CN106591221B - Method for separating and amplifying human vein endothelial cells in vitro - Google Patents
Method for separating and amplifying human vein endothelial cells in vitro Download PDFInfo
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Abstract
The invention relates to a method for obtaining human vein endothelial cells from human umbilical vein, which comprises the steps of separating the human vein endothelial cells and amplifying the human vein endothelial cells in vitro, wherein the separation of the human vein endothelial cells comprises the steps of fixing umbilical vein outer layer cells by alcohol, digesting the umbilical vein endothelial cells by collagenase, and collecting relatively more and purer umbilical vein endothelial cells; the platelet lysate is used in the in-vitro amplification culture process of the human vein endothelial cells without any animal source and serum components, so that the potential threat of the animal heterologous components and the serum to the cell application in the cell culture process is avoided. The human vein endothelial cell obtained by the method can be used for researching the neovascular mechanism and screening anti-tumor neovascular medicines.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for obtaining human vein endothelial cells, which comprises the steps of separation and in-vitro amplification.
Background
Endothelial cells have more biological functions, can be applied to vascular remodeling of atherosclerosis, relieve vascular injury and prevent cardiovascular diseases; relieving the lower limb ischemia symptom of patients with diabetes, and preventing the occurrence of non-traumatic amputation; in addition, endothelial cells have been reported in the research of angiogenesis-targeted drugs, tissue regeneration, and wound healing.
The method for separating the umbilical vein endothelial cells comprises the following steps: mechanical scraping, tissue block transplanting, a magnetic bead sorting method after tissue digestion and a collagenase grouting method. The first two methods have low cell positive rate, the third method has high cost, and the fourth method is mainly used at present, but the method fills enzyme liquid into umbilical vein, needs to maintain digestion temperature, and is inconvenient to operate, and requires the length of sample to be more than 20 cm, which causes too small number of collected cells, and in addition, other pollutants are often brought in during the operation process.
In the process of culturing endothelial cells, more animal-derived components such as fetal calf serum, equine calf serum and the like are added in the existing culture method so as to meet the requirement of nutrition in the process of growing the endothelial cells, but the problems of foreign proteins, unknown pollution sources and the like exist.
For example, in the Chinese patent application with the application number of 200810222274.X, the publication date of 2009, 1 month and 28 days, and the invention name of "a vascular endothelial cell and a preparation method and application thereof", a large amount of heterologous serum such as horse serum and fetal bovine serum is added in the later-stage endothelial cell culture process, so that the application of the endothelial cell has safety risk in later-stage endothelial cell use.
Disclosure of Invention
The invention aims to provide a method for separating human vein endothelial cells with high collection quantity and high purity.
The invention provides a method for separating human vein endothelial cells, which comprises the steps of firstly treating human umbilical vein with alcohol to fix cells on the outer layer of the vein, then digesting the endothelial cells on the inner layer of the vein with collagenase, removing residual tissues of the human umbilical vein after the digestion is finished, and centrifuging the digestive juice to obtain the human vein endothelial cells.
In the above method for isolating human venous endothelial cells, the endothelial cells are derived from human umbilical veins.
The method for obtaining the human umbilical vein comprises the following steps: the human umbilical cord is cleaned by normal saline to remove residual blood on the surface of the umbilical cord and in blood vessels, then forceps and a scalpel are used for stripping the amniotic membrane and the colloid of the umbilical cord, and then the human umbilical cord vein is picked.
The method comprises the following steps of (1) fixing the human umbilical vein by alcohol soaking, wherein the purpose of fixing the human umbilical vein by alcohol soaking is to inactivate cells on the outer layer of the umbilical vein, when the vein is digested by collagenase, the outer layer cells are fixed and are not easy to fall off or fall off to be inactivated cells, and endothelial cells on the inner layer of the vein are easily digested and obtained, so that more and purer endothelial cells are collected, and the method comprises the following steps: clamping both ends of the vein with hemostatic clamp, fixing human umbilical vein in 70-75% (preferably 75%, V/V) alcohol for 3-5s (preferably 3s), and washing with physiological saline.
