Abstract: Disclosed is a method of recovering tissue plasminogen activator from media and cell extracts. The method comprises contacting the tPA-containing solution with a silicaceous matrix material comprising covalently bound polymer having plural anionic groups. Selective elution of the tPA can produce eluants of high specific activity. The method can succeed in recovering greater than 90% of the tPA activity from the crude solution.

Abstract: A method for sustained release of molecules from gels or microcapsules has been developed. The method is based on controlling the gel state using chelating agents or ion transfer. The method is particularly useful for controlling the rate of release of insolubilized proteins into physiological solutions.

Abstract: Disclosed is a method of recovering urokinase compounds from media and cell extracts. The method comprises contacting the urokinase compound-containing solution with a silicaceous matrix material comprising covalently bound polymer having plural anionic groups. Selective elution of the urokinase compound can produce elutants of high specific activity. The method can succeed in recovering greater than 90% of the urokinase activity from the crude solution.

Abstract: Disclosed is a process for damage-free encapsulation of a variety of core materials including viable cells. The core material is suspended in an aqueous solution of a polymeric material containing cationic groups such as an aminated glucopolysaccharide, e.g. chitosan. A temporary matrix is formed by gelling droplets of the suspension with a divalent or multivalent anion. The temporary matrix is permanently cross-linked with a polymeric material containing plural anionic groups, e.g., polyaspartic or polyglutamic acid, to form a semipermeable membrane. The interior of the microcapsule may be resolublized by subjecting the capsule to a solution of low molecular weight cations. The process produces microcapsules which are not sticky, do not clump and allow viable cell growth and proliferation.

Abstract: Permeable capsules are loaded with a reservoir of the substance to be dispensed at a concentration sufficient to provide an osmotic pressure above threshold level, and the pore size of the capsule membrane is controlled so that the passage of the substance through the membrane becomes the rate-limiting factor in dispensing. Shape-retaining spheres are formed from a water-soluble polymer containing plural anionic or cationic groups and cross-linking surface layers of the spheres by contact with a polymer having plural groups of charge opposite that of the water-soluble polymer. After loading, the capsules are again treated with the same or a different cross-linking polymer to reduce the dimensions of the pores.

Abstract: Disclosed is a method of improving protein production in protein-producing cell cultures. The method comprises the steps of culturing protein-producing cells in a medium comprising essential nutrients, vitamins, salts and amino acids, modified by the addition of increased amounts of amino acids so that the osmolarity of the medium is hypertonic, i.e., above about 340 milliosmoles, preferably within the range of 340 to 450 milliosmoles and most preferably 360 milliosmoles. Cells grown in such media produce protein at levels on the order of four times the protein production in normal, isotonic media.

Abstract: A method and apparatus for forming microcapsules from a fluid medium containing living culture material by the atomization of the medium and the treatment of the atomized droplets with a treatment fluid. A medium under pressure enters a chamber having a wall with a plurality of orifices formed therein. A vibrator vibrates the chamber. As the medium passes through the orifices the exit stream vibrates and forms small droplets. The droplets fall into a collection vessel on the other side of the wall, which contains treatment fluid for hardening the droplets into microcapsules. A flow of treatment fluid may be maintained through the collection vessel to prevent clumping of the droplets and to transport the hardened microcapsules for harvesting. Preferred operating pressures, orifice size and chamber configuration are shown.

Abstract: Disclosed are dispensing systems for releasing a substance at a substantially constant rate and methods for its manufacture. The composition comprises semipermeable capsules containing the material to be released. The capsules comprise membranes having pores of dimensions sufficient to control the kinetics of release provided there is maintained a large intracapsular concentration of the substance relative to the concentration desired in the extracapsular environment. The compositions may be produced by forming permeable capsules, suspending the capsules in a solution containing a high concentration of the substance to load the intracapsular volume, and then post-treating the capsules to reduce the size of the pores.

Abstract: Disclosed is a method for loading capsules having a semipermeable membrane with a chemically active substance to be released over time. The method first involves gradually deflating and dehydrating the capsules and subsequently soaking the deflated capsules in a solution containing the substance to be encapsulated. After loading, the permeability of the capsule may be adjusted to accommodate the requirements of the end use. The loaded capsules of this invention may be used in a variety of applications, including use as a bioimplantable drug or biochemical delivery system.

