Your guide for 3D cell culture

For decades, isolated cells grown in 2D monolayers have been used to study a range of biological processes. While these culturing conditions are easy to control and are conducive to a variety of analytical techniques, they do not represent the complex 3D environments that cells are actually exposed to in vivo (1). In this article we will take a closer look at the properties of 3D cell culture, methods and the workflow.

Here are some reasons to use 3D cell culture

3D cultures better mimic tissue-like structures and conditions
Able to exhibit differentiated cellular functions
Possible to co-culture two or more different cell types
Can simulate microenvironment conditions such as hypoxia and nutrient gradients
Better predict in vivo responses to drug treatment (2)

Why 3D cell culture?

If you think about it, no cell types within our body grow as a monolayer independent of other cells or tissue. Instead, most cells naturally exist in complex 3D structures including different cell types within an extracellular matrix. The numerous cell-cell and cell-matrix interactions all have a profound effect on their behavior. In addition, 2D monolayers have uniform access to nutrients and oxygen, which is not the case in cell masses, such as tumors. 3D tumor spheroids are much more representative of in vivo tumors in which inner cells have less access to nutrients and oxygen compared to the outer layer, forming a natural gradient (2).

Spheroids are dense, 3D aggregates of cells that exhibit extensive cell-cell adhesion, typically retain their endogenous extracellular matrix, and have properties that closely mimic their in vivo tissue counterparts. (1)

The properties of spheroids in cell culture model make them well suited for use in drug development and tissue engineering studies, as well as for the study of fundamental biological principles (1).

Novel Workflows Solve Problems

Combining spheroids with bioinks and bioprinting has the potential for an effective workflow to generate 3D models. If consistent spheroids can be developed, adding them to a bioink to then be printed on a bioprinter gives researchers full control over the placement of these spheroids. Additionally, by selecting tunable biomaterials, the stiffness of these constructs can be better controlled. By controlling all these parameters, researchers can better replicate physiological conditions and produce models that yield more accurate results. CELLINK scientists suggest a workflow outlined in Figure 1 that demonstrates how these elements can be combined for consistent and effective 3D cell culture. (3)

Figure 1 - A proposed 4-step workflow for spheroid encapsulation and bioprinting

Step 1 - Spheroid formation

Spheroid formation is dependent on the tendency of cells in suspension to aggregate and self-assemble into multi-cellular groupings. This process likely involves the sequential induction of β1-integrin-dependent adhesion which facilitates cell aggregation, followed by strong homophilic E-cadherin-dependent cell-cell adhesion that drives spheroid compaction. There are a number of distinct methods for promoting spheroid formation in culture, including hanging drop, liquid overlay, pellet culture, spinner culture, microfluidics, and methods incorporating biomaterial scaffolds (1).

Figure 2 - Examples of the various techniques used for forming spheroids.

Hanging drop
Cells are placed in a suspended drop of medium, allowing cells to aggregate and form spheroids at the bottom of the droplet. The low cost and simplicity of this method makes it particularly amenable to high-throughput spheroid formation. (1).

Liquid overlay
In this case, the bottom of a cell culture dish is coated with a non-adhesive material, such as agarose, to inhibit cell adhesion and spreading. Suspension cells are then applied in a layer of media over the non-adhesive material, which drives their spontaneous aggregation and spheroid formation. While this technique is advantageous for certain cell types, the formation of tumor spheroids is inhibited by agarose. Alternative materials can be used to promote tumor spheroid development, such as hyaluronic acid, which drives signal transduction related to cell proliferation, angiogenesis, and survival (1).

Pellet culture
In this method, suspension cells are pelleted to the bottom of a tube through centrifugation, leading to cell aggregation as the cells are forced into contact. The pelleted cells are then resuspended in spheroid-promoting culture medium and placed in a plate with a non-adhesive surface coating (1).

Spinner culture
Suspension cells cultured in a flask with constant stirring will also form spheroids. The sizes of the resultant spheroids are partially dependent on the size of the container(1).

Biomaterials
Spheroids can also be formed using scaffolds made from biocompatible materials like alginate, collagen, and hyaluronic acid. Hydrogels, biofilms, and particles have all been shown to improve the viability and biological properties of spheroids. For example, hydrogels can provide an extracellular matrix-like environment with defined stiffness that can influence the malignancy of hepatocellular carcinoma cell spheroids (1).

