Perfusion Bioreactor Technology for Organoid and Tissue Culture: A Mini Review

Simple Summary

Cancer remains one of the leading causes of death globally. Due to its complexity and heterogeneity, traditional preclinical models, such as cell cultures and animal testing, often fail to replicate the true nature of tumors because they lack a realistic tumor microenvironment. Organoids and tissue cultured in perfusion bioreactors, which preserve the genetic and histological profiles of tumors, represent an innovative approach for studying cancer biology while improving diagnosis and treatment. This review discusses the promising results emerging from the latest studies that highlight the usefulness and advantages of perfusion bioreactors in supporting the growth of tissue specimens and organoids derived from various sources. Moving forward, the goal of researchers using perfusion bioreactors is to develop more accurate and effective preclinical models tailored to individual tumor characteristics, in order to reduce the high defeat rates in clinical trials and accelerate the advancement of specific and effective cancer therapies.

Abstract

Organoid culture is an emerging and promising 3D culture system by which three-dimensional cell aggregates have been produced from different organs and tissues. This new innovative culture technology preserves parental gene expression, as well as the biological features of parental cells in vitro and ensures maintenance of three-dimensional cell culture for prolonged periods, opening new encouraged scientific scenarios and making them a functioning and valid system for testing new drugs for tissue engineering studies and precision oncology medicine. Various research focused on organoids has been performed in perfusion bioreactors, an advanced device able to mimic the tumor environment, providing a physiological growth state and a long-term culture viability. Perfusion bioreactors have been used for the maintenance and growth of organoids as well as for tumor patient samples improving proliferation while supporting the development of extracellular matrix (ECM). The ability to mimic the tumor environment and to maintain patient-derived biopsies for a long time makes perfusion bioreactors an essential model for preclinical testing.

1. Introduction

Cancer is one of the leading causes of death in the world. It is a very complex and dynamic disease, and because of its multi-faceted nature, it becomes complicated in identifying a distinctive singularity of a specific tumor at specific times. The study of inherited genetic changes has provided insights into the molecular mechanisms that support and influence tumorigenesis and malignant progression. The complexity of this disease has become evident when we understand that the hallmarks of cancer are captive to inherited non-genetic mechanisms, including epigenetic changes and the interactions between tumor cells and the tumor microenvironment, which consists of several cellular phenotypes and the extracellular matrix. These different actors influence each other, shaping cancer cells’ evolution. Significant efforts have been made to develop effective approaches to cancer diagnosis and treatment. Preclinical cancer testing models that forecast the effectiveness, safety and pharmacokinetics of cancer treatments represent an important platform for mechanistic research and drug testing. To date, the poor data resulting from clinical studies highlight the failure of the conventional adopted models, including cell culture and animal models, which did not accurately depict the complexity and heterogeneity of tumors, characterized by a surrounding tumor microenvironment and extracellular matrix [1]. There is a need for new preclinical models to address the high failure rate observed in clinical trials. Although in vivo studies on mice models are an efficient system, they do not represent a valid method in some experimental contexts such as mass screenings or the design of personalized pharmacological therapies also due to the elevated experimental costs. Non-animal preclinical models should also be used to protect and ensure animal welfare. For this purpose, 3D culture techniques have been developed to bridge the gap between cell lines and in vivo models [2]. In the last few years, models recapitulating 3D co-culture attracted increasing interest in tumor research fields particularly for drug discovery and tissue engineering studies. Three-dimensional cell culture models emulate the in vivo microenvironment providing a proper cell–cell interaction, as well as interconnection and intercellular signalling networks between the cell and ECM components. Perfusion-driven flow from microcirculation is a critical element of the tumor microenvironment, influencing shear stress and nutrient supply. However, most studies on perfusion have primarily focused on the effects of fluid dynamics on tumor cell growth and migration [3].

Organoids are revolutionary new 3D cell culture tools that allow for the monitoring and studying of cancer progression, as well as testing new and old (drug-repurposing) drugs for personalized therapies. Many of the studies achieved on organoids have been performed in perfusion bioreactors that provide physiological growth and augmented viability for long-term studies.

Bioreactors are very valuable tools used to improve aeration and nutrient distribution in cells, as well as to promote the formation of complex 3D cell culture structures such as organoids. Bioreactors supply dynamic culture conditions that ensure appropriate oxygenation and transport of nutrients, metabolites, and waste products while maintaining a balanced cellular distribution and survival. Several 3D bioreactors have been designed to be applied in cancer research studies; therefore, in this review, we will discuss the results emerging from the latest published studies that confirm the advantages of perfusion bioreactors in supporting the growth of organoids derived from different sources (stem cells, tumor tissue, experimental tumor cell lines) and able to closely recapitulate the cellular composition and architecture of the derived organs or tissues.

