Distribution Of Viable Cells Research Articles

Viable tendon neotissue from adult adipose-derived multipotent stromal cells.

Background: Tendon healing is frequently prolonged, unpredictable, and results in poor tissue quality. Neotissue formed by adult multipotent stromal cells has the potential to guide healthy tendon tissue formation. Objectives: The objective of this study was to characterize tendon neotissue generated by equine adult adipose-derived multipotent stromal cells (ASCs) on collagen type I (COLI) templates under 10% strain in a novel bioreactor. The tested hypothesis was that ASCs assume a tendon progenitor cell-like morphology, express tendon-related genes, and produce more organized extracellular matrix (ECM) in tenogenic versus stromal medium with perfusion and centrifugal fluid motion. Methods: Equine ASCs on COLI sponge cylinders were cultured in stromal or tenogenic medium within bioreactors during combined perfusion and centrifugal fluid motion for 7, 14, or 21days under 10% strain. Viable cell distribution and number, tendon-related gene expression, and micro- and ultra-structure were evaluated with calcein-AM/EthD-1 staining, resazurin reduction, RT-PCR, and light, transmission, and scanning electron microscopy. Fibromodulin was localized with immunohistochemistry. Cell number and gene expression were compared between culture media and among culture periods (p < 0.05). Results: Viable cells were distributed throughout constructs for up to 21days of culture, and cell numbers were higher in tenogenic medium. Individual cells had a round or rhomboid shape with scant ECM in stromal medium in contrast to clusters of parallel, elongated cells surrounded by highly organized ECM in tenogenic medium after 21days of culture. Transcription factor, extracellular matrix, and mature tendon gene expression profiles confirmed ASC differentiation to a tendon progenitor-like cell in tenogenic medium. Construct micro- and ultra-structure were consistent with tendon neotissue and fibromodulin was present in the ECM after culture in tenogenic medium. Conclusion: Long-term culture in custom bioreactors with combined perfusion and centrifugal tenogenic medium circulation supports differentiation of equine adult ASCs into tendon progenitor-like cells capable of neotissue formation.

Increased Osteogenic Activity of Dynamic Cultured Composite Bone Scaffolds: Characterization and In Vitro Study

The purpose of this study was to develop and characterize beta-tricalcium phosphate (β-TCP)/polycaprolactone (PCL) scaffolds, with 2 different ratios (50/50% and 65/35%), using 3-dimensionally (3D) printed dissolvable molds, and to evaluate cellular growth and osteogenic differentiation of both groups seeded with porcine bone marrow stem cells (pBMSCs) under dynamic culture in vitro. Two different groups of scaffolds were produced: group 1 (n=40) with a ratio (wt%) of 50/50% and group 2 (n=40) with 65/35% of β-TCP/PCL. Physicochemical, morphological, and mechanical characterization of the scaffolds were performed. Scaffolds were seeded with pBMSCs and differentiated osteogenically in dynamic culture. Cell density, distribution, and viability were assessed. Osteogenic differentiation was examined through alkaline phosphatase (ALP) staining, immunofluorescence, and photospectrometry. Osteogenic differentiated constructs showed homogenous and viable cell distribution. Cell density was significantly higher (P < .05) for 65/35% scaffolds at 10 days postseeding, whereas at 6 weeks, cell number equalized for both groups. ALP activity increased over time and was significantly higher (P < .05) for 65/35% scaffolds at 14 days postseeding. The mechanical properties of the developed 65/35% scaffolds were within the range of natural trabecular bone. Moreover, the 65/35% scaffolds showed biological advantages, such as higher cell growth and higher ALP activity.

Extending Viability of Bifidobacterium longum in Chitosan-Coated Alginate Microcapsules Using Emulsification and Internal Gelation Encapsulation Technology.

