Microcarrier Culture System Research Articles

Microcarrier-based MDCK cell culture system for the production of influenza H5N1 vaccines

Current egg-based influenza vaccine production technology, which is labor intensive and slow, would not be able to meet demand during an influenza pandemic. Thus, interest in the emerging technology of using mammalian cells for vaccine production has been great. In this study, Madin–Darby canine kidney (MDCK) cells using microcarrier culture systems were established to produce inactivated whole-virus H5N1 vaccine. The current clade-1 influenza H5N1 vaccine virus (NIBRG-14) was provided by the UK National Institute for Biological Standards and Control. Various process parameters were first optimized in 100-mL scale spinner flasks then scaled up to a 1-L scale bioreactor system. In the 1-L scale bioreactor system, peak virus titer could reach 108–9TCID50/mL using serum-containing medium. After purification and inactivation, hemagglutinin (HA) protein content reached 31.56–43.96μg/mL in two different runs. In mice immunogenicity studies, two doses of the purified vaccine antigen adjuvanted with aluminum phosphate induced good immune responses in 0.2 and 1.0μg HA dosages (geometric mean titers of hemagglutination-inhibition antibody: 113 and 242, respectively). This study demonstrates the feasibility of the development of MDCK cell-based inactivated influenza H5 vaccines in microcarrier culture systems and could be valuable to many countries that are planning to establish manufacturing capacity for influenza vaccines.

Large scale isolation of non-uniform shear stress-responsive genes from cultured human endothelial cells through the preparation of a subtracted cDNA library

To investigate the molecular mechanisms responsible for the regional selectivity of early atherogenesis, we have applied a non-uniform shear stress to cultured human umbilical vein endothelial cells (HUVEC). We used a microcarrier culture system and a combination of subtraction and reverse-subtraction methods to isolate a number of genes upregulated by shear stress. The resultant subtracted library includes several known genes (e.g. MCP-1, TM) whose responsiveness to shear stress has been previously reported, indicating that the library is enriched for genes upregulated by shear stress. Also included are atherosclerosis-related genes (e.g. CTGF, IL-8) whose responsiveness to shear stress had not been demonstrated, other known genes whose relationship to atherosclerosis had not been reported, and novel genes. Some responsive to centrifugal force and shear stress ( RECS) genes are also upregulated following stimulation by steady laminar shear stress in a parallel plate chamber. Interestingly, the library includes ET-1 and PAI, which are well known atherogenic factors that are downregulated by laminar shear stress. This implies that turbulent shear stress has effects on HUVEC that are different from those elicited by laminar shear stress. Importantly, analysis of specimens taken from human aorta showed that several RECS genes are transcriptionally upregulated in atherosclerotic lesions, suggesting that the subtracted library includes novel therapeutic targets for the treatment of atherosclerosis.

Radio-frequency plasma treatment improves the growth and attachment of endothelial cells on poly(methyl methacrylate) substrates: implications in tissue engineering

The aim of this study was to improve the biocompatibility of poly(methyl methacrylate) (PMMA) substrates for possible applications in corneal prostheses or in micro-carrier cell culture systems. PMMA substrates were exposed to radio-frequency (RF) argon and nitrogen plasmas for 5 and 10 min each. The PMMA films were examined by Fourier transform infrared (FT-IR) spectroscopy, to characterize the surface changes after plasma exposure. Plasma treatment in general was found to decrease the water contact angle of PMMA, thus increasing its hydrophilicity. There was also an associated increase in the work of adhesion of plasma-treated PMMA substrates. PMMA substrates exhibited differential properties towards endothelial cell (CPA-47) growth. The untreated PMMA surface did not support endothelial growth, compared with both polystyrene (TCPS) and plasma-treated PMMA, while plasma (PL):PMMA exhibited growth rates slightly lower than the TCPS control, as assessed by [3H]thymidine incorporation profiles. The compatibility and growth supportive properties of PL: PMMA were further confirmed by an MTT assay, which showed preserved cellular viability and mitochondrial activity of the cells. Confocal microscopic visualization of cells with fluorescence-labeled vimentin showed normal organization of the cytoskeletal fibers, indicating appropriate attachment to the substrate. Cells growing on PL:PMMA maintained their functionality, as seen from Factor VIII expression. Taken collectively, the findings of this study point out the suitability of RF plasma treatment in inducing desirable changes in PMMA substrates, so as to improve their ability to support the growth and attachment of endothelial cells.

Attachment characteristics of normal human cells and virus-infected cells on microcarriers.

