225results about "Invertebrate cells" patented technology

Provided are compositions and methods of use for insect cells comprising baculovirus encoding non-surface expressed proteins and peptides. The claimed invention particularly relates to compositions comprising insect cells containing baculovirus that express cytokines. Such compositions may be administered by, for example, direct intratumoral injection into tumors in mammals, resulting in tumor reduction or recission. Another aspect of the claimed invention concerns methods of promoting resistance to the reoccurence of tumors in mammals who have undergone such tumor recission. In a specific aspect of the claimed invention, the mammals are human subjects presenting with various forms of cancer.

Disclosed and claimed is a new insect cell line, Sf900+, ATCC CRL-12579. The insect cell line was established from Lepidoptera, Noctuidae, Spodoptera frugiperda Sf-9 (ATCC CRL-1711) through multiple rounds of limiting dilution and selection in a serum-free insect medium supplemented with added human insulin. The insect cell line is useful in BEVS or as an adjuvant and has many characteristics and advantages. Also disclosed and claimed are recombinant proteins from recombinant baculovirus expression in insect cells such as Sf900+ cells, for instance, HA, NA, EPO, CD4, CEA, and thrombospondin.

T cell responses are often diminished in humans with a compromised immune system. We have developed a method to isolate, stimulate and expand naïve cytotoxic T lymphocyte precursors (CTLp) to antigen-specific effectors, capable of lysing tumor cells in vivo. This ex vivo protocol produces fully functional effectors. Artificial antigen presenting cells (AAPCs; Drosophila melanogaster) transfected with human HLA class I and defined accessory molecules, are used to stimulate CD8⁺ T cells from both normal donors and cancer patients. The class I molecules expressed to a high density on the surface of the Drosophila cells are empty, allowing for efficient loading of multiple peptides that results in the generation of polyclonal responses recognizing tumor cells endogenously expressing the specific peptides. The responses generated are robust, antigen-specific and reproducible if the peptide epitope is a defined immunogen. This artificial antigen expression system can be adapted to treat most cancers in a significant majority of the population.

The present invention generally provides a means to control stem cell self-renewal and differentiation. More particularly, the current invention is directed toward the elucidation of a signal transduction pathway that controls the expression of a gene necessary for differentiation of stem daughter cells. By modulating the levels of key regulatory molecules within this pathway, the fate of stem cell self-renewal and differentiation may be controlled. In addition, the present invention also provides mutant organisms, tissues, cells, as well methods where the level of key molecules within the pathway are modulated.

Methods for producing in-vitro cultured protein products that are enhanced with stem cells are disclosed. In-vitro cultured protein product compositions produced by said methods are also disclosed. The present invention also discloses methods of providing nutrients to an animal by feeding said animal with said in-vitro cultured protein products.

T cell responses are often diminished in humans with a compromised immune system. We have developed a method to isolate, stimulate and expand naïve cytotoxic T lymphocyte precursors (CTLp) to antigen-specific effectors, capable of lysing tumor cells in vivo. This ex vivo protocol produces fully functional effectors. Artificial antigen presenting cells (AAPCs; Drosophila melanogaster) transfected with human HLA class I and defined accessory molecules, are used to stimulate CD8⁺ T cells from both normal donors and cancer patients. The class I molecules expressed to a high density on the surface of the Drosophila cells are empty, allowing for efficient loading of multiple peptides that results in the generation of polyclonal responses recognizing tumor cells endogenously expressing the specific peptides. The responses generated are robust, antigen-specific and reproducible if the peptide epitope is a defined immunogen. This artificial antigen expression system can be adapted to treat most cancers in a significant majority of the population.

The invention provides a technology for dissociating and culturing prawn embryonic cells effectively, and applies the technology to research of prawn embryonic cell line establishment. According to the technology, the conventional sterile disinfection treatment technology for prawn embryos is optimized, that is, prawn eggs can be collected from a prawn breeding plant directly and transported to a laboratory for treatment; particularly, as iodophor treatment concentration is optimized, not only can pollution of microorganisms and protozoa be controlled effectively, but also toxicity of iodophor to the prawn embryos is reduced to the most extent; the formula of a prawn complete medium is optimized to provide a properer nutritional condition for adherence and growth of the prawn embryonic cells, so that adherence is faster, and growth is better; moreover, the formula of the medium is simplified, so as to reduce the cost of the medium; an embryonic cell collector made of a stainless-steel filter screen of a proper aperture is used for mashing the prawn embryos, so that a large quantity of living prawn embryos and cell clusters can be obtained in a short time; the operation is simple, convenient and fast, and lasts for only several seconds; pollution is reduced; the effect is far better than those of the other reported dissociating methods.

