A Robust Spontaneous Spheroid Generation Protocol

In the world of cellular biology, the generation of spheroids has become an essential method for studying cell behavior, drug responses, and tissue engineering. At Cellorama, we have honed a spontaneous spheroid generation protocol that emphasizes simplicity and reliability. This article outlines the features of our protocol, the steps involved, and the benefits that it provides for your research.

Understanding Spheroid Technology

Spheroids are three-dimensional cellular aggregates formed from single-cell suspensions. Unlike traditional two-dimensional cultures, spheroids more closely mimic in vivo events, providing a more accurate representation of cellular interactions and environments. With their ability to better replicate the cellular microenvironment, researchers have observed that spheroids can yield more relevant results in drug testing, cancer research, and regenerative medicine.

The need for effective spheroid generation techniques has become apparent, especially as scientists look for more reliable methods to create these structures. The spontaneous spheroid generation protocol developed by Cellorama offers a straightforward and efficient approach to this task. 

Advantages of Cellorama's Protocol

Here are the key advantages of Cellorama's spheroid generation protocol:

1. Simplicity: Our protocol prioritizes ease of use, unlike other spheroid generation methods that often require complex equipment and multi-step procedures. This is particularly beneficial for researchers without access to highly specialized lab equipment.

2. Reliability: Extensively tested, our protocol consistently delivers successful spheroid formation across various cell types. This reliability ensures reproducible outcomes, earning the trust of scientists. You can view light and electron microscopic images of spheroids from diverse experiments on our webpage at www.celloramatech.com.

3. Scalability: The protocol easily scales to fit diverse experimental designs, from small-scale studies to larger applications, making it an excellent choice for laboratories of any size.

4. Minimal Handling: The spontaneous nature of the protocol reduces the need for manual spheroid manipulation. This lowers the risk of contamination and improves the integrity of your experimental results.

5. Powerful Experimental Model: Our protocol provides a great experimental model for studying the interaction of cells in a 3D environment, invasion, metastasis, and for comparative studies to understand cell behavior in small or large spheroids.

Step-by-Step Protocol for Spontaneous Spheroid Generation

Understanding the steps involved in our spontaneous spheroid generation protocol is crucial for successfully implementing it in your laboratory. Below, you’ll find a clear and concise guide to the procedure.

This protocol outlines the steps for generating spheroids in a Hydrogel-Based System.  The use of a hydrogel provides a supportive 3D environment that encourages cell-cell aggregation and spheroid formation. The hydrogel serves as a scaffold, enabling cells to spontaneously organize into a spherical shape while mimicking key aspects of the extracellular matrix. With this protocol, cells naturally self-assemble into 3D structures under the right conditions. The protocol is highly reproducible, relatively quick, predictable, and yields high-quality spheroids across different experimental runs.

Step 1: Cell Preparation

• Begin by selecting appropriate cell lines. Cells can be derived from various sources, including tumor cells, stem cells, or other specific cell types.

• Collect and count the cells to achieve a desired seeding density. Typical concentrations range from 1 × 10^4 to 1 × 10^6 cells per milliliter, depending on the specific cell line and the experiment's goals.

  
  

Step 2: Medium Selection

• Select a suitable culture medium that supports cell growth and spheroid formation. We recommend using serum-free media. You may need to add some growth factors based on the cell type and the project. 

• A medium that maintains cell viability while facilitating spontaneous aggregation is critical.

  
  

Step 3: Generation of Spheroids

Day 0: Preparation

1. Thaw Hydrogel: Place the hydrogel on ice overnight in a refrigerator or a cold room to thaw completely.

2. Warm Your Cell-Culture Dish: Place the cell culture dish/plate, organonchip device, in which you will generate spheroids, in the incubator (37 °C, 5% CO2) one day before the experiment to warm up.

3. Chill Pipette Tips: Place sterile, wide-ended pipette tips in the refrigerator at 4 °C.

Day 1: Cell Seeding

1. Hydrogel Acclimation: Place the thawed hydrogel on ice in a laminar flow hood for 15 minutes.

2. Cell Pellet Preparation: Place the tube containing the cell pellet on ice.

3. Create Gel Drop: Plate 30–35 µL of hydrogel within the niche of the pre-warmed dish/plate to create a gel drop.

4. Seed Cells: Carefully place cell pellet in the middle of the top of each hydrogel drop ( Figure 1). Incubate for 15 minutes at 37 °C.

5. Add Medium: Gently cover the hydrogel drop with 200 µL of appropriate cell culture medium.

6. Incubate Dish/Plate: Incubate the cells under optimal culture conditions designed for your specific cell type, typically at 37 degrees Celsius in a 5 percent CO2 environment.

