Fluorescence optical detection in situ for real‐time monitoring of cytochrome P450 enzymatic activity of liver cells in multiple microfluidic devices

We describe an in situ fluorescence optical detection system to demonstrate real‐time and non‐invasive detection of reaction products in a microfluidic device while under perfusion within a standard incubator. The detection system is designed to be compact and robust for operation inside a mammalian cell culture incubator for quantitative detection of fluorescent signal from microfluidic devices. When compared to a standard plate reader, both systems showed similar biphasic response curves with two linear regions. Such a detection system allows real‐time measurements in microfluidic devices with cells without perturbing the culture environment. In a proof‐of‐concept experiment, the cytochrome P450 1A1/1A2 activity of a hepatoma cell line (HepG2/C3A) was monitored by measuring the enzymatic conversion of ethoxyresorufin to resorufin. The hepatoma cell line was embedded in Matrigel^TM construct and cultured in a microfluidic device with medium perfusion. The response of the cells, in terms of P450 1A1/1A2 activity, was significantly different in a plate well system and the microfluidic device. Uninduced cells showed almost no activity in the plate assay, while uninduced cells in Matrigel^TM with perfusion in a microfluidic device showed high activity. Cells in the plate assay showed a significant response to induction with 3‐Methylcholanthrene while cells in the microfluidic device did not respond to the inducer. These results demonstrate that the system is a potentially useful method to measure cell response in a microfluidic system. Biotechnol. Bioeng. 2009; 104: 516–525 © 2009 Wiley Periodicals, Inc.     

3D culture of osteoblast‐like cells by unidirectional or oscillatory flow for bone tissue engineering

A medium perfusion system is expected to be beneficial for three‐dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast‐like MC 3T3‐E1 cells were 3D‐cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell‐seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering. Biotechnol. Bioeng. 2009;102: 1670–1678. © 2008 Wiley Periodicals, Inc.     

Uniform tissues engineered by seeding and culturing cells in 3D scaffolds under perfusion at defined oxygen tensions

In this work, we assessed whether culture of uniformly seeded chondrocytes under direct perfusion, which supplies the cells with normoxic oxygen levels, can maintain a uniform distribution of viable cells throughout porous scaffolds several milimeters in thickness, and support the development of uniform tissue grafts. An integrated bioreactor system was first developed to streamline the steps of perfusion cell seeding of porous scaffolds and perfusion culture of the cell-seeded scaffolds. Oxygen tensions in perfused constructs were monitored by in-line oxygen sensors incorporated at the construct inlet and outlet. Adult human articular chondrocytes were perfusion-seeded into 4.5 mm thick foam scaffolds at a rate of 1 mm/s. Cell-seeded foams were then either cultured statically in dishes or further cultured under perfusion at a rate of 100 microm/s for 2 weeks. Following perfusion seeding, viable cells were uniformly distributed throughout the foams. Constructs subsequently cultured statically were highly heterogeneous, with cells and matrix concentrated at the construct periphery. In contrast, constructs cultured under perfusion were highly homogeneous, with uniform distributions of cells and matrix. Oxygen tensions of the perfused medium were maintained near normoxic levels (inlet congruent with 20%, outlet > 15%) at all times of culture. We have demonstrated that perfusion culture of cells seeded uniformly within porous scaffolds, at a flow rate maintaining a homogeneous oxygen supply, supports the development of uniform engineering tissue grafts of clinically relevant thicknesses.     

Perfusion enhanced polydimethylsiloxane based scaffold cell culturing system for multi‐well drug screening platform

Conventional two‐dimensional cultures in monolayer and sandwich configuration have been used as a model for in vitro drug testing. However, these culture configurations do not present the actual in vivo liver cytoarchitecture for the hepatocytes cultures and thus they may compromise the cells liver‐specific functions and their cuboidal morphology over longer term culture. In this study, we present a three‐dimensional polydimethylsiloxane (PDMS) scaffold with interconnected spherical macropores for the culturing of rat liver cells (hepatocytes). The scaffolds were integrated into our perfusion enhanced bioreactor to improve the nutrients and gas supply for cell cultures. The liver‐specific functions of the cell culture were assessed by their albumin and urea production, and the changes in the cell morphology were tracked by immunofluorescence staining over 9 days of culture period. N‐Acetyl‐Para‐Amino‐Phenol (acetaminophen) was used as drug model to investigate the response of cells to drug in our scaffold‐bioreactor system. Our experimental results revealed that the perfusion enhanced PDMS‐based scaffold system provides a more conducive microenvironment with better cell‐to‐cell contacts among the hepatocytes that maintains the culture specific enzymatic functions and their cuboidal morphology during the culturing period. The numerical simulation results further showed improved oxygen distribution within the culturing chamber with the scaffold providing an additional function of shielding the cell cultures from the potentially detrimental fluid induced shear stresses. In conclusion, this study could serve a crucial role as a platform for future preclinical hepatotoxicity testing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:418–428, 2014     

