Release of prostacyclin,endothelium-derived relaxing factor and endothelin by freshly harvested cells attached to microcarrier beads

Summary Cultured endothelial cells have been used in the past as a source of endothelium-derived relaxing factor (EDRF) and of prostacyclin (PGI₂). Although cell cultures are essential for observation of prolonged exposure to media or when there is delayed response, they are time consuming and sterile conditions are essential. In the present study, we report that endothelial cells, freshly harvested from bovine aortas, readily attached themselves to cytodex-3 microcarrier beads and released an endothelium-derived relaxing factor (EDRF), prostacyclin (PGI₂) and increased the amount of cyclic GMP in vascular smooth muscle. Attachment to microcarrier beads was essential since it increased the surface area and the number of attached cells and permited collection of cell free filtrates because of the formation of dense networks of cells and beads. As a result superfusion of cells and beads on the filter did not dislodge bound cells which remain on the filter. Conditioned filtrates from freshly harvested endothelial cells attached to microcarrier beads caused marked relaxation of endothelium-deprived bovine pulmonary artery strips. The degree of relaxation depended on the number of cells; maximal relaxation occurred with 50 million cells at ED₅₀ of 14 million. High values of cyclic GMP were found in vascular smooth muscle exposed to conditioned filtrate. The calcium ionophore A23187 further increased the amount of cyclic GMP. Large amounts of PGI₂ were released by freshly harvested endothelial cells particularly after stimulation with the calcium ionophore. In contrast, endothelin production by freshly harvested cells attached to microcarrier beads was barely detectable after 30 min incubation and was beyond the limit of detection by bioassay procedures. Freshly harvested endothelial cells attached to microcarrier beads appear to be a useful adjunct to tissue cultures under specific experimental conditions.Abbreviations EDRF Endothelium-Derived Relaxing Factor - PGI₂ Prostacyclin - K-H Krebs-Henseleit solution - cyclic GMP cyclic Guanosine Monophosphate - fmoles femtomoles - IB Ibuprofen     

The effect of immune mediators (cytokines) on the release of endothelium-derived relaxing factor (EDRF) and of prostacyclin by freshly harvested endothelial cells.

The effect of recombinant tumor necrosis factor and other cytokines stimulated by LPS (lipopolysaccharide), on the release of endothelial-derived relaxing factor and of prostacyclin was investigated using freshly harvested endothelial cells attached to plastic microcarrier beads. The results show that the cytokines failed to interfere with the release of EDRF and prostacyclin under the conditions of these experiments.     

Cytokine production from freshly harvested human mononuclear cells attached to plastic beads.

The release of tumor necrosis factor (TNF), interleukin-1 beta (IL-1) and granulocyte-macrophage colony-stimulating factor (GM-CSF) from freshly harvested monocytes and lymphocytes attached to plastic beads was investigated. Previous studies had shown that freshly harvested endothelial cells attached to microcarrier beads release an endothelium-derived relaxing factor. Attachment of freshly harvested lymphocytes and monocytes to plastic beads created a dense network, consisting of 25% monocytes and 75% lymphocytes as shown by flow cytometry. Viability of cells was 90%. Monocytes were characterized by phagocytosis and non-specific esterase stain. Freshly harvested cells stimulated with lipoprotein lipase (LPS) released TNF and IL-1. Non-stimulated cells also produced GM-CSF five hours after collection of blood.     

