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OBJECTIVE To evaluate differentiation of canine adipose–derived multipotent stromal cells (ASCs) into ligamentoblasts on tensioned collagen type I (Col1) templates in a perfusion culture system. SAMPLES Infrapatellar fat pad ASCs from healthy stifle joints of 6 female mixed-breed dogs. PROCEDURES Third-passage ASCs (6 × 106 cells/template) were loaded onto suture-augmented Col1 templates under 15% static strain in perfusion bioreactors. Forty-eight ASC-Col1 constructs were incubated with ligamentogenic (ligamentogenic constructs; n = 24) or stromal medium (stromal constructs; 24) for up to 21 days. Specimens were collected from each construct after 2 hours (day 0) and 7, 14, and 21 days of culture. Cell number, viability, distribution, and morphology; construct collagen content; culture medium procollagen-I-N-terminal peptide concentration; and gene expression were compared between ligamentogenic and stromal constructs. RESULTS ASCs adhered to collagen fibers. Cell numbers increased from days 0 to 7 and days 14 to 21 for both construct types. Relative to stromal constructs, cell morphology and extracellular matrix were more mature and collagen content on day 21 and procollagen-I-N-terminal peptide concentration on days 7 and 21 were greater for ligamentogenic constructs. Ligamentogenic constructs had increased expression of the genes biglycan on day 7, decorin throughout the culture period, and Col1, tenomodulin, fibronectin, and tenascin-c on day 21; expression of Col1, tenomodulin, and tenascin-c increased between days 7 and 21. CONCLUSIONS AND CLINICAL RELEVANCE Ligamentogenic medium was superior to stromal medium for differentiation of ASCs to ligamentoblasts on suture-augmented Col1 scaffolds. Customized ligament neotissue may augment treatment options for dogs with cranial cruciate ligament rupture.

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.

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.

Treatment of terminal heart failure still poses a significant clinical problem. Cardiac tissue engineering could offer autologous solutions for the replacement of nonfunctional myocardial tissue. So far, soft matrix construction and missing large-scale prevascularization prevented the application of sizeable cardiac repair patches. We developed a novel bioreactor system for semi-automatic compression of a collagen I hydrogel applying 16 times higher pressure than in previous studies. Resistance towards compression stress was investigated for multiple cardiac-related cell types. For scaffold prevascuarization, a tubular cavity was imprinted during the compaction process. Primary cardiac-derived endothelial cells (ECs) were isolated from human left atrial appendages (HLAAs) and characterized by fluorescence-activated cell sorting (FACS) and immunocytology. EC were then seeded into the preformed channel with dermal fibroblasts as interstitial cell component of the fully cellularized patch. After 8 days of constant perfusion culture within the same bioreactor, scaffold dynamic modulus and cell viability were analyzed. Endothelial proliferation and vessel maturation were examined by immunohistochemistry and transmission electron microscopy. Our design allowed for scaffold production and dynamic culture in a one-stop-shop model. Enhanced compression and cell-mediated matrix remodeling induced a significant increase in scaffold stiffness while ensuring excellent cell survival. For the first time, we could isolate HLAA-derived EC with proliferative potential. ECs within the central channel proliferated during flow culture, continuously expressing endothelial markers (CD31) and displaying basal membrane synthesis (collagen IV, ultrastructural analysis). After 7 days of culture, a complete endothelial monolayer could be observed. Covering cells aligned themselves in flow direction and developed mature cell-cell contacts.

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.

We demonstrate a new micro/nanofluidic system for continuous and automatic monitoring of protein product size and quantity directly from the culture supernatant during a high-cell-concentration CHO cell perfusion culture. A microfluidic device enables clog-free cell retention for a bench-scale (350 mL) perfusion bioreactor that continuously produces the culture supernatant containing monoclonal antibodies (IgG1). A nanofluidic device directly monitors the protein size and quantity in the culture supernatant. The continuous-flow and fully automated operation of this nanofluidic protein analytics reduces design complexity and offers more detailed information on protein products than offline and batch-mode conventional analytics. Moreover, chemical and mechanical robustness of the nanofluidic device enables continuous monitoring for several days to a week. This continuous and online protein quality monitoring could be deployed at different steps and scales of biomanufacturing to improve product quality and manufacturing efficiency.

Process intensification in mammalian cell culture-based recombinant protein production has been achieved by high cell density perfusion exceeding 10(8) cells/mL in the recent years. As the majority ...

Vero cells are considered as the most widely accepted continuous cell line by the regulatory authorities (such as WHO) for the manufacture of viral vaccines for human use. The growth of Vero cells is anchorage-dependent. Scale-up and manufacturing in adherent cultures are labor intensive and complicated. Adaptation of Vero cells to grow in suspension will simplify subcultivation and process scale-up significantly, and therefore reduce the production cost. Here we report on a successful adaptation of adherent Vero cells to grow in suspension in a serum-free and animal component-free medium (IHM03) developed in-house. The suspension adapted Vero cell cultures in IHM03 grew to similar or better maximum cell density as what was observed for the adherent Vero cells grown in commercial serum-free media and with a cell doubling time of 40–44 h. Much higher cell density (8 × 10 6 cells/mL) was achieved in a batch culture when three volume of the culture medium was replaced during the batch culture process. Both adherent and suspension Vero cells from various stages were tested for their authenticity using short tandem repeat analysis. Testing result indicates that all Vero cell samples had 100% concordance with the Vero DNA control sample, indicating the suspension cells maintained their genetic stability. Furthermore, suspension Vero cells at a passage number of 163 were assayed for tumorigenicity, and were not found to be tumorigenic. The viral productivity of suspension Vero cells was evaluated by using vesicular stomatitis virus (VSV) as a model. The suspension cell culture showed a better productivity of VSV than the adherent Vero cell culture. In addition, the suspension culture could be infected at higher cell densities, thus improving the volumetric virus productivity. More than one log of increase in the VSV productivity was achieved in a 3L bioreactor perfusion culture infected at a cell density of 6.8 × 10 6 cells/mL.

