Ales Prokop - Academia.edu (original) (raw)

Papers by Ales Prokop

An apparatus and methods for monitoring the status of a cell that consumes oxygen. In one embodim... more An apparatus and methods for monitoring the status of a cell that consumes oxygen. In one embodiment of the present invention, the method includes the steps of confining the cell in a sensing volume, measuring dynamically intracellular or extracellular signaling of the cell, and determining the status of the cell from the measured intracellular or extracellular signaling of the cell.

An apparatus and methods for using biological material to discriminate an agent. In one embodimen... more An apparatus and methods for using biological material to discriminate an agent. In one embodiment of the present invention, the method includes the steps of providing at least one cell, exposing at least one cell to an agent, measuring the response of the cell to the agent in terms of a physical quantity related to at least one of the cellular physiological activities of the cell, and identifying the agent from the measured response. The method further includes the step of quantifying the agent from the measured response.

Journal of industrial microbiology & biotechnology, 2010

Journal of Vascular Surgery, 2000

Journal of Electroanalytical Chemistry, 2006

Analytical Chemistry, 2004

A microphysiometer capable of measuring changes in extracellular glucose, lactate, oxygen, and ac... more A microphysiometer capable of measuring changes in extracellular glucose, lactate, oxygen, and acidification rate has been developed by incorporating modified electrodes into a standard Cytosensor Microphysiometer plunger. Glucose and lactate are measured indirectly at platinum electrodes by amperometric oxidation of hydrogen peroxide, which is produced from catalysis of glucose and lactate at films containing their respective entrapped oxidase. Oxygen is measured amperometrically at a platinum electrode coated with a Nafion film, while the acidification rate is measured potentiometrically by a Cytosensor Microphysiometer. Analytical information is obtained during the Cytosensor stop-flow cycles, where the electrodes measure changes in the extracellular medium corresponding to the consumption or production of the analyte by the cells. Modification of the Cytosensor plunger for multianalyte determination is described, and the operation of the technique is illustrated by the simultaneous measurement of all four analytes during the addition of fluoride and DNP to Chinese hamster ovary cells and fluoride and antimycin A to mouse fibroblast cells. Cell metabolic recovery and dynamics after exposure to agents can also be observed in specific cases.

Analytica Chimica Acta, 2003

The Cytosensor™ microphysiometer has been modified by incorporating several platinum electrodes i... more The Cytosensor™ microphysiometer has been modified by incorporating several platinum electrodes into the plunger head. The electrodes have been used to measure simultaneously the change in extracellular oxygen consumption rates and extracellular acidification rates during addition of various non-physiologic agents: 2-deoxy-d-glucose, sodium fluoride, paraoxon-ethyl, and antimycin A. Measurement of two parameters, oxygen and acidification, enables investigation of the aerobic and anaerobic metabolic consequences of these agents. Nafion membranes cast onto the platinum electrodes were useful in minimizing adsorption of cell-related interferents.

A bioreactor for cultivating living cells in a liquid medium. In one embodiment of the present in... more A bioreactor for cultivating living cells in a liquid medium. In one embodiment of the present invention, the bioreactor includes a first substrate having a first surface, an opposite second surface and edges. The bioreactor further includes a second substrate having a first surface and an opposite second surface, defining a cavity with a bottom surface, where the bottom surface is located therebetween the first surface and the second surface. The first surface of the first substrate is received by the second surface of the second substrate to cover the ...

