small spheroids and their tackling by employing low-melting point agarose and the CUBIC clearing technique are detailed, as well as key points for an efficient analysis of the 3D structure with different probes. Data indicate that NTPDASE-ectonucleotidase 3 does not colocalize with insulin in the spheroid model, suggesting varying maturity and functional levels of β-TC6 and that the complete procedure can also be applied to isolated pancreatic islets, with clear probing of intra-islet vessels. These findings underscore the effectiveness of the 3D imaging protocol in revealing complex pancreatic cell organization and interactions within the islet model.

Introduction

Pancreatic islet transplantation is often complicated by instant blood-mediated inflammatory reaction (IBMIR), which significantly damages the intra-islet endothelial cells (IEC) with an impact on graft perfusion and function. In the long run, a reduced mass of functional islets and the loss of insulin independence are described in patients, 4 years post-transplantation¹. While murine or rat models of islet transplantation have been developed, standardized models to investigate pharmacological targets for the pre-conditioning of islets, of particular relevance in the context of cytokine-driven graft damage induced by the IBMIR are lacking². Indeed, a spheroid construct is particularly suitable for the identification of new pharmacological targets and the evaluation of cytoprotective molecules against cytokine-induced cell dysfunction. Furthermore, it offers an alternate approach to limit the use of animal models of type 1 diabetes and islet transplantation.

Finding appropriate staining, optical clearing, and mounting conditions for small spheroids of an islet size range remains challenging. In the literature, very few studies describe the handling of small spheroids and their key structural characteristics. The difficulty also arises from the need to balance tissue penetration, signal preservation, and structural integrity while maintaining the delicate nature of these miniature 3D cell structures. In addition, conventional staining methods often struggle to achieve uniform labeling of spheroids. This issue is exacerbated by the dense cellular organization, which can hinder the diffusion of reagents because of the limited penetration of antibodies and dyes^3,4. Finally, the process of optical clearing, crucial for reducing the light scattering background and improving imaging depth, must be carefully optimized to avoid disrupting the structure of the spheroid or altering biological properties⁵.

Mounting small spheroids for 3D microscopy presents another challenge. Traditional mounting media may not provide adequate support for these delicate structures, potentially leading to their deformation or collapse during imaging. Furthermore, the refractive-index matching required for an optimal imaging quality must be precisely controlled to ensure accurate visualization throughout the depth of the spheroid^6,7. The development of protocols tailored to small spheroids is further complicated by the diversity of cell types and experimental conditions used in spheroid cultures³. Finally, the need for specialized approaches to maintain the integrity of multiple cell populations throughout the staining and clearing processes is critical for the assessment of cell responses.

To address the specific challenges of the study of IBMIR-mediated islet stress, a 2000 cell spheroid model was designed, combining murine IEC and insulin secreting β cells, with a 1:20 ratio of IEC to β cells similar to that observed in a pancreatic islet. In addition, previous experiments established that the structure maintains viability for 96 h, thus providing a physiologically relevant 3D model for the observation of short (hour range) or longer stress-induced cell responses (days) and their pharmacological modulation. The spheroid model is constituted of MS-1 murine intra-islet endothelial cells, characteristics of the islet microvasculature, and constitutive insulin secreting β cells, β-TC-6⁸. It was designed to closely mimic the smallest dimension range of pancreatic islets (50-200 µm in diameter) and their average cell number⁹.

A novel harvesting and staining methodology specifically optimized for small spheroids handling (150-200 µm in diameter) is reported, enabling optimal penetration of fluorescent dyes and antibodies throughout the spheroid structure and minimal background. The protocol incorporates a mounting procedure utilizing low melting point agarose (LMPA) to provide gentle structural support for delicate spheroids while allowing for optimal refractive index matching. LMPA is a modified form of agarose with reduced gelling (about 27 °C) and melting temperature (about 65 °C) that preserves sample integrity during embedding, and creates an optically clear matrix with minimal autofluorescence^4,6. Finally, the clear unobstructed brain/body imaging cocktails and computational analysis (CUBIC)¹⁰ method is employed for optical clearing to enable maximum visualization of internal structures and preservation of the integrity of delicate cell-cell interactions^5,6,10.

Protocol

The use of human pancreatic tissue and all experimental protocols was approved by the Ministry of Higher Education and Research of the French government (Directorate General for Research and Innovation, Bioethics Unit) and registered under number DC-2019-3439. Informed consent was obtained from all subjects' legal guardians.

