Online- and offline- monitoring of stem cell expansion on microcarrier.

In the biopharmaceutical industry, adherent growing stem cell cultures gain worldwide importance as cell products. The cultivation process of these cells, such as in stirred tank reactors or in fixed bed reactors, is highly sophisticated. Cultivations need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time on-line monitoring tools are recommended. A tool meeting the new requirements may be the dielectric spectroscopy for online viable cell mass determination by measurement of the permittivity. To establish these tools, proper offline methods for data correlation are required. The cell number determination of adherent cells on microcarrier is difficult, as it requires cell detachment from the carrier, which highly increases the statistical error. As an offline method, a fluorescence assay based on SYBR(®)GreenI was developed allowing fast and easy total cell concentration determination without the need to detach the cells from the carrier. The assay is suitable for glass carriers used in stirred tank reactor systems or in fixed bed systems, may be suitable for different cell lines and can be applied to high sample numbers easily. The linear dependency of permittivity to cell concentration of suspended stem cells with the dielectric spectroscopy is shown for even very small cell concentrations. With this offline-method, a correlation of the cell concentration grown on carrier to the permittivity data measured by the dielectric spectroscopy was done successfully.

Process control in cell culture technology using dielectric spectroscopy.

In the biopharmaceutical industry, mammalian and insect cells as well as plant cell cultures are gaining worldwide importance to produce biopharmaceuticals and as products themselves, for example in stem cell therapy. These highly sophisticated cell-based production processes need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time in-line monitoring tools are now recommended. Dielectric spectroscopy (DS) can serve as a tool to satisfy some PAT requirements. DS has been used in the medical field for quite some time and it may allow real-time process monitoring of biological cell culture parameters. DS has the potential to enable process optimization, automation, cost reduction, and a more consistent product quality. Dielectric spectroscopy is reviewed here as a tool to monitor biochemical processes. Commercially available dielectric sensing systems are discussed. The potential of this technology is demonstrated through examples of current and potential future applications in research and industry for mammalian and insect cell culture.

Use of Encapsulated Stem Cells to Overcome the Bottleneck of Cell Availability for Cell Therapy Approaches.

Nowadays cell-based therapy is rarely in clinical practice because of the limited availability of appropriate cells. To apply cells therapeutically, they must not cause any immune response wherefore mainly autologous cells have been used up to now. The amount of vital cells in patients is limited, and under certain circumstances in highly degenerated tissues no vital cells are left. Moreover, the extraction of these cells is connected with additional surgery; also the expansion in vitro is difficult. Other approaches avoid these problems by using allo-or even xenogenic cells. These cells are more stable concerning their therapeutic behavior and can be produced in stock. To prevent an immune response caused by these cells, cell encapsulation (e.g. with alginate) can be performed. Certain studies showed that encapsulated allo- and xenogenic cells achieve promising results in treatment of several diseases. For such cell therapy approaches, stem cells, particularly mesenchymal stem cells, are an interesting cell source. This review deals on the one hand with the use of encapsulated cells, especially stem cells, in cell therapy and on the other hand with bioreactor systems for the expansion and differentiation of mesenchymal stem cells in reproducible and sufficient amounts for potential clinical use.

Production process for stem cell based therapeutic implants: expansion of the production cell line and cultivation of encapsulated cells.

Cell based therapy promises the treatment of many diseases like diabetes mellitus, Parkinson disease or stroke. Microencapsulation of the cells protects them against host-vs-graft reactions and thus enables the usage of allogenic cell lines for the manufacturing of cell therapeutic implants. The production process of such implants consists mainly of the three steps expansion of the cells, encapsulation of the cells, and cultivation of the encapsulated cells in order to increase their vitality and thus quality. This chapter deals with the development of fixed-bed bioreactor-based cultivation procedures used in the first and third step of production. The bioreactor system for the expansion of the stem cell line (hMSC-TERT) is based on non-porous glass spheres, which support cell growth and harvesting with high yield and vitality. The cultivation process for the spherical cell based implants leads to an increase of vitality and additionally enables the application of a medium-based differentiation protocol.

Expansion and chondrogenic differentiation of human mesenchymal stem cells.