In order to facilitate digestion, the human umbilical vein after fixation needs to be cut into segments with a length of 3-8cm (preferably 5cm) with a scalpel.
The method for digesting umbilical vein tissue by collagenase comprises the following steps: digesting the fixed human umbilical vein segment with IMDM (or DMEM/F12, alpha-MEM or other cell culture medium) containing 0.1-0.5% (preferably 0.1%, M/V) collagenase I in a volume of 1mL of the digestion solution per 1cm of umbilical vein under digestion conditions of 35-38 deg.C (preferably 37 deg.C) for 20-40min (preferably 30min), and shaking for 1 time every 5-10min (preferably 10 min).
Human umbilical vein residual tissue can be removed using a 70-100 μm (preferably 100 μm) cell screen.
The centrifugation conditions are 1500-2000rpm (preferably 1500rpm) centrifugation for 8-10min (preferably 10 min).
Specifically, the method for separating human vein endothelial cells provided by the invention can comprise the following steps:
1) cleaning fresh human umbilical cord within 12 hours of natural labor with normal saline to remove blood, then stripping the amniotic membrane and the colloid of the umbilical cord with forceps and a scalpel, and picking human umbilical vein;
2) clamping two ends of human umbilical vein with hemostatic forceps, fixing human umbilical vein in 70-75% (preferably 75%, V/V) alcohol for 3-5s (preferably 3s), and washing with physiological saline;
3) cutting the fixed human umbilical vein tissue into segments with the length of 3-8cm (preferably 5cm) by using a scalpel;
4) digesting the human umbilical vein fragment with IMDM (or DMEM/F12, alpha-MEM and other cell culture media) digestive fluid containing 0.1-0.5% (preferably 0.1%, M/V) collagenase I, wherein the volume of the digestive fluid is 1mL of digestive fluid/1 cm of umbilical vein tissue, the digestion condition is 35-38 ℃ (preferably 37 ℃) for 20-40min (preferably 30min), and the digestion process is carried out for 1 shaking every 5-10min (preferably 10 min);
5) removing human umbilical vein tissue by using a 70-100 μm (preferably 100 μm) cell sieve, centrifuging at 1500-2000rpm (preferably 1500rpm) for 8-10min (preferably 10min), and collecting cell precipitate to obtain human vein endothelial cells.
Another object of the present invention is to provide a method for accomplishing the in vitro expansion of human venous endothelial cells without the use of heterologous elements.
The human vein endothelial cell in vitro amplification method provided by the invention is characterized in that human vein endothelial cells are re-suspended by using an amplification culture medium of an IMDM culture medium (or DMEM/F12, alpha-MEM and other cell culture media) containing human platelet lysate and VEGF (endothelial cell growth factor), the human vein endothelial cells are cultured, adherence is induced for 12-36 hours, the solution is completely changed, nonadherent cells are removed, fresh amplification culture medium is added, the solution is completely changed every other day, and when the cell fusion degree reaches 80-90%, the human vein endothelial cells are obtained by digestion and passage with pancreatin.
Preferred personThe in vitro amplification method of vein endothelial cells comprises resuspending human vein endothelial cells in IMDM medium containing 5-15% (V/V) platelet lysate and 8-15ng/mL VEGF, and culturing at 37 deg.C with 5% CO2Culturing under saturated humidity, inducing wall adhesion for 12-36 hours, changing liquid in full amount, removing non-wall adhesion cells, adding fresh amplification culture medium, changing liquid in full amount every other day, and digesting and passaging with 0.05-0.1% (M/V) pancreatin when the cell fusion degree reaches 80-90% to obtain the human vein endothelial cells.
The most preferred method for in vitro expansion of human venous endothelial cells is to resuspend human venous endothelial cells in IMDM medium containing 10% (V/V) platelet lysate, 10ng/mL VEGF, and 2 units of sodium heparin, at 37 deg.C, 5% CO2Culturing under saturated humidity, inducing wall adhesion for 24 hours, changing liquid in full quantity, removing non-wall-adhered cells, adding fresh amplification culture medium, changing liquid in full quantity every other day for 1 time, and digesting and passaging by 0.05% (M/V) pancreatin when the cell fusion degree reaches 90% to obtain the human vein endothelial cells.
