Perfusion Bioreactor System Research Articles

Design and evaluation of a 3D-printed, lab-scale perfusion bioreactor for novel biotechnological applications.

3D-printing increased in significance for biotechnological research as new applications like lab-on-a-chip systems, cell culture devices or 3D-printed foods were uncovered. Besides mammalian cell culture, only few of those applications focus on the cultivation of microorganisms and none of these make use of the advantages of perfusion systems. One example for applying 3D-printing for bioreactor development is the microbial utilization of alternative substrates derived from lignocellulose, where dilute carbon concentrations and harmful substances present a major challenge. Furthermore, quickly manufactured and affordable 3D-printed bioreactors can accelerate early development phases through parallelization. In this work, a novel perfusion bioreactor system consisting of parts manufactured by fused filament fabrication (FFF) is presented and evaluated. Hydrophilic membranes are used for cell retention to allow the application of dilute substrates. Oxygen supply is provided by membrane diffusion via hydrophobic polytetrafluoroethylene membranes. An exemplary cultivation of Corynebacterium glutamicum ATCC 13032 supports the theoretical design by achieving competitive biomass concentrations of 18.4gL-1 after 52h. As a proof-of-concept for cultivation of microorganisms in perfusion mode, the described bioreactor system has application potential for bioconversion of multi-component substrate-streams in a lignocellulose-based bioeconomy, for in-situ product removal or design considerations of future applications for tissue cultures. Furthermore, this work provides a template-based toolbox with instructions for creating reference systems in different application scenarios or tailor-made bioreactor systems.

Ex Vivo Preservation of Ovine Periosteum Using a Perfusion Bioreactor System.

Periosteum is a highly vascularized membrane lining the surface of bones. It plays essential roles in bone repair following injury and reconstruction following invasive surgeries. To broaden the use of periosteum, including for augmenting in vitro bone engineering and/or in vivo bone repair, we have developed an ex vivo perfusion bioreactor system to maintain the cellular viability and metabolism of surgically resected periosteal flaps. Each specimen was placed in a 3D printed bioreactor connected to a peristaltic pump designed for the optimal flow rates of tissue perfusate. Nutrients and oxygen were perfused via the periosteal arteries to mimic physiological conditions. Biochemical assays and histological staining indicate component cell viability after perfusion for almost 4 weeks. Our work provides the proof-of-concept of ex vivo periosteum perfusion for long-term tissue preservation, paving the way for innovative bone engineering approaches that use autotransplanted periosteum to enhance in vivo bone repair.

Mesenchymal Stem Cell Culture within Perfusion Bioreactors Incorporating 3D-Printed Scaffolds Enables Improved Extracellular Vesicle Yield with Preserved Bioactivity.

Extracellular vesicles (EVs) are implicated as promising therapeutics and drug delivery vehicles in various diseases. However, successful clinical translation will depend on the development of scalable biomanufacturing approaches, especially due to the documented low levels of intrinsic EV-associated cargo that may necessitate repeated doses to achieve clinical benefit in certain applications. Thus, here the effects of a 3D-printed scaffold-perfusion bioreactor system are assessed on the production and bioactivity of EVs secreted from bone marrow-derived mesenchymal stem cells (MSCs), a cell type widely implicated in generating EVs with therapeutic potential. The results indicate that perfusion bioreactor culture induces an ≈40-80-fold increase (depending on measurement method) in MSC EV production compared to conventional cell culture. Additionally, MSC EVs generated using the perfusion bioreactor system significantly improve wound healing in a diabetic mouse model, with increased CD31+ staining in wound bed tissue compared to animals treated with flask cell culture-generated MSC EVs. Overall, this study establishes a promising solution to a major EV translational bottleneck, with the capacity for tunability for specific applications and general improvement alongside advancements in 3D-printing technologies.