The potential of human mesenchymal stem cells (hMSC) to differentiate into various types of mesenchymal tissue, such as chondrocytes, makes them a potential cell source in cartilage tissue engineering. Because of the requirement of high cell amounts for the generation of cartilage implants or for the extensive experimental studies to investigate the culture parameters, the initial cells have to be expanded, which leads to high population doubling numbers. It is known that hMSC can differentiate into chondrocytes at least up to the 15th population doubling. To monitor the differentiation status, the protein MIA (melanoma inhibitory activity), which is only synthesized by malignant melanomas and chondrocytes, can be used. In this study the chondrogenic differentiation potential of hMSC beyond the 15th population doubling was investigated using MIA as a chondrocyte marker. A chondrogenic potential of hMSC at higher population doubling numbers may be of interest due to the requirement of less frequent isolations of cells. Therefore hMSC were cultured in a monolayer until the 37th population doubling. Cells of different passages were cultured as pellets for two weeks in transforming growth factor (TGF)-beta3 containing differentiation medium. The MIA contents in medium on the last three cultivation days were measured for each case using an MIA-ELISA-kit. A significant difference between MIA content in medium of the pellet and nonstimulated monolayer reference cultures was detectable until the 32nd population doubling. In addition, the hMSC were seeded at lower densities to investigate whether the cells may be expanded faster and with less amount of work due to higher population doubling numbers per passage. The reduced inoculation density led to an increased growth rate.

New generation ceramic membranes have the potential of removing endotoxins from dialysis water and dialysate.

Poor water properties, use of concentrated bicarbonate, and biofilm growth in pipes and storage tanks often cause dialysis water and dialysate contamination with bacteria and endotoxins. High-flux dialysis with bicarbonate may favor endotoxin transfer from the dialysate into the blood exposing patients to serious short-and long-term side effects. Ultrafiltration across hydrophobic synthetic membranes effectively removes endotoxins from dialysis water by combined filtration and adsorption. However, repeated sterilization worsens the membrane separation properties,and limits their use. Ceramic membranes are generally more resistant to harsh operating conditions than polymeric membranes, and may represent an alternative for endotoxin removal. Previously, we proved that the ceramic membranes commercially available at that time were not retentive enough to ensure production of endotoxin-free dialysis water. In this paper, we investigated the endotoxin removal capacity of new generation commercial ceramic membranes with nominal molecular weight cut-off down to 1,000. In dead-end filtration, all investigated membranes produced water meeting, the European standards, or close to,when challenged with low endotoxin concentrations, but only one membrane type succeeded at high endotoxin concentrations. In cross-flow filtration, none produced water meeting the European standard. Moreover, sterilization and rinsing procedures altered the separation properties of two out of three membrane types.

Feasibility of continuous CO2 removal with hydrophilic membranes at low blood flow rates.

In the conventional treatment of acute respiratory distress syndrome (ARDS), high O2 concentrations and mechanical ventilation may damage the lung tissue. Extracorporeal membrane oxygenation limits damage, provides the needed O2 supply and improves survival of ARDS neonates, but not of adults. Hydrophilic membranes used in hemodialysis are more non-thrombogenic and biocompatible than those used in blood oxygenation, but their O2 transport capacity is not as high. In recent years, CO2 removal at low blood flow rates combined with apneic oxygenation and low frequency ventilation has proved promising in the treatment of ARDS. This approach makes O2 supply across ECMO membranes unnecessary; it also makes hydrophilic membranes candidates for extracorporeal CO2 removal to minimize anticoagulation and immune system activation. This paper reports on the in vitro capacity of hydrophilic polysulphone membranes to remove CO2 from carbonated pig blood into an oxygen-rich gas stream. Experiments were performed on clinical-size dialysis modules and their capacity to remove CO2 as a function of blood flow rate and membrane surface area was investigated. Membranes effectively removed CO2 , more so at increasing blood flow rates and membrane surface areas, at rates of up to 15% of the CO2 metabolic production rate. The specific CO2 removal rate was comparable to that of blood oxygenators equipped with microporous hydrophobic membranes. It is concluded that CO2 removal from slowly flowing blood with hydrophilic membranes is feasible.

