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Background: Ex vivo cultures of retinal explants are appropriate models for translational research. However, one of the difficult problems of retinal explants ex vivo culture is that their nutrient supply needs cannot be constantly met., New Method: This study evaluated the effect of perfused culture on the survival of retinal explants, addressing the challenge of insufficient nutrition in static culture. Furthermore, exosomes secreted from retinal organoids (RO-Exos) were stained with PKH26 to track their uptake in retinal explants to mimic the efficacy of exosomal drugs in vivo., Results: We found that the retinal explants cultured with perfusion exhibited significantly higher viability, increased NeuN + cells, and reduced apoptosis compared to the static culture group at Days Ex Vivo (DEV) 4, 7, and 14. The perfusion-cultured retinal explants exhibited reduced mRNA markers for gliosis and microglial activation, along with lower expression of GFAP and Iba1, as revealed by immunostaining. Additionally, RNA-sequencing analysis showed that perfusion culture mainly upregulated genes associated with visual perception and photoreceptor cell maintenance while downregulating the immune system process and immune response. RO-Exos promoted the uptake of PKH26-labelled exosomes and the growth of retinal explants in perfusion culture., Comparison With Existing Methods: Our perfusion culture system can provide a continuous supply of culture medium to achieve steady-state equilibrium in retinal explant culture. Compared to traditional static culture, it better preserves the vitality, provides better neuroprotection, and reduces glial activation., Conclusions: This study provides a promising ex vivo model for further studies on degenerative retinal diseases and drug screening., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests Jiansu Chen and Yonglong Guo reports financial support was provided by National Natural Science Foundation of China. Jiansu Chen reports a relationship with National Natural Science Foundation of China that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The ever-stricter regulations on animal experiments in the field of cosmetic testing have prompted a surge in skin-related research with a special focus on recapitulation of the in vivo skin structure in vitro. In vitro human skin models are seen as an important tool for skin research, which in recent years attracted a lot of attention and effort, with researchers moving from the simplest 2-layered models (dermis with epidermis) to models that incorporate other vital skin structures such as hypodermis, vascular structures, and skin appendages. In this study, we designed a microfluidic device with a reverse flange-shaped anchor that allows culturing of an in vitro skin model in a conventional 6-well plate and assessing its barrier function without transferring the skin model to another device or using additional contraptions. Perfusion of the skin model through vascular-like channels improved the morphogenesis of the epidermis compared with skin models cultured under static conditions. This also allowed us to assess the percutaneous penetration of the tested caffeine permeation and vascular absorption, which is one of the key metrics for systemic drug exposure evaluation., (© 2024 IOP Publishing Ltd.)

Encapsulating multiple growth factors within a scaffold enhances the regenerative capacity of engineered bone grafts through their localization and controls the spatiotemporal release profile. In this study, we bioprinted hybrid bone grafts with an inherent built-in controlled growth factor delivery system, which would contribute to vascularized bone formation using a single stem cell source, human adipose-derived stem/stromal cells (ASCs) in vitro. The strategy was to provide precise control over the ASC-derived osteogenesis and angiogenesis at certain regions of the graft through the activity of spatially positioned microencapsulated BMP-2 and VEGF within the osteogenic and angiogenic bioink during bioprinting. The 3D-bioprinted vascularized bone grafts were cultured in a perfusion bioreactor. Results proved localized expression of osteopontin and CD31 by the ASCs, which was made possible through the localized delivery activity of the built-in delivery system. In conclusion, this approach provided a methodology for generating off-the-shelf constructs for vascularized bone regeneration and has the potential to enable single-step, in situ bioprinting procedures for creating vascularized bone implants when applied to bone defects.

Continuous manufacturing of lentiviral vectors (LVs) using stable producer cell lines could extend production periods, improve batch-to-batch reproducibility, and eliminate costly plasmid DNA and transfection reagents. A continuous process was established by expanding cells constitutively expressing third-generation LVs in the iCELLis Nano fixed-bed bioreactor. Fixed-bed bioreactors provide scalable expansion of adherent cells and enable a straightforward transition from traditional surface-based culture vessels. At 0.5 vessel volume per day (VVD), the short half-life of LVs resulted in a low total infectious titer at 1.36 × 10 4 TU cm -2 . Higher perfusion rates increased titers, peaking at 7.87 × 10 4 TU cm -2 at 1.5 VVD. The supernatant at 0.5 VVD had a physical-to-infectious particle ratio of 659, whereas this was 166 ± 15 at 1, 1.5, and 2 VVD. Reducing the pH from 7.20 to 6.85 at 1.5 VVD improved the total infectious yield to 9.10 × 10 4 TU cm -2 . Three independent runs at 1.5 VVD and a culture pH of 6.85 showed low batch-to-batch variability, with a coefficient of variation of 6.4% and 10.0% for total infectious and physical LV yield, respectively. This study demonstrated the manufacture of high-quality LV supernatant using a stable producer cell line that does not require induction., Competing Interests: This work was funded, in part, by the UCL-Cytiva Center of Excellence, as noted in the acknowledgments. N.B.J. was an employee of Cytiva at the time of submission., (© 2024 The Authors.)

To enhance the robustness and flexibility of biopharmaceutical manufacturing, a paradigm shift toward methods of continuous processing, such as perfusion, and fundamental technologies for high-throughput process development are being actively investigated. The continuous upstream process must establish an advanced control strategy to ensure a "State of Control" before operation. Specifically, feedforward and feedback control must address the complex fluctuations that occur during the culture process and maintain critical process parameters in appropriate states. However, control system design for industry-standard mammalian cell culture processes is still often performed in a laborious trial-and-error manner. This paper provides a novel control approach in which controller specifications to obtain desired control characteristics can be determined systematically by combining a culture model with control theory. In the proposed scheme, control conditions, such as PID parameters, can be specified mechanistically based on process understanding and control requirements without qualitative decision making or specific preliminary experiments. The effectiveness of the model-based control algorithm was verified by control simulations assuming perfusion Chinese hamster ovary culture. As a tool to assist in the development of control strategies, this study will reduce the high operational workload that is a serious problem in continuous culture and facilitate the digitalization of bioprocesses., (© 2023 Yokogawa Electric Corporation and The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Intensified fed-batch (IFB), a popular cell culture intensification strategy, has been widely used for productivity improvement through high density inoculation followed by fed-batch cultivation. However, such an intensification strategy may counterproductively induce rapidly progressing cell apoptosis and difficult-to-sustain productivity. To improve culture performance, we developed a novel cell culture process intermittent-perfusion fed-batch (IPFB) which incorporates one single or multiple cycles of intermittent perfusion during an IFB process for better sustained cellular and metabolic behaviors and notably improved productivity. Unlike continuous perfusion or other semi-continuous processes such as hybrid perfusion fed-batch with only early-stage perfusion, IPFB applies limited times of intermittent perfusion in the mid-to-late stage of production and still inherits bolus feedings on nonperfusion days as in a fed-batch culture. Compared to IFB, an average titer increase of ~45% was obtained in eight recombinant CHO cell lines studied. Beyond IPFB, ultra-intensified IPFB (UI-IPFB) was designed with a markedly elevated seeding density of 20-80 × 10 6 cell/mL, achieved through the conventional alternating tangential flow filtration (ATF) perfusion expansion followed with a cell culture concentration step using the same ATF system. With UI-IPFB, up to ~6 folds of traditional fed-batch and ~3 folds of IFB productivity were achieved. Furthermore, the application grounded in these two novel processes showed broad-based feasibility in multiple cell lines and products of interest, and was proven to be effective in cost of goods reduction and readily scalable to a larger scale in existing facilities., (© 2023 Wiley Periodicals LLC.)

