Microcarrier Cell Culture Research Articles

Characterization of the dynamic viscosity of cell cultures and its effect on mixing performance in a spinner flask bioreactor

Computational fluid dynamics (CFD) models have been developed to simulate cell culturing bioreactors but assume water-like viscosity properties due to significant data gaps. This study characterized the dynamic viscosity of HEK-293 cell cultures and evaluated its effect on mixing performance in a spinner flask bioreactor. Viscosity measurements indicated that the cell culture media, media with microcarriers, and cell cultures presented shear thinning behaviors within the measured shear rate range of 1–100 s−1. The viscosity also increased with the microcarrier concentrations and growth of cell culture. The CFD model, incorporating dynamic viscosity data, showed that shear stress and Kolmogorov length profiles are significantly influenced by microcarrier concentrations and cell culture growth. Higher microcarrier concentrations led to higher average shear stress and Kolmogorov values. The cultured HEK-293 cells after seven days of growth also had higher average shear stress and Kolmogorov values than at the day of seeding, indicating an impact caused by the cells’ metabolism and biomass. Overall, the results indicated that assuming water-like properties underestimates shear stress and Kolmogorov length scales, especially at zones of lower shear rates due to the observed shear thinning behavior. Thus, careful monitoring of dynamic viscosity of cell cultures and proper control of mixing parameters are critical to deliver the desired mixing conditions for optimized cell growth especially during scale-up production operations.

Glutathione affinity chromatography for the scalable purification of an oncolytic virus immunotherapy from microcarrier cell culture.

Therapeutic viral vectors are an emerging technology with several clinical applications in gene therapy, vaccines, and immunotherapy. Increased demand has required the redevelopment of conventional, low-throughput cell culture and purification manufacturing methods such as static cell stacks and ultracentrifugation. In this work, scalable methods were investigated for the manufacture of an oncolytic virus immunotherapy application consisting of a prototype strain of coxsackievirus A21 (CVA21) produced in adherent MRC-5 cells. Cell culture was established in stirred-tank microcarrier bioreactors, and an efficient affinity chromatography method was developed for the purification of harvested CVA21 through binding of the viral capsids to an immobilized glutathione (GSH) ligand. Bioreactor temperature during infection was investigated to maximize titer, and a decrease in temperature from 37°C to 34°C yielded a two-three-fold increase in infectivity. After purification of the 34°C harvests, the GSH affinity chromatography elution not only maintained a >two-fold increase in infectivity and viral genomes but also increased the proportion of empty capsids compared to 37°C harvests. Using material generated from both infection temperature setpoints, chromatographic parameters and mobile phase compositions were studied at the laboratory scale to maximize infectious particle yields and cell culture impurity clearance. Empty capsids that co-eluted with full capsids from 34°C infection temperature harvests were poorly resolved across the conditions tested, but subsequent polishing anion exchange and cation exchange chromatography steps were developed to clear residual empty capsids and other impurities. Oncolytic CVA21 production was scaled-up 75-fold from the laboratory scale and demonstrated across seven batches in 250L single-use microcarrier bioreactors and purified with customized, prepacked, single-use 1.5L GSH affinity chromatography columns. The large-scale bioreactors controlled at 34°C during infection maintained a three-fold increase in productivity in the GSH elution, and excellent clearance of host cell and media impurities was observed across all batches. This study presents a robust method for the manufacture of an oncolytic virus immunotherapy application that may be implemented for the scalable production of other viruses and viral vectors which interact with glutathione.

Recent advances in the use of microcarriers for cell cultures and their ex vivo and in vivo applications.

Microcarriers are 100- to 300-micron support matrices that permit the growth of adherent cells in bioreactor systems. They have a larger surface area to volume ratio in comparison to single cell monolayers, enabling cost-effective cell production and expansion. Microcarriers are composed of a solid matrix that must be separated from expanded cells during downstream processing stages. The detachment method is chosen on the basis of several factors like cell type, microcarrier surface chemistry, cell confluency and degree of aggregation. The development of microcarriers with a range of physiochemical properties permit controlled cell and protein associations that hold utility for novel therapeutics. In this review, we provide an overview of the recent advances in microcarrier cell culture technology. We also discuss its significance as an ex vivo research tool and the therapeutic potential of newly designed microcarrier systems in vivo.

