CN114292806A - Isolation culture method of primary spinal cord microvascular endothelial cells and spinal cord microvascular endothelial cells obtained by same - Google Patents
Isolation culture method of primary spinal cord microvascular endothelial cells and spinal cord microvascular endothelial cells obtained by same Download PDFInfo
- Publication number
- CN114292806A CN114292806A CN202210231390.8A CN202210231390A CN114292806A CN 114292806 A CN114292806 A CN 114292806A CN 202210231390 A CN202210231390 A CN 202210231390A CN 114292806 A CN114292806 A CN 114292806A
- Authority
- CN
- China
- Prior art keywords
- spinal cord
- microvascular endothelial
- endothelial cells
- culture
- cord microvascular
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a primary spinal cord microvascular endothelial cell isolation and culture method and a spinal cord microvascular endothelial cell obtained by the method, wherein the method comprises the following steps: (1) mixing spinal cord tissue with balanced salt solution, grinding, centrifuging, and collecting precipitate; (2) mixing the precipitate obtained in the step (1) with an enrichment reagent, centrifuging, and discarding the supernatant to obtain enriched spinal cord microvascular fragments; (3) mixing the obtained spinal cord microvascular fragments with enzyme, and digesting to obtain spinal cord microvascular endothelial cells; (4) mixing spinal cord microvascular endothelial cells with a proliferation culture medium, inoculating the mixture on a cell culture plate coated with protein and/or polypeptide, carrying out primary culture, and then carrying out secondary culture by using a selective culture medium. The method has the advantages of high stability, good repeatability, simplicity, rapidness, low cost, high purity and high yield of the obtained spinal cord microvascular endothelial cells, and has important application value.
Description
Technical Field
The invention belongs to the technical field of cell separation culture, and relates to a separation culture method of primary spinal cord microvascular endothelial cells and the spinal cord microvascular endothelial cells obtained by the same.
Background
Spinal Cord Microvascular Endothelial Cells (SCMECs) are important Cells involved in the formation of the blood-Spinal barrier, which plays an important role not only in the maintenance of Spinal Cord homeostasis, but also in the development of central nervous system diseases. The spinal cord microvascular endothelial cells are one of the very important cells in the spinal cord microenvironment, the key role of the spinal cord microvascular endothelial cells in the spinal cord physiological and pathological microenvironment is deeply explored from the cellular level, and the method has important significance for clarifying pathogenesis of spinal cord related diseases and developing corresponding treatment strategies. Unfortunately, however, no immortalized cell line is currently available for sale in association with spinal cord microvascular endothelial cells. Therefore, it is very important to construct a primary spinal cord microvascular endothelial cell isolation culture system. Further research on spinal cord microvascular endothelial cells will find out the unique characteristics of spinal cord microvascular endothelial cells compared with other microvascular endothelial cells, and the key role of the cells in the physiological and pathological microenvironment of the spinal cord.
At present, various methods for separating and culturing the endothelium of spinal cord microvasculature have been disclosed in the prior art, but the methods are relatively complex and the separation and culture effects are not good, for example, CN109234233A discloses a blood spinal cord barrier OGD/R injury model, a construction method thereof and application CN109142752A discloses application of a tight junction protein and an inflammation-causing factor in diagnosis of blood spinal cord barrier injury. In both of the above two patents, the method of mechanical disruption combined with twice accutase enzyme digestion is adopted to extract the spinal cord microvascular endothelial cells from 24h newborn rats, which takes long time and cannot effectively separate the spinal cord microvascular endothelial cells from adult rat spinal cord tissues. In the published journal literature, spinal cord microvascular endothelial cells are mostly separated by a two-step enzymatic hydrolysis method or a combination of the enzymatic hydrolysis method and a mechanical method, for example, Watson and the like enriches spinal cord microvascular fragments by an enzymatic hydrolysis combined centrifugation method, and then spinal cord microvascular fragments are further enzymatically hydrolyzed to separate spinal cord microvascular endothelial cells (Watson PM, Paterson JC, Thom G, Ginman U, Lundquist S, Webster CI. modeling the endovasculal block-CNS barriers: a method for the production of the protocol of the route in the visual modules of the rate-broad barrier and the bulk-biological barrier. BMC Neurosci 2013, 14: 59.), the whole process is time-consuming, the yield of the obtained cells is low, and the obtained cells are easily contaminated by other hetero-cells in the microvascular fragments; shujun Ge et al use syringe combine Dounce tissue homogenizer to dissociate spinal cord microvascular segment by mechanical method, spinal cord microvascular segment obtained is enriched by means of dextran et al and further enzymolysis to obtain spinal cord microvascular endothelial cell (Ge S, Patcher JS. Isolation and culture of microvascular endothelial cell from muscle mineral code. J neuro tissue 2006, 177(1-2): 209 cake 214.), the whole process is time consuming and needs special and expensive Dounce tissue homogenizer, and the required reagents are numerous and easily contaminated by miscellaneous cells.
