Cell culture technologies have provided biomedical researchers with fast and accessible tools to probe the breast tumor microenvironment. Exponential progress in fabrication methods combined with multiparametric approaches have enabled the development of cell culture model systems with enhanced biological complexity to identify key aspects that regulate breast cancer (BC) progression and therapeutic response. Yet, the culture parameters and conditions employed influence the behavior of tumor cells, thereby affecting its tissue biomimetic capabilities. In this chapter we review the wide range of culture platforms employed for the generation of breast tumor models and summarize their biomimetic capabilities, advantages, disadvantages and specific applications.
- culture platforms
- 3D bioprinting
- tumor microenvironment
Cell culture is an integral tool in biomedical research. It refers to the removal of cells from tissues or organs, into an artificial
Breast tumors are complex systems, composed of different cell subpopulations with distinct tumorigenic capabilities within the tumor.
The TME is heterogeneous and plays a significant role in tumor development, progression and metastasis . It is composed of multiple cell types such as fibroblasts, myoepithelial and endothelial cells, infiltrated immune cells (e.g., T cells, macrophages), adipocytes and mesenchymal stem cells (MSC), along with the extracellular matrix (ECM) and soluble factors [7, 8]. These cell types are important for modeling the disease as it has been shown that tumor prognosis is not solely based on the tumorigenic cells, but also on how those cells communicate with their environment . For example, cancer associated fibroblasts (CAFs) have been demonstrated to promote cancer cell aggressiveness and survival by the secretion of growth factors and cytokines and the creation of a “protective niche” against drugs [8, 10, 11]. Similarly, immune cells promote angiogenesis , immunosuppression, invasion and metastasis [13, 14]. Furthermore, adipocytes and MSCs have been shown to be involved in the secretion of factors related to matrix remodeling, invasion and survival of the tumor [15, 16]. Thereby, models that include multiple cell types are likely to be more mimetic of the pathology and predictive of responses in tissues. As such, custom microscale platforms have been developed to accommodate multiple cell types in spatially defined patterns and locations to enable examination of multi-cell type interactions. Such models include those related to angiogenesis and metastatic processes [17, 18, 19], and due to the lack of spatial control it would have been difficult to recreate such interactions in traditional culture platforms highlighting the applicability of custom platforms for multi-cell type interactions.
The identification of relevant parameters from the tumor microenvironment is imperative for proper assessment and predictability of efficacy of experimental therapies. For this reason, 3D cell culture systems have become more popular due to its potential to better mimic the complexity of the TME and thereby increase the physiological relevance of the study [20, 21]. This modality incorporates scaffolds and 3D cell constructs that have been shown to impact cell proliferation, morphology, signaling and drug resistance in a more physiologically relevant manner [22, 23, 24, 25].
Mimicking BC complexity is challenging, however, progress in microfabrication techniques, tissue engineering and cancer biology have paved the way to more sophisticated models with enhanced biomimetic capabilities that will help to elucidate the intricate nature of BC. In this chapter, we discuss the wide range of culture platforms employed for the generation of breast tumor models and summarize their biomimetic capabilities, advantages, disadvantages and specific applications.
2. Cell culture modalities
2.1 Two-dimensional and three-dimensional culture
The traditional cell culture methods for studying breast cancer employ two-dimensional monolayer cultures, where cells grow flat on a surface. Two-dimensional culture is still widely used, but with advances in microfabrication now surfaces can be modified with nanostructure topographies and different levels of stiffness to mimic to some extent the physical properties of the matrix surface. These topographies (e.g., roughness, surface geometry) have the capability of providing biomimetic surfaces that have been shown to modify the morphology, proliferation and signaling, among others, of cells . Similarly, changes in the mechanical properties of the ECM (e.g., stiffness) are related to increasing malignant phenotype , cancer progression, signaling [28, 29, 30] and drug sensitivity . Despite these technological advances in 2D cultures, multiple studies have shown that cell cultures in 2D felt short to mimic cell phenotypes associated with disease progress such as cell invasion, cell function and expression of pathological markers [4, 23, 32]. In some cases, utilizing 2D culture systems has resulted in the loss of essential cell signaling pathways, hence limiting the ability to fully evaluate cell–cell and cell-ECM interactions . Evidence has also shown that there are inconsistencies when comparing cell morphology, receptor expression, and polarity between cells grown in 2D and the
In order to bridge this gap in biological complexity, multiple methods employing 3D cell culture systems have emerged and continue to be steadily improving, aiming to produce the most
Cancer is a heterogeneous disease and even though there have been various advances in cell culture modalities, thorough comprehension of the crosstalk between cancer and non-cancer cells is still not fully understood . Co-culture and multi-culture models have been long established as appropriate tools for evaluating breast cancer heterotypic interactions
There have been an increasing number of studies looking to compare tri-culture models with the more traditional mono-culture or co-culture methods. With the intention of better understanding the bone microenvironment, Pagani
Co-culture models involve a cell growing arrangement, where two or more different cells are cultured with some amount of contact between them . The communication between the cells may be bi-directional or multi-dimensional, and it can happen at the macro-scale or at the micro-scale . The method of choice should be dependent on what is the focus of each individual study and can be grouped in: compartmentalized, conditioned media and mixed culture.
