Test of a novel transparent floating carrier by recombinant cho cells expressing sIL6 receptor protein.

We describe a carrier with some unusual properties."Cellrafts" increase yields from adherent cells in conventional static T150 flasks, by floating multilayer strips of transparent film at the oxygen-rich surface of unstirred medium. This new technique allowed microscopic inspection of cell growth inside the carriers during bulk culture. Individual carriers could be picked out for subculture of selected colonies. A novel surface treatment by hypochlorite/uv allowed recycling of used carriers. Cellrafts' open-deck structure facilitated trypsinization with90% release as viable single cells from bulk carriers. Macro size (10mm by 1 mm) enables retention in flask by a coarse sieve insert in its neck, facilitating separation of product cells or media. Residual cells in carriers regenerated repeated harvests without need for reseeding. Carriers were tested with shear-sensitive CHO clones expressing soluble human IL6 receptor (sIL6R). Control was monolayer bulk culture on trays. Floating multilayer cultures remained viable longer than monolayers, had higher cellular activity of protein expression, and were less serum dependent (resembling cells on porous carriers). Purity and anti-sIL6R binding were identical to control product. Cellrafts were also tested in a small spinner vessel, but for litre batches this proved less convenient than in T-flasks. Though yields are low compared to well established porous carrier technology (spinner or packed bed) static transparent carriers might provide transitional scaleup from normal cytogenetics laboratory culture.

Concentrations of endothelial-cell-stimulating angiogenesis factor, a major component of human uterine angiogenesis factor, in human and bovine embryonic tissues and decidua.

Embryonic development involves the establishment of new patterns of vascular growth in the fetus and within the lining of the womb. A factor, human uterine angiogenesis factor, has been purified from the decidua and stimulates the growth of blood vessels in collagen sponge implants and in the chick chorioallantoic membrane. Evidence is presented that suggests that a major active component of human uterine angiogenesis factor is an activator of latent matrix metalloproteinases, of low M(r), called endothelial-cell-stimulating angiogenesis factor and that this factor is present in substantial quantities in a number of embryonic tissues.

Effect of decidua angiogenic factors on experimental dermis allografts.

Meshed human dermis allografts containing human uterine angiogenic factor were implanted over full thickness surgical skin wounds in rats. Enhanced angiogenesis of the wound bed, three times more than that observed in control grafts, was found in experimental grafts at 3 days postimplantation. This was accompanied by a greater amount of granulation tissue formation, enhanced granulation tissue penetration into the dermal grafts and accelerated incorporation of the grafts. Angiogenesis of the wound bed regressed to control levels at 7 days postimplantation. The growth and penetration of the granulation tissue and the accelerated incorporation of the dermis grafts, however, continued ahead in the experimental grafts while necrosis appeared in the control grafts. The relevance of these results for acceleration of wound healing and improvement of the wound bed for subsequent application of cultured epidermal grafts is discussed. This method may be extended to the treatment of ulcers and burns.

Uterine angiogenic factor induces vascularization of collagen sponges in guinea-pigs.

The need for an adequate blood supply is of prime importance in successful skin grafting and in the take of keratinocyte cultures. Thus, the human uterine angiogenic factor (HUAF) extract, which induces neovascularization of the chorioallantoic membrane (CAM), was employed. The bioassay of HUAF was performed on an in vivo model of subdermally implanted collagen sponges and on sponges implanted into full skin thickness burn wounds in guinea-pigs. The HUAF extract was injected into the sponges every other day for 10 days. Each injection contained 10 micrograms decidual proteins with a total of 50 micrograms/sponge. The animals were sacrificed and the sponge together with the surrounding structures were extirpated, examined macro- and microscopically and by histological techniques. HUAF induced growth of blood vessels from the surrounding vascular bed into the implanted sponges. The angiogenesis was characterized by dense tortuous vessels with centripetal orientation. The control sponges exhibited only sporadic growth of blood vessels. This phenomenon repeated itself in the animals which were inflicted with burn wounds. The present study demonstrates that HUAF extract is also active on the in vivo model of experimental burns and wounds.

Polystyrene substratum for bulk culture of anchorage dependent cells.

Twisted ribbons made of polystyrene were used as a packing material for the cultivation of anchorage dependent cells. Normal human fibroblast cells grown on this support in a laboratory scale reactor reached densities of about 5-7×10(5) cells/ml. The cells adhered strongly to the carrier and no cell detachment was observed upon transfer to serum free medium. The properties of this packing material and its potential use are discussed.

Polymer treatments reduce adhesion of comminuted stone in rat bladder.

