Generation of Bone Marrow Mesenchymal Stem Cell-Derived Islet Organoid-like Structures From Fresh Pancreatic Tissue

Summary

Here, we present a protocol to differentiate bone marrow mesenchymal stem cells (BMSCs) into pancreatic islet organoid-like structures using porcine pancreatic tissue lysate. Organoid-like structures form after 22 days. This simple, cost-effective method produces structures morphologically similar to pancreatic islets for in vitro studies and drug screening.

Abstract

As a prevalent chronic illness across the globe, diabetes mellitus (DM) is marked by disrupted glucose balance in the body. In type 1 diabetes mellitus (T1DM), autoimmune responses destroy pancreatic β-cells entirely, resulting in total insulin insufficiency. Patients with this condition need lifelong supplemental insulin treatment. The present study intends to create a reliable and economical culture platform to generate pancreatic islet organoid-like structures from bone marrow mesenchymal stem cells (BMSCs). This platform can not only ease the shortage of donor islets for transplantation, but also serve as an in vitro model for diabetes-related research.

By optimizing the preparation process of porcine pancreatic tissue lysate (centrifugation at 12,000 rpm for 30 min at 4 °C, followed by 0.22 µm filtration for sterilization), BMSCs were successfully induced to differentiate into pancreatic islet organoid-like structures. Morphological validation confirmed that only induced BMSCs formed compact, plump, and highly transparent islet-like aggregates, whereas noninduced BMSCs exhibited significant vacuolization and reduced transparency.

Dynamic monitoring showed that cell aggregates (50–100 µm in diameter) formed on day 16 of culture and developed into capsule- like structures (200–300 µm in diameter) by day 22. This method is simple to operate, cost- effective, and does not require complex equipment. The resulting organoids are morphologically similar to pancreatic islets, providing a tool for in vitro studies and related drug screening.

Introduction

As three-dimensional (3D) in vitro model systems, pancreatic islet organoid-like structures have emerged as crucial tools in diabetes research and regenerative medicine, owing to their ability to closely mimic the multicellular composition and spatial structure of native pancreatic islets¹. Such models not only provide a unique platform for understanding islet developmental biology and the pathological mechanisms of diabetes^2,3 but also support the development of cell replacement therapies and drug screening systems⁴. With the global prevalence of diabetes continuing to rise—especially the absolute insulin deficiency caused by irreversible β-cell damage in type 1 diabetes—conventional insulin replacement therapy can control symptoms but cannot cure the disease. Meanwhile, islet transplantation faces a severe shortage of donors, making the development of scalable alternative islet sources an urgent priority^1,5. By leveraging the directed differentiation potential of pluripotent stem cells or adult stem cells, pancreatic islet organoid-like technology may help address this limitation^6,7,8.

In recent years, significant progress has been made in pancreatic islet organoid-like culture systems based on human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs)⁹. Multiple studies have confirmed that by optimizing induction protocols and culture conditions, stem cells can differentiate into islet-like cells with glucose-responsive insulin secretion functions¹⁰. Breakthroughs in chemical reprogramming technology have enabled the generation of hiPSCs without genetic manipulation, improving translational potential¹¹. In terms of culture systems, 3D culture combined with specific extracellular matrix and growth factor combinations can promote the self-organization of islet precursor cells into organoid structures composed of α, β, and δ cells¹². However, existing technologies still face numerous challenges, including unstable differentiation efficiency, insufficient functional maturity, and the lack of vascular networks and immune microenvironment—key limiting factors¹³ that restrict broader application.

Despite advances in pancreatic islet organoid-like structure research, several critical scientific issues in this field remain to be addressed¹⁴. First, the lack of standardized organoid definitions and construction procedures makes it difficult to directly compare results across different laboratories¹⁵. Second, current culture systems cannot fully replicate the complex cellular composition and precise spatial arrangement of native pancreatic islets¹⁶. Third, core issues such as functional stability during long-term organoid culture and posttransplantation survival remain insufficiently characterized¹⁷. In addition, most existing protocols still rely on animal-derived matrix materials, which suffer from undefined composition and large batch-to-batch variation, hindering quality control for clinical applications¹⁸. These knowledge and technical gaps seriously impede the translation of pancreatic islet organoid-like structures from basic research to clinical practice.

