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1.
Physiol Genomics ; 39(1): 72-81, 2009 Sep 09.
Article in English | MEDLINE | ID: mdl-19622797

ABSTRACT

Pancreatic islet transplantation as a potential cure for type 1 diabetes (T1D) cannot be scaled up due to a scarcity of human pancreas donors. In vitro expansion of beta-cells from mature human pancreatic islets provides an alternative source of insulin-producing cells. The exact nature of the expanded cells produced by diverse expansion protocols and their potential for differentiation into functional beta-cells remain elusive. We performed a large-scale meta-analysis of gene expression in human pancreatic islet cells, which were processed using three different previously described protocols for expansion and for which redifferentiation was attempted. All three expansion protocols induced dramatic changes in the expression profiles of pancreatic islets; many of these changes are shared among the three protocols. Attempts at redifferentiation of expanded cells induce a limited number of gene expression changes. Nevertheless, these fail to restore a pancreatic islet-like gene expression pattern. Comparison with a collection of public microarray datasets confirmed that expanded cells are highly comparable to mesenchymal stem cells. Genes induced in expanded cells are also enriched for targets of transcription factors important for pluripotency induction. The present data increase our understanding of the active pathways in expanded and redifferentiated islets. Knowledge of the mesenchymal stem cell potential may help development of drug therapeutics to restore beta-cell mass in T1D patients.


Subject(s)
Gene Expression Regulation , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Adult , Cell Proliferation , Embryonic Stem Cells/metabolism , Female , Gene Expression Profiling , Gene Regulatory Networks , Humans , Kinetics , Male , Mesenchymal Stem Cells/metabolism , Middle Aged , Oligonucleotide Array Sequence Analysis , Protein Binding
2.
Brain ; 131(Pt 8): 2106-26, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18669492

ABSTRACT

The current transplantation paradigm for Parkinson's disease that places foetal dopaminergic cells in the striatum neither normalizes neuronal activity in basal ganglia structures such as the substantia nigra (SN) and subthalamic nucleus (STN) nor leads to complete functional recovery. It was hypothesized that restoration of parkinsonian deficits requires inhibition of the pathological overactivity of the STN and SN in addition to restoration of dopaminergic activity in the striatum. To achieve inhibition, a multitargeted basal ganglia transplantation strategy using GABAergic cells derived from either foetal striatal primordia (FSP) cells or human neural precursor cells (hNPCs) expanded in suspension bioreactors was investigated. In hemiparkinsonian rats, transplantation of foetal rat dopaminergic cells in the striatum in conjunction with GABAergic grafts in the STN and/or SN promoted significant improvement in forelimb akinesia and motor function compared to transplantation of intrastriatal dopaminergic grafts alone or in conjunction with undifferentiated hNPCs. In culture, FSP cells exhibited neuronal electrophysiological properties. However, recordings from GABAergic hNPCs revealed limited ionic conductances and an inability to fire action potentials. Despite this, they were almost as efficacious as FSP cells in inducing functional recovery following transplantation, suggesting that such recovery may have been mediated by secretion of GABA rather than by functional integration into the host. Thus, restoration of dopaminergic activity to the striatum in concert with inhibition of the STN and SN by GABAergic grafts may be beneficial for improving clinical outcomes in patients with Parkinson's disease and potential clinical application of this strategy may be enhanced by the use of differentiated hNPCs.


Subject(s)
Basal Ganglia/metabolism , Fetal Stem Cells/transplantation , Neurons/transplantation , Parkinsonian Disorders/surgery , Animals , Dopamine/metabolism , Female , Humans , Immunohistochemistry , Microscopy, Confocal , Models, Animal , Neuropsychological Tests , Parkinsonian Disorders/metabolism , Random Allocation , Rats , Rats, Wistar , Substantia Nigra , Subthalamic Nucleus , Treatment Outcome , gamma-Aminobutyric Acid/metabolism
3.
Exp Cell Res ; 313(5): 931-42, 2007 Mar 10.
Article in English | MEDLINE | ID: mdl-17270172