Generation of colon cancer–derived tumor-infiltrating T cells (TILs) for adoptive cell therapy

Adoptive cell therapy (ACT) using specific immune cells and stem cells has emerged as a promising treatment option that could complement traditional cancer therapies in the future. In particular, tumor-infiltrating lymphocytes (TILs) have been shown to be effective against solid tumors in various clinical trials. Despite the enormous disease burden and large number of premature deaths caused by colorectal cancer (CRC), studies on TILs isolated from tumor tissue of patients with CRC are still rare. To date, studies on ACT often lack controlled and comparable expansion processes as well as selected ACT-relevant T-cell populations. We describe a procedure for generating patient-specific TILs, which are prerequisites for clinical trials of ACT in CRC. The manufacturing and characteristics of these TILs differ in important modalities from TILs commonly used for this therapeutic approach. Tumor tissue samples were obtained from 12 patients undergoing surgery for primary CRC, predominantly with low microsatellite instability (pMMR-MSI-L). Tumors in the resected specimens were examined pathologically, and an approved volume of tumor tissue was transferred to a disposable perfusion bioreactor. Tissue samples were subjected to an automatically controlled and highly reproducible cultivation process in a GMP-conform, closed perfusion bioreactor system using starting medium containing interleukin-2 and interleukin-12. Outgrowth of TIL from tissue samples was initiated by short-term supplementation with a specific activation cocktail. During subsequent expansion, TILs were grown in interleukin-2–enriched medium. Expansion of TILs in a low-scaled, two-phase process in the Zellwerk ZRP bioreactor under hyperoxic conditions resulted in a number of approximately 2 × 109 cells. The expanded TILs consisted mainly (73%) of the ACT-relevant CD3+/CD8+ effector memory phenotype (CD45RO+/CCR7−). TILs harvested under these conditions exhibited high functional potential, which was confirmed upon nonspecific stimulation (interferon-γ, tumor necrosis factor-α cytokine assay)

Cancer Exosomes: An Overview and the Applications of Flow

Cancer is one of the most prevalent and disruptive diseases affecting the population, and as such, is the subject of major research efforts. Recently, these efforts have been put towards understanding the role that exosomes can play in the progression of cancer. Exosomes are small extracellular vesicles ranging from 40–150 nm in size that carry bioactive molecules like proteins, DNA, RNA, miRNA, and surface receptors. One of the most important features of exosomes is their ability to easily travel throughout the body, extending the reach of parent cell’s signaling capabilities. Cancer derived exosomes (CDEs) carry dangerous cargo that can aid in the metastasis, and disease progression through angiogenesis, promoting epithelial to mesenchymal transition, and immune suppression. Exosomes can transport these molecules to cells in the tumor environment as well as distant premetastatic locations making them an extremely versatile tool in the toolbelt of cancer. This review aims to compile the present knowledge and understanding of the involvement of exosomes in the progression of cancer as well as current production, isolation, and purification methods, with particular interest on flow perfusion bioreactor and microfluidics systems, which allow for accurate modeling and production of exosomes.

On the quest of reliable 3D dynamic in vitro blood-brain barrier models using polymer hollow fiber membranes: Pitfalls, progress, and future perspectives.

With the increasing concern of neurodegenerative diseases, the development of new therapies and effective pharmaceuticals targeted to central nervous system (CNS) illnesses is crucial for ensuring social and economic sustainability in an ageing world. Unfortunately, many promising treatments at the initial stages of the pharmaceutical development process, that is at the in vitro screening stages, do not finally show the expected results at the clinical level due to their inability to cross the human blood-brain barrier (BBB), highlighting the inefficiency of in vitro BBB models to recapitulate the real functionality of the human BBB. In the last decades research has focused on the development of in vitro BBB models from basic 2D monolayer cultures to 3D cell co-cultures employing different system configurations. Particularly, the use of polymeric hollow fiber membranes (HFs) as scaffolds plays a key role in perfusing 3D dynamic in vitro BBB (DIV-BBB) models. Their incorporation into a perfusion bioreactor system may potentially enhance the vascularization and oxygenation of 3D cell cultures improving cell communication and the exchange of nutrients and metabolites through the microporous membranes. The quest for developing a benchmark 3D dynamic in vitro blood brain barrier model requires the critical assessment of the different aspects that limits the technology. This article will focus on identifying the advantages and main limitations of the HFs in terms of polymer materials, microscopic porous morphology, and other practical issues that play an important role to adequately mimic the physiological environment and recapitulate BBB architecture. Based on this study, we consider that future strategic advances of this technology to become fully implemented as a gold standard DIV-BBB model will require the exploration of novel polymers and/or composite materials, and the optimization of the morphology of the membranes towards thinner HFs (<50μm) with higher porosities and surface pore sizes of 1-2µm to facilitate the intercommunication via regulatory factors between the cell co-culture models of the BBB.

