216 results about "Erythrocyte lysate" patented technology

The invention provides a hemocyte analysis chip and an analysis system. The system comprises the chip, an electrical impedance detection unit and a signal processing system. The chip comprises a first detection chip and a second detection chip. The first detection chip comprises a blood sample channel, an erythrocyte lysate channel, a continuous phase channel and a first detection channel. The second detection chip comprises the blood sample channel, the continuous phase channel and a second detection channel. The blood sample channel, the erythrocyte lysate channel and the continuous phase channel of the first detection chip are communicated, and a communication position is connected with the first detection channel, the blood sample channel of the second detection chip is communicated with the continuous phase channel, and the communication position is connected with second detection channel, a small disperse aperture is provided between the connection positions of first detection channel and the second detection channel and the fluid passage, and a small detection aperture is provided at the position far from the small disperse aperture. The electrical impedance detection unit and the signal processing system use an electrical impedance method for analyzing hemocyte.

The invention relates to a method for freezing and thawing placental whole cells and separating and expanding stem cells. The method comprises the steps as follows: disinfecting and washing a placenta tissue; cutting off placental lobules from the tissue, and carrying out digestive treatment for 20 min; preparing a placenta tissue freezing solution for standby application; adding whole cells obtained by digestive treatment and the freezing solution into a freezing tube, refrigerating for 0.5 h at the temperature of 4 DEG C, freezing for 1 day at the temperature of subzero 80 DEG C, and freezing in liquid nitrogen for standby application; taking out the placental whole cells from the liquid nitrogen when needed, thawing in a thermostatic water bath, carrying out drop-method washing by a culture medium for mesenchymal stem cells, removing red cells by a red cell lysis solution, and expanding the mesenchymal stem cells by the thawed placental whole cells through cell culture and cell passage. According to the method, the frozen placenta tissue can be effectively protected and is convenient to thaw and use; and the method is in particular suitable for separating and expanding the mesenchymal stem cells after thawing the frozen placental tissue.

The invention provides a method for isolating and culturing human primary hepatocytes, comprising the following steps: cleaning blood clots on surfaces of hepatic tissues by using D-Hank's solution and cutting off connective tissues; using a needle syringe for puncturing on multipoints to perfuse anterior perfusion solution preheated to 38 DEG C so that a hepatic tissue block changes from dark red to gray and the effluent anterior perfusion solution becomes clear; using the needle for puncturing on multipoints to perfuse preheated recyclable type II collagenase solution until the hepatic tissue block is loosened and inelastic and has turtleback cracks on the surface; scissoring the hepatic tissue block, passively isolating the hepatic tissues, removing residual envelops and fibrillar connective tissues and continuously shaking and digesting in the type II collagenase solution at 37 DEG C; blowing and beating digest into hepatocyte suspension, filtering the suspension under ice bath condition, collecting filtered suspension, transferring the filtered suspension to a centrifuging tube and centrifuging at low speed; adding bottom precipitate produced from the primary low-speed centrifuging of the filtered suspension to erythrocyte lysate, blowing and beating, standing at room temperature, adding Dulbecco's modified eagle medium (DMEM), mixing the mixture uniformly, cleaning, centrifuging at low speed, adding DMEM again, mixing uniformly, centrifuging at low speed for 2-4 times and adding bottom precipitate produced from the last-time low-speed centrifuging to Williams' MediumE complete medium suspension; adjusting the hepatocytes density to 1*105 / ml, inoculating the hepatocytes in a culture bottle laid with rat tropocollagen, carrying out constant culturing in a CO2 incubator at 37 DEG C, changing the suspension to remove died hepatocytes and non-adherent hepatocytes after the hepatocytes are adhered to the wall and continually culturing the hepatocytes.

