112 results about "Ficoll" patented technology

The invention relates to a method for separating and extracting stem cells from a placenta, umbilical cord or adipose tissue, which comprises the following process steps: firstly mixing the placenta, umbilical cord or adipose tissue and a cell maintenance fluid according to the proportion by weight of 2.5-4:1, putting the mixture into a tissue crushing barrel, adding collagenase after crushing, uniformly mixing, hatching at the temperature of 37 DEG C, filtering, adding a precipitator, sucking a supernatant fluid after settling, centrifuging, removing the supernatant fluid, adding concentrated cells into a liquid of diatrizoate sodium-ficoll 400#, then centrifuging, collecting 10-15 ml of intermediate cell layer, washing with the cell maintenance fluid, counting the collected cells, and providing the cells for clinical use when the cell survival ratio is more than or equal to 95 percent. The invention not only realizes the separation and the extraction of all stem cells from the placenta, umbilical cord or adipose tissue, but also realizes industrialized production, and enables doctors to conveniently, safely and canonically obtain the adult stem cells in clinic and use the adult stem cells for treating the diseases of patients as using medicaments, thereby solving the bottleneck problem of difficult obtainment of adult stem cells in clinic and popularizing a cell treatment technology.

The invention relates the separating agent box and application. The agent box comprises erythrocyte precipitating agent, hypaque sodium- ficoll 400# or HISTOPAQUE1077 and cell maintenance liquid. The agent box can separate bone marrow and umbilical cord blood, on the surface of cell there is no any mark, and it doesn't change biological activity of cell. The invention can commercially manufacture, and the application is wide.

The present invention relates to a reagent kit of the isolation of a bone marrow umbilical cord blood stem cell in vitro and the application method thereof. The present invention is characterized in that Lin antibody red blood cell precipitant cell preserving solution, and sodium iothalamate-polysucrose 400 # or HISTOPAQUE1077 or Ficoll are contained in the reagent kit, and each reagent solution is preserved dividually. The present invention adopts the method which combines a negative collection method and a density method, thus the surface of the stem cell which is collected and abstracted has no any label, the cells which are isolated and abstracted are Lin antigenic negative cells, the method is simple, and the present invention can be directly used to isolate and abstract the stem cells clinically. The reagent kit of the present invention can operate the industrialized production, to realize the promotion of the isolation and purification technology of the human hematopoietic stem cell, leading the doctor to conveniently cure the patient with the stem cells like the medicine usage.

The invention relates to a simple and effective method for extracting blood immune cells, a cryopreservation and resuscitation method used with the former method and a method for proliferating the immune cells after resuscitation. The methods include the steps of whole-blood sample pretreatment, Ficoll separation, MNC (mononuclear cell) collection and washing, MNC counting, MNC cryopreservation, cell resuscitation and cell proliferation. By the aid of extraction, cryopreservation, resuscitation and proliferation with specific operation steps, the immune cells can be effectively obtained and used for treating tumors.

The invention discloses a method for inducing megakaryoblast and megakaryocyte in vitro and a special culture medium thereof. The special culture medium is a serum free medium containing 50ng / mL of SCF, 50ng / mL of TPO, 20ng / mL of IL-3 and 50ng / mL of IL-6. The method comprises the following steps of: 1) separating single karyocyte from umbilical cord blood by using the conventional Ficoll density gradient centrifugation method, wherein red blood cells in the umbilical cord blood are settled by 6 percent hydroxyethyl starch; and 2) culturing the single karyocyte in vitro for 4 to 14 days by using the special culture medium at 37 DEG C in the presence of 5 percent CO2 to obtain a mixture of the megakaryoblast and the megakaryocyte. The invention provides a source for supplementing the megakaryoblast and the megakaryocyte, and has the advantages of simple operation, low cost, high differentiation efficiency and the like. The method is about to play an important role in the field of medicaments, and has wide application prospect.

