Biomedical subjects

Mohamed Al-Rubeai

Publications and source records attributed to Mohamed Al-Rubeai.

17 recordsLinked to original sources

PubMed2007

A genome-wide transcriptional analysis of producer and non-producer NS0 myeloma cell lines.

'Genome-wide' or 'global' gene expression profiling provides a powerful approach to the characterization of a cell's transcriptional state. Such technology has been used in animal cell culture to create genome-wide snapshots of transcriptional activity in response to environmental factors or cellular triggers under bioprocessing conditions. Furthermore, it allows us to have a fundamental understanding of genetic mechanisms involved in recombinant protein production. One such mechanism adversely affecting the growth of recombinant bacteria is the increased metabolic burden resulting from the maintenance of plasmid copy number and heterologous protein expression. There have also been some reports on the effect of metabolic burden in mammalian cell systems. In the present study, we have used a mouse array representing 6400 genes to assess the expression profile of a WT (wild-type) mouse plasmacytoma cell line, NS0 WT, and a GS (glutamine synthetase)-NS0 6A1-100 cell line expressing chimaeric monoclonal antibody. The producer cells did not exhibit a slower growth as the result of any metabolic burden, but showed differences in metabolic activity. Gene expression profiling revealed that the producer cell line was selected for a higher expression of chromosomal genes, genes for zinc-finger proteins as well as cell-cycle-related events. On the other hand, protein synthesis is greater and ribosomal genes were more expressed in the WT cells. A possible shift from expressing antigen presenting proteins to recombinant protein could also be seen. Hence, gene expression profiling suggests that the effect of the metabolic burden in slowing growth can be mostly negated in producer cell lines by careful clonal selection, where stable transfected cells are selected for both high productivity as well as high growth rates.

Animals↗

PubMed2006

Modeling suppression of cell death by Bcl-2 over-expression in myeloma NS0 6A1 cells.

A novel population-balance model was employed to evaluate the suppression of cell death in myeloma NS0 6A1 cells metabolically engineered to over-express the apoptotic suppressor Bcl-2. The model is robust in its ability to simulate cell population dynamics in batch suspension culture and in response to thymidine-induced growth inhibition: 89% of simulated cell concentrations are within two standard deviations of experimental data. Kinetic rate constants in model equations suggest that Bcl-2 over-expression extends culture longevity from 6 days to at least 15 days by suppressing the specific rate of early apoptotic cell formation by more than 6-fold and necrotic cell formation by at least 3-fold, despite nearly a 3-fold decrease in initial cell growth rate and no significant change in the specific rate of late apoptotic cell formation. This computational analysis supports a mechanism in which Bcl-2 is a common mediator of early apoptotic and necrotic events occurring at rates that are dependent on cellular factors accumulating over time. The model has current application to the rational design of cell cultures through metabolic engineering for the industrial production of biopharmaceuticals.

Apoptosis↗

PubMed2006

Non-invasive flow cytometric monitoring of pHi in cell culture processes using EGFP.

We have described a new method for monitoring of cell culture processes using green fluorescent protein (GFP) fluorescent intensity. GFP has been used as a non-invasive fluorescent reporter for various cellular processes. In this study, enhanced (EGFP) was found to be a very sensitive indicator of pHi in in vitro cell culture, and responded rapidly to extracellular pH (pHe) changes. EGFP transfected cells were evaluated for pHi changes by flow cytometry, by measuring EGFP fluorescent intensity, and compared to that of the pH-sensitive fluoroprobe, SNARF. EGFP intensity was found to reflect pHi values of cells at different pHe in the presence of nigericin and was affected by the addition of HCl and NaOH. Significant changes in pHi were detected at different stages of batch culture and when using different cell density and media composition. The EGFP assay can be used to minimise the perturbation of cells and processes under study, thus leading to accurate information about the physiological state of single cells in a population. The results establish the application of EGFP as a non-invasive indicator of pHi for monitoring of mammalian cell culture processes.