Bifidobacteria are considered one of the most important intestinal probiotics because of their significant health impact. However, this ability is usually limited by gastrointestinal fluid and temperature sensitivity. Emulsification and internal gelation is an encapsulation technique with great potential for probiotic protection during storage and the gastrointestinal transit process. This study prepared microcapsules using an emulsification and internal gelation encapsulation method with sodium alginate, chitosan, and Bifidobacterium longum as wall material, coating material, and experimental strain, respectively. Optical, scanning electron, and focal microscopes were used to observe the microcapsule surface morphology and internal viable cell distribution, and a laser particle size analyzer and zeta potentiometer were used to evaluate the chitosan-coating characteristics. In addition, microcapsule probiotic viability after storage, heat treatment, and simulated gastrointestinal fluid treatment were examined. Alginate microcapsules and chitosan-coated alginate microcapsules both had balling properties and uniform bacterial distribution. The latter kept its balling properties after freeze-drying, verified by scanning electronic microscopy (SEM), and had a clear external coating, observed by an optical microscope. The particle size of chitosan-coated alginate microcapsules was slightly larger than the uncoated microcapsules. The zeta potential of alginate and chitosan-coated alginate microcapsules was negative and positive, respectively. Heat, acid and bile salt tolerance, and stability tests revealed that the decrease of viable cells in the chitosan-coated alginate microcapsule group was significantly lower than that in uncoated microcapsules. These experimental results indicate that the chitosan-coated alginate microcapsules protect B. longum from gastrointestinal fluid and high-temperature conditions.

Comparison of Techniques for Preimplantation Treatment of Osteochondral Allograft Bone.

Articular defects are a major problem with few effective treatment options. Osteochondral allograft (OCA) transplantation can be an effective treatment; however, lack of OCA bone integration can cause failure. This controlled laboratory study was designed to compare clinically applicable methods for marrow element removal and enhanced delivery of bone marrow aspirate concentrate (BMC) to OCA bone. We hypothesized that compressed carbon dioxide (CO2) treatment of OCA bone would result in significantly better marrow element removal, significantly more retention and distribution of viable osteoprogenitor cells, and significantly higher osteoinductive protein elution from OCAs compared with other preimplantation treatments. Fresh humeral heads (n = 24) were harvested and stored for 14 days, then randomly assigned to treatment based on marrow element removal and bone treatment: (standard of care [SOC]) (n = 4) - SOC high-pulse saline lavage, no BMC; (BMC) (n = 5) - saline lavage then canine BMC; (Drill + BMC) (n = 5) - 1.1 mm drill-hole immediately subchondral then saline lavage then BMC injection through drill hole; (Carb + BMC) (n = 5) - saline lavage then CO2 then BMC; or (Saline-Carb + BMC) (n = 5) - saline lavage and CO2 together then BMC. Treated OCAs were cultured for 14 days. On day 3, media were collected, centrifuged to isolate cells, and replaced. Cells were cultured for 11 days for colony forming unit (CFU) determination. OCA media were collected on days 7 and 14 of culture for analysis. On day 14, each graft was assessed for viable cell retention and distribution, and bone marrow element removal. BMC had significantly higher (p = 0.001) viable cell distribution compared with the SOC, Drill + BMC, Carb + BMC, and Saline-Carb + BMC groups. BMC and Drill + BMC had significantly higher (p < 0.05) CFUs than SOC, Carb + BMC, and Saline-Carb + BMC. Drill + BMC and Carb + BMC had the highest media concentrations of the osteoinductive biomarkers. The Carb + BMC and Saline-Carb + BMC groups were associated with significantly superior marrow element removal (p < 0.02) compared with the SOC, Drill + BMC, and BMC groups. Saline irrigation plus saturation with autogenous BMC appears to be the most advantageous preimplantation treatment for OCA transplantation.

Multiple Roles of Extracellular Polymeric Substances on Resistance of Biofilm and Detached Clusters

In this study, multiple roles of biofilm EPS were assessed with respect to the resistance of biofilm and detached biofilm clusters to chlorine disinfection. Strains from an opportunistic pathogen, Pseudomonas aeruginosa (wild type, EPS- and EPS+) with altered extracellular polymeric substances (EPS) secretion capabilities were tested. The impact of biofilm EPS quantity on disinfection was evaluated by monitoring biofilm viability, biofilm structure, removal of dissolved organic matter (DOM), and viability of detached biofilm simultaneously during chlorine disinfection. The obtained results suggested that the presence of EPS increased biofilm and detached biofilm resistance to chlorine in both presence and absence of DOM. The quantity of EPS had an effect on biofilm structure and the structural characteristics were closely related to both overall biofilm viability and the spatial distribution of viable cells within the biofilm. Additionally, the increased amount of EPS influenced selective removal of DOM with polar functional groups. However the DOM removal did not have a significant impact on the viability of biofilm cells during chlorine disinfection. Meanwhile, the viability of detached biofilm clusters, particularly the EPS overproducing strain, was significantly increased in the presence of DOM. The combined results suggested that biofilm EPS played multiple roles toward influencing the resistance of both biofilm and detached biofilm to disinfectant.