The invention discloses an aggregated white blood cell segmentation counting system. The system comprises an image acquisition module for dyeing white blood cells in a blood sample, dissolving red blood cells in the blood sample by using red blood cell lysate and acquiring a white blood cell image, an image preprocessing module used for performing image background removal on the white blood cellimage and obtaining an optimal segmentation threshold by using a maximum inter-class variance method and roughly segmenting a white blood cell region, an aggregated cell determination module used forobtaining a coarse segmentation image according to the rough segmentation of the white blood cell region, setting a discriminant function of a cell area and obtaining a multi-cell aggregation region,and an aggregated cell segmentation counting module used for extracting a cytoskeleton in each aggregation region and a gray curve at the cytoskeleton by using a morphological refinement method. According to the invention, by analyzing the gray scale characteristics of various white blood cell areas under a low power microscope, an adaptive threshold function is constructed, while a white blood cell count is obtained, the number of oxyphil cells is obtained, the cells in the aggregation region are quickly and accurately divided and counted, the method is quick and simple and is easy to implement.

The present invention relates to an integrated method for enriching and detecting rare cells in biological body fluid sample. The enriching method comprises: (a) removing plasma protein by centrifugation; (b) optionally adding a red cell lysis solution to carry out red cell lysis so as to remove the red blood cells; (c) adding immunomicrospheres or immunoadsorbent to incubate; and (d) carrying out density centrifugation based on a special cell separation medium for separating the circulating rare cells, residual red blood cells after removing red blood cells and the white blood cells combined on the immunomicrospheres. The method for detecting the enriched rare cells according to the present invention comprises combining immunohistochemistry based staining with immunofluorescence, or bicolor, tricolor or multicolor staining based on immunohistochemistry, and observing and identifying by fluorescence or ordinary optical microscope or a scanner based on microscope principle. The novel and unique method for enriching and staining has been proved to have low cost and can rapidly, effectively and high specifically enrich and quantitatively detect the rare cells in blood.

The invention discloses a detection kit and a detection method of circulating tumor cells. The kit comprises a PBS buffer solution, a density gradient separation medium, a red blood cell lysate, a CD45 immunomagnetic bead, an incubation solution, a fixing solution, a permeabilizing solution, a confining liquid, an antifade mounting medium, an antibody dilution solution and a fluorescent antibody concentrate, and the fluorescent antibody concentrate contains a fluorescent dye pan-CK-AF488, a fluorescent dye CD45-PE and a cell nucleus dye Hoechst33342. The detection method comprises the following steps: S10, centrifuging a blood sample, and separating and enriching the CTCs; and S20, carrying out dyeing identification on the enriched CTCs through the cell nucleus dye pan-CK-AF488, the fluorescent dye CD45-PE and Hoechst33342 fluorescence antibodies, wherein the detection is carried out for the purpose of non-treatment. When the detection kit and the detection method are adopted to perform detection, the detection result is accurate and reliable.

The invention relates to a method for quickly and efficiently extracting whole blood genome DNA (deoxyribonucleic acid), and belongs to the field of nucleic acid purification. The method comprises the following steps: adding a red cell lysis solution into whole blood, eddying until red cells are completely split, removing supernatant, and sucking all residual liquid; adding a white cell lysis solution and protease K, and splitting white cells in a water bath of 37 DEG C until the white cells are deposited and dissolved; adding sodium chloride and chloroform, carrying out centrifugal separation on DNA, and transferring the supernatant to another clean EP (epoxy) pipe; adding ammonium acetate and absolute ethanol for depositing DNA, washing by 75% of ethanol, naturally drying, and adding TE (Tris-EDTA(ethylene diamine tetraacetic acid)) for dissolving to obtain DNA. The method has the characteristics of high speed, high efficiency and non-toxicity. The extracted DNA can be used for various subsequent molecular biology studies such as polymerase chain reaction (PCR), methylation detection and gene cloning.

