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 diabet....

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-li....

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