ABSTRACT

We have previously identified a novel population of small cells in human and canine pancreas characterized by immature morphology, quiescence, and a glucose-responsive insulin secretion. Based on their immature phenotype and predominant presence in small islets, we have hypothesized that small cells serve as islet progenitors. This hypothesis remains untested, however, due to persistent quiescence and scarcity of small cells in vitro. We have recently developed a culture medium that allowed for modest small cell proliferation. In this study we characterized the expression of genes potentially involved in small cell growth regulation by Q-RT-PCR. Our results suggest that quiescence of small cells correlates with up-regulation of Cdk inhibitors p27(Kip1), p16(INK4a) and p21(CIP1), PTEN, Hep27 and Foxo1a and with down-regulation of c-Myc and the receptors for EGF, FGF2 and HGF. The exit from quiescence correlates with activation of EGFR expression and down-regulation of p27(Kip1) and p16(INK4a). We also report here that small cells can be maintained in long-term non-adherent cultures preserving insulin and glucagon production for up to 208 days. Therefore, expansion of small cells in vitro may have a significant potential for the treatment of diabetes. This study is an important step in understanding the mechanisms involved in small cell growth regulation, which is required to fully evaluate their functional potential.


Subject(s)
Homeodomain Proteins/metabolism , Insulin/metabolism , Islets of Langerhans/metabolism , Pancreas/metabolism , Trans-Activators/metabolism , Cell Count , Cell Proliferation , Cells, Cultured , Computer Systems , Culture Media, Serum-Free , Gene Expression Regulation , Humans , Pancreas/cytology , Pancreas/physiology , Polymerase Chain Reaction
4.
Cell Transplant ; 15(8-9): 689-97, 2006.
Article in English | MEDLINE | ID: mdl-17269440

ABSTRACT

The development of new cell replacement strategies using neural stem cells (NSC) may provide an alternative and unlimited cell source for clinical neural transplantation in neurodegenerative diseases such as Parkinson's and Huntington's disease. The clinical application of neural transplantation using NSC will therefore depend upon the availability of clinical grade NSC that are generated in unlimited quantities in a standardized manner. In order to investigate the utility of NSC in clinical neural transplantation, undifferentiated murine NSC were first expanded for an extended period of time in suspension bioreactors containing a serum-free medium. Following expansion in suspension bioreactors, NSC were still able to differentiate in vitro into both astrocytes and neurons after exposure to brain-derived neurotrophic factor (BDNF), suggesting that bioreactor expansion does not alter cell lineage potentiality. Undifferentiated bioreactor-expanded NSC were then transplanted into the rodent striatum. Immunohistochemical examination revealed undifferentiated bioreactor-expanded NSC survived transplantation for up to 8 weeks and expressed the astrocytic immunohistochemical marker glial fibrillary acidic protein (GFAP), suggesting that the host striatal environment influences NSC cell fate upon transplantation. Moreover, no tumor formation was observed within the graft site, indicating that NSC expanded in suspension bioreactors for an extended period of time are a safe source of tissue for transplantation. Future studies should focus on predifferentiating NSC towards specific neuronal phenotypes prior to transplantation in order to restore behavioral function in rodent models of neurodegenerative disease.


Subject(s)
Bioreactors , Brain/surgery , Neurons/transplantation , Stem Cell Transplantation/methods , Animals , Cell Differentiation , Cell Line , Cell Survival , Female , Glial Fibrillary Acidic Protein/analysis , Immunohistochemistry , Ki-67 Antigen/analysis , Mice , Microscopy, Confocal , Neurons/chemistry , Neurons/cytology , Rats , Rats, Wistar , Stem Cells/chemistry , Stem Cells/cytology , Time Factors
5.
Med Biol Eng Comput ; 41(3): 271-82, 2003 May.
Article in English | MEDLINE | ID: mdl-12803291

ABSTRACT

A relatively new approach to the treatment of neurodegenerative diseases is the direct use of neural stem cells (NSCs) as therapeutic agents. The expected demand for treatment from the millions of afflicted individuals, coupled with the expected demand from biotechnology companies creating therapies, has fuelled the need to develop large-scale culture methods for these cells. The rapid pace of discovery in this area has been assisted through the use of animal model systems, enabling many experiments to be performed quickly and effectively. This review focuses on recent developments in expanding human and murine NSCs on a large scale, including the development of new serum-free media and bioreactor protocols. In particular, engineering studies that characterise important scale-up parameters are examined, including studies examining the effects of long-term culture of NSCs in suspension bioreactors. In addition, recent advances in the human NSC system are reviewed, including techniques for the evaluation of NSC characteristics.


