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Spatial pattern formation is a critical step in embryogenesis. Bone morphogenetic protein 4 (BMP4) and its inhibitors are major factors for the formation of spatial patterns during embryogenesis. However, spatial patterning of the human embryo is unclear because of ethical issues and isotropic culture environments resulting from conventional culture dishes. Here, we utilized human pluripotent stem cells (hiPSCs) and a simple anisotropic (unidirectional perfusion) culture chamber, which creates unidirectional conditions, to measure the cell community effect. The influence of cell density on BMP4-induced differentiation was explored during static culture using a conventional culture dish. Immunostaining of the early differentiation marker SSEA-1 and the mesendoderm marker BRACHYURY revealed that high cell density suppressed differentiation, with small clusters of differentiated and undifferentiated cells formed. Addition of five-fold higher concentration of BMP4 showed similar results, suggesting that suppression was not caused by depletion of BMP4 but rather by high cell density. Quantitative RT-PCR array analysis showed that BMP4 induced multi-lineage differentiation, which was also suppressed under high-density conditions. We fabricated an elongated perfusion culture chamber, in which proteins were transported unidirectionally, and hiPSCs were cultured with BMP4. At low density, the expression was the same throughout the chamber. However, at high density, SSEA-1 and BRACHYURY were expressed only in upstream cells, suggesting that some autocrine/paracrine factors inhibited the action of BMP4 in downstream cells to form the spatial pattern. Human iPSCs cultured in a perfusion culture chamber might be useful for studying in vitro macroscopic pattern formation in human embryogenesis.

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 hybrid cell line, F2N78, was developed by somatic fusion of HEK293 and Namalwa cells for the production recombinant biopharmaceutical proteins. In this study, we performed perfusion culture to verify its potential in culture process used for human cell expression platform. Cell viability could be maintained over 90% and high viable cell density was obtained at higher than 1.0 × 10(7) cells/mL by bleeding process in perfusion culture. The cells were adapted well in both culture modes, but there were apparent differences in protein quality. Compared to fed-batch culture, degalactosylated forms such as G0F and G0 as well as Man5 showed no significant increases in perfusion culture. In terms of charge variants, acidic peaks increased, whereas main peaks constantly decreased according to the length of culture period in both methods.

A packed-bed (PB) bioreactor for bioartificial liver (BAL) was fabricated based on an optimum two-stage culture strategy and evaluated in vitro in this research. Human induced hepatocytes (hiHeps) were first expanded using Cytodex 3 microcarriers and the choice of microcarrier concentration and fetal bovine serum (FBS) content was optimized. Then, the cells expanded under the optimum expansion condition were perfused into a perfusion system containing Fibra-Cel (FC) disks to fabricate a PB bioreactor. Operating parameters including flow rate and seeding density for perfusion culture were optimized, respectively. Results indicated that during suspension culture, rapid cell proliferation and favorable amino acid metabolism were achieved at 3 mg/mL microcarriers combined with 1% FBS. While for the perfusion culture, the most effective flow rate and seeding density were 2 mL/min and 1 × 106 cells/mL, respectively. Under this optimum perfusion condition, hiHeps showed good proliferation ability, high viability, homogeneous distribution, high metabolism activities and efficient albumin secretion as well as high liver-specific genes expression. Therefore, the two-stage culture strategy based on operating parameters optimization provides a new method for the development of PB bioreactors.

We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm(2) in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 10(5)) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm(2). Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm(2).

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.

One major challenge in the field of tissue engineering was the creation of volumetric tissues and organs in vitro. To achieve this goal, the development of a three-dimensional vascular-like network that extended throughout the tissue-engineered construct was essential to supply sufficient oxygen and nutrients to all of the cells in the constructs. For sufficient oxygenation and nutrition of the tissue-engineered constructs, the distance between each microvessel-like channel in the network should ideally be within 100–200 μm. In addition, the medium or blood should be perfused through the microchannels as soon as possible after the seeding of cells into the templates (scaffolds) of the constructs. In the present study, we proposed a novel technique for fabricating an engineered vascular-like network that satisfied these two requirements. The network comprised assembled hollow alginate hydrogel microfibers with mammalian cells enclosed in the gel portions. We controlled the distance between each flow microchannel (hollow core portions and interspace of the microfibers) to be within 150 μm by using microfibers with a gel thickness of approximately 50 μm. Furthermore, we confirmed that medium could be perfused into the flow channels quickly (within 10 min) after immobilization of the cells in the assembly. A human hepatoblastoma cell line (HepG2) proliferated in the gel portions of the microfibers and maintained their specific function during perfusion culture for 7 days. These results showed that the novel vascular-like networks fabricated here had the potential to allow the creation of volumetric tissues in vitro.