The attachment kinetics of normal and virus-infected LuMA cells were studied to improve the production of live attenuated varicella viruses in human embryonic lung (LuMA) cells. Normal LuMA cells and LuMA cells infected by varicella virus at various cytopathic effects (CPE) were grown on microcarriers. Ninety-three percent of suspended LuMA cells attached to the solid surface microcarriers within fifteen minutes and cell viability was greater than 95% when the cell suspension was stirred. Low serum levels did not affect the attachment rate of virus-infected cells in the microcarrier culture system. Kinetic studies showed that varicella infected cells had a lower attachment rate than normal LuMA cells. Virus inoculum (= infected cells) at low CPE showed a relatively better attachment rate on cell-laden microcarriers than virus inoculum at a higher CPE. Maximum titers were obtained at 2 days post-infection. Based on cell densities, the use of viral inoculum showing a 40% CPE led to an approximately 2- and 1.2-fold increase in the cell associated and in cell free viruses, respectively, than a virus inoculum with a CPE of 10%.However, the ratio of cell-free to cell-associated virus in a microcarrier culture was very low, approximately0.04-0.06. These studies demonstrate that the virus inoculum resulting in a high CPE yielded a high production of cell-associated and cell-free virus in microcarrier cultures because of the high cellular affinity of the varicella virus.

A novel scale-up method for mammalian cell culture in packed-bed bioreactor

A novel method for the scale-up culture of Chinese hamster ovary (CHO) cells in a packed-bed bioreactor is developed wherein microcarriers, attached with CHO cells in a microcarrier culture system, are inoculated directly into the packed-bed bioreactor. Cells continue to grow after inoculation and the maximum cell density reaches about 2×107 cells ml−1. The method provides a new technique for the scale-up of a packed-bed culture while decreasing the labour cost and ensuring the safety of operation.

Development of high density mammalian cell culture system for the production of tissue-type plasminogen activator

A high cell density culture system for the anchorage dependent CHO cells was developed based on the combination ofin situ removal of ammonium ion and microcarrier culture system, and semi-fed-batch feeding of glucose and glutamine was employed to the developed culture system. The glass bead was selected as an optimum microcarrier in terms of cell growth. An ammonium ion selective zeolite, Phillipsite-Gismondine, was packed in a dialysis membrane and equipped on the agitator of spinner reactor forin situ removal of ammonium ion. The semi-fed-batch operation was employed to the novel culture system for the high density cell culture, and the results showed the cell growth was improved by 32% and tPA productivity by 250%.

Method for the passaging of microcarrier cultures to a production scale for producing high titre Disabled Infectious Single Cycle-Herpes Simplex Virus Type-2

A complementary cell line CR2 is currently used to propagate the Disabled Infectious Single Cycle Herpes Simplex Virus Type 2 (DISC HSV-2) on a small laboratory scale upto 15 L. These cultures are initiated by passaging the cells from roller bottle cultures. Whilst this is suitable for the laboratory scale it is totally impractical for use in seeding an industrial manufacturing scaled version of the culture. It is paramount to have a robust system for passaging cells from a small microcarrier culture system to a larger one by a serial subculturing regime. Here we report on the successes we have had in our laboratory in scaling up our production system for the DISC HSV-2 from small 1-L cultures to a 50-L vessel with the maintenance of the viral productivity. Ease of use, reproducibility and the need to minimise overall production times were factors which were taken into consideration whilst developing our procedures. We were aware of the need to keep a production train simple and as short as possible as this was the small scale study for an envisaged manfacturing process.

In vitro modulation of L‐arginine transport in trophoblast cells by glucose

The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.

Overexpression of the Human Lysosomal Enzyme α-L-Iduronidase in Chinese Hamster Ovary Cells

We developed a Chinese hamster ovary (CHO) cell line that produces and secretes large quantities of recombinant human α-L-iduronidase, the lysosomal hydrolase deficient in mucopolysaccharidosis I (Hurler, Hurler-Scheie, and Scheie syndromes). The α-L-iduronidase cDNA was introduced into a vector containing the cytomegalovirus immediate early gene promoter/enhancer, a murine immunoglobulin Cα region intron, and the bovine growth hormone polyadenylation signal. Following cotransfection with a plasmid containing the neomycin resistance gene, stably transfected lines were selected with G-418. The highest expressing CHO cell line contained 1400-6000 units of α-L-iduronidase per milligram of protein, or 0.6-2.4% of total cell protein. Secreted α-L-iduronidase was 3000- to 7000 fold increased, with about 5000 units accumulating in 24 h per 10 7 cells. The activity and distribution of five other lysosomal glycosidases were not significantly affected. Metabolic labeling showed that half of the newly synthesized α-L-iduronidase was secreted, but generally less was recovered due to its instability in the medium. It was post-translationally processed as previously shown for α-L-iduronidase of human fibroblasts. Recombinant alpha-L-iduronidase was efficiently endocytosed by Hurler fibroblasts utilizing a mannose 6-phosphate-dependent mechanism (half maximal uptake at 0.7 nM) and was "corrective" for abnormal glycosaminoglycan accumulation (half maximal correction at 0.7 pM). The half-life of the recombinant enzyme was 5 days following uptake into Hurler fibroblasts. Production in a 5-liter microcarrier culture system permitted the collection of 15 mg or more per day. Purification to apparent homogeneity was achieved by sequential chromatography on concanavalin A-Sepharose, heparin-Sepharose, and Sephacryl S-200. Amino acid sequencing of the purified protein proved its identity, and purity. The N-terminus contained alanine 26, inconsistent with previous conclusions regarding the site of signal peptide cleavage. Sufficient purified α-L-iduronidase can now be produced for biochemical studies and for therapeutic attempts in animal models.

