59 results about "Hepatic parenchymal cell" patented technology

The invention relates to the field of biotechnology and provides a method for long-time in-vitro culturing and proliferating hepatic cells. The method includes that mature hepatic parenchymal cells are induced to be hepatic cells with infinite proliferation capability in vitro. The hepatic cells obtained by the method can be used for toxicological and pharmacological evaluation of compounds and drug, study and diagnosis as well as treatment of hepatitis virus, hepatic cell transplantation therapy and preparation of bioartificial liver.

The invention discloses a parenchymal hepatic cell and preparation, identification and application methods thereof. The invention provides the method for preparing the parenchymal hepatic cell. The method comprises the following steps of: (1) culturing a pluripotent stem cell to obtain an endoderm cell; (2) culturing the endoderm cell obtained in the step (1) to obtain an original intestinal canal cell; (3) culturing the original intestinal canal cell obtained in the step (2) to obtain an adult hepatic cell; and (4) culturing the adult hepatic cell obtained in the step (3) to obtain a mature parenchymal hepatic cell, wherein the step (3) is characterized in that the cell obtained in the step (2) is subjected to passage and then transferred to a hepatic cell induction medium I containing fibroblast growth factors (FGF) and bone morphogenetic protein (BMP) for culturing to obtain the adult hepatic cell. The invention also relates to the parenchymal hepatic cell prepared by the method. The parenchymal hepatic cell prepared by the method has higher metabolic activity, high preparation efficiency and a wide application prospect than before.

The invention provides an artificial liver tissue and a preparation method thereof. The artificial liver tissue comprises a lower chip, a first microporous membrane, a middle chip, a second microporous membrane and an upper chip from the top to the bottom in sequence in a laminated manner. Three layers of cavities of a lower cavity, a middle cavity and an upper cavity are formed in the lower chip, middle chip and upper chip respectively. Three cavities are all grouted with culture solution containing growth factor, extracellular matrix containing hepatic parenchymal cells and suspension liquid containing endothelial cells. Adjacent cavities are separated by the microporous membrane. The microporous membrane can separate the matrix flow outside of the cell, and cause no influence to the cell migration. The culture solution and is fed through an inlet and an outlet of the upper cavity and lower cavity. Under the guidance of the growth factor, endothelial cells pass through the extracellular matrix of the middle cavity to form a lumen, and form the artificial liver tissue containing capillaries. The artificial liver tissue can simulate the human body liver tissue in a good way, and simulate the intrahepatic capillary functions of mass transfer, selective permeability, toxic reaction and the like.

The invention discloses an all-small molecular culture medium for culturing a liver organoid and an application of the all-small molecular culture medium. The culture medium comprises a basic culturemedium and a combination of small molecules, and the maintained combination of the small molecules is CBF, wherein C is CHIR-99021, B is Blebbistatin, C is Forskolin. The small molecules CHIR, Blebbistatin and Forskolin are added into the basic culture medium, so that the addition of a growth factor is replaced, a hpatic bile duct organoid can be cultured in vitro for a long time, and differentiation can be further induced to form a parenchymal hepatic cell. The method is beneficial to the in-vitro mass amplification of the liver organoid and provides the basis for researches and therapy of liver related diseases.

It is found that a biologically active substance contained in an endoplasmic reticulum can be delivered to a hepatocyte efficiently and specifically by adsorbing or chemically binding vitamin E to the surface of the endoplasmic reticulum.  This can be achieved by utilizing a serum component's property to deliver vitamin E to a hepatocyte.

