Seeing the full picture: advances in 3D cell culture microscopy

2D cell cultures have long been used as in vitro models to study cellular responses. While these approaches have significantly advanced our understanding of cell behavior, there are some areas in which they fall short. 2D cell culture is generally insufficient at reflecting the in vivo behavior of an organ, and it can be difficult to translate research conducted in 2D cultures from bench to bedside. As a result, animal models are often used; however, differences remain, and thus efforts to translate research to humans can still fail.

Research has seemed to shift towards the rapidly evolving field of 3D cell culture, allowing researchers to recreate organized, miniaturized versions of organs that can be used for various means, such as investigating disease mechanisms and developing new drugs that might be more likely to translate to the clinic than those discovered using other models.

3D cell culture represents a dramatic step forward; not only in the study of tissues and diseases, but also in the fields of drug discovery and analysis. This technology allows researchers to investigate causes of a disease, discover new drugs, test drug sensitivity and identify appropriate, patient-specific treatments. However, challenges still remain, for instance in high-throughput screening, 3D cell-culturing techniques and in maintaining cell viability, as we continue to work towards replicable, biologically accurate organ models.

Despite the dramatic increase in use and plethora of potential, the development of imaging and analytical methods to compliment this technology lags behind. As 3D cell models become more complex, they require more complex tools to analyze them. The majority of analysis techniques used today have originally been developed for 2D cell cultures, hence the transition to 3D is not ideal. This article will focus on the limitations associated with current imaging tools and the steps researchers are taking to develop more sophisticated techniques to tackle these challenges.

Don't put a label on it

Confocal fluorescence microscopy is the current gold standard for studying 3D culture systems. Unfortunately, limitations are still attached to this technique. Hence, there is a real need to develop and introduce a technique capable of studying cells in relevant 3D environments.

Importantly, conventional confocal microscopy is semi-quantitative and requires labeling that can impact cell function and intracellular processes.

Talking to BioTechniques at ASCB 2019 (DC, USA, 7–11 December 2019), Bruno da Rocha-Azevedo (UT Southwestern Medical Center, TX, USA) commented on the importance of the right level of labeling: “If you have too much labeling you're not able to detect single-molecule particles and then you lose the [ability to determine] what is a single molecule. On the other hand, if you label too few, you don't have enough particles to be detected and you cannot actually see the interactions of the receptors.” [1].

Berdeu et al. also previously attempted to address the labeling concerns highlighted by da Rocha-Azevedo through the development of a 3D + time lens-free microscopy technique, providing insights into both temporal and spatial aspects of 3D cell cultures. While the correct fluorescence labeling can provide higher resolution and specificity via confocal and light-sheet microscopy, the new technique favored ease of use [2].

There are a number of other imaging techniques that have previously been used to provide detailed visualization of the morphology and spatial distribution of biological structures. An example of this is Raman spectroscopy, which is an inelastic light-scattering technique that can provide label-free biochemical information.

Applying Raman technology, Kallepitis et al. sought to answer the need for an endogenous technique that can study cells in highly relevant 3D environments, while providing quantitative biomolecular information of multiple components simultaneously and nondestructively [3]. The team developed a quantitative, label-free Raman imaging approach for visualization of 3D cell morphology and volumetric quantification of biomolecular structures with submicron-size detail. Termed ‘quantitative volumetric Raman imaging’, the researchers believe this new method will open up new avenues for studying the complexities of cell–material interactions within a plethora of 3D culture systems, revealing new information about cell behavior and function in advanced biomaterials that has previously been difficult or impossible to measure.

There is, however, still a gap when it comes to Raman technology, and concerns arise around the lack of tools for the easy analysis and interpretation of spectral data focusing on biologically relevant information – a point that will be touched on later.

Aspects of da Rocha-Azevedo's concerns do seem to have been answered, but at a cost, be it resolution or specificity. There is still some way to go before dreams of high-level resolution 3D single-molecule imaging of live cells comes to fruition: “I would love to have a system where I can do single-molecule imaging and tracking in a 3D setting, that I can use to make blood vessels in vitro and image the cells to see how the receptors are behaving [in a situation that is] very close to the in vivo setting,” da Rocha-Azevedo remarked.

Clearing the cobwebs

Optical tissue transparency allows scalable cellular and molecular investigation of complex tissues in 3D. Tissue-clearing approaches have enabled observation of the cellular structures of transparent animal organs. However, adult human organs have proven more difficult due to the build-up of insoluble molecules in older tissues, causing human organs to be too ‘stiff’ for the animal approach.

To overcome these challenges, a team from Helmholtz Zentrum München (Germany) recently developed a new microscopic imaging approach that has made intact human organs transparent for the first time, raising hope for the development of 3D-printed human organs suitable for transplant [4].

First, the researchers discovered a detergent called CHAPS, which makes centimeters-deep small holes throughout the organ through which solutions can be administered to make the organ transparent. This is possible as CHAPS can aggregate into much smaller micelles compared with standard detergents, such as SDS.