3D Human iPSC Blood Vessel Organoids as a Source of Flow-Adaptive Vascular Cells for Creating a Human-Relevant 3D-Scaffold Based Macrovessel Model.

3D-scaffold based in vitro human tissue models accelerate disease studies and screening of pharmaceutics while improving the clinical translation of findings. Here is reported the use of human induced pluripotent stem cell (hiPSC)-derived vascular organoid cells as a new cell source for the creation of an electrospun polycaprolactone-bisurea (PCL-BU) 3D-scaffold-based, perfused human macrovessel model. A separation protocol is developed to obtain monocultures of organoid-derived endothelial cells (ODECs) and mural cells (ODMCs) from hiPSC vascular organoids. Shear stress responses of ODECs versus HUVECs and barrier function (by trans endothelial electrical resistance) are measured. PCL-BU scaffolds are seeded with ODECs and ODMCs, and tissue organization and flow adaptation are evaluated in a perfused bioreactor system. ODECs and ODMCs harvested from vascular organoids can be cryopreserved and expanded without loss of cell purity and proliferative capacity. ODECs are shear stress responsive and establish a functional barrier that self-restores after the thrombin challenge. Static bioreactor culture of ODECs/ODMCs seeded scaffolds results in a biomimetic vascular bi-layer hierarchy, which is preserved under laminar flow similar to scaffolds seeded with primary vascular cells. HiPSC-derived vascular organoids can be used as a source of functional, flow-adaptive vascular cells for the creation of 3D-scaffold based human macrovascular models.

Multi-Stream Perfusion Bioreactor Integrated with Outlet Fractionation for Dynamic Cell Culture.

Certain cell and tissue functions operate within the dynamic time scale of minutes to hours that are poorly resolved by conventional culture systems. This work has developed a low-cost perfusion bioreactor system that allows culture medium to be continuously perfused into a cell culture module and fractionated in a downstream module to measure dynamics on this scale. The system is constructed almost entirely from commercially available parts and can be parallelized to conduct independent experiments in conventional multi-well cell culture plates simultaneously. This video article demonstrates how to assemble the base setup, which requires only a single multichannel syringe pump and a modified fraction collector to perfuse up to six cultures in parallel. Useful variants on the modular design are also presented that allow for controlled stimulation dynamics, such as solute pulses or pharmacokinetic-like profiles. Importantly, as solute signals travel through the system, they are distorted due to solute dispersion. Furthermore, a method for measuring the residence time distributions (RTDs) of the components of the perfusion setup with a tracer using MATLAB is described. RTDs are useful to calculate how solute signals are distorted by the flow in the multi-compartment system. This system is highly robust and reproducible, so basic researchers can easily adopt it without the need for specialized fabrication facilities.

Personalized Volumetric Tissue Generation by Enhancing Multiscale Mass Transport through 3D Printed Scaffolds in Perfused Bioreactors.