The invention discloses an efficient CIK amplifying method, in particular to a method for cell populations, namely cytokine-induced killer cells utilizing in-vitro cell factors for efficiently inducing mononuclear cell expressions CD3 and CD56 of peripheral blood, wherein the cell populations have killing functions. The cultivation method for the CIK efficiently expressing CD3+CD56 comprises the following steps that the peripheral blood of a healthy person or a patient is collected in a sterile mode, after the peripheral blood is diluted through a saline solution with the same volume, a Ficoll lymphocyte separating medium is used for separating mononuclear cells, in the CIK cell inducing process, CD3 monoclonal antibodies (CD3mAb), CD28 monoclonal antibodies (CD28mAb), interferon-gamma (IFN-gamma), interleukin-2(IL-2) and interleukin 1alpha (IL-1alpha) are added, cultivation is carried out for 13-16 days to obtain cells, a CIK cell preparation is prepared, and flow cytometry detection and microorganism detection are carried out. According to the CIK cultivation method, the CIK number can be increased to be 6*10<9> or over 6*10<9> in two weeks, the cell survival rate can be increased to be 99% or over 99%, and the double-positive proportion of the CD3+CD56+cells reaches 30% or over 30%.

The invention discloses a separation method of human peripheral blood mononuclear cells. The separation method combines the advantages of a hydroxyethyl starch precipitation method and a Ficoll density gradient centrifugation method. The PBMC with a purity of greater than 95% and a vitality of greater than 85% is obtained by removing most of red blood cells by the hydroxyethyl starch precipitation method, then naturally settling in air, and then further purifying the mononuclear cells by the Ficoll density gradient centrifugation method, aiming at the separation characteristic of a high-capacity blood sample. Compared with a method without natural setting in air, the vitality and the purity of the cells obtained by the separation method are higher.

Provided herein are kits for processing stem cells from bone marrow or umbilical cord blood, comprising: a) a precipitation reagent comprising an aqueous solution of 0.1-30% hydroxyethyl starch or 0.1-30% methyl cellulose, and 0.1-20% of cerebroprotein hydrolysate; and b) a separation reagent comprising an aqueous solution comprising Ficoll and diatrizoate and having a density of 1.0-1.2 g / ml, and methods of use. Further provided herein is a collection of stem cells obtained by the kits and methods disclosed herein comprising stem cells from bone marrow or umbilical cord blood, and uses thereof.

The invention relates to preparation and a purification production process of mesenchymal stem cells (MSCs) derived exosomes, and application of the exosomes as a non-cell therapy in the treatment of acute lung injury. The MSCs deriving the exosomes belong to the wall adhesion type of stem cells, and the preparation and purification process have the characteristics that (1) parathyroid hormone is added specially for the preparation of MSCs condition medium, and (2) discontinuous Ficoll density gradient method and immunomagnetic beads separation and purification method are combined for separation and purification so as to obtain the exosomes. Rat acute lung injury (ALI) model experiments confirm that the exosomes can be used for treatment of ALI, so as to lay the foundation for the realization of non-stem cell therapies to substitute stem cells therapies.

The invention discloses a method for carrying out in-vitro efficient amplification on natural killer (NK) cells, and application of the method, aiming at solving the problems that the NK cells amplified by the traditional local method are small in amplification times, have cytokine dependency and cell aging, and the like. The method is characterized by comprising the following steps: 1, separating peripheral blood mononuclear cells (PBMCs) from human peripheral blood by using the traditional Ficoll-Paque density gradient centrifugation method; 2, carrying out 100 Grays irradiation treatment on artificial antigen presenting cells, and then storing by means of liquid nitrogen cryopreservation; 3, carrying out co-culture on the PBMCs and the artificial antigen presenting cells, subjected to the irradiation treatment, in a cell culture flask T75 of an RPMI 1640 medium, wherein the mass ratio of the PBMCs to the artificial antigen presenting cells is equal to 1: 2, and 50IU / mL of interleukin 2 is added into the RPMI 1640 medium; replacing the medium with a fresh medium every 2 to 3 days; 4, calculating the number of total cells in the cell culture flask T75 every 7 days, adding the same number of artificial antigen presenting cells subjected to the irradiation treatment into the cell culture flask T75 for stimulating again, and continuously culturing for 21 days to obtain the amplified NK cells. Therefore, after the method is adopted, a great deal of high-purity and high-quality NK cells can be obtained.

