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36 records · Page 2

Ultra-Sharp Nanowire Arrays Natively Permeate, Record, and Stimulate Intracellular Activity in Neuronal and Cardiac Networks

Intracellular access with high spatiotemporal resolution can enhance the understanding of how neurons or cardiomyocytes regulate and orchestrate network activity and how this activity can be affected with pharmacology or other interventional modalities. Nanoscale devices often employ electroporation to transiently permeate the cell membrane and record intracellular potentials, which tend to decrease rapidly with time. Here, one reports innovative scalable, vertical, ultrasharp nanowire arrays that are individually addressable to enable long-term, native recordings of intracellular potentials. One reports electrophysiological recordings that are indicative of intracellular access from 3D tissue-like networks of neurons and cardiomyocytes across recording days and that do not decrease to extracellular amplitudes for the duration of the recording of several minutes. The findings are validated with cross-sectional microscopy, pharmacology, and electrical interventions. The experiments and simulations demonstrate that the individual electrical addressability of nanowires is necessary for high-fidelity intracellular electrophysiological recordings. This work advances the understanding of and control over high-quality multichannel intracellular recordings and paves the way toward predictive, high-throughput, and low-cost electrophysiological drug screening platforms.

36 MATERIALS SCIENCE↗

Intrinsically stretchable electrode array enabled in vivo electrophysiological mapping of atrial fibrillation at cellular resolution

Electrophysiological mapping of chronic atrial fibrillation (AF) at high throughput and high resolution is critical for understanding its underlying mechanism and guiding definitive treatment such as cardiac ablation, but current electrophysiological tools are limited by either low spatial resolution or electromechanical uncoupling of the beating heart. To overcome this limitation, here we introduce a scalable method for fabricating a tissue-like, high-density, fully elastic electrode (elastrode) array capable of achieving real-time, stable, cellular level-resolution electrophysiological mapping in vivo. Testing with acute rabbit and porcine models, the device is proven to have robust and intimate tissue coupling while maintaining its chemical, mechanical, and electrical properties during the cardiac cycle. The elastrode array records epicardial atrial signals with comparable efficacy to currently available endocardial-mapping techniques but with 2 times higher atrial-to-ventricular signal ratio and >100 times higher spatial resolution and can reliably identify electrical local heterogeneity within an area of simultaneously identified rotor-like electrical patterns in a porcine model of chronic AF.

60 APPLIED LIFE SCIENCES↗

Evaluation of an in vitro three-dimensional HepaRG spheroid model for genotoxicity testing using the high-throughput CometChip platform

Three-dimensional (3D) culture systems are increasingly being used for genotoxicity studies due to improved cell-to-cell interactions and tissue-like structures that are limited or lacking in 2D cultures. The present study optimized a 3D culture system using metabolically competent HepaRG cells for in vitro genotoxicity testing. 3D HepaRG spheroids, formed in 96- or 384-well ultra-low attachment plates, were exposed to various concentrations of 34 test articles, including 8 direct-acting and 11 indirect-acting genotoxicants/carcinogens as well as 15 compounds that show different genotoxic responses in vitro and in vivo. DNA damage was evaluated using the high-throughput CometChip assay with concurrent cytotoxicity assessment by the ATP assay in both 2D and 3D cultures. 3D HepaRG spheroids maintained a stable phenotype for up to 30 days with higher levels of albumin secretion, cytochrome P450 gene expression, and enzyme activities compared to 2D cultures. 3D spheroids also demonstrated a higher sensitivity than 2D cultures for detecting both direct- and indirect-acting genotoxicants/carcinogens, indicating a better prediction of in vivo genotoxicity responses. When DNA damage dose-response data were quantified using PROAST software, 3D spheroids generally had lower or similar benchmark dose values compared to 2D HepaRG cells and were more comparable with primary human hepatocytes. These results demonstrate that 3D models can be adapted to the CometChip technology for high-throughput genotoxicity testing and that 3D HepaRG spheroids may be used as a reliable and pragmatic in vitro approach to better support the hazard identification and risk assessment of potential human genotoxic carcinogens.