Engineered tissues provide an alternative to graft material, circumventing the use of donor tissue such as autografts or allografts and non-physiological synthetic implants. However, their lack of vasculature limits the growth of volumetric tissue more than several millimeters thick which limits their success post-implantation. Perfused bioreactors enhance nutrient mass transport inside lab-grown tissue but remain poorly customizable to support the culture of personalized implants. Here, a multiscale framework of computational fluid dynamics (CFD), additive manufacturing, and a perfusion bioreactor system are presented to engineer personalized volumetric tissue in the laboratory. First, microscale 3D printed scaffold pore geometries are designed and 3D printed to characterize media perfusion through CFD and experimental fluid testing rigs. Then, perfusion bioreactors are custom-designed to combine 3D printed scaffolds with flow-focusing inserts in patient-specific shapes as simulated using macroscale CFD. Finally, these computationally optimized bioreactor-scaffold assemblies are additively manufactured and cultured with pre-osteoblast cells for 7, 20, and 24days to achieve tissue growth in the shape of human calcaneus bones of 13mL volume and 1cm thickness. This framework enables an intelligent model-based design of 3D printed scaffolds and perfusion bioreactors which enhances nutrient transport for long-term volumetric tissue growth in personalized implant shapes. The novel methods described here are readily applicable for use with different cell types, biomaterials, and scaffold microstructures to research therapeutic solutions for a wide range of tissues.

3D cell cultures, as a surrogate for animal models, enhance the diagnostic value of preclinical in vitro investigations by adding information on the tumour microenvironment: a comparative study of new dual-mode HDAC inhibitors

Anchorage-independent 3D-cultures of multicellular tumour spheroids (MCTS) and in vitro microtumours of cancer cells can provide upfront information on the effects of anticancer drug candidates, tantamount to that obtained from animal xenograft studies. Unlike 2D cancer cell cultures, 3D-models take into account the influence of the tumour microenvironment and the location dependence of drug effects and accumulation. We exemplified this by comparison of the effects of two new dual-mode anticancer agents, Troxbam and Troxham, and their monomodal congeners SAHA (suberoylanilide hydroxamic acid) and CA-4 (combretastatin A-4). We assessed the growth of MCTS of HCT116wt human colon carcinoma cells exposed to these compounds, as well as the spatial distribution of dead HCT116wt cells in these MCTS. Also, fluorescence imaging of live and fixed MCTS was used to assess the type of cellular death induced by test compounds. Furthermore, an innovative perfusion bioreactor system was used to grow microtumours in the presence or absence of test compounds. Both new investigational compounds led to significant reductions of the size of such MCTS and also of corresponding in vitro microtumours by inducing caspase-9 dependent apoptosis and elevated levels of reactive oxygen species. 3D multicellular tumour spheroids are easy to grow and employ for compound tests in the familiar well-plate set-up. Together with 3D microtumours grown at scaffolds in continuously perfused bioreactors they allow to study, early on in the course of drug evaluations, the communication of tumour cells with their microenvironment to an extent hitherto available only in animal experiments.

Synthetic vascular graft with spatially distinct architecture for rapid biomimetic cell organisation in a perfusion bioreactor

Access to lab-grown fully functional blood vessels would provide an invaluable resource to vascular medicine. The complex architecture and cellular makeup of native vessels, however, makes this extremely challenging to reproduce in vitro. Bioreactor systems have helped advanced research in this area by replicating many of the physiological conditions necessary for full-scale tissue growth outside of the body. A key element underpinning these technologies are 3D vascular graft templates which serve as temporary scaffolds to direct cell growth into similar cellular architectures observed in native vessels. Grafts further engineered with appropriate physical cues to accommodate the multiple cell types that reside within native vessels may help improve the production efficiency and physiological accuracy of bioreactor-grown vessel substitutes. Here, we engineered two distinct scaffold architectures into an electrospun vascular graft aiming to encourage the spatial organisation of human vascular endothelial cells (hCAECs) in a continuous luminal monolayer, co-cultured with human fibroblasts (hFBs) populating the graft wall. Using an electrospun composite of polycaprolactone and gelatin, we evaluated physical parameters including fibre diameter, fibre alignment, and porosity, that best mimicked the spatial composition and growth of hCAECs and hFBs in native vessels. Upon identifying the optimal scaffold architectures for each cell type, we constructed a custom-designed mandrel that combined these distinct architectures into a single vascular graft during a single electrospinning processing run. When connected to a perfusion bioreactor system, the dual architecture graft spatially oriented hCAECs and hFBs into the graft wall and lumen, respectively, directly from circulation. This biomimetic cell organisation was consistent with positive graft remodelling with significant collagen deposition in the graft wall. These findings demonstrate the influence of architectural cues to direct cell growth within vascular graft templates and the future potential of these approaches to more accurately and efficiency produce blood vessel substitutes in bioreactor systems.

