# New Islet Model for 3D Study of Endothelial and $\beta$ Cell Interactions: Relevance for the Screening of Cytoprotective Agents

Saidi Amira, Tollard Clémence, Liu Liyao, El Khallouqi, Sévigny Jean, Auger

Cyril, Kauffenstein Gilles, Fatiha El Ghazouani, Laurence Kessler, Florence

Toti

<figure><img src="image_1.png" /></figure>

► To cite this version:

Saidi Amira, Tollard Clémence, Liu Liyao, El Khallouqi, Sévigny Jean, et al.. New Islet Model for 3D Study of Endothelial and $\beta$ Cell Interactions: Relevance for the Screening of Cytoprotective Agents. Journal of visualized experiments: JoVE, In press, 10.3791/68220. http://hal-05224681

HAL Id: hal-05224681

https://hal.science/hal-05224681v1

Submitted on 26 Aug 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
1 New Islet Model for 3D Study of Endothelial and β Cell Interactions: Relevance for the Screening of Cytoprotective Agents
2
3
4 Saidi Amira¹, Tollard Clémence¹, Liu Liyao¹,⁵, El Khallouqi Yasmina¹, Sévigny Jean⁶,⁷, Auger Cyril¹, 5 Kauffenstein Gilles¹, Fatiha El Ghazouani¹, Laurence Kessler¹,²,³, Florence Toti ¹,⁴

6
7 ¹Regenerative Nanomedicine (RNM), UMR 1260, INSERM (French National Institute of Health and
8 Medical Research), University of Strasbourg, CRBS, 1 rue Eugène Boeckel, 67000 Strasbourg,
9 France

10 ²Department of Diabetes and Nutrition Endocrinology, University Hospital of Strasbourg,
11 Strasbourg, France

12 ³Faculty of Medicine, University of Strasbourg, Strasbourg, France

13 ⁴Faculty of Pharmacy, University of Strasbourg, Illkirch-Graffenstaden, France

14 ⁵Department of Emergency, The First Affiliated Hospital of Chongqing Medical University,
15 Chongqing, China

16 ⁶Research Center, CHU de Québec - Laval University, Québec City, QC G1V 4G2, Canada

17 ⁷Department of Microbiology-Infectiology and Immunology, Faculty of Medicine, Laval
18 University, Québec City, QC G1V 0A6, Canada

19
20
21 Email address of the corresponding author:

22 Saidi Amira saidiamira16@gmail.com

23
24 Email addresses of the co-authors:

25 Tollard Clémence clemence.tollard@gmail.com

26 Liu Liyao alicialiu1993@hotmail.com

27 El Khallouqi Yasmina y.elkhallouqi@gmail.com

28 Sévigny Jean jean.sevigny@crchudequebec.ulaval.ca

29 Auger Cyril Cyril.auger@unistra.fr

30 Kauffenstein Gilles Kauffenstein@unistra.fr

31 Fatiha El Ghazouani zobairi@unistra.fr

32 Laurence Kessler Laurence.kessler@chru-strasbourg.fr

33 Florence Toti toti@unistra.fr

34
35 SUMMARY:

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

38
39 ABSTRACT:

40 This protocol details an optimized method for the production of small stable spheroids, their
41 culture, and 3D imaging, for the study of the endothelial and insulin-producing β cells interactions
42 in a 3D model of pancreatic islets. The 150-200 µm spheroids, mirroring the lowest range of islet
43 sizes, were prepared from a selected ratio combining 1 intra-islet endothelial cells (MS-1 cells) to
44 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.

52

## 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.

64

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 dyes³,⁴. 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.

75

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 spheroid⁶,⁷. 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.

85

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 autofluorescence⁴,⁶. 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 interactions⁵,¹⁰,¹¹.

## 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).

1.1. Prepare the spheroid-culture plate

1.2. Wash the plate with 500 µL of anti-adherence rinsing solution provided by the manufacturer to prevent cell adhesion and to promote spheroid formation.
133
134.13. Centrifuge the plate at 1300 x g for 5 min in a swinging bucket rotor fitted with plate holders at room temperature.

136
137.1.4. Observe the plate under a light microscope to ensure that no bubbles remain in the microwells. If so, centrifuge a second time at 1300 x g for 5 min.

139
140.1.5. Discard anti-adherence rinsing and wash the well 3 times with 1 mL of warm HBSS.

141
142.1.6. 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.

144
145.1.7. Keep the plate at 37°C in a humid atmosphere while preparing spheroid suspension.

146
147.1.8. Incubate MS-1 and β-TC6 simultaneously with trypsin solution and count the living cells in each lineage.

149
150. 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.

155
156.1.9. 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.

158
159. NOTE: Use a 10 mL or 25 mL pipette in suction-flow motion to homogenize the suspension.

160
161.1.10. Pipette 1 mL for each well and proceed in a suction-flow motion to distribute the cell suspension evenly in the well.

163
164.1.11. Centrifuge the plate at 130 x g for 5 min in a swinging bucket rotor fitted with plate holders at room temperature.

166
167.1.12. Observe the plate under a microscope to ensure that the cells are distributed equally among the microwells.

169
170. 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.

172
173.1.13. Incubate the plate at 37°C, 5% CO₂.

174
175.1.14. Monitor spheroid formation and growth using a light microscope.