Perfusion affects the tissue developmental patterns of human mesenchymal stem cells in 3D scaffolds

Human mesenchymal stem cells (hMSCs) developed in three‐dimensional (3D) scaffolds are significantly affected by culture conditions. We hypothesized that the hydrodynamic forces generated in perfusion bioreactors significantly affected hMSC functionality in 3D scaffolds by shaping the extracellular matrix (ECM) proteins. In this study, hMSCs were grown in 3D poly(ethylene terephthalate) (PET) scaffolds in static and a parallel perfusion system under similar initial conditions for up to 35 days. Results demonstrated that even at very low media velocities (O [10^?4 cm/sec]), perfusion cultures affected the ability of hMSCs to form an organized ECM network as illustrated by the immunostaining of collagen I and laminin fibrous structure. The change in the ECM microenvironment consequently influenced the nuclear shape. The hMSCs grown at the lower surface of static culture displayed a 15.2 times higher nuclear elongation than those at the upper surface, whereas cells grown in the perfusion bioreactor displayed uniform spherical nuclei on both surfaces. The difference in ECM organization and nuclear morphology associated with gene expression and differentiation characteristics of hMSCs. The cells exhibited lower CFU‐F colony forming ability and decreased expressions of stem‐cell genes of Rex‐1 and Oct‐4, implying a less primitive stem‐cell phenotype was maintained in the perfusion culture relative to the static culture conditions. The significantly higher expression level of osteonectin gene in the perfusion culture at day 28 indicated an upregulation of osteogenic ability of hMSCs. The study highlights the critical role of dynamic culture conditions on 3D hMSC construct development and properties. J. Cell. Physiol. 219: 421–429, 2009. © 2009 Wiley‐Liss, Inc.     

From cells to embryos to rooted plantlets in a mist bioreactor

A mist bioreactor using a disposable bag as culture chamber was used to propagate carrot embryogenic cells into rooted plantlets. The best operating configuration was akin to a vertical hanging garden using 50–90 μm nylon mesh for explant attachment. Cells spray inoculated into the reactor were 51.2 % viable. Misting cycle and aeration conditions were studied and showed that under the same hourly volumetric nutrient feed and 0 VVM, embryo development in the reactor was best using a 0.3 min on/2.7 min off misting cycle, yielding about 23 % post heart stage embryos. Compared to 0 VVM, 3 % CO₂ enrichment improved embryo development in reactor culture. Spray inoculated cells also attached to several vertically hung poly-l-lysine coated strips and then developed in situ into embryos. Cell attachment was significantly improved when they were suspended in salt-free sucrose solution during spray inoculation. Almost 90 % of the originally attached cells remained on the nylon mesh 24 h later after spraying with B5 medium in the mist reactor. Strip grown embryos had the same post heart stage ratio but shorter overall length compared to those developed on a horizontal platform. Young plantlets developed uniformly up and down the hanging strips and did not detach after 3 weeks of culture suggesting this technology may prove useful for improving micropropagation.     

A noninvasive technique for the measurement of the energetic state of free‐suspension mammalian cells

A perfusion small‐scale bioreactor allowing on‐line monitoring of the cell energetic state was developed for free‐suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10‐mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min^?1 for high‐density cell suspensions (10⁸ cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo ³¹P NMR was performed and demonstrated the small‐scale bioreactor platform ability to monitor the cell physiological behavior for 30‐min experiments. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Suspended aggregates as an immobilization mode for high-density perfusion culture of HEK 293 cells in a stirred tank bioreactor

Cells of the human embryonic kidney cell line (HEK 293) grown in repeated suspension and perfusion systems were characterized and described. Cell aggregates that formed immediately after the HEK 293 cells were inoculated in stirred vessels in serum-containing Dulbecco’s modified Eagle’s medium (D-MEM)/F-12 medium. The mean diameter of the cell aggregates reflecting the aggregate size increased with culture time, shifting from 63 to 239 μm after 1 and 8 days of culture in spinner flasks, respectively. No significant differences in cell performance were observed between HEK 293 cell populations grown as suspended aggregates and those grown as anchored monolayers. Replacing the D-MEM/F-12 with CD 293 medium caused the compact spherical cell aggregates to dissociate into single cells and small irregular aggregates without any apparent effect on cell performance. Moreover, the spherical cell aggregates could reform from individual cells and small aggregates when exposed to the serum-containing D-MEM/F-12 dominant medium. Perfusion culture of HEK 293 cells grown as suspended aggregates in a 7.5-l stirred tank bioreactor for 17 days resulted in a maximum viable cell density of 1.2×10⁷ cells ml⁻¹. These results demonstrate the feasibility and proof-of-concept for using aggregates as an immobilization system in large-scale stirred bioreactors because a small-scale culture can be used as easily as the inoculum for larger bioreactors.The first two authors contributed equally to this work.     