Much of the biopharmaceutical industry's success over the past 30 years has relied on products derived from Chinese Hamster Ovary (CHO) cell lines. During this time, improvements in mammalian cell cultures have come from cell line development and process optimization suited for large-scale fed-batch processes. Originally developed for high cell densities and sensitive products, perfusion processes have a long history. Driven by high volumetric titers and a small footprint, perfusion-based bioprocess research has regained an interest from academia and industry. The recent pandemic has further highlighted the need for such intensified biomanufacturing options. In this review, we outline the technical history of research in this field as it applies to biologics production in CHO cells. We demonstrate a number of emerging trends in the literature and corroborate these with underlying drivers in the commercial space. From these trends, we speculate that the future of perfusion bioprocesses is bright and that the fields of media optimization, continuous processing, and cell line engineering hold the greatest potential. Aligning in its continuous setup with the demands for Industry 4.0, perfusion biomanufacturing is likely to be a hot topic in the years to come.

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 (VCDmax ), minimum cell-specific perfusion rate (CSPRmin ), 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 × 106 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.

Without a scale‐down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high‐capacity microscale system that is typically used in fed‐batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 106 mg/cell/day and 7 × 10 7 cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.

Hyperosmolality has been commonly investigated due to its effects on the production and quality characteristics of monoclonal antibodies (mAbs) produced in CHO cell fed-batch cultures. However, the application of hyperosmolality at different times and its effect on biopotency have seldom been researched, especially in perfusion culture. In our study, different degrees of hyperosmolality induced by sodium chloride were investigated in anti-IgE rCHO cell fed-batch cultures and anti-CD52 rCHO cell perfusion cultures during the initial and stable phases. The results showed that the initial hyperosmolality group (IHG) in fed-batch and early phase of perfusion cultures exhibited significant suppression of the viable cell density yet an enhancement in specific productivity, whereas the stable hyperosmolality group (SHG) achieved higher mAb production in both fed-batch and perfusion cultures. Additionally, the SHG produced less aggregates and acidic charge variants than IHG in fed-batch culture, which differed from perfusion cultures. However, the contents of non-glycosylation heavy chain (NGHC) and man5 were higher in SHG than in IHG in fed-batch cultures at plus 60 and 120 mOsm/kg, which was similar to perfusion cultures. Furthermore, the biopotency in the IHG was higher than in the SHG at plus 60 and 120 mOsm/kg in fed-batch cultures, which is similar to complement-dependent cytotoxicity (CDC) efficacy in perfusion cultures. The biopotency of all group was acceptable, except FI3. Thus, the study shows that hyperosmolality at a certain level could be beneficial for both mAb production, quality and biopotency, which could play an important role in process development for commercial production.

Ex vivo brain slice cultures are utilized as analytical models for studying neurophysiology. Common approaches to maintaining slice cultures include roller tube and membrane interface techniques. The rise of organ-on-chip technologies has demonstrated the value of microfluidic perfusion culture systems for sampling and analysis of complex biology under well-controlled in vitro or ex vivo conditions. A number of approaches to microfluidic brain slice culture have been developed, however these typically involve complex design, fabrication, or operational parameters in order to meet the high oxygen demands of brain slices. Here, we present proof-of-principle for a novel approach to microfluidic brain slice culture. In this system, which we term a microfluidic bubble perfusion device, principles of droplet microfluidics were employed to generate droplets of perfusion media dispersed between bubbles of carbogen gas, and brain tissue slices were perfused with the resulting monodispersed droplets and bubbles. The challenge of tissue immobilization in the flow system was addressed using a two-part cytocompatible carbohydrate-based tissue adhesive. Best practices are discussed for perfusion chamber designs that maintain segmented flow throughout the course of perfusion. Control of droplet and bubble volumes was possible across the range of ca. 4–15 μL, bubble generation frequency was well controlled in the range ca. 1–7 bubbles per min, and bubble duty cycle was well controlled across the range ca. 20–80%. Murine hypothalamic tissue slices containing the suprachiasmatic nuclei were successfully maintained for durations of 8–10 hours, with tissue remaining viable for the duration of perfusion as assessed by Ca2+ imaging and propidium iodide (PI) staining.