Step 2 - Choosing a Bioink

Choosing the right bioink for spheroid culture can pose a number of challenges. Bioinks often require intensive synthesis and preparation, extensive gelation times and cell specific protocols. Still, they are relevant for supporting long-term spheroid culture, and luckily, there are existing portfolios of bioinks and fine-tuned protocols that are suited for most cell types and applications. Readily sourced or commercially available bioinks like Collagen I and GelMA can make 3D spheroid culture easy and dependable (3).

Step 3 - Automating Spheroid Culture with Bioprinting

Today’s bioprinters are thoughtfully designed to provide a platform in which cell viability is the highest priority. High-end printers like CELLINK’s BIO X come equipped with temperature control features that allow spheroids to remain at homeostatic conditions during printing processes and enable researchers to use a wide range of low viscosity and temperature-sensitive biomaterials like Collagen I, GelMA, ColMA or alginate in their studies. Bioprinters with multiple methods of extrusion add an additional advantage to this workflow as they give researchers improved flexibility when it comes to printing spheroids. For instance, the droplet printhead, electromagnetic printhead, pneumatic printhead and syringe-pump printhead are all suited for printing low-viscosity, spheroid-laden bioinks. For more information on bioprinting, see related products (3).

Video - Spheroid 20x WI Deep Tissue 250um-Nuclei Mask-3Drecon

Step 4 - Quantifying Impact

Several methods of analysis can be implemented during or after spheroid culture, including viability checks, immunocytochemistry, live-cell imaging and rheological measurements. The latter methods can be used to assess spheroid growth, health and function, as well as spheroid-spheroid communication (3)

Cell-based assays
Although 3D cultures are designed to offer a more physiologically accurate environment, the added complexity of that environment can also present challenges to experimental design when performing cell-based assays. For example, it can be a challenge for assay reagents to penetrate to the center of larger microtissues and for lytic assays to disrupt all cells within the 3D system. Standard protocols are not likely to work with 3D cultures if you need to isolate a protein or a metabolite from the cytoplasm. The protocol and reagent(s) must be optimized for 3D cultures (4).

The CellTiter- Glo® 3D Assay is one of several Promega kits optimized with more detergent and a specialized protocol. Promega have developed protocols optimized for use with 3D culture for several assays typically used in monolayer culture, see related products below (4).

Figure 3 - the ImageXpress Confocal HT.ai High-Content Imaging System and its user interface

Live-cell imaging
Implementation of more complex assays and 3D models requires high resolution to capture publication-quality images and data. Enhanced assay sensitivity can be achieved by taking advantage of the optical properties of confocal imaging, capturing images with a high signal-to-noise ratio while reducing out-of-focus light for crisper images and accurate cellular detail (5).

Next-generation high-content, high-throughput tools for microscopy offer innovative and automated techniques for evaluating this complex biology. With new technology from Molecular Devices like the ImageXpress® Micro Confocal High-Content Imaging System and the MetaXpress® 3D Analysis Module with 3D Viewer, screening these models within a single, integrated interface will dramatically reduce the time to discovery (5).

Molecular Devices offers a complete solution for quantitative 3D assays with reliable, high-throughput imaging instrumentation, sophisticated image analysis tools, and comprehensive data analysis software designed for handling multi-plate screening data. For further information, see related products (5).

Figure 5 - Maximum projectionimages of spheroids representing various phenotypes: (A) image analysis read-outs derived as a result of Nuclei Count and Cell Scoring analysis. (B) Bar graphs: control (0.1% DMSO), paclitaxel 150 nM, etopocide 200µM, staurosporine 300nM, mitomycin C 1µM, doxorubicin 1µM, fluoroadenin 100µM. Geometric or avarage intensity values were noramlized to DMSO controls (set to 1000). (C) Concentration-dependent effects and 4-parameter curve fits pf seøected compunds. Red-paclitaxel, dark red-staurpsporine, blue-doxorubicin, green-mitymycin C, teal-etoposide, purple-fluoroadenine

Sources:
(1) https://cytosmart.com/resources/spheroids, (2) https://no.promega.com/resources/guides/cell-biology/3d-cell-culture-guide/, (3) https://www.cellink.com/the-role-of-spheroids-in-3d-cell-culture-methods/, https://www.promegaconnections.com/3d-cell-culture-models-challenges-for-cell-based-assays/, (4) https://www.moleculardevices.com/sites/default/files/en/assets/ebook/dd/img/acquire-and-analyze-3d-images-like-a-pro.pdf