2. Organoid Research and Clinical Translation

There are many ongoing efforts aimed to develop valid and reproducible organoid models, not only for scientific investigations, but also for different applications including clinical use. According to the innovative features of regenerative medicine, three-dimensional islet-like structures originating from stem cells could be used to replace tissue damage in patients with type 1 diabetes [4]. The complete and accurate data analysis published by Bjørn Hofmann et al. highlighted that although there are numerous clinical studies involving organoids underway or registered, there are, to date, no relevant clinical outcomes, even if promising results could soon arrive [5]. The authors described that all retrieved reviews, at that time, indicated that all results were in the experimental or preclinical phase. However, the authors highlighted results from two publications on tumor organoids [6,7], where experimental and preclinical data demonstrated potential therapeutic applications. Additionally, they referenced two studies involving healthy organoids, one on cardiac organoids for pharmacological testing [8] and another on liver organoids for transplants, both of which remain in the preclinical stage [9]. Other studies conducted on healthy organoids are at the experimental phase and concern liver organoids [10], thyroid organoids [11], testicular organoids [12], retinal organoids [13], lung-on-a-chip devices for examining lung models [14], organoids miming extracellular vesicles for cancer, stem cell studies, and cardiac repair [15].

Organoid Cultures Advantages and Limitations

Organoids have great potential in basic cancer research and represent a new preclinical model with promise for studying the genesis and physiology of human diseases, as they retain the histological and gene expression features of native tissue. Encouraging results could soon come from clinical applications, although, to date, there are still many limitations to overcome. The production and maintenance of organoids are expensive and their generation is influenced by several factors such as cellularity of primary tissues and intra- and intertumoral heterogeneity [16,17]. Therefore, according to tumor type, culture conditions should be standardized, validating experimental reproducibility in the generation of organoids. Many of the protocols include the use of Matrigel or other animal-based matrix extract in order to mimic the extracellular matrix and to promote organoids formation. Matrigel is a protein combination produced by Englebreth–Holm–Swarm mouse sarcoma cells and its use may be related with important variable factors, such as the introduction of xenogenic or viral contaminants that could activate immune responses. Furthermore, the use of different batches may be associated with inconsistent biochemical properties that may affect reproducibility of organoids. Indeed, for this reason, there are studies on the development of artificially synthesized hydrogels for organoid cultures [18]. An important advantage of using organoids, over the two-dimensional culture models, is the presence of a vascular system. In the last years, different techniques have been tested to enable the vascularization of organoids. Hsieh Y-K. et al. were able to produce a microvascular structure using laser ablation [19]. In a different study, the authors developed endothelialized organoids through a pioneering 3D bioprinting technology by which cells are conveyed into hydrogels and enclosed layer by layer [20]. Cakir B. et al. [21] have engineered embryonic stem cells to express human ETS variant 2 (ETV2), a transcription factor that contributes to the development of vascular endothelial cells. The increased ETV2 protein expression in human cortical organoids was associated with their functional maturation and the acquisition of different characteristics of the blood–brain barrier.

3. Bioreactors

Conventionally, “bioreactors” are defined as vessels designed to produce biological materials that can vary in complexity, functional capabilities, and size [22].

Commonly, bioreactors are distinguished by commercial device, which are generally big tanks used for large-scale protein and antibodies production from living cells and organisms [23] and bioreactors with smaller dimensions used for different types of cells or tissue cultures such as organoids [24]. Bioreactors represent a precious device for the culture of organoids in vitro since they can better support, compared to static culture conditions, the growth, differentiation and maturation of organoids from different tissues. Additionally, they enable the maintenance and expansion of pluripotent stem cells.

