Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering

This protocol describes an effective method for the production of spherical microtissues (microspheres), which can be used for a variety of tissue-engineering purposes. The obtained microtissues are well suited for the study of osteogenesis in vitro when multipotent stem cells are used. The dimensions of the microspheres can easily be adjusted according to the cell numbers applied in an individual experiment. Thus, microspheres allow for the precise administration of defined cell numbers at well-defined sites. Here we describe a detailed workflow for the production of microspheres using unrestricted somatic stem cells from human umbilical cord blood and adapted protocols for the use of these microspheres in histological analysis. RNA extraction methods for mineralized microtissues are specifically modified for optimum yields. The duration of running the complete protocol without preparatory cell culture but including 2 weeks of microsphere incubation, histological staining and RNA isolation is about 3 weeks.     

Fluorescent microsphere technique to measure cerebral blood flow in the rat

The present protocol describes a method for parallel measurement of cerebral blood flow (CBF) using fluorescent microspheres and structural assessment of the same material. The method is based on the standard microsphere technique, embolizing capillaries proportional to the blood flow, but requires dissolution of the tissue to retrieve the microspheres. To link the blood flow to the tissue morphology we modified the technique to fluorescent microspheres, which are quantified in cryo- or vibratome sections, allowing structural analysis by, for example, immunohistochemistry or standard histology. The protocol takes 8 h 50 min, without pauses, to complete, but additional flow measurements or specific protocols can increase the time needed.     

Novel, fluorescent, magnetic, polysaccharide-based microsphere for orientation, tracing, and anticoagulation: preparation and characterization

A fluorescent, magnetic composite poly(styrene-maleic anhydride) microsphere, suitable for conjugation with polysaccharide, was synthesized using magnetite/europium phthalate particles as seeds by copolymerization of styrene and maleic anhydride. The magnetite/europium phthalate particles were wrapped up by poly(ethylene glycol), which improved the affinity between the seed particles and the monomers. The composite microspheres obtained, with a diameter of 0.15-0.7 microm, contain 586-1013 microg of magnetite/g of microsphere and 0.5-16 mmol surface anhydride groups/g of microsphere. Heparin was conjugated with the reactive surface anhydride groups on the surface of the microspheres by covalent binding to obtain a fluorescent, magnetic, polysaccharide-based microsphere. The microspheres not only retain their bioactivities but also provide magnetic susceptibility and fluorescence. They can be used as a carrier with magnetic orientation and fluorescence tracer for potent drug targeting. The orientation, tracer, and anticoagulation of the fluorescence, magnetic, polysaccharide-based microspheres were studied. The anticoagulant activity of the microspheres and heparin binding capacity reached 54,212.8 U and 607.1 mg/g of dry microspheres. The activity recovery was 50.2%. The anticoagulant activity of the microspheres increases with the increase of the conjugated heparin on the surface of the microspheres and the decrease of the microsphere size. Furthermore, The fluorescent, magnetic, polysaccharide-based microspheres can be easily transported to a given position in a magnetic field and traced via their fluorescence.     

Stable labeled microspheres to measure perfusion: validation of a neutron activation assay technique

Neutron activation is an accurate analytic method in which trace quantities of isotopes of interest in a sample are activated and the emitted radiation is measured with high-resolution detection equipment. This study demonstrates the application of neutron activation for the measurement of myocardial perfusion using stable isotopically labeled microspheres. Stable labeled and standard radiolabeled microspheres (15 microm) were coinjected in an in vivo rabbit model of myocardial ischemia and reperfusion. Radiolabeled microspheres were detected with a standard gamma-well counter, and stable labeled microspheres were detected with a high-resolution Ge detection after neutron activation of the myocardial and reference blood samples. Regional myocardial blood flow was calculated from the deposition of radiolabeled and stable labeled microspheres. Both sets of microspheres gave similar measurements of regional myocardial blood flow over a wide range of flow with a high linear correlation (r = 0.95-0.99). Neutron activation is capable of detecting a single microsphere in an intact myocardial sample while providing simultaneous quantitative measurements of multiple isotope labels. This high sensitivity and capability for measuring perfusion in intact tissue are advantages over other techniques, such as optical detection of microspheres. Neutron activation also can provide an effective method for reducing the production of low-level radioactive waste generated from biomedical research. Further applications of neutron activation offer the potential for measuring other stable labeled compounds, such as fatty acids and growth factors, in conjunction with microsphere measured flow, providing the capability for simultaneous measurement of regional metabolism and perfusion.     