Chinese hamster ovary (CHO) cells have been the most commonly used mammalian host for large-scale commercial production of therapeutic proteins, such as monoclonal antibodies. Enhancement of productivity of these CHO cells is one of the top priorities in the biopharmaceutical industry to reduce manufacturing cost. Although there are many different methods (e.g. temperature, pH, feed) to improve protein production in CHO cells, the role of physiologically relevant hydrostatic pressure in CHO cell culture has not been reported yet. In this study, four different hydrostatic pressures (0, 30, 60, and 90 mmHg) were applied to batch CHO cells, and their cell growth/metabolism and IgG1 production were examined. Our results indicate that hydrostatic pressure can increase the maximum cell concentration by up to 50%. Moreover, overall IgG1 concentration on Day 5 showed that 30 mmHg pressure can increase IgG1 production by 26%. The percentage of non-disulphide-linked antibody aggregates had no significant change under pressure. Besides, no significant difference was observed between 30 mmHg and no pressure conditions in terms of cell clumping formation. All these findings are important for the optimization of fed-batch or perfusion culture for directing cell growth and improving antibody production.

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 107 cells per cm3 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

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.

A lack of perfusion has been one of the most significant obstacles for three-dimensional culture systems of organoids and embryonic tissues. Here, we developed a simple and reliable method to implement a perfusable capillary network in vitro. The method employed the self-organization of endothelial cells to generate a capillary network and a static pressure difference for culture medium circulation, which can be easily introduced to standard biological laboratories and enables long-term cultivation of vascular structures. Using this culture system, we perfused the lumen of the self-organized capillary network and observed a flow-induced vascular remodeling process, cell shape changes, and collective cell migration. We also observed an increase in cell proliferation around the self-organized vasculature induced by flow, indicating functional perfusion of the culture medium. We also reconstructed extravasation of tumor and inflammatory cells, and circulation inside spheroids including endothelial cells and human lung fibroblasts. In conclusion, this system is a promising tool to elucidate the mechanisms of various biological processes related to vascular flow.

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.

Bispecific protein scaffolds can be more complex than traditional monoclonal antibodies (MAbs) because two different sites/domains for epitope binding are needed. Because of this increased molecular complexity, bispecific molecules are difficult to express and can be more prone to physical and chemical degradation compared to MAbs, leading to higher levels of protein aggregates, clipped species, or modified residues in cell culture. In this study, we investigated cell culture performance for the production of three types of bispecific molecules developed at Amgen. In particular, we cultured a total of six CHO cell lines in both an approximately 12-day fed-batch process and an approximately 40-day high-density perfusion process. Harvested cell culture fluid from each process was purified and analyzed for product quality attributes including aggregate levels, clipped species, charge variants, individual amino acid modifications and host cell protein (HCP) content. Our studies showed that in average, the intensified perfusion process increased 15-fold the integrated viable cell density and the total harvested product (and fivefold the daily volumetric productivity) compared to fed-batch. Furthermore, bispecific product quality improved in perfusion culture (as analyzed in affinity-capture pools) with reduction in levels of aggregates (up to 72% decrease), clipped species (up to 75% decrease), acidic variants (up to 76% decrease), deamidated/isomerized species in complementarity-determining regions, and HCP (up to 84% decrease). In summary, the intensified perfusion process exhibited better productivity and product quality, highlighting the potential to use it as part of a continuous manufacturing process for bispecific scaffolds.

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.

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.

A human bone tissue model was developed by constructing ex vivo the 3D network of osteocytes via the biomimetic assembly of primary human osteoblastic cells with 20–25 µm microbeads and subsequent microfluidic perfusion culture. The biomimetic assembly: (1) enabled 3D-constructed cells to form cellular network via processes with an average cell-to-cell distance of 20–25 µm, and (2) inhibited cell proliferation within the interstitial confine between the microbeads while the confined cells produced extracellular matrix (ECM) to form a mechanically integrated structure. The mature osteocytic expressions of SOST and FGF23 genes became significantly higher, especially for SOST by 250 folds during 3D culture. The results validate that the bone tissue model: (1) consists of 3D cellular network of primary human osteocytes, (2) mitigates the osteoblastic differentiation and proliferation of primary osteoblast-like cells encountered in 2D culture, and (3) therefore reproduces ex vivo the phenotype of human 3D-networked osteocytes. The 3D tissue construction approach is expected to provide a clinically relevant and high-throughput means for evaluating drugs and treatments that target bone diseases with in vitro convenience.

As microbial secretory expression systems have become well developed for microbial yeast cells, such as Saccharomyces cerevisiae and Pichia pastoris, it is advantageous to develop high cell density continuous perfusion cultures of microbial yeast cells to retain the live and productive yeast cells inside the perfusion bioreactor while removing the dead cells and cell debris along with the secreted product protein in the harvest stream. While the previously demonstrated inclined or lamellar settlers can be used for such perfusion bioreactors for microbial cells, the size and footprint requirements of such inefficiently scaled up devices can be quite large in comparison to the bioreactor size. Faced with this constraint, we have now developed novel, patent-pending compact cell settlers that can be used more efficiently with microbial perfusion bioreactors to achieve high cell densities and bioreactor productivities. Reproducible results from numerous month-long perfusion culture experiments using these devices attached to the 5 L perfusion bioreactor demonstrate very high cell densities due to substantial sedimentation of the larger live yeast cells which are returned to the bioreactor, while the harvest stream from the top of these cell settlers is a significantly clarified liquid, containing less than 30% and more typically less than 10% of the bioreactor cell concentration. Size of cells in the harvest is smaller than that of the cells in the bioreactor. Accumulated protein collected from the harvest and rate of protein accumulation is significantly (> 6x) higher than the protein produced in repeated fed-batch cultures over the same culture duration. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:913-922, 2017.