Developing efficient bioreactor microcarrier cell culture system for large scale production of mesenchymal stem cells (MSCs)

Background & Aim A large-scale production of clinical-grade MSCs and their derived products is essential due to their immense therapeutic potential. Even though 3D bioreactors are cost efficient in scale up, the plastic adherence of MSCs, makes expansion in suspension cultures challenging. A variety of microcarriers (MCs) allow plastic adherent cells to grow on their surface while maintaining cells in suspension within a bioreactor for expansion. However, this leads to loss of cells, particularly during separation of cells from the carriers. To overcome this, we identified “dissolvable microcarriers” such as Corning Synthemax II dissolvable MCs, which removes the filtration step. Our aim was to develop an efficient bioreactor microcarrier cell culture system to improve the yield of MSCs. Methods, Results & Conclusion Cells from primary human Adipose derived cell line at passage 4 were seeded into a T175 flask (media: aMEM + 10% Human platelet lysate). The cells were expanded for 3 days, harvested by TryplE dissociation and seeded onto a Corning Dissolvable MCs and the control solid core MCs at 8000 cells/cm2, in two separate PBS mini 0.5L MagDrive bioreactors containing 350ml of pre-warmed media. Bioreactors were operated at 16 rpm for the first 12 hours, and 22 rpm for the remainder, in an incubator (37C, 5%CO2). Cells were harvested at 7 days, dissociated from the control MCs using TryplE (15-20 min) followed by passing through a 100um filter, to separate the cells from MCs. The Corning dissolvable MCs were digested with a manufacturer recommended Pectinase/EDTA/TryplE solution, without filtration. Cells from both cultures were washed in DPBS and analysed. A total of 3 replicate runs were performed. The post-harvest cell count (median± range) for dissolvable MCs was 104.6±39.6 million cells and for the control MCs was 76.04±6.28 million cells. The culture supernatant was assayed for pH and osmolality and were found to be similar for both MCs. Analysis of vascular endothelial growth factor(VEGF), showed that dissolvable MCs had a lower level of secreted VEGF (5690±476.4 pg/ml/million cells) compared to control MCs (10103±5384 pg/ml/million cells). Similar trend was noted for macrophage chemoattractant protein-1; 6302±2094 pg/ml/million cells in dissolvable MCs vs 9047±2621 pg/ml/million cells in control MCs. These results indicate that dissolvable MCs could lead to a higher yield from bioreactors (36% increase). However, it is important to further characterise cells and assess their therapeutic potential. A large-scale production of clinical-grade MSCs and their derived products is essential due to their immense therapeutic potential. Even though 3D bioreactors are cost efficient in scale up, the plastic adherence of MSCs, makes expansion in suspension cultures challenging. A variety of microcarriers (MCs) allow plastic adherent cells to grow on their surface while maintaining cells in suspension within a bioreactor for expansion. However, this leads to loss of cells, particularly during separation of cells from the carriers. To overcome this, we identified “dissolvable microcarriers” such as Corning Synthemax II dissolvable MCs, which removes the filtration step. Our aim was to develop an efficient bioreactor microcarrier cell culture system to improve the yield of MSCs. Cells from primary human Adipose derived cell line at passage 4 were seeded into a T175 flask (media: aMEM + 10% Human platelet lysate). The cells were expanded for 3 days, harvested by TryplE dissociation and seeded onto a Corning Dissolvable MCs and the control solid core MCs at 8000 cells/cm2, in two separate PBS mini 0.5L MagDrive bioreactors containing 350ml of pre-warmed media. Bioreactors were operated at 16 rpm for the first 12 hours, and 22 rpm for the remainder, in an incubator (37C, 5%CO2). Cells were harvested at 7 days, dissociated from the control MCs using TryplE (15-20 min) followed by passing through a 100um filter, to separate the cells from MCs. The Corning dissolvable MCs were digested with a manufacturer recommended Pectinase/EDTA/TryplE solution, without filtration. Cells from both cultures were washed in DPBS and analysed. A total of 3 replicate runs were performed.