176
177 NOTE: β-TC6 / MS-1 spheroids should begin to form after 24 h of culture, achieving a sphere
178 shape after 48 h. Viability can be monitored by trypan blue after spheroid disruption by trypsin
179 in duplicated samples and should maintain over 96 h (80% - 90%).

180
181 **2. Spheroid harvesting and staining**
182
183 2.1. Resuspend the spheroids gently with a P1000 pipette in their medium.
184
185 2.2. Eject the spheroids into small volumes in a 15 mL Falcon and centrifuge at 130 x g for 5
186 min at room temperature

187
188 2.3. Gently aspirate the medium while avoiding the pellet containing the spheroids.
189
190 2.4. Wash the pellet twice with 2 mL of HBSS, gently, to avoid any dissociation of the
191 spheroids, and centrifuge at 130 x g for 5 min.

192
193 2.5. Under a chemical hood, discard the HBSS and resuspend the spheroids in 1 mL of 4%
194 PFA solution overnight at 4°C to fixate.
195
196 2.6. Centrifuge the tube at 130 x g for 5 min, discard the 4% PFA solution, and wash 3 times
197 with 2 mL of 1x PBS (prepared in laboratory-grade ultrapure water).

198
199 2.7. Resuspend in 2.5 mL of HBSS.
200
201 2.8. Dispense the spheroid suspension into a 6-well plate.

202
203 2.9. Under a binocular magnifying glass, handpick 20 to 30 spheroids with a P200 pipette
204 and place them in a 1.5 mL tube.

205
206 NOTE: Each 1.5 mL tube is a staining condition containing 20 to 30 spheroids. Below 20 harvested
207 spheroids, it becomes difficult to see them when they are subsequently resuspended.

208
209 2.10. Equilibrate the volumes of the 1.5 mL tubes and centrifuge at 800 x g for 5 min.

210
211 NOTE: From this point on, all the solution aspiration steps are performed with the aid of a
212 magnifying glass. It is very difficult to see spheroids under artificial light. Always perform the
213 following steps during the daytime, with the tubes held against the daylight. All solutions are
214 aspirated with a P1000 tip topped with a P10 tip at its end.

215
216 2.11. Remove as much HBSS as possible without resuspending the spheroids.

217
218 2.12. Resuspend the spheroids in 400 µL of quenching solution: 0.5 M glycine in 1x PBS (pH 7-
219 7.4) to avoid any future background noise caused by remaining aldehydes from the PFA
220 fixation. Incubate at 37°C for 2 h.
221
222 2.13. Centrifuge 800 x g for 5 min and pipette off the quenching solution
223
224 2.14. Resuspend in 400 µL of penetration buffer: 0.2% Triton X-100, 0.3 M glycine, 20% DMSO
225 in 1x PBS. This will help improve the penetration of antibodies and nuclear probes. Incubate at
226 37°C for 2 h.
227
228 NOTE: From this stage onwards, spheroids settle on their own and no longer cling to the tube
229 wall. Centrifugation is no longer necessary if volumes are gently aspirated without disturbing the
230 spheroid pellet. If necessary, centrifuge at 800 x g for 5 min.

231
232 2.15. Discard the penetration buffer and incubate in 400 µL of blocking buffer: 0.2% Triton X-
233 100, 1% BSA, 10% DMSO, 5% Goat Serum in 1x PBS, overnight at 37°C.
234
235 2.16. Discard the blocking buffer and incubate overnight at 37°C in 300 µL of primary antibody
236 solution: 0.2% Tween 20, 1% BSA, 5% DMSO, 5% Goat Serum in 1x PBS.

237
238 NOTE: Used primary antibodies are: anti-CD31 (final concentration of 2.2 µg/mL), anti-Integrin
239 β1 (1.43 µg/mL), anti-Insulin (0.66 µg/mL), and anti-mouse NTPDase3¹² (dilution 1:1000) (see
240 Table of Materials). Control IgGs of respective identical isotypes should be validated on the same
241 structure to ascertain positive labeling by fluorescent microscopy.
242
243 2.17. Discard the primary antibody solution and wash 3 times with 400 µL of washing buffer:
244 0.2% Tween 20, 1% BSA in PBS 1X. Shake for 5 min between washes.
245
246 2.18. Incubate in 300 µL of secondary antibody solution: DAPI (2 µg/mL), 0.2% Tween 20, 1%
247 BSA, 5% DMSO, 5% Goat Serum in 1x PBS, overnight at 37°C. Protect the tubes with aluminum
248 foil.
249
250 NOTE: DAPI should always be added to the staining conditions as this makes it easier to find
251 spheroids during microscopy imaging and helps not to bleach the different fluorophores/dyes.
252 All secondary antibodies have a final concentration of 2 µg/mL (see Table of Materials).
253
254 2.19. Discard the secondary antibody solution and wash 3 times with 400 µL of a second washing
255 buffer: 0.2% Tween 20, 1% BSA in 1x PBS. Keep the last wash and store the tubes at 37°C till
256 mounting the same day.

257
258 3. Solution preparation
259
260 3.1. Optical clearing solution preparation
261
262 NOTE: The optical clearing solution follows the CUBIC¹⁰ procedure, and was prepared as
263 proposed by Gunasingh et al⁴. Component proportions are given in percentages of the final
264 solution weight (W/W), because Quadrol/N,N,N',N'-Tetrakis(2-Hydroxypropyl) ethylenediamine

265
266 3.1. Optical clearing solution preparation
267

268
269 2.19. Discard the secondary antibody solution and wash 3 times with 400 µL of a second washing
270 buffer: 0.2% Tween 20, 1% BSA in 1x PBS. Keep the last wash and store the tubes at 37°C till
271 mounting the same day.
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
265 is too viscous to pipette and must be weighed.