The invention belongs to the field of separation and culture of cells and in particular relates to a separation and culture method for dog hepatocytes. The separation and culture method comprises the following steps: S1, taking liver from a living body; S2, perfusing; S3, separating the hepatocytes; S4, culturing the hepatocytes. The perfusion is realized by the following steps: firstly, perfusing perfusate A preheated at the temperature of 37DEG C into a flushed liver at the flow speed of 50ml/min for 15 to 20 minutes; secondly, continuously perfusing perfusate B preheated at the temperature of 37DEG C at the flow speed of 50ml/min for 3 to 5 minutes; thirdly, perfusing perfusate C preheated at the temperature of 37DEG C at the flow speed of 20ml/min for 4 to 6 minutes; finally, pouring a basal culture medium which contains three antibodies and is precooled at the temperature of 4DEG C on the surface of the liver, wherein the volumetric ratio of the three antibodies to the basal culture medium is 1 to 99. The hepatocytes obtained by separating through the method are nearly pure parenchymal hepatic cells; various functions of the hepatocytes are retained; the hepatocytes have the advantages of high quantity, morphological integrity, good adherence and high activity.

The invention discloses application of adenosine and derivatives thereof in prevention and treatment of medicament-induced liver injury, relating to application of adenosine and derivatives thereof such as adenosine monophosphate, adenosine diphosphate and adenosine triphosphate in protection of acetaminophen-induced liver injury. A mouse model adopted in the application is an acetaminophen-induced mouse acute hepatic failure model, the dosage of the adenosine or derivatives thereof is 0.1 to 5mmol/kg, and the adenosine and derivatives thereof can reduce the activities of aspartate aminotransferase (AST) and alanine transaminase (ALT) in the medicament-induced liver injury, reduce the substantial cell necrosis area and improve the glutathione level so as to prevent and treat the acetaminophen-induced liver injury caused by liver toxic medicaments. Animal experiments prove that the adenosine and derivatives thereof have a significant effect on preventing and treating the medicament-induced liver injury, so the adenosine and derivatives thereof can be used for the preventing and treating processes of the medicament-induced liver injury, and a new method and means are provided for clinically treating the medicament-induced liver injury.

The invention relates to the technical field of cell separation and particularly discloses a separating and extracting method for primary liver parenchyma cells and application of the primary liver parenchyma cells. The method disclosed by the invention comprises the steps: injecting perfusate I and perfusate II into a hepatic tissue mass to digest and separate the liver parenchyma cells so as toobtain a cell suspension, then, subjecting filter liquor, obtained after filtering the cell suspension, to normal-temperature centrifugation for 5 times sequentially, wherein centrifugation conditionssequentially are 1000-1580rpm/min*3-5min, 500-650rpm/min*3-5min, 200-300rpm/min*4min, 200-300rpm/min*4min and 200-300rpm/min*4min. According to the method, the operation is simple, consumed time is short, low-temperature centrifugation equipment is not required, red blood cells and cell debris can be efficiently removed, and obtained liver parenchyma cells are relatively high in survival rate andpurity.

The invention relates to a separating pork liver stem cell method, comprising steps of : cutting off a majority of liver to promote liver regeneration, adding collagenase to digest liver tissue into single cell, centrifuging to remove fibroblast of liver, using prolease E specificly digesting liver parenchymall cell , keeping the liver nonparenchymall cell, Percoll purifying using density gradient centrifugation method. The inventive method can provide highly purified, large amount and viable pork liver stem cell, which provide large amount of cells for stem cell fundamental research and clinical uses.

The invention relates to the technical field of cell models, in particular to a preparation method for hepatic progenitor cells from different sources. After induced culture is performed on hepatocytes and/or non-parenchymal cells with a hepatocyte transition and expansion medium, the induced-cultured hepatocytes and/or non-parenchymal cells transition into the hepatic progenitor cells which can expand and passage in vitro. According to the preparation method, by applying a lineage tracing method, the hepatocytes and the non-parenchymal cells in a mouse liver are isolated; by micromolecule assembly induced culture in vitro, the hepatocytes and the non-parenchymal cells successfully transition into the respective hepatic progenitor cells having an expansion and regeneration function; and meanwhile, proved by three-dimensional culture and organoid culture, the hepatocytes and the non-parenchymal cells both keep the characteristics of the cell sources thereof. The preparation method is conducive to further researching biological and morphologic characteristics of the hepatic progenitor cells from different sources, and further explores the application prospect of the hepatic progenitor cells as new transplanted cell sources.