Optimization and Validation of a Custom-Designed Perfusion Bioreactor for Bone Tissue Engineering: Flow Assessment and Optimal Culture Environmental Conditions.

Various perfusion bioreactor systems have been designed to improve cell culture with three-dimensional porous scaffolds, and there is some evidence that fluid force improves the osteogenic commitment of the progenitors. However, because of the unique design concept and operational configuration of each study, the experimental setups of perfusion bioreactor systems are not always compatible with other systems. To reconcile results from different systems, the thorough optimization and validation of experimental configuration are required in each system. In this study, optimal experimental conditions for a perfusion bioreactor were explored in three steps. First, an in silico modeling was performed using a scaffold geometry obtained by microCT and an expedient geometry parameterized with porosity and permeability to assess the accuracy of calculated fluid shear stress and computational time. Then, environmental factors for cell culture were optimized, including the volume of the medium, bubble suppression, and medium evaporation. Further, by combining the findings, it was possible to determine the optimal flow rate at which cell growth was supported while osteogenic differentiation was triggered. Here, we demonstrated that fluid shear stress up to 15 mPa was sufficient to induce osteogenesis, but cell growth was severely impacted by the volume of perfused medium, the presence of air bubbles, and medium evaporation, all of which are common concerns in perfusion bioreactor systems. This study emphasizes the necessity of optimization of experimental variables, which may often be underreported or overlooked, and indicates steps which can be taken to address issues common to perfusion bioreactors for bone tissue engineering.

Selection of O-negative induced pluripotent stem cell clones for high-density red blood cell production in a scalable perfusion bioreactor system.

ObjectivesLarge‐scale generation of universal red blood cells (RBCs) from O‐negative (O‐ve) human induced pluripotent stem cells (hiPSCs) holds the potential to alleviate worldwide shortages of blood and provide a safe and secure year‐round supply. Mature RBCs and reticulocytes, the immature counterparts of RBCs generated during erythropoiesis, could also find important applications in research, for example in malaria parasite infection studies. However, one major challenge is the lack of a high‐density culture platform for large‐scale generation of RBCs in vitro.Materials and MethodsWe generated 10 O‐ve hiPSC clones and evaluated their potential for mesoderm formation and erythroid differentiation. We then used a perfusion bioreactor system to perform studies with high‐density cultures of erythroblasts in vitro.ResultsBased on their tri‐lineage (and specifically mesoderm) differentiation potential, we isolated six hiPSC clones capable of producing functional erythroblasts. Using the best performing clone, we demonstrated the small‐scale generation of high‐density cultures of erythroblasts in a perfusion bioreactor system. After process optimization, we were able to achieve a peak cell density of 34.7 million cells/ml with 92.2% viability in the stirred bioreactor. The cells expressed high levels of erythroblast markers, showed oxygen carrying capacity, and were able to undergo enucleation.ConclusionsThis study demonstrated a scalable platform for the production of functional RBCs from hiPSCs. The perfusion culture platform we describe here could pave the way for large volume‐controlled bioreactor culture for the industrial generation of high cell density erythroblasts and RBCs.