The invention discloses a method for preparing anti-tumor combined immune cells DC (dendritic cell)-CIKs (cytokine induced killers) and NKs (natural killers) simultaneously and the prepared combined immune cells. Ficoll density gradient centrifugation is used for efficient separation, a mononuclear cell is obtained, sufficient quantities of DCs, CIKs and NKs are obtained through cell culture bags and an immune cell induction culture system, and finally, the induced cells are cultured in a combined manner and applied to clinical treatment, so that a tumor killing effect is realized. The DCs, the CIKs and the NKs are subjected to induction culture respectively with adoption of TexMACS immune cell culture media produced by Miltenyi Biotec, autoserum, various cytokines and a combined culture technology, the cells are mixed for culture and application at certain point in time, application of fetal calf serum is avoided, the pollution rate of an exogenous pyrogen and an exogenous allergen is reduced, and the tumor killing activity of the finally mixed cells is enhanced simultaneously; with adoption of a cell culture bag technology, the cell contamination rate is reduced, and the method is suitable for clinical treatment and application.

The invention discloses a preparation method of human TSCMs (T memory stem cells), which comprises the following steps: (1) collecting peripheral venous blood with a blood cell separator or injector under aseptic conditions, and carrying out Ficoll-Hypaque density gradient centrifugation to obtain mononuclear cells; (2) putting the separated PBMCs (peripheral blood mononuclear cells) in a culture medium containing irritants and cell factors, regulating the cell density to (0.5-2)*10<6> / mL, transferring into a cell culture plate, culture bottle or culture bag, and culturing in an incubator; (3) after culturing the cells for 48-72 hours, replacing half of the culture solution, supplementing the equivalent culture solution to keep the cell density at (0.5-2)*10<6> / mL; and according to the cell growth state, harvesting the cells in the 10th-21st day. An anti-CD3 monoclonal antibody and an anti-CD28 monoclonal antibody are utilized as the irritants to activate the TSCMs, the cell factors rh IL-7, rh IL-15 and rh IL-2 are combined for irritation, and IM-12 is utilized to stop the stem cell differentiation, so that the cultured TSCMs have the common characteristics of both memory cells and stem cells, thereby providing a new optional scheme for clinical adoptive immunity cellular therapy.

This invention relates to a detection system for measuring a fluorescent signal in a fluorescent assay. The system comprises a probe having a small sensing surface bound with a fluorescent label, and a light source and a detector both mounted at the proximal side of the sensing surface of the substrate. The invention also relates to a method for detecting an analyte in a liquid sample using a probe tip having a small surface area (≦5 mm) and a high molecular weight polymer (≧1 MD) having multiple binding molecules and multiple fluorescent labels. The binding reaction is accelerated by flowing the reaction solutions laterally and moving the probe tip up and down in the reaction vessels. The invention furthers relates to a fluorescent labeling composition comprising a cross-linked Ficoll molecule having a plurality of binding molecules and a plurality of fluorescent labels.

The invention relates to an efficient CIK cell preparation and detection method and aims at solving the problems of few proliferation times, unstable preparation process and the like of the prior art. The efficient CIK cell preparation and detection method comprises the following steps that sterile acquisition is conducted peripheral blood of a healthy person or a patient according to a lymphocyte absolute value; whole blood testing and blood sample preservation are performed; separation and inactivation of blood plasma are performed; a density gradient centrifugation method is adopted, namely a single mononuclear cell is separated out by using lymphocyte separation liquid Ficoll; in the CIK cell induction process, a factor 1 and a factor 2 are added successively, collection can be performed only through serum-free induced cell multiplication culture for about 2-3 weeks, and a CIK cell preparation is prepared. The method has the advantages of being small in blood collecting quantity, stable in preparation process, simple and convenient to operate and high in cell proliferation times and cytotoxic activity.