Paper-supported 3D cell culture for tissue-based bioassays

Fundamental investigations of human biology, and the development of therapeutics, commonly rely on 2D cell-culture systems that do not accurately recapitulate the structure, function, or physiology of living tissues. Systems for 3D cultures exist but do not replicate the spatial distributions of oxygen, metabolites, and signaling molecules found in tissues. Microfabrication can create architecturally complex scaffolds for 3D cell cultures that circumvent some of these limitations; unfortunately, these approaches require instrumentation not commonly available in biology laboratories. Here we report that stacking and destacking layers of paper impregnated with suspensions of cells in extracellular matrix hydrogel makes it possible to control oxygen and nutrient gradients in 3D and to analyze molecular and genetic responses. Stacking assembles the "tissue", whereas destacking disassembles it, and allows its analysis. Breast cancer cells cultured within stacks of layered paper recapitulate behaviors observed both in 3D tumor spheroids in vitro and in tumors in vivo: Proliferating cells in the stacks localize in an outer layer a few hundreds of microns thick, and growth-arrested, apoptotic, and necrotic cells concentrate in the hypoxic core where hypoxia-sensitive genes are overexpressed. Altering gas permeability at the ends of stacks controlled the gradient in the concentration of the O(2) and was sufficient by itself to determine the distribution of viable cells in 3D. Cell cultures in stacked, paper-supported gels offer a uniquely flexible approach to study cell responses to 3D molecular gradients and to mimic tissue- and organ-level functions.

Engineered 3D tissue models for cell-laden microfluidic channels

Delivery of nutrients and oxygen within three-dimensional (3D) tissue constructs is important to maintain cell viability. We built 3D cell-laden hydrogels to validate a new tissue perfusion model that takes into account nutrition consumption. The model system was analyzed by simulating theoretical nutrient diffusion into cell-laden hydrogels. We carried out a parametric study considering different microchannel sizes and inter-channel separation in the hydrogel. We hypothesized that nutrient consumption needs to be taken into account when optimizing the perfusion channel size and separation. We validated the hypothesis by experiments. We fabricated circular microchannels (r = 400 microm) in 3D cell-laden hydrogel constructs (R = 7.5 mm, volume = 5 ml). These channels were positioned either individually or in parallel within hydrogels to increase nutrient and oxygen transport as a way to improve cell viability. We quantified the spatial distribution of viable cells within 3D hydrogel scaffolds without channels and with single- and dual-perfusion microfluidic channels. We investigated quantitatively the cell viability as a function of radial distance from the channels using experimental data and mathematical modeling of diffusion profiles. Our simulations show that a large-channel radius as well as a large channel to channel distance diffuse nutrients farther through a 3D hydrogel. This is important since our results reveal that there is a close correlation between nutrient profiles and cell viability across the hydrogel.

Rapid prototyping of anatomically shaped, tissue‐engineered implants for restoring congruent articulating surfaces in small joints

Preliminary studies investigated advanced scaffold design and tissue engineering approaches towards restoring congruent articulating surfaces in small joints. Anatomical femoral and tibial cartilage constructs, fabricated by three-dimensional fibre deposition (3DF) or compression moulding/particulate leaching (CM), were evaluated in vitro and in vivo in an autologous rabbit model. Effects of scaffold pore architecture on rabbit chondrocyte differentiation and mechanical properties were evaluated following in vitro culture and subcutaneous implantation in nude mice. After femoral and tibial osteotomy and autologous implantation of tissue-engineered constructs in rabbit knee joints, implant fixation and joint articulation were evaluated. Rapid prototyping of 3DF architectures with 100% interconnecting pores promoted homogeneous distribution of viable cells, glycosaminoglycan (GAG) and collagen type II; significantly greater GAG content and differentiation capacity (GAG/DNA) in vitro compared to CM architectures; and higher mechanical equilibrium modulus and dynamic stiffness (at 0.1 Hz). Six weeks after implantation, femoral and tibial constructs had integrated with rabbit bone and knee flexion/extension and partial load bearing were regained. Histology demonstrated articulating surfaces between femoral and tibial constructs for CM and 3DF architectures; however, repair tissue appeared fibrocartilage-like and did not resemble implanted cartilage. Anatomically shaped, tissue-engineered constructs with designed mechanical properties and internal pore architectures may offer alternatives for reconstruction or restoration of congruent articulating surfaces in small joints.