Subject(s)
Neurons/cytology , Stem Cell Transplantation , Stem Cells/cytology , Animals , Bioreactors , Cell Culture Techniques/methods , Cell Division , Disease Models, Animal , Humans , Mice , Neurodegenerative Diseases/therapy
6.
Proteins ; 41(1): 144-53, 2000 Oct 01.
Article in English | MEDLINE | ID: mdl-10944401

ABSTRACT

Production of recombinant proteins that are not secreted outside the producing cells usually requires purification steps that can result in significant yield reductions and loss of biological activity. Using insect cells as a model system to devise the means for secreting recombinant proteins that are not normally destined for secretion outside the producing cells, we initially examined the ability of an insect-specific signal peptide sequence to direct secretion of two intracellular proteins (the cytoplasmic enzyme chloramphenicol acetyl transferase [CAT] and the nuclear protein Bombyx mori chorion factor 1 [BmCF1]) expressed in transfected silkmoth cells. Although this signal sequence functioned efficiently as a chimera with normally secreted proteins, it failed to secrete CAT and BmCF1, suggesting that additional signals are required for passage of these polypeptides through the secretion pathway. For this reason, we also generated a secretion module consisting of the secreted protein juvenile hormone esterase (JHE), a spacer region containing a histidine tag and an endopeptidase cleavage site, to which coding sequences of choice can be cloned as C-terminal extensions. In C-terminal fusions with the CAT and BmCF1 open reading frames, the N-terminal JHE moiety was able to provide all the signals necessary for secretion of CAT and BmCF1 into the extracellular environment. The histidine tag present in the spacer region allowed purification of fusion proteins by metal affinity chromatography under nondenaturing conditions, and the enteropeptidase cleavage site was recognized and cleaved by the cognate protease causing the release of the intracellular proteins from the secretion module. We also show that another secreted protein, human granulocyte-macrophage colony stimulating factor (GM-CSF) can substitute for JHE in the secretion module and that these secretion modules can function in mammalian cells.


Subject(s)
Cytoplasm/metabolism , Nuclear Proteins/metabolism , Proteins/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cell Line , Chloramphenicol O-Acetyltransferase/genetics , Chromatography, Affinity , DNA Primers , Granulocyte-Macrophage Colony-Stimulating Factor/chemistry , Molecular Sequence Data , Nuclear Proteins/chemistry , Protein Sorting Signals/chemistry , Protein Sorting Signals/physiology , Proteins/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification
7.
Biotechniques ; 28(1): 148-54, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10649786

ABSTRACT

Plasmid transfection is the first step in the generation of stably transformed animal cells and is also a useful tool for analyzing transient gene expression. Maximizing the transfection efficiency and expression level from the introduced plasmid is critical to the success of these processes. By means of lipid-mediated transfection, a plasmid vector expressing the green fluorescence reporter protein has been coupled with flow cytometry to conveniently investigate those parameters that impact the efficacy of transfection of lepidopteran insect cells. The key feature of this technique is the rapid and simultaneous quantification of transfection efficiency and heterologous protein expression level per cell. Using this technique, we developed an optimized transfection protocol for insect cells by investigating the following parameters: lipid incubation time, lipid/DNA mixture incubation time, lipid and DNA concentration, incubation vessel and transfection duration. Following optimization, transfection efficiencies of 37%-40% were obtained for Bombyx mori Bm5 and Spodoptera frugiperda Sf-21 cells.


Subject(s)
Flow Cytometry/methods , Transfection , Animals , Bombyx/genetics , Cell Survival , Cells, Cultured , DNA/genetics , Gene Expression , Green Fluorescent Proteins , Lepidoptera , Lipid Metabolism , Luminescent Proteins/genetics , Phosphatidylethanolamines/metabolism , Plasmids , Spodoptera/genetics , Time Factors , Transformation, Genetic
8.
Biotechnol Prog ; 15(6): 1046-52, 1999.
Article in English | MEDLINE | ID: mdl-10585188

