Emulsion-based encapsulation of pluripotent stem cells in hydrogel microspheres for cardiac differentiation

Cardiovascular disease is the leading cause of death worldwide, and current treatments are ineffective or unavailable to majority of patients. Engineered cardiac tissue (ECT) is a promising treatment to restore function to the damaged myocardium; however, for these treatments to become a reality, tissue fabrication must be amenable to scalable production and be used in suspension culture. Here, we have developed a low-cost and scalable emulsion-based method for producing ECT microspheres from poly(ethylene glycol) (PEG)–fibrinogen encapsulated mouse embryonic stem cells (mESCs). Cell-laden microspheres were formed via water-in-oil emulsification; encapsulation occurred by suspending the cells in hydrogel precursor solution at cell densities from 5 to 60 million cells/ml, adding to mineral oil and vortexing. Microsphere diameters ranged from 30 to 570 μm; size variability was decreased by the addition of 2% poly(ethylene glycol) diacrylate. Initial cell encapsulation density impacted the ability for mESCs to grow and differentiate, with the greatest success occurring at higher cell densities. Microspheres differentiated into dense spheroidal ECTs with spontaneous contractions occurring as early as Day 10 of cardiac differentiation; furthermore, these ECT microspheres exhibited appropriate temporal changes in gene expression and response to pharmacological stimuli. These results demonstrate the ability to use an emulsion approach to encapsulate pluripotent stem cells for use in microsphere-based cardiac differentiation.     

Development of multilayered cell‐hydrogel composites using an acoustic focusing technique

Multilayered composites, composed of mammalian cells arranged in a hydrogel, have been prepared using an acoustic focusing technique. Acoustic focusing is a simple, nonchemical technique that allows for the fast arrangement of cells in matrices where the control of cell geometry is beneficial. Breast cancer cells (MDA‐MB231) were dispersed in a 30 wt % solution of poly(ethylene glycol) diacrylate (PEGDA) of molecular weight 400 at a density of 5 × 10⁶ cells/mL of PEGDA solution. An ultrasonic field was used to organize the cells before polymerization of PEGDA. Disk‐shaped hydrogel composites, typically 1 cm in diameter and 2‐mm thick were prepared based on a PEGDA solution volume of 130 μL. At an acoustic frequency of 2.32 MHz, composites having cells positioned within concentric cylindrical shells interspersed with zones of cell‐free hydrogel were produced. The cells were located in annuli approximately 80‐μm thick and about 300 μm apart. The structure and viability of the cells within these constructs were studied using a fluorescent LIVE/DEAD assay. The viability of the cells was on the order of 50%. For the conditions used in this study, cell death was primarily attributed to exposure of cells to the PEGDA solution prior to polymerization, rather than adverse effects of polymerization or the sound field itself. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Ethanol production by Kluyveromyces lactis immobilized cells in copolymer carriers produced by radiation polymerization

The conditions for batch and continuous production of ethanol, using immobilized growing yeast cells of Kluyveromyces lactis, have been optimized. Yeast cells have been immobilized in hydrogel copolymer carriers composed of polyvinyl alcohol (PVA) with various hydrophilic monomers, using radiation copolymerization technique. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol production of immobilized growing yeast cells with these hydrogel carriers was related to the monomer composition of the copolymers and the optimum monomer composition was hydroxyethyl methacrylate (HEMA). In this case by using batch fermentation, the superior ethanol production was 32.9 g L(-1) which was about 4 times higher than that of cells in free system. The relation between the activity of immobilized yeast cells and the water content of the copolymer carriers was also discussed. Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier, were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) g L(-1). For all lactose feed concentrations, an increase in dilution rates from 0.1 h(-1) to 0.3 h(-1) lowered ethanol concentration in fermented broth, but the volumetric ethanol productivity and volumetric lactose uptake rate were improved. The fermentation efficiency was lowered with the increase in dilution rate and also at higher lactose concentration in feed medium and a maximum of 70.2% was obtained at the lowest lactose concentration 50 g L(-1).     

