The present invention relates to adipose-derived stem cells (ASCs) and compositions, as well as methods for preparing and using such ASCs and compositions for therapy.

The present invention pertains to a method for the generation of neurotoxin-sensitive, neuronal differentiated cells comprising the steps of: a) cultivating tumor cells which are able to differentiate into neuronal cells in a culture medium under conditions and for a time which primes said tumor cells for neuronal differentiation; and b) cultivating the tumor cells primed for neuronal differentiation of a) in a differentiation medium having an osmolality of 100 to 270 mOsm/kg, and comprising (i) B27 supplement and/or (ii) N2 supplement, for at least 3 days, thereby obtaining neurotoxin-sensitive, neuronal differentiated cells. The invention further relates to neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention. In addition, the invention encompasses a method for determining the activity of a neurotoxin polypeptide comprising the steps of: a) contacting the neurotoxin-sensitive, neuronal differentiated cells obtainable by the method of the invention with a neurotoxin polypeptide; b) cultivating the neurotoxin-sensitive, neuronal differentiated cells of step a) for 3 to 74 hours or 72 hours under conditions which allow for the neurotoxin polypeptide to exert its biological activity; and c) determining the activity of the neurotoxin polypeptide in the said cells after cultivation according to step b). Finally, the invention provides for a medium comprising OptiMEM, FBS, B27 supplement, and N2 supplement.

The invention describes improved methods and compositions for producing a recombinant protein, e.g., an antibody, in mammalian cell culture. In addition, the invention provides improved cell culture media, including improved production media, feed solutions, and combination feeds, which may be used to improve protein productivity in mammalian cell culture.

In one embodiment, the present application discloses a cell culture medium for culturing cell lines suitable for producing a therapeutic protein, comprising an amino acid selected from a group consisting of L-arginine, L-asparagine, L-proline, L leucine and L hydroxyproline and a mixture thereof; a vitamin selected from a group consisting of ascorbic acid Mg.sup.2+ salt, biotin, pyridoxine HCL, folic acid, riboflavin and D-calcium pantothenate, and a mixture thereof; an element selected from a group consisting of ammonium meta vanadate, sodium meta vanadate, germanium dioxide, barium acetate, aluminum chloride, rubidium chloride, cadmium chloride, ammonium molybedate, stannous chloride, cobalt chloride, chromium sulfate, silver nitrate, sodium metasilicate, zinc sulfate, manganese sulfate H.sub.2O, manganous chloride, ferric nitrate 9H.sub.2O, ferrous sulfate 7H.sub.2O, ferric ammonium citrate, magnesium chloride anhydrous, and magnesium sulfate anhydrous, and a mixture thereof; a nucleoside selected from a group consisting of uridine and cystidine; a sugar selected from a group consisting of galactose, mannose and N-Acetyl-D-Mannosamine; and a triple buffering system comprising sodium carbonate, sodium bicarbonate and HEPES; wherein the cell culture medium is animal component-free, plant component-free, serum-free, growth factors-free, recombinant protein-free, lipid-free, steroid-free, and free of plant or animal hydrolysates and/or extracts.

The invention is in the field of cell culture. Particularly the invention relates to methods of culturing a mammalian host cell expressing a recombinant protein in perfusion mode, using a concentrated cell culture medium.

Herein is reported a method for obtaining a B-cell comprising the following steps a) labeling B-cells, b) depositing the labeled B-cells as single cells, c) co-cultivating the single cell deposited B-cells with feeder cells, d) selecting a B-cell proliferating and secreting IgG in step c) and thereby obtaining a B-cell. The labeling can be of IgG.sup.+CD19.sup.+-B-cells, IgG.sup.+CD38.sup.+-B-cells, IgG.sup.+CD268.sup.+-B-cells, IgG.sup.?CD138.sup.+-B-cells, CD27.sup.+CD138.sup.+-B-cells or CD3.sup.?CD27.sup.+-B-cells. The method can comprise the step of incubating said B-cells at 37? C. for one hour in EL-4 B5 medium prior to the depositing step. The method can also comprise the step of centrifuging said single cell deposited B-cells prior to the co-cultivation. In the co-cultivation a feeder mix comprising interleukin-1beta, and tumor necrosis factor alpha and Staphylococcus aureus strain Cowans cells or BAFF or interleukin-2 and/or interleukin-10 and/or interleukin-6 and/or interleukin-4 can be used.

Methods and compositions are provided for generating targeted genetic modifications on the Y chromosome or a challenging target locus. Compositions include an in vitro culture comprising an XY pluripotent and/or totipotent animal cell (i.e., XY ES cells or XY iPS cells) having a modification that decreases the level and/or activity of an Sry protein; and, culturing these cells in a medium that promotes development of XY F0 fertile females. Such compositions find use in various methods for making a fertile female XY non-human mammal in an F0 generation.

A composition for animal cell culture and techniques relating thereto are provided. A composition for animal cell culture, which contains an amino acid source and ?-ketoglutaric acid, and has the following properties: (A) the ?-ketoglutaric acid is a salt of ?-ketoglutaric acid; and/or (B) the amino acid source is free L-cysteine anhydrate is provided.

This disclosure relates to growth media and environments for in vitro culturing of cells that produce or are capable of producing antibodies. In certain embodiments, the media comprises IL-6, fibronectin, and typically a saccharide. In certain embodiments, the disclosure contemplates cell culture compositions comprising IL-6 and fibronectin that are derived from proteins secreted from mesenchymal stromal/stem cells (MSCs). In certain embodiments, the disclosure contemplates enclosures comprising culture compositions disclosed herein that are in ambient air or optionally in an environment wherein oxygen is absent or at a low concentration.

Described herein are cell culture media useful for the differentiation of human pluripotent stem cells into microglia. The methods described herein relate to in vitro generation of expandable, bankable, microglial cells by directed differentiation from human pluripotent stem cells (induced or embryonic). Using only defined cell culture media, differentiation of pluripotent stem cells is directed down a mesodermal path, in a rapid and scalable fashion, to generate cells adopting signatures of their in vivo counterparts, including gene expression, protein marker expression and functionality.