60 APPLIED LIFE SCIENCES↗

Growing And Assembling Cells Into Tissues

Laboratory process for growth and assembly of mammalian cells into tissue-like masses demonstrated with hamster and rat cells. New process better able to provide culture environment with reduced fluid shear stress, freedom for three-dimensional spatial orientation of particles suspended in culture medium, and localization of particles of different or similar sedimentation properties in similar spatial region.

Wolf, David A.↗

Neonatal rat heart cells cultured in simulated microgravity

In vitro characteristics of cardiac cells cultured in simulated microgravity are reported. Tissue culture methods performed at unit gravity constrain cells to propagate, differentiate, and interact in a two dimensional (2D) plane. Neonatal rat cardiac cells in 2D culture organize predominantly as bundles of cardiomyocytes with the intervening areas filled by non-myocyte cell types. Such cardiac cell cultures respond predictably to the addition of exogenous compounds, and in many ways they represent an excellent in vitro model system. The gravity-induced 2D organization of the cells, however, does not accurately reflect the distribution of cells in the intact tissue. We have begun characterizations of a three-dimensional (3D) culturing system designed to mimic microgravity. The NASA designed High-Aspect-Ratio-Vessel (HARV) bioreactors provide a low shear environment which allows cells to be cultured in static suspension. HARV-3D cultures were prepared on microcarrier beads and compared to control-2D cultures using a combination of microscopic and biochemical techniques. Both systems were uniformly inoculated and medium exchanged at standard intervals. Cells in control cultures adhered to the polystyrene surface of the tissue culture dishes and exhibited typical 2D organization. Cells in cultured in HARV's adhered to microcarrier beads, the beads aggregated into defined clusters containing 8 to 15 beads per cluster, and the clusters exhibited distinct 3D layers: myocytes and fibroblasts appeared attached to the surfaces of beads and were overlaid by an outer cell type. In addition, cultures prepared in HARV's using alternative support matrices also displayed morphological formations not seen in control cultures. Generally, the cells prepared in HARV and control cultures were similar, however, the dramatic alterations in 3D organization recommend the HARV as an ideal vessel for the generation of tissue-like organizations of cardiac cells in simulated microgravity.

Akins, Robert E.↗

Use of NASA Bioreactor in Engineering Tissue for Bone Repair

This study was proposed in search for a new alternative for bone replacement or repair. Because the systems commonly used in repair of bony defects form bone by going through a cartilaginous phase, implantation of a piece of cartilage could enhance the healing process by having a more advanced starting point. However, cartilage has seldom been used to replace bone due, in part, to the limitations in conventional culture systems that did not allow production of enough tissue for implants. The NASA-developed bioreactors known as STLV (Slow Turning Lateral Vessel) provide homogeneous distribution of cells, nutrients, and waste products, with less damaging turbulence and shear forces than conventional systems. Cultures under these conditions have higher growth rates, viability, and longevity, allowing larger "tissue-like" aggregates to form, thus opening the possibilities of producing enough tissue for implantation, along with the inherent advantages of in vitro manipulations. To assure large numbers of cells and to eliminate the use of timed embryos, we proposed to use an immortalized mouse limb bud cell line as the source of cells.

Duke, Pauline↗

Three Dimensional Primary Hepatocyte Culture

Our results demonstrated for the first time the feasibility of culturing PHH in microgravity bioreactors that exceeded the longest period obtained using other methods. Within the first week of culture, isolated hepatocytes started to form aggregates, which continuously increased in size (up to 1 cm) and macroscopically appeared as a multidimensional tissue-like assembly. To improve oxygenation and nutrition within the spheroids we performed experiments with the biodegradable nonwoven fiber-based polymers made from PolyGlycolic Acid (PGA). It has been shown that PGA scaffolds stimulate isolated cells to regenerate tissue with defined sizes and shapes and are currently being studied for various tissue-engineering applications. Our data demonstrated that culturing hepatocytes in the presence of PGA scaffolds resulted in more efficient cell assembly and formations of larger cell spheroids (up to 3 cm in length, see figure). The histology of cell aggregates cultured with PGA showed polymer fibers with attached hepatocytes. We initiated experiments to co-culture primary human hepatocytes with human microvascular endothelial cells in the bioreactor. The presence of endothelial cells in co-cultures were established by immunohistochemistry using anti-CD34 monoclonal Ab. Our preliminary data demonstrated that cultures of purified hepatocytes with human microvascular endothelial cells exhibited better growth and expressed higher levels of albumin MRNA for a longer period of time than cultures of ppfified, primary human hepatocytes cultured alone. We also evaluated microsomal deethylation activity of hepatocytes cultured in the presence of endothelial cells.In summary, we have established liver cell culture, which mimicked the structure and function of the parent tissue.