Based on the above analysis, the problems of the currently reported patents or journals about the isolated culture of spinal cord microvascular endothelial cells are mainly: complicated operation, need of special equipment or more types of required reagents, low cell yield, easy pollution of mixed cells and the like in the separation and culture process.
Therefore, how to provide a method for separating and culturing the spinal cord microvascular endothelial cells, which has the advantages of simple operation, low cost, high cell yield and high purity, becomes a problem to be solved in the field.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for separating and culturing primary spinal cord microvascular endothelial cells and the spinal cord microvascular endothelial cells obtained by the method.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for isolated culture of primary spinal cord microvascular endothelial cells, the method comprising the steps of:
(1) mixing spinal cord tissue with balanced salt solution, grinding, centrifuging, and collecting precipitate;
(2) mixing the precipitate obtained in the step (1) with an enrichment reagent, centrifuging, and discarding the supernatant to obtain enriched spinal cord microvascular fragments;
(3) mixing the obtained spinal cord microvascular fragments with enzyme, and digesting to obtain spinal cord microvascular endothelial cells;
(4) mixing spinal cord microvascular endothelial cells with a proliferation culture medium, inoculating the mixture on a cell culture plate coated with protein and/or polypeptide, carrying out primary culture, and then carrying out secondary culture by using a selective culture medium to obtain primary spinal cord microvascular endothelial cells;
the polypeptide comprises polylysine and the protein comprises any one of laminin, gelatin, collagen or fibronectin, or a combination of at least two of these.
Combinations of the at least two, such as a combination of laminin and gelatin, a combination of gelatin and collagen, a combination of laminin and collagen, and the like, and any other combination may be used.
The invention creatively provides a primary spinal cord microvascular endothelial cell separation culture method, which has the advantages of high stability, good repeatability, simplicity, convenience and rapidness, simple operation flow, short time consumption, strong operability, low cost, high purity and high yield of spinal cord microvascular endothelial cells obtained by separation culture and has important application value.
Preferably, the grinding of step (1) is performed using a tissue grinder, the tissue grinder comprising a glass tube and a plunger, the glass tube having a gauge greater than that of the plunger.
Preferably, the difference between the inner diameter of the glass tube and the outer diameter of the plunger is less than 1 mm, such as 0.001 mm, 0.005 mm, 0.01 mm, 0.03 mm, 0.05 mm, 0.08 mm, 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1 mm, and the like.
Preferably, the centrifugation speed in step (1) is 2000 g, such as 1000 g, 1100 g, 1200 g, 1300 g, 1400 g, 1500g, 1600 g, 1700 g, 1800 g, 1900 g, 2000 g, etc., and the centrifugation time is 3-10 min, such as 3 min, 4 min, 5min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.
Preferably, the enrichment reagent in step (2) comprises any one or a combination of at least two of bovine serum albumin, Dextran (Dextran), colloidal silica cell separation liquid (Percoll) or Ficoll cell separation liquid (Ficoll), for example, a combination of bovine serum albumin and Dextran, a combination of bovine serum albumin and Percoll, a combination of bovine serum albumin and Ficoll, and the like, and any other combination may be adopted.
Preferably, the enrichment reagent comprises bovine serum albumin at a concentration of 15-25% by mass, such as 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, etc.