Animals↗

PubMed2006

Optimal in-vitro expansion of chondroprogenitor cells in monolayer culture.

A continuous production of large quantities of chondroprogenitor cells for the manufacture of engineered cartilage tissue products is required. Expansion of the cell population in vitro has become an essential step in the process of tissue engineering of articular cartilage and the optimization of the culture conditions is a fundamental problem that needs to be addressed. The analysis of both seeding density and passage length was considered crucial in the optimization of expansion processes, and their correct selection should be taken as a requisite to establish culture conditions for monolayer systems. The determination of the optimal seeding density and the corresponding passage length for cell expansion in a serial passaging operation was found to be a compromise between growth kinetics and process time. This optimal determination was carried out using a mathematical approach that led to values of 10(4) cell/cm(2) for seeding density and 73 h for passage length. Additional considerations concerning the running cost of the process were introduced. Although the optimal passage length gave the desired expansion factor in a minimum process time, the selection of an alternative value of 120 h was shown to reduce the cost of the expansion process in more than 60%. The optimization approach presented will contribute to the development of feasible large scale expansion operations of chondroprogenitor cells required by the cartilage tissue engineering industry.

Animals↗

PubMed2006

Expansion of chondroprogenitor cells on macroporous microcarriers as an alternative to conventional monolayer systems.

Routine tissue culture methodologies can hardly cope with the scale of cell production required for the manufacture of engineered cartilage tissue products. In vitro cell expansion has become an essential step in the process of tissue engineering of articular cartilage and the optimization of expansion protocols is a fundamental issue that needs to be addressed. The expansion suitability of chondroprogenitor cells isolated from the superficial zone of articular cartilages was evaluated in both conventional monolayer and macroporous microcarrier in spinner flask cultures. Although monolayer systems promoted rapid in vitro expansion of undifferentiated cells, they present limited scalability. Alternatively, the use of CultiSpher-G microcarriers resulted in cell densities of 5.5 x 10(5) cell/ml, representing a 17-fold expansion in batch cultures. In addition, chondroprogenitor cells were capable of undergoing bead-to-bead migration, which allowed subcultivation to be performed without a harvesting step, thus improving the scalability of the expansion process. By employing macroporous microcarrier cultures it will be possible to obtain large number of chondroprogenitor cells for tissue engineering applications. Not only its satisfactory expansion potential, but more importantly the cost and operational advantages over traditional monolayer culture make this system a feasible alternative method for the extensive expansion of chondroprogenitor cells.

Animals↗

PubMed2006

Bioreactor systems for the production of biopharmaceuticals from animal cells.

The demand for biopharmaceutical products is set to see a significant increase over the next few years. As a consequence, the processes used to produce these products must be able to meet market requirements. The present paper reviews the current technologies available for animal cell culture and highlights the advantages and disadvantages of each method, while also providing details of recent case studies. Processes are described for both suspension and anchorage-dependent cell lines.

Animals↗

PubMed2005

Enhanced growth in NS0 cells expressing aminoglycoside phosphotransferase is associated with changes in metabolism, productivity, and apoptosis.