Abstract P469: Scale-up And Characterization Of Therapeutic Mesenchymal Stromal Cell-derived Extracellular Vesicles

Introduction: Early studies have shown therapeutic benefits of mesenchymal stromal cells (MSCs) in cardioprotection due to their angiogenic, proliferative, anti-apoptotic and anti-inflammatory properties, which are now attributed to secreted factors such as extracellular vesicles (EVs). While MSC-EVs have shown promise in small animals for cardiovascular therapies, large animal studies are required to evaluate the therapeutic benefit of MSC-EVs for clinical translation. One of the biggest challenges for large animal studies is the need to generate clinically-relevant quality and quantity of EVs without batch-to-batch variations that could compromise efficacy. This study aims to explore three different cell culture methods (traditionally-used tissue culture plates (TCP), 3-D printed bioscaffolds in a perfusion system (P), and microcarriers in dynamic spinner flask conditions (M)) to scale-up the production of MSC-EVs across four different biological donors and rigorously investigate EV yield, size, shape, and content. Methods: MSCs were isolated from the iliac crest of four different Yucatan minipigs using heparinized syringes, and cells were expanded to passage four, at which point they were seeded onto the respective cell culture methods. EVs were collected from conditioned medium (CM) via differential ultracentrifugation. EV size, distribution, yield, and protein concentration were studied using Nanoparticle Tracking Analysis (NTA) and microBCA assays. Results: Both perfusion bioreactor and spinner flask systems enabled sustained maintenance of large numbers of cells. Across biological donors and fabrication methods, modes remained within 50-150 nm and were not statistically different. Microcarrier-based spinner flasks and perfusion bioreactor set-ups both improved EV yield, up to 6 times in efficiency. Ongoing research focuses on examining differences in EV content across biological donors using RNA-sequencing and proteomics.

Using 3D perfusion bioreactor system to studying cell biology of ovarian cancer.

e17558 Background: Epithelial Ovarian Cancer (EOC) is the most common cause of death among gynecological malignancies. This is a result of the high rate of recurrence and chemo-resistance in EOC patients. Therefore, the development of new therapeutics is crucial. A major factor contributing to this is the lack of therapeutic candidates is lack of translational accuracy in preclinical models. Recently, 3-dimensional (3-D) models have aided in accurately recreating tumor biology. We have developed an EOC 3-D perfused bioreactor system that recapitulates EOC tumor biology and incorporates tumor biomechanical regulation. This model allows for us to more accurately predict the clinical response of new drug candidates, which aids in elimination of ineffective candidates prior to clinical trials. Methods: EOC cell lines (luciferase-taggedSKOV-3 and OVCAR-8) were embedded in a relevant extracellular matrix (ECM) and injected into a perfused, polydimethylsiloxane (PDMS) bioreactor. Microchannels were embedded in matrigel so that the cell culture media with or without chemotherapy could flow through the perfused PDMS to provide nutrient delivery and gas exchange enhancing viability and function of surrounding cells. The bioreactors were connected to a peristaltic pump that allowed for the cell culture media to perfuse over a 7-day period. We monitored cell viability using bioluminescence imaging (BLI), immunohistochemistry (IHC), and lactate dehydrogenase (LDH) release in media. Results: BLI showed a linear increase in SKOV-3 and OVCAR-8 cell growth over 7 days. These results were confirmed by IHC measuring the number of nucleated cells per micron2. Graphical representation of the region of interest (ROI) showed a high correlation between IHC staining of nucleated cells and BLI score. IHC analysis of PAX8 staining was positive and proved that the perfusion bioreactor system maintains EOC biology over time. In addition, our results suggest that the bioreactor is a suitable model for drug preclinical testing in both cell lines as well as in patients’ samples. Conclusions: Our preliminary results using the 3D EOC perfused, PDMS bioreactor model showed increased EOC cell growth overtime, while maintaining original EOC histology. Moreover, our results suggest that this model could provide a novel platform to study therapeutic interventions in EOC. Our ultimate goal is to implement ovarian cancer microenvironment components (e.g. immune cells) into bioreactor system to study different drug treatments to better determine drug candidate’s translational efficacy.