Preferably, the speed of centrifugation in step (2) is 2000-4000 g, such as 2000 g, 2100 g, 2200 g, 2300 g, 2400 g, 2500 g, 2600 g, 2700 g, 2800 g, 2900 g, 3000g, 3100 g, 3200 g, 3300 g, 3400 g, 3500 g, 3600 g, 3700 g, 3800 g, 3900 g, 4000 g, etc., and the time of centrifugation is 10-30 min, such as 10 min, 12 min, 15min, 18 min, 20 min, 22 min, 25 min, 28 min, 30 min, etc.
Preferably, the step (2) of discarding the supernatant further comprises mixing with a balanced salt solution, washing, centrifuging, and discarding the supernatant to obtain the washed spinal cord microvascular fragments. The centrifugation speed is 1000-2000 g, such as 1000 g, 1100 g, 1200 g, 1300 g, 1400 g, 1500g, 1600 g, 1700 g, 1800 g, 1900 g, 2000 g, etc., and the centrifugation time is 3-10 min, such as 3 min, 4 min, 5min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.
Preferably, the enzyme in step (3) includes any one or a combination of at least two of dnase, collagenase (colagenase IV, colagenase II, etc.) or neutral protease (Dispase), for example, dnase and collagenase IV, dnase and collagenase II, neutral protease and collagenase, dnase + neutral protease + collagenase, etc., and any other combination may be adopted.
Preferably, the temperature of the digestion in step (3) is 33-40 ℃, such as 33 ℃, 34 ℃, 35 ℃, 36 ℃, 37 ℃, 38 ℃, 39 ℃, 40 ℃ and the like, and the time of the digestion is 30-60 min, such as 30 min, 32 min, 35 min, 38 min, 40 min, 42 min, 45 min, 48 min, 50 min, 52 min, 55 min, 58 min, 60 min and the like.
Preferably, the method for coating the protein and/or polypeptide in step (4) comprises: inoculating protein and/or polypeptide to cell culture plate, and incubating at 33-40 deg.C for 8-24 h.
The specific value of 33-40 deg.C is 33 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C, etc.
Specific numerical values among the above-mentioned 8 to 24h are, for example, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24h and the like.
Preferably, the protein of step (4) comprises collagen and/or fibronectin.
Preferably, the protein of step (4) comprises collagen and fibronectin.
Preferably, the method of protein coating comprises: inoculating collagen to cell culture plate, incubating at 33-40 deg.C for 8-16 h, washing, inoculating fibronectin to cell culture plate, and incubating at 33-40 deg.C for 2-6 h.
Preferably, the amount of the collagen is 100-400 μ g/well.
Preferably, the amount of fibronectin is 10-40 μ g/well.
The specific value of 33-40 deg.C is 33 deg.C, 34 deg.C, 35 deg.C, 36 deg.C, 37 deg.C, 38 deg.C, 39 deg.C, 40 deg.C, etc.
Specific values in the above 8 to 16 h are, for example, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h and the like.
Specific values in the above 2 to 6 h are, for example, 2 h, 2.5 h, 3 h, 3.5 h, 4h, 4.5 h, 5 h, 5.5 h, 6 h and the like.
The specific value of 100-400 μ g/well is, for example, 100 μ g/well, 150 μ g/well, 200 μ g/well, 250 μ g/well, 300 μ g/well, 350 μ g/well, 400 μ g/well, etc.
Specific values of the above-mentioned 10 to 40. mu.g/well are, for example, 10. mu.g/well, 15. mu.g/well, 20. mu.g/well, 25. mu.g/well, 30. mu.g/well, 35. mu.g/well, 40. mu.g/well, etc.
Preferably, the time of the one-time culture in the step (4) is 30-40 h, such as 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h, 40 h and the like.
Preferably, the proliferation medium and the selection medium in step (4) each independently comprise any one of fetal bovine serum, penicillin, streptomycin, or an endothelial cell growth supplement, or a combination of at least two thereof. The combination of at least two of them may be, for example, a combination of penicillin and streptomycin, a combination of fetal bovine serum and penicillin, a combination of fetal bovine serum and streptomycin, or the like, and any other combination may be used.