Eudragit S100 entrapped insulin microspheres for oral delivery

The purpose of this research was to investigate whether Eudragit S100 microspheres have the potential to serve as an oral carrier for peptide drugs like insulin. Microspheres were prepared using water-in oil-in water emulsion solvent evaporation technique with polysorbate 20 as a dispersing agent in the internal aqueous phase and polyvinyl alcohol (PVA)/polyvinyl pyrrolidone as a stabilizer in the external aqueous phase. The use of smaller internal aqueous-phase volume (50 μL) and external aqueous-phase volume (25 mL) containing PVA in the manufacturing process resulted in maximum encapsulation efficiency (81.8%±0.9%). PVA-stabilized microspheres having maximum drug encapsulation released 2.5% insulin at pH 1.0 in 2 hours. In phosphate buffer (pH 7.4), microspheres showed an initial burst release of 22% in 1 hour with an additional 28% release in the next 5 hours. The smaller the volumes of internal and external aqueous phase, the lower the initial burst release. The release of drug from microspheres followed Higuchi kinetics. Scanning electron microscopy of PVA-stabilized microspheres demonstrated spherical particles with smooth surface, and laser diffractometry revealed a mena particle size of 32.51±20 μm. Oral administration of PVA stabilized microspheres in normal albino rabbits (equivalent to 6.6 IU insulin/kg of animal weight) demonstrated a 24% reduction in blood glucose level, with maximum plasma glucose reduction of 76±3.0% in 2 hours and effect continuing up to 6 hours. The area under the percentage glucose reduction-time curve was 93.75%. Thus, our results indicate that Eudragit S100 microspheres on oral administration can protect insulin from proteolytic degradation in the gastrointestinal tract and produce hypoglycemic effect.     

Interpenetrating polymer network (IPN) hydrogel microspheres for oral controlled release application

Interpenetrating polymer network (IPN) hydrogel microspheres of sodium carboxymethyl cellulose (NaCMC) and poly(vinyl alcohol) (PVA) were prepared by water-in-oil (w/o) emulsion crosslinking method for oral controlled release delivery of a non-steroidal anti-inflammatory drug, diclofenac sodium (DS). The microspheres were prepared with various ratios of NaCMC to PVA, % drug loading and extent of crosslinking density at a fixed polymer weight. The prepared microspheres with loose and rigid surfaces were evidenced by scanning electron microscope (SEM). Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis confirmed the IPN formation. Differential scanning calorimetry (DSC) study was performed to understand the dispersion nature of drug after encapsulation. The in vitro drug release study was extensively evaluated depending on the process variables in both acid and alkaline media. All the formulations exhibited satisfactory physicochemical and in vitro release characteristics. Release data indicated a non-Fickian trend of drug release from the formulations. Based on the results of this study suggest that DS loaded IPN microspheres were suitable for oral controlled release application.     

Quantification of a novel h-shaped ultrasonic resonator for separation of biomaterials under terrestrial gravity and microgravity conditions

A novel, h-shaped ultrasonic resonator was used to separate biological particulates. The effectiveness of the resonator was demonstrated using suspensions of the cyanobacterium, Spirulina platensis. The key advantages of this approach were improved acoustic field homogeneity, flow characteristics, and overall separation efficiency (sigma = 1 - ratio of concentration in cleared phase to input), monitored using a turbidity sensor. The novel separation concept was also effective under microgravity conditions; gravitational forces influenced overall efficiency. Separation of Spirulina at cleared flow rates of 14 to 58 L/day, as assessed by remote video recording, was evaluated under both microgravity (相似文献   

Process and formulation variables in the preparation of injectable and biodegradable magnetic microspheres