Prior work has reported that cotransfecting a gene of interest with the selectable marker neo can seriously perturb a number of cellular processes. In this study the influence of the neo gene on the growth, death, and metabolism of a murine myeloma NS0 cell line, expressing a chimeric antibody, was investigated. A pool of neo transfectants, 6A1-NEO, was selected with 500 microg/mL G418. Quantitative PCR analysis revealed that 6A1-NEO contained, on average, three copies of the neo gene per cell. Batch cultivation of 6A1-NEO showed that there was a 36% increase in maximum viable cell concentration, a 20% increase in the maximum apparent growth rate, and a 134% increase in cumulative cell hours as compared with the parent, 6A1-(100)3. Batch cultivation of five randomly selected clones illustrated that 6A1-NEO's advantage over the parent was not due to clonal variation. Neither the use of G418 during the selection process nor the cultivation of cells in the presence of G418 were responsible. This implied that the neo gene product, APH(3')-II, was causing the changes in proliferative capacity. Analysis of the cell cycle revealed that there were no differences in the distribution of cells in the G(1), S, and G(2) phases. When cell growth was synchronized, there were no observed differences in cell-cycle duration. 6A1-NEO resisted the onset of apoptosis during the growth phase. Consequently, there was a larger viable population of 6A1-NEO cells available for proliferation as compared with the parent. However, 6A1-NEO died at the same rate as the parent when resuspended in spent media or after treatment with staurosporin. Expression of the anti-apoptotic protein Bcl-2 was upregulated in 6A1-NEO, indicating that APH(3')-II could be acting by modulating endogenous gene expression. Analysis of key metabolites showed that 6A1-NEO's specific glucose consumption rate was 133% higher, whilst its specific glutamate consumption rate was 45% lower than the parent. 6A1-NEO's efficient utilization of glutamate and shift towards glucose metabolism may have contributed to the rise in proliferative capacity. However, this was accompanied by a 70% drop in the specific antibody production rate. These results show that the increase in growth rate and proliferative capacity caused by the expression of recombinant APH(3')-II was associated with changes in metabolism, apoptosis, and endogenous gene expression.

Animals↗

PubMed2005

Over-expression of hTERT in CHO K1 results in decreased apoptosis and reduced serum dependency.

The enzyme telomerase plays a crucial role in cellular proliferation. By adding hexameric repeats to the chromosome ends, it prevents telomeric loss and, thus entry into senescence of limited life span cells. It is unclear, however, what would be the effect of over-expressing telomerase in an immortalised cell line, characterised by unlimited life span and high levels of apoptosis under sub-optimal growth conditions. In order to address this question, we have transfected the immortal cell line CHO K1 with the human telomerase reverse transcriptase (hTERT) catalytic subunit. Differences in the growth profile and apoptosis levels between the cells over-expressing hTERT (Telo) and the cells containing mock vector were found under standard growth conditions. Similarly, the Telo cells showed lower levels of apoptosis, greater attachment tendency and higher viable cell density under serum-deprived conditions compared to the control cell line, suggesting a major role for hTERT over-expression in stressed cultures. Using a mouse cDNA microarray, the collagen type III and V genes were shown to have at least a 10-fold higher expression in the Telo cells than the control cells, suggesting a role of hTERT in the cell attachment pathways.

Animals↗

PubMed2005

Effect of Bcl-2 overexpression on cell cycle and antibody productivity in chemostat cultures of myeloma NS0 cells.

Chemostat cultures of NS0 cell lines were carried out at dilution rates ranging from 0.8 d(-1) to 0.2 d(-1). Compared with the control, the viable cell density of the Bcl-2 cell line was approximately 10% higher at 0.8 d(-1) and increased to 55% when the dilution rate was reduced to 0.2 d(-1). As the dilution rate was reduced, the viability of the two cultures diverged reaching a difference of 43% at 0.2 d(-1). The specific growth rate of the control cells was the same as the dilution rate down to a value of 0.6 d(-1). By contrast, the specific growth rate of Bcl-2 cells was parallel to the dilution rate down to a value as low as 0.3 d(-1). For both NS0 cell lines, the G1 cell population decreased, while the S and G2/M cell populations increased as the dilution rate was reduced. The antibody titer of the control cells increased from 7 to 21 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.2 d(-1). With an initial increase from 2 to 15 microg.ml(-1) as the dilution rate was reduced from 0.8 to 0.4 d(-1), the antibody titer of the Bcl-2 cells remained constant as the dilution rate was further reduced to 0.2 d(-1). A good correlation between specific antibody production rate and the percentage of G2/M cells was observed.

