Developments in 3D neural cell culture models: the future of neurotherapeutics testing?

KEYWORDS:

• 3D neural cell culture
• organotypic slice culture
• spheroids
• scaffold-based cell culture
• neurotherapeutics

Introduction

With diminishing returns from neurotherapeutics studies performed in animal models [1], researchers are increasingly relying on human cell culture platforms. Two-dimensional (2D) cell culture remains the predominant culture method despite incredible advances in bioengineered cell culture systems and tissue engineering over the past two decades. This may be because the simplicity of 2D culture allows cell samples and therapeutic agents to be easily screened. However, this simplicity has drawbacks. Cells grown in 2D do not accurately represent the three-dimensional (3D) characteristics of native tissue. In 2D cell culture, cells are forced to adhere to a flat, hard plastic, or glass surface. These unfamiliar mechanical cues and lack of 3D extracellular matrix (ECM) support can have a significant impact on cellular behavior, function, growth, and morphology [2–5]. With the complexities of the nervous system, these changes can greatly skew experimental outcomes. As animal models and 2D cell culture methods have reached a plateau, alternative methods have been developed. Many of these alternative techniques fall under the wide umbrella of 3D cell culture. Here, we will discuss a small selection of 3D cell culture methods used in modeling the nervous system. We will highlight some of the strengths and weaknesses of these methods and potential future directions of this growing field.

Methods for 3D cell culture

The main goal of 3D cell culturing is to bridge the gap between in vivo and artificial in vitro conditions. The more similar a cell culture system is to native tissue, the greater the potential for representative results. Techniques may be separated into categories including, but not limited to, explant culture, self-assembled aggregate culture, and scaffold-based cell culture.

Explant culture is a category of techniques that involves growing intact tissues in vitro. These techniques allow for the original complex 3D organization of the tissue to be preserved, which can offer unique insights into neurological diseases, nerve growth, and the effects of potential therapeutics. It is especially useful in studying effects of rare neuronal cells that cannot be harvested and do not have clear stem cell differentiation protocols. Explant cultures can be grown in cell culture dishes, microfluidic devices, or on sponge–gel matrices [6–10] Explant culturing on Gelfoam®, a sponge–gel matrix prepared from purified porcine skin gelatin, can support relatively long-term growth (over 1 month) of explanted nerves and ganglia [9,10]. However, brain or spinal cord slice cultures are more typically grown in cell culture dishes using supportive membrane inserts. Some success in culturing resected human brain tissue has been observed, but tissue supply is limited [11]. As most explant cultures are harvested from animals, these techniques suffer from similar limitations to animal models. Preparation of explant cultures can also be time consuming and costly. Despite these limitations, slice culture is an attractive 3D model that represents functioning natural tissue in vitro and remains a valuable tool.

Self-assembled aggregate cultures, which encompass spheroids, embryoid bodies, neurospheres, and microtissues, can be formed through suspension culture, in concave microwells, on sponge-gel matrices, and by a variety of other methods [12–18]. Neural spheroids can be produced using primary neural cells or neural cell lines such as the SK–N–SH neuroblastoma cell line for brain tumor studies [12,13]. Neural stem cell aggregates can also be used, but these aggregates have been criticized for being very sensitive to cell culture conditions, resulting in inconsistently differentiated cells even between experimental trials [14,15]. In addition, all spheroids are limited by the size of the aggregate. Excessively large spheroids can result in necrosis of internal cells due to poor diffusion of oxygen and nutrients to the center of the structure. Thus far, spheroids appear to be more popular for tumor studies than modeling neural physiology, because cell aggregates have strong similarities to actual tumor tissue [2]. However, our understanding and capabilities to manipulate stem cell aggregate development and differentiation has seen rapid advancement. Complex, patterned, and potentially transplantable formations of cortical and retinal tissue can be formed using self-organized aggregates of stem cells [16–18]. An important consideration when choosing a 3D culture model is that spheroids can be produced at a faster rate and may have greater potential for high-throughput pharmacological screening compared to other methods such as scaffold-based cell culture [12,14].