The development of a bioreactor to perfuse radially-confined hydrogel constructs: Design and characterization of mass transport properties

Limitations to nutrient transport provide a challenge to the development of 3D tissue-engineered constructs. A heterogeneous distribution of viable cells and functional matrix within the developing tissue is a common consequence. In the present study, a bioreactor was developed to perfuse fluid through cylindrical agarose constructs. The transport and distribution of dextran molecules (FD-4, FD-500, FD-2000) within the agarose was visualized in order to determine the bioreactors effectiveness for transport enhancement. By 24 h, the perfusion bioreactor achieved 529%, 395% and 294% higher concentrations of FD-4, FD-500 and FD-2000, respectively, than those solely due to diffusion. Of particular interest was the effectiveness of the bioreactor to transport molecules to the central region of the constructs. In this respect, the perfusion bioreactor was found to increase transportation of FD-4, FD-500 and FD-2000 by 30%, 291% and 222% over that of diffusion. Articular chondrocytes were cultured and perfused using the bioreactor. The improved molecular transport achieved led to an average 75% and 1340% increase of DNA and sulphated GAG, respectively at 20 days. More significantly was the 106% and 1603% increase of DNA and GAG, respectively, achieved at the central core of the 3D constructs.

FOAM VERSUS FIBRE SCAFFOLDS FOR MECHANICAL STIMULATION OF CELLS IN BONE TISSUE ENGINEERING

Introduction The morphology of a substrate on which cells are seeded is known to affect cells in multiple ways including morphology, proliferation differentiation and matrix production. However, it is difficult to characterise the effects of mechanical loading on cells seeded in scaffolds of different morphology but identical chemistry. The aim of this project is to create polyurethane foam and fibre scaffolds from the same linear polyurethane and to analyse the effect of scaffold morphology on mechanical properties, cell viability, and cell morphology. The current work aims to produce scaffolds of sufficient mechanical strength for mechanical stimulation experiments. Materials and Methods Foams were fabricated from a polypropylene oxide polyol and a toluene diisocyanate (TDI) by the direct foaming technique. Fibres were fabricated by dissolving the foam overnight in a 20wt% solution of dimethylacetamide under stirring conditions. The fibres were spun using an electrospinning rig with a 15kV electric field, 30cm working distance, flow rate of 3.5ml/hr and collected on a rotating drum. MC-3T3-E1 osteoblastic cells were seeded onto 2cm diameter, thin discs of each scaffold type at a density of 100 x 10 cells per sample. The seeded scaffolds were then left for 1hr to allow cell attachment before immersion in 3ml of media and culture for 21 days with a media change every 3 days. The distribution of viable cells was analysed by an assay of metabolic activity (MTS). Results Both foam and fibre morphologies are suitable for support of cell growth over medium term culture periods (21 days). Cell viability analysis showed no significant difference between the two morphologies. Microscopy analysis indicated cell attachment and survival within the pores of the foam scaffold with cells adhering to the struts. Both scaffolds can be cut into disk or dumbbell shapes suitable for mechanical stimulation in compression or tension respectively.

