Development and Experimental Validationof a Flmd/Structure-Interaction Finite Element Modelof a Vacuum-Driven Cell Culture Mechanostimulus System

A new fluid/structure-interaction finite element formulation is reported, by means of which reactive fluid stresses can be determined for what is currently the most widely used laboratory apparatus (; the Flexercell Strain Unit ) for delivering controlled in vitro mechanical stimuli to cultured cells. The apparatus functions by means of cyclic vacuum application to the undersurface of a membrane-like circular rubber substrate. When operated in Us original embodiment ( i.e., without axial constraint to substrate motion), the pulsatile vacuum causes appreciable pulsatile excursions ( often several millimeters) of the substrate. The mechanical stimuli experienced by cells attached atop the substrate include not only substrate distention, but also potentially confounding reactive fluid stresses due to coupled motions of the overlying liquid culture nutrient medium. Since it is impractical to directly measure reactive fluid stress in such environments, a corresponding mathematical model has been developed. The formulation involves transient continuum finite element solutions for the nutrient medium flow field and for the deformation of the substrate, coupled at their mutual interface ( the substrate culture surface) Besides the nonlinearities inherent in the flow field and substrate treatments per se, the numerical problem is complicated by the presence of moving boundaries at the nutrient free surface and at the nutrient/substrate interface, as well as by the need to enforce fluid/structure interaction throughout the duty cycle. Algorithmic considerations appropriate to achieving physically realistic numerical performance are reported, and a confirmatory laboratory validation experiment is described.     

Development and Experimental Validation of a Fluid/Structure-Interaction Finite Element Model of a Vacuum-Driven Cell Culture Mechanostimulus System

A new fluid/structure-interaction finite element formulation is reported, by means of which reactive fluid stresses can be determined for what is currently the most widely used laboratory apparatus (the Flexercell Strain Unit) for delivering controlled in vitro mechanical stimuli to cultured cells. The apparatus functions by means of cyclic vacuum application to the undersurface of a membrane-like circular rubber substrate. When operated in its original embodiment (i.e., without axial constraint to substrate motion), the pulsatile vacuum causes appreciable pulsatile excursions (often several millimeters) of the substrate. The mechanical stimuli experienced by cells attached atop the substrate include not only substrate distention, but also potentially confounding reactive fluid stresses due to coupled motions of the overlying liquid culture nutrient medium. Since it is impractical to directly measure reactive fluid stress in such environments, a corresponding mathematical model has been developed. The formulation involves transient continuum finite element solutions for the nutrient medium flow field and for the deformation of the substrate, coupled at their mutual interface (the substrate culture surface). Besides the nonlinearities inherent in the flow field and substrate treatments per se, the numerical problem is complicated by the presence of moving boundaries at the nutrient free surface and at the nutrient/substrate interface, as well as by the need to enforce fluid/structure interaction throughout the duty cycle. Algorithmic considerations appropriate to achieving physically realistic numerical performance are reported, and a confirmatory laboratory validation experiment is described.     

AN AUTOMATIC SYNCHRONIZER FOR SUSPENSION CULTURE OF L5178Y CELLS

The excess thymidine-colcemid synchronization method for mouse leukemia L5178Y was automated by deleting the removal of excess thymidine. The synchronizer adds thymidine and colcemid at predetermined times and makes the population accumulated at mitosis available at a designated time. The apparatus is inexpensive to build, easily operated and applicable to large volumes of a static suspension culture.     

A method for the control of the dissolved oxygen tension in microbial cultures

A method for the control of dissolved oxygen tension in growing microbial cultures is described. The apparatus consists of a motor-driven air sparge pipe which may be lowered or raised to give a variable point of entry of the air stream into the culture liquid and hence a variable gas dispersion and gas–liquid contact time. Control of the sparge pipe position is by means of a feedback control loop consisting of a dissolved oxygen probe, an on/off controller, and a reversing electric motor which drives the sparge pipe. The difficulty presented by the relatively slow response of the oxygen probe has been overcome by incorporating an adjustable rate of control action.     

A mechanical device for the rapid removal and freezing of liver or brain tissue from unanaesthetized and nonparalyzed rats.