Scaffold-based cell cultures attempt to use synthetic and/or natural biomaterials to reproduce neural tissue. Tissue-engineered structures are typically made from soft and porous matrices to allow for sufficient passage and supply of oxygen and nutrients to the cells. Synthetic materials such as polycaprolactone, polyethylene glycol, and even polystyrene have been used as scaffolds for cell adherence and support [5,19,20]. Another method for scaffold-based neural cell culture provides cells with natural ECM proteins such as laminin or collagen [3,21,22]. Type I collagen with hyaluronic acid and alginate with laminin are some examples of natural material combinations used as cell scaffolds [3,21]. Matrigel is another natural material that has been used extensively. Matrigel is a protein extract that contains a complex mix of ECM proteins including laminin, type IV collagen, and entactin. It should be noted, however, that Matrigel may contain low amounts of other proteins including various growth factors and has been criticized for batch-to-batch protein differences resulting in experimental inconsistency [23].

With the large range of materials available for scaffold-based cell culture, many factors must be considered. When using constructs that require a polymerization reaction to form a 3D matrix, care must be taken to select appropriate crosslinking agents to avoid negative effects on cell viability. Material type, size, structure, and mechanical properties can also influence cell culture outcomes, for example stem cell differentiation, neurite outgrowth, and cellular organization [3–5]. Mammary gland cells were observed to have differing morphologies and behaviors when grown in Matrigel versus type I collagen [24]. This effect does not appear to be present in growing neural cells, as investigators have used both Matrigel and type I collagen in separate scaffold-based cell culture studies without mention of significant anomalies [3,22]. However, there has yet to be any thorough comparison across scaffold-based models, which may vary considerably by scaffold fabrication method and materials. Although promising models have emerged using scaffold technology, scaffold-based cell culture remains in development and has yet to be optimized to the point where it can be used routinely for neurotherapeutics testing.

The future of 3D neural cell culture

A search on PubMed reveals that articles containing the search term, ‘3D cell culture,’ made up approximately 1/15 of the overall number of ‘cell culture’ papers in 2015 compared to approximately 1/30 in 2010. This fraction does not include many other studies involving 3D cell culture techniques that have not specifically noted the title of ‘3D’ in the paper, such as many self-assembled aggregate studies. Nonetheless, this doubling within the last 5 years reflects the growing interest in 3D cell culture techniques.

Even so, 2D cell culture remains the predominant cell culture method across all biologically related disciplines. With 2D cell culture, techniques have been well established, are faster and simpler to design, and are already well integrated with high-throughput screening. Moving to 3D adds increasing complexity that can be quite intimidating for many investigators new to the field. Increasing complexity can result in fewer samples being processed due to time constraints, but if results are more predictive of neurotherapeutic outcomes, then perhaps a balance can be found. So far, little attention has been directed toward the possibility of developing high-throughput spheroid assays of neural cell cultures. With much attention so far focused on scaffold-based cell cultures in the neural tissue engineering field, alternative 3D cell culture methods that are simpler to perform may be worth pursuing.

Explant culture and self-assembled aggregate models may also be able to contribute more toward our understanding of neural cell growth in scaffold-based designs. The majority of 3D cell culture studies have used 2D cell cultures as controls. Although this comparison is useful, what may be more helpful in the future will be to systematically compare 3D cell culture techniques with a 3D control such as an in vivo model or an organotypic slice culture. We already know in most cases 3D and 2D cell cultures grow quite differently. The next step is to actively compare current ‘promising’ 3D cell culture techniques to a 3D ‘gold standard’ in order to measure how representative these models are of actual tissue.

The future of 3D cell culture is rich with opportunities. For example, microfluidic systems are becoming more accessible and may be integrated with 3D cell culture to offer additional control over cell culture conditions. Microfluidics can be used to continuously replenish the culture medium to support growth of 3D cell cultures. Microfluidic systems can also be used to impart mechanical stimulus (e.g. shear flow), define chemical gradients (e.g. growth factors), and provide dynamic culture conditions to better model natural physiology [25,26]. In addition, advances in induced pluripotent stem cell technologies offer opportunities for studying adult disease pathologies, as well as for paving the path toward personalized medicine. As the 3D cell culture tool box continues to grow, 3D cultures may one day help to improve the success of clinical trials.

Declaration of interest

KR Ko has been supported by Canadian Institutes of Health Research (fellowship). J Frampton has been supported by the Canada Research Chairs Program. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.