Abstract: Cells such as genetically modified cells, e.g., hybridoma, which secrete an antigenic substance are encapsulated within capsule membranes having pores of dimensions sufficient to permit efflux of the antigens secreted by the cells but insufficient to permit traverse of the cells. The capsules are injected into an experimental animal where antigen passing through the pores of the capsule membrane induces lymphocytes to produce antibodies complementary to the antigen. The antibody may be harvested from the circulatory system of the animal. Preferably, lymphocytes are sampled from, e.g., the spleen of the animal, fused with a malignant cell line to produce a hybridoma which synthesizes the antibody in vivo, and the hybridoma is cultured to produce large quantities of monoclonal antibody.

Abstract: Disclosed is an assay procedure for detecting and quantifying interferon epsilon, a new composition of matter having selective antiviral activity on epithelial cells. The assay comprises incubating a preparation believed to contain interferon epsilon with human keratinocyte cells and with human fibroblast cells followed by a virus challenge. The presence of interferon epsilon is indicated if the preparation has antiviral activity on the keratinocytes but no detectable activity on the fibroblasts. The titer of the preparation may be determined by serially diluting it, incubating the dilution with subcultures of keratinocytes, challenging the subcultures with a virus, observing the viability of cells in the cultures, and comparing the results with a standard.

Abstract: Disclosed is a large scale apparatus for the harvesting cell products secreted from cells grown within semipermeable artificial membranes. The apparatus comprises a number of cooperating processing stations which perform the various steps necessary in the harvesting of cell product. Included within the apparatus is a holding tank for receiving mature encapsulated cells and cell medium, as well as any reagents, from separate storage sources, used in the process. Also included, and in fluid communication with the holding tank, is a shearing station where the capsule membranes are ruptured to free the encapsulated cell product, and a separating station where the liquid cell product is separated from solid components such as cell membrane debris and cells. The apparatus may include a microprocessor which generates control signals to enhance the level of automation of the apparatus.

Abstract: A core material such as viable cells is encapsulated by gelling an alginate polymer with a polyvalent cation to form shape-retaining gelled masses containing the core material, expanding and hydrating the gelled masses by contacting the masses with an aqueous saline solution, and forming a membrane about the expanded gelled massed to form capsules by contacting the gelled masses with a polycationic polymer having a molecular weight greater than 3,000 daltons. Expanding before membrane formation, permits better control of permeability properties and uniformity of the membrane. The gelled masses within the membrane may be liquified by contacting the capsules with a chelating agent which is preferably ethylene glycol bis-(.beta.-amino ethyl ether)-N,N-tetra-acetic acid. A second membrane layer may be formed by contacting the capsules with a second polycationic polymer. The second membrane may be coated with a polyanionic polymer such as alginate.

Abstract: Disclosed is a new material having antiviral activity designated interferon epsilon. The material may be produced, for example, by exposing primary, diploid human epithelial cells to a virus and then incubating the cells under conditions in which the new interferon is produced and is secreted into the culture medium. The material is antigenically distinct from interferon alpha, interferon beta, and interferon gamma, and displays marked antiviral activity in human epithelial cells but no detectable activity in other cell types.

Abstract: Disclosed is a process for recovering nonsecreted substances produced by cells. The process eliminates some of the high molecular weight contaminants thereby simplifying the purification process. The cells are encapsulated within a semipermeable membrane having properties which permit rapid passage of the relatively low molecular weight substances of interest but retard or prevent passage of higher molecular weight contaminants. The encapsulated cells are suspended in a culture medium and undergo normal cell growth and mitosis. The encapsulated cell culture grows to substantially fill the capsules but not rupture them. The cell membrane is then lysed without disrupting the capsule membrane. The permeability of the capsule membrane is such that the substances of interest diffuse rapidly through the capsule membrane into the extracapsular fluid while the higher molecular weight contaminants and cell fragments are retained within the capsule.

Abstract: Disclosed is a method of growing anchorage-dependent cells: cells of the type which normally undergo mitosis only when anchored on a substrate, e.g., fibroblasts or epithelial cells. The method comprises the steps of encapsulating a seed culture of the cells within a semipermeable membrane and suspending the capsules in a growth medium. The interior surfaces of the capsule membrane and/or collagen enclosed within the capsules serve as a substrate for the cells. The ratio of the available substrate surface area to the volume of the culture may be large, thereby allowing the cells to be grown substantially throughout the volume of the culture medium.