The anti-CD52 antibody has already been approved for the treatment of patients with resistant chronic lymphocytic leukemia, relapsing-remitting multiple sclerosis, and has demonstrable efficacy against stem cell transplantation rejection. A CHO cell line expressing a humanized anti-CD52 monoclonal antibody (mAb-TH) was cultivated in both fed-batch and perfusion modes, and then purified. The critical quality attributes of these mAb variants were characterized and the pharmacokinetics (PK) properties were investigated. Results showed that the perfusion culture achieved higher productivity, whereas the fed-batch culture produced more aggregates and acid components. Additionally, the perfusion culture produced similar fucose, more galactose and a higher proportion of sialic acid on the anti-CD52 mAb compared to the fed-batch culture. Furthermore, the perfusion process produced anti-CD52 mAb had higher complement-dependent cytotoxicity (CDC) efficacy than that produced by the fed-batch culture, a result probably linked to its higher galactose content. However, antibody produced by fed-batch and perfusion cultures showed similar PK profiles in vivo. In conclusion, perfusion is a more efficient method than fed-batch process in the production of functional anti-CD52 monoclonal antibody. Product quality variants of anti-CD52 mAb were found in different cell culture processes, which demonstrated different physiochemical and biological activities, but comparable PK properties. Whether these observations apply to all mAbs await further investigation.

Perfusion cell culture processes allow the steady-state culture of mammalian cells at high viable cell density, which is beneficial for overall product yields and homogeneity of product quality in the manufacturing of therapeutic proteins. In this study, the extent of metabolic steady state and the change of the metabolite profile between different steady states of an industrial Chinese hamster ovary (CHO) cell line producing a monoclonal antibody (mAb) was investigated in stirred tank perfusion bioreactors. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) of daily cell extracts revealed more than a hundred peaks, among which 76 metabolites were identified by tandem MS (MS/MS) and high resolution Fourier transform ion cyclotron resonance (FT-ICR) MS. Nucleotide ratios (Uridine (U)-ratio, nucleotide triphosphate (NTP)-ratio and energy charge (EC)) and multivariate analysis of all features indicated a consistent metabolite profile for a stable culture performed at 40 × 106 cells/mL over 26 days of culture. Conversely, the reactor was operated continuously so as to reach three distinct steady states one after the other at 20, 60, and 40 × 106 cells/mL. In each case, a stable metabolite profile was achieved after an initial transient phase of approximately three days at constant cell density when varying between these set points. Clear clustering according to cell density was observed by principal component analysis, indicating steady-state dependent metabolite profiles. In particular, varying levels of nucleotides, nucleotide sugar, and lipid precursors explained most of the variance between the different cell density set points. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:879-890, 2017.

Perfusion is a cell culture mode that is gaining popularity for the manufacture of monoclonal antibodies and their derivatives. The cell culture media supporting perfusion culture need to support higher cell densities than those used in fed-batch culture. Therefore, when switching from a fed-batch to a perfusion mode, a new medium need to be developed which supports high cell densities, high productivity, and favorable product quality. We have developed a method for deriving perfusion culture media based on existing fed-batch media and feeds. We show that we can obtain culture media that successfully support perfusion cultures in a single-use rocking bioreactor system at cell-specific perfusion rates below 25 pL-1 cell-1 day-1 . High productivities and favorable product quality are also achievable.

Microenvironmental control of interstitial flow around pancreatic islets effectively maintains islet function in vitro., Native pancreatic islets interact with neighboring cells by establishing three-dimensional (3D) structures, and are surrounded by perfusion at an interstitial flow level. However, flow effects are generally ignored in islet culture models, although cell perfusion is known to improve the cell microenvironment and to mimic in vivo physiology better than static culture systems. Here, we have developed functional islet spheroids using a microfluidic chip that mimics interstitial flow conditions with reduced shear cell damage. Dynamic culture, compared to static culture, enhanced islet health and maintenance of islet endothelial cells, reconstituting the main component of islet extracellular matrix within spheroids. Optimized flow condition allowed localization of secreted soluble factors near spheroids, facilitating diffusion-mediated paracrine interactions within islets, and enabled long-term maintenance of islet morphology and function for a month. The proposed model can aid islet preconditioning before transplantation and has potential applications as an in vitro model for diabetic drug testing.

Microcapsules for high cell density culture of mammalian cells have found an increasing interest, however, the poor stability of the microcapsules and the lack of characterisation methods led to few quantitative results. Alginate-poly-L-lysine (PLL) microcapsules have been studied in detail in order to form a basis for comparison of capsules made from different polymers. Since the microcapsules can be easily retained in the bioreactor without the need for a cell separation device, high cell densities were achieved with a maximum of 4 × 10(7) cell/ml(microcapsules), corresponding to a colonisation of 5% of the internal capsule volume. Measurement of microcapsule integrity and mechanical resistance showed that alginate-PLL microcapsules are not suitable for perfusion cultures since they are very sensitive to media composition, mainly the presence of non-gelling ions that have a higher affinity for alginate than PLL and Ca(2+), leading to the leakage of PLL and Ca(2+), and to microcapsule rupture.

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.