3.1. Perfusion Bioreactor Systems a Promising Platform for Cancer Research

Perfusion bioreactors are a device able to mimic and finely control the tumor environment consisting of nutrient supply, gas concentration and exchanges, and above all, waste removal. In fact, this instrument supports and promotes cell growth, providing a continuous and constant nutrient exchange and waste constituents removal. Perfusion bioreactors comprise several components with a very simple and linear working flow. The key structural elements are a glass vessel and scaffolds where cells are arranged and an inlet and outlet pipe which drive, at the same time, both the injection of different types of solution in the flow chamber and the removal of waste. These devices are generally equipped with a series of sensors and a control unit to detect and regulate chemical and physical parameters such as temperature, pH, and oxygenation [25]. In bioreactors, the perfusion of the cell culture medium may occur in an indirect manner when the medium flows partly through the biomaterial and partly around it, while it happens in a direct way when the medium flows only through the biomaterial [26]. In addition, perfusion provides to the system a more stable maintenance of the culture environment and an efficient renewal of cytokines and growth factors, making this model effective in reproducing the in vivo environment. To date, many published works demonstrate that perfusion bioreactors improve proliferation, differentiation and the formation of new ECM progression. In addition, prolonged culture patient-derived biopsies showed the same gene expression profile and drug resistance patterns recognized in vivo [27].

Although bioreactors are an innovative and promising technology for cancer research, their use and applicability face several limitations. These include variability in organoid growth, reproducibility issues, challenges in large-scale production and immune cell integration, lengthy validation processes, incomplete reconstruction of the tumor microenvironment, poor compatibility with advanced imaging, limited adaptability for certain tumor types, and high analytical costs. Organoids are intricate biological systems that can exhibit significant heterogeneity in their development [28]. This variability complicates the standardization and reproducibility of experimental results, both of which are critical factors for translational research [29]. Current methodologies for growing organoids are labor intensive and often lack the reliability mandatory for high-throughput applications, hindering their integration into drug screening and large-scale therapeutic trials. Furthermore, vasculature, extracellular matrix composition, and immune system interactions are often missing or poorly represented, affecting the accuracy of tumor modeling. Tumor organoids derived from stem cells often fail to fully replicate the intricate components of the cancer microenvironment, such as stromal, immune and blood cells. The development of advanced techniques could improve these models by incorporating more cell types and thus improving their physiological relevance [30]. Another important consideration is that diffusion of essential elements such as oxygen and nutrients may be uneven, leading to hypoxic regions that do not accurately reflect the tumor physiology observed in patients. This non uniformity may also affect drug distribution, potentially altering assessments of the therapeutic response [31]. For some cancer models, such as pancreatic tumors or glioblastomas, characterized by highly fibrotic or infiltrative structures, it is very difficult to replicate using current organoid technologies, which hinders the development and testing of targeted therapies [32].The three-dimensional architecture of organoids presents significant challenges for various microscopy techniques, including histology, immunohistochemistry and deep immunofluorescence imaging. This structural complexity can impede the effective penetration of imaging reagents, thus limiting both the depth and clarity of the resulting imaging data. Furthermore, this promising technology is very expensive due to high analytical costs, partly due to specialized bioreactor sensors and equipment that constantly monitors cell culture conditions, such as pH, ORP/redox, and CO₂ sensors, and partly due to culture maintenance, both in terms of consumables and culture media that often need to be enriched with growth factors or other very expensive components [33,34] (

Table 1. Advantages and limitations of perfusion bioreactors.

Advantages	Limitations
Mimic tumor environment	Increased analytical costs
Constant and continuous nutrient exchange and waste constituents’ removal	Long validation time
Constant and continuous control and monitoring of chemical–physical parameters	Variability in organoid growth
High cell density	Challenges in large-scale reproduction
Improve proliferation, differentiation, and the formation of new ECM progression	Incomplete tumor microenvironment reconstruction
Very convenient for long term study	Poor compatibility and integration with advanced imaging
	Challenges in integrating immune cells
	Limited adaptability to specific histological tumor type

).

3.2. Perfusion Bioreactors for the Maintenance and Growth of Organoids

Through perfusion bioreactors, several cancer phenotypes such as Erwin’s sarcoma [35], leukemia [36], glioblastoma [37], breast cancer [38,39], ovarian cancer [40], bone metastatic prostate cancer [41] and colon cancer have been examined and developed [42]. An effective 3D in vitro model of bone metastatic prostate cancer was established under dynamic conditions through a perfusion bioreactor system to analyze the influence of fluid-derived shear stress on the tumor progression and ability to metastasize. Results were also compared with static culture conditions. Under dynamic conditions, the shear stress supported hMSC growth and differentiation, induced cell morphology changes, and influenced MET biomarker expression such as E-cadherin and Vimentin [41].