Rotating-Wall Vessel Coculture of Small Intestine as a Prelude to Tissue Modeling: Aspects of Simulated Microgravity

A new low shear stress, low turbulence microcarrier culture system has been developed at NASA's Johnson Space Center that permits large-scale three-dimensional tissue culture. Tissue culture bioreactors called rotating-wall vessels were used in conjunction with multicellular cocultivation to develop a unique in vitro tissue-modeling system. Normal small intestine epithelium and mesenchymal cells were cocultivated on Cytodex-3 microcarriers and were initiated in two phases. Normal small intestine mesenchymal cells were inoculated into the rotating-wall vessel at 2 x 10(5) cells/ml and allowed to attach and proliferate for 2 to 3 days. Normal small intestine epithelium was then added at an innoculum of 2 x 10(5) cells/ml and cultivation continued for 30 to 40 days. These cocultures attained cell numbers of 4-6 x 10(6) cells/ml and differentiated to form tissue-like masses of 0.4-0.5 cm with minimal necrosis. The masses displayed apical brush borders, differentiated epithelial cells, cellular polarity, extracellular matrix, and basal lamina. Verification of mesenchymal and epithelial cell expression was determined by immunocytochemistry and scanning electron microscopy. These data suggest that the rotating-wall vessel affords a new tissue culture model for investigation of growth, regulatory, and differentiation processes within normal tissues.

Morphologic differentiation of colon carcinoma cell lines HT-29 and HT-29KM in rotating-wall vessels

A new low shear stress microcarrier culture system has been developed at NASA's Johnson Space Center that permits three-dimensional tissue culture. Two established human colon adenocarcinoma cell lines, HT-29, an undifferentiated, and HT-29KM, a stable, moderately differentiated subline of HT-29, were grown in new tissue culture bioreactors called Rotating-Wall Vessels (RWVs). RWVs are used in conjunction with multicellular cocultivation to develop a unique in vitro tissue modeling system. Cells were cultivated on Cytodex-3 microcarrier beads, with and without mixed normal human colonic fibroblasts, which served as the mesenchymal layer. Culture of the tumor lines in the absence of fibroblasts produced spheroidlike growth and minimal differentiation. In contrast, when tumor lines were co-cultivated with normal colonic fibroblasts, initial growth was confined to the fibroblast population until the microcarriers were covered. The tumor cells then commenced proliferation at an accelerated rate, organizing themselves into three-dimensional tissue masses that achieved 1.0- to 1.5-cm diameters. The masses displayed glandular structures, apical and internal glandular microvilli, tight intercellular junctions, desmosomes, cellular polarity, sinusoid development, internalized mucin, and structural organization akin to normal colon crypt development. Differentiated samples were subjected to transmission and scanning electron microscopy and histologic analysis, revealing embryoniclike mesenchymal cells lining the areas around the growth matrices. Necrosis was minimal throughout the tissue masses. These data suggest that the RWV affords a new model for investigation and isolation of growth, regulatory, and structural processes within neoplastic and normal tissue.

Structural characterization of recombinant human interferon-gammas derived from two different mammalian cells.

Recombinant human interferon-gammas (rHuIFN-gamma s) were obtained from two different mammalian cells (mouse C127 cells and Chinese hamster ovary, CHO, cells) cultured in a microcarrier culture system. Both rHuIFN-gamma s were purified using sequential chromatographies for their comparison of structural properties. The peptide maps of HuIFN-gamma s digested with V8 protease and Western blot analysis demonstrated that C127 cells yielded mainly about 25kDa component and CHO cells produced about 25kDa and about 20kDa components. By the identification of glycosylated peptides, it was suggested that 20kDa and 25kDa components are glycosylated at one and at two sites, respectively. C-terminal amino acid sequence analysis indicated that both rHuIFN-gamma s consisted of at least six different species lacking 2 to 16 amino acid residues from C-terminus, so that C-termini of both rHuIFN-gamma s were slightly different from each other. Amino acid sequence and composition analyses of N-terminal peptides demonstrated that N-termini of both rHuIFN-gamma s were blocked and were supposed to be identical with that of natural HuIFN-gamma. These results suggested that different molecular heterogeneities of rHuIFN-gamma s resulted from the difference of post-translational modifications of host cells.