Microcarrier culture enhances osteogenic potential of human periodontal ligament stromal cells

Regeneration of periodontal tissue represents a major challenge to modern tissue engineering, since cell-based therapies require large amounts of periodontal ligament stromal cells (PLSC), which can be obtained only by in vitro expansion. Ideally, the period of the in vitro expansion should be optimized for the generation of large enough numbers of pre-specified progenitor cells ready to contribute to the restoration of periodontal tissues.In the present study, we used a commercially available, three-dimensional culturing platform and alginate microcarrier cell culture system for the propagation of human PLSCs, which were derived using the explant outgrowth method. Induction of osteogenic differentiation resulted in rapid and robust mineralization of the extracellular matrix in PLSCs grown on microcarriers, but not in PLSCs grown under standard culture conditions. Gene expression studies revealed upregulation of osteogenesis-related genes, BMP2, ALP, RUNX2, MSX2, cementum protein 23, bone sialoprotein, osteopontin and periostin, in undifferentiated and differentiating microcarrier cultures of PLSCs. In addition, the microcarrier culture enhanced the expression of β-catenin, intermediate filament protein vimentin and focal adhesion proteins vinculin and paxillin.Our study shows that microcarrier culture allows rapid generation of large numbers of PLSCs pre-specified towards an osteogenic-like phenotype. This method may be useful for the development of new tissue engineering protocols for the reconstruction of periodontal tissues.

High quality in vitro expansion of human endothelial progenitor cells of human umbilical vein origin.

The limited availability of qualified endothelial progenitor cells (EPCs) is a major challenge for regenerative medicine. In the present study, we isolated human EPCs from human umbilical vein endothelial cells (HUVECs) by using magnetic micro-beads coated with an antibody against human CD34. Flow cytometric assay showed that majority of these cells expressed VEGFR2 (KDR), CD34 and CD133, three molecular markers for early EPCs. It was also found that a bioreactor micro-carrier cell culture system (bio-MCCS) was superior to dish culture for in vitro expansion of EPCs. It expanded more EPCs which were in the early stage, as shown by the expression of characteristic molecular markers and had better angiogenic potential, as shown by matrix-gel based in vitro angiogenesis assay. These results suggest that HUVECs might be a novel promising resource of EPCs for regenerative medicine and that a bio-MCCS cell culture system might be broadly used for in vitro expansion of EPCs.

An ovarian cell microcapsule system simulating follicle structure for providing endogenous female hormones

The aim of this study was to create a microcapsule system simulating native follicle structure by introducing microcarrier culture to microencapsulation for providing endogenous female hormones. Granulosa and theca cells of rat follicles were isolated. Granulosa cells were grown on microcarriers and enclosed together with theca cells in alginate-chitosan-alginate microcapsules. The cell viability and female hormone secretion were investigated in vitro. The microcapsules were transplanted to ovariectomized rats and the serum levels of estradiol and progesterone were measured for 60 days. The microencapsulated granulosa cells growing on microcarriers exhibited enhanced viability and promoted secreting ability of estradiol and progesterone compared with those without the microcarriers. Co-microencapsulation of granulosa cells and theca cells markedly elevated estradiol secretion in vitro. Transplantation of co-microencapsulated granulosa cells on microcarriers and theca cells maintained serum estradiol and progesterone at normal levels for 60 days. Microcarrier cell culture has been proved to be an effective method to enhance the viability of granulosa cells in microcapsules. Moreover, the transplantation of microcapsules enclosing granulosa cells on microcarriers and theca cells may be promising to provide endogenous female hormones for menopausal syndrome treatment.

Assessment of long-term effects of nanoparticles in a microcarrier cell culture system.

Nano-sized materials could find multiple applications in medical diagnosis and therapy. One main concern is that engineered nanoparticles, similar to combustion-derived nanoparticles, may cause adverse effects on human health by accumulation of entire particles or their degradation products. Chronic cytotoxicity must therefore be evaluated. In order to perform chronic cytotoxicity testing of plain polystyrene nanoparticles on the endothelial cell line EAhy 926, we established a microcarrier cell culture system for anchorage-dependent cells (BioLevitatorTM). Cells were cultured for four weeks and exposed to doses, which were not cytotoxic upon 24 hours of exposure. For comparison, these particles were also studied in regularly sub-cultured cells, a method that has traditionally been used to assess chronic cellular effects. Culturing on basal membrane coated microcarriers produced very high cell densities. Fluorescent particles were mainly localized in the lysosomes of the exposed cells. After four weeks of exposure, the number of cells exposed to 20 nm polystyrene particles decreased by 60% as compared to untreated controls. When tested in sub-cultured cells, the same particles decreased cell numbers to 80% of the untreated controls. Dose-dependent decreases in cell numbers were also noted after exposure of microcarrier cultured cells to 50 nm short multi-walled carbon nanotubes. Our findings support that necrosis, but not apoptosis, contributed to cell death of the exposed cells in the microcarrier culture system. In conclusion, the established microcarrier model appears to be more sensitive for the identification of cellular effects upon prolonged and repeated exposure to nanoparticles than traditional sub-culturing.