The invention discloses a method for preparing CIK cells with a killing effect on tumor cells. The CIK cells are obtained through induced culture of 17-20 days by using a ficoll mononuclear cell separation medium with a density of 1.084 in the PBS system to separate mononuclear cells in peripheral blood or umbilical cord blood. According to the present invention, mononuclear cells are separated and obtained efficiently by using the ficoll density gradient centrifugation method, and a sufficient amount of CIK is obtained by using cell culture bags and a CIK cell culture system to meet the needs of clinical treatment. The Takara lymphocyte culture medium and the autologous serum and cytokine co-culture technique are used in the method, thereby preventing application of fetal bovine serum, reducing contamination risks of exogenous pyrogen and sensitinogen, and while maintaining the advantage of CIK cell efficient proliferation. The cell culture bag technology is used to reduce risk of cell contamination, and is suitable for clinical therapeutic application.

The invention provides a method for separating total nucleated cells from mononuclear cells from umbilical cord blood. The method comprises the steps that firstly, TNCs is separated, wherein a PBS buffer solution containing 5% dextran and 2% human serum albumin is added into the umbilical cord blood, centrifugation is conducted to discard supernate, and TNCs precipitate I is obtained; after precipitating and washing, TNCs precipitate is obtained; the TNCs precipitate is subjected to density gradient centrifugation by using a Ficoll lymphocyte separation liquid, a mononuclear cell albuginea layer is collected, and mononuclear cell precipitate is obtained after centrifugation. The method is efficient, convenient to use, capable of saving time and high in repeatability, rich umbilical cord blood resources in an umbilical blood bank can be utilized, and the separation efficiency of derived stem cells and immune cells in the cryopreserved umbilical cord blood is improved.

Disclosed is an improved method for separation an purification of rat islet cells, which comprises pouring rat pancreas with novel Liberase RI enzyme, and centrifugalizing rat pancreas cells by employing Ficoll density gradient. The initially purified pancreas cells are cultured over night, and are selected one by one by means of glass needles connected with rubber pipes through the control of the mouth cavity and tongue of the operator.

The embodiment of the invention discloses a method for purifying cord blood mononuclear cells. The method comprises: step 1, centrifuging human umbilical cord blood to obtain blood cells and plasma; step 2, subjecting the blood cells to erythrocyte sedimentation with hydroxyethyl starch to obtain a lymphocyte suspension; and step 3, performing gradient centrifugal separation on the lymphocyte suspension by using a Ficoll reagent so as to obtain the cord blood mononuclear cells. The method can effectively reduce the number of erythrocyte and thrombocyte in the final product, and the number of the purified cord blood mononuclear cells is large, and the purity and the motility rate are high.

The invention discloses a method of carrying out isolated culture on immune cells by virtue of peripheral blood. The method comprises the following steps: centrifuging the peripheral blood for 10 min under a condition of 2000rpm and 4 DEG C, and collecting the upper-layer plasma to prepare a serum-free medium containing the plasma; adding Ficoll separation liquid and centrifuging; sucking a buffy coat, and washing to obtain PBMC; adding the serum-free medium in the PBMC, inoculating in the serum-free medium, and adding 20-300U / mL of IL-2 and 5-50ng / mL of IL-15; collecting the immune cells while culturing to the 13th to the 15th day. According to the method disclosed by the invention, the centrifuging time, the centrifuging rotational speed and the centrifuging temperature are adjusted, lots of the plasma can be obtained, and the stability of protein content in the plasma and no deficiencies of active factor ingredients can be ensured.

There is provided a method for the isolation and cultivation of mesenchymal stem / progenitor cells from umbilical cord blood, and also to a method for the differentiation of the umbilical cord blood-derived mesenchymal stem / progenitor cells into various mesenchymal tissues. The method include overlaying umbilical cord blood onto Ficoll-Hypaque solution; centrifuging the umbilical cord blood on the Ficoll-Hypaque solution to obtain a mononuclear cell layer; reacting cells obtained by monolayer culture of the mononuclear cells with antibodies to mesenchymal stem / progenitor cell-specific antigens for a predetermined period of incubation time; isolating only cells bound to their corresponding antibodies using a cell sorter; and cultivating the isolated cells, thereby obtaining mesenchymal stem / progenitor cells with high purity and excellent viability.