A stimulation unit for the application of mechanical strain on tissue engineered anulus fibrosus cells: a new system to induce extracellular matrix synthesis by anulus fibrosus cells dependent on cyclic mechanical strain.

A bioreactor system consisting of a multifunctional stimulation unit and common 6-well culture plate is introduced to activate extracellular matrix synthesis in intervertebral disc cells due to cyclic mechanical strain. The developed stimulation unit is sterilizable and reusable. It is viable for cultivation and mechanical stimulation of cartilage tissue and tissue engineered cell matrix constructs in combination with the common 6-well culture plate. The custom made device allows long-term cultivations in batch- or continuous operation mode. Manual handling and thereby the risk of contamination is reduced. Sampling, changing the medium, and addition of supplements are easily performed from the connected conditioning vessel. This bioreactor system enables stimulation of different samples independently during one run. For the work presented here anulus fibrosus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs. Over a period of 14 days the constructs were subjected to 10% compression under cyclic mechanical pressure with a frequency of 0.1 Hz. Afterwards the constructs were biochemically examined for hydroxyproline and sulphated glycosaminoglycanes. These proven constituents of extracellular matrix were found to be released depending on the applied compressive strain.

Hydrodynamic stimulation and long term cultivation of nucleus pulposus cells: a new bioreactor system to induce extracellular matrix synthesis by nucleus pulposus cells dependent on intermittent hydrostatic pressure.

A novel bioreactor system was constructed to induce extracellular matrix (ECM) synthesis by intervertebral disc (ID) cells due to intermittent hydrostatic pressure. The developed system is completely sterilizable and reusable. It is viable for cultivation, immobilization, and stimulation of various other cell types and tissues especially for cartilage. The custom made lid allows long-run cultivation through semi-continuous operation. Manual interferences and therefore the risk of contamination are reduced. Sampling, medium changing and addition of supplements are easily performed from the connected conditioning vessel, which could be placed in an incubator. For the present investigations nucleus pulposus cells from pigs were taken and immobilized in agarose to obtain three-dimensional cell matrix constructs which were subjected to intermittent hydrostatic pressure. Afterwards the construct was biochemically examined. The proven constituents of ECM were found to be released in dependence of the magnitude and profile of the applied pressure.

Lipase-catalyzed synthesis of geranyl acetate in n-hexane with membrane-mediated water removal.

The esterification of geraniol with acetic acid in n-hexane was investigated. A commercial lipase preparation from Candida antarctica was used as catalyst. The equilibrium conversion (no water removal) was found to be 94% for the reaction of 0.1 M alcohol and 0.1 M acid in n-hexane at 30 degrees C. This was shown by both hydrolysis and esterification reactions. The activation energy of reaction over the temperature range 10 degrees to 50 degrees C was found to be 16 kJ/mol. The standard heat of reaction was -28 kJ/mol. Membrane pervaporation using a cellulose acetate/ceramic composite membrane was then employed for selective removal of water from the reaction mixture. The membrane was highly effective at removing water while retaining all reaction components. Negligible transport of the solvent n-hexane was observed. Water removal by pervaporation increased the reaction rate by approximately 150% and increased steady-state conversion to 100%.

Membranes for endotoxin removal from dialysate: considerations on feasibility of commercial ceramic membranes.

As the quality of water in dialysis fluid varies considerably, dialysate is often contaminated by large amounts of bacteria and endotoxins. Membrane properties and operating pressures are acknowledged to give high-flux dialysis with bicarbonate the bacteriological potential to favor passage of endotoxin fragments from the dialysate into the blood stream. Therefore, a sterile dialysate will have to become a standard. Ultrafiltration across hydrophobic synthetic membranes was shown to remove endotoxins (and their fragments) from dialysis water by the combined effect of filtration and adsorption. However, each module can be used for a limited time only. Ceramic membranes may represent an alternative to polymeric membranes for endotoxin removal. In this article, we tested the capacity of different commercial ceramic membranes with nominal molecular weight cut-off down to 1,000 to retain endotoxins from Ps. aeruginosa. The tested membranes did not generally produce dialysate meeting the Association for the Advancement of Medical Instrumentation standard. When using aluminum-containing membranes, we detected aluminum leaking into the dialysate that could possibly be transported into the blood stream.