A method for inhibiting the adhesion of stone fragments in comminution is presented. Surfaces are coated with a molecular layer of so-called "excluding" polymer. In experimental tests, rat bladders were injected with comminuted urinary stone (phosphate or oxalate) in saline which contained small amounts of excluding polymer (0.1% hyaluronic acid or polyvinyl pyrrholidone). The urinary tract was then irrigated with fresh polymer solution and examined for residual stone. A ten-fold reduction in particle count was obtained, compared with irrigation with normal saline. The possible applications in percutaneous and extracorporeal lithotripsy are discussed. It seems that the "coagulum" and "exclusion" types of polymer irrigant might find complementary uses: the former for manual extraction of coarse particles and the latter for flushing out after fine comminution.

A chemical model for the cooperation of sulfates and carboxylates in calcite crystal nucleation: Relevance to biomineralization.

Acidic matrix macromolecules involved in regulation of biological crystal growth often contain aspartic acid-rich domains and covalently bound sulfated polysaccharides. We propose that sulfates and beta-sheet structured carboxylates cooperate in oriented calcite crystal nucleation. The sulfates concentrate calcium, creating the supersaturation necessary for nucleation on the structured carboxylate domains. An artificial model, composed of sulfonated polystyrene surfaces and adsorbed beta-sheet poly(aspartate), demonstrates that the two components indeed act cooperatively with respect to two independent assays, both by induction of calcite nucleation off the (001) plane and by calcium association. Evidence is presented that a purified organic matrix acidic glycoprotein from mollusk shells may behave in vitro in a similar way.

Activation of nuclear chromatin in stretch-dependent growth of tissues.

The growth of connective tissue cells can be controlled both by mechanical extension and by diffusion gradients. We have used nuclear fluorescence with acridine orange (AO) as a measure of nuclear activation, to study growth control in vivo in rat mesometrium and mouse spine. Frozen sections of spinal muscle taken from animals after two h exercise, revealed nuclear activation in peripheral cells. The number of active muscle nuclei decreased drastically with age. During mesometrial growth in pregnancy, AO fluorescence showed that activated cell nuclei occurred mainly near the capillaries. The width of the zone of activated nuclei was within 20 microns from the capillary walls. AO appears to be a sensitive stain for tracing gradients of growth in intact connective tissue.

Cell membrane entropy as regulator of receptor submergence.

An entropic mechanism, previously worked out to explain the low adhesiveness of fluid phospholipid, also seems to explain some effects of membrane fluidity on the "embedding" of receptor proteins. Water, at the phospholipid interface, has to compete with protein for the available area but is impeded in its Brownian motion by hydrogen bonding to the slow-moving phosphatide. Hence, any increase in phosphatide mobility (e.g. by lipid melting) is entropically favourable to water, which spreads by displacing receptor particles from the interface into the hydrocarbon phase. This reconciles the kinetic viewpoint of Shintzky (experimental correlation of submergence with microviscosity) with the thermodynamic theory of Gerson (submergence increases with phospholipid hydrophilicity). Because Brownian motion increases water entropy, it follows that a more fluid interface must have a more negative free energy i.e. become more hydrophilic.

Sulphonated polystyrene as an optimal substratum for the adhesion and spreading of mesenchymal cells in monovalent and divalent saline solutions.

Cell adhesion and spreading were studied on sulphonated polystyrene dishes in serum-free saline (Mn, Na, Cl, buffer) i.e., without an intervening protein layer. Spreading as a function of surface charge density, SCD, peaked around 2-10 negative charges per square nanometer, corresponding to a monomolecular layer of sulphonate ions. At optimal SCD, macrophages, BHK-C13 and whole mouse embryo secondary cells all showed considerable spreading, even in monovalent saline-more so than on a conventional tissue-culture surface. But outside this narrow range of SCD, or on protein-coated surfaces, the divalent cation was indispensable. The biphasic effect of sulphonation on cell adhesion is consistent with the theory that a substratum need not be biochemically specific, provided it is physiochemically polar, rigid and dense. According to this theory, polystyrene of sub-optimal SCD would not be sufficiently polar, while supra-optimal sulphonation would produce a hydrogel surface, lacking in local rigidity and density, due to osmotic swelling. The principle of polymer exclusion, by a surface hydrogel layer, is also consistent with observations on the inhibitory effects of adsorbed proteins-viz., albumin, collagen, serum and cellular exudate, respectively-contrasted with the ready attachment of cells to a bare, optimally charged substratum, in this minimal in vitro system.