Animals↗

PubMed2004

ACSD labelling and magnetic cell separation: a rapid method of separating antibody secreting cells from non-secreting cells.

Several new ways of selecting cells have recently been developed. These include magnetic separation of cells by labelling with magnetic beads against the recombinant product, gel microdrop technology which encapsulates the cells in gelatine beads and matrix-based secretion assays. Affinity capture surface display (ACSD) is a matrix-based assay for the enrichment of high producing cells and relies on the strong affinity between biotin and avidin derivatives. Matrix-based assays have previously only been used for the enrichment of recombinant cells. Here, we have optimised this assay and developed a method of separating antibody producing cells from non-producing cells in a recombinant myeloma cell line using ACSD combined with MACS magnetic separation. The method is rapid, simple enough to become routine and adaptable to many different secreted products from recombinant mammalian cells.

Animals↗

PubMed2004

Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells.

Chinese hamster ovary cells have been engineered to inducibly over-express the p21(CIP1) cyclin-dependent kinase inhibitor, to achieve cell cycle arrest and increase cell productivity. In p21(CIP1)-arrested cells production of antibody from a stably integrated lgG4 gene, was enhanced approximately fourfold. The underlying physiological basis for enhanced productivity was investigated by measuring a range of cellular and metabolic parameters. Interestingly, the average cell volume of arrested cells was approximately fourfold greater than that of proliferating cells. This was accompanied by significant increases in mitochondrial mass, mitochondrial activity, and ribosomal protein S6 levels. Our results suggest that p21(CIP1)-induced cell cycle arrest uncouples cell growth from cell-cycle progression, and provides new insight into how improved productivity can be achieved in a cell line commonly used for large-scale production of pharmaceutical proteins.

Animals↗

PubMed2004

The selection of high-producing cell lines using flow cytometry and cell sorting.

The selection of high-producing cell lines is usually time-consuming and labour-intensive. Following transfection, high-producing cells are selected using limiting dilution cloning to prevent non- and low-producing cells from outgrowing high-producing cells, a process that normally takes > 3 months. During this time, the cells have to be screened occasionally to ensure stability of the selected clone. Several new methods for selecting and screening cells using flow cytometry and cell sorting have recently been developed; these include gel microdrop technology, which encapsulates the cells in gelatine beads, and matrix-based secretion assays. This paper reviews these techniques for selecting high-producing cell lines and isolating rare cells.

Animals↗

PubMed2002

Regulation of cell cycle and productivity in NS0 cells by the over-expression of p21CIP1.

We have constructed NS0 myeloma cell lines that inducibly express the p21CIP1 cyclin dependent kinase inhibitor, using the Lacswitch system. Ectopic p21(CIP1) protein expression was rapidly induced within 12 h of addition of IPTG, causing G1-phase arrest and almost complete inhibition of cell proliferation. The production of a chimeric IgG4 antibody, expressed constitutively from an independent promoter, was found to be significantly increased by more than 4-fold in p21CIP1-arrested cells. This study demonstrates for the first time the successful construction of anchorage-independent and proliferation-controlled NS0 cell lines with enhanced secreted chimeric antibody production independent of the inducible promoter activity used to achieve cytostasis.

Animals↗

PubMed2002

Retroviral vectors for human gene delivery.

The potential for gene therapy to cure a wide range of diseases has lead to high expectations and a great increase in research efforts in this area. At present, viral vectors are the most efficient means of delivering a corrective gene into human cells. While a number of different viral vectors are under development, retroviral vectors are currently the most common type used in clinical trials today. However, the production of retroviral vectors for gene therapy applications faces a number of challenges. Of primary concern is the low titre of vector stocks produced by packaging cells in culture and the inherent instability of retroviral vector activity. The problems facing large-scale retroviral vector production are outlined in this review and the research efforts by a number of groups who have attempted to optimise production methods are presented.

Journal Article↗

PubMedDate not supplied

Modulation of cell cycle for enhancement of antibody productivity in perfusion culture of NS0 cells.

