Abstract: The method for the holding in flotation of a substance such as a tissue part in a bioreactor (61) is characterized in that the substance (73) is acted upon with a fluid; and in that the flow of the fluid acts counter to gravity in such a manner that the substance (73) is held in flotation. The bioreactor (61) with a container (62) for a substance (73) which is to be acted upon with fluid comprises a first flow chamber (66a) to which a flowing fluid can be supplied, with the first flow chamber (66a) being designed in such a manner that the fluid which flows upwardly therein has a lower speed with increasing height.

Abstract: An in vitro, three dimensional artificial tissue that resembles human skin has been developed. Microvascular endothelial cells from human adult lung were sandwiched between two layers of human dermal fibroblasts in three dimensional collagen gels. The sandwich was covered with keratinocytes. The cultures were self-maintained for prolonged periods of time without the addition of tumor promoters such as phorbol esters. Over a few days, the keratinocytes developed into a multilayered epithelium. Microvessels were produced in the support matrix. The microvessels were composed of a tight monolayer of endothelial cells surrounded by a continuous basal lamina, contacted by newly formed, sparse perioendothelial cells. The microvessels also contained newly formed blood cells. Human matrix molecules characteristic of skin were produced.

Abstract: The present invention relates to a medium for promoting the survival of islet cells, which comprises one or more growth factors in combination with FK506 in amounts having an anti-apoptotic effect on islet cells in a physiologically acceptable culture medium.

Abstract: The present invention relates to an apparatus and methods that immobilize one or more cells associated with magnetic material on a substrate on which are located one or more magnetic receptacle(s). Alternatively, in another aspect the present invention, the device arrays cells associated with magnetic material on a substrate having a pattern of magnetic receptacles disposed thereon. The size of the magnetic receptacle(s) determines the number of target cells that it is capable of immobilizing. The size of the magnetic receptacle is defined by the strength of a localized magnetic field gradient. The localized magnetic field gradient maybe derived from 1) permanent magnets embedded in the substrate or alternatively, the localized magnetic field gradient may be derived from an 2) external magnet whose strength is focused by objects of highly-permeable-magnetic material which create localized magnetic field gradients.

Abstract: Fibrin microbeads are prepared containing extensively cross-linked fibrin (ogen) without using glutaraldehyde. The fibrin microbeads are preferably prepared by containing an aqueous solution containing fibrinogen, thrombin and Factor XIII with an oil heated to about 50-80° C. to form an emulsion and mixing the emulsion at 50-80° C. until fibrin microbeads containing extensively cross-linked fibrin (ogen) are obtained. The fibrin microbeads may have a diameter of about 50-200 microns and can contain a bioactive agent. The fibrin microbeads are used for binding cells such as when culturing or separating one cell type from another, or when transplanting cells or engineering tissue.

Abstract: The invention provides a device and process for recellularizing essentially acellular or devitalized tissue grafts, including for example essentially acellular or devitalized vascular tissue grafts, derived from human or animal sources, or as constructed using any number of tissue engineering methodologies. The process includes repopulating and reendothelializing an essentially acellular or devitalized tissue graft. The device is useful for producing a repopulated tissue graft from an essentially acellular or devitalized tissue, as well as for producing an essentially acellular or devitalized tissue graft.

Abstract: Cell encapsulating devices capable of maintaining large numbers of viable cells are provided containing an inert, substantially cell-free core that displaces cells, a permeable membrane and a zone for maintaining cells. The permeable membrane surrounds the core such that the zone of cells is bounded by the core and the permeable membrane. A preferred device contains a polytetrafluoroethylene permeable membrane and a flexible polymer core having a plurality of ridges and valleys running lengthwise along the core. The cell zone may contain support means for cell attachment and the core may have an outer boundary containing a material that promotes cell adhesion. Preferably, the cell zone has a thickness such that at least about 10% of the cells, more preferably at least about 50% or 80%, in a cell layer located closest to the outer boundary of the core remain viable. The thickness is preferably less than 500 microns such as 25 to 250 microns or 50 to 100 microns.

Abstract: Methods and systems are provided for the selective expansion of target cell populations, and methods of treating patients with cell populations and products.

Abstract: The invention provides a method of selective expansion of a predetermined target population of cells, including (a) introducing a starting sample of cells into a growth medium; (b) causing cells of the predetermined target cell population to divide; and (c) contacting the starting cells with a selection element, comprising a plurality of selective binding molecules with specific affinity either for target cells or for non-target cells to select cells of said predetermined target population from other cells in the growth medium. The invention also provides systems for selective expansion and methods of treating patients with cell populations and products.