Preparation and adsorption characteristic of polymeric microsphere with strong adsorbability for creatinine

Cross-linking terpolymer microspheres (HEMA/NVP/MBA; it can also be designated as HEMA/NVP because HEMA and NVP are main components) with an average diameter of 180 microm, were prepared via inverse suspension copolymerization by using 2-hydroxyethyl methacrylate (HEMA) and N-vinylpyrrolidone (NVP) as monomers and N,N'-methylene bisacrylamide (MBA) as cross-linked agent. The microsphere HEMA/NVP was chemically modified with 3,5-dinitrobenzoyl chloride (DNBC), and the functional microsphere DNBZ-HEMA/NVP, on which a great number of 3,5-dinitrobenzoate group (DNBZ) were bound, was obtained. The functional microsphere DNBZ-HEMA/NVP were characterized with FTIR and the chemical analysis method. The adsorption characteristics and mechanism of the absorption of DNBZ-HEMA/NVP for creatine was mainly studied. The results of static adsorption experiments show that the functional microsphere DNBZ-HEMA/NVP has very strong adsorption ability for creatinine, and the saturated adsorption amount is 25 mg/g. The adsorption capacity of the functional microsphere DNBZ-HEMA/NVP for creatinine is enhanced 20 times as against unmodified microsphere HEMA/NVP. The adsorption capacity is smaller, at lower and higher pH, and has a maximum as pH 8.5. The higher the salinity of the medium, the smaller the adsorption capacity. The adsorption capacity decreases with increasing temperature. The study results show that the adsorption of the microsphere DNBZ-HEMA/NVP for creatinine is ascribed to a chemical adsorption by driving of electrostatic interaction.     

Direct fluorescent staining and analysis of proteins on microspheres using CBQCA.

BACKGROUND: General methods for accurate determination of microsphere surface protein loading are needed for applications from protein arrays to molecular assembly studies. Current methods include bulk absorption measurements of stained microspheres or use of known fluorescently tagged binding partners, which limit sensitivity and general applicability, respectively. METHODS: Microspheres bearing covalently coupled proteins were stained with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) using different incubation times and dye concentrations to determine optimal staining conditions. The CBQCA fluorescence of microspheres (measured by flow cytometry) bearing known amounts of protein were used to generate standard curves of CBQCA fluorescence response versus the amount of microsphere surface protein. CBQCA was also used to stain noncovalent protein interactions. RESULTS: Maximal labeling was attained within 1 h with 1 mM CBQCA. Linear fluorescence response occurred between 8 x 10(4) and 1 x 10(6) proteins/microsphere. CBQCA staining did not disturb noncovalent protein interactions. CONCLUSIONS: We have developed methods using CBQCA and flow cytometry to quickly and simply quantify the amount of protein on the surface of a microsphere. Importantly, this approach could be extended to other formats (e.g., chips). Further, because it does not disturb noncovalent protein-protein interactions, it may be possible to use this approach to detect protein interactions without the use of purified prestained probes.     

Hemodynamic effects of 15-microm-diameter microspheres on the rat pulmonary circulation.

The microsphere method has been used extensively to measure regional blood flow in large laboratory animals. A fundamental premise of the method is that microspheres do not alter regional flow or vascular tone. Whereas this assumption is accepted in large animals, it may not be valid in the pulmonary circulation of smaller animals. Three studies were performed to determine the hemodynamic effects of microspheres on the rat pulmonary circulation. Increasing numbers of 15-microm-diameter microspheres were injected into a fully dilated, isolated-lung preparation. Vascular resistance increased 0.8% for every 100,000 microspheres injected. Microspheres were also injected into an isolated-lung preparation in which vascular tone was increased with hypoxia. Microspheres did not induce vasodilatation, as reported in other vascular beds. Fluorescent microspheres were injected via tail veins into awake rats, and the spatial locations of the microspheres were determined. Regional distributions remained highly correlated when microspheres of one color were injected after microspheres of another color. This indicates that the initial injection did not alter regional perfusion. We conclude that, when used in appropriate numbers, 15-microm-diameter microspheres do not alter regional flow or vascular tone in the rat pulmonary circulation.     