The invention belongs to the field of biotechnology, and specifically relates to a method for separating, culturing and identifying primary hepatocytes of HBV transgenic mouse. The method comprises the following steps: performing separating and collecting primary hepatocytes by an improved collagenase two-step perfusion method; filtering the collected hepatocytes by a sterile cell filter; centrifuging and adding a culture medium for resuspension; performing cell counting and inoculated culture; the identification includes mouse primary hepatocyte albumin detection, mouse primary hepatocyte glycogen detection and expression analysis of HBV in the mouse primary hepatocyte. According to the method provided by the invention, the obtained HBV transgenic mouse primary hepatocyte has high cell viability as well as the functions of parenchymal hepatic cells, and the functions can be maintained for 8 days so as to more truly reflect the physiological metabolic activity of the hepatocytes in a HBV infection state, which is of great significance to in-vitro study of the physiological metabolic function of the hepatocytes as well as to the study of antiviral drugs.

The invention discloses a constructing device and a constructing method of an artificial liver lobule functional unit. The constructing device comprises a liver plate basal layer, a liver parenchyma cell zone and a fluid passageway layer, wherein a liver plate patterning groove is formed in the liver plate basal layer through processing; the fluid passageway layer comprises a fluid inlet passageway, an endothelial cell oriented growth zone and a fluid outlet passageway. The constructing method corresponding to the constructing device comprises the following steps: paving collagen containing liver parenchyma cells to the inner part of the liver plate patterning groove, bonding the liver plate basal layer with the fluid passageway layer after solidifying the collagen, pouring an endothelialcell suspension solution to the endothelial cell oriented growth zone, and enabling a cell culture medium solution to unidirectionally flow in the fluid inlet passageway, the endothelial cell orientedgrowth zone and the fluid outlet passageway after increasing endothelial cells to a certain amount. According to the constructing device and the constructing method, disclosed by the invention, reallayer-to-layer position relationship between endodermis and liver parenchyma cells is realized under dynamic culturing, the response of the liver parenchyma cells to physicochemical and biochemical irritation under the protection of the endodermis of blood vessels more tends to reality, and a more accurate result can be obtained when the artificial liver lobule functional unit is applied to drug screening, case research and therapy detection.

The invention relates to the field of biomedicine, in particular to a construction method of a liver unit stent, a liver unit stent and application of the liver unit stent in the field of drug testing. The invention aims to provide a construction method of a liver unit stent, a liver unit stent and application of the liver unit stent in the field of drug testing. The construction method of the liver unit stent comprises the following steps: a gelled first composition, which includes hepatic parenchymatous cells and a biodegradable material, is loaded into a biological 3D printer, and an intermediate is printed according to a preset path; a cross-linking agent is dropwise added to the intermediate such that the biodegradable material in the intermediate is cross-linked to form a liver unitstent, wherein a channel is formed in the liver unit stent. In the liver unit stent, cell variety, quantity and position are controllable and the channel is controllable, thus providing a microenvironment for different cells. In addition, the liver unit stent can be used for researching the drug hepatotoxicity of new drugs and is used for detecting drug metabolites, gene expression, protein expression, cell apoptosis and cell proliferation.