Preferably, the selection medium in step (4) further comprises puromycin 100-800 ng/mL, such as 100 ng/mL, 150 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 320 ng/mL, 350 ng/mL, 360 ng/mL, 370 ng/mL, 380 ng/mL, 390 ng/mL, 400 ng/mL, 410 ng/mL, 420 ng/mL, 430 ng/mL, 440 ng/mL, 450 ng/mL, 480 ng/mL, 500 ng/mL, 550 ng/mL, 600 ng/mL, 650 ng/mL, 700 ng/mL, 750 ng/mL, 800 ng/mL, etc., preferably 350-450 ng/mL.
In a second aspect, the present invention provides a spinal cord microvascular endothelial cell, wherein the spinal cord microvascular endothelial cell is prepared by the isolation culture method of primary spinal cord microvascular endothelial cells according to the first aspect.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has the following beneficial effects:
the invention creatively provides a primary spinal cord microvascular endothelial cell separation culture method, which has the advantages of high stability, good repeatability, simplicity, rapidness, simple operation process, short time consumption (about 1 h), no need of expensive or uncommon equipment such as a Doynes tissue homogenizer and the like, strong operability, few types of required reagents, low cost, high purity and high yield of spinal cord microvascular endothelial cells obtained by separation culture, and important application value.
Specifically, (1) the present invention is based on a tissue grinder for mechanically breaking cells, which is very common in the market or in the laboratory, creatively combines a glass tube of a tissue grinder with a larger size and a plunger of a tissue grinder with a smaller size, successfully obtains the dissociated microvascular fragments, and creatively finds that when the difference between the inner diameter of the glass tube and the outer diameter of the plunger is less than 1 mm, the number of the enriched microvascular fragments and the obtained cells is the largest. (2) Based on the creative design of the tissue grinder, the enrichment of the spinal cord microvascular fragments is realized only by one-time simple mechanical grinding, compared with the scheme of carrying out one-time mechanical treatment (such as mechanical pasteur mechanical blowing, shear shearing, Dorema tissue homogenizer and the like) and one-time enzymolysis in the prior art, the method disclosed by the invention is obviously simpler and more efficient, the cost of purchasing enzyme and expensive machines is saved, the steps of enzyme digestion and the like are omitted, a large amount of time is saved, and the advantages are obvious. (3) The invention creatively uses the combination of the rat tail collagen I type and fibronectin to coat the cell culture plate on the basis of a commercial cell culture plate, and the two proteins are mutually matched, thereby remarkably promoting cell adhesion, improving the cell adhesion amount after enzymolysis and being beneficial to cell proliferation. (4) The invention adopts endothelial cell proliferation culture medium (ECPM) to culture in the first 30-40 h after cell inoculation to promote the proliferation of the spinal cord microvascular endothelial cells as much as possible, and the liquid is changed to select culture medium (ECSM) for the endothelial cells after 30-40 h to remove the mixed cells such as pericytes, thereby obviously improving the yield and purity of the spinal cord microvascular endothelial cells. (5) The concentration of puromycin in ECSM is optimized, and when the concentration is 350-450 ng/mL, the obtained spinal cord microvascular endothelial cells have high purity (few hybrid cells) and high yield.
In conclusion, the invention successfully realizes the simple and high-efficiency extraction of the spinal cord microvascular endothelial cells from the spinal cord tissue through the design of the integral scheme, and the obtained spinal cord microvascular endothelial cells have high purity and high yield and have important application value.
Drawings
FIG. 1 is a photograph of a segment of spinal cord microvasculature taken by phase contrast microscopy at 200 microns;
FIG. 2 is a photograph taken by phase contrast microscopy of spinal cord microvascular endothelial cells at day 5 of culture, on a 100 micron scale;
FIG. 3 is a graph comparing the vascularization phenotype of spinal cord microvascular endothelial cells versus brain microvascular endothelial cells;
FIG. 4 is a graph of growth on day 3 of spinal cord microvascular endothelial cell culture obtained under different cell culture plate treatment conditions;
FIG. 5 is a graph showing the effect of puromycin concentration on purity and yield of spinal cord microvascular endothelial cells.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention. The following examples and comparative examples relate to methods for obtaining spinal cord tissue: removing spinal cord membrane and large blood vessel from 6-8 weeks old SD rat, and soaking in Ca-free solution at 4 deg.C2+，Mg2+HBSS solution (HBSS); cutting the spinal cord tissue into several sections with ophthalmic scissors. Type I rat tail collagen was obtained from Corning, fibronectin from Sigma, bovine serum albumin from Solambio, Collagenase IV from Gibco, and DNase from Sigma.