Development of a bioreactor for evaluating novel nerve conduits

We describe an experimental closed bioreactor device for studying novel tissue engineered peripheral nerve conduits in vitro. The system integrates a closed loop system consisting of one, two, or three experimental nerve conduits connected in series or parallel, with the ability to study novel scaffolds within guidance conduits. The system was established using aligned synthetic microfiber scaffolds of viscose rayon and electrospun polystyrene. Schwann cells were seeded directly into conduits varying from 10 to 80 mm in length and allowed to adhere under 0 flow for 1 h, before being cultured for 4 days under static or continuous flow conditions. In situ viability measurements showed the distribution of live Schwann cells within each conduit and enabled quantification thereafter. Under static culture viable cells only existed in short conduit scaffolds (10 mm) or at the ends of longer conduits (20-80 mm) with a variation in viable cell distribution. Surface modification of scaffold fibers with type-1 collagen or acrylic acid increased cell number by 17% and 30%, respectively. However, a continuous medium flow of 0.8 mL/h was found to increase total cell number by 2.5-fold verses static culture. Importantly, under these conditions parallel viability measurements revealed a ninefold increase compared to static culture. Fluorescence microscopy of scaffolds showed cellular adhesion and alignment on the longitudinal axis. We suggest that such a system will enable a rigorous and controlled approach for evaluating novel conduits for peripheral nerve repair, in particular using hydrolysable materials for the parallel organization of nerve support cells, prior to in vivo study.

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Optical computed tomography (OCT) and optical emission tomography (OET) are introduced as new 3D imaging techniques with unique capacity for imaging tumor microvasculature and viable cell distribution in whole unsectioned tumor tissue. OCT and OET can provide co-registered images relating 3D global structure with function, in high-resolution and with high contrast. The techniques address an ‘imaging-gap’ that exists between the relatively low-medium resolution of 3D imaging systems (e.g. micro-CT, micro-MRI and micro-PET) and the very high-resolution planar imaging techniques of light and confocal microscopy.

A comparison of non-radioactive methods for assessing viability in ex vivo cultured cancellous bone: Technical Note

Biocompatibility studies are carried out either in two dimensional monolayer culture or in animal studies. Bone organ cultures are therefore required in order to reduce the number of animal studies performed, while at the same time ensuring a more natural environment than that provided by monolayer culture of isolated cells. Due to the three dimensional nature of bone explants, assays that determine the distribution of viable cells are required, however dense mineralised bone is not easily penetrated by soluble factors. We sought to compare a number of non-radioactive viability methods in order to assess their suitability for use with cancellous bone. Fluorescent live/dead staining, MTT activity and lactate dehydrogenase detection were all investigated on either whole bone explants (9.5 mm in diameter, 5 mm high) or on sections of explants. All these assays are routinely used in 2 dimensional cell culture systems, yet each required modifications to be suitable for use with cancellous bone. Factors such as penetration of reagent, incubation time, assay temperature and ease of determining viable cells were all compared. It was demonstrated that penetration of the reagents into whole cores was a major problem which easily led to artefacts that could be overcome by preparing 250 mum unfixed sections. Fluorescent live/dead staining had extra complications caused by the autofluorescence of the bone generating a high signal to noise ratio, making assessment of osteocyte viability impossible. MTT staining was difficult to interpret due to the punctate nature of the stain. We found that lactate dehydrogenase staining of 250 mum thick unfixed sections led to excellent viability determination of osteocytes within the mineralised matrix. It also maintained marrow structure and enabled marrow viability to be assessed as a factor of volume occupied by viable marrow. Decreasing the viscosity of the LDH assay solution used in published methods led to a greatly improved penetration into the calcified matrix. Quantification of thick sections is aided by using the autofluorescence of the bone to highlight the darkly stained osteocytes against the fluorescing bone.

WE‐D‐330A‐01: 3D Optical Imaging of Tumor Microvasculature and Viable Cell Distribution