1. Spheroid preparation

NOTE: The murine IEC line MS-1 and murine β-pancreatic cell line β-TC6 were cultured simultaneously to obtain a sufficient cell quantity for spheroid culture. MS-1 were cultured in T75 adherent-cells flasks in DMEM High Glucose medium containing 4.5 g/L glucose, with 5% fetal bovine serum and 1% Penicillin/Streptomycin in a humid atmosphere at 37 °C. Passages were performed at confluency (2 days culture) after detachment using Trypsin EDTA 1x and three washes in Hank Balanced solution (HBSS). β-TC6 were cultured in T75 for difficult adherent-cells in DMEM High Glucose medium containing 4.5 g/L glucose, with 15% fetal bovine serum and 1% Penicillin/Streptomycin in a humid atmosphere at 37 °C. Passages were performed at β-TC6 confluency (10 days culture) after detachment by Trypsin EDTA 1x and 3 washings in Hank Balanced solution (HBSS).

1. Prepare the spheroid-culture plate
2. Wash the plate with 500 µL of anti-adherence rinsing solution provided by the manufacturer to prevent cell adhesion and to promote spheroid formation.
3. Centrifuge the plate at 1,300 x g for 5 min in a swinging bucket rotor fitted with plate holders at room temperature.
4. Observe the plate under a light microscope to ensure that no bubbles remain in the microwells. If so, centrifuge a second time at 1,300 x g for 5 min.
5. Discard anti-adherence rinsing and wash the well 3 times with 1 mL of warm HBSS.
6. Discard HBSS and replace with 500 µL of warm spheroid medium: DMEM High Glucose containing 4.5 g/L glucose, 10% fetal bovine serum, and 1% penicillin/streptomycin.
7. Keep the plate at 37 °C in a humid atmosphere while preparing the spheroid suspension.
8. Incubate MS-1 and β-TC6 simultaneously with trypsin solution and count the living cells in each lineage.
   NOTE: At seeding, the spheroids are formed using the 1 IEC to 20 β cell ratio typical of pancreatic islets. Use a 24-well spheroid-culture plate, with each well containing 300 microwells that could harbor one spheroid composed of 2000 cells, for a total of 600,000 cells per well. In the present investigation, measurements revealed identical spheroid volumes occupied by the selected β-TC6 and MS1 cell lines after the 48h spheroid culture.
9. Mix the two cell lines together to correspond to 1 to 20 ratio for 24 wells and then suspend them in 24 mL of spheroid medium.
   NOTE: Use a 10 mL or 25 mL pipette in suction-flow motion to homogenize the suspension.
10. Pipette 1 mL for each well and proceed in a suction-flow motion to distribute the cell suspension evenly in the well.
11. Centrifuge the plate at 130 x g for 5 min in a swinging bucket rotor fitted with plate holders at room temperature.
12. Observe the plate under a microscope to ensure that the cells are distributed equally among the microwells.
    NOTE: During this centrifugation step, use high acceleration and deceleration rates (70%-90% of the acceleration and deceleration scales), or the cell clumps will shift to the side of the microwell.
13. Incubate the plate at 37 °C, 5% CO₂.
14. Monitor spheroid formation and growth using a light microscope.
    NOTE: β-TC6 / MS-1 spheroids should begin to form after 24 h of culture, achieving a sphere shape after 48 h. Viability can be monitored by trypan blue after spheroid disruption by trypsin in duplicated samples and was maintained over 96 h (80%-90%).