A mechanical, pneumatically operated device has been developed that permits withdrawal and freezeclamping of either liver or brain tissue from unanaesthetized nonimmobilized rats. Furthermore, regional sampling from brain is possible. The apparatus works in the following way: By means of rotating knives, two cross-sections are made through a rat in such a way that the slice cut out of the animal between the two knives will contain the desired tissue. This slice is then freezeclamped between precooled aluminium blocks. The sampling procedure works automatically: i.e., when the rat is in position in the apparatus and the precooled aluminium blocks have been mounted on the arms of a pair of cooling tongs, the biopsy is made simply by pressing a button. The time for making a biopsy amounts to 0.10 sec. The brain or liver tissue can be isolated from the freezeclamped slices at the temperature of liquid N₂, using a small buzz saw developed for this purpose.     

Ran control of mitosis in human cells: gradients and local signals

Roles of the GTPase Ran in cell life and division rely on a largely conserved mechanism, i.e. Ran's ability to interact with transport vectors. Modes of control of downstream factors, however, are diversified at particular times of the cell cycle. Specificity and fine-tuning emerge most clearly during mitosis. In the present article, we focus on the distinction between global mitotic control by the chromosomal Ran gradient and specific spatial and temporal control operated by localized Ran network members at sites of the mitotic apparatus in human cells.     

The "rotary column" method for growth of large-scale quantities of cell monolayers

A method and an apparatus that reduce the heavy expenses involved in traditional methods for the large-scale monolayer production of primary and secondary, strain and line cells and of the biologicals derived therefrom are described. The method is based on the principle of gathering in a single unit a sheaf of columns by means of general manifolds fitted with cocks. The growth of cells on the glass walls is irrespective of the number, length, and diameter of the columns used. The apparatus, placed in a thermo-adjustable room and connected to adequate devices which allow it to rotate on its longitudinal axis and to be set in a vertical position, need not be dismounted nor transported since it can be connected by a number of tubes to the necessary services. Sterilization is carried out by flowing steam and fluids are poured in or drained off by vacuum or pressure. A microscope fitted to the bearing structure allows the operator to observe the cell monolayers and the cytopathic effect of viruses on the whole length of the outer columns. During the various working stages pH is under continuous control and automatically adjusted. The whole working cycle is extensively described (cleaning, sterilization, seeding, incubation, trypsinization of the monolayer, culture and harvesting of the virus) and results compared with those obtained by traditional methods.     

Dynamic concentration challenges for biosensor characterization

A method and apparatus are described for characterization of the steady state and dynamic response of biosensors. The apparatus produces a steady stream of homogeneously mixed analyte whose concentration can be fixed at discrete values or varied continously. The device is ideally suited for continously operated biosensors, but is also effective for biosensors that operate in discrete sampling modes. The system permits simultaneous testing of several sensors and determination of the accuracy, precision and repeatability of sensor response. The characteristics of this testing apparatus were validated with ferrocyanide and glucose as indicators. As an example of use of the apparatus, concentration ramps were created and used to complement conventional step changes for characterizing an implantable glucose sensor. The ramp rate can be adjusted easily by scaling the apparatus to simulate the rate of concentration change anticipated during actual monitoring situations.     

Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation

Migration and proliferation have been mostly explored in culture systems or fixed preparations. We present a simple genetic model, the chains of glia moving along fly wing nerves, to follow such dynamic processes by time-lapse in the whole animal. We show that glia undergo extensive cytoskeleton and mitotic apparatus rearrangements during division and migration. Single cell labelling identifies different glia: pioneers with high filopodial, exploratory, activity and, less active followers. In combination with time-lapse, altering this cellular environment by genetic means or cell ablation has allowed to us define the role of specific cell-cell interactions. First, neurone-glia interactions are not necessary for glia motility but do affect the direction of migration. Second, repulsive interactions between glia control the extent of movement. Finally, autonomous cues control proliferation.     

Mitotic apparatus-surface interaction and cell division

Summary

Results of recent investigations concerning the mechanisms of animal cell division are reviewed. The mitotic apparatus was aspirated from a blastomere of a sand dollar (Echinarachnius parma) egg before second cleavage, and the time interval between removal and the appearance of the furrow in the control companion blastomere was measured. When the mitotic apparatus is removed 4 min or less before the furrows appear in the controls, furrows also develop in the operated cells. These results show that 4 min before furrowing begins, the surface changes which lead to formation of the division mechanism have become irreversible. When the mitotic apparatus of a cylindrical cell is shifted by pushing in one of the poles when the furrow appears, a new furrow develops in association with the new position of the mitotic apparatus. The same mitotic apparatus could elicit as many as 13 furrows over a 24.5 min period following the appearance of the first furrow. The results show that, in the proper geometrical circumstances, the mitotic apparatus and the surface can interact over a longer period than they do in normal cells.

