3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Tae-Yun Kang; David Ellison; Sung Hoon Lee; Andrew  J. Ewald; Andre Levchenko

doi:10.3791/59226

Análisis 3D de las respuestas multicelulares a los degradados Quimioattractant

JoVE Journal
Bioengineering

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JoVE Journal Bioengineering

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

Análisis 3D de las respuestas multicelulares a los degradados Quimioattractant

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05:57 min

May 24, 2019

DOI: 10.3791/59226-v

Tae-Yun Kang¹, David Ellison², Sung Hoon Lee¹, Andrew J. Ewald^2,3, Andre Levchenko¹

¹Department of Biomedical Engineering and Yale Systems Biology Institute,Yale University, ²Department of Biomedical Engineering,Johns Hopkins University, ³Center for Cell Dynamics and Department of Cell Biology,Johns Hopkins University

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Overview

This article describes a method for constructing devices that facilitate 3D culture and experimentation with cells and multicellular organoids. The device enables the analysis of cellular responses to soluble signals in 3D microenvironments featuring defined chemoattractant gradients.

Key Study Components

Area of Science

Neuroscience
Cell Biology
3D Cell Culture

Background

2D in vitro cultures lack the spatial and chemical complexity of living tissues.
3D systems are gaining interest for their ability to better mimic in vivo environments.
The fabrication process of the 3D device does not require specialized facilities.
PDMS devices are suitable for physiological environment applications.

Purpose of Study

To develop a method for creating 3D culture devices.
To analyze cellular responses in a controlled 3D environment.
To demonstrate the advantages of organoids over single cells in detecting weak signals.

Methods Used

Utilization of 3D CAD software for mold design.
Printing molds using stereolithography with thermoresistant resin.
Mixing PDMS monomer solution with a curing agent.
Degassing the mixture in a vacuum desiccator.

Main Results

The 3D PDMS device allows for effective analysis of cellular responses.
Defined chemoattractant gradients enhance the study of cellular behavior.
Organoids demonstrate superior detection capabilities compared to single cells.

Conclusions

The developed method provides a robust platform for 3D cell culture.
This approach can advance research in cellular responses to environmental signals.
3D organoids are a promising tool for studying complex biological interactions.

Frequently Asked Questions

What are the advantages of 3D cultures over 2D cultures?

3D cultures better mimic the spatial and chemical complexity of living tissues, leading to more accurate biological responses.

How is the PDMS device fabricated?

The PDMS device is fabricated by designing a mold using CAD software, printing it, and then mixing and curing the PDMS solution.

What is the significance of chemoattractant gradients?

Chemoattractant gradients are crucial for studying how cells respond to soluble signals in a controlled environment.

Why are organoids preferred for certain experiments?

Organoids can detect weak signals more effectively than single cells, making them valuable for studying cellular responses.

What materials are used in the device construction?

The primary material used is polydimethylsiloxane (PDMS), known for its biocompatibility and ease of use.

Can this method be applied to other types of cells?

Yes, the method can be adapted for various cell types and applications in biological research.

Describimos un método para construir dispositivos para la cultura 3D y la experimentación con células y organoides multicelulares. Este dispositivo permite el análisis de las respuestas celulares a señales solubles en microambientes 3D con degradados de quimioattractant definidos. Los organoides son mejores que las células individuales en la detección de entradas ruidosas débiles.

Las diferencias en cultivos in vitro 2D simplificados frente a entornos similares a tejidos 3D han aumentado el interés en los sistemas 3D para representar la complejidad espacial y química de los tejidos vivos. El proceso de fabricación no requiere instalaciones ni técnicas de fotolitografía. Sin embargo, el dispositivo 3D PDMS incluye los vectores necesarios para aplicaciones de entorno fisiológico 3D.

Para la preparación de dispositivos mesofluídicos, utilice un programa de software de diseño tridimensional adecuado para diseñar la máscara del molde para el dispositivo polidimetilsiloxano o PDMS e imprimir el molde utilizando equipos de litografía estéreo con una resina termorresistente. Cuando el molde esté listo, mezcle aproximadamente tres mililitros de solución monórica PDMS por molde con un agente de curado en una proporción de 10 a 10 y utilice un vacío para desgasificar la mezcla en un desecador de vacío durante una hora. Al final de la desicación, utilice un pedazo de cinta adhesiva para eliminar cualquier polvo de la superficie del molde y llene cuidadosamente el molde con la solución PDMS desgasada.

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