Figure 1: Schematic Representation

This figure illustrates the process of generating spheroids using our protocol. It shows a schematic representation of seeding cells on top of a hydrogel drop, followed by the formation and presence of spheroids within that hydrogel.

Day 3 Onwards: Maintenance

1. Feed Cells: Feed the cells with the appropriate medium every other day. Be very gentle while aspirating and replacing the medium surrounding the gel to avoid disturbing the gel and the spheroids.

Step 4: Observation

Monitor spheroid formation over 24 to 72 hours. Depending on your cell line, you'll observe the cells aggregating into round structures (Figure 2).

Figure 2: Liver Cancer Spheroids

This figure presents various views of liver cancer spheroids generated using the protocol.

(A) Macroscopic view of liver cancer spheroids visible to the naked eye. This panel provides a low-magnification orunaided eye view, showing the overall appearance and presence of the spheroids.

(B) and (C) These panels present microscopic images, offering detailed views of the spheroids' morphology and structure at varying levels of magnification, 10 days after their formation in 3D culture.

TECHNICAL NOTES: 

Pre-warmed Cell Culture Dish/Plate: Warming the dish/plate for at least four hours before starting the experiment and placing the hydrogel within a specific, warmed niche helps create a consistent and controlled environment for initial cell seeding and aggregation. This standardization minimizes variability that could hinder spontaneous formation.

Controlled Cell Seeding: Placing a specific number of cells in the middle of the hydrogel drop ensures a consistent starting density and distribution, which is crucial for reproducible spontaneous aggregation. The spheroid generation time may vary depending on the specific cell type used and the number of cells seeded. As an example, we seed 1 x 10^4 HepG2 cells to generate liver cancer spheroids, and spheroids typically start to form within 3 to 5 days (Figure 2).

Choosing the Right Cell Culture Vessel: For imaging spheroids directly in your culture vessel, we highly recommend using one with a high-optical quality bottom. While any cell culture vessel can be used with this protocol, optimal imaging is achieved with options like glass coverslips, glass-bottomed dishes, microchambered slides, and CellO-M dishes.

Choosing the Right Hydrogel: This protocol has been tested with various commercial and customized hydrogels. It's essential to note that the nature of the hydrogel, including its composition and stiffness, has a significant impact on the results.

Practical Applications of Spheroids in Research

Spheroids have numerous applications in biomedical research that demonstrate their importance in real-world scenarios:

1. Drug Testing: Spheroids are increasingly used in preclinical drug testing, offering a more accurate prediction of drug responses compared to traditional two-dimensional cultures. This enhances the reliability of results in screening potential therapeutics.

   
   

2. Cancer Research: Tumor spheroids can replicate the growth patterns and behaviors of actual tumors, allowing researchers to study tumor microenvironments and how they respond to various treatments.

   
   

3. Regenerative Medicine: Spheroids are crucial for understanding tissue regeneration and healing. Researchers use them to create scaffolds for tissue engineering and to study cell differentiation.

   
   

As more laboratories adopt our spontaneous spheroid generation protocol, its applications in research continue to expand, making it a valuable asset in various fields.

Cellorama's Commitment to Cell Culture Excellence

At Cellorama, we are committed to providing innovative solutions that empower researchers. 

Making informed choices in the laboratory is crucial for successful research outcomes. By utilizing Cellorama's robust spontaneous spheroid generation protocol, researchers can simplify their processes while ensuring the reliability of their results. As you strive to advance your studies in cellular biology, leverage our expertise to enhance your understanding and application of spheroids.

Elevate Your Research with Cellorama

Explore the many possibilities that await you with Cellorama and take the next step towards improved research methodologies:

CellO-IF3 revolutionizes your workflow by enabling immunofluorescence labeling of your spheroids while they are still embedded in a hydrogel. This preserves the delicate 3D structure, leading to more accurate and insightful imaging.

Our CellO-M cell culture vessel creates an ideal environment for spheroid generation, microscopic analysis, and even electron microscopy of these delicate structures, all while they remain in hydrogel in a CellO-M. This integrated approach streamlines your research and maintains sample integrity.

We invite you to reach out for further information about our protocols and products, and to learn how Cellorama can support your scientific endeavors.

By adopting this reliable and straightforward method for spontaneous spheroid generation, you position your research for success, leading to more accurate insights and significant advancements in your field.

Images Credit: The spheroid images presented herein were generated by Gamze Demirel.

Reference: Tok, O. E., Demirel, G., Saatci, Y., Akbulut, Z., Kayalar, O., Aktas, R. G. Culturing, Freezing, Processing, and Imaging of Entire Organoids and Spheroids While Still in a Hydrogel. J. Vis. Exp. (190), e64563, doi:10.3791/64563 (2022).