Three hours of perfusion culture prior to 28 days of static culture, enhances osteogenesis by human cells in a collagen GAG scaffold

In tissue engineering, bioreactors can be used to aid in the in vitro development of new tissue by providing biochemical and physical regulatory signals to cells and encouraging them to undergo differentiation and/or to produce extracellular matrix prior to in vivo implantation. This study examined the effect of short term flow perfusion bioreactor culture, prior to long‐term static culture, on human osteoblast cell distribution and osteogenesis within a collagen glycosaminoglycan (CG) scaffold for bone tissue engineering. Human fetal osteoblasts (hFOB 1.19) were seeded onto CG scaffolds and pre‐cultured for 6 days. Constructs were then placed into the bioreactor and exposed to 3 × 1 h bouts of steady flow (1 mL/min) separated by 7 h of no flow over a 24‐h period. The constructs were then cultured under static osteogenic conditions for up to 28 days. Results show that the bioreactor and static culture control groups displayed similar cell numbers and metabolic activity. Histologically, however, peripheral cell‐encapsulation was observed in the static controls, whereas, improved migration and homogenous cell distribution was seen in the bioreactor groups. Gene expression analysis showed that all osteogenic markers investigated displayed greater levels of expression in the bioreactor groups compared to static controls. While static groups showed increased mineral deposition; mechanical testing revealed that there was no difference in the compressive modulus between bioreactor and static groups. In conclusion, a flow perfusion bioreactor improved construct homogeneity by preventing peripheral encapsulation whilst also providing an enhanced osteogenic phenotype over static controls. Bioeng. 2011; 108:1203–1210. © 2010 Wiley Periodicals, Inc.     

A multiparallel bioreactor for the cultivation of mammalian cells in a 3D‐ceramic matrix

For adherently growing cells, cultivation is limited by the provided growth surface. Excellent surface‐to‐volume ratios are found in highly porous matrices, which have to face the challenge of nutrient supply inside the matrices' caverns. Therefore, perfusion strategies are recommended which often have to deal with the need of developing an encompassing bioreactor periphery. We present a modular bioreactor system based on a porous ceramic matrix that enables the supply of cells with oxygen and nutrients by perfusion. The present version of the reactor system focuses on simple testing of various inoculation and operation modes. Moreover, it can be used to efficiently test different foam structures. Protocols are given to set‐up the system together with handling procedures for long‐time cultivation of a CHO cell line. Experimental results confirm vital growth of cells inside the matrices' caverns. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Fluid-induced low shear stress improves cartilage like tissue fabrication by encapsulating chondrocytes

In the recent years, there has been considerable development in the regenerative medicine, which aims to repair, regenerate, and improve injured articular cartilage. The aim of the present study was to investigate the effect of flow-induced shear stress in perfusion bioreactor on alginate encapsulating chondrocytes. The shear stress imposed on the cells in the culture chamber of bioreactor was predicted with computational fluid dynamic. Bovine nasal chondrocytes were isolated and expanded to obtain a pellet. The cell pellet was resuspends in alginate solution, transferred to the culture chamber, and dynamically cultured under direct perfusion. At the end of culture, tissue constructs were examined histologically and by immunohistochemistry. The results of computational fluid dynamic modeling revealed that maximum wall shear stress was 4.820 × 10^?3 Pascal. Macroscopic views of the alginate/chondrocyte beads suggested that it possessed constant shape but were flexible. Under inverted microscope, round shape of chondrocyte observed. Cell distribution was homogeneous throughout the scaffold. Tissue construct subjected to shear showed morphological features, which are characteristic for natural cartilage. Immunohistochemistry results revealed immunopositivity for type II collagens in tissue constructs samples. Flow induced shear stress in the perfusion bioreactor and chnondrocyte encapsulation provide environment to support cell growth, and tissue regeneration and improve cartilage like tissue fabrication.     