The culture of Chinese Hamster Ovary (CHO) cells for modern industrial applications, such as expression of recombinant proteins, requires media that support growth and production. Such media must support high viable cell densities while also stimulating the synthesis and extracellular transport of biologic products. Early media development efforts in this area yielded basic formulations to sustain growth, viability, and cellular function, albeit comprising animal sourced components, and complex constituents used in batch culture mode. Subsequent improvements included the development of serum-free and chemically defined (CD) media, the identification of critical nutrients, growth factors, and potentially inhibitory or toxic cellular metabolites, and the use of fed-batch and perfusion culture techniques to optimize nutrient delivery while minimizing accumulation of unwanted waste products. This review is comprised of sections covering milestones in the evolution of mammalian cell culture media, nutrient composition and formulation requirements, optimization strategies, consistency and scalability of powder and liquid media preparation for industrial applications, and key recent advances driving progress in CHO cell culture medium design and development. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1407-1426, 2018.

Although there is a great need for suitable vascular replacements in clinical practice, much progress needs to be made toward the development of a fully functional tissue-engineered construct. We propose a fabrication method of engineered tubular tissue for small blood vessels via a layer-by-layer cellular assembly technique using mouse smooth muscle cells, the construction of a poly-(l-lactide-co-e-caprolactone) (PLCL) scaffold, and integration in a microfluidic perfusion culture system. The cylindrical PLCL scaffold is incised, expanded, and its surface is laminated with the cell layers. The construct confirms into tubular structures due to residual stress imposed by the cylindrical PLCL scaffold. The perfusion culture system allows simulation of static, perfusion (laminar flow), and perfusion with pulsatile pressure (Pulsatile flow) conditions in which mimicking the in vivo environments. The aim of this evaluation was to determine whether fabricated tubular tissue models developed their mechanical properties. The cellular response to hemodynamic stimulus imposed by the dynamic culture system is monitored through expression analysis of fibrillin-1 and fibrillin-2, elastin and smooth muscle myosin heavy chains isoforms transcription factors, which play an important role in tissue elastogenesis. Among the available materials for small blood vessel construction, these cellular hybrid vascular scaffolds hold much potential due to controllability of the mechanical properties of synthetic polymers and biocompatibility of integrated cellular components.

Perfusion culture of mesenchymal stem cells (MSCs) seeded in biomaterial scaffolds provides nutrients for cell survival, enhances extracellular matrix deposition, and increases osteogenic cell differentiation. However, there is no consensus on the appropriate perfusion duration of cellular constructs invitro to boost their bone forming capacity invivo. We investigated this phenomenon by culturing human MSCs in macroporous composite scaffolds in a direct perfusion bioreactor and compared their response to scaffolds in continuous dynamic culture conditions on an XYZ shaker. Cell seeding in continuous perfusion bioreactors resulted in more uniform MSC distribution than static seeding. We observed similar calcium deposition in all composite scaffolds over 21 days of bioreactor culture, regardless of pore size. Compared to scaffolds in dynamic culture, perfused scaffolds exhibited increased DNA content and expression of osteogenic markers up to 14 days in culture that plateaued thereafter. We then evaluated the effect of perfusion culture duration on bone formation when MSC-seeded scaffolds were implanted in a murine ectopic site. Human MSCs persisted in all scaffolds at 2 weeks invivo, and we observed increased neovascularization in constructs cultured under perfusion for 7 days relative to those cultured for 1 day within each gender. At 8 weeks post-implantation, we observed greater bone volume fraction, bone mineral density, tissue ingrowth, collagen density, and osteoblastic markers in bioreactor constructs cultured for 14 days compared to those cultured for 1 or 7 days, and acellular constructs. Taken together, these data demonstrate that culturing MSCs under perfusion culture for at least 14 days invitro improves the quantity and quality of bone formation invivo. This study highlights the need for optimizing invitro bioreactor culture duration of engineered constructs to achieve the desired level of bone formation.

Adipose tissue engineered models are needed to enhance our understanding of disease mechanisms and for soft tissue regenerative strategies. Perfusion systems generate more physiologically relevant and sustainable adipose tissue models, however adipocytes have unique properties that make culturing them in a perfusion environment challenging. In this paper we describe the methods involved in the development of two perfusion culture systems (2D and 3D) to test their applicability for long term in vitro adipogenic cultures. It was hypothesized that a silk protein biomaterial scaffold would provide a 3D framework, in combination with perfusion flow, to generate a more physiologically relevant sustainable adipose tissue engineered model than 2D cell culture. Consistent with other studies evaluating 2D and 3D culture systems for adipogenesis we found that both systems successfully model adipogenesis, however 3D culture systems were more robust, providing the mechanical structure required to contain the large, fragile adipocytes that were lost in 2D perfused culture systems. 3D perfusion also stimulated greater lipogenesis and lipolysis and resulted in decreased secretion of LDH compared to 2D perfusion. Regardless of culture configuration (2D or 3D) greater glycerol was secreted with the increased nutritional supply provided by perfusion of fresh media. These results are promising for adipose tissue engineering applications including long term cultures for studying disease mechanisms and regenerative approaches, where both acute (days to weeks) and chronic (weeks to months) cultivation are critical for useful insight.