The aim of this study was to prepare biodegradable sustained release magnetite microspheres sized between 1 to 2 μm. The microspheres with or without magnetic materials were prepared by a W/O/W double emulsion solvent evaporation technique using poly(lactide-co-glycolide) (PLGA) as the biodegradable matrix forming polymer. Effects of manufacturing and formulation variables on particle size were investigated with non-magnetic microspheres. Microsphere size could be controlled by modification of homogenization speed, PLGA concentration in the oil phase, oil phase volume, solvent composition, and polyvinyl alcohol (PVA) concentration in the outer water phase. Most influential were the agitation velocity and all parameters that influence the kinematic viscosity of oil and outer water phase, specifically the type and concentration of the oil phase. The magnetic component yielding homogeneous magnetic microspheres consisted of magnetite nanoparticles of 8 nm diameter stabilized with a polyethylene glycole/polyacrylic acid (PEG/PAA) coating and a saturation magnetization of 47.8 emu/g. Non-magnetic and magnetic microspheres had very similar size, morphology, and size distribution, as shown by scanning electron microscopy. The optimized conditions yielded microspheres with 13.7 weight% of magnetite and an average diameter of 1.37 μm. Such biodegradable magnetic microspheres seem appropriate for vascular administration followed by magnetic drug targeting.     

Microflotation performance for algal separation

The performance of microflotation, dispersed air flotation with microbubble clouds with bubble size about 50 μm, for algae separation using fluidic oscillation for microbubble generation is investigated. This fluidic oscillator converts continuous air supply into oscillatory flow with a regular frequency to generate bubbles of the scale of the exit pore. Bubble characterization results showed that average bubble size generated under oscillatory air flow state was 86 μm, approximately twice the size of the diffuser pore size of 38 μm. In contrast, continuous air flow at the same rate through the same diffusers yielded an average bubble size of 1,059 μm, 28 times larger than the pore size. Following microbubble generation, the separation of algal cells under fluidic oscillator generated microbubbles was investigated by varying metallic coagulant types, concentration and pH. Best performances were recorded at the highest coagulant dose (150 mg/L) applied under acidic conditions (pH 5). Amongst the three metallic coagulants studied, ferric chloride yielded the overall best result of 99.2% under the optimum conditions followed closely by ferric sulfate (98.1%) and aluminum sulfate with 95.2%. This compares well with conventional dissolved air flotation (DAF) benchmarks, but has a highly turbulent flow, whereas microflotation is laminar with several orders of magnitude lower energy density.     

Characterization of photo-cross-linked oligo[poly(ethylene glycol) fumarate] hydrogels for cartilage tissue engineering

Photo-cross-linkable oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels have been developed for use in tissue engineering applications. We demonstrated that compressive modulus of these hydrogels increased with increasing polymer concentration, and hydrogels with different mechanical properties were formed by altering the ratio of cross-linker/polymer in precursor solution. Conversely, swelling of hydrogels decreased with increasing polymer concentration and cross-linker/polymer ratio. These hydrogels are degradable and degradation rates vary with the change in cross-linking level. Chondrocyte attachment was quantified as a method for evaluating adhesion of cells to the hydrogels. These data revealed that cross-linking density affects cell behavior on the hydrogel surfaces. Cell attachment was greater on the samples with increased cross-linking density. Chondrocytes on these samples exhibited spread morphology with distinct actin stress fibers, whereas they maintained their rounded morphology on the samples with lower cross-linking density. Moreover, chondrocytes were photoencapsulated within various hydrogel networks. Our results revealed that cells encapsulated within 2-mm thick OPF hydrogel disks remained viable throughout the 3-week culture period, with no difference in viability across the thickness of hydrogels. Photoencapsulated chondrocytes expressed the mRNA of type II collagen and produced cartilaginous matrix within the hydrogel constructs after three weeks. These findings suggest that photo-cross-linkable OPF hydrogels may be useful for cartilage tissue engineering and cell delivery applications.     