The invention relates to a simple and effective method for extracting blood immune cells, a cryopreservation and resuscitation method used with the former method and a method for proliferating the immune cells after resuscitation. The methods include the steps of whole-blood sample pretreatment, Ficoll separation, MNC (mononuclear cell) collection and washing, MNC counting, MNC cryopreservation, cell resuscitation and cell proliferation. By the aid of extraction, cryopreservation, resuscitation and proliferation with specific operation steps, the immune cells can be effectively obtained and used for treating tumors.

The invention relates to a method for separating and extracting stem cells from a placenta, umbilical cord or adipose tissue, which comprises the following process steps: firstly mixing the placenta, umbilical cord or adipose tissue and a cell maintenance fluid according to the proportion by weight of 2.5-4:1, putting the mixture into a tissue crushing barrel, adding collagenase after crushing, uniformly mixing, hatching at the temperature of 37 DEG C, filtering, adding a precipitator, sucking a supernatant fluid after settling, centrifuging, removing the supernatant fluid, adding concentrated cells into a liquid of diatrizoate sodium-ficoll 400#, then centrifuging, collecting 10-15 ml of intermediate cell layer, washing with the cell maintenance fluid, counting the collected cells, and providing the cells for clinical use when the cell survival ratio is more than or equal to 95 percent. The invention not only realizes the separation and the extraction of all stem cells from the placenta, umbilical cord or adipose tissue, but also realizes industrialized production, and enables doctors to conveniently, safely and canonically obtain the adult stem cells in clinic and use the adult stem cells for treating the diseases of patients as using medicaments, thereby solving the bottleneck problem of difficult obtainment of adult stem cells in clinic and popularizing a cell treatment technology.

The invention discloses a ficoll synthesis process method which comprises the following steps: (1) filling alkalic anion exchange resin into a fixed bed reactor; (2) adding a mixture prepared by sucrose, a cross-linking agent, distilled water, an oil phase and a dispersant into the fixed bed reactor, so that the mixture can conduct polyreaction on the alkalic anion exchange resin; (3) reacting for 4 to 5 hours at 22 to 28 DEG C, increasing the temperature to be 50 to 95 DEG C, and reacting for 2 to 5 hours under water bath again; (4) repeatedly washing by absolute ethyl alcohol and adsorbing a reaction product on the alkalic anion exchange resin, and eluting to obtain ficoll. The synthesis process has the characteristics of simple and convenient preparation process, mild reaction condition, less side reaction, repeated use of a catalyst, no problem of separation from the product, continuous production, cleanness, environmental protection and the like, and moreover, ficoll with good water-solubility and high reaction activity can be obtained. Therefore, the synthesis process has a wide application prospect.

The invention relates to a single-component TMB (Tetramethylbenzidine) developing solution and a preparation method thereof. The single-component TMB developing solution is prepared from the following components: TMB, dimethyl sulfoxide, glycerol, an oxidant, a sugar type compound or a derivative thereof and a buffering solution; the pH value of the single-component TMB developing solution is 3.0-7.0; the buffering solution is selected from a citric acid-sodium citrate buffering solution, a citric acid-EDTA (Ethylene Diamine Tetraacetic Acid) buffering solution, a citric acid-sodium acetate buffering solution or a citric acid-disodium hydrogen phosphate; and the sugar type compound is selected from at least one of glucoside or a derivative thereof, dextran or a derivative thereof and ficoll or a derivative thereof. The single-component TMB developing solution can be used for experiments including Western, IHC or in-situ hybridization and the like and is high in sensitivity, good in stability and long in preservation time.