Differentiation of HL-60 promyelocytic leukemia cells: simultaneous determination of phagocytic activity and cell cycle distribution by flow cytometry

Phagocytosis of fluorescent microspheres by HL-60 promyelocytic leukemia cells following induction of differentiation with dimethyl sulfoxide (DMSO) was monitored using flow cytometry. Initiation of phagocytic capability following initiation of differentiation with 1.5% DMSO coincided with the attainment of respiratory burst activity as measured by NBT (nitro blue tetrazolium) reduction; the degree of phagocytic activity was dependent upon parameters such as microsphere size, microsphere number, and exposure time. Ingestion of fluorescent microspheres did not interfere with the measurement of DNA content using propidium iodide; thus, simultaneous determination of phagocytic activity and the cell cycle phase was possible. Accumulation of cells in the G1/G0 phase of the cell cycle following DMSO treatment was correlated with the acquisition of the capacity to phagocytize. Analysis of two-parameter correlated data also indicated that phagocytosis is coupled with residence in the G1/G0 phase of the cell cycle, further suggesting that the ability to phagocytize fluorescent microspheres is associated with end-stage differentiation.     

Release of indomethacin from pH-sensitive pullulan acetate microsphere

We studied the pH-sensitive indomethacin (IND) delivery system using pullulan. Hydrophobic pullulan acetate was prepared by chemical modification of hydrophilic pullulan and pullulan acetate microsphere was made by a solvent evaporation method. The size of microspheres was below 5 μm, and the drug loading efficiencies of microspheres were approximately 78 and 65% at the initial amount of drug 40 and 80 mg, respectively. The microsphere showed pH-sensitive swelling behavior in PBS buffer. After 15 hrs, the swelling of the microsphere at pH 7.4 was approximately 20 times greater than that at pH1.2. The pH of the medium significantly influenced on thein vitro release rate. The released amount of drug at pH 7.2 was approximately 90 times greater than that at pH 1.2. The shape of microspheres at pH 1.2 were maintained sphere forms, but at pH 7.4 were disintegrated. The pH-sensitive IND release pattern was due both to the pH-sensitive diffusion of IND from the microspheres and to the release of the drug from the surface which underwent disintegration after swelling, due to the chemical composition of the microspheres and the pH of the release media.     

An automated analysis of highly complex flow cytometry-based proteomic data

The combination of color-coded microspheres as carriers and flow cytometry as a detection platform provides new opportunities for multiplexed measurement of biomolecules. Here, we developed a software tool capable of automated gating of color-coded microspheres, automatic extraction of statistics from all subsets and validation, normalization, and cross-sample analysis. The approach presented in this article enabled us to harness the power of high-content cellular proteomics. In size exclusion chromatography-resolved microsphere-based affinity proteomics (Size-MAP), antibody-coupled microspheres are used to measure biotinylated proteins that have been separated by size exclusion chromatography. The captured proteins are labeled with streptavidin phycoerythrin and detected by multicolor flow cytometry. When the results from multiple size exclusion chromatography fractions are combined, binding is detected as discrete reactivity peaks (entities). The information obtained might be approximated to a multiplexed western blot. We used a microsphere set with >1,000 subsets, presenting an approach to extract biologically relevant information. The R-project environment was used to sequentially recognize subsets in two-dimensional space and gate them. The aim was to extract the median streptavidin phycoerythrin fluorescence intensity for all 1,000+ microsphere subsets from a series of 96 measured samples. The resulting text files were subjected to algorithms that identified entities across the 24 fractions. Thus, the original 24 data points for each antibody were compressed to 1-4 integrated values representing the areas of individual antibody reactivity peaks. Finally, we provide experimental data on cellular protein changes induced by treatment of leukemia cells with imatinib mesylate. The approach presented here exemplifies how large-scale flow cytometry data analysis can be efficiently processed to employ flow cytometry as a high-content proteomics method.     