Additionally, Calamaio S. et al. [43] generated hepatic organoids starting from mesenchymal stem cells and hepatocytes both derived from hiPSCs, together with endothelial cells. Using both static and dynamic cultures, cell aggregates were grown in vitro for up to 1 week. The perfused bioreactor system, facilitating the constant recirculation of the culture medium, proved the effectiveness of dynamic culture in increasing and supporting hepatocytes maturation, as evidenced by the increased viability of organoids compared to spheroids. Through tissue examination and immunolocalization studies, the authors demonstrated the survival, aggregation, and structural organization of all cell types. Furthermore, RT-PCR analysis highlighted a notable increase in the expression levels of ALB mRNA, a marker of hepatic cell maturation.

Saggioro M. et al. [44] developed a three-dimensional model of alveolar rhabdomyosarcoma, culturing the RH30 cell line on a collagen sponge inserted in a perfusion-based bioreactor (U-CUP) for 7 days. Compared to static culture, perfusion conditions resulted in higher cell proliferation and greater cell dissemination. Furthermore, the developed organotypic tumor model simulated cell–ECM interactions and the expression of genes related to tumor progression and aggressiveness.

García-García A. et al. [36] detected in all experimental groups analyzed and grown under dynamic conditions, compared to static cultures, an increased cell proliferation and observed a higher cell content on scaffold with layers of smaller pore diameter and, hence, with a lower permeability. Human osteoblastic BM niches (O-N), engineered and grown in a bioreactor system, can become able to support malignant CD34+ cells from myeloproliferative tumors and acute myeloid leukemia patients for up to 3 weeks. Specifically, the stromal–vascular niche formed by human adipose tissue and stromal vascular fraction cells, along with O-N, regulated the cell growth, immunophenotype, and chemotherapy response of leukemic UCSD-AML1 cells.

A custom-made bioreactor was designed to study breast cancer behavior under static and dynamic culture conditions. MDA-MB-231 cells were grown in 3D collagen scaffolds and moved to a perfusion bioreactor which showed an augmented cell growth viability and distribution of cells within the scaffold together with changes in cell morphology. These data support a more aggressive phenotype for breast cancer cells cultured under dynamic conditions, as confirmed by the increased expression amount of some specific markers such as members of the matrix metalloproteinase group (MMP2, MMP3, and MMP9) and vimentin (VIM) [38].

Recently, prototypes of three-dimensional constructed perfusion bioreactors, made of polylactic acid, have been designed and produced. These new systems, which have the characteristic of being reusable and above all being customizable in both size and shape, have also been biologically confirmed for the perfusion-based culture of human mesenchymal stromal cells for a maximum of 2 weeks on collagen scaffolds. Images obtained by confocal microscopy, together with metabolic tests, confirmed a homogeneous distribution of mesenchymal cells within the pores of the material. Notably, the resultant human microenvironments were further utilized for 1 week to co-culture with human hematopoietic stem cells [45] (

Table 2. Selected studies using perfusion bioreactor for organoid culture.

Main Cancer Type	Maximun Culture Time	Bioreactor Effects	Reference
Prostate cancer	23 + 20 days	Increased proliferation and differentiation	[41]
Hepatic cancer	7 days	Increase and support hepatocytes maturation	[43]
Rhabdomyosarcoma	7 days	Increased tumor progression and aggressiveness	[44]
Ewing sarcoma	10 days	Enhanced cell proliferation	[35]
Myeloproliferative tumor and acute myeloid leukemia	3 weeks	Extended maintenance and expansion of patient-derived malignant HSPCs.	[36]
Breast cancer	7 days	More aggressive phenotype	[38]
Human mesenchymal stromal cells	2 weeks	Tumor cells and stromal structures retained their characteristics	[45]

).

3.3. Perfusion Bioreactors for the Maintenance and Growth of Tumor Patient Specimens

Glioblastoma explants of intra-operative pieces from the center and periphery of the cancer were cultured in a perfusion bioreactor to examine their composition and to evaluate their response to immunotherapy. For this purpose, the perfused samples were treated both at baseline and after 7 days targeting CD47 and/or PD-1. Central and periphery explants differed in cell type and composition of soluble factors and responses to immunotherapy as well as augmented levels of interferon-γ were detected in a subset of explants. Notably, the results obtained in this study revealed that ex vivo immunotherapy of glioblastoma tissue samples triggers an active antitumor immune response within the tumor center and provides a basis for the multidimensional personalized valuation of tumor response to immunotherapy [37].

