3D Cell Culture Assays
- Single Cells in a 3D Matrix
- Spheroid and Organoid Culture
- Chemotaxis and Migration Assays in 3D
- 3D Cell Culture Under Flow
Single Cells in a 3D Matrix
In many cases, a 3D environment more closely resembles an in vivo situation than a 2D cell culture. Single cells can be cultured and imaged in a 3D gel in order to analyze diverse biological questions, such as cell deformation, migration, tube formation, or ECM degradation. In addition to cultures with only one cell type, the invasion behavior of two different cell types (e.g., cancer cells and fibroblasts) can also be investigated by co-culturing them in the same vessel.
In order to isolate cells from the gel matrix, the matrix can be degraded enzymatically (e.g., collagen by collagenase). After this, the cells can be either expanded in a new gel matrix or further processed for DNA, RNA, or protein isolation.
LifeAct-expressing HT-1080 cells (green) in a Collagen Type I, Rat Tail layer in the µ-Slide Chemotaxis.
ibidi SolutionsThe ibidi Collagen Type I is a non-pepsinized, native collagen for modeling biological ECM in gel matrices. Its fast gelation facilitates optimal cell distribution in 3D gels. Read our Application Note about how to prepare a 3D gel using the ibidi Collagen Type I here: AN 26: Preparation of Collagen I Gels. |
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In the µ-Slide III 3D Perfusion, single cells are embedded in a 3D matrix. The special channel geometry allows for superfusion with a low flow rate (e.g., when utilizing the ibidi Pump System). Unlike in static cultures, the superfusion ensures optimal oxygen and nutrient supply. This setup makes long-term cultivation possible for up to several weeks. Additionally, the thin coverslip bottom allows for high-resolution imaging. |
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The µ-Slide 15 Well 3D and the µ-Plate 96 Well 3D allow for easy, cost-effective cultivation and microscopy of single cells and co-cultures on, or in, 3D gels. The gel layer is directly connected to the medium reservoir above, enabling fast and easy medium exchange by diffusion. For special applications, the µ-Slide 15 Well 3D Glass Bottom, with a No. 1.5H glass bottom, is also available. |
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The μ-Slide I Luer 3D is designed for culturing cells on, or in, a 3D gel matrix with defined flow. Each of the three wells can be filled with a gel, in which cells can be embedded. For defined flow application, the channel on top can be connected to a pump (e.g., to the ibidi Pump System) to ensure optimal oxygen and nutrient supply. |
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The µ-Slide Chemotaxis and the sticky-Slide Chemotaxis are ideal for analyzing single cell migration in 2D and 3D. Chemotactic gradients can be easily established in water-based 3D gels, such as Collagen I gels and Matrigel®, because the gel structure does not hinder the formation of a soluble gradient by diffusion. |
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Most of the ibidi labware, such as the µ-Dish 35 mm, high or the µ-Slide 8 Well high, can be used to culture single cells in a 3D matrix, and are ideal for high-end microscopy. |
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Spheroids are cells that adhere to each other under three-dimensional, non-adherent culture conditions. They lack stem cells, which means that they consist of fully differentiated cells. They can be generated by placing them into a scaffold-free suspension using the hanging drop or forced floating method, for example.
Spheroids are not capable of self-renewal and further differentiation. Tumor cell spheroids are an exception, because due to the unlimited proliferation capacities of the tumor cells, they are able to divide and renew. Therefore, spheroids are a useful model for examining tumor cell behavior, such as large-scale drug screenings.
Read here to see a detailed protocol for spheroid generation in the µ-Plate 96 Well 3D: AN 32: Generation of Spheroids (PDF)
NIH-3T3 cells forming defined spheroids on the ibidi µ-Pattern. Cells were seeded on 200 µm adhesion spots in a µ-Slide VI 0.4 and kept under flow (3 dyn/cm²) for 14 days.
Organoids are cultured "mini organs". They can be generated from adult stem cells (ASCs) or pluripotent stem cells (PSCs). When cultured in a three-dimensional matrix/scaffold (e.g., Matrigel® or collagen), these cells differentiate into organ-specific cell types that build small functional organs.
The first generation of intestinal organoids, created from an Lgr5+ stem cell by Sato et al., initiated many protocols for organoid generation from different organs, such as intestine, liver, brain, prostate, kidney, pancreas, lung, and thyroid. Importantly, they can be edited using technologies such as CRISPR, making them a powerful tool for studies about personal therapy, organogenesis, and drug screening.
Spheroids are cell aggregates, which are often generated from cancer cells.
Organoids are cultured miniature versions of organs, which are derived from stem cells.
Sato T, et al. (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459(7244):262–265. 10.1038/nature07935.
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Drost J, Clevers H (2018) Organoids in cancer research. Nat Rev Cancer 18:407–418. 10.1038/s41568-018-0007-6.
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Tuveson D, Clevers H (2019) Cancer modeling meets human organoid technology. Science 364(6444):952–955. 10.1126/science.aaw6985.