In collaboration with Miltenyi Biotec (Germany), a new laser-scanning microscope – the ‘Ultramicroscope Blaze’ – was developed that can image large organs.

While rapid progress in tissue-clearing methods has been made both here and elsewhere, this introduces a limitation – how do you analyze these large datasets collected? The team tackled this hurdle through the development of a deep-learning pipeline that can analyze the millions of cells cleared. Combined, this new technology was labeled SHANEL – small micelle-mediated human organ efficient clearing and labeling.

SHANEL is now being used to map some of the major organs of the human body, beginning with the pancreas, heart and kidney (Figure 1). These cellular maps of human organs could be used to engineer large-scale human tissues and organs with emerging 3D-bioprinting technologies, once again highlighting another technological advancement that researchers have developed to overcome existing problems in imaging 3D cell cultures.

3D cell cultures in tumor research

Understanding tumor characteristics by developing an accurate model is the key to understanding the link between various types of cancers. 3D tumor cells grown using 3D cell culture methods have claimed the spotlight in tumor cell biology research because of their innate ability to replicate the in vivo environment of a tumor cell in vitro.

Currently the gold standard for conducting brain tumor research is the animal model – as Guohao Dai (Northeastern University, MA, USA) described in an interview with BioTechniques: “You inject the patient's glioblastoma tumor cell into the mouse brain. This is called intracranial transplantation – then you can study how the tumor invades the brain and monitor its response to drugs.” Dai continued to explain that this is a very expensive and lengthy process [5].

Dai et al. recently focused on making improvements in glioblastoma modeling and imaging using 3D printing techniques [6]. By 3D modeling in vitro, tumor cell growth can be visualized in real time. In this case, the researchers were able to print a 3D vascular channel together with the 3D tumor model, so the tumor model is perfused with vascular channels. This allows researchers to test long term, which is much more reflective of treatment in real life. This is a key benefit of using a 3D model, as even the gold standard animal model has its pitfalls, as Dai explained: “During the 6 months after you inject the tumor cells into the mouse brain, you cannot tell much about what's happening in the brain and the tumor development until you open the cranial window. Once the cranial window is open you can observe what is happening there, but there is no real-time monitoring of how the tumor interacts with the brain tissue or how they grow.”

As mentioned previously, one of the well-known limitations of 3D models remains with the challenging imaging process. 3D models are commonly very deep, at least a few centimeters. Confocal microscopes can only image less than a millimeter. However, the limitations do not stop there – the reconstruction of 3D images can also take a few hours, leading to sample photobleaching. “That's a lot of interruption if you do that every day with a few hours of imaging time and it will also disrupt your image,” explained Dai.

To tackle this imaging problem, the researchers developed a novel imaging technique using a laser to scan the tumor, capturing and tracking all the scattered light from the tissue. A mathematical model that identifies the original location of the scattered photons is then combined to reconstruct the image. It is important to note that this process is conducted rapidly, meaning the sample is out of the incubator for just a few minutes, causing little disruption or damage to the cell.

While this technique does seem to provide an answer for the challenging imaging process of 3D models, this does come at a cost. Increasing both the depth and speed of imaging results in a loss of resolution. Dai and his team were able to visualize that the tumor had overall growth; however, they were unable to image to the cellular level, again answering part of da Rocha-Azevedo's dream for 3D modeling with the ability to monitor in 3D but without the desired resolution.

This combination of novel techniques – using fluorescence imaging to study the bioprinted tumor models – allowing reconstruction of the 3D shape of the tumor and visualization of how it grows in real time is obviously a massive step forward in the realm of imaging 3D cell cultures. There are still large strides to be made, particularly with tumor models. There are a lot of other complicated structures that cannot be incorporated into the 3D tumor model. Within an in vivo brain tumor there would be an abundance of other complicated structures such as neurons and microglia, the latter being shown to play a vital role in glioblastoma remission and immune response [7]. Incorporating these components into the model will prove very significant. Moreover, in terms of chemical composition, the hydrogels currently used are still far from actual brain tissue.

Despite it being much easier to form vascular channels accurately with 3D bioprinting, as described in Dai et al.‘s recent publication, a challenge still remains in how to recreate the extremely tight blood–brain barrier. “That's a very big challenge that would have a very significant impact in improving the tumor model,” concluded Dai.

Future perspective

The future significance of 3D cell cultures will not only depend on the advancement of microscopy technology, but also the development of tools for both analysis and interpretation of large spectral data. The research highlighted in this short piece represents just a fraction of the technological strides made in this field. Limitations in current methods are slowly being answered, albeit with some caveats.

3D cell culture techniques provide methods that are key to advancing research in many fields. As the imaging of these 3D cell cultures improve, consequently tumor models, cancer treatment therapies and disease-testing methodologies will follow suit.