Example 1
The embodiment provides a primary spinal cord microvascular endothelial cell isolation and culture method, which comprises the following steps:
(1) transferring spinal cord tissue to tissue grinder (glass tube with inner diameter of 12.4 mm and plunger with outer diameter of 12.0 mm) and adding appropriate amount (such as 3-5 mL) of HBSS, and grinding until there is no obvious large tissue to obtain tissue homogenate. The homogenate was then transferred to a 50 mL centrifuge tube and resuspended by adding the appropriate amount of HBSS to give a resuspension (total volume approximately 20 mL).
(2) Centrifuging the heavy suspension at 1500g at 4 ℃ for 5min, discarding the supernatant, adding 15 mL of 20% BSA (bovine serum albumin) solution, shaking vigorously to mix uniformly, centrifuging at 3000g at 4 ℃ for 15min, and discarding the supernatant to obtain enriched spinal cord microvascular fragments (at this time, the spinal cord microvascular fragments are at the bottom of the centrifuge tube).
(3) Adding an appropriate amount (such as 15 mL) of HBSS (hepatitis B Virus) into the enriched spinal cord microvascular fragments for resuspension, then centrifuging at 4 ℃ for 5min at 1500g, cleaning the spinal cord microvascular fragments, removing BSA (bovine serum albumin) components in the microvascular fragments, and discarding supernatant to obtain the cleaned spinal cord microvascular fragments.
(4) Adding 3 mL of spinal cord tissue digestive enzyme (collagenase IV 2 mg/mL + 50 mu g/mL of deoxyribonuclease) into the cleaned spinal cord microvascular fragment, digesting at 37 ℃ for 45 min, centrifuging at 37 ℃ for 5min at 300 g, and removing the supernatant to obtain the spinal cord microvascular endothelial cells.
(5) Adding a spinal cord microvascular endothelial cell proliferation culture medium (ECPM) into the spinal cord microvascular endothelial cells, uniformly suspending, inoculating the suspension on a cell culture plate treated by a protein combination (collagen 2+ fibronectin 2), changing a solution for a spinal cord microvascular endothelial cell selection culture medium (ECSM) at the 36 th hour, and carrying out further subculture when the cells grow to a 80-90% confluent state.
The formula of ECPM is as follows: DMEM basal medium +10% fetal bovine serum +1% penicillin-streptomycin +1% endothelial cell growth supplement (scienell).
The formula of the ECSM is as follows: DMEM basal medium +10% fetal bovine serum +1% penicillin-streptomycin +1% endothelial cell growth supplement (scienell) + 400 ng/mL puromycin (available from shanghai bi yunnan).
The steps of the protein combination coating treatment are as follows: dissolving 300 μ g/mL rat tail collagen type I (i.e. collagen 2) in 0.02M acetic acid aqueous solution, inoculating 1mL into 6-well plate, and incubating overnight at 37 deg.C; taking out collagen coating solution, and washing with PBS for 3 times; 1mL of 30. mu.g/mL fibronectin (i.e., fibronectin 2) was added to the corresponding 6-well plate and incubated at 37 ℃ for 4 h.
Example 2
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which is different from example 1 only in that the tissue grinder "a glass tube with an inner diameter of 12.4 mm and a plunger with an outer diameter of 12.0 mm" in step (1) is replaced with "a glass tube with an inner diameter of 13.0 mm and a plunger with an outer diameter of 12.0 mm", and other conditions refer to example 1.
Example 3
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which is different from example 1 only in that the tissue grinder "a glass tube with an inner diameter of 12.4 mm and a plunger with an outer diameter of 12.0 mm" in step (1) is replaced by "a glass tube with an inner diameter of 12 mm and a plunger with an outer diameter of 12 mm", and the other conditions refer to example 1.