Yoffe, Boris↗

Induction of Three-Dimensional Growth of Human Liver Cells in Simulated Microgravity

We previously reported that a NASA-developed bioreactor that simulates microgravity environment and creates the unique environment of low shear force and high-mass transfer is conducive for maintaining long term 3-D cell cultures of functional hepatocytes (60 days). However, significant further expansion of liver mass, or the remodeling of liver in vitro was jeopardized by the appearance of apoptotic zones in the center of large cell aggregates. To optimize oxygenation and nutritional uptake within growing cellular aggregates we cultured primary human liver cells (HLC) in a bioreactor in the presence or absence of microcarriers and biodegradable scaffolds. Also, to promote angiogenesis, HLC were cultured with or without microvascular endothelial cells. HLC were harvested from human livers by collagenase perfusion. While microcarriers did not affect cell growth, HLC cultured with biodegradable scaffolds made from polyglycolic acid (PGA) formed aggregates up to 3 cm in length. Culturing cells with PGA scaffolds increased the efficiency of cell self-assembly and the formation of larger cell aggregates. Based on histological evaluation it appears that the degree of apoptotic cells was diminished as compared to cultures without scaffolds. Histology of HLC with PGA-scaffolds revealed cell distribution between the fibers of the scaffolds, and cell-cell and cell-fiber interactions. Analyses of HLC spheroids revealed tissue-like structures comprised of hepatocytes, biliary epithelial cells and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes and bile canaliculi with multiple microvilli and tight cellular junctions. Hepatocytes were further organized into tight clusters surrounded by complex stromal structures and reticulin fibers. Also, we observed higher levels of albumin mRNA expression when hepatocytes were co-cultured with endothelial cells. To evaluate viability and microsomal deethylation activity of hepatocytes we assessed the metabolism of midazolam by gas chromatography. Samples that were collected from HLC cultured in the bioreactor contained higher levels of midazolam metabolites (1-OH-midazolam and 4-OH-midazolam) than samples collected from conventional cultures. In summary, we have shown that co-culture of HLC with endothelial cells and/or culturing in the presence of PGA scaffolds provides additional advantages in maintaining functional activity of 3-D hepatocyte cultures that resemble liver tissue. This cell culture system may facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo and suggest the feasibility of using this approach for pre-clinical metabolic screening of novel drug candidates.

Pellis, Neal R.↗

Multilineage differentiation of rhesus monkey embryonic stem cells in three-dimensional culture systems

In the course of normal embryogenesis, embryonic stem (ES) cells differentiate along different lineages in the context of complex three-dimensional (3D) tissue structures. In order to study this phenomenon in vitro under controlled conditions, 3D culture systems are necessary. Here, we studied in vitro differentiation of rhesus monkey ES cells in 3D collagen matrixes (collagen gels and porous collagen sponges). Differentiation of ES cells in these 3D systems was different from that in monolayers. ES cells differentiated in collagen matrixes into neural, epithelial, and endothelial lineages. The abilities of ES cells to form various structures in two chemically similar but topologically different matrixes were different. In particular, in collagen gels ES cells formed gland-like circular structures, whereas in collagen sponges ES cells were scattered through the matrix or formed aggregates. Soluble factors produced by feeder cells or added to the culture medium facilitated ES cell differentiation into particular lineages. Coculture with fibroblasts in collagen gel facilitated ES cell differentiation into cells of a neural lineage expressing nestin, neural cell adhesion molecule, and class III beta-tubulin. In collagen sponges, keratinocytes facilitated ES cell differentiation into cells of an endothelial lineage expressing factor VIII. Exogenous granulocyte-macrophage colony-stimulating factor further enhanced endothelial differentiation. Thus, both soluble factors and the type of extracellular matrix seem to be critical in directing differentiation of ES cells and the formation of tissue-like structures. Three-dimensional culture systems are a valuable tool for studying the mechanisms of these phenomena.