A thermosensitive hydrogel capable of releasing bFGF for enhanced differentiation of mesenchymal stem cell into cardiomyocyte-like cells under ischemic conditions

A thermosensitive hydrogel capable of differentiating mesenchymal stem cells (MSCs) into cardiomyocyte-like cells was synthesized. The hydrogel was based on N-isopropylacrylamide (NIPAAm), N-acryloxysuccinimide, acrylic acid, and hydroxyethyl methacrylate-poly(trimethylene carbonate). The hydrogel was highly flexible at body temperature with breaking strain >1000% and Young's modulus 45 kPa. When MSCs were encapsulated in the hydrogel and cultured under normal culture conditions (10% FBS and 21% O(2)), the cells differentiated into cardiomyocyte-like cells. However, the differentiation was retarded, and even diminished, under low nutrient and low oxygen conditions, which are typical of the infarcted heart. We hypothesized that enhancing MSC survival under low nutrient and low oxygen conditions would restore the differentiation. To enhance cell survival, a pro-survival growth factor (bFGF) was loaded in the hydrogel. bFGF was able to sustainedly release from the hydrogel for 21 days. Under the low nutrient and low oxygen conditions (1% O(2) and 1% FBS), bFGF enhanced MSC survival and differentiation in the hydrogel. After 14 days of culture, survival of 70.5% of MSCs remained in the bFGF-loaded hydrogel, while only 4.9% of MSCs remained in the hydrogel without bFGF. The differentiation toward cardiomyocyte-like cells was completely inhibited at 1% FBS and 1% oxygen. Loading bFGF in the hydrogel restored the differentiation, as confirmed by the expression of cardiac markers at both the gene (MEF2C and CACNA1c) and protein (cTnI and connexin 43) levels. bFGF loading also up-regulated the paracrine effect of MSCs. VEGF expression was significantly increased in the bFGF-loaded hydrogel. These results demonstrate that the developed bFGF-loaded hydrogel may potentially be used to deliver MSCs into hearts for regeneration of heart tissue.     

Preparation and in vitro evaluation of xanthan gum facilitated superabsorbent polymeric microspheres

Interpenetrating polymer network (IPN) hydrogel microspheres of xanthan gum (XG) based superabsorbent polymer (SAP) and poly(vinyl alcohol) (PVA) were prepared by water-in-oil (w/o) emulsion crosslinking method for sustained release of ciprofloxacin hydrochloride (CIPRO). The microspheres were prepared with various ratios of hydrolyzed SAP to PVA and extent of crosslinking density. The prepared microspheres with loose and rigid surfaces were evidenced by scanning electron microscope (SEM). Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) analysis confirmed the IPN formation. Differential scanning calorimetry (DSC) study was performed to understand the dispersion nature of drug after encapsulation. The in vitro drug release study was extensively evaluated depending on the process variables in both acidic and alkaline media. All the formulations exhibited satisfactory physicochemical and in vitro release characteristics. Release data indicated a non-Fickian trend of drug release from the formulations. Based on the results, this study suggest that CIPRO loaded IPN microspheres were suitable for sustained release application.     

基于液滴微流控的细胞凝胶微球研究进展

液滴微流控技术在微纳米尺度上对多种流体的流动进行精确控制,从而能够以高通量的方式生成结构可调和成分可控的微纳米液滴。通过结合合适的水凝胶材料和制造方法,可以将单个或多个细胞高效地封装进水凝胶中,制备细胞凝胶微球。细胞凝胶微球可以为细胞的增殖、分化等提供一个三维的、相对独立可控的微环境,在三维细胞培养、组织工程与再生医学、干细胞研究和单细胞研究等生命科学领域具有重要价值。本文主要综述了基于液滴微流控技术的细胞凝胶微球的制备及其在生物医学领域的应用,并对未来的研究工作提出了展望。     

Effects of methocel A15LV, polyethylene glycol, and polyvinyl alcohol on CD13 and CD33 receptor surface content and metabolism of HL60 cells cultured in stirred tank bioreactors