The invention provides a separation method for hepatic inherent and infiltrating macrophages. The separation method comprises the following steps of (1) establishing and identifying mouse allogeneicbone marrow transplantation model, wherein a surface marker of a mononuclear macrophage of a recipient mouse is CD45.2, and a surface marker of a mononuclear macrophage of a donor mouse is CD45.1; (2)observing and irradiating the influence on hepatic parenchymal cells and inherent macrophages with an IHC method and a flow cytometry; and (3) irradiating a mononuclear cell of a mouse liver in a transplantation group with primary isolation, and sorting the hepatic inherent and infiltrating macrophages with the flow cytometry according to the surface marker of the mononuclear macrophage of the donor mouse, and describing the quantities, distribution and phenotypes of the hepatic inherent and infiltrating macrophages. In the separation method provided by the invention, by removing the CD45.2 recipient mouse bone marrow derived macrophages by irradiation and supplementing and replacing bone marrow cells of the recipient mouse with CD45.1 donor mouse bone marrows, the bone marrow derived infiltrating macrophages and the bone marrow derived inherent macrophages have different molecular phenotypes and achieves the effect of accurately and efficiently separating different hepatic macrophages.

The invention relates to an establishment method of turbot hepatic parenchymal cell line and the turbot hepatic parenchymal cell line. The establishment method of the turbot hepatic parenchymal cell line comprises the following steps: obtaining turbot liver tissue; cutting the liver tissue into tissue blocks, and carrying out washing; carrying out digestion treatment by adopting type 4 collagenase, and carrying out washing again; carrying out digestion treatment on the tissue blocks obtained after the digestion treatment by adopting trypsin again; adding a primary complete medium to terminate digestion to obtain treated tissue blocks; uniformly attaching the treated tissue blocks into a culture bottle, placing the attached culture bottle into an incubator at 23 DEG C for 6 hours; inverting the culture bottle for 6 hours; adding 4 ml of a primary complete culture medium after the culture bottle is upright; performing primary culture; and then, performing subculture. The turbot hepatic parenchymal cell line established by the method can realize continuous passage so as to provide a large number of turbot hepatic cells; and the established stable and sustainable turbot hepatocyte line can be applied to the molecular cell biology research of turbot nutrition metabolism, thereby overcoming defects in the prior art at the cellular level.

The invention discloses a CPS1 reporter gene human liver cell line and its construction method and application. The human liver cell line is constructed by using aminoacyl phosphate synthase 1 (CPS1) and a reporter gene, and the obtained CPS1 reporter gene human liver cell has a human primary adult The functions of hepatic parenchymal cells, including albumin secretion, cytochrome P450 enzyme metabolism and ammonia metabolism, can be applied in artificial liver, drug metabolism research, drug toxicity evaluation or drug screening, and have broad application prospects.

The invention discloses a hepatoma targeting carbon nano tube loaded with doxorubicin hydrochloride and a preparation method thereof. Amidation reaction of amino in a chitosan structure and carboxyl in lactobionic acid is carried out; galactosyl is coupled to chitosan so as to obtain galactosylated chitosan; the galactosylated chitosan is modified onto the carbon nano tube; therefore, the water solubility and the biocompatibility of the carbon nano tube are increased; simultaneously, the specificity of non-reductive galactose or N-acetyl-galactose is identified and taken by utilizing an asialoglycoprotein acceptor on a liver parenchyma cell membrane, so that the active hepatoma targeting effect is realized; simultaneously, doxorubicin hydrochloride is combined with a vector in a non-covalent manner through phi-phi accumulation acting force; and therefore, the hepatoma targeting carbon nano tube loaded with doxorubicin hydrochloride is prepared. The hepatoma targeting carbon nano tube disclosed by the invention is simple in preparation process, moderate in experimental condition and easy to operate; release of the medicine loaded targeting carbon nano tube has pH dependency, good biocompatibility and obvious in-vivo tumour inhibition effect; and thus, the hepatoma targeting carbon nano tube has good practical value.