Conventional therapies to address critically sized defects in subcutaneous adipose tissue remain a reconstructive challenge for surgeons, largely due to the lack of graft pre-vascularization. Adipose tissue relies on a dense microvasculature network to deliver nutrients, oxygen, nonadipose tissue-derived growth factors, cytokines, and hormones, as well as transporting adipose tissue-derived endocrine signals to other organ systems. This chapter addresses these vascularization issues by combining decellularized lung matrices with a step-wise seeding of patient-specific adipose-derived stem cells and endothelial cells to develop large-volume, perfusable, and pre-vascularized adipose grafts., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Oncolytic viruses (OVs) have emerged as a novel cancer treatment modality, and four OVs have been approved for cancer immunotherapy. However, high-yield and cost-effective production processes remain to be developed for most OVs. Here suspension-adapted Vero cell culture processes were developed for high titer production of an OV model, herpes simplex virus type 1 (HSV-1). Our study showed the HSV-1 productivity was significantly affected by multiplicity of infection, cell density, and nutritional supplies. Cell culture conditions were first optimized in shake flask experiments and then scaled up to 3 L bioreactors for virus production under batch and perfusion modes. A titer of 2.7 × 10 8 TCID50 mL -1 was obtained in 3 L batch culture infected at a cell density of 1.4 × 10 6 cells mL -1 , and was further improved to 1.1 × 10 9 TCID50 mL -1 in perfusion culture infected at 4.6 × 10 6 cells mL -1 . These titers are similar to or better than the previously reported best titer of 8.6 × 10 7 TCID50 mL -1 and 8.1 × 10 8 TCID50 mL -1 respectively obtained in labor-intensive adherent Vero batch and perfusion cultures. HSV-1 production in batch culture was successfully scaled up to 60 L pilot-scale bioreactor to demonstrate the scalability. The work reported here is the first study demonstrating high titer production of HSV-1 in suspension Vero cell culture under different bioreactor operating modes., (© 2023 Wiley-VCH GmbH.)

We have developed a single process for producing two key COVID-19 vaccine antigens: SARS-CoV-2 receptor binding domain (RBD) monomer and dimer. These antigens are featured in various COVID-19 vaccine formats, including SOBERANA 01 and the licensed SOBERANA 02, and SOBERANA Plus. Our approach involves expressing RBD (319-541)-His6 in Chinese hamster ovary (CHO)-K1 cells, generating and characterizing oligoclones, and selecting the best RBD-producing clones. Critical parameters such as copper supplementation in the culture medium and cell viability influenced the yield of RBD dimer. The purification of RBD involved standard immobilized metal ion affinity chromatography (IMAC), ion exchange chromatography, and size exclusion chromatography. Our findings suggest that copper can improve IMAC performance. Efficient RBD production was achieved using small-scale bioreactor cell culture (2 L). The two RBD forms - monomeric and dimeric RBD - were also produced on a large scale (500 L). This study represents the first large-scale application of perfusion culture for the production of RBD antigens. We conducted a thorough analysis of the purified RBD antigens, which encompassed primary structure, protein integrity, N-glycosylation, size, purity, secondary and tertiary structures, isoform composition, hydrophobicity, and long-term stability. Additionally, we investigated RBD-ACE2 interactions, in vitro ACE2 recognition of RBD, and the immunogenicity of RBD antigens in mice. We have determined that both the monomeric and dimeric RBD antigens possess the necessary quality attributes for vaccine production. By enabling the customizable production of both RBD forms, this unified manufacturing process provides the required flexibility to adapt rapidly to the ever-changing demands of emerging SARS-CoV-2 variants and different COVID-19 vaccine platforms., Competing Interests: The Center of Molecular Immunology and the Finlay Vaccine Institute have filed a patent application related to the present results. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Boggiano-Ayo, Palacios-Oliva, Lozada-Chang, Relova-Hernandez, Gomez-Perez, Oliva, Hernandez, Bueno-Soler, Montes de Oca, Mora, Machado-Santisteban, Perez-Martinez, Perez-Masson, Cabrera Infante, Calzadilla-Rosado, Ramirez, Aymed-Garcia, Ruiz-Ramirez, Romero, Gomez, Espinosa, Gonzalez, Cabrales, Guirola, de la Luz, Pi-Estopiñan, Sanchez-Ramirez, Garcia-Rivera, Valdes-Balbin, Rojas, Leon-Monzon, Ojito-Magaz and Hardy.)

With various diseases ravaging internationally, the demands for recombinant adenoviral vector (Adv) vaccines have increased dramatically. To meet the demand for Adv vaccine, development of a new cell culture process is an effective strategy. Applying hyperosmotic stress in cells before virus infection could increase the yield of Adv in batch culture mode. Emerging perfusion culture can significantly increase the yield of Adv as well. Therefore, combining the hyperosmotic stress process with perfusion culture is expected to improve the yield of Adv at high cell density. In this study, a shake flask combined with a semi-perfusion culture was used as a scaled-down model for bioreactor perfusion culture. Media with osmotic pressure ranging from 300 to 405 mOsm were used to study the effect of hyperosmotic stress on cell growth and Adv production. The results showed that using a perfusion culture process with a hyperosmotic pressure medium (370 mOsm) during the cell growth phase and an isosmotic pressure medium (300 mOsm) during the virus production phase effectively increased the yield of Adv. This might be due to the increased expression of HSP70 protein during the late phases of virus replication. The Adv titer in a bioreactor with such a process reached 3.2×10 10 IFU/mL, three times higher than that of the traditional perfusion culture process. More importantly, this is the first time that a strategy of combining the hyperosmotic stress process with perfusion culture is applied to the production of Adv in HEK 293 cells. It also reveals the reason why the hyperosmotic stress process increased the yield of Adv, which may facilitate the process optimization of for producing other Adv in HEK 293 cells.

Precision-cut tumor slices (PCTS) maintain tissue heterogeneity concerning different cell types and preserve the tumor microenvironment (TME). Typically, PCTS are cultured statically on a filter support at an air-liquid interface, which gives rise to intra-slice gradients during culture. To overcome this problem, we developed a perfusion air culture (PAC) system that can provide a continuous and controlled oxygen medium, and drug supply. This makes it an adaptable ex vivo system for evaluating drug responses in a tissue-specific microenvironment. PCTS from mouse xenografts (MCF-7, H1437) and primary human ovarian tumors (primary OV) cultured in the PAC system maintained the morphology, proliferation, and TME for more than 7 days, and no intra-slice gradients were observed. Cultured PCTS were analyzed for DNA damage, apoptosis, and transcriptional biomarkers for the cellular stress response. For the primary OV slices, cisplatin treatment induced a diverse increase in the cleavage of caspase-3 and PD-L1 expression, indicating a heterogeneous response to drug treatment between patients. Immune cells were preserved throughout the culturing period, indicating that immune therapy can be analyzed. The novel PAC system is suitable for assessing individual drug responses and can thus be used as a preclinical model to predict in vivo therapy responses.