The invention relates to the field of cell culture medium, and in particular to a non-serum non-animal-origin-additive insect cell culture medium. The medium comprises mainly the following components: basic culture medium, glucose, inorganic salt, vitamins, L-arginine, L-agedoite, L-glycocoll, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-threonine, L-tryptophan, L-valine, L-proline, L-glutamine, yeast extracts, recombulin, malic acid, allomaleic acid, cholesterol, linoleic acid, granulesten, putrescine, glutathione, glycerol and fructosan. The culture medium can promote the growth of insect cell and is suitable for the large scale breeding of insect cell and the expression of recombination protein.

The invention relates to an insect cell line and discloses a Sf9 cell line free from Sf-RV pollution as well as a screening method and application of the Sf9 cell line. The cell line is screened fromcommercial Sf9 cells, named as an Sf9-ZY cell line and has the biological collection number of CCTCC NO:C201952. Detection of the proliferation level and protein expression quantity of baculovirus inthe Sf9-ZY cell line indicates that the Sf9-ZY cell line can replace Sf9 cells to serve as host cells of an insect cell-baculovirus expression vector system (BICS) to be applied to expression of recombinant protein for preparing biological products. The Sf9-ZY cell line provided by the invention solves the problem that host cells of the insect cell-baculovirus expression vector system (BICS) are polluted by Sf-RV and guarantees safety of the biological products.

The invention discloses a blood cell separation method for portunus trituberculatus. The method comprises the following steps: diluting iodixanol with an isotonic solution obtained by regulating an osmotic pressure of a PBS buffer solution by utilizing a sodium chloride solution, and performing gradient preparation to obtain three iodixanol solutions of different concentrations; forming gradient solutions of different densities by adopting a cushion technology, and standing to form a continuous density gradient solution to serve as a cell separation solution; collecting blood cells of portunustrituberculatus, and taking mixed cell suspension obtained by diluting the isotonic solution; slowly dropping the mixed cell suspension into the cell separation solution, centrifuging, and dividing the blood cells of portunus trituberculatus into three layers, wherein the uppermost layer refers to clear cells, the intermediate layer refers to small granular cells, and the lowest layer refers to large granular cells. The method has the advantages that only one-step density gradient centrifugation is adopted, the operation is simple, and experiments discover that the separated blood cells haveexcellent viabilities and can be used for cell culture.

A serum-free medium used for suspension culture of insect cells includes 5-10g/L of carbohydrates, 1-5g/L of amino acids, 5-10g/L of an inorganic salt, 0.01-0.05g/L of vitamins, 5-10g/L of yeast extract and 10-20ml/L of a lipid emulsifier. Compared with serum component-containing media, the serum-free medium has the advantages of low cost, easy preparation, no animal pollution, and high batch stability; and compared with general commercial media, the serum-free medium has the advantages of realization of suspension culture of cells, improvement of the expression level, suitableness for large-scale production application, and facilitation of stable obtaining of recombinant proteins.

The invention discloses a fusion protein of a rabies virus G protein expressing an Fc fragment and a preparation method thereof. A gene encoding the rabies virus G protein fusing and expressing the Fcfragment has the nucleotide sequence shown in SEQ ID NO:1, and the rabies virus G protein fusing and expressing the Fc fragment has the amino acid sequence shown in SEQ ID NO:2. The rabies virus G protein and the Fc fragment are fused and expressed for the first time. Recombinant baculovirus constructed by a baculovirus expression system has stable expression of foreign proteins and stable potency. Due to fusion with the Fc fragment, the fusion protein can cross a mucosal barrier, is transported to a lamina propria, is ingested by antigen-presenting cells, is presented to T cells to induce adaptive immunity of a body, a new strategy is provided for research and preparation of oral rabies vaccine and broad research prospects are achieved in prevention and treatment of rabies.

The invention provides a serum-free medium for culturing insect cells in a full suspension mode. The serum-free medium is prepared from a Grace's Insect Medium, a BFPM410 medium, 2-4 g/L of glucose, 1-3 g/L of lactalbumin hydrolysate, 1-3 g/L of PF68 and 2-5 g/L of yeast powder. The invention further provides a preparation method and application of the serum-free medium. As shown in experiments, the serum-free medium can be well used for culturing insect cells in the full suspension mode, the maximum cell proliferation concentration is 42.5*10<5>/mL when the serum-free medium is used for culturing Sf-21 cells, and the maximum cell proliferation concentration is 45.4*10<5>/mL when the serum-free medium is used for culturing Hi-five cells.

The invention discloses a bioreactor with a vessel defining an inner volume, agitation means and at least one addition tube, wherein a delivery orifice in the addition tube is located within the inner volume and a check valve is arranged in proximity of the delivery orifice for allowing flow of a fluid in the direction from the addition tube into the inner volume of the vessel and blocking flow in the reverse direction.