This study describes the development and experimental verification of a modified harvest system to enhance Factor VIII (FVIII) yield in an alternating tangential flow (ATF) perfusion culture. The main innovation of the modified harvest system is the use of check and pinch valves, eliminating the need of a peristaltic pump for harvest. The system was applied to perfusion cultures of Chinese hamster ovary cells, which co-express both recombinant human FVIII (rhFVIII) and von Willebrand factor (vWF). The modified harvest system showed comparable cell growth with the conventional harvest system using a peristaltic pump. The perfusion rate was successfully controlled using the system. In addition, the modified harvest system achieved an approximately 13.6-fold increase in the final concentration yield of FVIII activity and a 1.47-fold increase in the production yield of FVIII activity compared with a peristaltic pump. Enhancement of the yield of FVIII activity resulted from the reduction of FVIII antigen (FVIII:Ag) retention. As a result of transmembrane pressure (TMP) measurement, the reduction of the retained FVIII:Ag was due to the increased TMP, which was caused by the characteristic function of a check valve, compared with a peristaltic harvest system. The modified harvest system developed in this study could be useful to enhance the production yield of other recombinant proteins in ATF perfusion culture.

Based on the effects of temperature shift on the cell cycle, apoptosis and metabolism of a recombinant Chinese hamster ovary (rCHO) cell line (CL-11G) producing pro-urokinase (pro-UK) in batch cultures, the potential of temperature shift as a tool in the optimization of the perfusion culture of CL-11G cells for the production of pro-UK was examined. The proportion of CL-11G cells in the G0/G1 phase in static cultures increased from 56.4% to 82.8% following a temperature shift from 37 degrees C to 31 degrees C. Conversely, the proportion of CL-11G cells in the S phase decreased from 34.8% to 11.6%. The specific growth rate of CL-11G cells reflected the effect of temperature on the cell cycle and decreased from 0.024 h(-1) at 37 degrees C to 0.006 h(-1) at 31 degrees C. Continuous exposure to the non-permissive temperature of 31 degrees C led to a marginal increase in apoptosis. The specific pro-UK productivity of CL-11G cells increased by 74% at 34 degrees C compared with controls at 37 degrees C in batch cultures. CL-11G cells immobilized with Cytopore 1 in a 5-l bioreactor initiated at 37 degrees C and temperature shifted to 34 degrees C exhibited an average 17% increase in viable cell density and an average 47% increase in pro-UK production. These results demonstrated that temperature shift offers the prospect of enhancing the productivity of pro-UK in high-density perfusion culture.

The perfusion culture technique using a shaken ceramic membrane flask (SCM flask) was applied to the production of a secretory heterologous protein. A recombinant methylotrophic yeast strain, Pichia pastoris, was cultured aerobically on a reciprocal shaker using an SCM flask. High-level production of human serum albumin (HSA) was attempted by increasing both the cell concentration and the expression level of the recombinant gene. In the two-stage culture method, the cell concentration was first raised to 17 g/l by feeding glycerol, after which the expression of HSA was induced by feeding methanol. However, the concentration of HSA in the effluent filtrate was as low as 0.15 g/l, while the cell concentration continued to increase. In contrast, HSA was effectively produced by feeding methanol from an early stage of the culture. In this case, the HSA concentration reached 0.24 and 0.46 g/l, respectively, using the growth-associated production method without and with aeration into the head space of the SCM flask. The results showed that supplying sufficient oxygen together with the growth-associated induction method are effective for obtaining high-level expression of the methanol-inducible recombinant gene of P. pastoris. An HSA concentration in the filtrate of 1.5 g/l was finally achieved when the cell concentration was increased to 53 g/l by supplying oxygen-enriched gas to the SCM flask. The yield and productivity of HSA reached 2.6-fold and 10-fold those obtained in an ordinary fed-batch culture using a shake flask, and these levels were readily achieved by continuous replenishment of the culture supernatant. The achievements made in this study should contribute to the development of a handy bioreactor system for mini-scale mass production of target proteins with separation at high purity.