The perfusion medium is critical in maintaining high cell concentration in cultures for the production of monoclonal antibody by Chinese hamster ovary cells. In this study, the effects of perfusion culture strategies when using different media on the process stability, product titer, and product quality were investigated in 3-L bioreactor. The results indicated that continuous perfusion could maintain higher levels of cell density, product titer, and quality in comparison with those of the intermittent perfusion culture. Next, the perfusion culture conditions with different perfusion rates and temperature reduction methods were further optimized. When combining the high perfusion rates and delayed reduction of culture temperature at day 6, the product titer reached a higher level of 16.19 g/L with the monomer relative abundant of 97.6%. In this case, the main peak of the product reached 56.3% and the total N-glycans ratio was 95.2%. To verify the effectiveness of the optimized perfusion culture in a larger scale, a 200-L bioreactor was used to perform and the final product titer reached the highest level of 16.79 g/L at day 16. Meanwhile, the product quality (monomer abundant of 97.6%, main peak of 56.3%, and N-glycans ratio of 96.5%) could also be well maintained. This study provided some guidance for the high-efficient production of monoclonal antibody by CHO cells via optimized perfusion culture strategy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Liang, Luo and Li.)

This paper reports perfusion culture of human umbilical vein endothelial cells (HUVECs) on a microporous membrane in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. We designed fluidic culture unit with microporous membrane to culture HUVECs under fluidic shear stress and constructed a perfusion culture model in the PD-MPS platform. Four fluidic culture units were arranged in the microplate-sized device, which enables four-throughput assay for characterization of HUVECs under flow. Medium flow was generated above and below the membrane by sequential pneumatic pressure to apply physiological shear stress to HUVECs. HUVECs exhibited aligned morphology to the direction of the flow with shear stress of 11.5-17.7 dyn/cm 2 under the flow condition, while they randomly aligned under static culture condition in a 6 well plate. We also observed 3.3- and 5.0-fold increase in the expression levels of the thrombomodulin and endothelial nitric oxide synthase mRNAs, respectively, under the flow condition in the PD-MPS compared to the static culture in 6 well plate. We also observed actin filament aligned to the direction of flow in HUVECs cultured under the flow condition., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

This article describes fabrication of a customizable bioreactor, which comprises a perfusion system and coverslip-based tissue culture chamber that allow centimeter-scale vascularized or otherwise canalized tissue constructs to be maintained in weeks long static and/or perfusion culture at an exceptionally low cost, with intermittent live imaging and media sampling capabilities. The perfusion system includes a reusable polydimethylsiloxane (PDMS) lid generated from a three-dimensional (3D)-printed poly-lactic acid (PLA) mold and several lengths of perfusion tubing. The coverslip tissue culture chamber includes PDMS components built with 3D-printed PLA molds, as well as 3D-printed PLA frames and glass coverslips that house perfusable hydrogel constructs. As proof of concept, we fabricated a vascularized hydrogel construct, which was subjected to static and perfusion tissue culture, as well as flow studies using fluorescent beads and widefield fluorescent microscopy. This system can be readily reproduced, promoting the advancement of tissue engineering and regenerative medicine research.

Here we report the perfusion culture of a multi-layered tissue composed of HepG2 cells (a human hepatoma line) in a pressure-driven microphysiological system (PD-MPS), which we developed previously as a multi-throughput perfusion culture platform. The perfusion culture of multi-layered tissue model was constructed by inserting a modified commercially available permeable membrane insert into the PD-MPS. HepG2 cells were layered on the membrane, and culture medium was perfused both through and below the membrane. The seeded density (number of cells/cm 2 ) of the culture model is 70 times that of static culture in a conventional 35-mm culture dish. Pressure-driven circulation of the medium in our compact device (8.6 × 7.0 × 4.5 cm 3 ), which comprised two perfusion-culture modules and a pneumatic connection port, enabled perfusion culture of two multi-layered tissues (initially 1 × 10 5  cells). To obtain insight into the basic functionality of the multi-layered tissues as hepatocytes, we compared albumin production and urea synthesis between perfusion cultures and static cultures. The HepG2 cells grew and secreted increasing amounts of albumin throughout 20 days of perfusion culture, whereas albumin secretion did not increase under static culture conditions. In addition, on day 20, the amount of albumin secreted by the HepG2 cells in the microfluidic device was 68% of that in the conventional culture dish, which was seeded with the same number of cells but had a 70 times larger culture area. These features of high-density culture of functioning cells in a compact device support the application of PD-MPS in single- and multi-organ MPS., (Copyright © 2022 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Vessel-on-a-chips, which can be used to study microscale fluid dynamics, tissue-level biological molecules delivery and intercellular communication under favorable three-dimensional (3D) extracellular matrix microenvironment, are increasingly gaining traction. However, not many of them can allow for long-term perfusion and easy observation of angiogenesis process. Since angiogenesis is necessary for the expansion of tumor, antiangiogenic drugs play a significant role in cancer treatment. In this study, we established an innovative and reliable antiangiogenic drug screening chip that was highly modularly integrated for long-term perfusion (up to 10 days depending on the hydrogel formula) and real-time monitoring. To maintain an unobstructed flow of cell-laden tubes for subsequent perfusion culture on the premise of excellent bioactivities, a polycaprolactone stent inspired by coronary artery stents was introduced to hold up the tubular lumen from the inside, while the perfusion chip was also elaborately designed to allow for convenient observation. After 3 days of perfusion screening, distinct differences in human umbilical vein endothelial cell sprouting were observed for a gradient of concentrations of bevacizumab, which pointed to the effectiveness and reliability of the drug screening perfusion system. Overall, a perfusion system for antiangiogenic drug screening was developed, which can not only conduct drug evaluation, but also be potentially useful in other vessel-mimicking scenarios in the area of tissue engineering, drug screening, pharmacokinetics, and regenerative medicine., Competing Interests: The authors declare no conflicts of interest., (Copyright: © 2022 Gu et al.)

All forms of restriction, from caloric to amino acid to glucose restriction, have been established in recent years as therapeutic options for various diseases, including cancer. However, usually there is no direct comparison between the different restriction forms. Additionally, many cell culture experiments take place under static conditions. In this work, we used a closed perfusion culture in murine L929 cells over a period of 7 days to compare methionine restriction (MetR) and glucose restriction (LowCarb) in the same system and analysed the metabolome by liquid chromatography mass spectrometry (LC-MS). In addition, we analysed the inhibition of glycolysis by 2-deoxy-D-glucose (2-DG) over a period of 72 h. 2-DG induced very fast a low-energy situation by a reduced glycolysis metabolite flow rate resulting in pyruvate, lactate, and ATP depletion. Under perfusion culture, both MetR and LowCarb were established on the metabolic level. Interestingly, over the period of 7 days, the metabolome of MetR and LowCarb showed more similarities than differences. This leads to the conclusion that the conditioned medium, in addition to the different restriction forms, substantially reprogramm the cells on the metabolic level., Competing Interests: The authors declare no conflict of interest.