The invention discloses a method for measuring glycosylated hemoglobin and a kit. The kit comprises a kit body, reagent bottles, a bottle mat, a hinge and a kit cap, wherein the kit body is connected with the kit cap through the hinge; the four reagent bottles are respectively used for placing a magnetic particle suspension, an erythrocyte splitting liquor, a flushing liquid and signals. According to the invention, magnetic particles capable of quantitatively capturing glycosylated hemoglobin and non-glycosylated hemoglobin in a sample are adopted, and percentage of the glycosylated hemoglobin in the sample to the total hemoglobin can be calculated only by detecting the content of glycosylated hemoglobin on the magnetic particles and comparing the content with the standard work curve. According to the invention, the operation is simple, and the quantitative detection on the percentage composition of the glycosylated hemoglobin can be completed only with one operation; and biological raw materials such as antibodies are not needed, so that the price is low, and detecting instrument miniaturization is easy to realize.

The invention provides an erythrocyte lysis buffer. The erythrocyte lysis buffer comprises the following components: 4.5-5.5 mmol / L of sodium chloride, 0.8-1.3% (v / v) of triton 100, 300-340mmol / L of glucose and 0.008-0.012 mol / L of trishydroxymethyl aminomethane. The erythrocyte lysis buffer can effectively lyse the erythrocyte in a whole blood sample, separate and enrich leucocyte in the blood, and facilitate the subsequent nucleic acid extraction operation of the leucocyte, so as to obtain the leucocyte DNA in the whole blood sample and the DNA of virus in the leucocyte.

The invention provides a new primary culture method for liver cells of crucian carp, which mainly comprises the following steps: a, separating liver tissues from crucian carp; b, digesting cut liver tissues by using a stepwise digestion process and obtaining inner cell mass; and c, purifying and culturing obtained inner cell mass. In the method disclosed by the invention, the raw material is obtained from common crucian carp aquatic experimental animals, in-vitro tissue blocks are soaked in chlorhexidine gluconate (chlorhexidine) to be disinfected for reducing pollution. The tissues are digested by a stepwise digestion process, blood cell lysing solution is used to reduce the blood cell content in cells, and the liver cells are purified by percoll gradient centrifugation. The liver cells are purified by combined material preparation, the stepwise digestion process, blood cell lysing solution and percoll. The method can culture adequate liver cells, the survival rate of the liver cellsis over 90 percent, and the method meets requirements of primary culture.

The invention discloses a detecting method for a circulating tumor cell surface marker molecule PD-L1. The detecting method comprises the following steps: (1) treating whole blood with red blood celllysate to separate out karyocyte, and fixing with formaldehyde; (2) performing positive screening through tumor immune fluorescent marker cytokeratin antibody anti-CK, performing PD-L1 primary antibody incubation on all cells, and performing PD-L1 secondary antibody incubation with a fluorescence group marked with FITC, and marking all cells through cell nucleus fluorescent dye DAPI; and (3) adopting high-flux multi-color imaging analysis to select light filter plates of CY5, FITC and DAPI, observing fluorescent color on the surface of a channel, and finally realizing detection on the circulating tumor cell surface marker molecule PD-L1. The detecting method is simple and convenient, is quick, is economical, is non-invasive, is high in sensitivity and is good in specificity.

The invention relates to a homocysteine Hcy kit based on an aptamer fluorescence probe; at the same time, the invention also relates to a method for determination of the concentration of homocysteine Hcy, and a determination reagent composition and components, and belongs to the technical field of medical examination and determination. The kit comprises the main components: an erythrocyte lysate, a phosphate buffer solution PBS, a homocysteine Hcy standard substance, and the homocysteine Hcy aptamer fluorescent probe; through blood sample cracking and mixed egg fertility treatment, and with combination of a fluorescence spectrophotometer for detection, the value of the concentration of the homocysteine Hcy is measured and calculated. The kit has the advantages of simple sample treatment, simple and convenient operation, short detection time, strong detection specificity, high sensitivity, high repeatability of detection results and the like.