Spontaneous osteogenesis of MSCs cultured on 3D microcarriers through alteration of cytoskeletal tension

3-dimensional microcarrier (3D-MC) cell culture systems are often used for expansion of stem cells including mesenchymal stem cells (MSCs) for high cell volumes required in clinical applications. However, compared to 2-dimensional (2D) cell culture, effects of 3D-MC systems on MSC differentiation have not been well studied. In this study, the behavior of various sources of MSCs from two species was observed and compared on 3D collagen I-coated-MCs (COL-MC) versus 2D culture. Proliferation of all MSCs cultured on 3D COL-MC was much decreased compared to 2D culture. Unexpectedly, COL-MC-cultured MSCs underwent spontaneous osteogenesis without exogenous addition of biochemical factors, as evidenced by increased osteogenic genes expression, ALP activity, calcium deposition, and collagen I secretion. Furthermore, MSCs cultured on 3D-MC alone without collagen I coating is sufficient to induce osteogenesis. The spontaneous lineage commitment induced by 3D-MC culture was mediated by increased cytoskeletal tension and actomyosin contraction of MSCs, which could be prevented by latrunculin B and blebbistatin, inhibitors of cytoskeletal tension and actomyosin contraction respectively. Our findings show that the combination of bioengineered MC and MSCs alone can induce specific lineage commitment very efficiently. These data have strong implications in simplifying tissue engineering strategies for therapeutic applications.

Production of Inactivated Influenza H5N1 Vaccines from MDCK Cells in Serum-Free Medium

BackgroundHighly pathogenic influenza viruses pose a constant threat which could lead to a global pandemic. Vaccination remains the principal measure to reduce morbidity and mortality from such pandemics. The availability and surging demand for pandemic vaccines needs to be addressed in the preparedness plans. This study presents an improved high-yield manufacturing process for the inactivated influenza H5N1 vaccines using Madin-Darby canine kidney (MDCK) cells grown in a serum-free (SF) medium microcarrier cell culture system.Principal FindingThe current study has evaluated the performance of cell adaptation switched from serum-containing (SC) medium to several commercial SF media. The selected SF medium was further evaluated in various bioreactor culture systems for process scale-up evaluation. No significant difference was found in the cell growth in different sizes of bioreactors studied. In the 7.5 L bioreactor runs, the cell concentration reached to 2.3×106 cells/mL after 5 days. The maximum virus titers of 1024 Hemagglutinin (HA) units/50 µL and 7.1±0.3×108 pfu/mL were obtained after 3 days infection. The concentration of HA antigen as determined by SRID was found to be 14.1 µg/mL which was higher than those obtained from the SC medium. A mouse immunogenicity study showed that the formalin-inactivated purified SF vaccine candidate formulated with alum adjuvant could induce protective level of virus neutralization titers similar to those obtained from the SC medium. In addition, the H5N1 viruses produced from either SC or SF media showed the same antigenic reactivity with the NIBRG14 standard antisera.ConclusionsThe advantages of this SF cell-based manufacturing process could reduce the animal serum contamination, the cost and lot-to-lot variation of SC medium production. This study provides useful information to manufacturers that are planning to use SF medium for cell-based influenza vaccine production.

High‐density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin-Darby canine kidney cells in high-density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed-batch modes were applied using cell-specific feed rates. Cell densities up to 1.1 × 10(7) cells/mL were achieved. Cell-specific virus yields in high-density cultures were at similar levels compared with standard, low-density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8-National Institute for Biological Standards and Control (NIBSC) and PR8-Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8-NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.