There are significant challenges in developing in vitro human tissue and tumor models that can be used to support new drug development and evaluate personalized therapeutics. The challenges include: (1) working with primary cells which are often difficult to maintain ex vivo, (2) mimicking native microenvironments from which primary cells are harvested, and (3) the lack of culture devices that can support these microenvironments to evaluate drug responses in a high-throughput manner. Here we report a versatile well plate-based perfusion culture device that was designed, fabricated and used to: (1) ascertain the role of perfusion in facilitating the expansion of human multiple myeloma cells and evaluate drug response of the cells, (2) preserve the physiological phenotype of primary murine osteocytes by reconstructing the 3D cellular network of osteocytes, and (3) circulate primary murine T cells through a layer of primary murine intestine epithelial cells to recapitulate the interaction of the immune cells with the epithelial cells. Through these diverse case studies, we demonstrate the device's design features to support: (1) the convenient and spatiotemporal placement of cells and biomaterials into the culture wells of the device; (2) the replication of tissues and tumor microenvironments using perfusion, stromal cells, and/or biomaterials; (3) the circulation of non-adherent cells through the culture chambers; and (4) conventional tissue and cell characterization by plate reading, histology, and flow cytometry. Future challenges are identified and discussed from the perspective of manufacturing the device and making its operation for routine and wide use.

In vitro recapitulation of an organotypic stromal environment, enabling efficient angiogenesis, is crucial to investigate and possibly improve vascularization in regenerative medicine. Our study aims at engineering the complexity of a vascular milieu including multiple cell-types, a stromal extracellular matrix (ECM), and molecular signals. For this purpose, the human adipose stromal vascular fraction (SVF), composed of a heterogeneous mix of pericytes, endothelial/stromal progenitor cells, was cultured under direct perfusion flow on three-dimensional (3D) collagen scaffolds. Perfusion culture of SVF-cells reproducibly promoted in vitro the early formation of a capillary-like network, embedded within an ECM backbone, and the release of numerous pro-angiogenic factors. Compared to static cultures, perfusion-based engineered constructs were more rapidly vascularized and supported a superior survival of delivered cells upon in vivo ectopic implantation. This was likely mediated by pericytes, whose number was significantly higher (4.5-fold) under perfusion and whose targeted depletion resulted in lower efficiency of vascularization, with an increased host foreign body reaction. 3D-perfusion culture of SVF-cells leads to the engineering of a specialized milieu, here defined as an angiogenic niche. This system could serve as a model to investigate multi-cellular interactions in angiogenesis, and as a module supporting increased grafted cell survival in regenerative medicine.

International audience; 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.

Continuous production of biologics, a growing trend in the biopharmaceutical industry, requires a reliable and efficient cell retention device that also maintains cell viability. Current filtration methods, such as tangential flow filtration using hollow-fiber membranes, suffer from membrane fouling, leading to significant reliability and productivity issues such as low cell viability, product retention, and an increased contamination risk associated with filter replacement. We introduce a novel cell retention device based on inertial sorting for perfusion culture of suspended mammalian cells. The device was characterized in terms of cell retention capacity, biocompatibility, scalability, and long-term reliability. This technology was demonstrated using a high concentration (>20 million cells/mL) perfusion culture of an IgG1-producing Chinese hamster ovary (CHO) cell line for 18–25 days. The device demonstrated reliable and clog-free cell retention, high IgG1 recovery (>99%) and cell viability (>97%). Lab-scale perfusion cultures (350 mL) were used to demonstrate the technology, which can be scaled-out with parallel devices to enable larger scale operation. The new cell retention device is thus ideal for rapid perfusion process development in a biomanufacturing workflow.

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.

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 C6 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 13 C6 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 C6 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.

Intracranial pressure and volume vary considerably between hydrocephalic patients, and with age, health and haemodynamic status; if left untreated, intracranial pressure rises and the ventricular system expands to accommodate the excess cerebrospinal fluid, with significant morbidity and mortality. Cerebrospinal fluid shunts in use today have a high incidence of failure with shunt obstruction being the most serious. Conventional proximal shunt catheters are made from poly(dimethyl)siloxane, the walls of which are perforated with holes for the cerebrospinal fluid to pass through. The limited range of catheters, in terms of material selection and flow distribution, is responsible in large part for their poor performance. In this study, we present an alternative design of proximal catheter made of electrospun polyether urethane, and evaluate its performance in the presence of glial cells, which are responsible for shunt blockage. The viability and growth of cells on catheter materials such as poly(dimethyl)siloxane and polyurethane in the form of cast films, microfibrous mats and porous sponges were studied in the presence of proteins present in cerebrospinal fluid after 48 h and 96 h in culture. The numbers of viable cells on each substrate were comparable to untreated poly(dimethyl)siloxane, both in the presence and absence of serum proteins found in cerebrospinal fluid. A cell culture model of shunt obstruction was developed in which cells on electrospun polyether urethane catheters were subjected to flow during culture in vitro, and the degree of obstruction quantified in terms of hydraulic permeability after static and perfusion culture. The results indicate that a catheter made of electrospun polyether urethane would be able to maintain cerebrospinal fluid flow even with the presence of cells for the time period chosen for this study. These findings have implications for the design and deployment of microporous shunt catheter systems for the treatment of hydrocephalus.