The segregated or compartmentalized model consists of two or more physically separated cells, cultured in a shared environment . This type of culture is preferred when studying paracrine interactions of cells that are not located in close proximity in tissues. Also, this method is useful to identify target cells based on soluble factor signaling since the cells individual response can be examined, facilitating the identification of factors that may play a role in tumor growth and advancement. In compartmentalized co-cultures, one cell population is seeded in the bottom of the standard well, and the other is seeded on a top insert or in an adjacent compartment. By doing this, the cell types remain separated, while still being able to exchange soluble signals in their shared environment . Indirect cell culture eliminates heterotypic interactions mediated by contact between the cell types, which can be seen in direct cell culture. It also allows for cell type specific readouts, which are unachievable in direct cell culture . Such method has provided evidence on genes involved behind stromal invasiveness and metastasis, and the crucial role of fibroblasts in proliferation of estrogen-dependent human breast carcinomas [6, 49, 50]. Gonzalez
If multiple cells need to be examined, co-culture platforms, such as transwells, are not useful since they are limited to only two compartments. Hence, the use of customizable culture systems such as microscale devices, is warranted . Our group developed compartmentalized microwell culture platforms, in which we show the contribution of multiple cell types to the sensitivity to heat therapy in tumor cells . The data shown indicates that the presence of macrophages and fibroblasts had a significant protective effect against heat stress in BC cells, thus, perturbing the effectiveness of heat therapy. Others have employed multi-cell type cultures to deconvolute cell communication of metastatic breast tumors. Regier
2.2.2 Conditioned media
Conditioned media transfer utilizes two separately cultured cell populations, where one culture medium is utilized to nourish the other . This type of method is simple and allows one-way signaling from effector to responder . The advantage of utilizing this method is that conditioned media can be profiled for the identification of secreted soluble factor-related effects is possible . Consequently, the role of signaling molecules could be tested in a specific response . Also, this method is useful when the cells of interest cannot be cultured together such as studies involving tumor cells and microbes . However, when employing multiple cell types, the method becomes a bit more complex since identification of the secretor and recipient cells can be complicated. Additionally, when this type of method is utilized, there is no cross-communication within the cells and it is not possible to study bi-directional signals . For this reason, this type of method would not be ideal if the goal is to study multi-cell type interactions that naturally occur in the
2.2.3 Mixed co-culture
In mixed cell culture, different types of cells are cultured together. Just as with conditioned media transfer, this type of method is accessible and simple. It can be done in 2D or 3D using traditional well plates . If the cells are cultured together in a standard plate, the method is referred to as direct or mixed cell culture. However, if a transwell insert or adjacent compartments are utilized, the method is denoted as indirect or compartmentalized cell culture. Unlike the conditioned media method, mixed co-culture does allow for bi-directional paracrine and juxtacrine signaling, which is of great importance when studying multi-cell type interactions in breast cancer . Because of the cellular arrangement, this method is also ideal for studying how cell–cell contact affects cell behavior . When performing multi-cell type studies, the direct method simply requires the inclusion of the additional cell lines mixed.
Mixed co-culture experiments shed light on distinct microenvironment features based on cancer subtype; and potential mechanisms behind invasive phenotypes [55, 56]. Camp
3. Culture platforms for enhanced biomimetic capabilities
Despite the development and application of the aforementioned cell culture methods, thorough understanding of cancer development and progression continues to be a challenge. As shown in Figure 2,
|3D Microfluidics||Small size samples, spatial and temporal control, reduced reagent volumes, controlled gradients, high-throughput||Mechanical stress, complicated set-ups, material fabrication||Invasion, metastasis, vasculature, modeling TME||[20, 37, 59, 60]|
|Bioreactors||Long term culture, effective nutrient distribution, large scale||Contamination risk, expensive, specialized equipment, low throughput, limited spatial resolution, high cell numbers needed||Metastasis, drug discovery||[61, 62, 63]|
|3D bioprinting||Controlled spatial arrangement of cells and matrix, biomolecular gradients, high-throughput||Lower cell viability, material challenges, lack of standardized methods, high cell numbers needed||Migration, angiogenesis, drug discovery, modeling TME||[64, 65, 66]|
|Organoids||Small size samples, retain parental tumor phenotype, can be preserved as biobanks, mimetic of tissue function||Lack of standardized methods, heterogeneous cell samples, high variability across replicates||Drug discovery, invasion, metastasis||[67, 68, 69]|