In this study, we demonstrated the first, to our knowledge, integrated continuous bioprocess (ICB) designed for the production of acid-sensitive monoclonal antibodies, prone to aggregate at low pH, on pilot scale. A high cell density perfusion culture, stably maintained at 100 × 10 6  cells/ml, was integrated with the downstream process, consisting of a capture step with the recently developed Protein A ligand, Z Ca ; a solvent/detergent-based virus inactivation; and two ion-exchange chromatography steps. The use of a mild pH in the downstream process makes this ICB suitable for the purification of acid-sensitive monoclonal antibodies. Integration and automation of the downstream process were achieved using the Orbit software, and the same equipment and control system were used in initial small-scale trials and the pilot-scale downstream process. High recovery yields of around 90% and a productivity close to 1 g purified antibody/L/day were achieved, with a stable glycosylation pattern and efficient removal of impurities, such as host cell proteins and DNA. Finally, negligible levels of antibody aggregates were detected owing to the mild conditions used throughout the process. The present work paves the way for future industrial-scale integrated continuous biomanufacturing of all types of antibodies, regardless of acid stability., (© 2022 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Infliximab is a mouse/human chimeric IgG1 monoclonal antibody which recognizes the proinflammatory cytokine, tumor necrosis factor α (TNFα), and inhibits receptor interactions, thereby decreasing inflammation and autoimmune response in patients. This monoclonal antibody has been successfully used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the high treatment cost limits patient access to this biotherapy. One alternative to this problem is the use of biosimilars. In this work, we describe the stable expression and physicochemical characterization of an anti-TNFα antibody. While infliximab is produced in recombinant murine SP2/0 cells, our anti-TNFα IgG antibody was expressed in recombinant murine NS0 myeloma cells. The best anti-TNFα antibody-expressing clone was selected from three clone candidates based on the stability of IgG expression levels, specific productivity as well as TNFα-binding activity compared to commercial infliximab. Our results indicate that the selected cell clone, culture medium, and fermentation mode allowed for the production of an anti-TNFα antibody with similar characteristics to the reference commercially available product. An optimization of the selected culture medium by metabolomics may increase the volumetric productivity of the process to satisfy the demand for this product. Further experiments should be performed to evaluate the biological properties of this anti-TNFα antibody. KEY POINTS: • An anti-TNFα antibody was produced in NS0 cells using perfusion culture. • A proprietary chemically defined culture medium was used to replace commercially available protein-free medium. • The purified anti-TNFα antibody was comparable to the reference marketed product., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: In vitro generation of three-dimensional vessel network is crucial to investigate and possibly improve vascularization after implantation in vivo. This work has the purpose of engineering complex tissue regeneration of a vascular network including multiple cell-type, an extracellular matrix, and perfusability for clinical application., Methods: The two electrospun membranes bonded with the vascular network shape are cultured with endothelial cells and medium flow through the engineered vascular network. The flexible membranes are bonded by amine-epoxy reaction and examined the perfusability with fluorescent beads. Also, the perfusion culture for 7 days of the endothelial cells is compared with static culture on the engineered vascular network membrane., Results: The engineered membranes are showed perfusability through the vascular network, and the perfused network resulted in more cell proliferation and variation of the shear stress-related genes expression compared to the static culture. Also, for the generation of the complex vascularized network, pericytes are co-cultured with the engineered vascular network, which results in the Collagen I is expressed on the outer surface of the engineered structure., Conclusion: This study is showing the perfusable in vitro engineered vascular network with electrospun membrane. In further, the 3D vascularized network module can be expected as a platform for drug screening and regenerative medicine., (© 2021. The Author(s).)

The widespread adoption of long-term organs-on-a-chip culture necessitates both active perfusions that mimic physiological flow conditions and minimization of the complexity of microfluidic system and fluid handling. In particular, flow in microtissue such as microvascular is free of pulsation and backflow. The refreshable Braille actuator-based integrated microfluidic system can be employed with simple microchannels and setups. However, due to high pulsatile flow and backflow, ordinary Braille-driven micropumps generate non-physiological flow conditions. We have described a simple method for creating steady flow employing Braille actuators driven with a high-voltage analog waveform, called "constant flow waveform", without incorporating complicated structures into the microchannel or actuator. We determined the constant flow waveform by measuring volume change of microchannel caused by actuated Braille pins using a conventional fluorescent dye and microscope. Using the constant flow waveform, we demonstrated that a Braille-driven pump reduced pulsating flow by 79% and backflow by 63% compared to conventional Braille-driven pump. Furthermore, we demonstrated that a parallel pair of three-stranded pin pumps effectively eliminated backflow by driving two pumps with the constant flow waveform half-cycle shifted to each other. Moreover, by raising the driving frequency, we could increase the average flow rate to ~2× higher than previously reported flow rate of a typical Braille-driven micropump.

To address the remaining issue of poor cell immobilization and insufficient mass transfer in scaffold-based tissue engineering approach for future islet transplantation, we employed a macro-porous poly-l-lactide (PLLA) scaffold immobilizing mouse insulinoma cells and studied its function toward an implantable pancreatic tissue in 7-day perfusion culture. The murine pancreatic β cells could be immobilized in the PLLA scaffold at a high density of 10 7 cells per cm 3 close to the estimated range in normal pancreas. The perfusion culture promoted the 3D cellular organization as observed with live/dead staining and histological staining. The insulin production was significantly enhanced in comparison with static 2D culture and 3D rotational suspension culture by two and six folds, respectively ( p  < 0.001). As enhanced insulin response was only observed where both the perfusion and 3D cellular organization were present, this could represent important elements in engineering a functional bioartificial pancreas.

The liver microphysiological system (MPS) model is an in-vitro culture method that mimics physiological blood flow, which enhances basal cellular functions. However, the liver MPS model has not been tested in the preclinical stage because of its obscure utility. It can overcome the major problem of conventional systems-rapid loss of mitochondrial activity in cultured hepatocytes due to limited oxygen supply-by supplying oxygen to cultured hepatocytes using a perfusion device. In this study, we developed a new perfusion culture system that can detect mitochondrial toxicity. Primary mouse hepatocytes were cultured under perfusion condition for 48 hr. The hepatocytes showed increased oxygen consumption and reduced lactate release. These results indicated that the ATP-production pathway was switched from glycolysis to mitochondrial oxidative phosphorylation in the perfusion culture system. Furthermore, ATP levels were considerably reduced in the perfusion culture system after exposure to phenformin, a mitochondrial complex I inhibitor. To summarize, the perfusion culture system could improve the mitochondrial activity in primary mouse hepatocytes, and thus, has potential implications in the detection of mitochondrial toxicity.

Critically sized defects in subcutaneous white adipose tissue result in extensive disfigurement and dysfunction and remain a reconstructive challenge for surgeons; as larger defect sizes are correlated with higher rates of complications and failure due to insufficient vascularization following implantation. Our study demonstrates, for the first time, a method to engineer perfusable, pre-vascularized, high-density adipose grafts that combine patient-derived adipose cells with a decellularized lung matrix (DLM). The lung is one of the most vascularized organs with high flow, low resistance, and a large blood-alveolar interface separated by a thin basement membrane. For our work, the large volume capacity within the alveolar compartment was repurposed for high-density adipose cell filling, while the acellular vascular bed provided efficient graft perfusion throughout. Both adipocytes and hASCs were successfully delivered and remained in the alveolar space even after weeks of culture. While adipose-derived cells maintained their morphology and functionality in both static and perfusion DLM cultures, perfusion culture offered enhanced outcomes over static culture. Furthermore, we demonstrate that endothelial cells seamlessly integrate into the acellular vascular tree of the DLM with adipocytes. These results support that the DLM is a unique platform for creating vascularized adipose tissue grafts for large defect filling.

Previous studies have proved that dynamic culture could facilitate nutrients transport and apply mechanical stimulation to the cells within three-dimensional scaffolds, thus enhancing the differentiation of stem cells towards the osteogenic phenotype. However, the effects of macropore size on osteogenic differentiation of stem cells under dynamic condition are still unclear. Therefore, the objective of this study was to investigate the effects of macropore size of hydroxyapatite (HAp) scaffolds on osteogenic differentiation of bone mesenchymal stem cells under static and perfusion culture conditions. In vitro cell culture results showed that cell proliferation, alkaline phosphate (ALP) activity, mRNA expression of ALP, collagen-I (Col-I), osteocalcin (OCN) and osteopontin (OPN) were enhanced when cultured under perfusion condition in comparison to static culture. Under perfusion culture condition, the ALP activity and the gene expression of ALP, Col-I, OCN and OPN were enhanced with the macropore size decreasing from 1300 to 800 µm. However, with the further decrease in macropore size from 800 to 500 µm, the osteogenic related gene expression and protein secretion were reduced. Computational fluid dynamics analysis showed that the distribution areas of medium- and high-speed flow increased with the decrease in macropore size, accompanied by the increase of the fluid shear stress within the scaffolds. These results confirm the effects of macropore size on fluid flow stimuli and cell differentiation, and also help optimize the macropore size of HAp scaffolds for bone tissue engineering., (© The Author(s) 2021. Published by Oxford University Press.)