Martinez A. et al. [40] demonstrated the validity and effectiveness of a 3D perfused bioreactor as a preclinical system to investigate epithelial ovarian cancer and ovarian carcinosarcoma in response to treatment outcomes. Specifically, authors showed that the perfused bioreactor significantly increased the density of the ovarian tumor cell compared to the solid and non-perfused systems. Importantly, patient-derived tumor samples cultured for 7 days in the perfused bioreactor kept their histologic features, immune profiles, and viability. This system has also proven to be valid for analyzing archived patient specimens that had been frozen for <1 year, since the samples maintain an acceptable morphology and proliferation markers. The latter is an achievement of great importance since fresh tissue is not always available. The authors also provided further evidence supporting the reliability of the perfused bioreactor as a preclinical model, testing chemotherapeutic agents (cisplatin and paclitaxel), both in patient-derived tumor samples and in epithelial ovarian cancer cell lines.

A perfusion bioreactor was also used to evaluate the system’s ability to preserve the cellular elements within the tumor microenvironment (TME) in primary colorectal cancer (CRC) tissues. For this purpose, tumor specimens from freshly resected human colorectal cancer samples were placed among two collagen scaffolds in a sandwich formation and grown under static or dynamic conditions for up to 3 days. Compared to static culture, perfused tissues maintain their architecture and cellular density. Furthermore, stromal and immune cells have proven to be vital as they were reactive to microenvironmental stimuli. Interestingly, cell cultures maintained under perfusion were demonstrated to be appropriate for assessing the sensitivity of primary tumor cells to chemotherapies currently in use for CRC, as perfusion-based primary cultured CRC samples recapitulate key features of the TME [42].

Muraro M.G. et al. [27], using a perfusion bioreactor, kept tumor breast specimens in a sandwich culture system for up to 14 days. Additionally, using next-generation sequencing, the authors observed a strong correlation between clinical samples and those cultured in the bioreactor.

A perfusion bioreactor was also used to grow and monitor neuroblastoma tissue. This system proved to be a valid approach since the tissue structure appeared to be well organized even after 7 days of culture, where the tumor cells and stromal structures retained their characteristics intact. Furthermore, the system was used for the consecutive monitoring of drug response with isothermal microcalorimetry, confirming that the system is efficient and sensitive and therefore could represent an excellent method for developing personalized pharmacological therapies for neuroblastoma treatment [46] (

Table 3. Selected studies using perfusion bioreactor for tissue culture.

Main Cancer Type	Maximun Culture Time	Bioreactor Effects	Reference
Glioblastoma	7 days	Antitumor immune response activation	[37]
Epithelial ovarian cancer	7 days	Increased ovarian cancer cell density	[40]
Colorectal cancer	3 days	Conservation and maintenance of cellular architecture and density	[42]
Breast cancer	14 days	Preservation of cellar composition and interaction of cancer cells and TME	[27]
Neuroblastoma	7 days	The system enables the preservation of high-quality tissue, maintaining both intact tumor cells and stromal structure	[46]

).

4. Conclusions

Organoids represent the evolutionary leap beyond 2D cell culture and are characterized by the ability to recapitulate the morpho-functional features of human organs and tissues. These new experimental models hold significant potential for studying and treating human diseases including cancer and its essential microenvironment. With recent advances in organoid culture, they have become a critical tool in drug development, particularly for expanding therapeutic options for rare tumors that lack targeted therapy [47,48]. However, their full potential relies on the development of specialized devices capable of supporting organoid growth, differentiation and maintenance while preserving fidelity to the original tissue. In this context, perfusion bioreactors (Figure 1) have emerged as a promising platform for cancer research and tissue engineering. These systems offer key advantages by providing a highly controlled and reproducible environment that closely mimics vivo conditions. The perfusion system enables efficient nutrient exchange and waste removal, improving cell viability and promoting proliferation and differentiation; moreover, this technology allows for the real-time monitoring of critical parameters such as pH, temperature and oxygenation, which are essential for maintaining optimal culture conditions and cellular functions. The dynamic flow in perfusion bioreactors also supports the spatial distribution of cells and extracellular matrix, enabling the more accurate modeling of tumor microenvironments and improved responses to therapeutic challenges. Additionally, they have been successfully employed for the maintenance and growth of both organoids and patient-derived biopsies, retaining their gene expression profiles and drug resistance patterns, making them precious for preclinical testing.

Despite their promise, several limitations hinder this new technology, including high analytical costs, long validation times, growth variability, imperfect recapitulation of the tumor microenvironment, compatibility issues and integration with advanced imaging and omics technologies, and feasibility constraints for certain tumors types. Addressing these challenges requires a multidisciplinary approach to translate individual research findings into clinically effective therapies.