Pluripotent Stem Cells/cytology/physiology↗

Induction of three-dimensional assembly of human liver cells by simulated microgravity

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

NASA Center JSC↗

Studies of chondrogenesis in rotating systems

A great deal of energy has been exerted over the years researching methods for regenerating and repairing bone and cartilage. Several techniques, especially bone implants and grafts, show great promise for providing a remedy for many skeletal disorders and chondrodystrophies. The bioreactor (rotating-wall vessel, RWV) is a cell culture system that creates a nurturing environment conducive to cell aggregation. Chondrocyte cultures have been studied as implants for repair and replacement of damaged and missing bone and cartilage since 1965 [Chesterman and Smith, J Bone Joint Surg 50B:184-197, 1965]. The ability to use large, tissue-like cartilage aggregates grown in the RWV would be of great clinical significance in treating skeletal disorders. In addition, the RWV may provide a superior method for studying chondrogenesis and chondrogenic mutations. Because the RWV is also reported to simulate many of the conditions of microgravity it is a very useful ground-based tool for studying how cell systems will react to microgravity.

Non-NASA Center↗

Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor-mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three-dimensional ductal-type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low-shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid-filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth-based culture of three-dimensional cell assemblies, tissue-like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid-mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research.

NASA Program Space Biology↗

Glucocorticoids promote development of the osteoblast phenotype by selectively modulating expression of cell growth and differentiation associated genes

To understand the mechanisms by which glucocorticoids promote differentiation of fetal rat calvaria derived osteoblasts to produce bone-like mineralized nodules in vitro, a panel of osteoblast growth and differentiation related genes that characterize development of the osteoblast phenotype has been quantitated in glucocorticoid-treated cultures. We compared the mRNA levels of osteoblast expressed genes in control cultures of subcultivated cells where nodule formation is diminished, to cells continuously (35 days) exposed to 10(-7) M dexamethasone, a synthetic glucocorticoid, which promotes nodule formation to levels usually the extent observed in primary cultures. Tritiated thymidine labelling revealed a selective inhibition of internodule cell proliferation and promotion of proliferation and differentiation of cells forming bone nodules. Fibronectin, osteopontin, and c-fos expression were increased in the nodule forming period. Alkaline phosphatase and type I collagen expression were initially inhibited in proliferating cells, then increased after nodule formation to support further growth and mineralization of the nodule. Expression of osteocalcin was 1,000-fold elevated in glucocorticoid-differentiated cultures in relation to nodule formation. Collagenase gene expression was also greater than controls (fivefold) with the highest levels observed in mature cultures (day 35). At this time, a rise in collagen and TGF beta was also observed suggesting turnover of the matrix. Short term (48 h) effects of glucocorticoid on histone H4 (reflecting cell proliferation), alkaline phosphatase, osteopontin, and osteocalcin mRNA levels reveal both up or down regulation as a function of the developmental stage of the osteoblast phenotype. A comparison of transcriptional levels of these genes by nuclear run-on assays to mRNA levels indicates that glucocorticoids exert both transcriptional and post-transcriptional effects. Further, the presence of glucocorticoids enhances the vitamin D3 effect on gene expression. Those genes which are upregulated by 1,25(OH)2D3 are transcribed at an increased rate by dexamethasone, while those genes which are inhibited by vitamin D3 remain inhibited in the presence of dexamethasone and D3. We propose that the glucocorticoids promote changes in gene expression involved in cell-cell and cell-extracellular matrix signaling mechanisms that support the growth and differentiation of cells capable of osteoblast phenotype development and bone tissue-like organization, while inhibiting the growth of cells that cannot progress to the mature osteoblast phenotype in fetal rat calvarial cultures.