Periodic harvesting of embryonic stem cells from a hollow‐fiber membrane based four‐compartment bioreactor

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale‐up of stem cell culture is necessary. Bioreactors for dynamic three‐dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow‐fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10⁶ mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10⁶ mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four‐compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:141–151, 2016     

无载体固定化培养模式下HEK293细胞的生长和代谢特征

利用HEK293细胞在悬浮培养体系中下具有聚集成团的体外培养特性,在250ml的spinner flask搅拌式细胞培养瓶中以悬浮细胞团的形式实施HEK293细胞的无载体固定化培养,以细胞密度、细胞活力、细胞团粒径分布和葡萄糖比消耗率 (qglc)、乳酸比产率 (qlac)、乳酸转化率 (Ylac/glc)、氨基酸消耗为观察指标,同时设置静止培养体系作为参照,考察无载体固定化培养模式下的HEK293细胞生长和代谢特征。观察结果表明,HEK293细胞在搅拌式细胞培养瓶中无载体固定化培养和在组织培养瓶中静止贴壁培养表现为基本相同的细胞生长和代谢特征,平均粒径小于300μm的细胞团中的物质传递能够满足HEK293细胞维持正常生长和代谢的基本需要。HEK293细胞的无载体固定化培养便于实施灌注操作、提高生物反应器单位体积的生产效率。     

A study of the Coriolis effect on the fluid flow profile in a centrifugal bioreactor

Increasing demand for tissues, proteins, and antibodies derived from cell culture is necessitating the development and implementation of high cell density bioreactors. A system for studying high density culture is the centrifugal bioreactor (CCBR), which retains cells by increasing settling velocities through system rotation, thereby eliminating diffusional limitations associated with mechanical cell retention devices. This article focuses on the fluid mechanics of the CCBR system by considering Coriolis effects. Such considerations for centrifugal bioprocessing have heretofore been ignored; therefore, a simpler analysis of an empty chamber will be performed. Comparisons are made between numerical simulations and bromophenol blue dye injection experiments. For the non‐rotating bioreactor with an inlet velocity of 4.3 cm/s, both the numerical and experimental results show the formation of a teardrop shaped plume of dye following streamlines through the reactor. However, as the reactor is rotated, the simulation predicts the development of vortices and a flow profile dominated by Coriolis forces resulting in the majority of flow up the leading wall of the reactor as dye initially enters the chamber, results are confirmed by experimental observations. As the reactor continues to fill with dye, the simulation predicts dye movement up both walls while experimental observations show the reactor fills with dye from the exit to the inlet. Differences between the simulation and experimental observations can be explained by excessive diffusion required for simulation convergence, and a slight density difference between dyed and un‐dyed solutions. Implications of the results on practical bioreactor use are also discussed. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Visualizing medium and biodistribution in complex cell culture bioreactors using in vivo imaging

There is a dearth of technology and methods to aid process characterization, control and scale‐up of complex culture platforms that provide niche micro‐environments for some stem cell‐based products. We have demonstrated a novel use of 3d in vivo imaging systems to visualize medium flow and cell distribution within a complex culture platform (hollow fiber bioreactor) to aid characterization of potential spatial heterogeneity and identify potential routes of bioreactor failure or sources of variability. This can then aid process characterization and control of such systems with a view to scale‐up. Two potential sources of variation were observed with multiple bioreactors repeatedly imaged using two different imaging systems: shortcutting of medium between adjacent inlet and outlet ports with the potential to create medium gradients within the bioreactor, and localization of bioluminescent murine 4T1‐luc2 cells upon inoculation with the potential to create variable seeding densities at different points within the cell growth chamber. The ability of the imaging technique to identify these key operational bioreactor characteristics demonstrates an emerging technique in troubleshooting and engineering optimization of bioreactor performance. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:256–260, 2014     

Development and validation of a novel bioreactor system for load- and perfusion-controlled tissue engineering of chondrocyte-constructs