The invention relates to a cell separation culture method of biological tissues and use thereof, in particular to a method for separating and culturing hepatic cells of a newborn rat (or mouse) and using the hepatic cells as a substitutional animal experiment model for toxicological evaluation of exogenous compounds. The method for separating and culturing the hepatic cells of the newborn rat (or mouse) of the invention has simple operation, does not need a liver perfusion process and expensive instruments, equipment and reagents, and can obtain more than 90% of liver parenchyma cells which have good uniformity and stability and high sensitivity and are easy for manual control. The hepatic cells separated and cultured by the invention are very sensitive to the toxicity of the exogenous compounds, have some functions and activity of in vivo hepatic cells, especially reserve the activity of drug metabolic enzyme, can well simulate the physiological environment of the in vivo liver, have the advantage of combining in vitro and integral animal toxicity tests, and can be expected to be used in high flux rapid detection of the toxicity of the exogenous compounds.

The invention discloses functional liver parenchyma cells and a preparation method therefor. According to the preparation method, potential-extended multipotential stem cells, i.e., EPS cells are subjected to directed differentiation, so as to prepare the functional liver parenchyma cells (or called liver cells). Furthermore, before directed differentiation of the EPS cells, the EPS cells is firstly pretreated in an iPSCs/ESCs culture medium. According to the preparation method disclosed by the invention, human EPS cells can be more efficiently differentiated into the functional liver parenchyma cells, and the functional liver parenchyma cells EPS-Heps obtained through EPS differentiation are more similar to fresh-separated primary liver cells in a full-gene transcription level.

The invention relates to the field of biological materials and biomedical engineering, and particularly relates to an artificial liver organ and a preparation method and application thereof. The artificial liver organ comprises an endothelial cell layer, a hepatic stellate cell layer and a hepatic parenchyma cell layer; the different cell layers are arranged in a bionic and close mode, and the distance between different kinds of adjacent cells is smaller than 30 micrometers; the mechanical property of the artificial liver organ is 500Pa-1MPa; and the characteristic length of the artificial liver organ is 50-1000 micrometers. The constructed liver organ comprises liver cells, endothelial cells and hepatic stellate cells, the structure and physiological function of the liver Disse gap can be simulated, and the requirements of in-vitro liver physiological/pathological research, drug testing and drug development, tissue engineering and regenerative medicine are met. The artificial liver organ can be induced into various liver disease models at the same time, and corresponding pathological models can be constructed according to requirements.

The invention provides a cryopreservation method of primary-generation mouse parenchymal liver cells. The method includes the steps of firstly, preparing mouse parenchymal liver cell cryopreservationliquid; secondly, using a two-step perfusion method to separate the mouse parenchymal liver cells; thirdly, using the cryopreservation liquid to resuspend the mouse parenchymal liver cells, and then adding the mouse parenchymal liver cells into a cryopreservation tube; fourthly, using a step-by-step temperature reduction method to cryopreserve the mouse parenchymal liver cells; fifthly, resuscitating the mouse parenchymal liver cells; sixthly, using a dye exclusion method to measure the survival rate of the mouse parenchymal liver cells; seventhly, determining enzyme index activity; eighthly,observing the morphology of the mouse parenchymal liver cells; ninthly, performing ELISA method detection; tenthly, performing RT-PCR detection. The cryopreservation method has the advantages that themethod is simple to operate, many cells can be obtained after cryopreservation resuscitating, high survival rate is achieved, and the method is an ideal primary-generation mouse parenchymal liver cell cryopreservation method and capable of providing reliable cell resources for experiments.

The invention provides a method for cultivating a mouse model of primary biliary cholangitis, the method comprising: (1) perfusing the mouse liver with collagenase IV for 10 minutes through the hepatic portal vein, removing hepatic parenchymal cells, and obtaining Complete bile ducts; (2) shredding the bile ducts obtained in step (1), further using ultrasonic waves for homogenization, and quantifying the protein concentration of the obtained bile duct protein homogenate; (3) using the obtained bile duct proteins to The source mice were subcutaneously immunized to establish primary biliary cholangitis model mice induced by bile duct antigen immunization. The mouse obtained by the method of the invention can be used as a new animal model for screening drugs for primary biliary cholangitis.