Biomedical Microdevices, 2004

There is a need for microminiaturized cell-culture environments, i.e. NanoLiter BioReactors (NBRs... more There is a need for microminiaturized cell-culture environments, i.e. NanoLiter BioReactors (NBRs), for growing and maintaining populations of up to several hundred cultured mammalian cells in volumes three orders of magnitude smaller than those contained in standard multi-well screening plates. These devices would enable the development of a new class of miniature, automated cell-based bioanalysis arrays for monitoring the immediate environment of multiple cell lines and assessing the effects of drug or toxin exposure. We fabricated NBR prototypes, each of which incorporates a culture chamber, inlet and outlet ports, and connecting microfluidic conduits. The fluidic components were molded in polydimethylsiloxane (PDMS) using soft-lithography techniques, and sealed via plasma activation against a glass slide, which served as the primary culture substrate in the NBR. The input and outlet ports were punched into the PDMS block, and enabled the supply and withdrawal of culture medium into/from the culture chamber (10-100 nL volume), as well as cell seeding. Because of the intrinsically high oxygen permeability of the PDMS material, no additional CO 2 /air supply was necessary. The developmental process for the NBR typically employed several iterations of the following steps: Conceptual design, mask generation, photolithography, soft lithography, and proof-of-concept culture assay. We have arrived at several intermediate designs. One is termed "circular NBR with a central post (CP-NBR)," another, "perfusion (grid) NBR (PG-NBR)," and a third version, "multitrap (cage) NBR (MT-NBR)," the last two providing total cell retention. Three cells lines were tested in detail: a fibroblast cell line, CHO cells, and hepatocytes. Prior to the culturing trials, extensive biocompatibility tests were performed on all materials to be employed in the NBR design. To delineate the effect of cell seeding density on cell viability and survival, we conducted separate plating experiments using standard culture protocols in well-plate dishes. In both experiments, PicoGreen assays were used to evaluate the extent of cell growth achieved in 1-5 days following the seeding. Low seeding densities resulted in the absence of cell proliferation for some cell lines because of the deficiency of cell-cell and extracellular matrix (ECM)-cell contacts. High viabilities were achieved in all designs. We conclude that an instrumented microfluidics-based NanoBioReactor (NBR) will represent a dramatic departure from the standard culture environment. The employment of NBRs for mammalian cell culture opens a new paradigm of cell biology, so far largely neglected in the literature.

Principles of Tissue Engineering, 2014

There are significant engineering challenges in translating gene and nucleic acid delivery from c... more There are significant engineering challenges in translating gene and nucleic acid delivery from cell and animal models into the clinic. Off-target effects and inefficient delivery to the proper intracellular compartment of the targeted cells are major obstacles to success. Systemic delivery of any viral or nonviral vector requires an appreciation of the adverse physiological barriers that exist in vivo and incorporation of well-designed vector components that limit nonspecific uptake and accelerated clearance. In addition, the genetic engineer should consider the design criteria related to cell-specific action. For example, cell-surface recognition can increase the therapeutic index of a DNA- or RNA-based medication. Finally, one must consider the mechanism of cellular internalization, and investigators should attempt to target pathways and incorporate vector functionalities that will mediate trafficking to the subcellular compartment that is optimal for activity of the nucleic acid...

Growth in the pharmaceutical market has slowed down almost to a standstill. One reason is that go... more Growth in the pharmaceutical market has slowed down almost to a standstill. One reason is that governments and other payers are cutting costs in a faltering world economy. But a more fundamental problem is the failure of major companies to discover, develop and market new drugs. Major drugs losing patent protection or being withdrawn from the market are simply not being replaced by new therapies the pharmaceutical market model is no longer functioning effectively and most pharmaceutical companies are failing to produce the innovation needed for success. This multi-authored new book looks at a vital strategy which can bring innovation to a market in need of new ideas and new products: Systems Biology (SB). Modeling is a significant task of systems biology. SB aims to develop and use efficient algorithms, data structures, visualization and communication tools to orchestrate the integration of large quantities of biological data with the goal of computer modeling. It involves the use of computer simulations of biological systems, such as the networks of metabolites comprise signal transduction pathways and gene regulatory networks to both analyze and visualize the complex connections of these cellular processes. SB involves a series of operational protocols used for performing research, namely a cycle composed of theoretical, analytic or computational modeling to propose specific testable hypotheses about a biological system, experimental validation, and then using the newly acquired quantitative description of cells or cell processes to refine the computational model or theory.

