35 results about "LYMPHOCYTE SEPARATION MEDIUM" patented technology

The invention provides a preparation method of clinical application-level placental hematopoietic stem cells. The preparation method comprises the steps of pretreating a fresh delivered placenta immediately and then separating out umbilical arteries and umbilical veins, pouring a cleaning fluid into the chorionic vessels of the placenta and colleting the cleaning fluid after pouring, pouring an enzyme digestion fluid into the chorionic vessels of the placenta and digesting at 37 DEG C for 5-20min, pouring the collected fluid into the placenta and collecting the liquid after pouring, centrifuging the obtained fluid and re-suspending cells after removing the supernatant, and separating the resuspended cell suspension through a two-step process with hydroxyethyl starch and a lymphocyte separation medium to obtain the clinical application-level placental hematopoietic stem cells. The method is capable of increasing the number and the activity of the prepared placental hematopoietic stem cells and the content of CD34 positive cells, and the prepared placental hematopoietic stem cells are at the clinical application level and free of potential pathogenic contamination, and moreover, the method is low in preparation cost.

The invention discloses a composition for separating a mononuclear cell and a compound lymphocyte separation medium. All raw materials in a formula of the compound lymphocyte separation medium respectively meet the standard of intravenous injection-grade bulk drugs and are high in safety; the purity of the recovered lymphocyte and the recovery rate of the lymphocyte have no remarkable difference from those of a conventional separation medium control group; and the lymphocyte obtained by separating respectively has equivalent effects to those of the control group in four indexes, namely multiplication, morphology, a surface marker and cytotoxicity activity, and is favorable in clinical application prospect.

The invention discloses a method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media. A lymphocyte separation medium density gradient centrifugation method is used for separating umbilical cord blood mononuclear cells, a dulbecco modified eagle medium (DMEM) / F12 is used for primary culture of the mesenchymal stem cells of the human umbilical cord blood, and the method increases adherence of the cells, reduces growth of osteoclast like cells remarkably, facilitates formation of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) colonies, and greatly improves successful rate of culture of the hUCB-MSCs. The DMEM / F12 is used continuously for subculturing to a P2 generation, the method is combined by a using enzymatic digestion and differential velocity adherent method simultaneously, and the method facilitates purification of P1-P2 generation cells remarkably. A P3 generation and the following generations are cultured by using an Oricell human umbilical cord mesenchymal stem cell culture medium, marker proteins and good morphological characteristics and growth characteristics of the hUCB-MSCs are maintained, and multilineage differentiation potential of the hUCB-MSCs is maintained. In addition, costs of the culture media adopted by the method are reduced remarkably.

The invention belongs to the technical field of blood products and particularly relates to a preparation method for platelet rich plasma (PRP). The method comprises a blood collection step, a lymphocyte separation medium treatment step, a first-time centrifugation step, a second-time centrifugation step and the like. According to the preparation method disclosed by the invention, on the basis of the existing PRP preparation technology, firstly, collected blood is treated by adopting a lymphocyte separation medium, and then, by optimizing a centrifugal force and time of centrifugation, the PRP concentration effect of the prepared PRP is further promoted, and the number of red blood cells is greatly reduced; the preparation method has better practical application value.

A highly efficient method of making a primary cell derived biologic by purifying mononuclear cells (MNCs) in a automated cell processor to remove contaminating cells by loading leukocytes onto lymphocyte separation medium (LSM) and centrifuging the medium to obtain purified MNCs, storing the MNCs overnight in a closed sterile bag system, stimulating an induction mixture of the MNCs with phytohemagglutinin (PHA) or other mitogen and ciprofloxacin in a scalable cell culture device and producing a primary cell derived biologic from the MNCs, removing the mitogen from the induction mixture by filtering, incubating the induction mixture, clarifying the induction mixture by filtering to obtain a primary cell derived biologic supernatant, and clearing the primary cell derived biologic supernatant from adventitious agents by anion exchange chromatography, filtration. A closed system prevents contamination of the resulting primary cell derived biologic. An automated method of purifying cells. A method of scalably inducing cells.