Conventional static culture fails to replicate the physiological conditions that exist in vivo. Recent advances in biomedical engineering have resulted in the creation of novel dynamic culturing systems that permit the recapitulation of normal physiological processes ex vivo. Whilst the physiological benefit for its use in the culture of two-dimensional cellular monolayer has been validated, its role in the context of primary human tissue culture has yet to be determined. This systematic review identified 22 articles that combined dynamic physiological culture techniques with primary human tissue culture. The most frequent method described (55%) utilised dynamic perfusion culture. A diverse range of primary human tissue was successfully cultured. The median duration of successful ex vivo culture of primary human tissue for all articles was eight days; however, a wide range was noted (5 h-60 days). Six articles (27%) reported successful culture of primary human tissue for greater than 20 days. This review illustrates the physiological benefit of combining dynamic culture with primary human tissue culture in both long-term culture success rates and preservation of native functionality of the tissue ex vivo. Further research efforts should focus on developing precise biochemical sensors that would allow for real-time monitoring and automated self-regulation of the culture system in order to maintain homeostasis. Combining these techniques allows the creation of an accurate system that can be used to gain a greater understanding of human physiology.

The perfusion culture of primary hepatocytes has been widely adopted to build bioreactors for various applications. As a drug testing platform, a unique vertical-flow bioreactor (VfB) array was found to create the compaction culture of hepatocytes which mimicked the mechanic microenvironment in vivo while maintaining the 3D cell morphology in a 2D culture setup and enhancing the hepatic functions for a sustained culture. Here, we report the methodology in designing and fabricating the VfB to reach ideal bioreactor requirements, optimizing the VfB as a prototype for drug testing, and to demonstrate the enhanced hepatic function so as to demonstrate the performance of the bioreactor. This device enables the modular, scalable, and manufacturable construction of a functional drug testing platform through the sustained maintenance of model cells.

The N-linked glycosylation pattern is an important quality attribute of therapeutic glycoproteins. It has been reported by our group and by others that different carbon sources, such as glucose, mannose and galactose, can differently impact the glycosylation profile of glycoproteins in mammalian cell culture. Acting on the sugar feeding is thus an attractive strategy to tune the glycan pattern. However, in case of feeding of more than one carbon source simultaneously, the cells give priority to the one with the highest uptake rate, which limits the usage of this tuning, e.g. the cells favor consuming glucose in comparison to galactose. We present here a new feeding strategy (named 'TAFE' for targeted feeding) for perfusion culture to adjust the concentrations of fed sugars influencing the glycosylation. The strategy consists in setting the sugar feeding such that the cells are forced to consume these substrates at a target cell specific consumption rate decided by the operator and taking into account the cell specific perfusion rate (CSPR). This strategy is applied in perfusion cultures of Chinese hamster ovary (CHO) cells, illustrated by ten different regimes of sugar feeding, including glucose, galactose and mannose. Applying the TAFE strategy, different glycan profiles were obtained using the different feeding regimes. Furthermore, we successfully forced the cells to consume higher proportions of non-glucose sugars, which have lower transport rates than glucose in presence of this latter, in a controlled way. In previous work, a mathematical model named Glycan Residues Balance Analysis (GReBA) was developed to model the glycosylation profile based on the fed carbon sources. The present data were applied to the GReBA to design a feeding regime targeting a given glycosylation profile. The ability of the model to achieve this objective was confirmed by a multi-round of leave-one-out cross-validation (LOOCV), leading to the conclusion that the GReBA model can be used to design the feeding regime of a perfusion cell culture to obtain a desired glycosylation profile., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Today's biologics manufacturing practices incur high costs to the drug makers, which can contribute to high prices for patients. Timely investment in the development and implementation of continuous biomanufacturing can increase the production of consistent-quality drugs at a lower cost and a faster pace, to meet growing demand. Efficient use of equipment, manufacturing footprint, and labor also offer the potential to improve drug accessibility. Although technological efforts enabling continuous biomanufacturing have commenced, challenges remain in the integration, monitoring, and control of traditionally segmented unit operations. Here, we discuss recent developments supporting the implementation of continuous biomanufacturing, along with their benefits.

The great saphenous vein (GSV) has served as a coronary artery bypass graft (CABG) conduit for over 50 years. Despite prevalent use, first-year failure rates remain high compared to arterial autograft options. Amongst other factors, vein graft failure can be attributed to material and mechanical mismatching that lead to apoptosis, inflammation, and intimal-medial hyperplasia. Through the implementation of the continuum mechanical-based theory of "stress-mediated growth and remodeling," we hypothesize that the mechanical properties of porcine GSV grafts can be favorably tuned for CABG applications prior to implantation using a prolonged but gradual transition from venous to arterial loading conditions in an inflammatory and thrombogenic deficient environment. To test this hypothesis, we used a hemodynamic-mimetic perfusion bioreactor to guide remodeling through stepwise incremental changes in pressure and flow over the course of 21-day cultures. Biaxial mechanical testing of vessels pre- and post-remodeling was performed, with results fit to structurally-motivated constitutive models using non-parametric bootstrapping. The theory of "small-on-large" was used to describe appropriate stiffness moduli, while histology and viability assays confirmed microstructural adaptations and vessel viability. Results suggest that stepwise transition from venous-to-arterial conditions results in a partial restoration of circumferential stretch and circumferential, but not axial, stress through vessel dilation and wall thickening in a primarily outward remodeling process. These remodeled tissues also exhibited decreased mechanical isotropy and circumferential, but not axial, stiffening. In contrast, only increases in axial stiffness were observed using culture under venous perfusion conditions and those tissues experienced moderate intimal resorption.

Liver disease and the subsequent loss of liver function is an enormous clinical challenge. A severe shortage of donor liver tissue greatly limits patients' options for a timely transplantation. Tissue engineering approaches offer a promising alternative to organ transplantation by engineering artificial implantable tissues. We have established a platform of cell-laden microbeads as basic building blocks to assemble macroscopic tissues via different mechanisms. This modular fabrication strategy possesses great potential for liver tissue engineering in a bottom-up manner. In this study, we encapsulated human hepatocytes into microbeads presenting a favorable microenvironment consisting of collagen and mesenchymal stem cells, and then we perfused the beads in a three-dimensional printed tubular perfusion bioreactor that promoted oxygen and medium diffusion to the impregnated cells. We noted high cell vitality and retention of parenchymal cell functionality for up to 30 days in this culture system. Our engineering-based approach led to the advancement in tissue size and long-term functionality of an artificial liver tissue in vitro .