Abstract: The invention referres to a method for the preparation and selective replication of deoxyribonucleic acid (DNA) from biomaterial using PCR (polymerase chain reaction) for the analysis in time-of-flight mass spectrometers (TOF) with matrix-assisted laser desorption and ionization (MALDI) for determining specific genetic features in biomaterial. The invention consists, in a first step, of replicating the selected DNA segments by the PCR method in the usual unmodified fashion, and in a further enzymatic replication process, to replicate the DNA segments using modified substrates (the nucleic acids used for PCR) and specially prepared primers to such DNA analogs as are especially suitable for ionization by MALDI. Preparation of the primers particularly consists of introducing a charged group, which improves the ionization, and modification of the substrates is used to neutralize the negative charge on the phosphoric acid group of the DNA backbone.

Abstract: The present invention describes synthetic human monoclonal antibodies that immunoreact with and neutralize human immunodeficiency virus (HIV). The synthetic monoclonal antibodies of this invention exhibit enhanced binding affinity and neutralization ability to gp120. Also disclosed are immunotherapeutic and diagnostic methods of using the monoclonal antibodies, as well as cell lines for producing the monoclonal antibodies.

Abstract: The invention concerns an implant consisting of a carrier material containing medical substances such as pharmaceuticals, antibiotics, cytotstatic agents, hormones or the like made out of organic matter, preferably of a biological tissue of human, animal or plant origin, which before being incubated preferably in vacuum with one or the substances, is broken up, cleaned and freeze-dried.

Abstract: There is provided a method of making microcarrier beads having the steps of forming a bead made of a lightly crosslinked styrene copolymer core and also having functional groups on the surface of the bead and washing the microcarrier beads with basic and acidic solutions to make the beads compatible for cell culture. Also provided is a microcarrier bead made of a styrene copolymer core with a tri-methylamine exterior which has been washed in basic and acidic solutions to make the beads compatible for cell culture. The method of using microcarrier beads for increased growth of anchorage dependent cells having the steps of washing the microcarrier bead with basic and acidic solutions and mixing the microcarrier bead with an anchorage dependent cell containing culture medium is also provided.

Abstract: A microcarrier based process to produce viral vaccines, of which one example is hepatitis A virus (HAV), is composed of an aggregated microcarrier system of glass coated polystyrene microcarriers and MRC-5 cells which creates a stable environment for the propagation of the virus over even extended infection periods. The microcarrier aggregates formed according to this process eliminate the sloughing of cells from the beads during long cultivations seen in other systems, allowing high virus productivity in microcarrier culture. The methodology is applicable where virus production can be enhanced by creating a stable culture during an extended infection period. Scalable stirred bioreactors are used instead of multiple parallel stationary surface bioreactors.

Abstract: This invention relates to methods for the cultivating cells, and in particular to methods for propagating recombinant viruses for gene therapy.

Abstract: This invention relates to methods for the cultivating cells, and in particular to methods for propagating viruses.

Abstract: A porous inorganic oxide carrier body for immobilizing living cells is prepared from inorganic oxide particles such as clay particles. The particles have an average particle size of from 0.01 to 20 microns, and the carrier body has a total pore volume of 0.05 to 1.0 cc/g and an average pore diameter of 50 to 700 .ANG., and has substantially no pore volume in the range of 800 .ANG. or greater. The carrier is prepared by forming a mixture containing the particles, a liquid medium and optional ingredients including zeolite and activated carbon, and forming the mixture into a shaped carrier body which may be optionally dried and calcined. Typical shapes of the carrier include spheres, cylinders, rings, honeycombs and shaped monoliths. Bacteria and other microorganisms immobilized on the carrier are useful for treatment of contaminated waste streams or contaminated vapors, or for other uses for which microorganisms are used such as the synthesis of chemicals.

Abstract: A bioreactor and cell culturing method using the bioreactor have been developed to enhance cell production and growth and decrease the shear forces on the cells and multicellular aggregates. The bioreactor contains a dome-shaped culture vessel having walls defining an interior volume, an apex, a bottom circular edge, and a axis of symmetry perpendicular to the bottom circular edge; and a gas-permeable membrane fluidtightly integrated with the bottom circular edge of the culture vessel. The bioreactor may further contain a rotating base fluidtightly integrated with the gas-permeable membrane, wherein the rotating base is fixed to rotate about the axis of symmetry of the dome-shaped culture vessel set on a substantially horizontal axis.

Abstract: The present invention is directed to a method of producing recombinant viral vectors at high titers incorporating a variety of important advancements over the art. The method of the present invention incorporates multiple features which provide enhanced production of viruses, particularly those viruses encoding exogenous transgenes. The specifically illustrated method describes a method for the high titer serum-free media production of recombinant replication defective adenoviruses containing an exogenous transgene. The invention provides methods of preparing microcarriers, methods for seeding bioreactors at high cell density, increasing the infectivity of the producer cells to the virus, methods to increase product yield through synchronization of the cell cycle of the producer cells, and methods to minimize the deleterious effects of exogenous transgenes. The invention further provides producer cells prepared by the process of the invention. The invention further provides viruses produced by the process.