ABSTRACT

Nine insect cell lines were evaluated for their potential as host systems for recombinant protein production using a new expression vector permitting the continuous high-level expression of secreted glycoproteins by transformed insect cells (Farrell et al., 1998). As a means of preliminary screening, all nine insect cell lines were transfected with the green fluorescence protein. Growth in static and suspension culture was then examined as a further method of screening. On the basis of their transfection efficiencies and cell growth characteristics, five insect cell lines, Bm5, High Five, IPLB-LdFB, IZD-MB-0503, and Sf-21, were selected for stable transformation to produce granulocyte-macrophage colony-stimulating factor (GM-CSF). These five cell lines were stably transformed using an antibiotic resistance scheme and evaluated as a polyclonal population. Increasing the antibiotic concentration was found to cause not only a decrease in the specific growth rate but also an increase in the specific protein production rate and final GM-CSF concentration. The transformed High Five cells exhibited by far the greatest specific protein production rate of 5.1 x 10(-)(6) microgram/(cell.h), resulting in the highest final GM-CSF concentration of 22.8 mg/L when grown in static culture. One cloned High Five cell line produced a GM-CSF concentration of 46 mg/L in static culture and 27 mg/L in suspension culture.


Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/biosynthesis , Insecta , Luminescent Proteins/biosynthesis , Recombinant Proteins/biosynthesis , Transfection/methods , Animals , Biotechnology/methods , Cell Culture Techniques/methods , Cell Division , Cell Line , Cell Line, Transformed , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Green Fluorescent Proteins , Luminescent Proteins/genetics , Plasmids
9.
Biotechnol Bioeng ; 65(5): 589-99, 1999 Dec 05.
Article in English | MEDLINE | ID: mdl-10516585

ABSTRACT

Neural stem cells (NSCs) are primitive cells that are the "parent" cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10(7) with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS-neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.


Subject(s)
Bioreactors , Cell Culture Techniques/methods , Central Nervous System/cytology , Stem Cells/cytology , Animals , Astrocytes/cytology , Cell Differentiation , Cell Division , Cell Line , Cell Separation/methods , Mice , Neurons/cytology , Oligodendroglia/cytology
10.
Biotechnol Bioeng ; 64(4): 426-33, 1999 Aug 20.
Article in English | MEDLINE | ID: mdl-10397881

ABSTRACT

Stable transformation was used to generate a cloned insect cell line (Bm5 silkmoth cells) over-expressing human tissue plasminogen activator (tPA). This cell line expressed 135 microg/mL single chain tPA in serum-free medium in static culture with a maximum specific activity of 120 IU/microg. In serum-containing medium, this line expressed 160 microg/mL of combined single-chain tPA, two-chain tPA, and a higher molecular weight SDS-stable tPA complex in suspension cultures with a maximum specific activity of 255 IU/microg. Approximately 100 copies of the tPA cDNA were randomly integrated into each Bm5 cell. For secretion of recombinant tPA from Bm5 cells, the native human tPA signal peptide is as effectively recognized as an insect specific signal peptide derived from a silkmoth chorion gene. Finally, stably transformed polyclonal populations of Bm5, High Five, and Sf21 cells expressing tPA were generated and compared for relative tPA expression.


Subject(s)
Bombyx/genetics , Spodoptera/genetics , Tissue Plasminogen Activator/genetics , Animals , Blood , Bombyx/cytology , Cell Line, Transformed , Cloning, Molecular , Culture Media, Serum-Free , Humans , Protein Sorting Signals/genetics , Protein Sorting Signals/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spodoptera/cytology , Tissue Plasminogen Activator/chemistry , Tissue Plasminogen Activator/metabolism
11.
Biotechnol Bioeng ; 63(4): 473-83, 1999 May 20.
Article in English | MEDLINE | ID: mdl-10099628

ABSTRACT

Inoculation and growth conditions for the large-scale expansion of mammalian neural stem cells (NSC) have been determined. We examined suspension culture bioreactors of murine NSC, and concluded that the oxygen level should be kept high (20%), and the osmolarity of the medium should be kept low (below 400 mOsm/kg). The pH of the medium was found to have a large effect on cell proliferation, and the best growth characteristics were obtained within an optimum pH range of 7. 1 to 7.5. The inoculation conditions were also seen to have a large effect not only on the growth characteristics, but also on the number of cells that die in the initial stages of the culture. For large expansion of cells, low inoculum levels (10(4) cells/mL) and single-cell suspensions proved superior, whereas, for fast expansion of cells, higher inoculum levels (10(5) cells/mL) and spheroid inoculum forms were preferred. The inoculum temperature of the medium did not have a large effect on growth characteristics, but the pH greatly influenced cell proliferation. Inoculum pH levels should also be kept between 7.1 and 7.5. If these protocols are followed, high multiplication ratios and viabilities can be obtained in a 5-day batch suspension culture bioreactor run. A large number of cells could then be used in animal models for testing of neural drugs and in research and development toward cures for neurodegenerative disorders such as multiple sclerosis (MS) and Huntington's and Parkinson's disease. The results presented here also point the way toward studies on in vitro expansion of human neural stem cells.