Background: In liver tissue engineering, co-culturing hepatocytes with typical non-parenchymal hepatic cells to form cell aggregates is available to mimic the in vivo microenvironment and promote cell biological functions. With a modular assembly approach, endothelialized hepatic cell aggregates can be packed for perfusion culture, which enables the construction of large-scale liver tissues. Since tightly packed aggregates tend to fuse with each other and block perfusion flows, a loosely packed mode was introduced in our study., Methods: Using an oxygen-permeable polydimethylsiloxane (PDMS)-based microwell device, highly dense endothelialized hepatic cell aggregates were generated as hepatic tissue elements by co-culturing hepatocellular carcinoma (HepG2) cells, Swiss 3T3 cells, and human umbilical vein endothelial cells (HUVECs). The co-cultured aggregates were then harvested and applied in a PDMS-fabricated bioreactor for 10 days of perfusion culture. To maintain appropriate interstitial spaces for stable perfusion, biodegradable poly-L-lactic acid (PLLA) scaffold fibers were used and mixed with the aggregates, forming a loosely packed mode., Results: In a microwell co-culture, Swiss 3T3 cells significantly contributed to the formation of hepatic cell aggregates. HUVECs developed a peripheral distribution in aggregates for endothelialization. In the perfusion culture, compared with pure HepG2 aggregates, HepG2/Swiss 3T3/HUVECs co-cultured aggregates exhibited a higher level of cell proliferation and liver-specific function expression (i.e., glucose consumption and albumin secretion). Under the loosely packed mode, co-cultured aggregates showed a characteristic histological morphology with cell migration and adhesion to fibers. The assembled hepatic tissue elements were obtained with 32% of in vivo cell density., Conclusions: In a co-culture of HepG2, Swiss 3T3, and HUVECs, Swiss 3T3 cells were observed to be beneficial for the formation of endothelialized hepatic cell aggregates. Loosely packed aggregates enabled long-term perfusion culture with high viability and biological function. This study will guide us in constructing large-scale liver tissue models by way of aggregate-based modular assembly., Competing Interests: Conflicts of Interest: All of the authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-1598). The authors have no conflicts of interest to declare., (2020 Annals of Translational Medicine. All rights reserved.)

In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.

Perfusion is considered as the preferable unit operation mode for fully integrated continuous bioprocessing. However, the inherent complex process control, long process development times, and lack of suitable scale-down models for high-throughput screening are reasons why perfusion processes are still not routinely applied in cell culture technology. Advantages of perfusion are maintenance of a consistent cellular environment, a constant high-quality product flow, enhanced volumetric bioreactor productivity, and small lab footprint. Here, we provide guidelines for screening different proprietary but commercially available HyClone™ Cell Boost™ supplements in a Design of Experiment (DoE) approach to spike the HyClone™ CDM4NS0 basal media for enhanced product titers in small-scale TubeSpin models. These surrogate semi-perfusion cultures were successfully realized by a daily complete media exchange routine resulting in high viable cell densities for extended time periods at minimal media consumption. This technique was leveraged to define the potential of different perfusion media formulations.

Introduction: The definitive treatment for severe heart failure is transplantation. However, only a small number of heart transplants are performed each year due to donor shortages. Therefore, novel treatment approaches based on artificial organs or regenerative therapy are being developed as alternatives. We have developed a technology known as cell sheet-based tissue engineering that enables the fabrication of functional three-dimensional (3D) tissue. Here, we report a new technique for engineering human cardiac tissue with perfusable blood vessels. Our method involved the layering of cardiac cell sheets derived from human induced pluripotent stem cells (hiPSCs) on a vascular bed derived from porcine small intestinal tissue., Methods: For the vascular bed, a segment of porcine small intestine was harvested together with a branch of the superior mesenteric artery and a branch of the superior mesenteric vein. The small intestinal tissue was incised longitudinally, and the mucosa was resected. Human cardiomyocytes derived from hiPSCs were co-cultured with endothelial cells and fibroblasts on a temperature-responsive dish and harvested as a cardiac cell sheet. A triple-layer of cardiac cell sheets was placed onto the vascular bed, and the resulting construct was subjected to perfusion culture in a bioreactor system., Results: The cardiac tissue on the vascular bed pulsated spontaneously and synchronously after one day of perfusion culture. Electrophysiological recordings revealed regular action potentials and a beating rate of 105 ± 13/min (n = 8). Furthermore, immunostaining experiments detected partial connection of the blood vessels between the vascular bed and cardiac cell sheets., Conclusions: We succeeded in engineering spontaneously beating 3D cardiac tissue in vitro using human cardiac cell sheets and a vascular bed derived from porcine small intestine. Further development of this method might allow the fabrication of functional cardiac tissue that could be used in the treatment of severe heart failure., Competing Interests: Kazunori Sano is an employee of Tokai Hit Co., Ltd (Japan). Eiji Kobayashi is a medical adviser for Sysmex Corp. (Japan) and Regience Ltd. (Japan). Tatsuya Shimizu is a member of the scientific advisory board and a shareholder of CellSeed Inc. (Japan). Tokyo Women's Medical University received research funds from CellSeed Inc. Tatsuya Shimizu and Katsuhisa Matsuura are inventers of the bioreactor system for the differentiation and culture of pluripotent stem cells, the patent of which is held by Able Corp. (Japan) and Tokyo Women's Medical University (Cell culture apparatus and cell culture method using the same, US9574165B2). Tokyo Women's Medical University received a research grant from Tokaihit Co., Ltd, Japan., (© 2019 The Japanese Society for Regenerative Medicine. Production and hosting by Elsevier B.V.)

There is a great deal of demand for the construction of transplantable liver grafts. Over the last decade, decellularization techniques have been developed to construct whole liver tissue grafts as potential biomaterials. However, the lack of intact vascular networks, especially sinusoids, in recellularized liver scaffolds leads to hemorrhage and thrombosis after transplantation, which is a major obstacle to the development of transplantable liver grafts. In the present study, we hypothesized that both mechanical (e.g., fluid shear stress) and chemical factors (e.g., fibronectin coating) can enhance the formation of hierarchical vascular networks including sinusoid-scale microvessels. We demonstrated that perfusion culture promoted formation of sinusoid-scale microvessels in recellularized liver scaffolds, which was not observed in static culture. In particular, perfusion culture at 4.7 ml/min promoted the formation of sinusoid-scale microvessels compared to perfusion culture at 2.4 and 9.4 ml/min. In addition, well-aligned endothelium was observed in perfusion culture, suggesting that endothelial cells sensed the flow-induced shear stress. Moreover, fibronectin coating of decellularized liver scaffolds enhanced the formation of sinusoid-scale microvessels in perfusion culture at 4.7 ml/min. This study represents a critical step in the development of functional recellularized liver scaffolds, which can be used not only for transplantation but also for drug screening and disease-modeling studies. STATEMENT OF SIGNIFICANCE: Decellularized liver scaffolds are promising biomaterials that allow production of large-scale tissue-engineered liver grafts. However, it is difficult to maintain recellularized liver grafts after transplantation due to hemorrhage and thrombosis. To overcome this obstacle, construction of an intact vascular network including sinusoid-scale microvessels is essential. In the present study, we succeeded in constructing sinusoid-scale microvessels in decellularized liver scaffolds via a combination of perfusion culture and surface coating. We further confirmed that endothelial cells in decellularized liver scaffolds responded to flow-derived mechanical stress by aligning actin filaments. Our strategy to construct sinusoid-scale microvessels is critical for the development of intact vascular networks, and addresses the limitations of recellularized liver scaffolds after transplantation., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

The development of mammalian cell perfusion cultures is still laborious and complex to perform due to the limited availability of scale-down models and limited knowledge of time- and cost-effective procedures. The maximum achievable viable cell density (VCD max ), minimum cell-specific perfusion rate (CSPR min ), cellular growth characteristics, and resulting bleed rate at steady-state operation are key variables for the effective development of perfusion cultures. In this study, we developed a stepwise procedure to use shake tubes (ST) in combination with benchtop (BR) bioreactors for the design of a mammalian cell perfusion culture at high productivity (23 pg·cell -1 ·day -1 ) and low product loss in the bleed (around 10%) for a given expression system. In a first experiment, we investigated peak VCDs in STs by the daily discontinuous medium exchange of 1 reactor volume (RV) without additional bleeding. Based on this knowledge, we performed steady-state cultures in the ST system using a working volume of 10 ml. The evaluation of the steady-state cultures allowed performing a perfusion bioreactor run at 20 × 10 6 cells/ml at a perfusion rate of 1 RV/day. Constant cellular environment and metabolism resulted in stable product quality patterns. This study presents a promising strategy for the effective design and development of perfusion cultures for a given expression system and underlines the potential of the ST system as a valuable scale-down tool for perfusion cultures., (© 2019 Wiley Periodicals, Inc.)