The invention relates to a kit of extracting a whole genome DNA (Deoxyribonucleic Acid) from blood and a using method thereof. The kit is characterized by comprising a red blood cell lysate, a white blood cell scrubbing solution, digestive juice, proteinase K, a purifying liquid, gDNA salting out liquid, a gDNA scrubbing solution, a gDNA eluant and the like. The using method of the whole genome DNA extraction kit for blood is characterized by comprising the following steps: washing the red blood cell split to obtain the white blood cell; splitting the white blood cell by the digestive juice containing the proteinase K; and further purifying by an improved lithium chloride purifying liquid, salting out the liquid layer, and carrying out chromatography to obtain the high purity whole genome DNA. When the kit provided by the invention is used to extract the whole genome DNA in blood, plasma and serum in blood are not separated in advance but fresh or frozen anti-freezing whole blood is taken, wherein the lowest blood volume required reaches 20 microliters or blood cakes are required. According t the kit provided by the invention, the whole genome DNA with high purity can be fully unlinked and the PCR (Polymerase Chain Reaction) amplification is efficiently carried out, so that the kit is used for scientific research or clinical diagnostic analysis such as PCR amplification, gene expression, gene sequencing, whole genome sequencing, exome sequencing, gene mutation and single nucleotide polymorphism.

The invention discloses a method for separating primary adult hepatocytes. The method comprises the following steps: (1) carrying out multi-point puncture on surface of isolated adult hepatic tissue through a needle syringe, and injecting a preperfusate, wherein connective tissues are removed from the isolated adult hepatic tissue; (2) carrying out the multi-point puncture on the surface of the isolated adult hepatic tissue through the needle syringe again, and injecting a IV collagenase solution; (3) separating the isolated adult hepatic tissue, followed by adding the IV collagenase solution and carrying out digesting through vibration at a temperature of 37 DEG C to obtain digest; (4) carrying out filtering for the digest, followed by centrifuging and collecting cell aggregate in the underlayer, then resuspending the adult hepatocyte aggregate through a hepatocyte wash buffer, followed by filtering and centrifuging, then abandoning supernatant and collecting the adult hepatocyte aggregate in the underlayer; (5) washing the adult hepatocyte aggregate in the underlayer from the step (4) through a serum-free DMEM medium to obtain the primary adult hepatocytes. The invention further discloses a disposable special sterile apparatus box for separating the primary adult hepatocytes. With the present invention, the disposable special sterile apparatus box is adopted, the primary adult hepatocytes are separated through the multi-point puncture on the surface of the tissue and the injection, such that operation is simplified, cost is reduced, and the method and the apparatus box are applicable for extracting the hepatocytes from small pieces of the irregular isolated adult hepatic tissues of recovery of liver resection.

The invention provides a method for culturing umbilical cord mesenchymal stem cells in a separated mode from umbilical cord outer layer amnion tissue. The method includes the steps that umbilical cord tissue of a healthy newborn is used, washed, disinfected and mechanically cut into pieces to separate an outer layer amnion, and the outer layer amnion is cultured in a suspension mode with a serum-free culture medium after being treated with erythrocyte lysate; medium change is carried out every three to five days, after the attachment rate in a plate reaches 30-70%, trypsinization is used, cells are collected centrifugally for passage amplification, and confluence is carried out when the cell confluence rate reaches 80-90% to obtain the high-purity umbilical cord mesenchymal stem cells.

The invention discloses a kit detecting hot spot mutation sites in a colorectal cancer PIK3CA gene. The kit comprises an erythrocyte lysate, a whole blood DNA extraction reagent, an anhydrous ethanol, a PCR amplification reaction solution, a positive control, a negative control and a pyrosequencing reaction solution. The kit is characterized by comprising upstream and downstream primers 9F-Biotin and 9R for detecting exon 9 of the target gene and a sequencing primer 9RCX, and upstream and downstream primers 20F-Biotin and 20R for detecting exon 20 of the target gene and a sequencing primer 20RCX. The kit can be used to detect polymorphism of hot spot mutation sites (E542K, E545K and H1047R) in the colorectal cancer related PIK3CA gene, and has good specificity and high accuracy.