By artificially constricting sand dollar eggs with a glass loop, the normal distance relations between the astral centers and the polar and equatorial surfaces can be reversed. Constricted cells cleave normally. The blocking effect of ethyl urethane can be reversed by moving the equatorial surface closer to the spindle portion of the mitotic apparatus. Relocation of other parts of the surface closer to the mitotic apparatus was ineffective. These results help elucidate the geometrical relations that are essential for furrow formation between the mitotic apparatus and the surface.

In cylindrical sand dollar eggs, single asters and the widely separated asters of a broken mitotic apparatus can cause furrow-like constrictions in the adjacent cylindrical surface. This reaction can be blocked by treating cells with ethyl urethane, which reduces astral size. The nature of the shape change that the aster causes depends upon the surface region affected. These results aid in understanding the nature of the change in surface physical activity caused by the mitotic apparatus.     

A versatile primate cage for a multiple use facility.

A mobile-transport-squeeze cage for primates was constructed. The cage contained a clear plastic guillotine door, squeeze apparatus, ventilation holes and locking casters. The squeeze apparatus could be operated by one person.     

The cyclone column culture vessel for batch and continuous,synchronous or asynchronous,culture of microorganisms

A simple inexpensive apparatus with a working volume of 10 liters of culture is described. Details of construction and procedures for operation of the cyclone column vessel are given. The vessel is self-contained, so that experimental parameters of temperature and aeration are individually controlled; homogeneous mixing and representative sampling of the culture, besides control of foam without need for antifoam, are obtained. The vessel may be used in single or multistage systems for aerobic or anaerobic cultivation of organisms in batch, chemostat, or phased cultures.     

Continuous Culture of Ruminal Microorganisms in Chemically Defined Medium: I. Design of Continuous-Culture Apparatus

An apparatus is described which has been used for successful continuous culture of the ciliates from the rumen of cattle. Automatic control of feeding rate, pH, oxidation-reduction potential, temperature, stirring rate, aeration rate, salinity, and volume of the culture is provided for, using standard commercial equipment, whenever possible. The operation of this apparatus is described.     

Analytical scanning isoelectrofocusing. 4. Design and operation of a column-filling apparatus

An apparatus was designed and constructed for filling an analytical isoelectrofocusing column in sequence with electrolytes, sucrose density gradient and sample. The column-filling apparatus (CFA) consists of a series of valves and syringes, a density gradient mixer, a syringe microburet operated at constant speed, and Teflon tubing. The sample is injected at any position of the density gradient with a microliter syringe through an injection port. Possibilities for complete automation of this device are discussed.     

On-line HPLC-measurement and control of substrate in a continuously operated biological tank reactor

This paper presents a concept for the measurement and control of the substrate concentration in a continuously operated biological tank reactor (CBTR). The concept was successfully applied in the degradation of Naphthalenesulfonic acid by the culture Pseudomonas testosteroni. The measurement of the substrate concentration was carried out by means of an automated HPLC (high performance liquid chromatography) analysis of the contents of the reactor. The methods of cyclic sampling, sample filtration and evaluation of the chromatograms during the process control are explained. In addition the characteristics of the measuring system (linearity of measurement, noise disturbance data, sampling rates, transient behavior) are described in detail. The feed-back control of the substrate concentration was carried out by variations in the dilution rate with a constant substrate feed concentration. The control results were achieved with a controller which was specially developed to be used in connection with automated cyclic analysis processes. The control algorithm is explained and the conditions necessary for maintaining stability of the control loop are given.     

A Rocking Table for Staining Electrophoretic Gels, Nitrocellulose Blots and Histological Slides

A rocking table has been designed for gentle cyclic agitation of electrophoretic gels, nitrocellulose blots and microscope slides during staining (Fig. 1). It is particularly useful for long staining periods or when a small volume of staining solution is to be used. Even under conditions where gels or slides, laid flat, are not fully submerged when the staining vessel is horizontal, staining is consistently even and satisfactory. The apparatus is simple and inexpensive to construct, easily operated and quiet during use. It can be operated over a temperature range from —20 to +60 C. The rocking speed is continuously variable from 4 to 14 cycles per minute. This allows precise control to avoid gross disturbance of the materials being stained, or splashing of the stain solution in different containers.     