Purpose: The ability to image 3D global structure (e.g. microvasculature) and function (e.g. gene expression) in whole unsectioned tumors, in high‐resolution and with high‐contrast, is of significant present interest in cancer research. We have developed novel optical imaging techniques capable of reconstructing the 3D distribution of absorbance or fluorescence‐emitting sources in tumor tissue. Primary advantages include high spatial resolution and contrast, and ability to image a wide range of molecular probes.Method and Materials: Constitutive RFP expressing HCT116 (human colon carcinoma) xenograft tumors were implanted in hind legs of nude BALBC mice. The tumor microvasculature was dual‐stained by in vivo tail vein injections of light attenuating isotonic ink and FITC lectin. The tumor was excised post‐staining and subjected to a fluorescence‐friendly clearing procedure to render it amenable for optical imaging. Quantitative images of microvascular density and viable‐cell distribution were reconstructed using tomographic algorithms from a set of transmission and fluorescent projection images acquired at multiple angles using appropriate filters, focusing optics and a CCD camera.Results: Co‐registered, multimodal, high‐resolution and high‐contrast 3D reconstructions were achieved through large, (∼1cm) whole mount, murine tumor samples. Quality assurance tests indicated encouraging accuracy of reconstructed attenuation coefficients and geometry. The 3D reconstructed slices were juxtaposed with histological slices of the same tumor to reveal striking correlation between viable‐cell distribution and well‐perfused regions. A fluorescence friendly clearing procedure was identified.Conclusion: We present a novel technique for multi‐modality optical imaging of 3D microvasculature and/or viable cell distribution in tumor tissue with unprecedented image resolution and contrast. The technique has potential to image a wide variety of tumor and normal tissue structure and function. Of particular interest is the potential to analyze the effect of anti‐angiogenic agents and radiation therapy on the response of the entire 3D tumor microvasculature network.

Cardiac tissue engineering: effects of bioreactor flow environment on tissue constructs

AbstractThe limited ability of cardiac muscle to regenerate after injury and the small number of organs available for transplantation motivate studies aimed at curative treatment options. Tissue engineering based on the integrated use of cells on biomaterial scaffolds in bioreactors may offer cardiac grafts suitable for surgical attachment to the myocardium or for basic research. In one of the current approaches, neonatal rat cardiomyocytes are combined with collagen sponges, gels or polyglycolic acid scaffolds (PGA). Cultivations performed in dishes, static or mixed flasks or rotating bioreactors yield constructs with a thin (100–200 µm) peripheral layer of tissue expressing markers of cardiac differentiation and able to propagate electrical signals. The non‐uniform cell distribution is a result of oxygen diffusional limitations within the constructs. Cultivations with perfusion of culture medium through the construct enhance the convective‐diffusive oxygen supply and yield 1–2 mm thick constructs with physiologically high and spatially uniform distribution of viable cells expressing cardiac markers. We review here a series of studies we conducted using cells seeded on three‐dimensional scaffolds and cultured in several different bioreactors, to demonstrate that the bioreactor flow environment can have substantial effects on structural and functional properties of cardiac constructs. Copyright © 2006 Society of Chemical Industry

Modeling mass transfer in hepatocyte spheroids via cell viability, spheroid size, and hepatocellular functions

Hepatocyte aggregation into spheroids attributes to their increased activity, but in the absence of a vascular network the cells in large spheroids experience mass transfer limitations. Thus, there is a need to define the spheroid size which enables maximal cell viability and productivity. We developed a combined theoretical and experimental approach to define this optimal spheroid size. Hepatocyte spheroids were formed in alginate scaffolds having a pore diameter of 100 microm, in rotating T-flasks or spinners, to yield a maximal size of 100, 200, and 600 microm, respectively. Cell viability was found to decrease with increasing spheroid size. A mathematical model was constructed to describe the relationship between spheroid size and cell viability via the oxygen mass balance equation. This enabled the prediction of oxygen distribution profiles and distribution of viable cells in spheroids with varying size. The model describes that no oxygen limitation will take place in spheroids up to 100 microm in diameter. Spheroid size affected the specific rate of albumin secretion as well; it reached a maximal level, i.e., 60 microg/million cells/day in 100-microm diameter spheroids. This behavior was depicted in an equation relating the specific albumin secretion rate to spheroid size. The calculated results fitted with the experimental data, predicting the need for a critical number of viable hepatocytes to gain a maximal albumin secretion. Taken together, the results on mass transport in spheroids and its effects on cell viability and productivity provide a useful tool for the design of 3D scaffolds with pore diameters of 100 microm.

Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique

In this study, we present and characterize a fiber deposition technique for producing three-dimensional poly(ethylene glycol)-terephthalate—poly(butylene terephthalate) (PEGT/PBT) block co-polymer scaffolds with a 100% interconnecting pore network for engineering of articular cartilage. The technique allowed us to “design-in” desired scaffold characteristics layer by layer by accurately controlling the deposition of molten co-polymer fibers from a pressure-driven syringe onto a computer controlled x– y– z table. By varying PEGT/PBT composition, porosity and pore geometry, 3D-deposited scaffolds were produced with a range of mechanical properties. The equilibrium modulus and dynamic stiffness ranged between 0.05–2.5 and 0.16–4.33 MPa, respectively, and were similar to native articular cartilage explants (0.27 and 4.10 MPa, respectively). 3D-deposited scaffolds seeded with bovine articular chondrocytes supported a homogeneous cell distribution and subsequent cartilage-like tissue formation following in vitro culture as well as subcutaneous implantation in nude mice. This was demonstrated by the presence of articular cartilage extra cellular matrix constituents (glycosaminoglycan and type II collagen) throughout the interconnected pore volume. Similar results were achieved with respect to the attachment of expanded human articular chondrocytes, resulting in a homogenous distribution of viable cells after 5 days dynamic seeding. The processing methods and model scaffolds developed in this study provide a useful method to further investigate the effects of scaffold composition and pore architecture on articular cartilage tissue formation.

Critical period-dependent reduction of the permissiveness of cat visual cortex tissue for neuronal adhesion and neurite growth.

During postnatal development, the visual cortex undergoes an experience-dependent refinement of its circuitry. This process includes synapse formation, as well as synapse elimination. Both mechanisms appear to be restricted to a limited 'critical period' which lasts for approximately 2 months in cats. We tested whether the termination of the critical period for cortical malleability is paralleled by changes in the growth permissiveness of the tissue. These changes may inhibit progressive reorganization of functional circuitries mediated by axon growth. Embryonic cortical neurons were cultured on unfixed cryostat sections of the visual cortex obtained from cats aged 2-50 weeks. After 2-3 days in vitro the distribution of viable cells and the percentage of neurite-bearing cells were determined and analysed with respect to the developmental age and subdivisions of the underlying tissue substrate. It was shown that cell adhesion and neurite formation are correlated with the developmental age of the substrate tissue and the time period of myelination. While embryonic neurons adhered and survived on grey and white matter tissue from 2- and 4-week-old kittens, there was a significant reduction in cell adhesion on the myelinated white matter regions of the tissue sections of older animals. Quantitative analyses showed that neurite formation by cultured neurons also became successively impaired on grey and white matter areas of tissue substrates, corresponding to the time course of the critical period for cortical malleability. On grey matter tissue this effect was most pronounced between the second and sixth postnatal weeks. The effects were not antagonized by coating the substrate sections with the growth-promoting molecule laminin. It is therefore proposed that neurite growth-inhibiting factors, most probably associated with central nervous system myelin, are gradually expressed postnatally and may contribute to the termination of the critical period in the visual cortex of cats.

Detection of Drug-induced Apoptosis by Flow Cytometry after Alkaline Extraction of Ethanol Fixed Cells.

A new flow cytometric method was developed to detect apoptotic cells with fragmented DNA and to determine cell cycle distribution of viable cells, in the same sample, by propidium iodide staining. Apoptosis, in HT58 human B lymphoma cells, was induced by etoposide and/or by staurosporine. Using appropriate alkaline solutions (between 1-10 mN NaOH in 150 mM saline) followed by neutralization with buffer solution, the fragmented DNA can be extracted quantitatively from ethanol fixed cells. Further, good resolution of the cell cycle distribution can be obtained in unimpaired cells without RNase treatment. Furthermore, unlike the widely used hypotonic-detergent extraction of unfixed cells, the suggested extraction method can prevent drug-induced disintegration of dead cells when karyorrhexis occurs.

Immobilized Coffea arabica cell culture using a bubble‐column reactor with controlled light intensity

Coffea arabica cells immobilized by calcium alginate gel were photocultured using a bubble-column reactor under controlled light intensity. This process was carried out after their alkaloid productivity was improved by increasing the cell density in the initial gel matrix and preculturing the immobilized cells in the dark prior to light irradiation. The cells were grown in the form of a biofilm on gel beads, producing 100 mg/L of purine alkaloids in a 24-day batch culture. Alkaloid production was relatively constant with respect to light intensity changes, and also cell growth was not suppressed much at high light intensity, with these behaviors being different from those obtained using suspended cells. These phenomena are explained by estimating the light intensity gradient within the cell-immobilizing particles and by measuring the viable cell distribution within them. It subsequently suggests that the subsurface cells affect both the production and growth behaviors.