Aligned Silk Sponge Fabrication and Perfusion Culture for Scalable Proximal Tubule Tissue Engineering.

Chronic kidney disease and kidney failure are on the rise globally, yet there has not been a corresponding improvement in available therapies. A key challenge in a biological approach to developing kidney tissue is to identify scaffolding materials that support cell growth both in vitro and in vivo to facilitate translational goals. Scaffolds composed of silk fibroin protein possess the biocompatibility, mechanical robustness, and stability required for tissue engineering. Here, we use a silk sponge system to support kidney cells in a perfused bioreactor system. Silk fibroin protein underwent directional freezing to form parallel porous structures that mimic the native kidney structure of aligned tubules and are able to support more cells than nonaligned silk sponges. Adult immortalized renal proximal tubule epithelial cells were seeded into the sponges and cultured under static conditions for 1 week, then grown statically or with perfusion with culture media flowing through the sponge to enhance cell alignment and maturation. The sponges were imaged with confocal and scanning electron microscopies to analyze and quantify cell attachment, alignment, and expression of proteins important to proximal tubule differentiation and function. The perfused tissue constructs showed higher number of cells that are more evenly distributed through the construct and increased gene expression of several key markers of proximal tubule epithelial cell function compared to sponges grown under static conditions. These perfused tissue constructs represent a step toward a scalable approach to engineering proximal tubule structures with the potential to be used as in vitro models or as in vivo implantable tissues to supplement or replace impaired kidney function.

A combined computational-fluid-dynamics model and control strategies for perfusion bioreactor systems in tissue engineering

Abstract This work sets the foundations for the design of control algorithms to facilitate manufacturing of a cell growth process using a continuous perfusion bioreactor. The algorithms are designed to work with different types of cell cultures and deal with major disturbances that might appear in the process. Different types of control strategies are designed, implemented and tested. First, a comprehensive mathematical model of convection and diffusion in a perfusion bioreactor, combined with cell growth kinetics, is developed and implemented using Computational Fluid Dynamics. The model describes the spatio-temporal evolution of glucose concentration and cell density within a 3D polymeric scaffold. Since such a model is too complex to be used directly for control studies, a simplified version is used for the design of the controllers. Finally, the performances of the control strategies are validated against the original high-fidelity CFD model, thus closing the loop. The simulations show good performances and satisfactory behavior.

Global Sensitivity Analysis for a perfusion bioreactor system in tissue engineering

Abstract This work presents a global sensitivity analysis and simulations of a perfusion bioreactor process using the method of high-dimensional model representation (HDMR). This method was developed to express the input–output relationships of a complex model with a high dimensional input space. The comprehensive mathematical model of convection and diffusion in a perfusion bioreactor, combined with cell growth kinetics, is developed and implemented using Computational Fluid Dynamics (with the commercial software COMSOL Multiphysics v5.5). The model describes the spatio-temporal evolution of glucose concentration, oxygen concentration, lactate concentration and cell density within a 3D polymeric scaffold. A quantitative analysis of the complex kinetic mechanisms using recent development of advanced mathematical approaches to global sensitivity and uncertainty analysis through HDMR can be exploited to investigate the important features of the perfusion bioreactor process as well as possible factors underlying qualitative discrepancies.

Perfusion bioreactor culture of human adipose-derived stromal cells on decellularized adipose tissue scaffolds enhances in vivo adipose tissue regeneration.