Microfluidic platforms can be utilized to scale down the traditional culture modalities, yet they enable to customize the culture environments to examine more complex interactions . This technology employs microsystems that allow the manipulation of small fluid volumes and control over the spatial location of cell clusters . Its application to improve 3D cell culture models has been increasing since 2012, particularly in BC research . In comparison to macroscopic culture, microfluidic cell culture models have several significant advantages that, when employed, lead towards better biomimetic models. Firstly, cells may be cultured in a spatially controlled environment by controlling fluid patterns and proximity across culture compartments [72, 73, 74, 75, 76, 77]. This technology permits the combination of multiple cell types and to control cell patterning, to recapitulate to some extent tissue observations. For example, microfluidic devices permit the study of angiogenesis while also allowing the study of endothelial migration and evaluation of cell response in co-culture [71, 78]. Also, microfluidics can implement continuous perfusion conditions, and controlled gradients, which are both characteristics that also resemble the cancerous
Recent studies in microfluidic systems have highlighted their capability to recreate and profile some of the biological complexity of the tumor microenvironment. Such studies have revealed important information regarding the processes involved in metastasis and how the tumor microenvironment contributes. For example, single cell RNA sequencing using microfluidic devices have revealed the diversity of the breast epithelium, which sheds light about early tumorigenesis and tumor progression [79, 80]. In addition, microfluidic devices pose as an advantage to personalized medicine by aiding in the selection of appropriate pharmacologic agents. In this regard, Lanz
Even though microfluidic devices have given the opportunity to better replicate the tumor environment, there are still some caveats to its use. Silicone-based devices have been shown to sequester small hydrophobic molecules, which can compromise the results of some studies , yet researchers have been addressing this by modifying the material to make it more hydrophilic and reduce molecule sequestration . Also, microfluidic devices in some cases can induce mechanical stress to the cells , which can modulate cell responses in an unpredictable manner, and are often limited by complicated set-ups , which limits their broad adoption by the scientific and clinical community. As such, simpler fabrication methods and commercial availability of customizable microscale platforms is desirable to overcome such limitations.
Despite the numerous advantages of the aforementioned 3D culture methods, the duration of culture and nutrient availability can be a limitation in static cultures particularly to enable observations that occur in cells over periods of several weeks. In this case, perfusive systems, such as bioreactors, are more appropriate. A bioreactor is a canister that allows the 3D culture of cell clusters for extended periods of time. It is coupled to sensors and actuator components allowing for the controlled delivery of oxygen, nutrients and other parameters . Goliwas
3.3 Three-dimensional (3D) bioprinting
Another technology that has emerged in recent years and that is being applied to 3D culture technology is 3D bioprinting. Its development has been possible thanks to advances in 3D printing technology, biomaterials and tissue engineering methods. Three-dimensional (3D) bioprinting consists of printing cells together with ECM components, biomaterials and bioactive factors . It has been shown that bioprinting techniques can be used to generate 3D tumor models that can better resemble the TME [90, 91]. This has been achieved as bioprinting provides the ability of controlling the spatial arrangement of cells, creating biomolecular gradients and well-organized vessel-like structures (vasculature) within a micron scale resolution [92, 93]. Therefore, bioprinted tumor models are used for angiogenesis, migration and drug development and screening studies as well as TME models [65, 94]. Although 3D bioprinting is widely used in tumor research, very few studies use bioprinted models for BC. Yet, most of these studies are focused on BC metastasis and drug resistance. A study performed by Zhou
The most recent 3D cell culture modality are organoids. These are 3D heterotypic
Studies with BC organoids are limited, since this modality has just started to be explored. However, they have gained more popularity in the last few years. Cheung
4. Concluding remarks
Breast cancer is an evolutionary disease and cell culture modalities should continue to evolve concomitantly. Even though traditional 2D co-culture methods have provided valuable insights on disease development and progression, there is a need for more heterotypic biomimetic models that can replicate the tumor environment more closely. Some of the consequences of limited biomimetic models has been the large number of investigational drugs that never make it past clinical trials and the lack of clear understanding on the foundations of breast cancer malignant transformation. Aside from the need for more biomimetic models, most of the current research has been focused on the metastatic stage of the disease. Even though understanding tumor progression and the role of its microenvironment is of utmost importance, understanding the early and localized stages of breast cancer is also imperative. Not having an explicit grasp on the biological processes behind progression from early stage to invasive to metastasis has hindered the ability to make a predictive diagnosis in patients with early disease that have a greater probability of invasive cancer progression. Hence, designing new targeted pharmacologic agents becomes a challenge. Despite the continuous development of innovative cell culture modalities, there are still many unanswered questions. However, the hope is that with the emergence of the new methods (bioreactors, organoids, etc.), many of these questions can be interrogated in a controlled and user friendly cell culture environment.
This publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award number SC1 GM131967 and partial support from the Puerto Rico Idea Network for Biomedical Research Excellence (PR-INBRE) under Grant No. P20-GM103475.
Conflict of interest
The authors declare no conflict of interest.