The invention discloses a preparation method of a Car-NK cell for small cell lung cancer. The method comprises the following steps: collecting peripheral blood of a patient and isolating an NK cell with an Ficoll method; placing a peripheral blood mononuclear cell in an NK medium, adjusting the cell concentration, placing the cell in an incubator for continuous culture for 48-72 h, and collectingan adherent cell; adding the cell to the NK medium for continuous culture for 12-15 days, and supplementing a culture solution once every three days; transfecting the NK cell with lentivirus and performing multiplication culture to obtain the Car-NK cell; preparing a small cell lung cancer cell preparation.

The invention provides a rapid amplification method of natural killer cells. The rapid amplification method of the natural killer cells comprises the following steps: A, preparing a culture medium ofthe natural killer cells; B, carrying out isolation and culture of the natural killer cells; B1, removing anticoagulant peripheral blood into a centrifuge tube, performing centrifugation, absorbing upper-layer plasma into another centrifuge tube for later use; B2, diluting the plasma with physiological saline of which the volume is 0.5 to 2 times that of the plasma to obtain diluted blood; B3, separating PBMCs from the diluted blood with Ficoll-Paque; B4, resuspending 3 to 5*10<7> / ml PBMCs in a 40-60 ml culture vessel of a completely activated culture medium, and placing in an incubator of which the temperature is 35 to 40 DEG C and the CO2 concentration is 2 to 8% to be cultured for 2 to 4 days; B5, supplementing a cell expansion culture medium in the completely activated culture medium,and continuously carrying out culture to obtain the natural killer cells. The culture method of the invention can amplify a large number of the natural killer cells in a short time, and is lower in amount of the culture medium, and simple, convenient and safe in culture.

The invention provides a method for separating and primary culture of grass carp dendritic cells, which comprises the following steps: step 1. separation of grass carp spleen and head kidney: anesthetizing grass carp to death, removing spleen and head kidney, and removing adherent connective tissues and adipose tissues and performing immersion; step 2: preparation of grass spleen and head kidney single cell suspension: performing continuous mesh filtration to obtain the cell suspension; step 3. separation of grass spleen and head kidney mononuclear cells: performing Ficoll separation, after centrifugation, and taking the cells of a white film layer and washing the material; step 4. static culture: adjusting the concentration of monocytes and culturing the material in an incubator; and step5. enriching and purifying of the dendritic cells of grass carp: collecting the suspension cells after a period of culture, and purifying the dendritic cells of the grass carp by a density gradient centrifugation method. The method can effectively prepare a large number of grass carp dendritic cells, and the obtained grass carp dendritic cells have a purity of more than 80%; the operation processis simple and clear, and the time consuming is short.

The invention relates to a peripheral blood mononuclear cell separation tube and a preparation method thereof, comprising a tube body and a tube plug, wherein the inner part of the tube body comprisesFicoll-hypaque stratified liquid layer, separating the gel layer and the anticoagulant layer; in the tube body, an upper space of the separating glue layer is used for accommodating sample blood. Compared with the prior art, the use of the peripheral blood mononuclear cell separation tube of the present invention eliminates the need for transferring blood from the vacuum collection tube into theconical tube, and eliminates the need for transferring Ficoll-hypaque stratification fluid is carefully injected into the lower part of the blood, and the PBMCs layer is sucked from the solid separating gel interface and yellow plasma without being carefully sucked from the liquid interface. Thus, the experimental apparatus is reduced, the operation process is simplified, the operation difficultyis reduced, and the inconsistency among different operators is reduced.

The invention discloses a novel cervical exfoliated cell separation method, which is characterized by: placing cervical exfoliated cells in phosphate buffer solution (PBS) and uniformly mixing by whirling to obtain cell suspension; adding the cell suspension into mucilage separating solution; centrifuging, removing supernate, adding the phosphate buffer solution (PBS) again and re-suspending sediment to obtain re-suspended cell suspension; and adding the re-suspended cell suspension into neutral granular cell separating solution Ficoll, centrifuging, removing supernate and obtaining the settlement which is pure cells after centrifugation. The method of the invention greatly purify clinically extracted cervical exfoliated cell specimens, adopts simple and convenient operation steps, is suitable for large-scale cell separation with stable and reliable effect, can obtain high-purity target cells, has high actual use value and a promising market prospect.