A prolonged period of high productivity at high cell density is desirable for industrial production of biopharmaceuticals. Previous efforts have shown that cessation of cell proliferation in low cell density culture results in increased productivity. We report here further results on multigenic manipulation of cell cycle and apoptosis to enhance productivity at high cell density. The NS0 6A1/4-9F myeloma cell line, which constitutively expresses a chimeric IgG4 antibody and inducibly expresses the p21(CIP1) cyclin-dependent kinase inhibitor has been further engineered to constitutively overexpress the Y28 mutant Bcl-2 anti-apoptotic protein. The effects of overexpression of p21(CIP1) and Bcl-2 on cell proliferation, cell viability, and antibody production has been investigated in batch and continuous perfusion cultures. In both cultures the p21(CIP1) protein arrested cell proliferation, confirming the previous results in low-density culture of 4-fold increase in antibody production, whereas mutant Bcl-2 expression has not resulted in any significant improvement in cell viability of arrested cells. This study demonstrates that it is possible to enhance the productivity of relatively high-density continuous mammalian cell cultures by arresting the cell cycle in G1 phase.

Animals↗

PubMedDate not supplied

Regulation of cell proliferation and apoptosis in CHO-K1 cells by the coexpression of c-Myc and Bcl-2.

Proliferation and cell death are regarded as key targets for the optimization of animal cell culture processes and for the maximization of product yield. Although chemical and physical factors are vitally important, of primary interest is the utilization of genetic engineering to regulate cellular processes. CHO cells were first genetically modified to enhance proliferation rate in both suspension and monolayer cultures. Under the constitutive control of c-myc overexpression the CHO cultures showed an increase in growth rate and maximum cell number accompanied by a similar decrease in specific glucose consumption rate. Although the c-myc transfected cell line exhibited apoptosis at much lower rates than is widely reported and associated with the overexpression of c-Myc, it was nevertheless apparent that c-Myc was responsible for the induction of higher apoptotic rates when compared with the control cell line. Hence, the anti-apoptotic gene bcl-2 was also used to transfect the c-Myc CHO cell line, to reduce cell death. Overexpression of both oncoproteins resulted in a cell line that exhibited higher proliferation rates and maximum cell numbers, with a decrease in apoptosis when compared to the parental cell line. In conclusion, it was shown that Bcl-2 protein overexpression specifically abrogates c-Myc-induced apoptosis without affecting the c-Myc mitogenic function.

Animals↗

PubMedDate not supplied

Cryopreservation and in vitro expansion of chondroprogenitor cells isolated from the superficial zone of articular cartilage.

Understanding the proliferation mechanisms of chondroprogenitor cells and their influence on cell differentiation is crucial in order to develop large-scale expansion processes for tissue engineering applications. Proliferation control mechanisms were mainly attributed to substrate limitation and cell-cell contact inhibition. The limiting substrates were found to be components of the FCS, with an optimal proliferation rate achieved in the presence of 40% FCS. In addition, the medium supply rate was found to be essential in reducing substrate limitation. In terms of FCS, 10 microL FCS cm(-2) h(-1) was the threshold feed rate required to prevent substrate limitation. Above this rate, maximum cell densities of 5.3 x 10(5) cells/cm2 were achieved, representing a 53-fold expansion. To reduce the need for high supply rates, the effect of specific growth factors was also investigated. Cell densities of 3.3 x 10(5) cells/cm2 were achieved in batch cultures using 40% FCS and 1 ng/mL TGF-beta1. Chondroprogenitor cells were expanded in this medium up to three passages without compromising their ability to differentiate and produce cartilage-like matrix in pellet cultures. In addition to substrate limitation, cell-cell contact, even at very sparse subconfluent densities, appeared capable of exerting some degree of growth inhibition. The cells exhibited deceleratory growth kinetics, characterized by a decrease of specific growth rates over time.

Animals↗