Abstract: Epithelial cells are attached to microcarriers to form a transplantation material for treatment of skin wounds. Preferably, keratinocyte-covered microcarriers are prepared using microcarriers having a diameter of 50 to 500 .mu.m, and obtaining a coverage of keratinocytes of 30 to 100% of the maximum coverage. An optimum coverage is between 50 and 80% and preferably between 60 and 70%. Keratinocytes are selected from autologous cells, allogenic cells and a combination of these cells. The microcarriers may be pre-coated with eukaryotic cells or an extracellular matrix protein such as collagen before keratinocytes are attached. The keratinocyte-covered microcarriers may be combined with a cryoprotective substance for storage at +3.degree. C. to -196.degree. C. prior to use. Keratinocytes are attached to the microcarriers by pre-culturing the keratinocytes, and then culturing the keratinocytes in the presence of the microcarriers while intermittently stirring. A serum free culture medium may be used.

Abstract: Cell encapsulating devices capable of maintaining large numbers of viable cells are provided containing an inert, substantially cell-free core that displaces cells, a permeable membrane and a zone for maintaining cells. The permeable membrane surrounds the core such that the zone of cells is bounded by the core and the permeable membrane. The cell zone may contain a support means for cell attachment and the core may have an outer boundary containing a material that promotes cell adhesion. Preferably, the cell zone has a thickness such that at least about 10% of the cells, more preferably at least about 50% or 80%, in a cell layer located closest to the outer boundary of the core remain viable. The thickness is preferably less than 500 microns such as 25 to 250 microns or 50 to 100 microns. The devices are suitable for implantation into an individual in need of treatment and are capable of supplying therapeutic substances to such individuals.

Abstract: A method is provided for selective expansion of a predetermined target population of cells, including (a) introducing a starting sample of cells into a growth medium; (b) causing cells of the predetermined target cell population to divide; and (c) concurrently with, intermittently during, or following step (b) contacting the starting cells with a selection element, comprising a plurality of selective binding molecules with specific affinity either for target cells or for non-target cells to select cells of said predetermined target population from other cells in the growth medium. Systems are provided for selective expansion and methods of treating patients with cell populations and products.

Abstract: Stem cells or other organ-function cells are cultivated in a fluidized bed system on macroporous glass carrier bodies treated with a structure protein such as gelatin and an extracellular matrix protein such as fibronectin, and coated with a stroma cell layer. Glass carriers coated with gelatin are added to a fluidized bed reactor, and a culture medium containing an extracellular matrix protein is added to bind the protein to the gelatin. Stromal cells are then added and the cells are cultured to immobilize the cells on the carriers containing the bound protein. Immature organ-function cells are added to the reactor, and while generating a fluidized bed of the carriers in the culture medium, the culture medium is recirculated from and to the reactor in a recirculation loop. Bubble-free aeration of the culture medium is effected to cultivate the immature organ-function cells on the carriers to obtain both mature differentiated organ-function cells and progenitor organ-function cells.

Abstract: A method for large scale cultivation and attenuation of L. intracellularis bacteria by inoculating cells with L. intracellularis bacteria to infect the cells, incubating the infected cells in a reduced oxygen concentration and maintaining the infected cells in suspension. Anti-L. intracellularis vaccines are prepared from cultures grown in suspension. Diagnostic agents are also disclosed.

Abstract: Methods and compositions are provided for controlling cell distribution within an implantable bioartificial organ by exposing the cells to a treatment that inhibits cell proliferation, promotes cell differentiation, or affects cell attachment to a growth surface within the bioartificial organ. Such treatments include (1) genetically manipulating cells, (2) exposing the cells to a proliferation-inhibiting compound or a differentiation-inducing compound or removing the cells from exposure to a proliferation-stimulating compound or a differentiation-inhibiting compound; exposing the cells to irradiation, and (3) modifying a growth surface of the bioartificial organ with extracellular matrix molecules, molecules affecting cell proliferation or adhesion, or an inert scaffold, or a combination thereof. These treatments may be used in combination. The bioartificial organ typically has a semipermeable membrane encapsulating a cell-containing core, and is preferably immunoisolatory.

Abstract: In vitro production of clinically useful quantities of single species of mature, differentiated human blood cells is carried out by a method in which human pluripotent hematopoietic stem cells, preferably from a universal donor, are incubated in a bioreactor in a growth medium also containing specific combinations of recombinant human growth and maturation promoting polypeptide factors that expand stem cell cultures and promote the maturation and differentiation of stem cells into single species of erythroid, thrombocytic or granulocytic human blood cells, and harvesting the mature cells. The growth and maturation promoting polypeptides employed include SCGF, Interleukins 1,3,4,5,6, and 11, GM-CSF, M-CSF, G-CSF and EPO. Stem cells may be preliminarily genetically modified so as to remove histocompatibility or blood group antigens with which a recipient may be incompatible, or the stem cells may be genetically altered by transfection with appropriate DNA-containing vectors, prior to addition to the bioreactor.