$Research paper thumbnail of Developing an objective function to characterize the tradeoffs in salting out and the foam and droplet fractionation processes$

Brazilian Journal of Chemical Engineering, 2000

There are many methods for separating and purifying proteins from dilute solutions, such as salti... more There are many methods for separating and purifying proteins from dilute solutions, such as salting out/precipitation, adsorption/chromatography, foam fractionation, and droplet fractionation. In order to determine the optimal condition for a selected separation and purification process, an objective function is developed. The objective function consists of three parameters, which are the protein mass recovery, the separation ratio, and the enzymatic activity ratio. In this paper the objective function is determined as a function of the pH of the bulk solution for egg albumin, cellulase, and sporamin (for foam fractionation) and invertase ( for droplet fractionation). It is found that the optimal pH for all the systems except for cellulase is near their isoelectric point.

Life

These days many leading scientists argue for a new paradigm for cancer research and propose a com... more These days many leading scientists argue for a new paradigm for cancer research and propose a complex systems-view of cancer supported by empirical evidence. As an example, Thea Newman (2021) has applied “the lessons learned from physical systems to a critique of reductionism in medical research, with an emphasis on cancer”. It is the understanding of this author that the mesoscale constructs that combine the bottom-up as well as top-down approaches, are very close to the concept of emergence. The mesoscale constructs can be said to be those effective components through which the system allows itself to be understood. A short list of basic concepts related to life/biology fundamentals are first introduced to demonstrate a lack of emphasis on these matters in literature. It is imperative that physical and chemical approaches are introduced and incorporated in biology to make it more conceptually sound, quantitative, and based on the first principles. Non-equilibrium thermodynamics is...

Advances in Polymer Science, 1998

The use of polymeric nanoparticles as drug carriers is receiving an increasing amount of attentio... more The use of polymeric nanoparticles as drug carriers is receiving an increasing amount of attention both in academia and industry. The development of suitable delivery systems for protein drugs with high molecular weights and short half-lives is of current interest. In addition, nanoparticles have a number of potential applications in drug and vaccine delivery as well as gene therapy applications. This article features a new production technology for nanoparticles comprised of multicomponent polymeric complexes that are candidates for delivery vehicles of biological molecules such as proteins and drugs. Materials science theory and practice provide the basis for the development of highly compacted structures that are insoluble in water and buffered media. Biocompatible and mostly natural polymers are fabricated into thermodynamically stable nanoparticles, in the absence of organic solvents, using two types of processing: batch and continuous. Careful choice of construction materials and the superposition of several interacting principles during their production allow for the customization of the physicochemical properties of the structures. Among the typical polymers used to assemble nanoparticles, different polysaccharides, natural amines and polyamines were investigated. The entrapped substances tested included proteins, antigens and small drug molecules. The size and charge of nanoparticles is considered to be of primary importance for application in biological systems. Detailed experiments in batch and continuous systems allowed time-dependent stoichiometric characterization of the production process and an understanding of fundamental assembly principles of such supramolecular structures. Continuous-flow production is shown to provide more consistent data in terms of product quality and consistency, with further possibilities of process development and commercialization. To control permeability, polydextran aldehyde, incorporated into the particle core, was used to enable physiologic cross-linking and long-term retention of substances that would otherwise rapidly leak out of the nanoparticles. Results of cross-linking experiments clearly demonstrated that the release rate could be substantially reduced, depending on the degree of cross-linking. For vaccine antigen delivery tests we measured an antibody production following subcutaneous and oral administration. The data indicated that only the cross-linked antigen was immunogenic when the oral route of administration was used. The data presented in this paper address primarily the utility of nanoparticulates for oral delivery of vaccine antigen. This novel technology is extensively discussed in contrast to other technologies, primarily water-and organic solvent-based. The usefulness is demonstrated using several examples, evaluating protein and small drug delivery.

Advances in Polymer Science

Microcapsules have been prepared via a complex coacervation reaction from polyanion and polycatio... more Microcapsules have been prepared via a complex coacervation reaction from polyanion and polycation mixtures. Multicomponent blends of synthetic, semi-synthetic and naturally occurring macromolecules have been evaluated with a particular interest in the preparation of immunoisolation barriers for pancreatic islets. A screening has resulted in thirty three polymeric systems which have been compared according to their mechanical strength, capsule characteristics (such