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 with the introduction of 3D culture systems. 3D systems vary considerably in complexity depending on cellular composition and cytoarchitecture. Generally, two types of structures are generated, namely: spheroids and organoids. Spheroids are described as simple clusters of cells obtained from normal or tumor tissues, embryoid bodies, and cell lines. The formation of 3D structures is influenced by various factors, including cell-cell interactions and signaling pathways mediated by components of the extracellular matrix (ECM), which provide structural support and biochemical cues. These elements regulate interactions that contribute to tissue organization and function³. The spheroid culture system was first described in the early 1970s, using V79 Chinese hamster lung cell lines as a model of nodular carcinomas, growing under non-adherent conditions and forming perfect spheres⁴. Organoids are described as clusters of organ-specific cell types derived from stem or progenitor cells, which self-organize through processes, such as cell sorting and lineage specification, in a spatially confined manner, mirroring the in vivo development⁵.

Several available methods and materials are available to culture cells under 3D conditions. The main methods currently employed for generating 3D cultures are: 1) hanging drops; 2) rotating cell culture and low-attachment plastics; 3) pyramid plates containing conical wells; 4) macroporous scaffolds; 5) magnetic beads; and 6) scaffold-free hydrogels.

Hanging drops is the method used to obtain scaffold-free 3D cultures. This method presents certain limitations, including the need for extensive handling, low production efficiency, spherical geometry, and exposure to high shear forces. Moreover, specific procedures, such as medium replacement or compound addition, can be challenging and may result in material loss. Furthermore, literature reports indicate that some cell lines fail to produce tightly packed spheroids when employing this approach⁶.

Rotating cell culture and low-attachment plastics are used to prevent cells from attaching to the substrate, causing them to aggregate and form spheroids. This process requires specific flasks and/or agitation/rotation. Although this is one of the most straightforward approaches for large-scale spheroids or organoids production, it is not without drawbacks, such as the need for specific equipment, low culture longevity, size variation in spheroids, mechanical damage to cells, and low efficiency⁶.

Pyramid plates containing conical wells are commercially available plates impacting costs, in addition to the fact that some manipulations may hinder the formation of spheroids/organoids⁷.

Macroporous scaffolds are also employed for 3D culturing; however, a major obstacle lies in achieving effective cell seeding and uniform distribution. This issue arises because the pore sizes may either be too small for cell penetration or too large to securely retain the cells. To address this issue, several strategies have been explored⁸, which directly impact the complexity and cost of this technique.

The magnetic beads methodology generates a small number of spheroids/organoids, has a high cost, and may leave nanoparticle residues inside the cells⁹.

Among the systems for cultivating spheroids, non-adhesive agarose hydrogels are available, representing a scaffold-free hydrogel. This approach offers notable benefits, such as precise control over the size of the 3D structures and the capacity to generate a substantial number of these structures per plate. In this method, cells are introduced into a hydrogel with preformed wells, in which they sink and self-assemble into 3D spheroids¹⁰.

In this study, we present a device and methodology for generating agarose microwells using a micropattern mold in a simple, efficient, reproducible, and low-cost manner.

The use of this stamp as a mold to generate microwells in agarose, aided by gravity, aims to enhance cell interaction within the microwell and cellular organization, generating 3D structures (spheroids/organoids) in vitro in a simple, efficient, reproducible, and low-cost manner, thus saving research time and laboratory resources.

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 stamp device was developed using the referenced software, widely recognized for its precision and advanced three-dimensional modeling capabilities. 

1. Ensure that the designed prototypes feature 663 micropins arranged to enable the generation of an equivalent number of three-dimensional cultures, such as spheroids or organoids. To ensure the stability of the stamp on T6 tray surfaces, position five support points strategically along the device's circumference.
2. Ensure that the micropins have a conical design with adjustable dimensions, ranging from 1 to 3 mm in length. The base diameter of the cone varies between 0.7 and 1.5 mm, while the taper angle ranges from 5° to 10°.
3. Make sure that the "stop" structure of the device, which defines the depth of the micropins, has an adjustable length of 10 to 19 mm and a diameter of 20 to 40 mm, with micropins uniformly distributed across its surface.
4. Additionally, design the device to have an ergonomic handle, suitable for one-handed operation, to enhance practicality and precision during use.
   NOTE: To facilitate large-scale production of the device, the digital file in .stl format is provided, being essential for the 3D printing process.
5. Simply slice it using appropriate software and print it with a compatible 3D printer. Use the following printing dimensions: X - 68 mm; Y - 120 mm; Z - 150 mm, ensuring an ideal balance between functionality and structural quality.
6. For the device's fabrication, use a photocurable 3D printer, which is compatible with the UV-curable polymer resin that must have high resolution and uniform surface finish, essential characteristics for optimal device's performance.
7. Slice the digital model for printing using software compatible with the selected 3D printer.