266
267 3.1.1. In a glass bottle, weigh 9% (w/w) of N,N,N',N'-Tetrakis(2-Hydroxypropyl) ethylenediamine, 268 22% (w/w) of urea, 44% (w/w) of sucrose, 0.1% (w/w) of Triton X-100, and 24.9% (w/w) of 269 laboratory-grade ultrapure water.

270
271 NOTE: About 60 g of solution is necessary to clear a 24-well plate.

272
273 3.1.2. Add a magnetic bar adapted to the diameter of the bottle.

274
275 3.1.3. Heat and stir the solution to 56 °C. Keep a control glass bottle filled with water on the same 276 plate to monitor temperature rise.

277
278 3.1.4. Control the stirring speed to maintain a smooth liquid vortex and stir the solution until all 279 compounds are completely dissolved. The solution should be completely liquid and clear. This 280 may take up to 1 h.

281
282 3.1.5. Keep at room temperature, protected from the light.

283
284 NOTE: The clearing solution must be prepared at least 24 h before optical clearing, to allow the 285 solution to degas and eliminate any bubbles it may contain. To avoid introducing bubbles, never 286 shake the solution again. Never store in cold storage.

287
288 3.2. 2% LMPA solution preparation

289
290 3.2.1 Weigh 0.5g of LMPA on a precision balance and place in a 100 mL Erlenmeyer flask.

291
292 CAUTION: Do not take a smaller volume Erlenmeyer, there is a risk of burns if it overflows.

293
294 3.2.2. Dissolve in 25 mL of 1x PBS at room temperature.

295
296 3.2.3. Thoroughly incorporate the powder into the 1x PBS by gently stirring the Erlenmeyer flask.

297
298 CAUTION: For the next step, it is essential to use protective equipment: gloves and a mask for 299 the eyes and face, to protect against splashes and burns.

300
301 3.2.4. Heat the Erlenmeyer flask in the microwave.

302
303 3.2.5. Observe when the solution starts to boil (presence of bubbles in the liquid), and remove 304 immediately before it boils over.

305
306 3.2.6. Gently stir the Erlenmeyer to incorporate the powder.

307
308 CAUTION: Do not stir too hard, as the liquid may overflow/over-boil and splash.
309
310 3.2.7. Return the Erlenmeyer to the microwave, repeat until the LMPA is completely dissolved.

311
312 3.2.8. Remove the Erlenmeyer and, before the LMPA solidifies, quickly aliquot 1mL of 2% LMPA
313 solution into 1.5 mL tubes. Store at room temperature.

314
315 NOTE: The process is time-consuming. Prepare the solution in advance and store it for later use.

316
317 4. Mounting and optical clearing

318
319 4.1. Preheat the plate centrifuge to 40 °C, running a long cycle to prepare the sample plate.

320
321 NOTE: LMPA gels at 30°C. Do not run the centrifuge below 40 °C. If the centrifuge allows it,
322 increase to 50°C.

323
324 4.2. Liquefy aliquots of 2% LMPA in a water bath or tube heater at 70°C for 5 to 10 min.

325
326 4.3. Lower and maintain the temperature of the 2% LMPA at 55 °C.

327
328 4.4. Place the imaging plate gently on the plate warmer preheated to 50 °C.

329
330 NOTE: Be careful not to break or scratch the fragile polymer used as a base for the wells.

331
332 4.5. Be careful that the plate is hot before adding the 2% LMPA. It must not gel at this stage.

333
334 4.6. Pipette 200 µL of 2% LMPA into each well. Cover the entire bottom of the support to
335 obtain a first liquid base, which allows a more fluid distribution of the spheroids.

336
337 4.7. Discard the washing buffer from the tubes, resuspend the spheroids in 300 µL of 2%
338 LMPA, and pipette them directly into their well. Always perform gentle resuspension to avoid
339 bubbles.

340
341 4.8. Cover the plate with aluminum foil to protect the fluorophores.

342
343 4.9. Quickly weigh the plate without the aluminum foil and return it to the plate warmer.

344
345 4.10. Prepare a 24-well plate, adding water for equilibration.

346
347 4.11. Quickly remove the plate with the aluminum foil from the plate warmer to protect the
348 fluorophores coupled to the secondary antibodies. Keep in hand, do not place on a cold surface.

349
350 4.12. Remove the aluminum foil and place it in the plate centrifuge.

351
352 4.13. Centrifuge at 1000 x g for 15 min at 40 °C. Check the temperature of the rotor, which
353 should be at RT.

354
355 4.14. During centrifugation, prepare an ice tray: flatten the ice so that the plate can be placed
356 on a flat surface.

357
358 4.15. Remove the plate and place it on the ice, covering the tray or plate with aluminum foil.
359 Leave the plate on ice for 10 min.

360
361 4.16. Remove the plate from the ice and leave it in the aluminum to stabilize at room
362 temperature for 20 min.

363
364 4.17. Pipette 1 to 1.5 mL of the clearing solution per well, cover with aluminum, and leave to
365 optically clear at room temperature, protected from light, for at least 48 h.