A comparative study of cell growth and adenovirus production using suspension and stationary cell culture

The growing demand of adenoviral vectors for gene therapy, especially for cancer treatment, creates the need for large scale production of these vectors [2, 4]. In order to do this, characterization and optimization of culture conditions for HEK293 cells is essential to improve productivity. This study has two main objectives: characterizing growth and adenovirus productivity of HEK293 cells in three different culture systems: stationary, microcarriers and suspension; and determining media composition that is best suited for cell growth and adenovirus production under low serum conditions in terms of glucose and glutamine concentrations. Culture parameters were determined for HEK293 cells growing attached in basal media supplemented with 10% FBS. HEK293 cells were gradually adapted to grow in suspension in the absence of serum in supplemented media. We compared cell growth in stationary culture with 10% serum, Cytodex 3 with 10% serum and suspension culture without serum. The infectivity of adenovirus and final viral titer were also assessed in all systems. We applied metabolic flux analysis to all culture systems pre and post infection, considering the main carbon metabolism pathways, a biomass production term based on the reported stoichiometric composition of mammalian cells and a virus production equation based on protein and nucleic material content of the virus [1, 3]. This allows us to characterize and compare the metabolic state of cells and resource distribution during cell growth and virus production. Based on metabolic demand, media composition that was best suited for cell growth and adenovirus production in terms of glucose and glutamine concentrations was determined. In microcarriers we obtained a higher cell density than in stationary and suspension cultures. However, a different metabolic flux distribution, lower specific lactate production and a lower lactate produced to glucose consumed ratio was observed in suspension cultures. Results obtained can provide further insight into the metabolism of HEK293 cells during growth and virus production stages and evaluate the use of suspension and microcarrier cell culture technology for adenoviral vector production.

Scalable culture systems using different cell lines for the production of Peste des Petits ruminants vaccine

Peste des Petits ruminants (PPR) is considered as one of the major constraints to the productivity of small ruminants in Africa and Asian countries. Currently PPR control is done by vaccination with an attenuated PPR strain (Nigeria 75/1) produced in monolayers of Vero cells grown in roller bottles or static flasks. This work focuses on the production of a PPR vaccine strain using stirred conditions as an advanced option for process scale-up. Non-porous microcarriers (Cytodex-1 ®) were used to support Vero cell growth in suspension cultures. The use of Ex-Cell™ medium could improve cell specific productivities obtained with standard serum containing medium, independently of the type of system used, i.e. static as well as suspension stirred cultures. As an alternative, several cell lines adapted to grow as single cells in suspension (CHO-K1, BHK-21A and 293) and another anchorage-dependent (MRC-5) were evaluated in their capacity to produce a PPR vaccine. BHK-21A and 293 cells grown as single-cell suspension in serum free medium were both suited to produce PPR vaccine with productivities similar to Vero cells, namely 10 6 TCID 50/mL. However, for the 293 cells, these results were only obtained 2–3 days later. CHO-K1 and MRC-5 cells have shown not to be suitable to adequately produce this virus. These results provide further insights into the feasibility of applying microcarrier cell culture technology to produce PPR vaccine in Vero cells as well as in the alternative use of single-cell suspension cultures of BHK-21A, significantly simplifying the existing production process.

Development and optimisation of a procedure for the production of Parapoxvirus ovis by large-scale microcarrier cell culture in a non-animal, non-human and non-plant-derived medium

For the production of a chemically inactivated Parapoxvirus ovis (PPVO), an adherent bovine kidney cell line was cultivated on Cytodex-3 microcarriers in suspension culture. The inactivated and purified virus particles have shown immune modulatory activity in several animal models. PPVO was produced by a biphasic batch process at the 3.5 and 10 L scale. Aeration was realised by bubble-free membrane oxygenation via a tube stator with a central two-blade anchor impeller. In order to increase efficiency, process robustness and safety, the established process was optimised. The cell line was adapted to a protein-free medium (except recombinant insulin) in order to increase biosafety. A scale up to a 50 L pilot plant with direct cell expansion was performed successfully. In parallel, the biphasic batch process was optimised with special emphasis on different operating conditions (cell number, Multiplicity of Infection (MOI), etc.) and process management (fed-batch, dialysis, etc.). The quality and concentration of the purified virus particles was assessed by quantitative electron microscopy, residual host cell protein and DNA-content and, finally, biologic activity in a transgenic mouse model. This integrated approach led to a new, safe, robust and highly productive large-scale production process, called "Volume-Expanded-Fed" Batch with cell densities up to 6-7e06 cells/mL. By subsequent dilution of infected cells into the next process scale, an increase in total productivity by a factor of 40 (related to an established biphasic batch process) was achieved.