The invention provides a kit for marine mollusk primary cell separation and culture and uses thereof, and relates to marine mollusk cell culture. The kit is provided with a packing box, washing liquid I bottles, washing liquid II bottles, washing liquid III bottles, a solution A bottle, a solution B bottle, a solution C bottle and an operating instruction. The washing liquid I bottles, the washing liquid II bottles, the washing liquid III bottles, the solution A bottle, the solution B bottle, the solution C bottle and the operating instruction are in the packing box. The kit is applied in sterile separation and in-vitro primary culture of a plurality of somatic cells of a plurality of marine mollusks. Through optimizing conditions for separation and primary culture of marine mollusk cells, proper conditions for separation and primary culture of the marine mollusk cells are acquired. A problem that rapid construction of marine mollusk cell rapid separation and primary culture sterile culture systems cannot be achieved in the prior art is overcome. A method for marine mollusk primary cell rapid separation and primary cell sterile culture is established. The kit for marine mollusk primary cell separation and culture is provided.

The present invention belongs to the technical field of cell culture and particularly relates to a Chinese mitten crab blood cell culture medium and a culturing method. The Chinese mitten crab blood cell culture medium comprises a basic culture medium and added components, wherein the basic culture medium is selected from a TC-100 basic culture medium; and the added components and concentrations thereof are as follows: 1%-10% of a buffer solution, 1g/L-2g/L of calcium chloride, 1g/L-2g/L of alkali metal chloride, 1.5g/L-2.5g/L of glucose and 1%-1.5% of antibiotic. The culture medium significantly improves survival rate of Chinese mitten crab blood cells in in-vitro culture and prolongs duration of the blood cell culture in vitro, and the blood cells cultured in vitro can maintain good cellmorphology.

The present invention provides a process for large scale in vitro production of amoebocytes of Indian Horseshoe Crab (Tachypleus gigas) from dissected gill flaps of T. gigas, in Leibovitz culture medium concentration (2xL-15), having enhanced generation of amoebocytes, said process comprising the steps of: dissecting gill flaps of T. gigas; washing the gill flaps with an antibiotic solution followed by alcohol; culturing the gill flaps in tissue culture plates of sterile saline on a Rocker platform; culturing further the gill flapsin Leibovitz culture medium (2xL-15); purging the gill flaps with Tween 80 solution; and purging again the gill flaps with horse crab serum, keeping the culture viable for 90 days by feeding with fresh medium at an interval of 10-15 days to enable the enhanced release of amoebocytes both within and outside the gill flaps.

The invention relates to a method for labeling heterologous protein expressed by insect protein or an insect expression system. The method comprises the following steps: doping azido-doped peracetylated N-acetylglucosamine into glycosyl of protein expressed by insects under the assistance of a metabolic pathway of the insects; subsequently, carrying out click chemistry reaction, and coupling a detachable marker to the protein, thereby realizing labeling of the protein from the insects. The method can be used for in-situ labeling of insect cells or labeling of the heterologous protein expressedby the insect expression system.

The invention provides a continuous culture technology of prawn cells by establishing an effective prawn cell 3D culture and subculture method. The continuous culture technology can be applied to establishment of prawn immortal cell lines. The addition ratio of matrigel is optimized, and thus, a preparation method of the matrigel for 3D culture of the prawn cells is provided. A formula of a complete culture medium of the prawn cells is further optimized, the complete culture medium of the prawn cells is selected as an anticoagulant and a diluent of prawn blood lymphocytes, a 3D culture mode of attached growth on the surface of the matrigel is selected, thus, the separation and 3D culture technology of the prawn peripheral blood lymphocytes is provided, the prawn peripheral blood lymphocytes are attached to the surface of the matrigel for growth by a mode of round single cell and cell clusters/balls, and the survival and growth abilities of the prawn peripheral blood lymphocytes are higher than those of the prawn peripheral blood lymphocytes subjected to 2D culture. The invention also provides a passaging method for culturing the prawn cells in a 3D mode, the passaged prawn blood lymphocytes can be well attached to the surface of the matrigel for growth, and the survival rate of the passaged prawn blood lymphocytes can reach up to 90% or above.

The invention provides a haliotis discus hannai cell culture medium, which is based on an L-15 culture medium and is added with inorganic salt, growth factors, L-glutamine, astaxanthin, gamma-aminobutyric acid, carboxymethyl glucan, taurine, hydrocortisone, a haliotis discus hannai shell aqueous extract, fetal calf serum, antibiotics and other components. The invention also provides a constructionmethod of the haliotis discus hannai cell line, which comprises the following steps: by using haliotis discus hannai heart tissues as a material, obtaining single cells by a mechanical dissociation method, and resuspending the single cells in the cell culture medium for primary culture, thereby successfully constructing the haliotis discus hannai heart cell line. The culture medium and the methodprovided by the invention can well maintain survival and growth of haliotis discus hannai heart cells in vitro, and the constructed cell line has continuous passage capacity. According to the method,the survival rate of the haliotis discus hannai heart cells in-vitro culture is remarkably increased, the in-vitro culture time is prolonged, and the method has the characteristics of quickness, easiness in operation, stability and easiness in repetition.