Bioreactors provide controlled delivery of nutrients and biomimetic stimuli in order to influence cell growth, differentiation, and tissue formation. They have been extensively used to promote the expansion of red blood cells, chimeric antigen receptor (CAR) T cells, induced pluripotent stem cells, and mesenchymal stem cells. Additionally, the ability to control the spatiotemporal delivery of the biological, biochemical, and biophysical signals that regulate tissue development confers a number of advantages for engineering 3D tissues relative to standard cell culture techniques by providing well-defined conditions to regulate cell behaviors. These advantages include (i) improved standardization and reproducibility, (ii) scale-up to larger, clinically relevant tissue grafts or cell expansion scales, (iii) superior functionality compared with 3D grafts cultured in tissue culture flasks, and (iv) improved systems for testing cell responses to a range of experimental parameters. As the field of regenerative medicine has matured, the number of applications has increased and the roles that bioreactors play in enabling the commercialization and clinical translation of stem cell-based technologies have become more defined. In this review, we will provide a critical overview of biomedical applications of bioreactors and discuss current trends and recent advances that promote the application of bioreactor technologies for single-cell manufacture, production of engineered tissue grafts, and drug screening.

The therapeutic promise of stem cell-based technologies for the treatment of pathologies ranging from hair loss ¹ to blindness ² has precipitated the need for a cell-manufacturing sector to provide therapeutic allogeneic cells. Owing to the extensive infrastructure requirements and rigorous standards defined by regulatory agencies, the cost will likely be too burdensome for traditional hospitals and treatment centers and will manifest as centralized facilities that specialize in providing high-quality cells with verifiable characteristics. However, cell-based therapies often require the application of vast quantities of cells (10 ⁸–10 ¹⁰) in order to be effective. A practical limitation arises as the amount of space required to grow these large quantities of cells using standard cell culture apparatus is prohibitive. This has spawned a demand for bioreactors capable of supporting industrial-scale, ultra-high-density cell suspension cultures with controlled microenvironments, standardization, and uniformity of culture conditions in order to generate homogenous populations of stem or lineage-specific cells. A few types of bioreactors have been employed to generate large populations of phenotypically defined cells. Variable designs have been employed for adherent versus non-adherent cells and to account for differences in cellular responses to microenvironmental cues. Table 1 summarizes several bioreactor types that have been used for cell expansion.

In contrast to bioreactors that produce single cells, tissue engineering bioreactors have supported the development of large 3D tissue grafts. To produce large (centimeter-sized) viable grafts, these systems often use convective flow to provide crucial mass transport regimes, overcome the diffusional limitations of nutrients and oxygen, and prevent the accumulation of metabolic waste products that otherwise induce starvation and death of the cells in the inner regions of the construct. Tissue engineering bioreactors can also enhance the functionality of grafts through the application of biomimetic physiological stimuli as well as the incorporation of sensors that give real-time feedback of culture conditions. After incubation, the mature, functional cellular constructs can be transplanted in vivo to regenerate damaged tissues. Tissue engineering bioreactors will likely play a significant role in translating engineered grafts to the clinic as the potential automation renders them economically efficient and amenable to mass production for larger populations of patients.

Cutting-edge research in this field continues to focus on the improved application of biophysical stimuli to optimize functional tissue assembly ^{30– 35} and computational modeling to improve predictability of the outcomes ^{19, 36, 37– 40} . Additionally, notable efforts to enhance the clinical applicability of these grafts have focused on engineering grafts that are similar in size to critical-sized bone defects in humans and are tailored to the patient ^{20, 41– 42} . Nguyen et al. recently demonstrated the ability to culture a 200 cm ³ cell-based construct in vitro without the development of necrotic centers ^{20, 42} . In this approach, bone marrow-derived mesenchymal stem cells were encapsulated in hydrogel beads and placed in a tubular perfusion bioreactor. Three-dimensional-printed molds that could be anatomically shaped were used to direct the flow through the hydrogel beads. The space between the hydrogel beads enhanced mass transport to the cells throughout the entire construct, allowing the stem cells to remain viable and undergo osteogenic differentiation. Although this approach represents a critical advancement in the culture of clinically sized constructs, it remains limited by the use of hydrogel beads that minimize cell–cell interactions and inhibit paracrine signaling between cells, which are important factors in bone formation. In contrast, Bhumiratana et al. directly seeded adipose-derived stem cells into the pore spaces of anatomically shaped, porcine temporomandibular joint scaffolds ⁴¹ . They cultured the adipose-derived stem cell-seeded scaffolds in perfusion bioreactors for 3 weeks in vitro before using the bioreactors to maintain their viability during transport to an on-site animal facility. The grafts—customized for each pig—were implanted and cultured for up to 6 months in vivo. This was a foundational, proof-of-concept study and clearly demonstrated the feasibility of using this strategy as a treatment for humans. However, in general, there remains a huge gap in the growth of 3D engineered tissues in bioreactors and demonstration of in vivo functional integration and potency.