366
367 NOTE: Never store the plate at 4°C. The LMPA may contract, detach from the bottom of the well,
368 and float in the clearing solution. Image within one week of clearing.

369
370 5. Imaging

371
372 5.1. Use a confocal spinning disk microscope, choose (at a minimum) the 30X objective.

373
374 5.2. Keep the clearing solutions, it serves to homogenize refractive indexes and make the 3D
375 imaging easier.

376
377 5.3. Find the spheroids by selecting the DAPI channel to avoid bleaching the other
378 fluorophores.

380
381 5.4. Acquire images by using the Z-stack function. Select the top and bottom of the spheroid
382 and space the optical slices for 10 µm between each other.

383
384 NOTE: Image acquisition and image analysis were performed on the Imaging Platform of the
385 CRBS, PIC-STRA UMS 38, Inserm, Unistra.

386
387
388 REPRESENTATIVE RESULTS:
389

390
391 To assess the effectiveness of the labeling and optical clearing, several models were subjected to
392 the same protocol and subsequently fixed (Figure 1). Spheroids with ratios of 1 IEC to 20 β-cells
393 and 1 IEC to 50 β-cells were cultured according to the above-described method. Of note, the 1
394 IEC to 20 β-cells ratio approximates that of a human pancreatic islet. Using trypan blue to stain
395 disrupted spheroids has confirmed that they maintain viable cells (80-90% living cells) over 96 h.
396 This observation is of particular importance for the study of the progressive cell alterations
396 induced by the IBMIR cytokines in the context of islet transplantation and the identification of
397 cytoprotective agents.

398
399 Because one of the consequences of cytokine stress is the alteration of IEC morphology and
400 function, which can impact β-cell survival and insulin function, we further substantiated the
401 feasibility of a 3D pharmacological modulation of cytokine-induced stress using dulaglutide, a
402 GLP1 analogue. In previous experiments, its cytoprotective effects on IEC monolayers submitted
403 to TGFβ-2 or IL-1β-induced stress have been demonstrated.13
404

405 The investigation addressed 3 different questions: Does the protocol 1) enable homogeneous
406 staining without compromising the model's structure, even after stress or pharmacological
407 treatment; 2) facilitate imaging and distinction of the different cell types for subsequent
408 mapping, utilizing specific markers for each cell type (CD31 for IEC and insulin or NTPDase 3 for
409 β-cells); 3) allows the evaluation of the morphological impact of the cell proportions?

410
411 To achieve these goals, 2 different IEC proportions were compared and cell-cell interactions were
412 evaluated using integrin β-1 probing. Indeed, Integrin β-1 was demonstrated to contribute to the
413 cohesion of IEC and β-cells within the islet structure and maintain the physiological endocrine
414 function of β-cells via the polarization of insulin secretion towards the endothelial bed¹⁴.

415
416 To better define the benefits of a pharmacological modulation, spheroids comprising 1 IEC to 20
417 β-cells ratio were treated with 1 µM dulaglutide, 24 h after initiation of their culture and prior to
418 complete spheroid formation. In these selected experiments, potential consequences on 3D
419 morphological modifications were investigated.

420
421 To ensure the transposability of the method to other biological models, the staining and optical
422 clearing protocol were applied to a human pancreatic islet freshly prepared and fixed within 48
423 h after isolation.

424
425
426 [Place Figure 1 here]
427

428 Uniform labelling across entire spheroid surface enabled by staining and optical clearing
429 protocol.

430 To enable comparison between different conditions, the 6th optical cut was selected. It
431 represents the median of a stack of approximately 10 to 14 optical cuts. The CD31 and insulin
432 labelling precisely outlines the two distinct cell lineages, IEC and β-cells, with conserved signal
433 intensity compared to more external cuts (Figure 1). This observation demonstrates an efficient
434 diffusion of the probes to the core of the spheroid, at an approximate thickness of 75 µm on the
435 Z axis, and therefore enables a reliable three-dimensional localization of cell populations. Indeed,
436 analysis of the median cut in the 1 IEC to 20 β-cells spheroids shows a homogeneous distribution
437 of both lineages across the entire surface of the section. No observable differences between
438 labelling were noted among the 1 IEC to 20 β-cells, 1 IEC to 20 β-cells dulaglutide, and 1 IEC to 50
439 β-cells conditions, indicating a robustness with respect to spheroid cell composition and
treatment. The distribution of IECs and β-cells in the 1 IEC to 20 β-cells model and in the pancreatic islet can be visualized as videos in **Supplementary Figure 1** and **Supplementary Figure 2**, respectively.

Of note, flow cytometry measurements would eventually bring complementary quantitative information to the 3D analysis of the cell type distribution, on the number and proportion of IEC to 20 β-cells constitutive of the spheroids at different time courses and treatments.

**Staining and optical clearing protocol discrimination at membrane and sub-cellular levels across cell lineages.**

NTPDASE-ectonucleotidase 3 (NTPDase 3) is a plasma membrane enzyme that characterizes β-cells. As an ectonucleotidase, it contributes to the control of the relative proportion of extracellular ATP and metabolites, and modulates insulin secretion by controlling the activation of the β-cell purinergic receptors¹⁵, ¹⁶. NTPDase 3 is expressed on the cell surface of all adult human β-cells¹⁷ rendering it a useful marker to delineate the β-cells.