The invention relates to the field of biology and discloses a placental hematopoietic stem cell and a preparation method thereof and a placental hematopoietic stem cell injection. The preparation method comprises the following steps of: after pretreating a placenta, digesting the treated placenta by using collagenase type IV digestive juice and collagenase type II digestive juice, then washing and filtering and respectively collecting first filtrate and first residue; digesting the first filter residue by using collagenase type I digestive juice and the collagenase type II digestive juice in the first step and then washing, filtering and collecting second filtrate; combining the filtrate from two times, centrifuging and discarding supernate, re-suspending and precipitating; adding a resuspension solution into a lymphocytes separation medium; and carrying out density gradient centrifugation, collecting a buffy coat cell solution on an intermediate layer and centrifuging and discarding supernate to obtain the placental hematopoietic stem cell. According to the placental hematopoietic stem cell and the preparation method thereof disclosed by the invention, collagenase type IV and collagenase type II are combined, collagenase type I and collagenase type II are combined and the hematopoietic stem cell is prepared by fully digesting the placenta according to the characteristics of different collagenases, so that the quantity and the vitality of the prepared hematopoietic stem cell are improved.

The invention provides a separation culture method of bone marrow mesenchymal stem cells (BMSCs) by a density gradient centrifugation method. The separation culture method comprises the steps of BMSCs separation, BMSCs primary culture, BMSCs subculture and amplification, cultured cell surface antigen detection and growing curve determination. According to the method, a density gradient centrifugation method is adopted, a lymphocyte separation medium with density of 1.077 is used for separating BMSCs, and most erythrocyte, adipocyte, soterocyte and the like are removed after gradient centrifuge to obtain BMSCs cells with relatively high purity; and the BMSCs cells are cultured for 2 to 3 days to find that the BMSCs basically grow by adhering walls according to the characteristic that the BMSCs grows by adhering to walls; therefore, BMSCs with good uniformity can be obtained in the final phase of primary culture. The method has the advantages of simpleness in operation, quickness and practicality and the like, and is an effective BMSCs separation purification method.

The invention provides a kit for detecting NK (natural killer) cell activity. The kit includes a PBS buffer solution, a lymphocyte separation medium, a RPMI1640 culture solution, a fluorescent dye EGFP, an antibody 7-AAD-percp-cy5.5, and a fluorescent dye's carrier pEGFP-N1 plasmid. The invention also provides a method for detection of NK cells by the kit. The method provided by the invention can save detection time and detection cost, and has high sensitivity.

The invention relates to a method for amplifying a gamma-delta T cell by induction in vitro, and belongs to the technical field of biology. The method comprises the following steps: acquiring peripheral blood mononuclear cells (PBMCs) through a lymphocyte separation medium; performing stimulation culturing for 3 days by utilizing a serum-free medium containing zoledronic acid, IL-2, IL-7 and IL-15; performing stimulation amplifying culturing with a serum-free medium containing IL-2, IL-7 and IL-15; and adding nicotinamide when culturing is performed for 7-9 days to improve the anti-tumor activity of the gamma-delta T cells. According to the method, the PBMCs do not needed to be purified, and the gamma-delta T cells having the purity of 90 percent or more can be obtained when culturing is performed for 14 days; and the anti-tumor activity of the gamma-delta T cells can be remarkably improved by adding the nicotinamide. The gamma-delta T cells can be amplified for 1500 times or more, which can meet the requirement for the number of gamma-delta T cells in clinical application. The method is simple in operation steps and strong in operability, and has a good popularization value.

The invention belongs to the field of biotechnology, and particularly relates to a method for separating bone marrow mononuclear cells. The separation of the mononuclear cells in bone marrow is carried out by using one point that a gelatin solution can destroy red blood cell surface charges, accelerates the deposition of red blood cells and has no influence on other principal components covering the mononuclear cells through density difference between the mononuclear cells in the bone marrow and the other components according to the principle that when a lymphocyte separation medium is used for density gradient centrifugation, various cellular components are redistributed and gathered by density gradient, thus obtaining the mononuclear cells. By using the method for separating the bone marrow mononuclear cells, the loss of the MNCs (mononuclear cells) is not increased, and bone marrow hematopoietic stem cells and mesenchymal stem cells can be reserved very well; and the method is simple and convenient to operate and can be popularized clinically.