The invention provides a mammal blood genome DNA extraction kit which is composed of an erythrocyte lysis buffer, a leukocyte lysis buffer, a protein precipitation liquid and DNA precipitation liquid. Compared with the prior art, the mammal blood genome DNA extraction kit has the following technical effects: (1) no organic reagent is used, and the mammal blood genome DNA extraction kit is friendly to the environment and harmless to a human body; (2) DNA with relatively high concentration and purity can be obtained, and stretches of DNA are preserved; (3) one-step removing method is used, and is simple to operate and applicable to a wide crowd; (4) the operation time is short, a DNA sample needed by an experiment can be obtained in a short time, so that the efficiency of the experiment is greatly improved; and (5) the cost is low and the price is low.

The invention relates to a method for rapidly extracting total ribonucleic acid from blood, belonging to the field of nucleic acid purification. The method comprises the following steps of: firstly, adding a rapid erythrocyte lysing solution into whole blood; performing centrifugal separation on a mixture of the rapid erythrocyte lysing solution and the whole blood; taking precipitate formed by the centrifugal separation out; adding suspension into the precipitate; adding a lysing solution and a protease K solution in sequence; uniformly mixing and then adding absolute ethyl alcohol; uniformly mixing and transferring into a silicon membrane adsorption column to perform adsorptive centrifugation; adding a deproteinizing solution and a deoxyribonuclease solution; performing deproteinizing centrifugation twice; adding a rinsing solution to perform desalting centrifugation; and finally performing drying centrifugation and adding water without ribonuclease, and eluting and centrifuging so as to obtain a ribonucleic acid solution. The method provided by the invention has the characteristics of high efficiency, rapidness and simplicity; and purified RNA (Ribonucleic Acid) can be used for various downstream experiments such as chip analysis, in vitro translation, molecular cloning and the like.

The invention provides a method for rapidly separating and extracting hUC-MSC (human Umbilical Cord mesenchymal stem cells). The method comprises the following steps: taking the freshly collected umbilical cord tissue of a healthy newborn baby, carrying out on-ice transportation on the freshly collected umbilical cord tissue in umbilical cord storage transportation liquid containing double antibodies, carrying out cleaning and disinfection by adopting 75% alcohol and normal saline, removing blood vessels, carrying out blunt dissection on wharton jelly, carrying out mechanical pulverization, treating the obtained product I by adopting red blood cell lysis buffer for 3 min, digesting the obtained product II by adopting IV collagenase, screening the obtained product III by adopting a 100-200-mesh sieve, carrying out suspension culture on the obtained product IV by adopting a serum-free medium, wherein the liquid is changed every 3-5 days, taking supernatant, detecting cell pollution, after the adherent rate in a plate reaches 30-70%, carrying out trypsinization, carrying out centrifugation, collecting cells, carrying out passage amplification, carrying out merging when the cell merging rate reaches 90% or above, collecting the cells, carrying out cryopreservation on the cells, and detecting the biological characteristics of hUC-MSC.

The invention provides a diagnostic kit for distinguishing active and latent mycobacterium tuberculosis infection with cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) serving as a diagnosis marker and a method for distinguishing active and latent mycobacterium tuberculosis infection by means of the diagnostic kit. The diagnostic kit comprises a CTLA-4, CD3, CD4, CD25, FoxP3 fluorescent antibody, erythrocyte lysate, membrane breaking liquid, washing buffer, phosphate buffer, fetal calf serum, stationary liquid and a streaming pipe. The diagnostic kit can be used for detecting the CTLA-4 expression quantity of peripheral venous blood of a patient and distinguishing active and latent mycobacterium tuberculosis infection, sensitivity and accuracy are high, and a strong basis is provided for clinic differential diagnosis.