In the method for in vitro amplification of human vein endothelial cells, platelet lysate is used for providing nutrients and factors necessary for cell attachment and growth, and VEGF is used for stimulating endothelial cells to proliferate growth factors.
Human venous endothelial cells expanded by the above method, which do not contain heterologous serum, also belong to the present invention.
The invention further aims to provide a method for obtaining human vein endothelial cells from human umbilical vein, which comprises the processes of the separation of the human vein endothelial cells and the in vitro expansion of the human vein endothelial cells. The human vein endothelial cells obtained by the method have high purity and large yield and do not contain heterologous serum.
The method for obtaining the human vein endothelial cells from the human umbilical vein specifically comprises the following steps:
1) picking human umbilical vein from human umbilical tissue;
2) clamping two ends of human umbilical vein with hemostatic forceps, soaking human umbilical vein in 70-75% (preferably 75%, V/V) ethanol for 3-5s (preferably 3s), and washing with normal saline;
3) cutting the fixed human umbilical vein tissue into segments with the length of 3-8cm (preferably 5cm) by using a scalpel;
4) soaking human umbilical vein fragment in IMDM (or DMEM/F12, alpha-MEM or other cell culture medium) containing 0.1-0.5% (preferably 0.1%, M/V) collagenase I in a volume of 1mL of digestive juice/1 cm of umbilical vein tissue under 35-38 deg.C (preferably 37 deg.C) for 20-40min (preferably 30min), and shaking for 1 time every 5-10min (preferably 10min) during digestion;
5) removing human umbilical vein tissue by using a 70-100 μm (preferably 100 μm) cell sieve, centrifuging the digestive juice at 1500-2000rpm (preferably 1500rpm) for 8-10min (preferably 10min), and collecting cell precipitate to obtain separated human vein endothelial cells;
6) resuspending human venous endothelial cells in an expansion medium containing 5-15% (preferably 10%, V/V) platelet lysate and 8-15ng/mL (preferably 10ng/mL) of IMDM medium, 5% CO at 37 deg.C2Culturing under saturated humidity, inducing adherence for 12-36 hours (preferably 24 hours), changing liquid in full quantity, removing nonadherent cells, adding fresh amplification culture medium, changing liquid in full quantity every other day, and digesting and passaging with 0.05-0.1% (preferably 0.05%, M/V) pancreatin when the cell fusion degree reaches 80-90% (preferably 90%), so as to obtain the amplified human vein endothelial cells.
The intravenous endothelial cell digest used in the above-described method, i.e., a solution containing 0.1 to 0.5% (preferably 0.1%, M/V) collagenase I in IMDM (or other cell culture media such as DMEM/F12, alpha-MEM) is also included in the present invention.
The human venous endothelial cell expansion medium used in the above described method, i.e. IMDM medium containing 5-15% (preferably 10%, V/V) platelet lysate and 8-15ng/mL (preferably 10ng/mL) VEGF (preferably with the addition of an anticoagulant such as heparin sodium) is also within the present invention.
By adopting the scheme, the invention has the following advantages:
1) the method adopts mechanical separation and enzyme digestion to collect the human vein endothelial cells, has simple operation and short consumed time: the method comprises the steps of mechanically stripping umbilical cord colloid to obtain human umbilical vein, soaking and fixing cells on the outer layer of the umbilical vein in alcohol, soaking the umbilical vein in collagenase for digestion, adding sufficient enzyme liquid, maintaining the temperature easily, and enabling the endothelial cells of the umbilical vein to fall off easily;
2) the platelet lysate is used in the in-vitro amplification culture process of the human vein endothelial cells, so that the endothelial cell amplification culture medium does not contain any animal source and serum components, and potential threats of animal heterologous components and serum to cell application in the cell culture process are avoided.
The human vein endothelial cells separated by the invention can be used for research and design of a new vessel mechanism, screening of tumor angiogenesis inhibiting drugs and the like, and have wide application prospect.