The invention discloses a method for preparing Car-NK cells for breast cancer. The method comprises the following steps: collecting peripheral enriched blood from a patient, filtering out leukocytes, slowly moving it into a centrifuge tube containing lymphocyte separation liquid, and using a density gradient Centrifuge, absorb the buffy coat, and obtain peripheral blood mononuclear cells; take 90 mL of the resuspension, add inactivated autologous serum 10 mL and IL-2 10 ng / mL, inoculate into six-well culture plates, and in six-well culture plates Add 0.9% sodium chloride injection and Lymactin-NK; after culturing for 36-48h, add IL-2, IL-15 and IL-21 to the culture plate at a final concentration; transfect and amplify NK cells with lentivirus cultured to obtain CAR-NK cells. The invention discloses a preparation method of Car-NK cells, which comprises separating nuclear cells from peripheral blood of breast cancer patients by Ficoll method, and amplifying NK cells in vitro through IL-2+IL-15+IL-21 combined culture scheme, The preparation method is convenient and easy to operate, and the obtained Car-NK cells can be injected into the patient's body through the method of preparing cell preparations, which is of great help to the treatment of breast cancer.

The invention discloses a human adipose derived stromal cell separation and culture method which includes the steps: firstly, fat collection; secondly, removing impurities in fat; thirdly, digestion by mixed enzyme; fourthly, collecting and centrifuging; fifthly, culture and passage; sixthly, digestion and freezing. According to the human adipose derived stromal cell separation and culture method,separation is implemented by the mixed enzyme, digestion time is short, acquired cells cannot be further purified by a lymphocyte separation medium and a filter screen, and liquid needs to be changedafter the cells fits with wall. According to the method, a separation process is low in cost, used reagents and materials are less in separation, operation processes are simplified, pollution risks in the operation process are reduced, animal source serums are omitted in the whole separation and culture process, cell toxicity is reduced, and a culture period is short by the aid of the mixed enzyme and a serum-free culture system.

The invention discloses an inducing culture reagent for CIK (Cytokines Induced Killer) cells. The reagent is prepared from a reagent A: 50ng / ml anti-CD3 monoclonal antibody; a reagent B: 1000IU / ml IFN-gamma; a reagent C: 1000IU / ml IL-2 and 20ng / ml IL-15; a reagent D: 100IU / ml IL-1; a reagent E: 40ml of lymphocyte separation medium. The invention also discloses an inducing culture method for the CIK cells by using the reagent. The reagent and the method, provided by the invention have the advantages that simple, high-efficient and stable induced CIK cells can be realized; by adding IL-15 in a classic preparation solution, multiplication number of the CIK cells and secretion of gamma-IFN can be better improved, and the anti-tumor effect of the CIK cells is improved; the cell inducing quality during CIK cell culture is high and the stability of cell multiplication number is good.

The invention discloses a method for preparing a soluble human recombinant MICA protein, which comprises the following steps of: a step 1 of sampling 30ml of in-vitro peripheral venous blood donated by a healthy adult volunteer, diluting the in-vitro peripheral venous blood by physiological saline and separating out an offwhite lymphocyte layer by a lymphocytes separation medium; a step 2 of extracting an RNA (Ribose Nucleic Acid) of processed offwhite lymphocytes by a TRIZOL, carrying out sequence screening on the RNA, planting the RNA into a rhabdovirus genome, carrying out recombination, collection, dialysis and centrifugation to obtain an MICA protein and filtering the MICA protein by a CENTRICON ultrafilter; a step 3 of purifying a target protein; and a step 4 of identifying the MICA target protein. The soluble human recombinant MICA protein prepared by the preparation method is the MICA protein of which a molecular structure domain is closer to the natural state and has the advantages of convenience, rapidness, sensitivity, good repetitiveness and the like. Due to successful application of the soluble human recombinant MICA protein in an in-vitro immunological rejection model, practical support is provided and a solid foundation is laid for in-vivo experiments and clinical application of animals.