Flow cytometry was used to examine the effect of hydrodynamic forces in a stirred tank bioreactor on the CD13 and CD33 receptor surface content of HL60 (human promyelocytic leukemia) cells. A step increase in agitation rate from 80 to 400 rpm reduced the HL60 cell apparent growth rate and increased the CD13 receptor surface content per cell, on average, by 95%. In contrast, this step increase in agitation rate to 400 rpm decreased the CD33 receptor surface content per cell, on average, by 10%. The protective effects of 0.1% Methocel A15LV, polyethylene glycol (PEG), and polyvinyl alcohol (PVA) on CD13 and CD33 receptor surface content were examined under agitation at 300 rpm in parallel 2 L bioreactor runs. The average CD33 receptor surface content was unaffected by the presence of Methocel A15LV or PEG, while PVA had a slight protective effect. In contrast, in terms of CD13 receptor content, HL60 cells agitated at 300 rpm with Methocel A15LV, PEG, or PVA behaved like cells agitated at 80 rpm with no media additives (McDowell and Papoutsakis, 1998). That is, Methocel A15LV, PEG, and PVA prevented the transduction of mechanical forces which affect CD13 cell content. HL60 cells cultured with 0.1% A15LV, PEG or PVA under conditions of mild agitation (60 rpm) in spinner flasks exhibited glucose consumption and lactate production rates that were approximately 20% lower than values of cultures containing no additive. Under conditions of agitation at 300 rpm in the 2 L bioreactor, the presence of A15LV, PEG, and PVA reduced the HL60 glucose consumption and lactate production rates by approximately 50%. Thus, media additives can dramatically reduce lactate accumulation in agitated bioreactors due to cell growth, in addition to providing protection from cellular injury.     

Intracellular degradation of microspheres based on cross-linked dextran hydrogels or amphiphilic block copolymers: a comparative raman microscopy study

Micro- and nanospheres composed of biodegradable polymers show promise as versatile devices for the controlled delivery of biopharmaceuticals. Whereas important properties such as drug release profiles, biocompatibility, and (bio)degradability have been determined for many types of biodegradable particles, information about particle degradation inside phagocytic cells is usually lacking. Here, we report the use of confocal Raman microscopy to obtain chemical information about cross-linked dextran hydrogel microspheres and amphiphilic poly(ethylene glycol)-terephthalate/poly(butylene terephthalate) (PEGT/PBT) microspheres inside RAW 264.7 macrophage phagosomes. Using quantitative Raman microspectroscopy, we show that the dextran concentration inside phagocytosed dextran microspheres decreases with cell incubation time. In contrast to dextran microspheres, we did not observe PEGT/PBT microsphere degradation after 1 week of internalization by macrophages, confirming previous studies showing that dextran microsphere degradation proceeds faster than PEGT/PBT degradation. Raman microscopy further showed the conversion of macrophages to lipid-laden foam cells upon prolonged incubation with both types of microspheres, suggesting that a cellular inflammatory response is induced by these biomaterials in cell culture. Our results exemplify the power of Raman microscopy to characterize microsphere degradation in cells and offer exciting prospects for this technique as a noninvasive, label-free optical tool in biomaterials histology and tissue engineering.     

Confocal measurement of the three-dimensional size and shape of plant parenchyma cells in a developing fruit tissue

Parenchyma cells from the inner mesocarp of a grape berry (Vitis vinifera L. cv. Chardonnay) were visualised in three-dimensions within a whole mount of cleared, stained tissue using confocal laser scanning microscopy and digital image reconstruction. The whole berry was fixed, bisected longitudinally, cleared in methyl salicylate, stained with safranin O and mounted in methyl salicylate. Optical slices were collected at 1.0 μm intervals to a depth of 150 μm. Neighbouring z-series were joined post-collection to double the field-of-view. Attenuation at depth of the fluorescent signal from cell walls was quantified and corrected. Axial distortion due to refractive index mismatch between the immersion and mounting media was calibrated using yellow-green fluorescent microspheres and corrected. Transmission electron microscopy was used to correct fluorescent measurements of cell wall thickness. Digital image reconstructions of wall-enclosed spaces enabled cells to be rendered as geometric solids of measurable surface area and volume. Cell volumes within the inner mesocarp tissue of a single grape berry exhibited a 14-fold range, with polysigmoidal distribution and groupings around specific size classes. Cell shape was irregular and the planes of contact were rarely flat or simple. Variability in cell shape was indicated by the range in surface area to volume ratios, from 0.080 to 0.198 μm^–1. Structural detail at the internal surface of the cell wall was apparent. The technique is applicable to a wide range of morphometric analyses in plant cell biology, particularly developmental studies, and reveals details of cell size and shape that were previously unattainable.     