Measurement of bronchial blood flow with radioactive microspheres in awake sheep

Distribution of bronchial blood flow was measured in unanesthetized sheep by the use of two modifications of the microsphere reference sample technique that correct for peripheral shunting of microspheres: 1) A double microsphere method in which simultaneous left and right atrial injections of 15-microns microspheres tagged with different isotopes allowed measurement of both pulmonary blood flow and shunt-corrected bronchial blood flow, and 2) a pulmonary arterial occlusion method in which left atrial injection and transient occlusion of the left pulmonary artery prevented delivery to the lung of microspheres shunted through the peripheral circulation and allowed systemic blood flow to the left lung to be measured. Both methods can be performed in unanesthetized sheep. The pulmonary arterial occlusion method is less costly and requires fewer calculations. The double microsphere method requires less surgical preparation and allows measurement without perturbation of pulmonary hemodynamics. There was no statistically significant difference between bronchial blood flow measured with the two methods. However, total bronchial blood flow measured during pulmonary arterial occlusion (1.52 +/- 0.98% of cardiac output, n = 9) was slightly higher than that measured with the double microsphere method (1.39 +/- 0.88% of cardiac output, n = 9). In another series of experiments in which sequential measurements of bronchial blood flow were made, there was a significant increase of 15% in left lung bronchial blood flow during the first minute of occlusion of the left pulmonary artery. Thus pulmonary arterial occlusion should be performed 5 s after microsphere injection as originally described by Baile et al. (1).(ABSTRACT TRUNCATED AT 250 WORDS)     

A polystyrene microsphere assay for detecting surface hydrophobicity variations within Candida albicans populations

Adherence of microbial pathogens to host cell surfaces may involve hydrophobic interactions. Here, we describe the development of an assay for detecting cell surface hydrophobicity of populations and individual cells of the opportunistic fungal pathogen Candida albicans. The assay involves mixing polystyrene latex microspheres with cells and subsequent enumeration of cell-attached microspheres. Similar levels of hydrophobicity within a population of yeast cells were obtained with the microsphere assay and with a commonly used aqueous-hydrocarbon biphasic partitioning assay. Various buffers were found to support detection of surface hydrophobicity with the microsphere assay. Complex fungal growth media did not. Serum in test media prevented microsphere attachment. A unique advantage of the assay compared to others is that individual cells can be assessed for surface hydrophobicity. Within a population of C. albicans yeast cells, strongly, moderately and weakly hydrophobic cells were observed. Within some pairs of mother-daughter cells, only one cell was hydrophobic. Germ tbes and hyphae were hydrophobic regardless of the hydrophobic status of the parent cell. These results indicate that the microsphere assay is a useful test evaluating cell surface hydrophobicity of C. albicans.     

Effects of Protoceratium reticulatum yessotoxin on feeding rates of Acartia hudsonica: A bioassay using artificial particles coated with purified toxin

Paralytic shellfish toxins produced by dinoflagellates are known to deter copepod grazing. Dinoflagellate species, including Protoceratium reticulatum, also produce disulfated polyether yessotoxins that were previously referred to as diarrheic shellfish toxins. However, the role of yessotoxins in predator–prey relationships is not yet clear. In the present study, the effects of purified yessotoxin (YTX) on feeding activities of Acartia hudsonica (Copepoda, Calanoida) were experimentally investigated. Polystyrene fluorescent microspheres (10 μm in diameter) colored bright blue or yellow-green were coated with cell extracts of P. reticulatum that do not produce yessotoxins. The bright blue microspheres were further coated with YTX, and the yellow-green microspheres were used as the reference. The microspheres were then given to the copepods separately or in combination to measure clearance rates and feeding selectivity. A. hudsonica was found to feed on the yellow-green microspheres without YTX at twice the rate of the bright blue microspheres with YTX. We also confirmed that microsphere color per se did not affect the feeding rates. The bright blue microspheres adsorbed 1.8–43.3 pg of YTX per microsphere, which is similar to the cell-specific yessotoxin content of toxic P. reticulatum found in natural environments. These results suggest that production of yessotoxin is advantageous for P. reticulatum by deterring predation by copepods.     

Development of AC microelectrophoresis for rapid protein affinity evaluation

We have developed a new method for evaluating the affinity interactions between two different proteins by applying an alternating current (AC) voltage to a micro-flow channel. An AC voltage was applied to the protein-modified microspheres in the micro-flow channel, which resulted in the oscillation of the microspheres owing to their surface charges. The oscillation amplitude showed a linear relationship with the charge density of the microspheres. As an example for protein affinity measurement, the amplitude changes of a profilin-modified microsphere were measured by the addition of actin. In the same electrical condition, the oscillation amplitude of the profilin-modified microsphere increased by ≈175% by binding with actin. Similar results in the principle were obtained for the affinity interaction between biotin and streptavidin. The results showed that the higher the charge density of the microspheres induced by binding with different proteins, the higher the oscillation amplitude of the microspheres, thus, suggesting a possible application of the micro-flow channel and AC voltage on the protein property study, as well as on the biosensor application using the oscillation amplitude changes.     