The invention discloses a method for sequential culture of human umbilical cord blood mesenchymal stem cells by using two culture media. A lymphocyte separation medium density gradient centrifugation method is used for separating umbilical cord blood mononuclear cells, a dulbecco modified eagle medium (DMEM) / F12 is used for primary culture of the mesenchymal stem cells of the human umbilical cord blood, and the method increases adherence of the cells, reduces growth of osteoclast like cells remarkably, facilitates formation of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) colonies, and greatly improves successful rate of culture of the hUCB-MSCs. The DMEM / F12 is used continuously for subculturing to a P2 generation, the method is combined by a using enzymatic digestion and differential velocity adherent method simultaneously, and the method facilitates purification of P1-P2 generation cells remarkably. A P3 generation and the following generations are cultured by using an Oricell human umbilical cord mesenchymal stem cell culture medium, marker proteins and good morphological characteristics and growth characteristics of the hUCB-MSCs are maintained, and multilineage differentiation potential of the hUCB-MSCs is maintained. In addition, costs of the culture media adopted by the method are reduced remarkably.

The invention relates to a separation method and a separation kit for neutrophil in blood, and belongs to the technical field of neutrophil separation. The invention provides the separation method for the neutrophil in blood, which comprises the following steps: sequentially adding a neutrophile granulocyte separating medium, a lymphocyte separating medium and whole blood into a centrifugal tube, enabling the lymphocyte separating medium to be positioned above the neutrophile granulocyte separating medium and the whole blood to be positioned above the lymphocyte separating medium, then centrifuging, and separating to obtain the neutrophile granulocytes. The invention further provides a separation kit for the neutrophil in the blood. The separation kit contains a neutrophil separation medium and a lymphocyte separation medium which are independently packaged respectively. By adopting the separation method or the separation kit provided by the invention, the neutrophil with the purity of about 80% can be rapidly obtained through one-time centrifugation, and the neutrophil and the red blood cells can be separated at the same time through one-time centrifugation, so that the separation method or the separation kit has a very good practical value.

The invention discloses a lymphocyte separating medium and a preparation method thereof, the lymphocyte separating medium is characterized by comprising the following components: glycerin, ethanol, sucrose, meglumine, diatrizoic acid and epichlorohydrin; the sucrose and the diatrizoic acid are added into an ethanol solution to synthesize a water-soluble polysucrose ethanol solution as a molecular sieve separating medium; the method also includes adding meglumine and epichlorohydrin into glycerol, preparing a clear biological solution at room temperature, putting the biological solution into a dialysis paper bag, putting the dialysis paper bag into deionized water, carrying out ion replacement culture for a period of time, and detecting that the dialysis paper bag does not contain redundant foreign ions; then adjusting the pH value of the two solutions to a proper range by using sodium hydroxide and hydrochloric acid; finally, mixing and stirring the two solutions with the pH value adjusted to be stable to be uniform, and obtaining the lymph separation medium. The lymphocyte separation efficiency is high, the separation purity is high, the quality of lymphocytes is improved, high-quality lymphocytes can be obtained, and the lymphocyte separation medium is suitable for clinical application and popularization.

The invention discloses a method for quickly separating a large quantity of PBMCs (peripheral blood mononuclear cells) from human peripheral blood. The method comprises the following steps: the human peripheral blood is transferred to a centrifuge tube for centrifugation; a grey yellow layer is absorbed, and normal saline is added to the layer for dilution; a product is sent to a centrifuge tube containing a lymphocyte separation medium, and the product obtained in the S2 is located on an upper layer of the lymphocyte separation medium; centrifugal separation is performed, an intermediate albugineous coat is absorbed, washed and centrifuged, supernatant is removed, and the PBMCs are obtained. For separation of a large quantity of the PBMCs from the human peripheral blood, the operation steps can be reduced, the operation time and reagent supplies are saved, reagent consumption is reduced, and PBMC separation and cell viability improvement are facilitated.

