Automating Tumor Implantation in Zebrafish Larvae for Cancer Research and Medicine

Summary

Here, we present a detailed protocol for automated tumor xenograft implantation into zebrafish larvae using a state-of-the-art robotic injection system. Furthermore, we present injection videos and validation data demonstrating the system's successful performance.

Abstract

Zebrafish larval injections, including subcutaneous and brain-orthotopic tumor xenografting as well as intravenous drug administration, have become central techniques in zebrafish-based research. These procedures, however, remain tedious, technically challenging, and highly dependent on operator skill, limiting throughput and reproducibility. To address these limitations, we introduce an advanced robotic system that automates microinjections into zebrafish larvae, significantly improving efficiency, precision, and reproducibility over manual injections. Here, we provide a detailed protocol for operating the automated injector, a high-throughput system capable of delivering liquids, particles, or cancer cells at three different anatomical sites with superior accuracy and speed compared to trained zebrafish researchers. We outline the essential steps, including setup, calibrations, injection procedures, and troubleshooting, to ensure optimal performance. Furthermore, we provide representative examples of results from using the robot and discuss its potential to facilitate large-scale larval injections, including for drug screens and precision medicine. By integrating an automated injector into the workflows, researchers can overcome limitations of manual injections and accelerate preclinical drug discovery and personalized treatment selection.

Introduction

As our understanding of cancer behavior and drug resistance improves, it has become increasingly clear that there is a need for better diagnosis, characterization, and therapeutic targeting of cancer subtypes. During the last twenty years, publications in the field of personalized medicine (also known as precision medicine) have been on the rise^1,2. Indeed, precision medicine is defined as the practice of tailoring medical treatment based on the individual patient's disease characteristics³. This follows our current understanding that there is no one-drug-fits-all approach, whi....

Protocol

Zebrafish lines were maintained according to standard protocols (https://zfin.org) and handled in compliance with local animal welfare regulations. These regulations implement the Council of Europe's Guidelines for the Protection of Experimental Animals (Directive 86/609/EEC), which permit the use of zebrafish larvae up to the onset of free feeding (5 days post-fertilization). As all larvae used in this study were no more than 5 days old, no ethical license was required under Directive 86/609/EEC.

NOTE: Dechorionated 48 hpf zebrafish larvae and the material to be injected in these larvae should be prepared prior to starting the pro....

Results

Here, we present the automated injection process for the three most used injection sites for cancer cell xenografts.

Video 1 demonstrates the automatic injection of the duct of Cuvier (DoC) using the robotic system. The robot first scans the plate systematically, moving from top to bottom and left to right, to locate a zebrafish larva. Once a larva is identified, the needle automatically approaches it, adjusting to the predetermined starting position and needle angle based on .......

Discussion

The development and implementation of the automated injector represent a significant advancement in the field of zebrafish tumor xenografting for anti-cancer drug screening and precision medicine. This is, to the best of our knowledge, the first commercially available robot for automated injection of cell suspensions into zebrafish larvae, and this protocol is therefore relevant for all research that involves such injections in high throughput. By automating the injection process, this system furthermore minimizes operat.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
6-well plate	Greiner bio-one cellstar	ref 657160
Agarose	VWR	#N605-100G
Automated injection system + consumables	Life Science Methods	https://www.lifesciencemethods.com/microinjection_system_for_zebrafish_larvae.html?nocache=1752058717774
Borosilicate glass capillaries	World Precision Instruments	N/A	1.0 mm OD, 0.78 mm ID
Cell counter	ThermoFisher	Countess 3
Commercial needles	Clunbury Scientific LLC	#B100-58-20
DNase type I	Roche	#11284932001
Filter for cancer cells	Sysmex CellTricsTM	04-0042-2316	30 µm
Mineral oil	Diapharma	#330779-1L
Needle grinder ()	Narishige	EG-401
Polyvinylpyrrolidone 40	Sigma	#102420477
GentleMACS Octo Dissociator	Miltenyi Biotec	https://www.miltenyibiotec.com/US-en/products/gentlemacs-octo-dissociator-with-heaters.html