Subject(s)
Neurons/cytology , Stem Cells/cytology , Animals , Bioreactors , Biotechnology/instrumentation , Biotechnology/methods , Cell Culture Techniques/instrumentation , Cell Culture Techniques/methods , Cell Differentiation , Cell Division , Cell Line , Cell Transplantation , Humans , Mammals , Mice , Neurodegenerative Diseases/therapy
12.
Cytotechnology ; 30(1-3): 133-42, 1999 Jul.
Article in English | MEDLINE | ID: mdl-19003362

ABSTRACT

Dielectrophoresis is a well established and effective means for the manipulation of viable cells. However, its effectiveness greatly depends upon the utilization of very low electrical conductivity media. High conductivity media, as in the case of cell culture media, result only in the induction of weaker repulsive forces (negative dielectrophoresis) and excessive medium heating. A dielectrophoresis-based cell separation device (DEP-filter) has been recently developed for perfusion cultures that successfully overcomes these obstacles and provides a very high degree of viable cell separation while most of the nonviable cells are removed from the bioreactor by the effluent stream. The latter results in high viabilities throughout the culture period and minimization of lysed cell proteases in the bioreactor. However, an important question that remains to be answered is whether we have any adverse effects by exposing the cultured cells to high frequency electric fields for extended periods of time. A special chamber was constructed to quantitate the effect under several operational conditions. Cell growth, glucose uptake, lactate and monoclonal antibody production data suggest that there is no appreciable effect and hence, operation over long periods of time of the DEP-filter should not have any adverse effect on the cultured cells.

13.
Biotechnol Bioeng ; 54(3): 239-50, 1997 May 05.
Article in English | MEDLINE | ID: mdl-18634090

ABSTRACT

Cost-effective production of biopharmaceuticals on a large scale can be carried out by perfusion cultures of mammalian cells. One problem with this mode of operation for submerged free-cell cultures is the requirement for an efficient cell separation device located in the effluent stream. The present work investigates the potential for the development of a novel dielectrophoresis-based cell separator, capable of providing selective retention of viable cells in cell culture media, which are highly conductive. Predictions of the dielectrophoretic (DEP) response in culture media were first obtained through a series of DEP-levitation experiments. Subsequently, a prototype microelectrode "filter" was microfabricated and tested with C174 myeloma cell suspensions of density 1 x 10(6) cells/mL. The optimum frequency range for selective retention of viable cells was found in the range 5-15 MHz. A maximum separation efficiency of 98% was achieved at 10 MHz, with an applied peak-to-peak voltage of 30 V (maximum field strength of 10(5) V/m) and a flow rate of 30 mL/h which corresponds to a superficial velocity of 5.23 cm/h through the DEP-filter channels.

14.
Biotechnol Prog ; 10(6): 636-43, 1994.
Article in English | MEDLINE | ID: mdl-7765698

ABSTRACT

The baculovirus/insect cell expression system is an efficient and practical method for the production of many active therapeutic proteins on a large scale. The advantages of suspension cultures have been demonstrated with the study of a baculovirus/insect cell (BmNPV/Bm5) expression system for the production of recombinant chloramphenicol acetyltransferase (CAT), a model heterologous protein. Key infection parameters such as infection time and multiplicity of infection were examined systematically for the maximization of protein production. Furthermore, emphasis was placed on the development of possible medium replenishment strategies, which were necessary to achieve higher volumetric protein production from the infection of high-density cell cultures without sacrificing specific protein productivity. The highest protein production was achieved with the infection of suspended cells in the mid to late exponential growth phase.