The invention belongs to the technical field of lymphocyte separation, and particularly relates to a production method and formula of a lymphocyte separation medium. According to the formula of the lymphocyte separation medium, the lymphocyte separation medium is specifically prepared from the following components by mass: 8-12kg of sucrose, 3-3.5kg of meglumine, 3-6kg of amidotrizoic acid and 8-10kg of chloropropylamine. The production method and formula of the lymphocyte separation medium have the beneficial effects that the production method is simple, the extracted cells are twice more than a conventional method, the production cost is low, the effect is good, and the production method and formula are in the leading position in the industry.

The invention provides a kit for detecting NK (natural killer) cell activity. The kit includes a PBS buffer solution, a lymphocyte separation medium, a RPMI1640 culture solution, a fluorescent dye EGFP, an antibody 7-AAD-percp-cy5.5, and a fluorescent dye's carrier pEGFP-N1 plasmid. The invention also provides a method for detection of NK cells by the kit. The method provided by the invention can save detection time and detection cost, and has high sensitivity.

The invention discloses a preparation method of NK cells. The preparation method of the NK cells comprises the following steps: (1) adding a blood sample and an isovolumetric lymphocyte separation medium in a separation culture tank for centrifugal separation so as to obtain single karyocytes; (2) sorting the single karyocytes to purify NK cells; (3) carrying out induced culture on the NK cells in the separation culture tank; (4) carrying out amplification culture on the NK cells in the separation culture tank to obtain an NK cell final product, and 5) washing packing the NK cell final product. In the preparation method of the NK cells, the NK cells are prepared by the separation culture tank. Compared with a mode of culturing the NK cells in vitro by using a culture dish in the prior art, the preparation method of the NK cells is standard in steps, and large-scale production of the NK cells is realized conveniently.

The invention discloses an ADSCs and EPCs stem cell system capable of promoting recovery of microcirculation blood supply of a graft. The ADSCs and EPCs stem cell system capable of promoting recoveryof microcirculation blood supply of the graft is composed of adipose-derived stem cells (ADSCs) derived from adipose tissue and endothelial progenitor cells (EPCs) derived from peripheral blood. The ADSCs are extracted by adopting an enzymolysis method for culture; and the peripheral blood is collected, after heparin is used for anticoagulation and methylcellulose is used for sedimentation of redblood cells, gradient centrifugation is conducted by adopting a lymphocyte separation medium, and the EPCs are collected and purified by adopting an adherence method. The ADSCs and the EPCs which arecultured separately are subjected to co-culture according to a cell ratio of the ADSCs to the EPCs of 1:1 and a total cell concentration of 20000 / cm<2>. According to the ADSCs and EPCs stem cell system capable of promoting recovery of microcirculation blood supply of the graft, nutrition supply of the graft is improved, transplanted adipose tissue survival and adipose cell differentiation are promoted, the transplantation efficiency is improved, and a treatment cycle is shortened. On the other hand, all the cells are extracted from the body of a patient, injury is minor, and pain is little, sothat the ADSCs and EPCs stem cell system capable of promoting recovery of microcirculation blood supply of the graft is liable to be accepted by the patient.

The invention relates to a kit for diagnosing or detecting leukemia, which contains a lymphocytes separation medium, total RNA extracting solution, reverse transcription buffer solution, M-MLV reverse transcriptase, a reverse transcription primer Oligo(dT)18, an RNA enzyme inhibitor, DEPC water, quantitative PCR buffer solution, Taq DNA polymerase, dNTPs solution, a Wnt5a gene reference standard, an internal reference GAPDH gene reference standard, a primer and a fluorescent probe for detecting a Wnt5a gene and a primer and a fluorescent probe for detecting an internal reference GAPDH gene. The kit for diagnosing or detecting the leukemia has the advantages of simple preparation, convenient detection, rapidness, sensitivity, accurate quantification, high repetitiveness and specificity and suitability for clinical application.