A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR

The development of a rapid and sensitive assay for detection of replication-competent adenoviruses (RCAs) is described. This RCA assay consists of an incubation step of 4 days of adenoviral vectors on A549 cells in a microcarrier cell culture system followed by detection of amplified RCAs by E1-specific quantitative PCR. The detection limit of this assay is 3 RCAs in 1 × 10 10 vector particles per 70 ml of microcarrier cell culture. The main advantage of the combination of cell culture and PCR detection is that replicated virus can be detected long before cytopathic effects become visible and therefore, it is much faster than conventional cell culture-based assays. This assay was validated by spiking replication-incompetent adenoviral vectors with wild-type adenovirus serotype 5 (wt Ad5) as a positive control for RCA. It was found that the replication of wt Ad5 is hampered above a vector particle per cell ratio of 50. However, if microcarrier beads are used, many cells can be grown in a small suspension culture and consequently a large number of vector particles can be tested for contamination with RCA.

A microcarrier cell culture process for propagating rabies virus in Vero cells grown in a stirred bioreactor under fully animal component free conditions

Rabies virus strain production in Vero cells grown on Cytodex 1 in a 2 L stirred tank bioreactor and in a medium free of components of human or animal origin (VP-SFM) is described. Cell banking procedure in VP-SFM supplemented with an animal components free mixture (10%DMSO + 0.1%methylcellulose) was reported and cell growth after revitalization was assessed. Vero cells exhibited growth performances (specific growth rate and cell division number) similar to that obtained in serum containing medium. To design a scalable process that is totally free of animal-derived substances, two proteases: TrypLE Select and Accutase, were assessed as an alternative to trypsin which is routinely used for cell passage. Growth performance of Vero cells grown in VP-SFM and MEM + 10% fetal calf serum (FCS) over four passages and subcultivated with either TrypLE Select or Accutase was evaluated. TrypLE Select showed the best performance in terms of specific growth rate and cell division number. Kinetics of cell growth and rabies virus production (LP2061/Vero strain) were investigated in spinner flask and in a 2 L bioreactor. In spinner flask, a maximal cell density level of 1.85 × 10 6 cells/mL was achieved when the cells were grown in VP-SFM on 2 g/L Cytodex 1. Cell infection experiments conducted at an MOI of 0.3 and without the medium exchange step, typically needed for serum containing rabies virus production, resulted in a maximal virus titer equal to 2 × 10 7 (Fluorescent Focus Unit) FFU/mL. In stirred tank bioreactor, Vero cell growth in VP-SFM on 3 g/L Cytodex 1 was shown to be sensitive to the aeration mode. Sparging the culture was detrimental for cell growth, whereas cell density level was greatly enhanced when only headspace aeration was used. A cell density level of 2.6 × 10 6 cells/mL was obtained when the cells were grown on 3 g/L Cytodex 1 and in batch culture mode. Cell infection at an MOI of 0.1 without any medium exchange, yielded a maximal rabies virus titer of 2.4 × 10 7 FFU/mL. Furthermore, Vero cell growth in a 2 L bioreactor using recirculation culture mode during cell proliferation step and perfusion for virus multiplication phase was investigated. In comparison to batch culture, a higher cell density level that was equal to 5 × 10 6 cells/mL was reached. Cell infection under conditions similar to batch culture, resulted in a maximal virus titer equal to 1.38 × 10 8 FFU/mL. The potency of the pooled inactivated virus harvests showed an activity of 2.58 IU/mL which was comparable to that obtained in serum supplemented medium.

High immunogenic enterovirus 71 strain and its production using serum-free microcarrier Vero cell culture

Developing an effective vaccine against enterovirus 71 (EV71) infection provides the best means to control the disease. We have previously reported that large-scale preparation of a low immunogenic EV71 strain can be achieved using serum free microcarrier Vero cell culture in a 2-l bioreactor [Wu SC, Liu CC, Lian WC. Optimization of microcarrier cell culture process for the inactivated enterovirus type 71 vaccine development. Vaccine 2004;22:3858–64]. This present work further investigated the virus growth and the immunogenicity of two high immunogenic strains (EV71-075 and EV71-117) prepared in serum-free microcarrier cell cultures. Our results showed that serum free culture increased cell death rate after infection, reduced the virus specific productivity, but resulted in elicitation of higher neutralizing titers in immunized mice as compared to that parallel obtained in serum-containing cultures. Therefore, serum-free microcarrier culture is a valuable process for developing inactivated EV71 vaccines.