A culture apparatus for maintaining H2 at sub-nanomolar concentrations

We devised a microbial culture apparatus capable of maintaining sub-nanomolar H2 concentrations. This apparatus provides a method for study of interspecies hydrogen transfer by externally fulfilling the thermodynamic requirement for low H2 concentrations, thereby obviating the need for use of cocultures to study some forms of metabolism. The culture vessel is constructed of glass and operates by sparging a liquid culture with purified gases, thereby removing H2 as it is produced. We used the culture apparatus to decouple a syntrophic association in an ethanol-consuming, methanogenic enrichment culture, allowing ethanol oxidation to dominate methane production. We also used the culture apparatus to grow pure cultures of the ethanol-oxidizing, proton-reducing Pelobacter acetylenicus (WoAcy 1), and to study the bioenergetics of growth.     

Oxygen-absorption rate-controlled feeding of substrate into aerobic microbial cultures

A system of automatic control of substrate inflow into an aerated culture of microorganisms which depend on oxygen-absorption rate (OAR) has been devised and tested. As the control variable, dissolved oxygen concentration (DOC), which shows the equilibrium between OAR and oxygen-uptake rate in the microbial culture, was chosen. If the equilibrium is disturbed by changes in OAR, then the oxygen-uptake rate is changed by substrate limitation. The DOC is measured by means of a Clark-type polarographic electrode, and the signal is used to actuate the substrate inflow valve or pump. The oxygen-uptake rate changes of microorganisms, after the addition or exhaustion of substrate in the medium, are so rapid that they can be used for this type of control. Fundamental equations were derived and graphical solutions for the control system parameters were suggested for the steady-state relations between DOC, oxygen-uptake rate, specific growth rate, substrate concentration, K_La, and concentration of microorganisms. The system is stable in the entire range of the uptake rates up to nearly the maximum attainable in unlimited substrate conditions, and can be operated in batch feed or continuous flow modifications. It was experimentally tested with the yeast Saccharomyces cerevisiae. The complete utilization of aeration-system capacity of the fermentor was achieved with high yeast yield and no alcohol formation. The quality of the product was excellent.     

A system identification approach for developing model predictive controllers of antibody quality attributes in cell culture processes

As the biopharmaceutical industry evolves to include more diverse protein formats and processes, more robust control of Critical Quality Attributes (CQAs) is needed to maintain processing flexibility without compromising quality. Active control of CQAs has been demonstrated using model predictive control techniques, which allow development of processes which are robust against disturbances associated with raw material variability and other potentially flexible operating conditions. Wide adoption of model predictive control in biopharmaceutical cell culture processes has been hampered, however, in part due to the large amount of data and expertise required to make a predictive model of controlled CQAs, a requirement for model predictive control. Here we developed a highly automated, perfusion apparatus to systematically and efficiently generate predictive models using application of system identification approaches. We successfully created a predictive model of %galactosylation using data obtained by manipulating galactose concentration in the perfusion apparatus in serialized step change experiments. We then demonstrated the use of the model in a model predictive controller in a simulated control scenario to successfully achieve a %galactosylation set point in a simulated fed‐batch culture. The automated model identification approach demonstrated here can potentially be generalized to many CQAs, and could be a more efficient, faster, and highly automated alternative to batch experiments for developing predictive models in cell culture processes, and allow the wider adoption of model predictive control in biopharmaceutical processes. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 33:1647–1661, 2017     

Space bioreactors: their use,their future

The use of bioreactors during space flight is discussed. The major elements of a bioreactor are a culture chamber, sensors, a control unit with feedback, as gas exchange system, a pump, fresh culture medium, and a waste reservoir. Types of bioreactors developed for use in space include the Woodlawn Wanderer 9 apparatus, the Space tissue loss system, rotating wall vessel, dynamic cell culture system and the SBR I. Future development for space bioreactors include improvements for cultivation of mammalian cells and tissue engineering and the transfer of bioreactor technology for earth-bound instruments.