Neovascular responses induced by cultured aortic endothelial cells

Neovascularization was studied in the chorioallantoic membrane of the chick embryo after implantation of bovine aortic endothelial and smooth muscle cells, Swiss and BALB/c 3T3 cells and human diploid fibroblasts cultured separately on microcarrier beads. Quantitative analysis of neovascularization indicated a 3 1/2-fold increase in the number of blood vessels responding to endothelial cells while smooth muscle cells induced a twofold increase when compared to the response of beads without cells. Skin fibroblasts and Swiss 3T3 cells did not elicit a comparable response. The marked angiogenic response induced by endothelial cells was characterized by a 137% increase in total vessel length and a 35% increase in average vessel area when compared to controls. Two of the properties required for an angiogenesis factor--stimulation of cellular migration and proliferation--can also be demonstrated using endothelial cell-conditioned medium in cell culture systems. Medium from cultured bovine aortic endothelium stimulates DNA synthesis, proliferation, and migration of smooth muscle cells. In addition, conditioned media from both endothelial cells and smooth muscle cells produced an angiogenic response in the chorioallantoic membrane assay, which was comparable to that produced by intact cells growing on microcarrier beads. Similar responses were not evident with medium conditioned by other cell types. These results indicate the potential importance of endothelial cells and endothelial cell products in regulating blood vessel growth.     

Pure gelatin microcarriers: Synthesis and use in cell attachment and growth of fibroblast and endothelial cells

Summary A new type of microcarrier was described using bead emulsion-polymerization techniques. An aqueous solution of gelatin and glutaraldehyde was dispersed in a hydrophobic phase of mineral oil, using Triton X-114 as an emulsifier, and polymerization was initiated. The resultant spherical beads, composed entirely of gelatin, showed excellent mechanical stability to ethanol drying, sterilization, and long-term use in microcarrier spinner cultures. The solid gelatin microcarriers supported the growth of L-929 fibroblast, swine aorta endothelial, human umbilical endothelial, and HeLa-S₃ cultures with no adverse effects on cell morphology or growth. The beads were transparent in growth medium and attached cells were clearly visualized without staining. The beads were also compatible with techniques for scanning electron microscopy. Collagenase could be used to entirely digest the gelatin beads, leaving the cells free from microcarriers and suspended in solution while retaining 98% cell viability. The results further showed that after collagenase treatment the cells would populate fresh gelatin microcarriers and grow to confluence. Cell attachment kinetics revealed that the endothelial cells attached to the gelatin beads at the same rate as to tissue culture plates, whereas the fibroblast cells attached to the beads more slowly. However, once the fibroblast cells were attached to the gelatin microcarriers they spread and grew normally. This research was supported in part by the National Institutes of Health (GN 29127) and Ventrex Laboratories, Portland, Maine.     

Release and properties of endothelium-derived relaxing factor (EDRF) from endothelial cells in culture

Cultured bovine endothelial cells were seeded onto the intimal surface of endothelium-denuded rings of canine coronary artery. These rings did not previously relax to acetylcholine, substance P, bradykinin, and A23187. After seeding, the same rings relaxed to bradykinin and A23187, but not to acetycholine or substance P. Indomethacin pretreatment did not affect these responses. Cells from the same source were then grown to confluence on microcarrier beads, poured into small columns, and perfused with Krebs' solution. The perfusate from the columns was bioassayed on endothelium-denuded rings of coronary artery from either the dog or pig. Challenge of the column in the presence of indomethacin with either bradykinin or A23187 as well as acetylcholine or substance P caused release of a substance that relaxed both types of artery. Its activity half-life was 6.4 +/- 0.4 sec at 37 degrees C and it was hydrophilic and negatively charged. Prostacyclin (PGI2) as a candidate for EDRF was ruled out because 1) indomethacin failed to block its release and 2) the pig coronary artery, although insensitive to PGI2, relaxed to the endothelium-derived substance. These results show that, in response to a number of dilator drugs, cultured endothelial cells release a vascular relaxing substance (EDRF) that has characteristics similar to the EDRF of normal endothelium. The chemical nature of EDRF awaits clarification.     