Limited adhesion of biodegradable microspheres to E- and P-selectin under flow

In a variety of disease settings the expression of the endothelial selectins E- and P-selectin appears to be increased. This feature makes these molecules attractive targets around which to design directed drug-delivery schemes. One possible approach for achieving such delivery is to use polymeric biodegradable microspheres bearing a humanized monoclonal antibody (MAb) for E- and P-selectin, MAb HuEP5C7.g2. Perhaps the simplest technique for "coupling" HuEP5C7.g2 to the microspheres is via nonspecific adsorption. Previous studies suggest, however, that the adsorption of proteins onto microspheres fabricated in the presence of a stabilizer such as poly(vinyl alcohol) (PVA) is limited. It is unclear to what extent this limited level of adsorbed HuEP5C7.g2 would be able to support adhesion to E- and P-selectin under flow conditions. To explore this issue, we prepared microspheres from the biodegradable polymer, poly(epsilon-caprolactone) (PCL), using a single emulsion process and PVA as a stabilizer. We then incubated the PCL microspheres with HuEP5C7.g2 and studied the adhesion of the resulting HuEP5C7.g2 microspheres to E- and P-selectin under in vitro flow conditions. We found that the HuEP5C7.g2 PCL microspheres exhibit specific adhesion to Chinese hamster ovary cells stably expressing P-selectin (CHO-P) and 4-h IL-1beta-activated human umbilical vein endothelial cells (HUVEC). In contrast, HuEP5C7.g2 PCL microspheres exhibit little adhesion to parental CHO cells or unactivated HUVEC. The attachment efficiency to the selectin substrates was quite low, with appreciable attachment occurring only at low shear (0.3 dyn/cm(2)). Other supporting data strongly suggest that the limited attachment efficiency is due to a low level of HuEP5C7.g2 adsorbed to the PCL microspheres. Although the attachment was limited, a significant percentage of the HuEP5C7.g2 PCL microspheres were able to remain adherent at relatively high shear (8 dyn/cm(2)). Combined, our data suggest that HuEP5C7.g2 PCL microspheres exhibit selective limited adhesion to cellular substrate expressing E- and P-selectin.     

The COX-2 inhibitor Celecoxib enhances the sensitivity of KB/VCR oral cancer cell lines to Vincristine by down-regulating P-glycoprotein expression and function

Previous studies have indicated that long-term chemotherapy decreases the sensitivity of oral cancer cells to chemotherapeutics while simultaneously increasing resistance to these drugs. COX-2 inhibitors are known to enhance the toxic action of anti-tumor drugs against cancer cells. Using the MTT method, we investigated the influence of the COX-2 selective inhibitor Celecoxib on the proliferation of KB/VCR oral cancer cell lines and analyzed the effect of Celecoxib on the regulation of P-glycoprotein (P-gp) expression and function. Western blot analysis was employed to detect the expression of P-gp, and flow cytometry was used to evaluate P-gp function by detecting the accumulation of the active P-gp functional fluorescence substrate within KB/VCR cells. The results revealed that a low dose of Celecoxib (10 μmol/L) showed no growth inhibitory effects on KB/VCR cell lines. When the concentration of Celecoxib was greater than or equal to 20 μmol/L, the inhibitory effect on KB/VCR cells was significantly enhanced in a time- and dose-dependent manner. The lower dose of Celecoxib (10 μmol/L) significantly enhanced the toxicity of Vincristine (VCR) against KB/VCR cell lines. After the application of Celecoxib plus VCR (10 μmol/L+1.5μmol/L, respectively) treatment for 24, 48 or 72 h, the growth inhibition rates of KB/KBV cells were 37.82 ± 1.60%, 47.84 ± 1.29% and 54.43 ± 2.35%, respectively, which were significantly higher than the rates in the cells treated only with Celecoxib (10 μmol/L) or VCR (1.5 μmol/L) (all P<0.01). P-gp expression levels in KB/KBV cells treated with Celecoxib plus VCR (10 μmol/L+1.5 μmol/L, respectively) were markedly lower than the levels in control cells and those treated with VCR (1.5 μmol/L) (all P<0.01). In addition, the intensity of Rho123 fluorescence of KB/KBV cells in cells treated with Celecoxib plus VCR (10 μmol/L+1.5 μmol/L, respectively) or Celecoxib alone (10 μmol/L) was significantly higher than the intensity observed in control cells and those treated with VCR alone (1.5 μmol/L) (all P<0.01). The underlying mechanism of these phenomena is likely correlated with the down-regulation of the expression and function of P-gp due to Celecoxib, thereby increasing the amount of VCR accumulated in KB/VCR cells.     