The present invention will be described in further detail with reference to specific examples.
Drawings
FIG. 1 shows the morphology of human vein endothelial cells
FIG. 2 shows the flow cytometry detection results of the surface markers of human vein endothelial cells
FIG. 3 Effect of melatonin on endothelial cell proliferation
Detailed Description
The invention obtains human vein endothelial cells from human umbilical vein, which comprises two parts of separation and in-vitro amplification of the human vein endothelial cells.
Isolation of human venous endothelial cells
Treating human umbilical vein with alcohol to fix cells on outer layer of vein, treating with collagenase to digest endothelial cells on inner layer of vein, removing residual tissue of human umbilical vein after digestion, and centrifuging digestive liquid to obtain human vein endothelial cells.
Here, the endothelial cells are derived from human umbilical veins. The human umbilical vein may be isolated from the umbilical tissue by: the human umbilical cord is cleaned by normal saline to remove residual blood on the surface of the umbilical cord and in blood vessels, then the umbilical cord amnion and colloid are stripped by forceps and a scalpel, and then the human umbilical cord vein is picked.
Here, the treatment of the human umbilical vein with alcohol first means that both ends of the human umbilical vein are tied up and soaked in alcohol for several seconds, with the purpose of fixing or inactivating cells in the outer layer of the umbilical vein. Thus, when the collagenase is used for digesting the vein, because the outer layer cells are fixed and are not easy to fall off or fall off are inactivated cells, the inner layer endothelial cells of the vein are easy to digest and obtain, and more and purer endothelial cells can be collected. The operation can be as follows: clamping both ends of human umbilical vein with hemostatic forceps, fixing human umbilical vein in 70-75% (preferably 75%, V/V) alcohol for 3-5s (preferably 3s), and washing with physiological saline.
Herein, the treatment of the fixed human umbilical vein with collagenase means that the human umbilical vein is soaked with collagenase to digest endothelial cells in the inner layer of the vein. In order to facilitate digestion, the fixed human umbilical vein is cut into segments with the length of 3-8cm (preferably 5cm), and the specific operation can be as follows: digesting the fixed human umbilical vein segment with IMDM (or DMEM/F12, alpha-MEM or other cell culture medium) containing 0.1-0.5% (preferably 0.1%, M/V) collagenase I by using a scalpel, wherein the volume of the digestive juice is 1 mL/1 cm umbilical vein, the digestion condition is 35-38 deg.C (preferably 37 deg.C), and the digestion is performed for 20-40min (preferably 30min), and the digestion process is performed by shaking for 1 time every 5-10min (preferably 10 min).
Here, human umbilical vein residual tissue can be removed using a 70-100 μm (preferably 100 μm) cell sieve.
Here, the digested solution from which the residual tissue of the human umbilical vein was removed was centrifuged, and the precipitate was taken to obtain human vein endothelial cells. The centrifugation conditions may be 1500-2000rpm (preferably 1500rpm) for 8-10min (preferably 10 min).
Expansion of human venous endothelial cells
The invention relates to in vitro amplification of vein endothelial cells, which is to use an amplification medium of IMDM medium (or DMEM/F12, alpha-MEM and other cell culture media) containing human platelet lysate and VEGF (endothelial cell growth factor) to resuspend the human vein endothelial cells, induce adherent culture and use pancreatin to digest and passage to obtain the human vein endothelial cells.
Here, the amplification medium used contains human platelet lysate and no horse bloodThe platelet lysate is used to provide necessary nutrients and factors for cell attachment and growth, and VEGF is used to stimulate endothelial cell to proliferate growth factor, human platelet lysate is obtained by collecting platelet 1500-1500 rotation centrifugal collector, mixing, and regulating platelet concentration to 1 × 109Freezing and thawing the cells/ml for 3 times at minus 80 ℃ and 37 ℃, centrifuging the cells for 30min at 900g, taking supernatant, subpackaging and freezing the supernatant in a refrigerator at minus 80 ℃ for later use, wherein the technical index of the human platelet lysate used in the invention is that the concentration of platelets in the human platelet lysate is 1 × 109Per ml, human platelet lysate is commercially available according to the above criteria.