Background: Organoids derived from induced pluripotent stem (iPS) or embryonic stem (ES) cells have been evaluated as in vitro models of development and disease. However, maintaining these cells under long-term static culture conditions is difficult because of nutrition shortages and waste accumulation. To overcome these issues, perfusion culture systems are required for organoid technology. A system with a stable microenvironment, nutrient availability, and waste removal will accelerate organoid generation. The aim of this study was to develop a novel perfusion system for renal organoids by maintaining the air-liquid interface with a device fabricated using a 3D printer., Results: Our results revealed slow flow at the organoid cultivation area based on microbead movement on the membrane, which depended on the perfusion rate under the membrane. Moreover, the perfused culture medium below the organoids via a porous membrane diffused throughout the organoids, maintaining the air-liquid interface. The diffusion rates within organoids were increased according to the flow rate of the culture medium under the membrane. The perfused culture medium also stimulated cytoskeletal and basement membrane re-organization associated with promotion tubular formation under 2.5 μL/min flow culture. In contrast, tubules in organoids were diminished at a flow rate of 10 μL/min., Conclusions: Our liquid-air interface perfusion system accelerated organization of the renal organoids. These results suggest that suitable perfusion conditions can accelerate organization of epithelial cells and tissues in renal organoids in vitro., Competing Interests: Competing interestsT.S. is a member of the scientific advisory board and a stakeholder of CellSeed, Inc. Tokyo Women’s Medical University received a research fund from CellSeed, Inc., (© The Author(s) 2019.)

A Quality by Design (QbD) concept was applied to characterize a cell culture process for production of the recombinant Factor VIII (rFVIII). We characterized the production bioreactor process and defined the design space by applying risk assessment to determine potential critical process parameters (CPPs) impacting critical quality attributes (CQAs). Characterization studies were subsequently performed using a qualified scaled-down model (SDM) and a multi-factorial design of experiment (DOE) approach to determine both the individual and combined impacts of the potential CPPs on CQAs. Among the operating parameters characterized, production temperature, production pH and a shift in the timing of production affected rFVIII activity and tyrosine sulfation level. Finally, we identified CPPs and established a design space for the cell culture process to identify appropriate conditions for routine manufacturing., (Copyright © 2019. Published by Elsevier Ltd.)

Microfluidic devices have emerged as a new cell culture tool, which can mimic the structure and physiology of living human organs. However, no standardized culture method for a microfluidic device has yet been established. Here, we describe the effects of various conditions on cell proliferation in a microchannel with a depth smaller than 100 μm. Primary endothelial cell proliferation was suppressed with a decrease in the culture medium volume per cell culture area. Moreover, cell growth was compared with or without medium flow, and the optimum culture condition was determined to be 1 μL/h flow in a 65-μm-deep microchannel. In addition, glucose consumption was greater under fluidic conditions than under static conditions, and the ability of tumor (HeLa) cells to convert glucose into lactate appeared to be higher in a static culture than that in a fluidic culture. Overall, our results will serve as a useful guide for designing a microfluidic cell culture platform in a channel smaller than 100 μm.

The cultivation of retinal pigment epithelium (RPE)-choroid explants gives the opportunity to study the RPE and Bruch's membrane in its natural environment. Porcine eyes are easily available and an excellent model for human RPE. Explants are prepared less than 4 h postmortem from cooled eyes and are transferred in fixation rings. The tissues held between rings are cultured in a perfusion organ culture system for up to a week. Viability of the explants can be investigated by calcein staining.

Stem cell cultures within perfusion bioreactors, while efficient in obtaining cell numbers, often lack the similarity to native tissues and consequently cell phenotype. We develop a three-dimensional (3D)-printed fluidic chamber for dynamic stem cell culture, with emphasis on control over flow and substrate curvature in a 3D environment, two physiologic features of native tissues. The chamber geometry, consisting of an array of vertical cylindrical pillars, facilitates actin-mediated localization of human mesenchymal stem cells (hMSCs) within ∼200 μm distance from the pillars, enabling spatial patterning of hMSCs and endothelial cells in cocultures and subsequent modulation of calcium signaling between these two essential cell types in the bone marrow microenvironment. Flow-enhanced osteogenic differentiation of hMSCs in growth media imposes spatial variations of alkaline phosphatase expression, which positively correlates with local shear stress. Proliferation of hMSCs is maintained within the chamber, exceeding the cell expansion in conventional static culture. The capability to manipulate cell spatial patterning, differentiation, and 3D tissue formation through geometry and flow demonstrates the culture chamber's relevant chemomechanical cues in stem cell microenvironments, thus providing an easy-to-implement tool to study interactions among substrate curvature, shear stress, and intracellular actin machinery in the tissue-engineered construct.

For the fabrication of appropriate bone tissue-engineered constructs several prerequisites should be fulfilled. They should offer long-term stability, allow proper cell attachment and proliferation and furthermore be osteoinductive and easy to be vascularized. Having these requirements as background, we fabricated a novel porous 3D-printed hydroxyapatite (HA) scaffold and treated it with oxygen plasma (OPT). MG-63 pre-osteoblast-seeded bone constructs allowed good cell attachment and proliferation, which was even better when cultivated in a perfusion flow bioreactor. Moreover, the deposition of extracellular matrix (ECM) on the otherwise inorganic surface changed the mechanical properties in a favourable manner: elasticity increased from 42.95±1.09 to 91.9±5.1 MPa (assessed by nanoindentation). Compared to static conditions, osteogenic differentiation was enhanced in the bioreactor, with upregulation of ALP, collagen I and osteocalcin gene expression. In parallel experiments, primary human bone marrow mesenchymal stromal cells (hBMSCs) were used and findings under dynamic conditions were similar; with a higher commitment towards osteoblasts compared to static conditions. In addition, angiogenic markers CD31, eNOS and VEGF were upregulated, especially when osteogenic medium was used rather than proliferative medium. To compare differently fabricated ECMs in terms of vascularization, decellularized constructs were tested in the chorioallantoic membrane (CAM) assay with subsequent assessment of the functional perfusion capacity by MRI in the living chick embryo. Here, vascularization induced by ECM from osteogenic medium led to a vessel distribution more homogenous throughout the construct, while ECM from proliferative medium enhanced vessel density at the interface and, to a lower extent, at the middle and top. We conclude that dynamic cultivation of a novel porous OPT HA scaffold with hBMSCs in osteogenic medium and subsequent decellularization provides a promising off-the-shelf bone tissue-engineered construct., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The use of benchtop bioreactors (BRs) for the development of mammalian cell perfusion cultures is expensive and time consuming, given its complexity in equipment and operation. Scale-down models, going from liter to milliliter scale, are needed to support the rapid determination of suitable operating conditions in terms of viable cell density (VCD), perfusion rate, and medium composition. In this study, we compare the performance of steady-state perfusion cultures in orbitally shaken tube and BR systems for a given Chinese hamster ovary cell line. The developed scale-down model relied on a daily workflow designed to keep the VCD constant at specific target values. This includes: cell count, removal of excessive cells (bleeding), spin down of remaining cells, harvest of cell-free supernatant, and resuspension in fresh medium. Steady-state cultures at different VCD values, medium exchange rates and working volumes were evaluated. Shake-tube perfusion cultures allowed the prediction of cell-specific growth, glucose consumption, ammonia, and monoclonal antibody production rates for much larger BRs, but not lactate (LAC) production rates. Although charge variant profiles remained comparable, different glycosylation patterns were obtained. The differences in LAC production and glycosylation probably resulted from the discontinuous medium exchange, the poor carbon dioxide removal, and the deficient pH control. Therefore, if requested by the specific process to be developed, product quality has to be fine-tuned directly in the BR system. Altogether, the developed strategy provides a useful scale-down model for the design and optimization of perfusion cultures with strong savings in time and media consumption., (© 2018 Wiley Periodicals, Inc.)

