Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Summary

This study presents a cost-effective and efficient methodology for generating 3D cell structures using a stamp-based system to create microwells in agarose molds. The system promotes the formation of uniform spheroids/organoids, thus improving cell interactions. This approach reduces experimental variability and supports applications in drug testing and tissue engineering.

Abstract

Three-dimensional (3D) cell cultures provide a more accurate representation of the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix. This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 °C under 5% CO₂, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization. The method successfully generated uniform and high-quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and cost-effective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings.

Introduction

Over the past 50 years, numerous cell biology investigations have demonstrated that two-dimensional (2D) cultures fail to accurately replicate the in vivo conditions observed in animal models¹. Structurally, 2D cell cultures do not allow cells to organize three-dimensionally and replicate the situation observed in in vivo systems. Furthermore, cellular signaling pathways are altered in 2D cultures compared to three-dimensional (3D) cultures, which could likely explain why certain types of drug screening using 2D cultures are so discrepant². A significant advancement in cell culture techniques emerged wi....

Protocol

This protocol follows the guidelines of the Human Research Ethics Committee of our Institution CEUA-FMUSP: 1699/2021, approved on September 8, 2021 -"Isolation and Encapsulation of Porcine Pancreatic islets" and is part of the Thematic Project of our Cell and Molecular Therapy NUCEL Group (www.usp.br/nucel), FAPESP Grant No. 2016/05311-2, entitled: “Regenerative Medicine aiming at therapy of chronic-degenerative diseases (cancer and diabetes)”.

1. Fabrication of the stamp device

NOTE: This Stamp is custom-made by the NUCEL group (https://w3nucel.webhostusp.sti.usp.br/). The stam....

Results

The cell culture used in this study was derived from porcine pancreatic islets. The islet preparation used in this study had an 80 ± 5% purity based on dithizone staining, and >80% islet cell viability based on the detection of fluorescein diacetate in live cells or propidium iodide in dead cells (the Live/Dead fluorescent method). Ensure that the porcine pancreatic islet preparation is at least 80% pure (e.g., by dithizone staining) and >80% viable. Upon isolation, maintain adherent cultures in CMRL 1066 me.......

Discussion

Although various 3D culture protocols exist in the literature, a study conducted by Wassmer et al.¹³ tested several methodologies for generating 3D structures using pancreatic islets. The authors observed that native islets and self-aggregated spheroids exhibited considerable heterogeneity with respect to size and shape and were larger than those obtained using other methods. Based on their findings, they concluded that spheroids can be generated using different techniques, each with its own advan.......

Materials

Name	Company	Catalog Number	Comments
31L Microwave	Electrolux	78965840 6699 9	Equipment used to heat the agarose solution, facilitating its dissolution and ensuring greater homogeneity. It allows the solution to reach the ideal liquid state for the formation of the wells.
3DFila Gray Opaque Photosensitive 3D Resin			UV-curable polymer resin
3D Printer - Creality Halot One	Creality	N/A	3D printer used for printing the stamp device
Agarose	UNISCIENCE	UNI-R10111	To form the gel, dissolve 1 to 2% in Saline Phosphate Buffer (PBS) or appropriate medium.
Autodesk Fusion 360			3D modeling
BB15 CO2 Incubator	Thermo Fisher	51023121	Equipment used to incubate cultured cells in a suitable and controlled environment.
Chitubox	Chitubox	N/A	Software used for slicing the part for printing
Class II Biological Safety Cabinet	Grupo VECO	N/A	Ensures a sterile environment for performing cell culture within established parameters and protocols.
Culture medium	USBiological/Life Sciences	C5900-03A	Contains additives for proper cell cultivation.
Culture plates (P6)	SARSTEDT	1023221	Used to shape the agarose and culture the cells.
Erlenmeyer Flask (25 mL)	Laborglas	91 216 14	A container used for dissolving 1–2% agarose in Phosphate Buffered Saline (PBS) or another suitable medium, typically heated in a microwave.
Falcon 15 mL Polystyrene Centrifuge Tube	Corning	352099	Used to keep cells in suspension and perform possible dilutions.
Fetal bovine serum (FBS)	Vitrocell Embriolife	005/19	Additive for culture medium.
PBS solution (Saline Phosphate Buffer)	Lab made	N/A	Diluted 1x with MiliQ ultrapure water. Used to dissolve agarose 1 to 2% and to wash wells already produced.
Reagent bottle with blue cap - Schott	Laborglas	21801545	Used for preparing and storing culture medium.
Stamp device	NUCEL Group	N/A	Link- This link provides access to the .stl file of the stamp device. Simply slice it using appropriate software and print it with a compatible 3D printer. https://drive.google.com/drive/folders/1gTYComnJWzHpN6ZKOyK EChKS3Qns0rOA?usp=sharing
Treated culture flask with filter 25 cm²	Corning	430639	Used for the cultivation and maintenance of adherent cells.
Trypsin	Merck	07-07-9002	For dissociation of cells before seeding.
Ultra violet light (UV)	N/A	N/A	Used to sterilize the stamp and plates.