The invention provides a separation method of PBMCs (peripheral blood mononuclear cells) and relates to the field of regenerative medicines. The separation method comprises steps as follows: pretreatment: acquired peripheral blood is centrifuged, upper-layer blood and lower-layer blood cells are separated, the lower-layer blood cells and a hydroxyethyl starch solution are mixed uniformly and placed to be settled, a liquid supernatant after settlement is removed, and lower-layer precipitates are reserved; separation: a red blood cell lysis buffer is added to the lower-layer precipitates, uniform mixing and centrifuging are carried out, cell precipitates are taken, PBS is added for re-suspension, the cell suspension is added to a lymphocyte separation liquid, centrifugation is carried out,a liquid supernatant is removed, cell precipitates are reserved, PBS, heparin and human serum albumin are added, mixing and centrifuging are carried out, and PBMC precipitates are obtained; and culture: the PBMC precipitates are resuspended with a culture medium and sub-packed in a culture bottle for culture. With the adoption of the method, the number of the separated PBMCs is large, and the PBMCs are high in purity and activity.

The invention provides a kit for detecting basophil activation and a using method of the kit. The kit comprises a scrubbing solution, an irritant reference substance, fluorescently-labeled basophil recognition antibodies, red blood cell lysis buffer and an antibody staining solution, wherein the fluorescently-labeled basophil recognition antibodies comprise the following basophil recognition antibodies: Anti human CD 11b, Anti human CD 123, Anti human CD 203c and Anti human CD 63; different basophil recognition antibodies have different fluorescent markers. After the kit is used for detecting the basophil activation situation, the known influence factors of the basophil activation and detection can be avoided, the basophil activation situation can be comprehensively and accurately identified and detected, and a better service is provided for the detection of the basophil activation in the aspect of clinical application and scientific research.

The invention relates to a detection kit for detecting the relative expression level of PML-RAR (alpha) fusion gene by use of fluorescent quantitative PCR (polymerase chain reaction) technology. The detection kit comprises the following materials in part ratio: red blood cell lysis buffer, TRIzol, chloroform, absolute ethyl alcohol, reverse transcription PCR reagent, detection system PCR reaction liquid, positive control substance and negative control substance. The kit provided by the invention has the advantages that the precision is high, the result is convenient to read, both the amplification efficiency and the rate reach the best level and the like; the complicated condition grope link is omitted; and the experimental efficiency is greatly improved. The kit has good specificity and high sensitivity and is easy and convenient to operate according to the test. The detection kit is favorable for the trace residue detection of the PML-PAR (alpha) (type L / type S) fusion gene in a clinical APL (acute promyelocytic leukemia) patient, and has great importance in performing timely intervention therapy to avoid hematological recurrence, adjusting the therapeutic schedule, evaluating the therapeutic effect, predicting the prognosis and preventing clinical recurrence.

The present invention relates to a new cell-free translation system. In particular, the invention relates to a cell-free reaction system for translating in vitro a RNA into a protein, said reaction system comprising a ribosome-depleted red blood cell lysate and ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes. The invention also pertains to a method for translating in vitro a ribonucleic acid template into an amino acid sequence of interest using the cell-free reaction system of the invention. The invention also relates to the use of (i) a ribosome-depleted red blood cell lysate, and (ii) ribosomes isolated from eukaryotic cells, with the proviso that (1) when the ribosome-depleted red blood cell lysate is obtained from a nuclease untreated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease untreated rabbit reticulocytes, and (2) when the ribosome-depleted red blood cell lysate is obtained from a nuclease treated rabbit reticulocyte lysate, the eukaryotic cells from which ribosomes are isolated are not nuclease treated rabbit reticulocytes, for producing a cell-free translation system.