Subject(s)
Baculoviridae/genetics , Baculoviridae/metabolism , Biotechnology/methods , Bombyx/metabolism , Bombyx/virology , Recombinant Proteins/biosynthesis , Animals , Bombyx/cytology , Cell Division/physiology , Cells, Cultured , Cellular Senescence/physiology , Chloramphenicol O-Acetyltransferase/biosynthesis , Chloramphenicol O-Acetyltransferase/genetics , Culture Media , Female , Recombinant Proteins/genetics
15.
J Biotechnol ; 34(2): 133-47, 1994 May 15.
Article in English | MEDLINE | ID: mdl-7515626

ABSTRACT

In anchorage-dependent cell microcarrier cultures knowledge of the cell's growth kinetics is necessary in order to design and successfully operate bioreactors, particularly on a large scale. However, in addition to growth kinetics, an understanding of the physiological state of the culture is also important. In this paper the cell cycle progression of Vero and MRC-5 microcarrier cultures have been observed utilizing a flow cytometer. Flow cytometry analysis enabled the differentiation of the various phases of the cell cycle as the culture moved from initial inoculation to the stationary, or confluent stage. Not only was the flow cytometer able to distinguish contact inhibited cells from noncontact inhibited cells, but the measured fraction of contact inhibition cells were found to be in agreement with fractions predicted from a previously developed cellular automation model for microcarrier cultures. Further, the data from the stationary phase was used to quantify the death rate in microcarrier cultures.


Subject(s)
Cell Cycle , Culture Techniques/methods , Animals , Automation , Cell Division , Cell Line , DNA/analysis , Fetus , Flow Cytometry/methods , Humans , Kinetics , Lung , RNA/analysis , Time Factors , Vero Cells
16.
J Biotechnol ; 33(3): 249-58, 1994 Apr 15.
Article in English | MEDLINE | ID: mdl-7764737

ABSTRACT

Silkworm (Bombyx mori, Bm5) cell growth at different environmental conditions was systematically studied in controlled bioreactors. The physiochemical parameters, which affect the growth of Bm5 cells, were defined and optimized. The growth rate was maximal at a medium osmolality of about 370 mosm kg-1; and more than 90% of the maximum growth rate was achieved with osmolality between 350 and 385 mosm kg-1. The optimum pH for Bm5 cell growth was from 6.10 to 6.30. Moreover, the effect of dissolved oxygen concentrations (higher than 20% air saturation) on growth was minimal. However, the maximum cell density decreased markedly with dissolved oxygen tensions less than 20%. The shear protection additive Pluronic F-68 had a very important effect on the growth of Bm5 cells in bioreactors with an optimum concentration of 0.2% (w/v). Compared to the original culture condition, the optimized conditions resulted in a twofold increase in cell yield and a reduced population doubling time from 48 to 36 h. Furthermore, the yield of a recombinant protein (CAT) was increased substantially in the optimized cultures.


Subject(s)
Recombinant Proteins/biosynthesis , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Bombyx/cytology , Cell Division , Cells, Cultured , Chloramphenicol O-Acetyltransferase/biosynthesis , Hydrogen-Ion Concentration , Osmolar Concentration , Oxygen/pharmacology , Poloxalene/pharmacology
17.
Cytotechnology ; 15(1-3): 95-102, 1994.
Article in English | MEDLINE | ID: mdl-7765957

ABSTRACT

It has been shown that some B-cell hybridomas secrete autocrine factors in vitro which can influence cell metabolic processes. Rather than screen specifically for suspected cytokines, that may or may not affect our cell line, we have examined the lumped effects of intracellular and secreted factors on cell proliferation and monoclonal productivity in hybridoma batch cultures. Firstly, supplements of total soluble intracellular proteins combined with other intracellular metabolites were found to both decrease the specific growth rate and increase the antibody production rate at higher concentrations in batch culture. This is an important consideration in high cell density cultures, such as perfusion systems, where a reduction of growth by the presence of intracellular factors may be compensated by an increase in MAb production. In addition, flow cytometry data revealed that the average cell cycle G1 phase fraction was unaffected by the variation in the maximum specific growth rates during the exponential growth phase, caused by the addition of intracellular factors; this suggests that higher MAb productivity at lower growth rates are not a result of cell arrest in the G1 phase. Secondly, secreted extracellular proteins larger than 10,000 Daltons, which were concentrated from spent culture supernatant, were shown to have no significant effect on growth and specific MAb productivity when supplemented to batch culture at levels twice that encountered late in normal batch culture. This indicates that endogenous secreted cytokines, if at all present, do not play a major autocrine role for this cell line.


