5 Pipetting Tips for Reproducible, Picture-Perfect Organoid Cultures

3D cell models like organoids are an exciting area of research because they can bridge the gap between traditional 2D cell cultures and complex human tissues. But for those working in this area, pipetting organoids can be a source of frustration. Here are our top five best practices for pipetting organoids.

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What are organoids and why are they exciting?

Inside the body, cells are organized into three-dimensional tissues and organs, where they interact with a complex microenvironment. Capturing cellular biology at this level is simply not possible with traditional 2D cell cultures.   

3D cell models, on the other hand, are cultivated in an environment that’s more like the conditions found in living organisms.

Organoids , a type of 3D model, are essentially miniaturized versions of organs. They mimic the architecture, function, and even some of the complexities of real organs, making them a powerful tool for various fields, like disease modeling, drugs testing, and personalized medicine.

How are organoids cultivated in the lab?

All 3D cultures need an initial cell source – for organoids, that source can be stem cells. Growing organoids in the lab involves coaxing stem cells to self-organize and differentiate into 3D structures.

The process starts by culturing stem cells, like induced pluripotent stem cells (iPSCs), in a specialized medium. This rich medium provides the stem cells with growth factors, nutrients, and everything else they need to grow and differentiate into a specific cell type.

Growing the organoids is a long process with many adjustments to culture conditions, which encourage organoid formation and maturation.

Pipetting in 3D organoid culture workflows

Pipetting is a fundamental part of working with 3D cell culture at various stages. While each touchpoint may last only a few seconds, using the right equipment and technique is important for creating a successful organoid culture and maintaining consistency.

Growing 3D cultures, like organoids, is fundamentally different than 2D cultures. Seeding the source cells within the special growth medium requires more skill and finesse to ensure an even distribution of cells. Accurate pipetting also comes into play when changing the medium, adding nutrients, or testing for drug response.

Gentle and consistent pipetting technique are also crucial for preserving the integrity of 3D organoid structures during cell harvesting for analysis.

Why are organoid cultures hard to pipette?

3D cell models are more fragile compared to cells spread out in 2D. Pipetting too vigorously can disrupt these structures and affect cell behavior and survival, so it’s important to go slow during media exchanges and other manipulations.

Also, for the organoids to grow evenly and produce reliable data, you need an even distribution of cells within the 3D structure. Pipetting inconsistently can lead to clumps of cells in one area and fewer in another.

Matrix mediums used to support organoid formation, like Matrigel or synthetic hydrogels, are also tricky to work with. These liquids are very viscous and difficult to pipette in general, so you need to adjust your pipetting technique accordingly.

Five pipetting tips for working with organoids

Pipettes are complicated instruments, but most scientists jump right in without much training. Aside from cleaning and maintenance, there are so many variations on the basic pipetting technique that can make a huge difference when working with challenging samples, like 3D cell cultures.

Here are our top 5 pipetting tips when handling 3D cell cultures:

1-    Adjust your technique

By adjusting your pipetting technique, you can pipette 3D cultures more gently and more reproducibly. Speed is important; pipette slowly to avoid disturbing the delicate structure of 3D cell models. For viscous solutions, like hydrogel, reverse pipetting is the way to go. Also, some organoid preparations need mixing; an electronic pipette   can help you find and program the perfect mixing time and speed for consistent results.

2-    Use the right pipette tips

The pipette and tip work as one system— this point was also emphasized in the latest ISO 8655:2022 guidelines for pipette calibration . For the best results, you should always use both pipettes and tips from the same manufacturer. Also consider different tip options; wide-bore tips, for example, are great for some 3D cell culture applications.

3-    Standardize your protocols

Small differences in pipetting technique can cause variability in the data. Develop standardized pipetting protocols for your 3D cell culture workflow, so that everyone in the lab follows the same procedures. With electronic pipettes, like Picus^® 2, it’s possible to connect to any workflow management system and execute pipetting steps the same, independent of the user.

4-    Don’t ignore ergonomics

Pipetting for long periods can get tiring. An ergonomic setup and proper pipetting technique can reduce fatigue and improve your overall precision. Ergonomically designed pipettes   are noticeably more comfortable to use and really helpful at preventing hand strain.

5-    Maintain your pipette

Pipettes that are out of calibration can introduce variability into your cell experiments. Follow the manufacturer’s recommended maintenance schedule and regularly calibrate your pipettes to keep them in top working order.

Continue your learning about best pipetting practices by checking out these three great resources on preparing 3D cell culture, proper pipetting technique for different scenarios, and improving reproducibility.