In spheroids, the NTPDase 3 labeling never colocalizes with the CD31 staining of IEC. This spatial separation confirms the specificity of the markers and the feasibility of the study of cell distribution using our staining technique. As illustrated in the 1 IEC to 20 β-cells spheroids, data also confirms that NTPDase 3 is a reliable marker of the β-cell membrane, which is consistent with the expectations from the literature¹⁷, ¹⁸ (**Figure 2A**).

Our observation of the spatial distribution of NTPDase-3 provides further insights into the organization, function, and potential maturity¹⁷ of β-cells. Indeed, as illustrated in **Figure 2B**, the NTPDase-3 and insulin staining of β-cells are not fully colocalized. One possible explanation relies on the number of β-TC6 cell culture passages that may have been too important. Indeed, murine β-cell lines have been reported to develop unstable insulin production with the number of passages⁸, ¹⁹. Another interesting consideration would be that the incomplete overlap between NTPDase-3 labeling and insulin in β-cells provides valuable information on β-cell maturation. Indeed, the insulin staining is mainly associated with the cytoplasmic content of certain β-cells, while the NTPDase 3 probing delineates all β-cells. In the present investigation, the distinct distribution of the two markers suggests that not all β-TC6 cells within the spheroid are at the same maturity state for insulin production.

**Staining and optical clearing protocol for analyzing β1 integrin-mediated cell-cell cohesion in spheroids and islets.**

The uniform distribution of integrin β1 staining observed in the spheroid models, particularly in the 1 IEC to 20 β-cells ratio, confirms robust interactions between the two cell types, of particular physiological relevance¹⁴. Furthermore, a consistent integrin β1 probing across different spheroid compositions and in the human pancreatic islet sample is shown to indicate successfully preserved cell-cell connections, even in the presence of the 4 different lineages constitutive of the more complex structure of the human islet. This preservation is particularly significant in the context of islet transplantation. Indeed, the maintenance of such connections is key to the long-
term viability and function of transplanted islets.

The protocol also demonstrates staining efficacy on human pancreatic islets. In the islet, β-cells are clearly discernible, as is the morphology of the micro-vessel lined with endothelial cells.

Altogether, our findings indicate that the staining and optical clearing methods are fully suitable for 3D analysis of distinct biomarkers and cell lineages. Nevertheless, the development of mathematical models appears necessary to analyze cell distribution. These preliminary data also show that variation in integrin β-1 signal intensity could serve as an indicator of the IEC-β-cell junction integrity. Indeed, variations in these junction patterns could serve as early indicators of stress-induced alterations in islet structure and function, potentially offering new insights into the mechanisms of islet damage and protection in transplantation scenarios.

[Place Figure 2 here]

FIGURE AND TABLE LEGENDS:

**Figure 1:** Staining and optical clearing of spheroids composed of varying IEC to β-cell ratios and of a human pancreatic islet. From top to bottom: 48 h culture of 1 IEC to 20 β-cells spheroid; 48 h culture of 1 IEC to 20 β-cells spheroid treated for 24 h with dulaglutide, a GLP1-analog; 48 h culture of 1 IEC to 50 β-cells spheroid; human pancreatic islet fixed 48 h after isolation. Models were imaged in Z-stack using a spinning-disk microscope with a 30X magnification. The sixth optical section of each model is selected, representing approximately its middle. DAPI labeling for nuclei in blue, CD31 for IECs in green, Integrin β-1 for cell-cell connections in red, and Insulin for β-cells in magenta.

**Figure 2:** Three-dimensional spatial distribution of NTPDASE-ectonucleotidase 3 in a 48 h culture of a spheroid (1 IEC to 20 β-cells) imaged in Z-stack using a spinning-disk microscope with a 30X magnification. (A) CD31 co-labeling in green / NTPDASE-ectonucleotidase 3 in red on 12 optical sections. (B) Insulin co-labeling in magenta / NTPDASE-ectonucleotidase 3 in cyan on 12 optical sections.

**Supplementary Figure 1:** Video of a Z-stack of a 48 h culture of 1 IEC to 20 β-cells spheroids after staining and optical clearing. Model was imaged in Z-stack using a spinning-disk microscope with a 30X magnification. CD31 labeling for IECs in green and Insulin for β-cells in magenta.

**Supplementary Figure 2:** Video of a Z-stack of a fixed human pancreatic islet. After staining and optical clearing, the islet structure was imaged in Z-stack using a spinning-disk microscope with a 30X magnification. CD31 labeling for IECs in green and Insulin for β-cells in magenta.

**Supplementary Figure 3:** MS-1 cells and β-TC6 have similar individual volumes within a spheroid. The cross-sectional areas of 30 IECs and 30 β-cells were measured using ImageJ/Fiji in the representative section shown in Figure 1 (1 IEC to 20 β-cells, no treatment). The volumes of
each lineage were calculated from their respective surface assuming that each cell is a sphere.
Mean volumes of each lineage were compared with an unpaired T-test (p=0.9564). The absence
of statistical significance suggests that at each time point, the two lineages constitutive of the
spheroid would occupy a volume proportional to their ratio.

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 achievement is the development and validation of a protocol for harvesting, staining,
and optically clearing small spheroids (150-200 µm), addressing challenges in three-dimensional
imaging, including enhanced dye penetration, structural support with LMPA, and the CUBIC
optical clearing technique for improved visualization while maintaining cellular integrity. The
protocol's efficacy was demonstrated in spheroids with varying IEC to β-cell ratios (1:20 and 1:50)
and in isolated human pancreatic islets.