Size Effects on the Uptake of Particles by Populations of Tetrahymena pyriformis Cells

Flow cytometry has been used to make direct measurements of rates of uptake of latex microspheres from dilute, monodisperse suspensions by Tetrahymena pyriformis. Measurements were made for five different sizes of microspheres, ranging from 1.09 to 6.17 μm diameter. Fractions of cells in the population that did not ingest the microspheres offered were also determined. In addition, the size distributions, as indicated by the forward angle light scattering intensity which is measured by the instrument, were determined for the whole population and for the subpopulations of cells that did and did not ingest the particles, for each particle size used. It was found that the fraction of cells that did not ingest the particles was small and independent of particle size when this was less than about 2.7 μm, but increased with particle size when particle size was increased above this value. The so-called maximum clearance rate, which can be calculated from the data, was found to increase monotonically with particle size if it were based only on those cells which actually ingested the particles offered. However, a plot of maximum clearance rate vs. particle size exhibited a maximum if the clearance rate were based on all cells present in the population.     

Biomimetic carbohydrate substrates of tunable properties using immobilized dextran hydrogels

Glycidylmethacrylate-modified dextran macromers (Dex-GMA) of different degrees of substitution (DS) were synthesized. The elastic modulus of the hydrogels produced using one-component and two-component macromer systems was measured using rheometry. When one macromer of DS 1/10 was used, a hydrogel modulus in the range of 0.2 Pa to 42 kPa was obtained by varying the Dex-GMA concentration from 80 to 200 mg/mL. When a mixture of two macromers of different DS (1/10 and 1/23) was used, a more uniform variation of modulus in the range of 0.4 Pa to 42 kPa was achieved by controlling the ratio of the two macromers. When dextran hydrogels were functionalized with fibronectin and immobilized onto glass substrates, the attachment, spreading, and growth of human aortic smooth muscle cells were modulated by the elastic properties of the dextran matrix. The dextran hydrogel system with tunable mechanical and biochemical properties appears promising for applications in cell culture and tissue engineering.     

The VX2 carcinoma in the rabbit auricle as an experimental model for intra-arterial embolization of head and neck squamous cell carcinoma with dextran microspheres

A head and neck cancer model is developed using the VX2 carcinoma cell line injected s.c. in both ears of New Zealand White (NZW) rabbits. The study is focused on the effects of intraarterial embolization of the carcinomas with a new type of dextran hydrogel microspheres. During the phase of exponential growth the tumour-surface doubling-time was 7.1+/-2.0 days. Standard deviation in growth of the tumours was significantly larger between separate animals than between tumours growing in the left and right auricle of each individual animal (2.0 versus 0.65 days). A fresh cell suspension containing at least 10 x 10(6) vital tumour cells was necessary to yield a tumour-take of 85%. The caudal auricular artery perfuses the caudal half of the external ear and is very suitable for macroscopic cannulation. Histological evaluation shows, that the use of dextran hydrogel microspheres of at least 25 microm in combination with ligation of non-tumour perfusing branches of the central auricular artery yields diffuse embolization of the VX2 carcinoma. This tumour model can be of use in further studies to optimize particle size and dosage for embolization as well as to evaluate the effect of different anti-neoplastic drugs, slowly released by controlled degradation of dextran microspheres.