The growth inhibition of Pyrococcus furiosus by acetic acid was stronger than that by hydrogen and could be described by a non-competitive inhibition model in which the inhibition constants of undissociated acetic acid, K p and n , were estimated to be 0.69 mM (25 mM total acetic acid at pH 6.5; pKa=4.96; 98°C) and 1.0, respectively. In order to reduce the acetic acid inhibition, repeated-batch culturing was performed using a filtration module. This yielded 0.49 g of dry cells l −1 after growth for 12 h after inoculation. It became impossible, however, to continue repeated-batch culturing manually because the time intervals for medium replacement became too short. In order to automatically maintain a low concentration of acetic acid, a perfusion culture was carried out in which medium feeding coupled to a pH-auxostat was performed. In this perfusion culture, it was possible to maintain the acetic acid concentration below 7.6 mM during exponential growth of P. furiosus , resulting in 1.8 g of dry cells l −1 at 15 h after inoculation.

Cells of the human embryonic kidney cell line (HEK 293) were grown as suspended aggregates in stirred vessels and infected with a recombinant adenovirus vector (Ad-TH-GFP). Regular spherical aggregates with the mean diameter less than 300 μm and a viable cell density greater than 5 × 10 6 cells·ml − 1 were readily achieved after 9 day culture in spinner flasks. The HEK 293 cells growing as suspended aggregates could be efficiently infected by Ad-TH-GFP at an MOI of 10 with a prolonging infection time up to 144 hour post-infection (hpi). The time profile of Ad-TH-GFP production was strongly corresponding to the infection process with a virus concentration peak occurred consistently at 144 h after infection. And the infected aggregates essentially maintained spherical in shape, the portion of dissociated cells from the infected aggregates was less than 5% at 144 hpi. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5 L stirred tank bioreactor and infected with Ad-TH-GFP at a density higher than 1 × 10 7 cells·ml − 1 resulted in a similar Ad-TH-GFP production kinetics, but a much higher virus yield approximately at 5.7 × 10 11 GTU ml − 1 at 144 hpi to that of the infected spinner flask cultures. These results demonstrate the feasibility for using suspended cell aggregates as an immobilization system to facilitate perfusion in stirred tank bioreactors, and improve volumetric productivities by eliminating the cell density effect.

Recombinant Chinese hamster ovary (rCHO) cells capable of producing a prourokinase mutant (mPro-uk) grown as suspended aggregates in stirred vessels were described and characterized. The addition of chitosan to a mixture of DMEM and Ham's F12 (D-MEM/F-12) medium promoted cell aggregation and spheroid formation efficiently. Multicellular aggregates formed immediately after the rCHO cells were inoculated into the chitosan-added medium, and the mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 65 to 163 mum after 2 and 9 d of culture in spinner flasks. No significant difference in the metabolism performance of the rCHO cells was observed between suspended aggregates and anchored monolayers. However, the cells cultured as suspended aggregates showed a marked decrease in growth rate as evaluated from specific growth rate (mu). Replacing D-MEM/F-12 medium with CD 293 medium caused compact spherical cell aggregates to dissociate into small irregular aggregates and single cells without apparent effects on cell performance in subcultures. The perfusion culture of the rCHO cells grown as suspended aggregates in a 2-l stirred tank bioreactor for 15 d resulted in a maximum viable cell density of 5.6 x 10(6) cells ml(-1) and an mPro-uk concentration of about 2.6 x 10(3) IU ml(-1), and cell viability was remained at roughly 90% during the entire run.

A galactose moiety was introduced into the fiber surface of a vegetable sponge by the covalent binding of lactobionic acid. The galactosylated sponge was used as scaffold for the culture of rat hepatocytes in a packed-bed bioreactor. Hepatocytes could be dynamically seeded into and uniformly distributed throughout the scaffold, and the immobilized cells maintained high albumin and urea production rates during long-term perfusion culture. The hepatocytes showed an increasing albumin production rate from 49 to 109 microg/10(6) cells/d over the 7-d culture.

Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase productivity that reached a plateau fluctuating around 490 units/l/d.

The effect of centrifugal force applied for cell separation at the medium change on the growth, metabolism and tissue plasminogen activator (tPA) productivity of Chinese hamster ovary (CHO) cells suspension culture was investigated. The viability of the precipitated cells increased exponentially as the centrifugal force decreased. However, the cell recovery was lower than 91% when centrifugal forces applied for 5 min was less than 67 x g. In cultures incubated for 474 h with 7 medium changes employing centrifugal forces ranging from 67 to 364 x g, a centrifugal force lower than 119 x g resulted in higher specific rates of growth, glucose consumption, and lactate and tPA production during the whole culture period. On the other hand, daily centrifugation at 67 to 537 x g without discarding the supernatant had no effect on the specific rates. The cultures inoculated with cells precipitated at a centrifugal force of 67 x g showed apparently higher specific rates of metabolism compared to those inoculated with cells in the supernatant. The cells in the supernatant and the precipitate obtained following centrifugation at 67 x g have average diameters of 15.5 and 17.4 microm, respectively. The intracellular contents of amino acids, especially nonessential amino acids, of the precipitated cells were markedly higher than those of the cells in the supernatant. These results indicate that large cells with high amino acid content and metabolic activity were selectively retained in the culture by means of centrifugation at low forces such as 67 x g. Consequently, application of a low centrifugal force is recommended for medium change in order to maintain higher specific productivity of suspended mammalian cells in perfusion culture.

The perfusion culture system using a shaken ceramic membrane flask (SCMF) was employed to accumulate microorganisms separated from river water and to produce poly-beta-hydroxybutyric acid (PHB). Using a two-step culture method with a single SCMF, river microorganisms were cultured by separately feeding four representative carbon sources, n-propanol, lactic acid, methanol, and formic acid. After 140 h culture, the cell concentration and PHB content respectively reached 43 g/l and 35% when a propanol medium was fed. Using a two-stage perfusion culture with twin SCMFs, the seed cell mass was increased in the first SCMF and then supplied to the second flask for PHB production. As a consequence, the cellular PHB content rose to 51% in the second SCMF, while the cell concentration gradually increased to 25 g/l after 175 h perfusion culture. These results demonstrated the utility of the two-stage perfusion culture system for developing a cheap means of producing PHB coincident with wastewater treatment.

A perfusion culture system was used for continuous production of lactic acid by retaining cells at a high density of Lactococcus lactis in a stirred ceramic membrane reactor (SCMR). After the cell concentration increased to 248 g/l, half of the culture broth volume was replaced with the fermentation medium. Subsequently, a substrate solution containing glucose (run 1) or molasses (run 2) was continuously supplied to the cells retained in the SCMR. Simultaneously, the culture supernatant was extracted using a ceramic filter with a pore size of 0.2 mum. The dilution rate was initially set at 0.4 h(-1) and gradually decreased to 0.2 h(-1) due to reduction in the permeability of the filter. The concentration of glucose in the substrate solution was adjusted to 60 g/l for the transition and the first period until 240 h, 90 g/l for the second period from 240 h to 440 h, and 70 g/l for the third period from 440 h to 643 h. The average concentration of lactic acid in the filtrate reached 46 g/l in the first period, 43 g/l in the second period, and 33 g/l for the third period. The productivity obtained for the first period reached 15.8 g.l(-1).h(-1), twice as much as that achieved in repeated batch fermentations. Based on the results obtained in run 1, the substrate solution containing 120 g/l of molasses was continuously supplied for 240 h in run 2. The concentration and productivity of lactic acid reached 40 g/l and 10.6 g.l(-1).h(-1), respectively, by continuously replenishing the culture medium at a dilution rate of 0.26 h(-1). These results demonstrated that the filtration capacity of the SCMR was sufficient for a continuous and rapid replenishment of molasses solution from the dense cell culture and, therefore, the perfusion culture system is considered to provide a low-cost process for continuous production of lactic acid from cheap resources.