This protocol is designed to build a stable, affordable and repeatable approach to generate pancreatic islet organoid-like constructs using bone marrow mesenchymal stem cells (BMSCs). The established method is intended for laboratory experimental research and preclinical drug screening applications. Current strategies for BMSC-induced islet differentiation suffer from multiple limitations, including excessive costs, inconsistent operational standards and limited applicability. Most existing induction systems rely on costly recombinant cytokines, professional culture matrices and high-precision culture devices, which greatly restrict their widespread use. To solve these drawbacks, the present protocol adopts porcine pancreatic tissue lysate as the core induction factor. This optimized and simplified culture strategy enables the efficient differentiation of BMSCs into islet-like structures with only routine laboratory equipment and conditions.

Compared with conventional methods relying on hESCs, iPSCs, or high-cost growth factor cocktails, the present strategy offers several distinct practical advantages. It eliminates the need for expensive reagents and complex culture setups, reduces experimental costs, and enables consistent results with minimal inter-batch variability^10,11,12,13. Unlike xenogeneic matrix-based systems, this protocol uses a simplified tissue lysate, improving experimental controllability and reproducibility across laboratories^15,16,17,18. Furthermore, BMSCs are easily accessible, stable in culture, and free of ethical constraints associated with pluripotent stem cells, making this approach particularly suitable for routine laboratory use.

Existing research approaches are polarized in the field of pancreatic organoid modeling: advanced organoid platforms for clinical transformation are costly and technically intricate, while simplified alternative models fail to support in-depth functional analysis. The protocol proposed in this work bridges this research gap by offering a standardized, intermediate technical scheme. This approach preserves the core physiological properties of pancreatic islets and is fully compatible with conventional basic laboratory settings. It serves as a practical tool for researchers focusing on islet developmental mechanisms, diabetic disease modeling and preliminary drug candidate assessment, particularly for teams equipped with routine cell culture infrastructure but lacking professional stem cell culture platforms. Although this method cannot generate fully vascularized or immunologically competent organoids for specific advanced research, it is well-suited for preliminary experimental screening, mechanistic exploration and academic training in islet biological research.

This work focuses on constructing an economical and standardized culture system for pancreatic islet organoid-like structures, aiming to offer a credible three-dimensional research model for diabetes-related studies. By systematically analyzing how diverse induction regimens and culture environments affect organoid formation efficiency and morphological features, this research attempts to break through the major technical obstacles that restrict progress in this field. The core research objectives cover three key aspects: establishing a universal culture protocol applicable to ordinary basic laboratories without sophisticated instruments, formulating quantitative evaluation standards for assessing organoid quality, and optimizing culture strategies to sustain the morphological and functional stability of organoids during long-term incubation. The findings of this study can provide solid technical support for in vitro experimental exploration, while building a practical and reliable model for research on islet development mechanisms and the screening of hypoglycemic drugs.

Protocol

All experimental procedures adhered to the guidelines for laboratory animal welfare approved by the Ethics Committee of Guangxi Medical University (Approval No.: SYXK 2014-0003), and complied with the ethical codes for animal care and use specified in the International Guiding Principles for Biomedical Research Involving Animals formulated by the Council for International Organizations of Medical Sciences (CIOMS).

All operations involving tissue processing and reagent preparation must be performed in a Class II biosafety cabinet to prevent contamination. Allow at least 8 h to complete fresh tissue processing and pancreatic islet organoid-like structure inducer preparation. Keep all samples and reagents on ice throughout the experiment.

1. Experimental preparation

1. Euthanize 7-day-old Sprague Dawley (SD) suckling rats via cervical dislocation. Anesthetize domestic pigs via intravenous injection of pentobarbital sodium at a dose of 20 mg/kg, and confirm adequate anesthesia by disappearance of the pedal withdrawal reflex and stable respiratory rate.
2. Process porcine pancreatic tissue in a Class II biosafety cabinet under sterile conditions. Process fresh porcine pancreatic tissue within 24 h of collection. Store collected tissue samples at 4 °C in complete DMEM medium (as shown in Table 1) until processing.
3. Preset the refrigerated centrifuge to 4 °C and select the low-speed centrifugation mode to avoid damaging cell preparations. Store 1000 µL pipette tips, 200 µL pipette tips, and 50 mL centrifuge tubes in a −20 °C freezer until use in the experiment. Prepare sufficient 0.22 µm sterile filters and 20 mL disposable sterile syringes. Sterilize ophthalmic scissors, curved forceps, glass Petri dishes, and ampoules by high-temperature autoclaving (121 °C, 20 min).
4. Prior to experimental use, aliquot the fully prepared pancreatic tissue lysate. For optimal experimental consistency, pipette 100 μL of the homogenate into individual 1.5 mL microcentrifuge tubes for separate storage and subsequent use.