2. Preparation of agarose microwells

1. Prepare the agarose solution.
   1. Weigh the pure agarose powder and dissolve it in 1x phosphate buffered saline (PBS) to achieve a final concentration of 1-2% (w/v).
   2. Heat the solution in a microwave or water bath until fully dissolved, ensuring it is homogeneous and transparent.
      NOTE: Avoid overheating or excessive reheating, as this can degrade the agarose. Do not prepare large volumes of stock solution.
   3. Allow the solution to cool to ~40 °C, the ideal temperature for pipetting .
2. Prepare the custom-made stamp.
   1. Wash the stamp with a soft-bristle sponge and distilled water to remove residues.
   2. Expose the cleaned stamp to UV light for 5-10 min to ensure sterility.
3. Form the microwells.
   1. Pour/pipette ~3 mL of the 40 °C agarose solution into each well of a 6-well plate, ensuring a uniform depth of 2-3 mm.
   2. Position the custom-made stamp in the center of the well while the agarose is still warm.
      NOTE: The stamp has lateral supports that ensure alignment and stability, eliminating the need for manual support. Each stamp creates ~663 microwells (~2 mm in height) per well. Ensure there are no bubbles on the micropins; if bubbles form, gently move the stamp to eliminate them.
   3. Allow the agarose to solidify for 5-10 min at room temperature without moving the plate.
      NOTE: Do not disturb the agarose during solidification to avoid variations in the microwell structure.
   4. Remove the stamp with gentle back-and-forth motions to release the vacuum without damaging the microwells.
      NOTE: This movement facilitates air entry, allowing smooth stamp removal without deforming the gel. The microwells must remain intact.
   5. Wash the microwells 3x with PBS solution to remove residues.
   6. Expose the plate to UV light for 10 min to eliminate microbial contamination.
   7. Add 3 mL of culture medium and incubate the plate in a CO₂ incubator at 37 °C overnight.
      NOTE: This incubation verifies the absence of contamination before cell seeding.

3. Cell seeding in microwells

1. Prepare the cell suspension.
   1. Collect the cells via trypsinization or another dissociation method to obtain a homogeneous suspension.
   2. Count the cells and adjust the concentration according to the desired number per microwell.
      ​NOTE: To seed 5,000 cells per microwell in 663 microwells, prepare a suspension containing at least 3.315 × 10⁶ total cells. Optimize the cell concentration to prevent central necrosis and to ensure adequate nutrient and oxygen diffusion.
2. Seed the cells.
   1. Pipette ~3 mL of the prepared cell suspension over the microwells, ensuring even distribution by gently swirling the plate.
   2. Allow the cells to settle by gravity (~10-15 min) or perform gentle centrifugation at 100 × g for 5 min.
      ​NOTE: Centrifugation can be used to ensure the cells concentrate in the microwells.
   3. Transfer the plate to a CO₂ incubator at 37 °C with a humidified atmosphere.

4. Maintenance of 3D cell cultures

1. Replace the culture medium.
   1. Change the medium every 2-3 days, removing 50% of the old medium and adding fresh medium.
      NOTE: Ensure the microwells remain submerged throughout the culture period to prevent dehydration.
2. Monitor the formation of 3D cultures.
   1. Continue culturing until the 3D structures are fully formed and compact (e.g., 2-3 days for primary pancreatic islet cultures).
      NOTE: For cultures requiring a longer formation time, perform partial medium changes every 2-3 days.

5. Collection and characterization of 3D structures

1. Collect the spheroids/organoids.
   1. Remove the old medium and collect the 3D structures via vigorous pipetting using a wide-diameter or cut pipette tip to prevent compression or disaggregation.
      NOTE: Vigorous pipetting must be controlled to dislodge the aggregates without deforming them.
2. Prepare for analysis.
   1. Use the collected spheroids/organoids immediately or fix them for future analysis.
      ​NOTE: The association and stability of spheroids depend on cell type, formation time, and culture conditions. Take care is essential to avoid dissociation.

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 medium, s...

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

List of materials used in this article

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.