Uniform staining, precise cell group delimitation, preservation of cell interactions, and successful
application to human islets, highlight its versatility. The present study provided insights into cell
distribution and function within spheroids, including homogeneous IEC and β-cell distribution in
the 1:20 model, micro-vessel morphology visualization in human islets, and NTPDASE-
ectonucleotidase 3 distribution patterns relative to insulin-producing cells, strongly suggesting
varying β-cell maturity levels. Indeed, the observation of a distinct distribution of NTPDASE-
ectonucleotidase 3 in relation to insulin-producing cells highlights the importance of advanced
3D imaging techniques.

Several technical challenges were addressed to eventually circumvent specific difficulties and
should be taken into consideration in future studies. First, the small size of the spheroids (150-
200 µm) makes them delicate to handle, which could affect reproducibility if not done carefully.
One of the common techniques for large spheroid isolation and washing is decantation. However,
this method is not adapted for the isolation of smaller spheroids as they don't precipitate in
aqueous buffers. The alternate method, centrifugation, was found easy to modulate for optimal
recovery, while strainers failed to reliably isolate and maintain spheroid integrity. Interestingly,
CUBIC, a procedure initially set up for imaging of mice brains¹⁰, is demonstrated herein to be fully
suitable for very small structures like 200 µm spheroids, provided an adapted clearing time is
defined. The LMPA mounting coupled to a microscopy plate was designed to accommodate a
spinning disk confocal microscope by setting the spheroid at the bottom of the plate close to the
microscope objective. However, the choice of mounting is a key aspect as it is microscope-
dependent. Future applications would therefore require protocol adjustments to facilitate
observation by other microscopy methods, such as light-sheet or two-photon microscopes,
suited for visualizing deep structures in 3D models²⁰⁻²². Notably, those microscopy techniques
572 would differ in acquisition time and handling of the sample, leading to an obligatory optimization
573 study. Despite these challenges, the protocol offers significant advantages in terms of usability
574 and efficiency.

575
576 **Limitation and necessary adaptations in other models:**
577

578 The applicability to spheroids and primary human islets demonstrates versatility. The procedure
579 allows uniform 3D staining and imaging of small spheroids, enabling detailed analysis of cell
580 distribution and interactions. Yet transposition to other cell lineages, will require careful
581 adaptation to the respective cell volumes occupied by each constitutive cell lineage. In the
582 present bi-lineage spheroid, the volume ratio between IEC and β-cells and its reproducibility
583 constitute key expected outcomes for quality management. In any case, rudimentary image
584 analysis by the software Image J/Fiji, would allow the evaluation of the volume ratio by compiling
585 the areas occupied by each cell line at specific Z-stack optical cut, assuming that each cell is
586 simplified as a sphere (Supplementary Figure 3). Once optimized, the technique should also
587 provide high-quality 3D images, enabling the analysis of an eventual necrotic core in larger
588 pseudo-islets. Indeed, cell viability in larger spheroids might be critical with respect to the risk of
589 uncontrolled insulin leakage from the necrotic core. Future integration with automated image
590 analysis tools could further improve the efficiency of data extraction from 3D images, especially
591 when combining more than 2 lineages. Indeed, automation will rely on the identification of
592 fluorescent probes from center, border and in all the layers of spheroids for the determination
593 of relevant thresholds and optimal cellular mapping through the whole structure.

594
595 The validated model and three-dimensional imaging methods herein show potential for islet
596 transplantation research. Pharmacological screening of cytoprotective treatments preserving the
597 three-dimensional structure of the islet, the IEC morphology and function, and cell-cell
598 interactions affecting the islet function and survival peri- and post-transplantation are the short-
599 term developments of this screening tool including the study of membrane-borne proteins and
600 enzyme activities as modulators of the 3D cohesion.

601
602 While the goal of the study was to provide researchers with a screening tool, it is tempting to
603 speculate that the model can be expanded to the study of the role of glucose-induced responses
604 within the spheroid. Yet, *in-vitro* or *in-vivo* assessment of the spheroid function would require a
605 murine cell-line that maintains its sensitivity to glucose elevation over passages. Nevertheless,
606 such future adaptations of the murine model will require new cell ratio studies and hand-picking
607 experimentations.

608
609 Future research directions include developing mathematical models for the automated analysis
610 of cell distribution in the optical cuts, investigating CD31, insulin, and integrin β1 signal intensity
611 variations, and further exploring the significance of NTPDASE-ectonucleotidase 3 distribution in
612 β-cell maturation and function.

613
614 These advancements in imaging and analysis techniques have the potential to enhance the
615 understanding of islet biology, contributing to improved knowledge in the field.
616
617 ACKNOWLEDGMENTS:
618 Authors are indebted to Pascal Kessler, from the PICSTRA imaging platform, for expert assistance
619 in spinning disk microscopy and extraction of the raw labelling data from the Z stack. Authors
620 wish to acknowledge the kind gift of purified human islets by Dr. W. Bietiger, Dr. A. Langlois and
621 Dr. K. Bouzakri from the European Center for the Study of Diabetes (CEED, Strasbourg France)

622
623 DISCLOSURES:
624 Authors declare they have no conflict of interest

625
626
627 REFERENCES:
628 1. Langlois, A., Pinget, M., Kessler, L., Bouzakri, K. Islet Transplantation: Current Limitations and Challenges for Successful Outcomes. *Cells*. **13** (21), 1783, doi: 10.3390/cells13211783 (2024).
629 2. Li, X., Meng, Q., Zhang, L. The Fate of Allogeneic Pancreatic Islets following Intraportal Transplantation: Challenges and Solutions. *Journal of Immunology Research*. **2018**, 1–13, doi: 10.1155/2018/2424586 (2018).