We recently developed a polydimethylsiloxane (PDMS)-based three-compartment microfluidic cocultivation device enabling real-time interactions of different cell populations as an advanced physiologically-relevant cell-based assay. This device had valves and small magnetic stirrer-based internal pumps for easy and flexible perfusion operations. In this study, we applied this device for the evaluation of Irinotecan (CPT-11) toxicity to the lung, because it is detoxified by the liver and accumulated in the fat in humans. We successfully cultured representative three different tissue model cells in each compartment under the individual culture conditions and also in entire perfusion. Growth inhibition of rat lung epithelial cell line L-2, was measured when administered with 50 μM CPT-11 under various cocultivation conditions with respect to the presences and absence of primary rat hepatocytes (liver tissue model) and adipocyte-like cells (fat tissue model) induced from a mouse fibroblast cell line, 3T3-L1. Although CPT-11 showed moderate toxicity to the pure culture of L-2 cells in the device after 72 h of perfusion culture, this was lowered mainly in the presence of the liver tissue. Inhibition of the L-2 cell growth agreed with the area under curve (AUC) values obtained from fluorescent image-based analyses in each compartment. These results demonstrate that developed simple and flexible microfluidic cocultivation device, with appropriate image-based analyses, can be used in evaluating toxicokinetic behaviors of drug candidates in systemic levels.

Compelling evidence suggests the limitation and shortcomings of the current and well established cell culture method using multi-well plates, flasks and Petri dishes. These are particularly important when cell functions are sensitive to the local microenvironment, cell–cell and cell–extracellular matrix interactions. There is a clear need for advanced cell culture systems which mimic in vivo and more physiological conditions. This review summarises and analyses recent progress in three dimensional (3D) cell culture with perfusion as the next generation cell culture tools, while excluding engineered tissue culture where three dimensional scaffold has to be used for structural support and perfusion for overcoming mass transfer control. Apart from research activities in academic community, product development in industry is also included in this review.

Developmental cell biology requires technologies in which the fate of single cells is followed over extended time periods, to monitor and understand the processes of self-renewal, differentiation, and reprogramming. A workflow is presented, in which single cells are encapsulated into droplets (Ø: 80 µm, volume: ≈270 pL) and the droplet compartment is later converted to a hydrogel bead. After on-chip de-emulsification by electrocoalescence, these 3D scaffolds are subsequently arrayed on a chip for long-term perfusion culture to facilitate continuous cell imaging over 68 h. Here, the response of murine embryonic stem cells to different growth media, 2i and N2B27, is studied, showing that the exit from pluripotency can be monitored by fluorescence time-lapse microscopy, by immunostaining and by reverse-transcription and quantitative PCR (RT-qPCR). The defined 3D environment emulates the natural context of cell growth (e.g., in tissue) and enables the study of cell development in various matrices. The large scale of cell cultivation (in 2000 beads in parallel) may reveal infrequent events that remain undetected in lower throughput or ensemble studies. This platform will help to gain qualitative and quantitative mechanistic insight into the role of external factors on cell behavior.

In vitro perfusable 3D tissue models mimic in vivo tissues and have several benefits in drug testing. However, processes used to fabricate these models often tend to be complicated. Here, we present a double-layer perfusable collagen tube device for multilayered in vitro 3D cell culture. The device is simply made by the repetition of a molding process. The thicknesses of the collagen layers in the tube device can be flexibly designed, and heterogeneous cell types can be co-cultured in/on each collagen layer. Moreover, while our collagen tube is directly attached to silicone tubes, the collagen tube can easily be connected to an external pump system for perfusion culture. We fabricated six different sizes of collagen devices (inner diameter approximately 300-1000 μm) using different molds, and successfully controlled the coefficients of variation to be below 5% for the diameters of each layer for all six device sizes. The device is strong enough to manipulate with tweezers, and can remain stable for more than 3 months in a medium. For the cell culture, we successfully and correctly encapsulated cells in the layer shape at the desired position, and confirmed cell migration. Using the perfusion culture, we demonstrated that the alignments of the HUVEC actin filaments become parallel to the flow direction. We believe that our device could advance the easy fabrication of various tissue models (that is, models mimicking in vivo tissues). In particular, the device could help fabricate vascularized tissue models, and contribute to the development of pharmacokinetic testing platforms and regenerative medicine.

In an attempt to support robust automated sampling and analysis of mammalian cell bioreactors, an integrated platform, BaychroMAT®, was developed which includes an innovative sterile sampling device, automated sample transport, a sample preparation module, online analyzers, and communication interfaces to process automation systems. The robustness of this platform was verified by applying it to a laboratory-scale perfusion bioreactor that was operated for over 100 days. Both manual and automated samples were collected over the course of the run and a comparison was made for cell density, viability, glucose, and lactate concentrations. The highest variability (14.4%) was seen for cell density estimates while those for viability, glucose, and lactate were 0.7, 12.9, and 8.2%, respectively. In addition, cell density set-point changes were made towards the end of the perfusion culture and the high frequency automated samples provided a higher resolution description of the dynamics of cell density change compared to less frequent manual sampling. Overall, our results indicate stable and robust operation of the BaychroMAT® platform in a long-term perfusion culture. This success should readily translate to shorter duration fed-batch cultures thereby enabling feed-back control based on real-time nutrient measurements.

