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

3D-Analyse von multizellulären Reaktionen auf tschoattraktive Verläufe

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Bioengineering

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JoVE Journal Bioengineering

3D Analysis of Multi-cellular Responses to Chemoattractant Gradients

3D-Analyse von multizellulären Reaktionen auf tschoattraktive Verläufe

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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.

Wir beschreiben eine Methode, um Geräte für die 3D-Kultur zu konstruieren und mit Zellen und multikellulären Organoiden zu experimentieren. Dieses Gerät ermöglicht die Analyse von zellulären Reaktionen auf lösliche Signale in 3D-Mikroumgebungen mit definierten Chemoattractged-Steigungen. Organoide sind besser als einzelne Zellen, wenn es um die Erkennung schwacher lauter Eingänge geht.

Unterschiede in vereinfachten 2D-In-vitro-Kulturen im Vergleich zu 3D-Gewebe-ähnlichen Umgebungen haben das Interesse an 3D-Systemen erhöht, um die räumliche und chemische Komplexität lebender Gewebe darzustellen. Der Herstellungsprozess erfordert keine Einrichtungs- oder Photolithographietechniken. Das 3D-PDMS-Gerät enthält jedoch die notwendigen Vektoren für 3D-Anwendungen in physiologischer Umgebung.

Verwenden Sie für die mesofluidische Gerätevorbereitung ein geeignetes dreidimensionales computergestütztes Design-Softwareprogramm, um die Maske der Form für das Polydimethylsiloxan- oder PDMS-Gerät zu entwerfen und die Form mit Stereo-Lithographiegeräten mit einem thermoresistenten Harz zu drucken. Wenn die Form fertig ist, mischen Sie grob drei Milliliter PDMS-Monomerlösung pro Form mit einem Härtungsmittel im Verhältnis 10 zu eins und verwenden Sie ein Vakuum, um die Mischung in einem Vakuum-Austrocknungsor für eine Stunde zu entgasen. Am Ende der Austrocknung verwenden Sie ein Stück Klebeband, um Staub von der Oberfläche der Form zu entfernen und die Form vorsichtig mit der entgasten PDMS-Lösung zu füllen.

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