2. Processing fresh pancreatic tissue to prepare pancreatic islet organoid-like structure inducer

1. Harvest fresh porcine pancreatic specimens and transfer them onto dry ice immediately to sustain tissue bioactivity. The acquired tissues are first rinsed repeatedly with sterile normal saline via pipetting. Following the initial cleaning steps, 3 mL of sterile phosphate-buffered saline (PBS) is used for further thorough washing to completely eliminate residual blood stains and tissue contaminants.
2. Remove and discard any residual liquid from sterile culture plates, then supplement the plates with 2 mL of DMEM medium. The pretreated pancreatic tissues are minced into uniform 1 mm³ fragments with sterile ophthalmic scissors  (Figure 1A,B). All minced tissue pieces are collected into a 50 mL centrifuge tube, followed by gentle up-and-down pipetting for 10 times to achieve homogeneous mixing.
3. Tissue homogenization is conducted with a sterile glass pestle  (Figure 1C). During the grinding process, pancreatic tissue fragments are blended evenly with DMEM medium at a 1:1 volume ratio. The fully homogenized tissue suspension is transferred into a 50 mL centrifuge tube and subjected to high-speed refrigerated centrifugation at 21,600 × g for 30 minutes under 4 °C conditions.
4. After centrifugation, the clarified supernatant is slowly extracted with a sterile pipette. The harvested supernatant is then passed through a 0.22 μm sterile filter to eliminate tiny tissue debris and undissolved impurities (Figure 1D). The purified liquid is finally divided into 1.5 mL microcentrifuge tubes with a fixed volume of 100 μL per tube for sealed preservation and subsequent experiments (Figure 1E).
5. Store the aliquoted pancreatic islet organoid-like structure inducer at −20 °C.
   NOTE: The inducer remains stable for 1 year under proper storage conditions. Evaluate the induction activity of the prepared lysate by visual inspection and functional induction. Identify active lysate by its clear, pale pink appearance with no obvious precipitation; recognize loss of activity by visible precipitation and failure to induce typical islet organoid formation.

3. Preparation of induction medium

1. Prepare the induction medium freshly right before cell culture operations. Combine the self-prepared pancreatic islet organoid inducer with high-glucose DMEM medium containing 10% fetal bovine serum (FBS-H-DMEM) strictly in accordance with the proportion listed in Table 1.
2. Place the cultured pancreatic organoid samples in a cell incubator maintained at a constant temperature of 37 °C with 5% CO₂ saturation for continuous cultivation.

4. Morphological observation and growth monitoring of organoids

1. Microscopic observation is performed at scheduled time points throughout the culture period, including day 1, day 3, day 7, day 10 and day 15 after cell seeding.
2. A light microscope is adopted to track the morphological changes, growth conditions and cellular viability of pancreatic organoid-like structures during each detection session, so as to record the dynamic growth characteristics of the constructs.

5. Passage and cryopreservation of organoids

1. Seed passage 3 BMSCs at a density of 1 × 10⁵ cells per well in 6-well culture plates and culture in complete medium overnight to allow cell attachment before induction.
2. Add 1 mL of trypsin containing 0.02% EDTA to the culture system. Gently pipette the surface of the culture dish 5–8x to detach pancreatic organoids from the dish.
3. Add 2 mL of 10% H-DMEM-F12 medium to terminate trypsin digestion. Centrifuge the mixture at 1,500 × g for 5 min at 4 °C using a refrigerated centrifuge. Aspirate and discard the supernatant after centrifugation.
4. Add twice the volume of fresh 10% FBS-H-DMEM medium to the cell pellet to achieve a 1:2 passage ratio.
5. Perform cryopreservation of organoid cultures as follows: Resuspend the obtained cell pellet in 1 mL of serum-free cryopreservation solution. Store the resuspended organoids at −80 °C for short-term preservation (up to 1 year) and in liquid nitrogen for long-term storage.
   NOTE: Adjust the volume of fresh medium to achieve different passage ratios. For example, use the same volume of medium as the initial volume for a 1:1 passage, or add 3x the initial volume of medium for a 1:3 passage.