NASA Discipline Musculoskeletal↗

An Optimization of Pulsed ElectroMagnetic Fields Study

To date, in our research we have focused on the use of normal human neuronal progenitor (NHNP) cells because of their importance in human nervous system regeneration, development and maintenance, but we have developed 2-D and 3-D bioreactors that can accommodate any cell line. In this Project, we will include the use of tissues important for physiological regeneration: Human osteoblasts or chondrocytes, and vascular cells. Our initial results with the NHNP cells were quite startling using extremely low-level electromagnetic fields (5 microtesla at 10Hz; 6mA). The low-amplitude, rapidly time-varying electromagnetic fields exert a very potent effect on the proliferation, morphology, and gene expression of the cells in culture, both in standard 2-dimensional culture plates as well as cells organized into 3-dimensional tissue-like assemblies (TLAs) in a 3D bioreactor. We have replicated our preliminary results many, many times, have analyzed the gene expression using gene arrays (followed by Luminex analysis for protein production), and have monitored cell proliferation, orientation, morphology, and glucose metabolism, and we are confident that we have a stable and reliable model to study the control of high-level cellular processes by application of low-amplitude, time varying electromagnetic fields (TVEMF) (1, 2). In additional studies at the University of Michigan, we have been able to generate functional in vitro engineered mammalian skeletal muscle, and have employed nerve-muscle co-culture techniques to promote axonal sprouting. We believe that nearly all tissues, in particular, neural, are susceptible to the influences of low-level TVEMF.

Goodwin, Thomas J.↗

NASA Tech Briefs, March 2010

Topics covered include: Software Tool Integrating Data Flow Diagrams and Petri Nets; Adaptive Nulling for Interferometric Detection of Planets; Reducing the Volume of NASA Earth-Science Data; Reception of Multiple Telemetry Signals via One Dish Antenna; Space-Qualified Traveling-Wave Tube; Smart Power Supply for Battery-Powered Systems; Parallel Processing of Broad-Band PPM Signals; Inexpensive Implementation of Many Strain Gauges; Constant-Differential-Pressure Two-Fluid Accumulator; Inflatable Tubular Structures Rigidized with Foams; Power Generator with Thermo-Differential Modules; Mechanical Extraction of Power From Ocean Currents and Tides; Nitrous Oxide/Paraffin Hybrid Rocket Engines; Optimized Li-Ion Electrolytes Containing Fluorinated Ester Co-Solvents; Probabilistic Multi-Factor Interaction Model for Complex Material Behavior; Foldable Instrumented Bits for Ultrasonic/Sonic Penetrators; Compact Rare Earth Emitter Hollow Cathode; High-Precision Shape Control of In-Space Deployable Large Membrane/Thin-Shell Reflectors; Rapid Active Sampling Package; Miniature Lightweight Ion Pump; Cryogenic Transport of High-Pressure-System Recharge Gas; Water-Vapor Raman Lidar System Reaches Higher Altitude; Compact Ku-Band T/R Module for High-Resolution Radar Imaging of Cold Land Processes; Wide-Field-of-View, High-Resolution, Stereoscopic Imager; Electrical Capacitance Volume Tomography with High-Contrast Dielectrics; Wavefront Control and Image Restoration with Less Computing; Polarization Imaging Apparatus; Stereoscopic Machine-Vision System Using Projected Circles; Metal Vapor Arcing Risk Assessment Tool; Performance Bounds on Two Concatenated, Interleaved Codes; Parameterizing Coefficients of a POD-Based Dynamical System; Confidence-Based Feature Acquisition; Algorithm for Lossless Compression of Calibrated Hyperspectral Imagery; Universal Decoder for PPM of any Order; Algorithm for Stabilizing a POD-Based Dynamical System; Mission Reliability Estimation for Repairable Robot Teams; Processing AIRS Scientific Data Through Level 3; Web-Based Requesting and Scheduling Use of Facilities; AutoGen Version 5.0; Time-Tag Generation Script; PPM Receiver Implemented in Software; Tropospheric Emission Spectrometer Product File Readers; Reporting Differences Between Spacecraft Sequence Files; Coordinating "Execute" Data for ISS and Space Shuttle; Database for Safety-Oriented Tracking of Chemicals; Apparatus for Cold, Pressurized Biogeochemical Experiments; Growing B Lymphocytes in a Three-Dimensional Culture System; Tissue-like 3D Assemblies of Human Broncho-Epithelial Cells; Isolation of Resistance-Bearing Microorganisms; Oscillating Cell Culture Bioreactor; and Liquid Cooling/Warming Garment.

