Fundamentals of 3D Cell Culture

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Advancing research with 3D cell culture systems

3D cell culture systems are environments where cells and tissues are grown in three dimensions, offering great potential to advance our understanding of complex biological processes. These models are increasingly being used in many research applications, including drug discovery, toxicology, disease modeling, and regenerative medicine. These models offer an opportunity to better understand complex biology in a physiologically relevant context where 2D models have known limitations.

 

3D cell culture is a method where cells are grown in a way that allows them to self-organize into three-dimensional structures. This approach places the cells in a more physiologically relevant geometry, as cells in vivo naturally grow in a three-dimensional context and do not encounter the hard substrates typical of traditional 2D cell culture. Additionally, the three-dimensional geometry creates physiologically relevant nutritional gradients, where cells closer to the center of the 3D structures have less access to nutrients than the external cells. This mimics the nutrient gradients associated with vasculature in vivo. These gradients are particularly important when cells are treated with stimuli (e.g., anti-cancer compounds) and can significantly influence the response compared to cells grown in 2D.

 

There are several methods to culture cells in 3D, including hanging drops, low attachment vessels, suspension flasks, bioreactors, and extracellular matrix mimics. The choice of culture method depends on the cell type being grown, the culture media system, and the application (e.g., assay versus long-term culture). Here, we define the different types of 3D models, explain the cell types for which 3D cell culture is particularly applicable, and describe the considerations and benefits of utilizing 3D model systems.

Spheroids vs organoids and tumoroids


Organoids are typically derived from normal (non-malignant) human stem/progenitor cells (e.g., adult stem cells (ASCs), induced pluripotent stem cells (iPSCs)) and self-organize into tissue-like 3D structures when they are embedded in an extracellular matrix mimic. They often contain both stem cells and differentiated progeny and are usually grown in specialized serum-free media systems. ASC-derived organoids are generally grown exclusively in 3D from ASC derivation, and iPSCs may be expanded in 2D or 3D prior to organoid differentiation and culture exclusively in 3D.


Spheroids are models in which immortalized cell lines (e.g. MCF7, LNCaP, Hep G2) are grown in suspension or within an extracellular matrix mimic, forming roughly spherical clusters. 3D cell culture spheroids generally have simple media requirements; they are typically grown in a serum-based media system. Generally plated for expansion in 2D and plated in 3D only for assays.


Tumoroids (a.k.a cancer organoids, PDTO) comprise primary cancer cells derived from human tumor tissue and have only ever been grown in 3D self-organized multicellular structures. Tumoroids are typically grown embedded in extracellular cellular matrix mimics, but may be grown in suspension with specific culture conditions. These models are typically grown in specialized serum-free media systems.

Cell types for building 3D cell culture systems

Induced pluripotent stem cells

Induced pluripotent stem cells (iPSCs) are derived from somatic cells and have the ability to differentiate into any cell type in the body, making them a powerful tool for creating 3D models of various tissues and organs. iPSCs can serve as a particularly valuable platform for disease modeling, drug discovery, and regenerative medicine.

 

Adult stem cells

Adult stem cells, also known as somatic stem cells, are undifferentiated cells found throughout the body that can divide to replenish dying cells and regenerate damaged tissues. These cells are more limited in their differentiation potential compared to iPSCs, but are crucial for modeling specific tissues such as bone marrow, skin, and muscle as well as organ systems in 3D environment.

 

Cancer cells

Cancer cells are cells that divide uncontrollably due to accumulated oncogenic mutations. They are used in 3D models to study tumor growth, metastasis, and response to treatments, e.g. chemotherapeutics, immune cell therapies. By culturing cancer cells in 3D, researchers can better model tumors in vitro, with a more physiologically relevant microenvironment than traditional 2D culture.