The invention provides a tuberculous infection T cell detection kit and a detection method thereof. The kit comprises seven polypeptides with amino acid sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 7. The detection method includes the steps: (1) separating a sample to be detected by a Ficoll lymphocyte separation medium, and re-suspending separated cells in a serum-free medium; (2) adding the seven polypeptides with the sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 7 into the re-suspended cells, and then adding gamma interferon antibodies into a co-incubation system to perform antigen antibody reaction; (3) washing the reacted system, adding a chromogenic substrate for chromogenic reaction and counting spots generated by reaction. The kit can be artificially synthesized in a complete sequence manner, is convenient and rapid to produce and high in stability and specificity and has a good commercial application prospect, and cost is greatly reduced.

The invention belongs to the field of molecular biological detection and in particular relates to a method for simultaneously detecting immune repertoires of a T cell and a B cell on the basis of high-throughput sequencing. The method for simultaneously detecting the immune repertoires of the T cell and the B cell on the basis of high-throughput sequencing comprises the steps of obtaining 10 mL human blood sample; putting the 10 mL human blood sample in an EDTA anticoagulant tube; separating peripheral blood monouclear cells (PBMC) by using a lymphocyte separation medium Ficoll-1077; extracting total RNA of the PBMC by using a Trizol method, wherein a reagent used is cDNA reversely transcribed from RNAzolRT and RNA; adding a linker, PCR1 and PCR2 to 5' end of the cDNA; carrying out purification and high-throughput sequencing on the cDNA and the like. Total length information of gene sequences of TCR (Alpha chain or Beta chain) and BCR (Heavy chain or Light chain) in lymphocyte are obtained from an upstream primer of the linker and a downstream primer of a C-region.

The invention provides an isolation and cultivation method of prokaryotic cells of human umbilical cord blood mesenchyme stem cells, which is applied to a dimethyl sulfoxide induction process which enables the human umbilical cord blood mesenchyme stem cells to be differentiated into neuronal cells. The induction process uses serum-free dulbecco modified eagle medium (DMEM) containing dimethyl sulfoxide and butylated hydroxyanisole to induce the human umbilical cord blood mesenchyme stem cells to be differentiated into the neuronal cells. The isolation and cultivation method includes: collecting umbilical cord blood of a normal full-term cesarean section fetus under an aseptic condition, anti-freezing through heparin, separating single prokaryotic cell of the umbilical cord blood by using lymphocyte separation medium, cultivating and purifying by using partial acidic culture medium to obtain anchorage-dependent cells, obtaining application dimethyl sulfoxide which is amplified by a third generation to induce the mesenchymal stem cells (MSCs) to be differentiated towards the neuronal cells, and marking and displaying the induced MSCs in immunohistochemical mode to express a neurofilament protein (NF) and neuron specific endase (NSE). The human umbilical cord blood MSCs can be cultivated and amplified in vitro, can be differentiated towards neuron-like cells by using induction of the dimethyl sulfoxide, and provides a novel cell source for wound repair of central nervous systems.

The invention discloses a method for extracting high-quality B cells from bone marrow, peripheral blood and lymphoma tissue, which comprises the steps of respectively taking the peripheral blood and the bone marrow of a lymphoma patient, adding hydroxyethyl starch, standing and taking supernatant; taking a lymphocyte separation medium with the same volume as the supernatant, adding the supernatant into the lymphocyte separation medium, and centrifuging; taking the centrifuged white film layer, washing and resuspending; splitting tumor tissue of the lymphoma patient, cutting the tissue and incubating; transferring the supernatant into PBS containing 10% of serum, and repeatedly washing the tissue; and enabling the obtained supernatant to pass through a cell sieve, centrifuging, collecting cell precipitate, washing and resuspending. By changing the experimental method for separating and purifying the bone marrow and the peripheral blood, the B cells from the bone marrow and the peripheral blood are extracted more efficiently, and impurity cells are reduced; and by changing a lysis method of lymphoma tissues, more lymphoma cells are mildly and effectively extracted, and a necessary cell basis is provided for subsequent cell culture and single cell sequencing.