Specific procedure for in vitro amplification of human venous endothelial cells, human venous endothelial cells can be resuspended in IMDM medium containing 5-15% (preferably 10%, V/V) platelet lysate and 8-15ng/mL (preferably 10ng/mL) VEGF (to avoid platelet aggregation, an anticoagulant such as heparin sodium can be added) at 37 deg.C and 5% CO2Culturing under saturated humidity, inducing adherence for 12-36 hours (preferably 24 hours), changing liquid in full quantity, removing nonadherent cells, adding fresh amplification culture medium, changing liquid in full quantity every other day, and digesting and passaging with 0.05-0.1% (preferably 0.05%, M/V) pancreatin when the cell fusion degree reaches 80-90% (preferably 90%), so as to obtain the human vein endothelial cells.
In the present invention, the percentage concentration is a mass/mass (W/W, unit g/100g) percentage concentration, a mass/volume (W/V, unit g/100mL) percentage concentration, or a volume/volume (V/V, unit mL/100mL) percentage concentration unless otherwise specified.
The various biological materials described in the examples are obtained by way of experimental acquisition for the purposes of this disclosure and should not be construed as limiting the source of the biological material of the invention. In fact, the sources of the biological materials used are wide and any biological material that can be obtained without violating the law and ethics can be used instead as suggested in the examples. For example, the embodiment of the invention obtains human umbilical vein from human umbilical cord, the human umbilical cord is medical waste and can be recycled in large quantity, and an HLA matched endothelial cell bank can be established according to umbilical cord blood bank resources, so that a large quantity of endothelial cell resources for preclinical research can be provided.
The embodiments are provided in order to provide detailed embodiments and specific procedures, which will help understanding of the present invention, but the scope of the present invention is not limited to the following embodiments.
Example 1 isolation of human venous endothelial cells
This example uses veins from human umbilical cord to effect isolation of human venous endothelial cells, including the following specific procedures, it being understood that the following procedures are for clarity of disclosure of the continuous procedure and are not intended to limit the invention:
1) taking a fresh umbilical cord of a birth-oriented infant in a sterile biological safety cabinet (within 12 hours, if the time is too long, endothelial cells die in vitro and are not easy to survive), cleaning the umbilical cord for 2 times by using 250mL of physiological saline, extruding umbilical cord tissues by using forceps, removing residual blood in the umbilical cord, stripping umbilical cord amniotic membrane and colloid by using the forceps and a scalpel, and then picking a human umbilical vein for about 10 cm;
2) clamping two ends of human umbilical vein with hemostatic forceps, soaking vein in 75% (70-75% all, V/V) alcohol for 3s (3-5s all), and washing with normal saline for 1 time (1-2 times all);
3) cutting the fixed human umbilical vein into segments with the length of 5cm (3-8 cm);
4) digesting human umbilical vein fragments with IMDM digestive juice containing 0.1% (0.1-0.5% of all, M/V) collagenase I (purchased from sigma), wherein the added volume of the digestive juice is 1mL of digestive juice/1 cm of umbilical vein, the digestion conditions are 37 ℃ (35-38 ℃) and 30min (20-40 min), and the digestion process is carried out for 1 time every 10min (5-10 min);
5) removing human umbilical vein by using 100 μm (70-100 μm) cell sieve, centrifuging the digestive fluid at 1500rpm (both 1500 and 2000 rpm) for 10min (both 8-10 min), and collecting cell precipitate to obtain human vein endothelial cells.
Example 2 in vitro expansion of human venous endothelial cells
This example of in vitro expansion of human venous endothelial cells includes the following specific operations: it contains 10% (5-15%) of the active componentV/V), 10ng/mL (all 8-15 ng/mL) of VEGF (endothelial growth factor, available from peprotech), 2 units of heparin sodium (available from Wanbang Biochemical medicine) of IMDM medium (available from gibco) (or other cell culture media such as DMEM/F12, α -MEM), resuspending human vein endothelial cells in expanded medium, 5% CO at 37 deg.C2Culturing under saturation humidity, inducing wall adhesion for 24 hours (12-36 hours), changing liquid in full quantity, removing non-wall adhesion cells, adding fresh amplification culture medium, changing liquid in full quantity every other day for 1 time, digesting and passaging by 0.05% (0.05-1%, M/V) pancreatin (purchased from sigma) when the cell fusion degree reaches 90% (80-90%), and obtaining human vein endothelial cells.
