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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 13C6 glucose containing feed media during constant operation at 20 × 106 cells and a perfusion rate of 1 reactor volume per day, isotopic steady state was studied. A step change to the 13C6 glucose medium in spin tubes allowed the determination of characteristic times for the intracellular turnover of unlabeled metabolites pools [ABSTRACT FROM AUTHOR]

Small-scale devices enable rapid development and optimization, and while many exist for fed-batch cultures, there are few comparable devices for high cell density perfusion cultures. To address this gap, pseudo-perfusion methodologies were developed in microwell plates at mL-scale capable of achieving many of the specific characteristics of perfusion culture, including high cell density, cell retention and increased productivity. Pseudo-perfusion was achieved via sedimentation or centrifugation of 24 microwell plates prior to media exchange 1–2 times daily, generating separation efficiencies higher than 90%. Media exchanges commenced on day 3 and achieved perfusion rates of 0.5–1.8 vessel volumes per day (VVD). Pseudo-perfusion methodologies resulted in maximum viable cell densities (VCDs) of up to 42 × 106 cells mL-1, 4.2-fold greater than fed-batch cultures. Volumetric productivities increased by 1.9-fold, generating industrially relevant productivities. Similar performance was observed between sedimentation and centrifugation methodologies, with minor deviations attributed to elongated manipulation times and lower packed cell density prior to separation when sedimentation was used. The microwell plate (MWP) experiments were validated at the 5 L scale and showed comparability in metabolite and growth profiles. The work presents the development of a robust tool for high-throughput development studies which is sensitive to changes in media composition and exchange rate, which could be reliably used for initial screening of high cell density perfusion cultures. • Design and characterisation of a pseudo-perfusion microwell plate methodology for optimisation of perfusion bioreactors. • Both sedimentation and centrifugation were explored as methods of cell retention. • Results were compared to fed-batch and achieved over 3-fold improvement in maximum viable cell densities. • This method can be used to optimise media blends and clone selection for perfusion operations. [ABSTRACT FROM AUTHOR]

For a high production rate of microalgae, a high-cell-density culture is an effective strategy for reducing the light intensity per dry weight in the culture to avoid photoinhibition under high light conditions. However, under high cell density microalgae are exposed to nutrient-deficient conditions, resulting in a reduced biomass production rate. To enhance the production rate of high-value bioactives such as fucoxanthin and eicosatetraenoic acid (EPA) by the diatom Chaetoceros gracilis , a perfusion culture was operated at harvesting rates of 10–40 % under light intensities of 300–2000 μmol m−2 s−1. At a harvesting rate of 20 % under a high light intensity of 2000 μmol m−2 s−1, the dry weight increased, and the areal productivity reached 15.6 ± 1.3 g-dry weight (dw) m−2 d−1, which is comparable to the maximum value previously reported in diatoms. The high biomass production, even at a low harvesting rate of 20 %, could result from sufficient nutrient supply by perfusion culture. At a harvesting rate of 20 %, the EPA areal productivity achieved 183 mg-dw m−2 d−1 even under 2000 μmol m−2 s−1 due to the increase in the dry weight using the perfusion culture. However, the fucoxanthin content decreased under 2000 μmol m−2 s−1. A strong relationship was observed between the specific light intensity per dry weight and fucoxanthin content in C. gracilis. This relationship indicates that the fucoxanthin content does not increase unless the specific light intensity per dry weight is adjusted below 20 μmol g-dw−1 s−1. Thus, perfusion culture proved to be useful to control the biomass-specific light intensity for the effective production of EPA and fucoxanthin in C. gracilis under high light conditions expected for outdoor culture. 8Marine diatom Chaetoceros gracilis was cultivated in perfusion culture. • Areal production rate reached 15.6 ± 1.3 g-dw m−2 d−1 under 2000 μmol m−2 s−1. • EPA production rate achieved 183 mg m−2 d−1 even under 2000 μmol m−2 s−1. • There was a correlation between specific light intensity and fucoxanthin content. • Fucoxanthin increased only when specific light is adjusted below 20 μmol g-dw−1 s−1. [ABSTRACT FROM AUTHOR]

Cell culture medium replacement is necessary to replenish nutrients and remove waste products, and perfusion and batch media exchange methods are available. The former can establish an environment similar to that in vivo, and microfluidic devices are frequently used. However, these methods are hampered by incompatibility with commercially available circular culture dishes and the difficulty in controlling liquid flow. Here, we fabricated a culture dish adapter using polydimethylsiloxane that has a small recess structure for flow control compatible with commercially available culture dishes. We designed U-shaped and I-shaped recess structure adapters and we examined the effects of groove structure on medium flow using simulation. We found that the U-shaped and I-shaped structures allowed a uniform and uneven flow of medium, respectively. We then applied these adaptors to 293T cell culture and examined the effects of recess structures on cell proliferation. As expected, cell proliferation was similar in each area of a dish in the U-shaped structure adapter, whereas in the early flow area in the I-shaped structure adapter, it was significantly higher. In summary, we succeeded in controlling liquid flow in culture dishes with the fabricated adapter, as well as in applying the modulation of culture medium flow to control cell culture.