Membrane anchored protein production from spheroid, porous, and solid microcarrier chinese hamster ovary cell cultures

The influence of the microcarrier type on the performance of a controlled release process used to produce a recombinant glycosyl-phosphatidylinositol anchored protein was investigated. Chinese hamster ovary (CHO) cells expressing the human melanoma tumor antigen (p97) were cultured in 10% serum on Cultispher-GH porous microcarriers and then, for protein production, maintained in 2% serum. Cells were harvested every 48 h and p97 was recovered at 90 mug/mL and 40% purity. Harvested p97 concentrations were increased by harvestingfrom spheroid (241 mug/mL) and smaller porous microcarrier, Cultispher-G (167 mug/mL) cultures. The low total cell specific p97 production of cells cultured on Cultispher-GH was due to necrosis of cells within the beads, decreased p97 expression of the immobilized cells, dilution by the liquid (up to 40% volume) associated with settled beads, and incomplete recovery of p97 from within the beads. Cells cultured on solid microcarriers, Cytodex-1, had the highest cell viability and cell specific p97 production, It is recommended that a two-stage cyclic harvesting process of cells cultured on small Cultispher-G or on Cytodex-1 beads would minimize protein loss and maximize cell specific protein recovery. (c) 1995 John Wiley & Sons Inc.     

Simulated conditions of microgravity suppress progesterone production by luteal cells of the pregnant rat

The purpose of this study was to assess whether simulated conditions of microgravity induce changes in the production of progesterone by luteal cells of the pregnant rat ovary using an in vitro model system. The microgravity environment was simulated using either a high aspect ratio vessel (HARV) bioreactor with free fall or a clinostat without free fall of cells. A mixed population of luteal cells isolated from the corpora lutea of day 8 pregnant rats was attached to cytodex microcarrier beads (cytodex 3). These anchorage dependent cells were placed in equal numbers in the HARV or a spinner flask control vessel in culture conditions. It was found that HARV significantly reduced the daily production of progesterone from day 1 through day 8 compared to controls. Scanning electron microscopy showed that cells attached to the microcarrier beads throughout the duration of the experiment in both types of culture vessels. Cells cultured in chamber slide flasks and placed in a clinostat yielded similar results when compared to those in the HARV. Also, when they were stained by Oil Red-O for lipid droplets, the clinostat flasks showed a larger number of stained cells compared to control flasks at 48 h. Further, the relative amount of Oil Red-O staining per milligram of protein was found to be higher in the clinostat than in the control cells at 48 h. It is speculated that the increase in the level of lipid content in cells subjected to simulated conditions of microgravity may be due to a disruption in cholesterol transport and/or lesions in the steroidogenic pathway leading to a fall in the synthesis of progesterone. Additionally, the fall in progesterone in simulated conditions of microgravity could be due to apoptosis of luteal cells.     

A low-serum medium with BSA and ferrous sulfate for the production of tissue plasminogen activator by human embryo lung cells

A low-serum medium containing bovine serum albumin (BSA) was investigated with respect to the growth of and tissue plasminogen activator (TPA) production by human embryo lung (HEL) cells on microcarrier beads and in collagen gel. BSA and ferrous sulfate were chosen as substitutes for fetal calf serum (FCS) through a simple screening test involving many substances. The growth promoting effects of BSA and ferrous sulfate were independent of each other and from the FCS concentration. Though BSA inhibited initial cell attachment to the carrier surface, it did promote the growth of cells attached to microcarrier beads. Cells grown on microcarrier beads in the low-serum medium containing BSA, ferrous sulfate and 3% FCS produced an amount of TPA similar to that produced by ones grown in the 10% FCS medium. Although cells on the dish surface did not grow at all on serum-free media containing BSA and ferrous sulfate, cells in the collagen gel were able to grow slightly on the serum-free medium. Cells grown on the low-serum medium in collagen gel produced more TPA over a long period than those in the microcarrier beads using the low-serum medium. The optimum concentration of proteose peptone in the TPA production medium for the collagen gel culture was similar to that for the dish surface culture.     