A Novel Bioreactor Microcarrier Cell Culture System for High Yields of Proliferating Autologous Human Keratinocytes

Rapid and efficient resurfacing of various skin defects by autologous keratinocyte transplantation is significant in skin wound healing. We developed a novel bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous human keratinocytes on a large scale. In this Bio-MCCS we used porcine gelatin microbeads as microcarriers for autolgous keratinocytes and spinning bottles as fermentation tanks. First, the microbeads were modified by culturing them with autologous dermal fibroblasts that were subsequently killed when they proliferated to confluence on the microbeads. We then performed the Bio-MCCS by expanding ketatinocytes on the microbeads in spinning bottles at 37 degrees C, 5% CO2. Our results showed that keratinocytes rapidly attached to and actively proliferated on the modified microbeads in the Bio-MCCS, achieving high cell densities on the modified microbeads (MTT assay and PI staining). Keratinocytes cultured on the modified microbeads in the Bio-MCCS remained proliferating potentials as shown by positive PCNA staining and BrdU labeling. In contrast, keratinocytes cultured on nonmodified microbeads in the Bio-MCCS proliferated slowly, rapidly ceased to proliferate, and finally dislodged from the microbeads. When removed from the Bio-MCCS and cultured under static conditions, keratinocytes were able to leave the modified microbeads and formed a multilayered epidermal equivalent on the culture surfaces. While stored at room temperature, keratinocytes remained at higher viabilities on the modified microbeads when compared to those on nonmodified microbeads. The achievement of high yields of proliferating autologous keratinocytes by this Bio-MCCS offers a practical potential of resurfacing various skin defects by direct administration of autologous keratinocyte microbeads on various skin defects.

High Yields of Autologous Living Dermal Equivalents Using Porcine Gelatin Microbeads as Microcarriers for Autologous Fibroblasts

Permanent skin replacement requires a dermal component to ensure adequate long-term graft stability and to prevent wound contraction. This study was to construct a bioreactor microcarrier cell culture system (Bio-MCCS) to produce autologous living dermal equivalents on a large scale. Autologous fibroblasts were isolated from split-thickness skin biopsy from a leg ulcer patient, inoculated onto macroporous porcine gelatin microbeads, and incubated in a bioreactor (Cellspin) in serum-free fibroblast growth medium or in DMEM medium containing 10% fetal calf serum (FCS). Fibroblasts rapidly adhered to and actively proliferated on the microbeads in the bioreactor in both serum-free and serum-containing medium. MTT assay showed the number of fibroblasts on the microbeads reached up to 5.3- or 4.0-fold the cells seeded in DMEM medium containing 10% FCS or serum-free medium, respectively. When removed from Bio-MCCS and cultured under static conditions, fibroblasts were able to leave the microbeads and proliferate to confluence on the bottom of tissue culture flasks. When stored at room temperature in DMEM containing 10% FBS, fibroblast cultured on the microbeads retained highest viabilities for at least 3 weeks, up to 82% of originals. This Bio-MCCS using porcine gelatin microbeads as carriers for fibroblasts offers a new option of mass production of autologous living dermal equivalents.

Propagation kinetics of retrovirus transgene vector and replication-competent retrovirus in static and microcarrier cell culture systems using different medium exchange strategies

While retrovirus vector (RV) is the main virus vector used in human gene therapy trials, the biosafety issues that surround currently used RVs have become a matter of concern. Similar to the insertional mutagenesis in the therapeutic target cells, the generation of replication-competent retrovirus (RCR) must be minimized during the manufacture of the virus vector. This work investigated the kinetics of RV and RCR production in PA317-RCM1 producer cells in static and microcarrier cell culture systems. RCR in the progeny of transduced Mus dunni cells was detected by the PCR method and the titer of RCR was quantified by cell-based S+/L− assay. The specific rates of RCR production in microcarrier cultures were 271–462% higher than those in the static well-plate cultures. Increased medium exchange operations yielded higher specific rates of RCR production in both static and microcarrier cultures. The optimal medium exchange strategy was on an every 2-day schedule, yielding the highest RV/RCR ratio in static culture but not microcarrier culture. Results of this study presented the difficulty for gene therapy processes that together with the product RV also unwanted RCR produced in two different cell culture systems.