2. Spheroid harvesting and staining

1. Resuspend the spheroids gently with a P1000 pipette in their medium.
2. Eject the spheroids into small volumes in a 15 mL Falcon and centrifuge at 130 x g for 5 min at room temperature
3. Gently aspirate the medium while avoiding the pellet containing the spheroids.
4. Wash the pellet twice with 2 mL of HBSS, gently, to avoid any dissociation of the spheroids, and centrifuge at 130 x g for 5 min.
5. Under a chemical hood, discard the HBSS and resuspend the spheroids in 1 mL of 4% PFA solution overnight at 4 °C to fixate.
6. Centrifuge the tube at 130 x g for 5 min, discard the 4% PFA solution, and wash 3 times with 2 mL of 1x PBS (prepared in laboratory-grade ultrapure water).
7. Resuspend in 2.5 mL of HBSS.
8. Dispense the spheroid suspension into a 6-well plate.
9. Under a binocular magnifying glass, handpick 20 to 30 spheroids with a P200 pipette and place them in a 1.5 mL tube.
   NOTE: Each 1.5 mL tube is a staining condition containing 20 to 30 spheroids. Below 20 harvested spheroids, it becomes difficult to see them when they are subsequently resuspended.
10. Equilibrate the volumes of the 1.5 mL tubes and centrifuge at 800 x g for 5 min.
    NOTE: From this point on, all the solution aspiration steps are performed with the aid of a magnifying glass. It is very difficult to see spheroids under artificial light. Always perform the following steps during the daytime, with the tubes held against the daylight. All solutions are aspirated with a P1000 tip topped with a P10 tip at its end.
11. Remove as much HBSS as possible without resuspending the spheroids.
12. Resuspend the spheroids in 400 µL of quenching solution (0.5 M glycine in 1x PBS (pH 7-7.4)) to avoid any future background noise caused by remaining aldehydes from the PFA fixation. Incubate at 37 °C for 2 h.
13. Centrifuge at 800 x g for 5 min and pipette off the quenching solution.
14. Resuspend in 400 µL of penetration buffer (0.2% Triton X-100, 0.3 M glycine, 20% DMSO in 1x PBS). This will help improve the penetration of antibodies and nuclear probes. Incubate at 37 °C for 2 h.
    NOTE: From this stage onwards, spheroids settle on their own and no longer cling to the tube wall. Centrifugation is no longer necessary if volumes are gently aspirated without disturbing the spheroid pellet. If necessary, centrifuge at 800 x g for 5 min.
15. Discard the penetration buffer and incubate in 400 µL of blocking buffer (0.2% Triton X-100, 1% BSA, 10% DMSO, 5% Goat serum in 1x PBS) overnight at 37 °C.
16. Discard the blocking buffer and incubate overnight at 37 °C in 300 µL of primary antibody solution (0.2% Tween 20, 1% BSA, 5% DMSO, 5% Goat serum in 1x PBS).
    NOTE: Used primary antibodies are: anti-CD31 (final concentration of 2.2 µg/mL), anti-Integrin β1 (1.43 µg/mL), anti-Insulin (0.66 µg/mL), and anti-mouse NTPDase3¹¹ (dilution 1:1000) (see Table of Materials). Control IgGs of respective identical isotypes should be validated on the same structure to ascertain positive labeling by fluorescent microscopy.
17. Discard the primary antibody solution and wash 3 times with 400 µL of washing buffer (0.2% Tween 20, 1% BSA in 1x PBS). Shake for 5 min between washes.
18. Incubate 300 µL of the secondary antibody solution (DAPI (2 µg/mL), 0.2% Tween 20, 1% BSA, 5% DMSO, 5% Goat Serum in 1x PBS) overnight at 37 °C. Protect the tubes with aluminum foil.
    NOTE: DAPI should always be added to the staining conditions, as this makes it easier to find spheroids during microscopy imaging and helps not to bleach the different fluorophores/dyes. All secondary antibodies have a final concentration of 2 µg/mL (see Table of Materials).
19. Discard the secondary antibody solution and wash 3 times with 400 µL of a second washing buffer: 0.2% Tween 20, 1% BSA in 1x PBS. Keep the last wash and store the tubes at 37 °C till mounting the same day.