Microcarrier culture of vascular endothelial cells on solid plastic beads

The culture of vascular endothelial cells on solid plastic beads is described. A greater than 30-fold increase in cell numbers was achieved in stationary culture medium. The inclusion of fibroblast growth factor slightly improved the rate of growth from low densities. Addition of fresh beads to colonized beads resulted in colonization of the newly introduced microcarrier. In common with the behaviour of endothelium in conventional culture, the cells cultured on beads changed from a fusiform to a polygonal shape after reaching confluence. Cell proliferation was also observed by [³H]thymidine autoradiography of DNA. The fraction of radiolabelled nuclei declined at confluence on each bead, indicating density-inhibition of growth. By electron microscopy, the cells displayed the typical ultrastructural appearance of endothelium. Following transfer of colonized beads to a chromatography column with slow perfusion of the bead bed, cell viability was maintained over a 24 h period and proportional synthesis of prostaglandin I₂ upon stimulation by ionophore A23187 was demonstrated. This simple microcarrier technique allows the generation of large numbers of vascular endothelial cells for subcellular fractionation with economical use of space and medium. When set up as a perfused bead bed, it offers possibilities for the short-term collection of concentrated endothelial metabolites.     

Endothelium-derived nitric oxide: actions and properties

Vascular smooth muscle relaxation in response to chemically diverse naturally occurring neurotransmitters and autacoids has been attributed to the formation and/or release of one or more vascular endothelium-derived relaxing factors (EDRFs) distinct from prostacyclin. The chemical, biochemical, and pharmacological properties of one such EDRF resemble closely the properties of nitric oxide (NO). Thus, both arterial and venous EDRFs as well as authentic NO cause heme-dependent activation of soluble guanylate cyclase, endothelium-independent vascular and nonvascular smooth muscle relaxation accompanied by tissue cyclic GMP formation, and inhibition of platelet aggregation and adhesion to endothelial cell surfaces. EDRF from artery, vein, and freshly harvested and cultured aortic endothelial cells was recently identified as NO or a labile nitroso species as assessed by chemical assay and bioassay. Endothelium-derived NO (EDNO) has an ultrashort half-life of 3-5 s due to spontaneous oxidation to nitrite and nitrate, both of which have only weak biological activity. EDNO can be synthesized from L-arginine and possibly other basic amino acids and polypeptides, perhaps by oxidative metabolic pathways that could involve polyunsaturated fatty acid-derived oxygen radicals. Inorganic nitrite could serve as both a stored precursor and an inactivation product of EDNO. EDNO and related EDRFs may serve physiological and/or pathophysiological roles in the regulation of local blood flow and platelet function.     

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive,negative or collagen coated

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen, was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins adsorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.     

Quantitative and kinetic characterization of nitric oxide and EDRF released from cultured endothelial cells

Endothelial cells (EC) contribute to the control of local vascular diameter by formation of an endothelium derived relaxant factor (EDRF) (1). Whether nitric oxide (NO) is identical with (EDRF) or might represent only one species of several EDRFs has not been decided as yet (2-5). Therefore, we have directly compared in cultured EC the kinetics of NO formation determined in a photometric assay with the vasodilatory effect of EDRF and NO in a bioassay. Basal release of NO was 16, 4 pmol/min/ml packed EC column. After stimulation with bradykinin (BK) and ATP onset of endothelial NO release and maximal response preceded the EDRF-mediated relaxation. Concentrations of NO formed by stimulated EC were quantitatively sufficient to fully explain the smooth muscle relaxation determined in the bioassay. Our data provide convincing evidence that under basal, BK and ATP-stimulated conditions 1. endothelial cells release nitric oxide as free radical, 2. nitric oxide is solely responsible for the vasodilatory properties of EDRF.     

Cell growth optimization in microcarrier culture

Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephandex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per milliliter of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for scale up into large volume production units.     