 

Neural cells

Neural cells, including neurons and glial cells, are essential for creating 3D models of the nervous system. These models are used to study brain development, neurodegenerative diseases, and the effects of drugs on neural tissues. Advanced 3D models such as unguided cerebral organoids and guided neural organoids and assembloids, provide insights into the complex interactions within the brain and nervous system.

 

Hepatocytes

Hepatocytes are the main functional cells of the liver, responsible for a wide range of metabolic, detoxification, and synthetic activities. 3D models using hepatocytes are crucial for studying liver function, disease, and drug metabolism. These models help in understanding liver-specific responses and the impact of various substances on liver health.

3D Cell Culture Crash Course on-demand webinar

Join our on-demand 3D cell culture crash course webinar to gain a foundational understanding of this innovative technology, which better mimics the in vivo environment compared to 2D methods, enabling more accurate models for studying cell behavior, disease mechanisms, and drug responses. By the end, participants will be equipped to integrate 3D cell culture techniques into their research, leading to more relevant and innovative studies.

2D vs 3D cell culture

2D cell culture models grown from immortalized cell lines have served as a foundation for disease modeling and drug development for decades. These cell cultures are generally easy to grow and maintain but typically lack biological complexity and physiological relevance.

 

3D cell culture models were developed to imitate tissue-like or organ-like characteristics, to better recapitulate the cellular microenvironment in vivo as compared to 2D models. While typically more complex to culture than their 2D counterparts, 3D models they generally exhibit gene and protein expression signatures closer to those observed in vivo. 3D models, such as organoids and spheroids, are now being broadly adopted for in neurobiology, stem cell biology, regenerative medicine, and cancer biology research.

 

Why are researchers moving to 3D models?

  • Spatial complexity: 3D models more accurately replicate intercellular interactions in vivo by enabling spatial complexity with self-assembly in three-dimensions.
  • Lack of hard substrates: 3D models generally lack direct cellular contact with hard plastic substrates which are common in 2D culture. These hard substrates which have stiffness not commonly found in the body can impact cell signaling and lead to non-physiologically relevant responses to stimuli.
  • Gradients: 2D models have all cells are exposed to the same concentration of nutrients and compounds at the same time due to monolayer culture, which is not representative of in vivo dynamics. 3D models are subject to diffusion of nutrients and compounds from the exterior of the multicellular structures to the interior, which means cells within the structure are exposed to differing concentrations dependent on their positions. This is more representative of the cell stimulation in vivo which is governed by the relative location of a cell to the nearest blood vessel and how the characteristics of a given tissue type impact diffusion.
  • Representative gene and protein expression patterns: These models show gene and protein expression patterns that more closely resemble those found in living organisms than traditional cell lines, enhancing the reliability of experimental results.

Looking for more information to on how to choose between cancer models, read our Cancer organoids vs cancer spheroids blog. 

 

Spheroid

Organoid

Tumoroid

Cell source

Immortalized cell lines or primary cell

Stem cells (adult or pluripotent)

Patient-derived tumor cells

Example cell types

Cancer cell line, hepatocytes, iPSCs

Differentiated iPSCs and ASCs

Patient-derived tumor cells (e.g., epithelial cancers, neural cancers)

Media

Basal media (RPMI, DMEM, DMEM/F-12) or specialty media

Specialized media

OncoPro medium, homebrew methods

Culture format

3D (embedded or suspension)

3D (embedded or suspension)

3D (embedded or suspension)

Biological relevance

Low-to-moderate: low for immortalized cell lines, moderate for primary cells

Moderate-to-high: depending on model/complexity

High: highly correlated with patient genotype and phenotype, long term stability in vitro (reference)

Ease of use

Easy

Moderate-to-difficult

Moderate-to-difficult

Cost

Low: simple, standard workflows, reagents, consumables

Moderate-to-high: requirement for specialized media and cells

 

Extended culture periods

Moderate-to-high: requirement for specialized media and cells

 

Extended culture periods

For Research Use Only. Not for use in diagnostic procedures.