Although the reconstruction of functional 3D liver tissue models in vitro presents numerous challenges, it is in great demand for drug development, regenerative medicine, and physiological studies. Here we propose a new approach to perform perfusion cultivation of liver cells by assembling cell-laden hydrogel microfibers. HepG2 cells were densely packed into the core of sandwich-type anisotropic microfibers, which were produced using microfluidic devices. The obtained microfibers were bundled up and packed into a perfusion chamber, and perfusion cultivation was performed. We evaluated cell viability and functions, and also monitored the oxygen consumption. Furthermore, fibers covered with vascular endothelial cells were united during the perfusion culture, to form vascular network-like conduits between fibers. The presented technique can structurally mimic the hepatic lobule in vivo and could prove to be a useful model for various biomedical research applications., (Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Tissue engineering strategies have been developed to optimize osseointegration in dental implant surgery. One of the major problems is the non-homogeneous spatial cell distribution in the scaffold, as well as subsequent matrix production. Insufficient nutrient and oxygen supplies inside the scaffold are factors in this phenomenon. To mediate this gradient formation, we have implemented a perfusion culture method to seed human bone marrow mesenchymal stem cells (MSCs) into three-dimensional (3-D)-allogenic bone scaffolds in combination with a marine haemoglobin, HEMOXCell ® , for oxygen delivery. Cell culture was performed under static and perfusion conditions, with standard and osteogenic media, with and without HEMOXCell ® . The cell seeding efficiency, as well as MSC/scaffold cytocompatibly were assessed using viability and proliferation assays. Scaffolds' cellularization and extracellular matrix (ECM) formation were analyzed using scanning electron microscopy and histological staining. Cell differentiation was investigated with osteogenic biomarkers gene expression analysis. The perfusion culture was observed to significantly promote MSC proliferation and differentiation throughout the scaffolds, especially when using the induction medium w/HEMOXCell ® . Our data suggest that perfusion culture of MSC into allogenic bone substitute with HEMOXCell ® as a natural oxygen carrier is promising for tissue engineering applications to oxygenate hypoxic areas and to promote cellular proliferation.

3D spheroid or organoid culture is becoming mainstream method for studying physiologically relevant cell behaviors, and used in applications such as cell-based diagnostic, therapy, disease modeling and drug screening. Organoids/spheroids function best when maintaining size of <200μm diameter and in perfusion culture. To achieve this, we describe in this chapter various methods of constraining spheroid size during the formation and culture processes such that the spheroids can maintain high-level cell functions for characterization and applications. We describe methods based on substrate tethering, physical-constraints such as covering spheroids under porous membranes, inside macroporous sponges, or in microfluidic channels. The chemical and physical properties of the cell-contacting surfaces and devices are carefully engineered to enable the formation and maintenance of spheroid functions over time. Pitfalls of these methods and proposed solutions in the contexts of respective applications will also be discussed., (© 2018 Elsevier Inc. All rights reserved.)

The steady-state operation of Chinese hamster ovary (CHO) cells in perfusion bioreactors requires the equilibration of reactor dynamics and cell metabolism. Accordingly, in this work we investigate the transient cellular response to changes in its environment and their interactions with the bioreactor hydrodynamics. This is done in a benchtop perfusion bioreactor using MALDI-TOF MS through isotope labeling of complex intracellular nucleotides (ATP, UTP) and nucleotide sugars (UDP-Hex, UDP-HexNAc). By switching to a 13 C 6 glucose containing feed media during constant operation at 20 × 10 6 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13 C 6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools, τST (≤0.56 days), which were confirmed in the bioreactor. On the other hand, it is shown that the reactor residence time τR (1 day) and characteristic time for glucose uptake τGlc (0.33 days), representative of the bioreactor dynamics, delayed the consumption of 13 C 6 glucose in the bioreactor and thus the intracellular 13 C enrichment. The proposed experimental approach allowed the decoupling of bioreactor hydrodynamics and intrinsic dynamics of cell metabolism in response to a change in the cell culture environment. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1630-1639, 2017., (© 2017 American Institute of Chemical Engineers.)

Lab-on-a-chip systems are increasingly used as tools for cultures and investigation of cardiac cells. In this article, we present how the geometry of microsystems and microenvironmental conditions (static and perfusion) influence the proliferation, morphology, and alignment of cardiac cells (rat cardiomyoblasts-H9C2). Additionally, studies of cell growth after incubation with verapamil hydrochloride were performed. For this purpose, poly(dimethylsiloxane) (PDMS)/glass microfluidic systems with three different geometries of microchambers (a circular chamber, a longitudinal channel, and three parallel microchannels separated by two rows of micropillars) were prepared. It was found that static conditions did not enhance the growth of H9C2 cells in the microsystems. On the contrary, perfusion conditions had an influence on division, morphology, and the arrangement of the cells. The highest number of cells, their parallel orientation, and their elongated morphology were obtained in the longitudinal microchannel. It showed that this kind of microsystem can be used to understand processes in heart tissue in detail and to test newly developed compounds applied in the treatment of cardiac diseases.

Microfluidic perfusion systems enable small-volume cell cultures under precisely controlled microenvironments, and are typically developed for cell-based high-throughput screening. However, most such systems are designed to manipulate dissociated single cells, not cell aggregates, and are thus unsuitable to induce differentiation in human induced pluripotent stem cells (hiPSCs), which is conventionally achieved by using cell aggregates to increase cell-cell interactions. We have now developed a compartmentalized microfluidic perfusion system with large flow channels to load, culture, and observe cell aggregates. Homogeneously sized cell aggregates to be loaded into the device were prepared by shredding flat hiPSC colonies into squares. These aggregates were then seeded into microchambers coated with fibronectin and bovine serum albumin (BSA) to establish adherent and floating cultures, respectively, both of which are frequently used to differentiate hiPSCs. However, the number of aggregates loaded in fibronectin-coated microchambers was much lower than in BSA-coated microchambers, suggesting that fibronectin traps cell aggregates before they reach the chambers. Accordingly, hiPSCs that reached the microchambers subsequently adhered. In contrast, BSA-coated microchambers did not allow cell aggregates to adhere, but were sufficiently deep to prevent cell aggregates from flowing out during perfusion of media. Immunostaining for markers of undifferentiated cells showed that cultures on both fibronectin- and BSA-coated microchambers were successfully established. Notably, we found that floating aggregates eventually adhered to surfaces coated with BSA upon differentiation, and that differentiation depends on the initial size of aggregates. Collectively, these results suggest that the microfluidic system is suitable for manipulating hiPSC aggregates in compartmentalized microchambers., (Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)