Tissue-engineering approaches hold promise to address the need in plastic and reconstructive surgery for new therapies that promote stable adipose tissue regeneration. Previous studies have demonstrated the potential of combining decellularized adipose tissue (DAT) scaffolds with adipose-derived stromal cells (ASCs) for volume augmentation applications. With the goal of enhancing in vivo angiogenesis and adipogenesis, this study evaluated the effects of culturing human ASCs on DAT scaffolds within a perfusion bioreactor. Using this system, the impact of both dynamic culture and hypoxic preconditioning were explored in vitro and in vivo. Initial studies compared the effects of 14 days of culture within the perfusion bioreactor under hypoxia (2% O2 ) or normoxia (~20% O2 ) on human ASC expansion and expression of hypoxia inducible factor-1 alpha (HIF-1α) in vitro relative to static cultured controls. The findings indicated that culturing within the bioreactor under hypoxia significantly increased ASC proliferation on the DAT, with a higher cell density observed in the scaffold periphery. Subsequent characterization in a subcutaneous implant model in athymic nude mice revealed that in vivo angiogenesis and adipogenesis were markedly enhanced when the ASCs were cultured on the DAT within the perfusion bioreactor under hypoxia for 14 days prior to implantation relative to the other culture conditions, as well as freshly seeded and unseeded DAT control groups. Overall, dynamic culture within the perfusion bioreactor system under hypoxia represents a promising approach for preconditioning ASCs on DAT scaffolds to enhance their capacity to stimulate angiogenesis and host-derived adipose tissue regeneration.

Abstract B12: 3D perfusion bioreactor system as a model for studying cell biology of ovarian cancer

Abstract Introduction: Epithelial ovarian cancer (EOC) is the most common cause of death among gynecologic malignancies. This is a result of challenging clinical management due to the high rate of recurrence and the development of chemoresistance in EOC patients. Therefore, the development of new therapeutics is crucial. Unfortunately, the success rate of establishing new drugs is very low. A major factor contributing to this is the lack of preclinical models that accurately predict in vivo efficacy of candidate therapeutics. Preclinical models do not accurately mimic the biomechanical regulation of the tumor microenvironment (TME) and cannot predict in vivo efficacy of candidate therapeutics. We have developed an EOC 3D perfused bioreactor system that recapitulates EOC tumor biology; better predicts the clinical response of new drug candidates, thereby eliminating ineffective candidates prior to clinical trials; and incorporates tumor biomechanical regulation. Materials and Methods: EOC cell lines (luciferase-tagged SKOV-3 and OVCAR-8) were embedded in a relevant ECM (5.25 × 105 total cells/100 uL ECM) containing 90% bovine collagen I + 10% basement membrane (Matrigel) and injected into a polydimethylsiloxane bioreactor (PDMS). The volume was then perforated by five wires and the bioreactor was placed in the incubator (37C, 5%CO2) for polymerization. After that, the wires were removed to create microchannels so that the cell culture media (RPMI 10% FBS, 1% penicillin-streptomycin-gentamicin) can flow through the perfusion bioreactor system and provide nutrient delivery and gas exchange, which helps maintain viability and function of surrounding cells. Finally, the bioreactors were connected to a peristaltic pump that allows the cell culture media to run over 14 days, along the channels and through the matrix. We monitored cell growth over 14 days using bioluminescence (BLI) and these results were validated with histologic analysis of the cell density. In order to prove that the bioreactor keeps EOC biology, by immunohistochemical (IHC) analysis with p53 and PAX8, clinically used ovarian cancer markers. Results and Discussion: For our preliminary data utilizing the 3D perfusion bioreactor system in ovarian cancer, we first used the luciferase-tagged OVCAR-8 cell line to evaluate cell growth. The bioluminescence signal showed a linear increase over 14 days. These results were validated histologically with cell density by measuring the number of nucleated cells per micron2. Graphical representation of the region of interest (ROI) showed a high correlation between manual determination of cell number and bioluminescence score. In our second experiment, we used the luciferase-tagged SKOV-3 cell line (high-grade serous origin) and we also monitored cell growth over 14 days using BLI and histologic analysis of the cell density as described for OVCAR-8 using the same conditions in the bioreactor. IHC analysis with p53 and PAX8 was positive and proved that the perfusion bioreactor system keeps EOC biology. Citation Format: Alba Martinez, Molly Buckley, Joel Berry, Rebecca Arend, Michael Birrer. 3D perfusion bioreactor system as a model for studying cell biology of ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B12.