3. Solution preparation

1. Optical clearing solution preparation
   NOTE: The optical clearing solution follows the CUBIC¹⁰ procedure, and was prepared as proposed by Gunasingh et al⁴. Component proportions are given in percentages of the final solution weight (w/w), because Quadrol/N,N,N′,N′-Tetrakis(2-Hydroxypropyl) ethylenediamine is too viscous to pipette and must be weighed.
   1. In a glass bottle, weigh 9% (w/w) of N,N,N′,N′-Tetrakis(2-Hydroxypropyl) ethylenediamine, 22% (w/w) of urea, 44% (w/w) of sucrose, 0.1% (w/w) of Triton X-100, and 24.9% (w/w) of laboratory-grade ultrapure water.
      NOTE: About 60 g of solution is necessary to clear a 24-well plate.
   2. Add a magnetic bar adapted to the diameter of the bottle.
   3. Heat and stir the solution to 56 °C. Keep a control glass bottle filled with water on the same plate to monitor temperature rise.
   4. Control the stirring speed to maintain a smooth liquid vortex and stir the solution until all compounds are completely dissolved. The solution should be completely liquid and clear. This may take up to 1 h.
   5. Keep at room temperature, protected from the light.
      NOTE: The clearing solution must be prepared at least 24 h before optical clearing, to allow the solution to degas and eliminate any bubbles it may contain. To avoid introducing bubbles, never shake the solution again. Never store in cold storage.
2. 2% LMPA solution preparation
   1. Weigh 0.5 g of LMPA on a precision balance and place it in a 100 mL Erlenmeyer flask.
      CAUTION: Do not take a smaller volume Erlenmeyer, as there is a risk of burns if it overflows.
   2. Dissolve in 25 mL of 1x PBS at room temperature.
   3. Thoroughly incorporate the powder into the 1x PBS by gently stirring the Erlenmeyer flask.
      CAUTION: For the next step, it is essential to use protective equipment: gloves and a mask for the eyes and face, to protect against splashes and burns.
   4. Heat the Erlenmeyer flask in the microwave.
   5. Observe when the solution starts to boil (presence of bubbles in the liquid), and remove immediately before it boils over.
   6. Gently stir the Erlenmeyer to incorporate the powder.
      CAUTION: Do not stir too hard, as the liquid may overflow/over-boil and splash.
   7. Return the Erlenmeyer to the microwave, repeat until the LMPA is completely dissolved.
   8. Remove the Erlenmeyer and, before the LMPA solidifies, quickly aliquot 1 mL of 2% LMPA solution into 1.5 mL tubes. Store at room temperature.
      NOTE: The process is time-consuming. Prepare the solution in advance and store it for later use.

4. Mounting and optical clearing

1. Preheat the plate centrifuge to 40 °C, running a long cycle to prepare the sample plate.
   NOTE: LMPA gels at 30 °C. Do not run the centrifuge below 40 °C. If the centrifuge allows it, increase to 50 °C.
2. Liquefy aliquots of 2% LMPA in a water bath or tube heater at 70 °C for 5 to 10 min.
3. Lower and maintain the temperature of the 2% LMPA at 55 °C.
4. Place the imaging plate gently on the plate warmer preheated to 50 °C.
   NOTE: Be careful not to break or scratch the fragile polymer used as a base for the wells.
5. Be careful that the plate is hot before adding the 2% LMPA. It must not gel at this stage.
6. Pipette 200 µL of 2% LMPA into each well. Cover the entire bottom of the support to obtain a first liquid base, which allows a more fluid distribution of the spheroids.
7. Discard the washing buffer from the tubes, resuspend the spheroids in 300 µL of 2% LMPA, and pipette them directly into their well. Always perform gentle resuspension to avoid bubbles.
8. Cover the plate with aluminum foil to protect the fluorophores.
9. Quickly weigh the plate without the aluminum foil and return it to the plate warmer.
10. Prepare a 24-well plate, adding water for equilibration.
11. Quickly remove the plate with the aluminum foil from the plate warmer to protect the fluorophores coupled to the secondary antibodies. Keep in hand, do not place on a cold surface.
12. Remove the aluminum foil and place it in the plate centrifuge.
13. Centrifuge at 1000 x g for 15 min at 40 °C. Check the temperature of the rotor, which should be at room temperature.
14. During centrifugation, prepare an ice tray: flatten the ice so that the plate can be placed on a flat surface.
15. Remove the plate and place it on the ice, covering the tray or plate with aluminum foil. Leave the plate on ice for 10 min.
16. Remove the plate from the ice and leave it in the aluminum to stabilize at room temperature for 20 min.
17. Pipette 1 to 1.5 mL of the clearing solution per well, cover with aluminum foil, and leave to optically clear at room temperature, protected from light, for at least 48 h.
    NOTE: Never store the plate at 4 °C. The LMPA may contract, detach from the bottom of the well, and float in the clearing solution. Image within one week of clearing.