Source record↗

Next Generation Respiratory Viral Vaccine System: Advanced and Emerging Bioengineered Human Lung Epithelia Model (HLEM) Organoid Technology

Acute respiratory infections, including pneumonia and influenza, are the S t" leading cause of United States and worldwide deaths. Newly emerging pathogens signaled the need for an advanced generation of vaccine technology.. Human bronchial-tracheal epithelial tissue was bioengineered to detect, identify, host and study the pathogenesis of acute respiratory viral disease. The 3-dimensional (3D) human lung epithelio-mesechymal tissue-like assemblies (HLEM TLAs) share characteristics with human respiratory epithelium: tight junctions, desmosomes, microvilli, functional markers villin, keratins and production of tissue mucin. Respiratory Syntial Virus (RSV) studies demonstrate viral growth kinetics and membrane bound glycoproteins up to day 20 post infection in the human lung-orgainoid infected cell system. Peak replication of RSV occurred on day 10 at 7 log10 particles forming units per ml/day. HLEM is an advanced virus vaccine model and biosentinel system for emergent viral infectious diseases to support DoD global surveillance and military readiness.

Goodwin, Thomas J.↗

Hydrofocusing Bioreactor Produces Anti-Cancer Alkaloids

A methodology for growing three-dimensional plant tissue models in a hydrodynamic focusing bioreactor (HFB) has been developed. The methodology is expected to be widely applicable, both on Earth and in outer space, as a means of growing plant cells and aggregates thereof under controlled conditions for diverse purposes, including research on effects of gravitation and other environmental factors upon plant growth and utilization of plant tissue cultures to produce drugs in quantities greater and at costs lower than those of conventional methodologies. The HFB was described in Hydro focus - ing Bioreactor for Three-Dimensional Cell Culture (MSC-22358), NASA Tech Briefs, Vol. 27, No. 3 (March 2003), page 66. To recapitulate: The HFB offers a unique hydrofocusing capability that enables the creation of a low-shear liquid culture environment simultaneously with the herding of suspended cells and tissue assemblies and removal of unwanted air bubbles. The HFB includes a rotating cell-culture vessel with a centrally located sampling port and an internal rotating viscous spinner attached to a rotating base. The vessel and viscous spinner can be made to rotate at the same speed and direction or different speeds and directions to tailor the flow field and the associated hydrodynamic forces in the vessel in order to obtain low-shear suspension of cells and control of the locations of cells and air bubbles. For research and pharmaceutical-production applications, the HFB offers two major benefits: low shear stress, which promotes the assembly of cells into tissue-like three-dimensional constructs; and randomization of gravitational vectors relative to cells, which affects production of medicinal compounds. Presumably, apposition of plant cells in the absence of shear forces promotes cell-cell contacts, cell aggregation, and cell differentiation. Only gentle mixing is necessary for distributing nutrients and oxygen. It has been postulated that inasmuch as cells in the simulated microgravitation of an HFB do not need to maintain the same surface forces as in normal Earth gravitation, they can divert more energy sources to growth and differentiation and, perhaps, to biosynthesis of greater quantities of desired medicinal compounds. Because one can adjust the HFB to vary effective gravitation, one can also test the effects of intermediate levels of gravitation on biosynthesis of various products. The potential utility of this methodology for producing drugs was demonstrated in experiments in which sandalwood and Madagascar periwinkle cells were grown in an HFB. The conditions in the HFB were chosen to induce the cells to form into aggregate cultures that produced anti-cancer indole alkaloids in amounts greater than do comparable numbers of cells of the same species cultured according to previously known methodologies. The observations made in these experiments were interpreted as suggesting that the aggregation of the cells might be responsible for the enhancement of production of alkaloids.

Gonda, Steve R.↗