Abstract: Permeable substantially spherical hollow particles of 1-5000 .mu.m in size are formed having an outer shell of a mechanically rigid porous material. Pores of the shell are through-going pores that connect the inside of the particles to surroundings and allow chemical species to traverse the outer shell. To prepare the particles, impermeable hollow particles are treated with an acid or base under reflux conditions to form pores. The outer shell is formed of anhydrous forms of silicon dioxide, metal silicates, metal borosilicates, metal oxides or boric oxide. Metals and metal alloys are not used in forming the outer shell. Particles containing a polymer are formed by immersing the permeable hollow particles in a solution of components that polymerize to form the polymer, allowing the solution to partially fill the particles via the pores and polymerizing the components.

Abstract: A method for grafting a cell in the brain of a mammalian subject is accomplished by attaching the cell to a support matrix so that the cell attaches to the matrix surface, and implanting the support matrix with the attached cell into the brain. A syringe containing viable cells that are attached to a matrix surface may be used to transplant the cells into the brain or spinal cord of a mammalian subject. Preferred support matrices are glass or plastic microbeads, either solid or porous, having a diameter from about 90 to about 125 .mu.m. The method employs cells of different types, preferably cells of neural or paraneural origin, such as adrenal chromaffin cells. Also useful are cell lines grown in vitro. Cells not of neural or paraneural origin, such as fibroblasts, may also be used following genetic alteration to express a desired neural product such as a neurotransmitter or a neuronal growth factor.

Abstract: A porous carrier for biocatalysts (a) comprising a water-insoluble inorganic filler and a polyolefine binder selected from polyethylene and polypropylene, (b) having open pores allowing cells to penetrate and grow within its pores, and (c) having a density above 1 g/cm.sup.3.

Abstract: A method for large scale cultivation and attenuation of IS intracellularis bacteria by inoculating cells with IS intracellularis bacteria to infect the cells, incubating the infected cells in a reduced oxygen concentration and maintaining the infected cells in suspension. Anti-IS intracellularis vaccines are prepared from attenuated strains. Diagnostic agents are also disclosed.

Abstract: Reactor-type device for placing in contact solid particles with a liquid. The device comprises an enclosure, delimited by walls, to be filled with the liquid and containing the solid particles in contact with the liquid, and an arrangement for supplying and evacuating the liquid from the enclosure. At least one of the walls of the enclosure can be moved, thereby creating a variable volume. The invention also concerns reaction or culture processes using the device.

Abstract: A system is provided for selective clonogenic expansion of relatively undifferentiated cells, including (a) a tube containing a plurality of beads of a size which permits a plurality of the undifferentiated cells to grow thereon, the beads bearing on their surfaces a selective binding molecule which binds to a surface antigen present on the relatively undifferentiated cells, wherein the antigen is not present on the surfaces of the relatively differentiated cells; (b) means for continuously providing nutrients to the relatively undifferentiated cells growing on the beads, wherein the nutrients are delivered via a fluid which flows through the tube and past the beads; and (c) means for continuously harvesting the relatively undifferentiated cells downstream of the beads.

Abstract: The present invention provides an improved method and apparatus for growing biomass particles, particularly microcarrier-bound cells, in an agitated suspension culture vessel in which fresh culture medium is added and spent culture medium is withdrawn continuously or semi-continuously. The improvement comprises withdrawing the spent culture medium through a particle settling chamber located within the vessel and at least partially immersed in the agitated culture medium therewithin. The particle settling chamber comprises a hollow container with a bottom opening through which biomass particles, such as microcarrier-bound cells, settle by gravity back into the agitated culture medium and a top opening through which particle-free spent culture medium is withdrawn form the vessel. The settling chamber is configured such that the fluid velocity of culture medium entering the settling chamber through the bottom opening is significantly less than the biomass particle settling velocity.

Abstract: Methods for increasing the number of human hematopoietic precursor cells in vitro are provided. The methods generally comprise (a) separating human hematopoietic precursor cells from mature hematopoietic cells present in a blood product; (b) inoculating the separated precursor cells into a culture vessel containing a culture medium comprising a nutritive medium and a source of growth factors at a density of between 1.times.10.sup.3 cells/ml and 4.times.10.sup.6 cells/ml; and (c) culturing the cells under conditions and for a time sufficient to increase the number of precursor cells relative to the number of such cells present in the blood product. The culture medium may also include a suitable amount of microcarrier beads. Suitable blood products include bone marrow, umbilical cord blood, and peripheral blood. A device for carrying out such methods is also provided.