630 3. Nürnberg, E. et al. Routine Optical Clearing of 3D-Cell Cultures: Simplicity Forward. *Frontiers in Molecular Biosciences*. **7**, doi: 10.3389/fmolb.2020.00020 (2020).

635 4. Gunasingh, G., Browning, A.P., Haass, N.K. Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids. *Journal of Visualized Experiments*. **2022** (186), doi: 10.3791/64103 (2022).

638 5. Paysan, J. *The Art of Tissue Clearing Essential Knowledge Briefings First Edition 2021*. at <www.essentialknowledgebriefings.com>. (2021).

640 6. Costa, E.C., Silva, D.N., Moreira, A.F., Correia, I.J. Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids. *Biotechnology and Bioengineering*. **116** (10), 2742–2763, doi: 10.1002/bit.27105 (2019).

643 7. Vieites-Prado, A., Renier, N. Tissue clearing and 3D imaging in developmental biology. *Development*. **148** (18), dev199369, doi: 10.1242/dev.199369 (2021).

645 8. Poitout, V., Stout, L.E., Armstrong, M.B., Walseth, T.F., Sorenson, R.L., Robertson, R.P. Morphological and functional characterization of βTC-6 cells - An insulin-secreting cell line derived from transgenic mice. *Diabetes*. **44** (3), 306–313, doi: 10.2337/diab.44.3.306 (1995).

648 9. Ionescu-Tirgoviste, C. et al. A 3D map of the islet routes throughout the healthy human pancreas. *Scientific Reports*. **5** (1), 14634, doi: 10.1038/srep14634 (2015).

650 10. Susaki, E.A., Tainaka, K., Perrin, D., Yukinaga, H., Kuno, A., Ueda, H.R. Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging. *Nature Protocols*. **10** (11), 1709–1727, doi: 10.1038/nprot.2015.085 (2015).

653 11. Costa, E.C., Silva, D.N., Moreira, A.F., Correia, I.J. Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids. *Biotechnology and Bioengineering*. **116** (10), 2742–2763, doi: 10.1002/bit.27105 (2019).

656 12. Munkonda, M.N. et al. Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase-3: Partial characterization of the inhibitory epitope and potential applications. *The FEBS Journal*. **276** (2), 479–496, doi: 10.1111/j.1742-4658.2008.06797.x (2009).
660 13. Sbat, N. Étude de la transition endothélio-mésenchymateuse des cellules endothéliales intra-insulaires de souris et modulation pharmacologique : intérêt dans la greffe d'îlots 661 662 pancréatiques. at <https://theses.hal.science/tel-03873595> (2021).

663 14. Barillaro, M., Schuurman, M., Wang, R. β1-Integrin - A Key Player in Controlling Pancreatic 664 Beta-Cell Insulin Secretion via Interplay With SNARE Proteins. *Endocrinology (United States)*. **164** 665 (1), doi: 10.1210/endocr/bqac179 (2023).

666 15. Lavoie, E.G. *et al*. Identification of the ectonucleotidases expressed in mouse, rat, and human Langerhans islets: potential role of NTPDase3 in insulin secretion. *American Journal of Physiology-Endocrinology and Metabolism*. **299** (4), E647-E656, doi: 667 668 10.1152/ajpendo.00126.2010 (2010).

670 16. Giuliani, A.L., Sarti, A.C., Di Virgilio, F. Ectonucleotidases in Acute and Chronic 671 Inflammation. *Frontiers in Pharmacology*. **11**, doi: 10.3389/fphar.2020.619458 (2021).

672 17. Saunders, D.C. *et al*. Ectonucleoside Triphosphate Diphosphohydrolase-3 Antibody 673 Targets Adult Human Pancreatic β Cells for In Vitro and In Vivo Analysis. *Cell Metabolism*. **29** (3), 674 745-754.e4, doi: 10.1016/j.cmet.2018.10.007 (2019).

675 18. Syed, S.K. *et al*. Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and 676 regulates glucose-induced insulin secretion. *American Journal of Physiology-Endocrinology and 677 Metabolism*. **305** (10), E1319-E1326, doi: 10.1152/ajpendo.00328.2013 (2013).

678 19. Cheng, K. *et al*. High passage MIN6 cells have impaired insulin secretion with impaired 679 glucose and lipid oxidation. *PLoS ONE*. **7** (7), doi: 10.1371/journal.pone.0040868 (2012).

680 20. Rane, T.D., Armani, A.M. Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake 681 in 3D Cell Spheroids. *PLOS ONE*. **11** (12), e0167548, doi: 10.1371/journal.pone.0167548 (2016).

682 21. Alzeeb, G. *et al*. Gastric cancer multicellular spheroid analysis by two-photon microscopy. 683 *Biomedical Optics Express*. **13** (5), 3120, doi: 10.1364/BOE.450518 (2022).