Here, the human vein endothelial cells can be obtained by the method of example 1, but not limited thereto, and the in vitro amplification method of example 2 can be used alone to achieve in vitro amplification of all human vein endothelial cells, or can be combined with example 1 to achieve the purpose of obtaining human vein endothelial cells with high purity, large harvest yield and good safety by separation and amplification from human umbilical vein.
Example 3 identification of human venous endothelial cells
Growth characteristics and morphological characteristics of human vein endothelial cells
Human vein endothelial cells induced to adhere for 2 days in example 2 are observed by a microscope (the human vein endothelial cells isolated in example 1 are amplified), as shown in fig. 1, adherent cells with a scattered paving stone shape can be seen under the microscope, monoclonal cells can be formed after the cells are cultured for 3-4 days after the liquid is completely changed (fig. 1A), and when the cells are cultured to about 90% of cell fusion degree (fig. 1B), the cells are in a typical paving stone shape, have a high proliferation speed and relatively uniform morphology and can be used for cell biological characteristic identification.
Flow cytometry for identifying human vein endothelial cell surface marker
Respectively taking 3 rd generation human vein endothelial cells of different umbilical cord sources (R1 is a target cell, R2 is a CD34 positive cell, R3 is a CD31 positive cell, and R4 is a CD105 positive cell), detecting cell surface markers by flow cytometry, and observing the change of the cell surface markers of the different umbilical cord sources. The specific method comprises the following steps: digestion and harvestCollecting cells, counting, and collecting 1 × 106Washing with PBS for 1 time, centrifuging at 1500rpm for 10min, discarding supernatant, leaving 200 μ L, blowing and mixing the cells, adding FITC labeled CD31 (purchased from BD), PERCP labeled CD105 (purchased from BD) and PE labeled CD34 antibody (purchased from BD) each 10 μ L, setting 1 tube as blank control, reacting at 4 deg.C in the dark for 30min, washing with PBS for 1 time, centrifuging at 1500rpm for 10min, discarding supernatant, leaving 200 μ L, and detecting on machine.
The flow cytometry detection result of the human vein endothelial cell surface marker is shown in fig. 2, the change of the human vein endothelial cell surface markers from different umbilical cords is not large, each umbilical cord source cell highly expresses CD105, CD31 and CD34, the cell components are uniform, and the purity is more than 95%.
Example 4 application of human vein endothelial cells
The human vein endothelial cells separated in the embodiment 1 and the human vein endothelial cells amplified in vitro in the embodiment 2 can be used for screening the medicines for inhibiting the tumor angiogenesis. This example illustrates an example of one of the drugs melatonin for inhibiting tumor angiogenesis.
Melatonin is capable of inhibiting the formation of tumor neovessels by endothelial cells, mimicking the effects of melatonin on the growth state of endothelial cells in vitro, and reflecting laterally the biocidal effect of melatonin on tumor cells.
Human venous endothelial cells (obtained in example 1 or example 2) were resuspended in the endothelial cell in vitro amplification medium described in example 2, seeded at a concentration of 3 ten thousand cells/well in 6-well plates at 3mL per well of medium, and control, experimental 1 and experimental 2 were set up at 37 ℃ with 5% CO2Culturing under saturated humidity, inducing and fusing for 12 hours, after 12 hours, not processing a control group, sucking all culture media from experimental groups 1 and 2, adding fresh amplification culture medium into the experimental group 1, adding amplification culture medium containing 1.5mM melatonin into the experimental group 2, continuing to induce and culture for 48 hours, and determining the cell proliferation condition.