Immobilized fluorogenic substrates for proteinases: a method for visualization and quantitation of elastase release from human monocytes

An elastase-specific fluorogenic substrate, 6-(N-carbo-benzoxy-L-alanyl-L-alanyl-L-alanylamido)-qu inoline, was synthesized and immobilized via the fluorophoric group to an alkylatable derivative of polyacrylamide microspheres. Upon hydrolysis by elastase, the proteolytic product of the reaction fluorescences with a characteristic greenish-yellow light corresponding to the presence of the 1-alkyl-6-aminoquinolinium ion. This method has been applied to detect the elastase activity released from monocytes grown on the microspheres. Because the fluorescent product is covalently attached to the microsphere and cannot diffuse away from the site of reaction, it is possible to identify individual cells releasing the proteinase molecules. These experiments demonstrate that covalently immobilized fluorogenic substrates can be used for direct visualization and quantitation of proteinase activity from individual cells in culture.     

Application of bead-based assays for flow cytometry analysis

To date microsphere-based assays in flow cytometry have focused on the detection of antibody or antigen. Most studies have been research based to evaluate the performance of the technique relative to conventional techniques. However, there have not been any carefully controlled studies of the sensitivity and specificity, as well as analytic sensitivity of the FMIA technique. As such, it is difficult to document advantages of this tecnique clearly. The data suggest that FMIA is considerably more sensitive than conventional techniques, and the ability to analyze for multiple analytes in one sample dilution is attractive. This ability to simultaneously analyze for multiple samples is primarily dependent upon the size difference as sensed by FALS of the microspheres. However, it is also possible to use microspheres of the same size but that differ in either fluorescence or RALS signal. If microspheres of the same size are used but one fluoresces red and the signal in the assay uses a green fluorochrome, then the two microspheres can be separated by their red fluorescence. Using this technique, one can increase the number of microspheres that can be used in an assay. It is also possible to use microspheres of the same size but with different abilities to scatter the incident light at right angles. The use of these microspheres is then similar to the nonfluorescent versus red microspheres. By the judicious combination of microsphere size, it is possible to easily differentiate eight different microspheres. With the addition of a fluorescebt dye and/or differences in right-angle light-scatter capabilities, the number of different microspheres that can be used simultaneously becomes quite large. In practice, the number of microspheres that can be differentiated is no doubt greater than the number of analytes that need to be assayed in one assay.Although the apparent increase in sensitivity and the ability to simultaneously detect and quantitate numerous analytes are important attributes of FMIA, there are drawbacks to this method. Although the FMIA lends itself well to one-step no-wash procedures, when wash steps are necessary they are time-consuming and ineffecient. Most wash steps in FMIA use centrifugation of the microspheres to remove them from the reagent. There is a significant loss of microspheres in these wash steps, which are time-consuming. There are studies ussing vacuum filtration of the suspension to separate the microspheres from the reagents. A number of different groups are pursuing an automated or semiautomated method for the efficient washing and reagent delivery system for FMIA. Commercial systems are being developed that may allow for the easier handling of these reagents.Numerous groups are investigating the use of microspheres and flow cytometry primarily in immunoassay development. The procedure has the advantages of the simultaneous yet discrete analysis of multiple analytes and the inherent increase in sensitivity using fluorescence over other signals. There will no doubt be wider applications     

A Simple Method for Encapsulating Single Cells in Alginate Microspheres Allows for Direct PCR and Whole Genome Amplification

Microdroplets are an effective platform for segregating individual cells and amplifying DNA. However, a key challenge is to recover the contents of individual droplets for downstream analysis. This paper offers a method for embedding cells in alginate microspheres and performing multiple serial operations on the isolated cells. Rhodobacter sphaeroides cells were diluted in alginate polymer and sprayed into microdroplets using a fingertip aerosol sprayer. The encapsulated cells were lysed and subjected either to conventional PCR, or whole genome amplification using either multiple displacement amplification (MDA) or a two-step PCR protocol. Microscopic examination after PCR showed that the lumen of the occupied microspheres contained fluorescently stained DNA product, but multiple displacement amplification with phi29 produced only a small number of polymerase colonies. The 2-step WGA protocol was successful in generating fluorescent material, and quantitative PCR from DNA extracted from aliquots of microspheres suggested that the copy number inside the microspheres was amplified up to 3 orders of magnitude. Microspheres containing fluorescent material were sorted by a dilution series and screened with a fluorescent plate reader to identify single microspheres. The DNA was extracted from individual isolates, re-amplified with full-length sequencing adapters, and then a single isolate was sequenced using the Illumina MiSeq platform. After filtering the reads, the only sequences that collectively matched a genome in the NCBI nucleotide database belonged to R. sphaeroides. This demonstrated that sequencing-ready DNA could be generated from the contents of a single microsphere without culturing. However, the 2-step WGA strategy showed limitations in terms of low genome coverage and an uneven frequency distribution of reads across the genome. This paper offers a simple method for embedding cells in alginate microspheres and performing PCR on isolated cells in common bulk reactions, although further work must be done to improve the amplification coverage of single genomes.     