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ibidi SolutionsThe µ-Slide Spheroid Perfusion is a specialized flow chamber for long-term spheroid culture. Each of the 3 x 7 wells forms its own niche, in which the specimen is cultured. The application of perfusion through the channel on top of the wells ensures optimal nutrition and oxygen diffusion throughout the experiment, without exposing the specimen to significant shear forces. |
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The µ-Slides With Multi-Cell µ-Pattern enable spatially defined cell adhesion for spheroid and organoid generation, long-term culture, and high resolution imaging. Defined adhesion spots are able to catch all adherent single cells from a cell suspension. The surrounding Bioinert surface is fully non-cell-attachable. This forces all cells to aggregate to each other at the adhesion spots, thus forming spheroids in a defined and controllable way. |
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Bioinert is a stable, biologically inert surface for long-term culture and high-resolution microscopy of spheroids, organoids, and suspension cells on a non-adherent surface without any cell or biomolecule adhesion. It is currently available as the µ-Dish 35 mm, high Bioinert, the µ-Slide 8 Well high Bioinert, the µ-Slide 4 Well Bioinert, and the µ-Slide VI 0.4 Bioinert. |
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In the µ-Slide III 3D Perfusion, spheroids or organoids can be cultivated in or on a gel layer or embedded in a 3D matrix. The special channel geometry allows for superfusion with a low flow rate (e.g., when utilizing the ibidi Pump System). This setup makes long-term cultivation possible for up to several weeks. Additionally, the thin coverslip bottom allows for high-resolution imaging. |
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The µ-Slide 15 Well 3D and the µ-Plate 96 Well 3D are easy, cost-effective solutions for the 3D cultivation and microscopy of spheroids and organoids on, or in, gel matrices. The gel layer is directly connected to the medium reservoir above, which enables fast and easy medium exchange by diffusion. For special applications, the µ-Slide 15 Well 3D Glass Bottom, with a No. 1.5H glass bottom, is also available. |
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The ibidi Collagen Type I is a non-pepsinized, native collagen for modeling ECM in gel matrices. Its fast gelation facilitates optimal cell distribution in 3D gels. Read our Application Note about how to prepare a 3D gel using the ibidi Collagen Type I here: AN 26: Preparation of Collagen I Gels. |
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A chemotaxis assay is used to analyze directed cell migration towards a chemoattractant. Culturing cells in a 2D environment during a chemotaxis assay might not reflect the in vivo situation, resulting in an altered cell behavior and migration. To overcome this issue, cells can be embedded in a 3D matrix that mimics their natural environment, such as collagen, Matrigel®, or other hydrogels.
Advantages of 3D Chemotaxis Assays
- More in vivo-like setting for most cell types
- Highly defined environment (e.g., fibers or matrix)
- Chemotaxis assays with suspension cells possible
Limitations of 3D Chemotaxis Assays
- Gel handling: more parameters to control during the experiment
- Cells might attach to 2D surface, thus creating 2.5D conditions
- Cells might go out of focus during 3D tracking
Microscopy and schematic of adherent HT-1080 cancer cells on a 2D surface (left), and embedded into a 3D Collagen I gel (right) in the µ-Slide Chemotaxis.
Spinning disk confocal time-lapse microscopy of LifeAct TagRFP transfected HT-1080 cancer cells, which are migrating in a 3D Collagen matrix in the µ-Slide Chemotaxis, 63x oil immersion.
Find more information about 2D and 3D chemotaxis assays in the following Application Notes:
- AN 17: Chemotaxis 2D and 3D (PDF)
- AN 23: 3D Chemotaxis Protocol for Non-Adherent Cells in a Gel Matrix (PDF)
- AN 24: Chemotaxis of HT-1080 Cells in 2D and 3D (PDF)
- AN 26: Collagen I Gel for 3D Cell Culture (PDF)
- AN 34: Chemotaxis of HUVEC Cells in 2D and 3D (PDF)
Biswenger V, et al. Characterization of EGF-guided MDA-MB-231 cell chemotaxis in vitro using a physiological and highly sensitive assay system. PLoS One, 2018, 10.1371/journal.pone.0203040.
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ibidi SolutionsThe µ-Slide Chemotaxis and the sticky-Slide Chemotaxis are ideally suited for both 2D and 3D experiments. Chemotactic gradients can be easily established in water-based 3D gels, such as Collagen I gels and Matrigel®, because the gel structure does not hinder the formation of a soluble gradient by diffusion. |
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In vivo, many cell types are constantly exposed to liquid flow. When culturing them in an in vitro 3D matrix, a soft interstitial flow can be applied by perfusing them with growth medium or any reagent or drug of choice. By doing this, conditions close to the cells' natural environment can be established.
Perfusion
The combination of cells inside a 3D matrix and a channel above allows for an easy application of flow. This experimental setup passively feeds the cells inside the in vitro 3D matrix by diffusion through the gel. Oxygen and nutrients are supplied by the gentile flow. The adjustable flow rate defines the level of nutrition, enabling long-term live cell experiments.
ibidi SolutionsThe ibidi Channel Slides, including the µ-Slide III 3D Perfusion, the µ-Slide I Luer 3D and the µ-Slide VI families, allow for the seeding of the cells in a 3D matrix and the application of flow (e.g., using the ibidi Pump System). |
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Read on and have a look at Experimental Examples.