684 22. Diosdi, A. *et al*. Single-cell light-sheet fluorescence 3D images of tumour-stroma spheroid 685 multicultures. *Scientific Data*. **12** (1), 492, doi: 10.1038/s41597-025-04832-0 (2025).

686
<figure><img src="image_3.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>Figure 2</figcaption></figure>

<figure><img src="image_5.png" /></figure>
<table><thead><tr><th>Name of Material/ Equipment</th><th>Company</th><th>Catalog Number</th><th>Comments/Description</th></tr></thead><tbody><tr><td>μ-plate 24 well Black ID 14 mm</td><td>IBIDI</td><td>82426</td><td></td></tr><tr><td>AggreWell 800 (24 wells)</td><td>StemCell</td><td>34815</td><td></td></tr><tr><td>Anti-Adherence Rinsing solution</td><td>StemCell</td><td>7010</td><td></td></tr><tr><td>Anti-CD31 antibody [P2B1]</td><td>abcam</td><td>ab24590</td><td>Final concentration 2.2μg/mL</td></tr><tr><td>Anti-Integrin β1 Antibody, clone MB1.2</td><td>Merck</td><td>MAB1997</td><td>Final concentration 1.43μg/mL</td></tr><tr><td>Cell culture flask, T-75, surface, surface: Cell+, filtre cap</td><td>Sarstedt</td><td>83.3911.302</td><td>β-TC6 culture</td></tr><tr><td>Cell culture flask, T-75, surface: Standard, 2-position screw cap</td><td>Sarstedt</td><td>83.3911.02</td><td>MS-1 culture</td></tr><tr><td>DAPI dihydrochloride</td><td>Thermofisher</td><td>D1306</td><td>Final concentration 2μg/mL</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma</td><td>D8418-250ML</td><td></td></tr><tr><td>DMEM, high glucose, GlutaMAX™ Supplement, pyruvate</td><td>Thermofischer</td><td>31966047</td><td></td></tr><tr><td>Dulaglutide</td><td></td><td></td><td></td></tr><tr><td>Dulaglutide</td><td>Lilly</td><td>Trulicity 1,5 mg pen</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Biowest</td><td>S1810-500</td><td></td></tr><tr><td>Glycine</td><td>Sigma</td><td>G7126-1KG</td><td></td></tr><tr><td>Goat anti-Guinea Pig IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555</td><td>Thermofischer</td><td>A21435</td><td>Final concentration 2μg/mL</td></tr><tr><td>Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555</td><td>Thermofischer</td><td>A-11029</td><td>Final concentration 2μg/mL</td></tr><tr><td>Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555</td><td>Thermofischer</td><td>A-21071</td><td>Final concentration 2μg/mL</td></tr><tr><td>Goat anti-Rat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 555</td><td>Thermofischer</td><td>A21434</td><td>Final concentration 2μg/mL</td></tr><tr><td>Goat serum</td><td>Sigma</td><td>S26-100ML</td><td></td></tr><tr><td>HBSS without Calcium & magnesium & phenol red</td><td>Corning</td><td>21-022-CV</td><td></td></tr><tr><td>Insulin (C27C9) Rabbit mAb</td><td>Cell signaling</td><td>3014S</td><td>Final concentration 0.66μg/mL</td></tr><tr><td>Isolated human pancreatic islet</td><td>Centre Européen d'Etude du Diabete (CEDI)</td><td></td><td>Kind gift from Dr Karim Bouzakri. Clinical grade, isolated according to guidelines.</td></tr><tr><td>Low geling temperature Agarose</td><td>Merck</td><td>A9414-10G</td><td></td></tr><tr><td>MS-1 cells</td><td>ATCC</td><td>CRL-2279</td><td></td></tr></tbody></table>
<table><tr><td>Paraformaldehyde Solution, 4% in PBS 1x</td><td>Fischer scientific</td><td>15670799</td><td></td></tr><tr><td>PBS 10X</td><td>Euromedex</td><td>ET330-A</td><td>Diluted 1x in laboratory-grade ultrapure water</td></tr><tr><td>Penicillin-Streptomycin</td><td>PAN Biotech</td><td>P06-07100</td><td></td></tr><tr><td>Polyclonal guinea pig anti-mouse NTPDase3 antibodies</td><td>mN3-1CI4; mN3</td><td>https://ectonucleotidas</td><td>Dilution 1:1000</td></tr><tr><td>Quadrol/N,N,N',N'-Tetrakis(2-Hydroxypropyl) ethylenediamine</td><td>Sigma</td><td>122262-1L</td><td></td></tr><tr><td>Spinning-disk Olympus IXplore SPIN</td><td>Olympus Scientific Solutions</td><td></td><td></td></tr><tr><td>Sucrose for molecular biology</td><td>Sigma</td><td>S0389-1KG</td><td></td></tr><tr><td>Triton X-100 Bioquality Laboratory Grade</td><td>Euromedex</td><td>2000-A</td><td></td></tr><tr><td>Trypsin/EDTA (10x)</td><td>PAN Biotech</td><td>P10-025100</td><td></td></tr><tr><td>Tween-20</td><td>Euromedex</td><td>//</td><td></td></tr><tr><td>Urea</td><td>Sigma</td><td>U-5128</td><td></td></tr><tr><td>β-TC6 cells</td><td>ATCC</td><td>CRL-11506</td><td></td></tr></table>
<figure><img src="image_6.png" /></figure>