3D cell culture illustration

Culturing Cells in 3D: A Step-by-Step Guide

Soraya Malik in Science & Nature December 2025 • 4 min read.

"Unlock the secrets to establishing and characterizing primary cell cultures in three dimensions, and discover how this innovative approach is revolutionizing biomedical research."

Traditional two-dimensional (2D) cell cultures have long been the cornerstone of biological and medical research. However, these simplified systems often fail to accurately replicate the intricate environment found within living tissues. This limitation has spurred the development of three-dimensional (3D) cell culture techniques, offering a more realistic and physiologically relevant platform for studying cell behavior.

3D cell cultures provide cells with a more natural environment, allowing them to interact with each other and the surrounding matrix in a way that closely mimics in vivo conditions. This leads to more accurate results in studies of cell growth, differentiation, drug response, and disease mechanisms. The shift towards 3D cultures is particularly relevant in fields like cancer research, regenerative medicine, and drug discovery, where understanding complex tissue interactions is crucial.

This article dives into the methods for establishing and characterizing primary cell cultures in a 3D environment. Drawing from research on innovative approaches, we will provide a step-by-step guide, highlight the benefits of 3D cultures over traditional 2D methods, and discuss the potential of this technology to revolutionize biomedical research. Whether you're a seasoned researcher or new to the field, this guide will equip you with the knowledge to harness the power of 3D cell cultures in your own work.

Setting Up Your 3D Cell Culture: A Practical Guide

Creating a successful 3D cell culture requires careful consideration of several factors, including the choice of scaffold material, cell seeding density, and culture conditions. The following steps will guide you through the process:

Selecting the Right Scaffold: Scaffolds provide structural support and promote cell attachment and growth in 3D cultures. Common scaffold materials include:

Collagen: A natural extracellular matrix protein that provides excellent cell adhesion and biocompatibility.
Alginate: A polysaccharide derived from seaweed that forms a gel-like matrix, allowing for easy cell encapsulation.
Synthetic hydrogels: These materials can be tailored to mimic specific tissue properties and provide controlled degradation.

Cell Seeding and Culture Conditions: The initial number of cells seeded into the scaffold and the subsequent culture conditions play a crucial role in the success of your 3D culture. Here are some key considerations:

The Future of 3D Cell Cultures: Transforming Biomedical Research

3D cell culture is more than just a laboratory technique; it represents a paradigm shift in how we approach biomedical research. By bridging the gap between traditional 2D cultures and complex in vivo models, 3D cultures offer a powerful platform for studying cell behavior in a more realistic and physiologically relevant context.

As technology advances, we can expect to see even more sophisticated 3D culture systems emerge, incorporating microfluidics, biosensors, and advanced imaging techniques. These innovations will enable researchers to precisely control and monitor the cellular microenvironment, leading to new insights into disease mechanisms and the development of more effective therapies.

Whether you're exploring new drug candidates, investigating complex tissue interactions, or developing personalized medicine strategies, 3D cell cultures hold the key to unlocking new frontiers in biomedical research. Embrace this technology and join the growing community of scientists who are shaping the future of healthcare.

About this Article -

This article was crafted using a human-AI hybrid and collaborative approach. AI assisted our team with initial drafting, research insights, identifying key questions, and image generation. Our human editors guided topic selection, defined the angle, structured the content, ensured factual accuracy and relevance, refined the tone, and conducted thorough editing to deliver helpful, high-quality information.See our About page for more information.

This article is based on research published under:

DOI-LINK: 10.1242/bio.023911, Alternate LINK

Title: Correction: Innovative Approaches To Establish And Characterize Primary Cultures: An Ex Vivo 3D System And The Zebrafish Model

Subject: General Agricultural and Biological Sciences

Journal: Biology Open

Publisher: The Company of Biologists

Authors: Chiara Liverani, Federico La Manna, Arwin Groenewoud, Laura Mercatali, Gabri Van Der Pluijm, Federica Pieri, Davide Cavaliere, Alessandro De Vita, Chiara Spadazzi, Giacomo Miserocchi, Alberto Bongiovanni, Federica Recine, Nada Riva, Dino Amadori, Ennio Tasciotti, Ewa Snaar-Jagalska, Toni Ibrahim

Published: 2017-02-08

Everything You Need To Know

What are 3D cell cultures?

3D cell cultures are a method of growing cells in a three-dimensional environment, which more closely resembles the natural environment found within living tissues compared to traditional 2D cell cultures. The significance lies in providing a more realistic platform for studying cell behavior, allowing for more accurate results in studies of cell growth, differentiation, drug response, and disease mechanisms. Implications include advancements in cancer research, regenerative medicine, and drug discovery due to a better understanding of complex tissue interactions.

What are scaffolds in 3D cell culture, and why are they important?

Scaffolds are crucial in 3D cell cultures because they provide structural support and promote cell attachment and growth. Common scaffold materials include Collagen, Alginate, and Synthetic hydrogels. Collagen is a natural extracellular matrix protein offering excellent cell adhesion and biocompatibility. Alginate, derived from seaweed, forms a gel-like matrix for easy cell encapsulation. Synthetic hydrogels can be tailored to mimic specific tissue properties and offer controlled degradation. The choice of scaffold influences the success of the 3D culture by affecting cell behavior and the overall environment.

What is cell seeding density in 3D cell culture?

In 3D cell culture, cell seeding density refers to the initial number of cells introduced into the scaffold. This, along with culture conditions, plays a key role in the success of the 3D culture. Maintaining optimal conditions is essential for cells to thrive in the 3D environment and to ensure the experiment's validity. Poor cell seeding or inadequate conditions may lead to inaccurate results. The specific cell seeding density is determined by the cell type and the scaffold type used.

Why are 3D cell cultures significant in biomedical research?

3D cell cultures are important in biomedical research because they offer a more realistic and physiologically relevant platform for studying cell behavior compared to traditional 2D cultures. This leads to more accurate results, especially in fields such as cancer research, regenerative medicine, and drug discovery. By mimicking the complex interactions found in living tissues, 3D cultures allow researchers to gain a better understanding of disease mechanisms and develop more effective therapies. The implications are a shift towards more predictive and effective research models.

What is the difference between 2D and 3D cell cultures?

The primary difference is that 2D cultures grow cells on a flat surface, such as a petri dish, while 3D cultures provide an environment where cells can interact in all three dimensions. 2D cultures often fail to accurately replicate the complex environment found within living tissues, leading to less realistic results. In contrast, 3D cell cultures allow cells to interact with each other and the surrounding matrix in a way that closely mimics in vivo conditions, leading to more accurate data regarding cell growth, differentiation, drug response, and disease mechanisms. This shift towards 3D cultures is therefore pivotal for more relevant and successful biomedical research outcomes.