Results

Recording the growth dynamics of pancreatic islet organoid-like cultures over time is crucial, especially in the first few weeks of culture, as it allows predicting the performance of the culture system in subsequent experiments. Figure 2 shows a 22-day culture example, where BMSCs were efficiently isolated from the bone marrow of 7-day-old suckling rats and successfully used to generate pancreatic islet organoid-like structures. The identity of BMSCs was confirmed by trilin...

Discussion

Diabetes mellitus is a global metabolic disorder, mainly classified into type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM is an autoimmune disease characterized by the selective destruction of pancreatic β-cells, resulting in absolute insulin deficiency¹⁹. Patients require lifelong exogenous insulin therapy but still face the risks of severe hypoglycemia and microvascular complications²⁰. Islet transplantation is currently the only therapeutic ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.22 μm Sterile Filter	Merck Millipore	SLGP033RB	Sterile filtration of pancreatic tissue lysate to remove impurities and debris
0.25% Trypsin(within 0.02% EDTA)	Wisent	325-043-CL	Mild dissociation of pancreatic islet organoids for passage operation
1.5 mL microcentrifuge Tubes	Corning Axygen	AXY-20-1500	Aliquoting and storage of pancreatic tissue lysate (100 μL per tube)
10 μL Pipette Tips	Eppendorf	22491653	Liquid transfer for antibodies and small-volume enzyme solutions
1000 μL Pipette Tips	Eppendorf	22491651	Liquid transfer for reagents and cell suspensions
-20°freezer	Haier	HYC-326	Storage of reagents (e.g., FBS, trypsin) and pipette tips
200 μL Pipette Tips	Eppendorf	22491652	Precise liquid transfer for small-volume reagents (e.g., lysate aliquoting)
10 μL Pipette Tips	Haier	DW-86L626	Long-term storage of pancreatic tissue lysate and cryopreserved organoids
50 mL Centrifuge Tubes	Corning	430829	High-speed low-temperature centrifugation of pancreatic tissue lysate and cell suspension
6-Well Culture Plates	Corning	3516	Culture of pancreatic islet organoids and BMSCs
Class II Biosafety Cabinet	Haier	HR40-IIA2	Sterile processing of porcine pancreatic tissue and organoid culture operations
CO2 Incubator	Thermo Fisher Scientific	3111	Incubation of pancreatic islet organoids at 37°C, 5% CO2 environment
Curved Forceps	Shanghai Huaqi Medical Instruments Co., Ltd.	HQ-1302	Handling of pancreatic tissue during processing, high-temperature sterilized
DMEM medium (high glucose)	Wisent	318-025-CL	Used for pancreatic islet organoid induction culture
DMEM medium (low glucose)	Wisent	319-015-CL	Basal medium for BMSC culture and tissue processing
Fetal Bovine Serum (FBS)	Gibco (Thermo Fisher Scientific)	10099141C	Added to DMEM at 10% volume ratio for cell culture and digestion termination
H-DMEM-F12 medium	Wisent	315-080-CL	Used to terminate trypsin digestion during organoid passage
High-Temperature Autoclave	Haier	GR80DA	Sterilization of instruments (121°C, 20 min) and glassware
Light Microscope	Olympus	CX43	Observation and imaging of pancreatic islet organoids at different magnifications (×4, ×10, ×20)
Ophthalmic Scissors	Shanghai Huaqi Medical Instruments Co., Ltd.	HQ-1201	Cutting porcine pancreatic tissue into 1 mm³ fragments, sterilized by high-temperature autoclaving
Penicillin-Streptomycin Solution	Thermo Fisher Scientific	15140122	Used at 1% volume ratio to prevent bacterial contamination in culture medium
Phosphate-Buffered Saline (PBS)	Gibco (Thermo Fisher Scientific)	10010023	Washing buffer for porcine pancreatic tissue and cell rinsing
Refrigerated Centrifuge	Eppendorf	5810R	High-speed centrifugation at 4°C for pancreatic lysate preparation and cell pellet collection
Serum-Free Cryopreservation Solution	Suzhou Xinsaimai Biotechnology Co., Ltd.	#C40050	Used for cryopreservation of pancreatic islet organoids, resuspending cell pellets before -80°C storage