The invention discloses a method for separating adipose-derived stem cells from fat and a preparation in use. On one hand, the method for separating the adipose-derived stem cells from fat comprises the steps of conducting liposuction to obtain a fat sample doped with inflation liquid to be subjected to centrifugation; removing the uppermost-layer fat, and removing suspension fat particles and bottom-layer cell sediment respectively; adding the fat particles into digestive enzyme mixed liquid, conducting vibration digestion, stopping the digestion, conducting centrifugation, and re-suspendingcell sediment; re-suspending the cell sediment at the bottom layer, adding erythrocyte pyrolysis liquid to conduct pyrolysis, conducting centrifugal, and re-suspending the cell sediment; merging to obtain cell suspension, filtering, and separating out the adipose-derived stem cells serving as primary passage. The invention further relates to a method for separating and cultivating the adipose-derived stem cells. The prepared adipose-derived stem cell preparations are used for separating the digestive enzyme mixed liquid of the adipose-derived stem cells, and are used for cultivating a culturemedium of the adipose-derived stem cells. The method for separating the adipose-derived stem cells from fat has various advantages as shown in the description.

Reagent kit for predicting 5-hydroxytryptamine re-ingestion inhibitor medicinal effect contains erythrocyte lytic liquid, leukocyte lytic liquid, protein deposit, nucleic acid reserve liquid, PCR reactive mixed liquid with MgC12, dNTP and PCR cloning primer and indicator, DNA polymerase, positive proton, negative control, endonuclease buffer system, limited endonuclease, PCR water, 10X electrophoretic buffer liquid, bromphenol blue 0.25% and sugar solution w / v40%. It extracts gene DNA in biological sampler and has polymerase chain-reaction-limited enzyme fragment polymorphism PCR-RFLP analysis.

The invention provides a construction method of a human adipose derived stem cell bank. The construction method comprises the following steps: (1) collecting adipose tissues, adding a saline solution and centrifuging; (2) removing an oil layer and washing the adipose tissues; (3) inoculating the washed adipose tissues into a complete culture medium and culturing under the conditions that the temperature is 37 DEG C and the concentration of CO2 is 5 percent; (4) replacing the culture medium for one time every 5 to 8 days; when the cell confluence is 70 percent to 80 percent, digesting cells with TrypLETM Select; (5) carrying out subculture; (6) detecting the quality; (7) cryopreserving; (8) establishing the bank. According to the construction method provided by the invention, oil and red blood cells are removed by adopting a centrifugal method and collagenase is not used for digesting; the operation time can be shortened and a process is simple and feasible; a lot of the red blood cells are not generated, and the red blood cells do not need to be removed by adopting a red blood cell lysis solution or low-molecular-weight heparin calcium; potential risks on human adipose derived stem cells are not caused; meanwhile, the cost is saved.

The invention discloses a method for utilizing a malignant pleural effusion for separately culturing primary cancer cells. The method comprises the steps of putting a pleural effusion sample into a sample collecting liquid, and centrifuging to remove a supernatant; adding a red blood cell lysis buffer for removing a red blood cell, adding a PBS buffer solution for washing the cells, and centrifuging to remove a supernatant; using a KL culture medium for resuspending a cell precipitate, inoculating in a culture flask, and culturing in a culture box. When the cells are proliferated to more than85 percent of abundance, the cells are washed through the PBS buffer solution, pancreatin-EDTA is digested, and a stop buffer neutralizes digestion reaction; the cells are centrifugally collected, areresuspended through the KL culture medium, and inoculated in the cell flask according to the proportion so as to be subcultured. The primary cancer cells of the pleural effusion obtained by separatedculture through the method provided by the invention can be applied to related research of physiology, pharmacy, new drug research and development and the like, exploration of pathogenesis of relateddiseases of a lung cancer, detection of drug sensitivity of different drugs, screening of anti-cancer drugs, new drug research and development, and establishment of a personalized treatment program aiming at a patient.