Spheroids - 3D Cell Culture

Get Better Results by Optimizing Your Image Capture Steps

Furthermore, cells in multiple planes, whether floating or living within a large organoid, can cause inconsistency with autofocus methods, bringing into question the reliability and reproducibility of later analysis steps. Autofocus failings can also be prevalent with gel-based scaffolds and other tools frequently used in 3D experiments. So how can you optimize your image capture steps for best results?

1. Slow the Carrier Speed: New in Gen5 3.08 is the ability to slow the speed with which the carrier enters the imager. This option is available in both Manual Mode and Experiment Mode, and is particularly recommended when imaging suspension or spheroid-based cultures during extended kinetics utilizing the BioSpa 8 Automated Incubator.

2. Add a Delay After Plate Movement: By default, Gen5 builds in a 300ms delay between carrier movement and image capture. This value can be reduced for faster acquisition OR increased to allow suspension cultures or spheroids to resettle after movement.

3. Optimize the Autofocus Method: Even for spheroid and suspension cultures, the laser autofocus provides superior speed and performance; however the unique conditions inherent in these cultures sometimes require further optimization. By de-selecting the Default focus method and settings, you gain additional freedom in defining parameters such as the scan distance and increment, or utilizing alternative methods, such as user-trained autofocus. Read more about Autofocus Methods in the Imaging Training Guide that was included with your BioTek Lionheart FX Automated Microscope or Cytation Cell Imaging Multi-Mode Reader.

4. Adjust the Correction Collar: While we're on the topic of focusing, don't forget to increase the correction collar settings on your 20x, 40x, and 60x objectives when working with thicker biology!

5. Call your Field Applications Scientist (FAS): When you're feeling stumped or frustrated or just want to brainstorm ideas to improve your assay (3D or anything else), contact BioTek's FASTeam for scientific support!

Automated Media Exchange of Non-Adherent Cells and Spheroids

Media exchange is an essential element of successful long-term cell culture in both high and low throughput experiments. Automated Media Exchange streamlines this process by reducing workflow duration, increasing reproducibility and therefore saving time and labor. However, some cell culture systems are sensitive to the disruptive effects of media exchange requiring specialized liquid handling equipment with optimized parameters for successful analysis post exchange. BioTek's patent-pending Automated Media Exchange (AMX) module, available on the MultiFlo FX, is designed to gently and reliably exchange media for biology sensitive to the disruptive forces of fluid movement. The AMX module uses two peristaltic pumps to gently aspirate, and subsequently dispense, media from up to eight wells simultaneously.

Here we demonstrate the ability of the AMX module to gently perform media exchange of spheroids. Importantly, the AMX module is able to exchange media with no discernable displacement of the well biology, as well as complete retention of suspension cell numbers. Technical tips are provided regarding media exchange settings and guidelines for optimization using the MultiFlo FX with AMX.

3D Spheroid media exchange in 96-well, round bottom ULA plate:

• Define the aspiration height for your vessel
  Careful empirical experimentation needs to be performed when working with suspension or non-adherent cells such as the formation of spheroids in ULA plates. The plate dimensions are required to determine a starting point for both aspiration and dispense heights as described above as well as substantially more residual volume in comparison to adherent cells. The composition of the spheroids, and subsequent level of cellular cohesion between cells in the spheroid, will determine the residual volume required. We found the 50% residual volume is a good starting point in a standard 96-well microplate format. For tightly formed, structurally stable spheroids, we found up to ~ 75% of media can be exchanged per cycle. The exact aspiration height required to leave behind a similar volume in your experiment is best determined empirically using your microplate, and your media.
• Use “slow carrier mode” feature for washing and imaging of suspension cells
  Normal handling including transporting the plate can disturb non-adherent cells. The slow carrier feature available in both Liquid Handling Control (LHC) and Gen5 software provides slow, smooth plate movements throughout the liquid handling and imaging processes. This feature was designed specifically to minimize disruption of sensitive cells and structures.
• Determine additional aspiration and dispense parameters
  The AMX module takes advantage of two peristaltic pumps: one for aspiration and one for dispense. The speeds can be individually optimized for each step. Begin by using the slowest aspiration and dispense rates and verify that your biology is undisturbed. Here we demonstrate spheroid media exchange using the slowest aspiration and dispense rates. As described above, the aspirate and dispense parameters will need to be optimized for the plate type and biological system. It is recommended to aspirate directly above spheroids when spheroid positioning needs to remain constant; centered in the well of a round bottom, ULA plate in this case. Visualization of this process is depicted in a short video found here: Automated Gentle Media Exchange for Spheroid Assays using the AMX module for MultiFlo FX.
• Dispense near the well edge
  The position of dispense and aspiration needles within the well is fully adjustable using LHC software. Dispensing media near the well edge allows a liquid bridge to form against the sidewall, and avoids droplets hitting the well media surface.
• Minimize the distance between the dispense needle and the final liquid surface
  Dispense needle height is fully customizable in LHC software. To avoid cross-contamination between wells, it is important that dispense needles are not submerged below the final media level in the well. Dispensing from far above the surface interface, however, encourages droplet formation and increases mixing forces. The dispense height was optimized to just above the final well volume of 200 mL for this 96-well vessel.

Best practices and tips:
Below is a short list of tips to consider when setting up AMX module media exchange for suspension cells:

• Start with the slowest possible aspiration and dispense settings, and modify as needed
  If total washing time is a factor for your experiments, increase dispense or aspiration speeds incrementally and always verify cell retention.
• Allow non-adherent cells to settle to the bottom of the well before washing
  Ensure cells have had adequate opportunity to settle to the bottom of the vessel before attempting a media exchange to avoid directly aspirating cells near the top of the liquid column. Typically, a media exchange will occur days after initial plating of cells, so most non-adherent cells will be well settled to the bottom of the vessel. Spheroids typically settle to the bottom relatively quickly once formed, on the order of minutes.
• Validate your exchange settings using relevant experimental liquids
  Although tempting to use less expensive and more abundant liquids such as saline or water; liquids demonstrate different liquid handling characteristics due to differences in physical properties such as surface tension. Therefore, the use of the final experimental liquid is recommended for validating settings, typically complete media.
• Leave more media in the well, and run more wash cycles
  As a general principle to avoid disturbing the biology at the bottom of the well, minimize the amount of media aspirated per wash cycle, and simply repeat the wash cycle more times to achieve a complete media exchange.

Application Notes

• Automated Media Exchange for Spheroid Cultures Using a Novel MultiFlo FX Accessory
• 3D Spheroid-Based Tumor Invasion Assay
• Brightfield and Fluorescence Imaging using 3D PrimeSurface® Ultra-Low Attachment Microplates
• An Image-Based Method to Detect and Quantify T Cell Mediated Cytotoxicity of 2D and 3D Target Cell Models
• Long-Term Hepatotoxicity Studies using Cultured Human iPSC-Derived Hepatocytes