A poly(dimethylsiloxane) biophotonic lab-on-a-chip (bioPhLoC) containing two chambers, an incubation chamber and a monitoring chamber for cell retention/proliferation and pH monitoring, respectively, is presented. The bioPhLoC monolithically integrates a filter with 3 mu m high size-exclusion microchannels, capable of efficiently trapping cells in the incubation chamber, as well as optical elements for real-time interrogation of both chambers. The integrated optical elements made possible both absorption and dispersion measurements, which were comparable to those made in a commercially available cuvette. The size-exclusion filter also showed good and stable trapping capacity when using yeast cells of variable size (between 5 and 8 mu m diameter). For cell culture applications, vascular smooth muscle cells (VSMC), with sizes between 8 and 10 mu m diameter, were used as a mammalian cell model. These cells were efficiently trapped in the incubation chamber, where they proliferated with a classical spindle-shaped morphology and a traditional hill-and-valley phenotype. During cell proliferation, pH changes in the culture medium due to cell metabolism were monitored in real time and with high precision in the monitoring chamber without interference of the measurement by cells and other (cell) debris.

Hand, foot and mouth diseases (HFMD) are mainly caused by Enterovirus A71 (EV-A71) infections. Clinical trials in Asia conducted with formalin-inactivated EV-A71 vaccine candidates produced from serum-free Vero cell culture using either roller bottle or cell factory technology, are found to be safe and highly efficacious. To increase vaccine yields and reduce the production costs, the bioprocess improvement for EV-A71 vaccine manufacturing is currently being investigated. The parameters that could affect and enhance the production yields of EV-A71 virus growth in the microcarrier bioreactor were investigated. The medium replacement culture strategy included a multi-harvested semi-batch process and perfusion technology and was found to increase the production yields more than 7-14 folds. Based on the western blot and cryo-EM analyses of the EV-A71 virus particles produced from either the multi-harvested semi-batch (MHSBC) or perfusion cultures were found to be similar to those virus particles obtained from the single batch culture. Mouse immunogenicity studies indicate that the EV-A71 vaccine candidates produced from the perfusion culture have similar potency to those obtained from single batch bioprocess. The physical structures of the EV-A71 particles revealed by the cryo-EM analysis were found to be spherical capsid particles. These results provide feasible technical bioprocesses for increasing virus yields and the scale up of EV-A71 vaccine manufacturing using the bioreactor cell culture methods.

Shear stress caused by fluid flow is known to promote tissue development from cells in vivo. Therefore, perfusion cultures have been studied to investigate the mechanisms involved and to fabricate engineered tissues in vitro, particularly those that include blood vessels. Microfluidic devices, which function with fine machinery of chambers and microsyringes for fluid flow and have small culture areas, are conventionally used for perfusion culture. In contrast, we have developed a self-driven perfusion culture system by using a paper-based double-layered scaffold as the fundamental component. Gelatin microfibers were electrospun onto a paper material to prepare the scaffold system, in which the constant perfusion of the medium and the scaffold for cell adhesion/proliferation were functionally divided into a paper and a gelatin microfiber layer, respectively. By applying both the capillary action and siphon phenomenon of the paper-based scaffold, which bridged two medium chambers at different height levels, a self-driven medium flow was achieved and the flow rate was also stable, constant, and quantitatively controllable. Moreover, the culture area was enlargeable to the cm(2) scale. The endothelial cells cultivated on this system oriented along the medium-flow direction, suggesting that the shear stress caused by medium flow was effectively applied. This perfusion culture system is expected to be useful for fabricating three-dimensional and large engineered tissues in the future.

Articular cartilage has a limited capacity for self-repair after damage. Engineered cartilage is a promising treatment to replace or repair damaged tissue. The growth of engineered cartilage is sensitive to the extracellular culture environment. Chondrocytes were seeded into alginate beads and agarose scaffolds at 4 millions/mL, and the response to static and perfusion culture was examined over period of up to 12 days. For both types of scaffolds, the chondrocytes kept their differentiated morphology over 12 days in all culture conditions. In alginate beads, more glycosaminoglycans (GAGs) were produced in perfusion culture than in static conditions. GAG distribution in alginate constructs was more uniform in perfusion culture than in static culture. However, in agarose constructs there was no significant difference in GAG production between static culture and perfusion culture. Under perfusion culture, the retention rate of GAG in alginate was higher than in agarsoe. It is suggested that the positive effect of perfusion culture only can be achieved by an appropriate choice of other factors such as scaffold materials.

Hepatocyte and various hepatic stem cell transplantations have been studied as alternative therapies to orthotopic liver transplantation for liver injury. The engraftment of transplanted cells into the parenchyma requires transmigration through sinusoidal endothelial cells (SECs), the only cellular barrier. In this study, we constructed a SEC-imaging perfusion culture system that mimics sinusoids with respect to hemorheologic properties. SECs were successfully maintained for 24 hours. Human liver stem cells (HLSCs) were used as a model of transplanted cells for in vitro engraftment to SECs under perfusion culture conditions. Conditions of high shear stress perfusion with 0.34 dyne/cm2 significantly reduced cell adhesion in contrast to lower shear stress conditions of 0.1 and 0.03 dyne/cm2. Among the biologic perfusion fluids, namely, fetal bovine serum (FBS), pig plasma, and 5% human albumin solution, HLSCs showed significantly greater attachment to SECs when perfused with FBS, which is well known to contain abundant amounts of adhesion molecules. This biomimetic SEC perfusion culture system may provide a useful tool to study engraftment mechanisms and to evaluate the effects of various enhancers as an alternative to animal models.