Homogeneous cultivation of animal cells for the production of virus and virus products

Homogeneous technique facilitates the cultivation of large quantities of cells, reduces the risk of contamination by eliminating many manipulations, and makes practical the control of conditions such as pH and oxygen tension. Although most animal cells will not multiply in free suspension, certain cell lines have lost the requirement of being attached to a solid surface. These cells can be subcultured indefinitely but have some resemblance to cancer cells such as their abnormal karyotype. Certain cell linen developed from human embryonic tissue maintain their diploid character after repeated subculture and would seem to be ideal for the production of vaccines. However, strict regulations exist for viral products for human injection in that only cells taken from normal tissue and subcultured but once may be used. A microcarrier method in which cells adhere to DEAE-Sephadex beads permits a suspension culture which may be termed quasihomogeneous. The attached cells may be retained by sedimentation or by screening as the medium is replaced. Cell debirs from the original tissue is difficult to remove from microcarrier cultures; modifications of the trypsinization technique have alleviated but not solved this problem. Conditions for virus replication can be less critical than those for cell growth in that oxygen tension seems to have little influence on virus production. In cases where rate of virus production increases with specific growth rate of cells, homogeneous culture would have a advantage in maintaining a high cell mogeneous culture would have a valuble advantage in maintaining a high cell growth rate for a longer time. Some virus infections destroy cells, but others cause little change in cellular mteabolism except that virus is continually produced. The latter type can be conducted with a microcarrier in continuous culture with a virus titer exceeding 10⁷ plaque forming units per milliliter for over 50 days with Rubella-infected BHK cells.     

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.3 and 500 degrees C/min and stored below -180 degrees C. Samples were thawed in a 37 degrees C water bath and the cryoprotectant was removed by serial dilution at 22 degrees C over 6 min. The recovery of cell suspensions was assayed by culturing aliquots in 24-well plates for 7-9 days and counting the number of colonies that contained >25 cells. Maximum survival was 45-50% at cooling rates of 0.3, 1.0, and 10 degrees C/min, but decreased to 20% at 50 degrees C/min and to <1% at 500 degrees C/min. Biosilon microcarrier beads were used for the attached cells. Confluent beads were cryopreserved by exactly the same technique and cell function was assayed by measuring active amino acid (leucine) transport at 37 degrees C. Control, untreated confluent beads gave approximately 73% of control uptake and negative controls (frozen without cryoprotectant) gave approximately 4% uptake. The cells attached to beads showed percentage uptakes that were numerically similar to the survival of cells in suspension at cooling rates between 10 and 500 degrees C/min, but at lower cooling rates the recovery of attached cells increased to 70% at 1 degrees C/min and to 85% at 0.3 degrees C/min. These results indicate a marked difference in the effect of cooling rate on ECV304 cells depending upon attachment.     

Neonatal rat heart cells cultured in simulated microgravity

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

Cell growth optimization in microcarrier culture

Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm² of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.     

Isolation of cell plasma membranes on microcarrier culture beads

A simple, efficient method for the purification of plasma membranes from cultured cells is presented. Membrane purification is effected by attachment of viable cells to commercially available microcarrier culture beads, followed by lysis of the cells, agitation on a vortex mixer and sonication. Optimal conditions for each step of the procedure are described. Enzyme markers from plasma membranes are purified 10–20-fold relative to whole cell homogenates while internal membrane markers are depleted 10–20-fold.     

Attachment of marine sponge cells of Hymeniacidon perleve on microcarriers

Toward the development of an in vitro cultivation of marine sponge cells for sustainable production of bioactive metabolites, the attachment characteristics of marine sponge cells of Hymeniacidon perleve on three types of microcarriers, Hillex, Cytodex 3, and glass beads, were studied. Mixed cell population and enriched cell fractions of specific cell types by Ficoll gradient centrifugation (6%/8%/15%/20%) were also assessed. Cell attachment ratio (defined as the ratio of cells attached on microcarrier to the total number of cells in the culture) on glass beads is much higher than that on Cytodex 3 and Hillex for both mixed cell population and cell fraction at Ficoll 15-20% interface. The highest attachment ratio of 41% was obtained for the cell fraction at Ficoll 15-20% interface on glass beads, which was significantly higher than that of a mixed cell population (18%). The attachment kinetics on glass beads indicated that the attachment was completed within 1 h. Cell attachment ratio decreases with increase in cell-to-microcarrier ratio (3-30 cells/bead) and pH (7.6-9.0). The addition of serum and BSA (bovine serum albumin) reduced the cell attachment on glass beads.