Kinetic analysis of unpurified native antigens available in very low quantities and concentrations

Affinity measurements of antigen–antibody interactions are generally performed using known concentrations of purified or recombinant materials. In addition, many technologies that measure affinity require the interacting components to be present in at least microgram quantities. Specifically, if the antigen is either available only in low quantities or unable to be purified, or if the quantity is unknown, then the measurement of affinity can be very difficult. Using the Kinetic Exclusion Assay (KinExA) technology, here we describe a method that overcomes the requirement for large amounts of purified and known quantities of antigen. We used this method to precisely measure the affinity of fully human anti-human interleukin 13 (IL13) monoclonal antibodies to IL13 produced in native form from primary T cells derived from a variety of species, including human. These antigens were available only in the limited quantities present in the conditioned cell culture medium, and the affinity was measured directly without further purification.     

Investigations on the Physical Structure and the Mechanism of Drug Release from an Enteric Matrix Microspheres with a Near-Zero-Order Release Kinetics Using SEM and Quantitative FTIR

The objectives of this study were to evaluate the physical structure and the release mechanisms of theophylline microspheres made of Eudragit S 100 polymer as an enteric polymer, combined with a nonerodible polymer, Eudragit RL 100. In the preparation process, polymer combinations (1:1) were dissolved in an organic solvent mixture composed of acetone and methanol at a specific ratio containing a theoretical drug loading of approximately 15%. Two microsphere formulations (LS1 and LS2) were prepared at two different total polymer concentrations (10% in LS1 and 12.7% in LS2). Dissolution studies were carried out using US Pharmacopeia Dissolution Apparatus II in an acidic medium for 8 h and in an acidic medium (2 h) followed by a slightly basic-buffered medium for 10 h. Both LS1 and LS2 microsphere formulations produced particles that were spherical in shape and had very narrow size distributions with one size fraction comprising 70–80% of the yield. Scanning electron microscopy and quantitative Fourier transform infrared were used for microsphere physical structure evaluation. Except for the absence of drug crystals, photomicrographs of both LS microspheres after dissolution in pH 1.2 and 7.2 buffer solutions were similar to those before dissolution. Dissolution results indicated the ability of LS microspheres to minimize drug release during the acid stage. However, in the slightly basic medium that followed the acidic stage, the drug release was sustained and controlled in its kinetics and data fitted to Peppas equation indicated a case II transport suggesting that the drug release is mainly through swelling/erosion mechanism.     

The blood supply to the abdominal organs of the fetal guinea-pig

Guinea-pigs near term of pregnancy were anaesthetized with diazepam and sodium pentobarbitone. A fetus was exposed and the vitelline artery catheterized to measure blood pressure and heart rate or to render a reference sample of blood for the determination of organ blood flow by the microsphere technique. The radioactive microspheres were injected through a catheter in the right atrium. Mean arterial blood pressure was 4.0 kPa and heart rate was 261 beats min-1. The liver, spleen, pancreas and gut receive most of their blood supply from the same trunk as the vitelline artery. The sample from this vessel was also used to calculate blood flow to the adrenal glands, kidneys, urogenital tract, and placenta, assuming even mixing of microspheres and blood in the abdominal aorta. Umbilical blood flow, corrected to a fetal weight of 100 g, averaged 7.5 ml min-1. The adrenal glands, which are known to increase their cortisol secretion near term, had a very high rate of perfusion. If the microspheres were injected in the umbilical vein, almost all were trapped in the liver, confirming the absence of a ductus venosus in the guinea-pig fetus. Most of these microspheres were found in the quadrate lobe of the liver. Hepatic arterial blood flow was also unequally distributed, with more than two-thirds going to the right lobe of the liver. Although the distribution of portal venous blood flow is not known, it is evident that different areas of the liver are presented with blood of greatly varying oxygen saturation.