5. Imaging

1. Use a confocal spinning disk microscope, choose (at a minimum) the 30x objective.
2. Keep the clearing solutions, as they serve to homogenize refractive indexes and make the 3D imaging easier.
3. Find the spheroids by selecting the DAPI channel to avoid bleaching the other fluorophores.
4. Acquire images by using the Z-stack function. Select the top and bottom of the spheroid and space the optical slices 10 µm apart.
   NOTE: Image acquisition and image analysis were performed on the Imaging Platform of the CRBS, PIC-STRA UMS 38, Inserm, Unistra.

Results

To assess the effectiveness of the labeling and optical clearing, several models were subjected to the same protocol and subsequently fixed (Figure 1). Spheroids with ratios of 1 IEC to 20 β-cells and 1 IEC to 50 β-cells were cultured according to the above-described method. Of note, the 1 IEC to 20 β-cells ratio approximates that of a human pancreatic islet. Using trypan blue to stain disrupted spheroids has confirmed that they maintain viable cells (80-90% living cells) over 96 h. This obs...

Discussion

A spheroid model incorporating MS-1 murine IEC and murine β cells β-TC6 has been developed mimicking the three-dimensional structure of pancreatic islets. This model is suitable for the analysis of stress-induced IEC dysfunction over 72 h and its impact on β-cell viability and function, with relevance to islet transplantation research. Importantly, the spheroid constituted of murine cells was homogenous in nature and with a relevant intra-islet endothelial lineage.

A significant...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
µ-plate 24 well Black ID 14 mm	IBIDI	82426
AggreWell 800 ( 24 wells)	StemCell	34815
Anti-Adherence Rinsing solution	StemCell	7010
Anti-CD31 antibody [P2B1]	abcam	ab24590	Final concentration 2.2µg/mL
Anti-Integrin β1 Antibody, clone MB1.2	Merck	MAB1997	Final concentration 1.43µg/mL
Cell culture flask, T-75, surface , surface : Cell+, filtre cap	Sarstedt	83.3911.302	β-TC6 culture
Cell culture flask, T-75, surface: Standard, 2-position screw cap	Sarstedt	83.3911.02	MS-1 culture
DAPI dihydrochloride	Thermofisher,	D1306	Final concentration 2µg/mL
Dimethyl Sulfoxide	Sigma	D8418-250ML
DMEM, high glucose, GlutaMAX™ Supplement, pyruvate	Thermofischer scientific	31966047
Dulaglutide
Dulaglutide	Lilly	Trulicity 1,5 mg pen
Fetal Bovine Serum	Biowest	S1810-500
Glycine	Sigma	G7126-1KG
Goat anti-Guinea Pig IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555	Thermofischer	A21435	Final concentration 2µg/mL
Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488	Thermofischer	A-11029	Final concentration 2µg/mL
Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 633	Thermofischer	A-21071	Final concentration 2µg/mL
Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555	Thermofischer	A21434	Final concentration 2µg/mL
Goat serum	Sigma	S26-100ML
HBSS without Calcium & magnesium  &phenol red	Corning	21-022-CV
Insulin (C27C9) Rabbit mAb	Cell signaling	3014S	Final concentration 0.66µg/mL
Isolated human pancreatic islet	Centre Européen d'Etude du Diabete (CeeD)		Kind gift from Dr Karim Bouzakri. Clinical grade , isolated according to guidelines.
Low geling temperature Agarose	Merck	A9414-10G
MS-1 cells	ATCC	CRL-2279
Paraformaldehyde Solution, 4% in PBS 1x	Fischer scientific	15670799
PBS 10x	Euromedex	ET330-A	Diluted 1x in laboratory-grade ultrapure water
Penicillin-Streptomycin	PAN Biotech	P06-07100
Polyclonal guinea pig anti-mouse NTPDase3 antibodies	mN3-1CI4; mN3-2C(I4,I5); mN3-3C(I4,I5)	https://ectonucleotidases-ab.com/Antibodies.php	Dilution 1:1000
Quadrol/N,N,N′,N′-Tetrakis(2-Hydroxypropyl) ethylenediamine	Sigma	122262-1L
Spinning-disk Olympus IXplore SPIN	Olympus Scientific Solutions
Sucrose for molecular biology	Sigma	S0389-1KG
Triton X-100 Bioquality Laboratory Grade	Euromedex	2000-A
Trypsin/EDTA (10x)	PAN Biotech	P10-025100
Tween-20	Euromedex	//
Urea	Sigma	U-5128
β-TC6 cells	ATCC	CRL-11506