Osteoarthritis is a severe socio-economical disease, for which a suitable treatment modality does not exist. Tissue engineering of cartilage transplants is the most promising method to treat focal cartilage defects. However, current culturing procedures do not yet meet the requirements for clinical implementation. This article presents a novel bioreactor device for the functional tissue engineering of articular cartilage which enables cyclic mechanical loading combined with medium perfusion over long periods of time, under controlled cultivation and stimulation conditions whilst ensuring system sterility. The closed bioreactor consists of a small, perfused, autoclavable, twin chamber culture device with a contactless actuator for mechanical loading. Uni-axial loading is guided by externally applied magnetic fields with real-time feedback-control from a platform load cell and an inductive proximity sensor. This precise measurement allows the development of the mechanical properties of the cultured tissue to be monitored in real-time. This is an essential step towards clinical implementation, as it allows accounting for differences in the culture procedure induced by patient-variability. This article describes, based on standard agarose hydrogels of 3 mm height and 10 mm diameter, the technical concept, implementation, scalability, reproducibility, precision, and the calibration procedures of the whole bioreactor instrument. Particular attention is given to the contactless loading system by which chondrocyte scaffolds can be compressed at defined loading frequencies and magnitudes, whilst maintaining an aseptic cultivation procedure. In a "proof of principle" experiment, chondrocyte seeded agarose gels were cultured for 21 days in the bioreactor system. Intermittent medium perfusion at a steady flow rate (0.5 mL/min) was applied. Sterility and cell viability (ds-DNA quantification and fluorometric live/dead staining) were preserved in the system. Flow induced shear stress stimulated sGAG (sulfated glycosaminoglycan) content (DMMB assay) after 21 days, which was confirmed by histological staining of Alcian blue and by immunostaining of Aggrecan. Experimental data on mechanotransduction and long-term studies on the beneficial effects of combined perfusion and different mechanical loading patterns on chondrocyte seeded scaffolds will be published separately.     

Perfusion seed cultures improve biopharmaceutical fed‐batch production capacity and product quality

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof‐of‐concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high‐seed fed‐batch production cultures. First, we optimized the perfusion N‐1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N‐1 duration, reaching >40 × 10⁶ vc/mL at the end of the perfusion N‐1 stage. The cultures were subsequently split into high‐seed (10 × 10⁶ vc/mL) fed‐batch production cultures. This strategy significantly shortened the culture duration. The high‐seed fed‐batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low‐seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N‐1 and high‐seed fed‐batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low‐seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:616–625, 2014     

Compact Cell Settlers for Perfusion Cultures of Microbial (and Mammalian) Cells

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     

Culturing and differentiation of murine embryonic stem cells in a three-dimensional fibrous matrix

Embryonic stem (ES) cells have indefinite self-renewal ability and pluripotency, and can provide a novel cell source for tissue engineering applications. In this study, a murine CCE ES cell line was used to derive hematopoietic cells in a 3-D fibrous matrix. The 3-D matrix was found to maintain the phenotypes of undifferentiated ES cells as indicated by alkaline phosphatase (ALP) activity and stage specific embryonic antigen-1 (SSEA-1) expression. In hematopoietic differentiation, cells from 3-D culture exhibited similar cell cycle distribution and SSEA-1 expression to those in the initial cell population. The Oct-4 expression was significantly down-regulated, which indicated the occurrence of differentiation, although the level was slightly higher than that in Petri dish culture. The expression of c-kit, cell surface marker for hematopoietic progenitor, was higher in the 3-D culture, suggesting a better-directed hematopoietic differentiation. Cells in the 3-D matrix tended to form large aggregates associated with fibers. For large-scale processes, a perfusion bioreactor can be used for both maintenance and differentiation cultures. As compared to the static culture, a higher growth rate and final cell density were resulted from the perfusion bioreactor due to better control of the reactor environment. At the same time, the differentiation capacity of ES cells was preserved in the perfusion culture. The ES cell culture in the fibrous matrix thus can be used as a 3-D model system to study effects of extracellular environment and associated physico-chemical parameters on ES cell maintenance and differentiation.     

A novel alginate hollow fiber bioreactor process for cellular therapy applications

Gel‐matrix culture environments provide tissue engineering scaffolds and cues that guide cell differentiation. For many cellular therapy applications such as for the production of islet‐like clusters to treat Type 1 diabetes, the need for large‐scale production can be anticipated. The throughput of the commonly used nozzle‐based devices for cell encapsulation is limited by the rate of droplet formation to ～0.5 L/h. This work describes a novel process for larger‐scale batch immobilization of mammalian cells in alginate‐filled hollow fiber bioreactors (AHFBRs). A methodology was developed whereby (1) alginate obstruction of the intra‐capillary space medium flow was negligible, (2) extra‐capillary alginate gelling was complete and (3) 83 ± 4% of the cells seeded and immobilized were recovered from the bioreactor. Chinese hamster ovary (CHO) cells were used as a model aggregate‐forming cell line that grew from mostly single cells to pancreatic islet‐sized spheroids in 8 days of AHFBR culture. CHO cell growth and metabolic rates in the AHFBR were comparable to small‐scale alginate slab controls. Then, the process was applied successfully to the culture of primary neonatal pancreatic porcine cells, without significant differences in cell viability compared with slab controls. As expected, alginate‐immobilized culture in the AHFBR increased the insulin content of these cells compared with suspension culture. The AHFBR process could be refined by adding matrix components or adapted to other reversible gels and cell types, providing a practical means for gel‐matrix assisted cultures for cellular therapy. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009