In spite of the inherent advantages of using tissue engineering bioreactors to grow entire grafts that are primed for implantation into defect sites, there are a number of practical barriers to clinical translation associated with extended ex vivo culture. One major limitation is that the large, volumetric grafts often lack an intact vasculature, which consequently hampers their post-transplantation viability. To overcome these limitations, an alternative approach known as “ in vivo bioreactors” has been employed. Unlike the systems described above, the in vivo bioreactor, despite the use of the “bioreactor” terminology, does not incorporate robust design principles or the development of new equipment. There is no hardware, and the success of the strategy is highly dependent on surgical expertise and manipulation. Rather, it primarily refers to a pocket within the body into which biomaterials or immature tissue engineered constructs are surgically implanted and incubated for an extended period of time. Within these pockets (for example, omentum or muscle flap), the grafts harness the regenerative capacity of the body to become fully vascularized. Key advantages of this method include the presence of naturally occurring cytokines and other factors, the establishment of neovasculature and nervous tissue within the implant, and immune compatibility ⁴³ . The primary application of the in vivo bioreactor principle has been for the development of critical-sized bone grafts. Several recent studies have demonstrated the use of prefabricated bone grafts which are either incubated in situ or vascularized by extended implantation in muscle or omentum or anastomosed with large arteries ^{43– 53} and may be feasible even without the use of transplanted stem cells or growth factors.

As the previous examples illustrate, bioreactors typically have been employed to address challenges of scale-up. However, miniaturized tissues created by using microfluidic bioreactors facilitate efficient, inexpensive, high-throughput drug screening or disease modeling. Microfluidic bioreactors—often referred to as lab-on-a-chip systems—use minute quantities of cells grown together in micrometer-scaled wells. Microliter volumes of fluid are pumped to the cells through channels that allow the effects of multiple concentrations of growth factors or pharmacological agents to be rapidly tested. Often, modified cells are used to permit easily monitored parameters (such as fluorescence ⁵⁴ ) to be used as read-outs of cellular responses. Early modifications to these systems enabled the use of high-density 3D cell culture using multi-cell aggregates, microspheres, and cell encapsulation to better recapitulate the cell–cell interactions of native tissues in ways not possible in 2D culture. Even so, it is challenging to replicate the impact of pharmacological agents on the complex functions of tissues, such as the lung or heart, in these simplified systems. Hence, more recent versions of lab-on-a-chip bioreactors have incorporated physiological factors such as airflow and mechanical stimulation that mimic breathing ^{55, 56} or have integrated vasculature and direct blood flow with contractile cardiac cells ⁵⁷ . These two technologies, which are currently being commercialized, more accurately capture physiological responses to specific stimuli while retaining the benefits of simplicity and low cost.

The most recent developments in the field of lab-on-a-chip technology have focused on increasing the ease of use. For example, researchers are investigating methods to 3D print and seed an entire chip in a single pass ^{54, 58} . Other teams have developed smartphone-based systems to monitor the internal environment ⁵⁹ and hybrid materials which allow point-by-point manipulation of the cells within the bioreactor ⁶⁰ . Perhaps one of the most significant advantages of lab-on-a-chip systems is their ability to capture complex physiology of multiple organ systems. Lee et al. ⁶¹ and Shirure and George ⁶² reported on the development of pumpless, dual-organ bioreactor systems. Current trends portend the advent of more advanced human-on-a-chip systems, which will test on- and off-target effects of drugs on multiple organ systems.

Bioreactors fill a critical niche in the commercialization and clinical translation of cell-based therapies and drug-testing platforms. Current trends suggest an increased emphasis on manufacturing needs. This includes scaling up of suspension culture bioreactors to industrial sizes and modifications of tissue engineering bioreactors to enable the formation of patient-specific grafts that are of therapeutically relevant sizes. In spite of the many scientific and technical advantages of these systems, regulatory requirements may prove to be significant barriers to their clinical application. For lab-on-a-chip systems, major advancements in monitoring, control, and fabrication techniques are resulting in progressively more complex systems that more closely mimic human physiology and capture the interactions of multiple organs. The establishment of low-cost platforms will have a significant beneficial impact on the future of disease modeling and drug testing.