Development of a new type of drug by using cell technology; preparation of human interferon-beta from human diploid fibroblast cultures.

Several strains of human fibroblast were identified as good producers of human interferon-beta (HuIFN-beta), among DIP-2 cell was one of the best. We have developed an improved microcarrier culture system for both the mass culture of such cells and the large scale HuIFN-beta production. A routine pilot plant, and successively a large plant, have been accomplished for preparation, purification and preclinical or clinical trials of HuIFN-beta. The purified and lyophilized HuIFN-beta was assayed for its safety for clinical use under the regulation of the National Institute of Health of Japan. Using this HuIFN- preparation, the clinical trials on various viral diseases and malignant tumors were started from the middle of 1979. More recently, the Ministry of Health and Welfare approved this HuIFN-beta as a new drug against melanoma, glioblastoma and chronic active B type hepatitis.

Constitutive long-term production and characterization of recombinant human interferon-gammas from two different mammalian cells.

Two expression plasmids (pSVIFN gamma/BPV97 and pSVIFN gamma/AdDHFR) for constitutive production of human interferon-gamma (HuIFN-gamma) were constructed and introduced into the two different mammalian cell lines, mouse C127 cells and Chinese hamster ovary (CHO) cells. Genetically engineered C127 and CHO cells grew on microcarriers having high productivity of HuIFN-gammas for at least six months. Isoelectric focusing patterns and molecular weight analyses suggest that C127- and CHO-HuIFN-gammas are glycoproteins and that both HuIFN-gammas have different molecular structures. The development of a microcarrier culture system for genetically engineered mammalian cells has enabled us to prepare glycosylated HuIFN-gammas on a large scale.

Effect of glucocorticoid hormones on growth of human fibroblast cells and interferon production in a microcarrier culture system.

Glucocorticoid hormones promoted the growth of fibroblast cells derived from human neonatal foreskins and prolonged their life span in a microcarrier culture system that used Eagle's minimum essential medium (MEM) supplemented with fetal calf serum (FCS). But, these hormones suppressed cell growth in conventional monolayer cultures. Precolostrum newborn calf serum (PNCS) was the only species that supported the serial propagation of fibroblast cells on microcarriers, possibly because of its high content of hydrocortisone (HC). Fibroblast cells grown on microcarriers in the presence of glucocorticoid hormones maintained their ability to produce interferon (IFN)-beta in a superinduction method with poly I: poly C and antimetabolites. These cells had more than 93% diploidy and no chromosomal aberration or translocation. Use of PNCS for the cultivation of human fibroblast cells has high potential for providing a microcarrier culture system for the mass production of human IFN-beta.

Microcarriers in combination with enzyme immunofiltration and immunofluroescence for the detection of herpes simplex virus antigens in culture.

A microcarrier culture system in combination with enzyme immunofiltration with a herpes simplex virus (HSV) group monoclonal antibody was found to be as sensitive as immunofluorescence for the detection of HSV type 1 (HSV-1) in cell cultures and to give specific identification at the same time as the appearance of the cytopathic effect with very high infectious inocula and within 10 to 24 h of the appearance of the cytopathic effect with very high infectious inocula and within 10 to 24 h of the appearance of the cytopathic effect with low to high inocula. Multiplicities of infection from 10(-5) to 10(1) were tested at 8 to 96 h postinfection. When applied to the identification of HSV-1 and HSV-2 in cultures of clinical samples, the system detected HSV antigens in 50% of the samples after 2 days and in 100% of the samples after 3 days. With 2 ml of microcarrier suspension and with 50 to 300 microliter per sample, several portions are available for replicate and sequential sampling without destroying the culture. The system requires only that 2 X 10(5) microcarriers be added to the culture tube at the time or before it is seeded with cells, at an extra cost of 6 cents (U.S.) per tube and little extra labor.

Propagation of poliovirus inmicrocarrier cultures of three monkey kidney cell lines

The microcarrier culture system was applied to the propagation of polioviruses in three established monkey kidney cell lines, Vero, LLC-MK 2 and CV-1, and the yields were compared with that obtained in roller bottle and stationary cultures. The yield of the three types of polioviruses in the various cell lines and culture systems were similar. However, the use of the microcarrier culture system for anchorage-dependent cells together with the use of continuously propagating cell lines offer great advantage for large-scale cultivation of poliovirus for the preparation of killed poliovirus vaccines.

Cell growth optimization in microcarrier culture.

Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephandex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per milliliter of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for scale up into large volume production units.