Mastering Spheroid Culture: A Complete Guide to Cultivating Round and Uniform Spheroids

Mastering Spheroid Culture: A Complete Guide to Cultivating Round and Uniform Spheroids

Mastering Spheroid Culture: A Complete Guide to Cultivating Round and Uniform Spheroids

Spheroids have emerged as powerful tools for advancing biomedical research, with applications ranging from drug screening and toxicity testing to disease modeling and tissue engineering. To achieve reliable and reproducible results with spheroids, it is crucial to use the right culture system and follow proper techniques. In this guide, we will cover the best methods and protocols for culturing perfect spheroids from start to finish.

What is a Spheroid in cell culture?

A spheroid is a three-dimensional (3D) structure composed of cells that form when grown in culture plates. Spheroids are commonly used in cell assays, offering numerous physiological advantages which are impaired by traditional two-dimensional monolayer cultures. They effectively mimic the 3D microenvironment experienced physiologically, and therefore can more accurately predict drug responses and cellular behaviors.

The Steps Involved in Culturing Spheroids

Spheroid culture can be divided into four main steps – seeding, aggregation, maintenance, and harvesting. Seeding is the process of placing single cells or clumps of several cells on the surface of a culture plate and allowing them to attach to each other to form clumps. The cells then aggregate together to form spheroids that are visible after 2-5 days in culture. Once formed, regular medium changes should be performed to keep the spheroids fresh and healthy. Finally, when spheroids are big enough they can be harvested using methods such as pipetting.

Protocol for Successful Spheroid Formation

To ensure successful spheroid formation and maintenance, it’s important to focus on all four stages of the process: seeding, aggregation, maintenance, and harvesting. Seeding is traditionally achieved using lab-made or commercially available cell seeding plates and tools for uniform, high-density seeding. Additionally, selecting a plate with low adherence properties can reduce unspecific attachment and ultimately yield improved spheroids.

Aggregation is typically done at 33-37°C in incubators that provide optimal conditions for cell growth and survival. Aggregation can be challenging if the cells are not prone to aggregation or have low adhesion properties. This step is also crucial for determining the size and shape of the spheroids. It is important that the spheroids be uniform in size and shape to be useful for many applications, but achieving this can be difficult, especially with cell types that tend to form irregular shapes or large spheroids.

Additionally, maintaining an appropriate rate of medium circulation can help keep the contents fresh while providing ample nutrition for the continued development of the spheroids. Finally, careful harvesting techniques need to be employed in order to preserve the structural integrity of the spheroids prior to downstream applications.

Factors Affecting Spheroid Characteristics

When it comes to optimizing spheroid culture, there are many factors that can affect the growth of spheroids. These include reagents and media composition, cell type and number, culturing/incubator conditions (such as temperature, humidity, CO2 levels), culture microenvironment, and adding specific culture reagents like growth factors or extracellular matrix. Additionally, the choice of culture vessel is important as well it can impact spheroid size and shape, with specialized spheroid culture plates designed to promote spheroid formation and uniformity. For example, using a spinner flask or rotating vessels may improve morphology compared to static plates since rotation allows for better nutrient accessibility and increased oxygen diffusion. Finally, manipulating the surface properties of common tissue culture plastics via chemical treatments or protein coating may also help increase the viability and yield more uniform spheroids.

Methods of Observing and Measuring Spheroid Quality

The success of spheroid culture can be measured by assessing the quality of the produced spheroids. Quality control is usually regarded as a combination of size, shape, and cell viability. Measurement techniques used to observe these include bright field microscopy, phase contrast imaging (PCI), and fluorescence microscopy (FM). Bright-field microscopy is a widely used method because it is non-destructive and allows for qualitative information to assess the uniformity in size and shape of spheroids. PCI has better resolution than BR but suffers from a lack of contrast in tuning an image or quantifying individual or population values. FM provides quantitative results but requires pre-labeling with fluorescent probes which may lead to increased cell death due to probe uptake or attachment to cell surface receptors.

Tips to select the right culture vessel for your 3D culture.

Selecting the right spheroid culture plate is crucial for achieving successful spheroid culture experiments. There are various types of spheroid culture plates available in the market, with different features and benefits. When selecting a spheroid culture plate, consider factors such as the cell type, the intended application, and the culture method. For instance, some spheroid culture plates are optimized for hanging drop culture, while others are designed for rotary culture or static culture. Specialized spheroid culture plates can also have surface coatings or grooves to promote spheroid formation and uniformity. Additionally, the size and number of wells in the culture plate can impact spheroid size and yield. It’s essential to choose a spheroid culture plate that is compatible with your cell type and culture method and has the necessary features to promote spheroid formation and uniformity.

SphericalPlate 5D technology for high throughput production of speroids

As we are focused on helping researchers easily adapt and improve their work from bench to bed, we have been on the lookout for 3D culture vessels that can easily be used for translational science applications. We found a specialized spheroid culture plate by Kugelmeiers Ltd a spin-off from the University of Zurich in Switzerland focusing on developing regenerative medicine, cell therapy, developmental biology, and transplant surgery technology. Their Sphericalplate 5D allows easy control and formation of uniform round spheroids with undisturbed cell-to-cell communication to prevent expression of unwanted genes & differentiation.



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