Subject(s)
Antibodies, Monoclonal/biosynthesis , Cell Cycle , Hybridomas/cytology , Hybridomas/immunology , Proteins/metabolism , Animals , B-Lymphocytes/immunology , Culture Techniques/methods , Cytokines/physiology , Extracellular Space/physiology , Flow Cytometry , G1 Phase , Hybridomas/metabolism , Immunoglobulin M/biosynthesis , Kinetics , Mice , Time Factors
18.
Biotechnol Bioeng ; 43(1): 90-100, 1994 Jan 05.
Article in English | MEDLINE | ID: mdl-18613314

ABSTRACT

In order to achieve high cell densities anchoragedependent cells are commonly cultured on microcarriers, where spatial restrictions to cell growth complicates the determination of the growth kinetics. To design and operate large-scale bioreactors for microcarrier cultures, the effect of this spatial restriction to growth, referred to as contact inhibition, must be decoupled from the growth kinetics. In this article, a cellular automaton approach is recommended to model the growth of anchorage-dependent cells on microcarriers. The proposed model is simple to apply yet provides an accurate representation of contact-inhibited cell growth on microcarriers. The distribution of the number of neighboring cells per cell, microcarrier surface areas, and inoculation densities are taken into account with this model. When compared with experimental data for Vero and MRC-5 microcarrier cultures, the cellular automaton predictions were very good. Furthermore, the model can be used to generate contact-inhibition growth curves to decouple the effect of contact-inhibition from growth kinetics. With this information, the accurate determination of kinetic parameters, such as nutrient uptake rates, and the effects of other environmental factors, such as toxin levels, may be determined. (c) 1994 John Wiley & Sons, Inc.

19.
Biotechnol Bioeng ; 42(3): 357-66, 1993 Jul.
Article in English | MEDLINE | ID: mdl-18613020

ABSTRACT

A two-stage bioreactor scheme was developed for the large-scale production of recombinant proteins using a genetically engineered baculovirus/insect cell system. The first bioreactor was employed for cell growth and the second for cell infection. Silkworm Bm5 cells were infected with a recombinant baculovirus, BmNPV/P5.cat, containing a bacterial chloramphenicol acetyltransferase (CAT) gene under the control of the polyhedrin gene promoter of Bombyx mori nuclear polyhedrosis virus (BmNPV). This recombinant baculovirus has been used as an expression vector for the production of recombinant CAT enzyme. A specific productivity of 82 to 90 microg CAT/(10(6) cells) was obtained using the BmNPV/Bm5 expression system, a yield similar to that achieved using the AcNPV/Sf expression system. Repeated infection of high-density cell cultures did not reduce the specific productivity of the CAT enzyme. Most importantly, the problems associated with the infection of high-density cell cultures were resolved by means of controlled infection conditions and appropriate replenishment of spent culture medium following infection. The glucose uptake rate by the cells following infection was 50% higher than that by the cells before infection. Not only did the infection of high-density cell cultures result in consistent yields of 250 mg/L of CAT enzyme, but also the two-stage bioreactor system was proven to be reliable for a long-term operation beyond 600 h.

20.
Biotechnol Bioeng ; 40(10): 1165-72, 1992 Dec 05.
Article in English | MEDLINE | ID: mdl-18601067

ABSTRACT

An experimental study has been carried out to investigate the effectivenes of several reduced serum and serum-free media for the cultivation of an ovarian cell line, Bm5, of the lepidopteran insect Bombyx mori. Bm5 cell were successfully adapted to grow in a medium containing 5% serum and a serum-free medium (EX-CELL 400). On the other hand, this cell line could not be adapted to grow in several other media suggested in the literature, including IPL-41 + 2% fetal bovine serum (FBS), SF-900, and a serum-free medium (ISFM). Furthermore, a comparative study was conducted to determine the production levels of B. mori nuclear polyhedrosis virus (BmNPV) in Bm5 cells cultured in three different medium formulations. The production levels of BmNPV in adapted Bm5 cells grown in a 5% serum-supplemented medium and a serum-free medium (EX-CELL 400) were comparable to those obtained in Bm5 cells grown 10% serum-supplemented medium.

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