As shown in fig. 3, it can be seen that the data of group 2 shows that 1.5mM melatonin has a significant inhibitory effect on endothelial cell proliferation, and clinically, melatonin can be targeted to migrate to a tumor tissue site, inhibit angiogenesis of the tumor tissue, cut off the nutrient supply of the tumor tissue, and promote the tumor tissue to diminish or stop growth, thereby killing tumor cells.
Therefore, the human vein endothelial cell can be used for establishing a screening system of various drugs for inhibiting tumor angiogenesis, and has a very wide application prospect.
Claims (10)
1. A method for obtaining human venous endothelial cells from a human umbilical vein, comprising: the method comprises the following steps:
1) picking human umbilical vein from human umbilical tissue;
2) clamping two ends of a human umbilical vein by using hemostatic forceps, soaking the human umbilical vein in 70-75% alcohol for 3-5s, and washing with normal saline;
3) cutting the fixed human umbilical vein tissue into segments with the length of 3-8cm by using a scalpel;
4) soaking human umbilical vein fragment in IMDM or DMEM/F12 containing 0.1-0.5% (M/V) collagenase I and alpha-MEM cell culture medium digestive solution at 35-38 deg.C for 20-40min, and shaking for 1 time every 5-10min during digestion;
5) removing human umbilical vein tissue by using a 70-100 mu m cell sieve, centrifuging the digestive juice at 1500-2000rpm for 8-10min, and collecting cell sediment to obtain separated human vein endothelial cells;
6) resuspending human vein endothelial cells by using an amplification culture medium containing 5-15% (V/V) platelet lysate and 8-15ng/mL VEGF-containing IMDM culture medium, inducing adherence for 12-36 hours, changing the solution at all times, removing nonadherent cells, and adding a fresh amplification culture medium;
7) when the cell fusion degree reaches 80-90%, using 0.05-0.1% (M/V) pancreatin to digest and passage to obtain the expanded human vein endothelial cell.
2. The method of claim 1, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: the digest was a solution of IMDM cell culture medium containing 0.1% (M/V) collagenase I.
3. The method of claim 1, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: the volume of the added digestive juice is 1mL of digestive juice/1 cm of umbilical vein, the digestion condition is that the umbilical vein is soaked for 30min at 37 ℃, and the digestive juice is shaken for 1 time every 10min in the process of digestion.
4. The method of claim 1, wherein the expansion medium is IMDM medium containing 10% (V/V) platelet lysate with platelet concentration of 1 × 10 and 10ng/mL VEGF9One per ml.
5. The method of claim 4, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: heparin sodium is also added into the amplification culture medium.
6. The method of claim 5, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: 2 units of heparin sodium was also added to the amplification medium.
7. The method of claim 1, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: the process of treating human umbilical vein with alcohol is as follows: clamping two ends of human umbilical vein with hemostatic forceps, soaking human umbilical vein in 75% (V/V) alcohol for 3s, and washing with normal saline.
8. The method of claim 1, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: removing residual tissues of human umbilical vein by using 100 μm cell sieve; the centrifugation condition is 1500rpm centrifugation for 10 min.
9. The method of claim 1, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: in the process of in vitro expansion of human vein endothelial cells, the expansion medium is used for resuspending the human vein endothelial cells at 37 DEG C、5％CO2Culturing under saturated humidity, inducing wall adhesion for 24 hr, changing liquid completely, removing non-wall-adhered cells, adding fresh amplification culture medium, and changing liquid completely every other day.
10. The method of claim 9, wherein the step of obtaining human venous endothelial cells from a human umbilical vein comprises: in the process of in vitro amplification of the human vein endothelial cells, when the cell fusion degree reaches 90%, the cells are digested and passaged by 0.05% (M/V) pancreatin to obtain the human vein endothelial cells.
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| CN105969720A (en) * | 2016-07-29 | 2016-09-28 | 上海瑞鹿生物技术有限公司 | Human vascular endothelial cell culture solution and culture method |
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| CN104673747A (en) * | 2015-02-27 | 2015-06-03 | 中国人民解放军第三军医大学第一附属医院 | Method for preparing platelet lysate and application of platelet lysate |
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