In the method of this embodiment, the glass tube with the same specification is used with the plunger, which directly results in the disruption of tissue cells during the grinding process, and the viable cells or the dissociated microvascular fragments cannot be obtained, so the method of this embodiment is not feasible.
Example 4
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which differs from example 1 only in that the "protein combination" of step (5) is replaced with 50 μ g/mL of rat tail collagen type I (i.e. collagen 1), and other conditions refer to example 1.
Example 5
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which differs from example 1 only in that the "protein combination" of step (5) is replaced with 300 μ g/mL of rat tail collagen type I (i.e. collagen 2), and other conditions refer to example 1.
Example 6
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which differs from example 1 only in that the "protein combination" of step (5) is replaced with 5 μ g/mL fibronectin (i.e., fibronectin 1), and the other conditions refer to example 1.
Example 7
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which differs from example 1 only in that the "protein combination" of step (5) is replaced with 30 μ g/mL fibronectin (i.e., fibronectin 2), and the other conditions refer to example 1.
Example 8
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which differs from example 1 only in that "400 ng/mL puromycin" in the formulation of ECSM of step (5) is replaced with "800 ng/mL puromycin", and other conditions refer to example 1.
Example 9
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which differs from example 1 only in that "400 ng/mL puromycin" in the formulation of ECSM of step (5) is replaced with "200 ng/mL puromycin", and other conditions refer to example 1.
Example 10
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which is different from example 1 only in that "400 ng/mL puromycin" is replaced with "100 ng/mL puromycin" in the formulation of ECSM of step (5), and other conditions refer to example 1.
Example 11
This example provides a primary spinal cord microvascular endothelial cell isolation culture method, which is different from example 1 only in that the formulation of ECSM of step (5) does not contain puromycin, and other conditions refer to example 1.
Comparative example 1
This comparative example provides a primary spinal cord microvascular endothelial cell isolation culture method, which differs from example 1 only in that the "protein combination-coated cell culture plate" of step (5) is replaced with an "untreated cell culture plate", and other conditions refer to example 1.
Test example 1
Characterization of purity
Observing the spinal cord microvascular fragments obtained in the step (3) of the example 1 and the spinal cord microvascular endothelial cells obtained by culturing the primary spinal cord microvascular endothelial cells obtained in the step (5) for 5 days by using a phase contrast microscope to perform purity characterization.
The specific experimental method comprises the following steps: cells were fixed with 4% PFA; washing with PBS for three times, 5min each time; treating with 0.5% triton ice for 15 min; washing with PBS for 5min three times; blocking with 5% BSA for 1 h; adding primary antibody, and standing overnight at 4 deg.C; washing with PBS for three times, 5min each time; adding a secondary antibody and DAPI, and incubating for 1h at room temperature; and (6) taking a picture.
The pictures are shown in fig. 1 and fig. 2. FIG. 1 is a photograph of a segment of spinal cord microvasculature taken by phase contrast microscopy at 200 microns. FIG. 2 is a photograph taken by phase contrast microscopy of spinal cord microvascular endothelial cells at day 5 of culture, on a 100 micron scale.
As shown in the figure, the spinal cord microvascular segments are clearly visible, the number of the spinal cord microvascular segments is large, impurities are few, the morphology of the spinal cord microvascular endothelial cells is good, and the spinal cord microvascular endothelial cells are almost free of impurity cells, so that the separation culture method provided by the invention can well remove other tissue impurities and impurity cells, and the purity of the extracted spinal cord microvascular segments and the spinal cord microvascular endothelial cells is high.
Test example 2
Specificity test
Respectively obtaining rat brain tissues and spinal cord tissues, respectively obtaining brain-derived microvascular endothelial cells and spinal cord-derived microvascular endothelial cells by adopting the separation culture method of example 1, culturing until 80-90% of cells are fused, respectively inoculating the cells on the same substrate, culturing for 12 h by using ECPM, and photographing by using a phase contrast microscope.
The results are shown in fig. 3 (scale is 100 microns), and as shown in the figure, under the same culture conditions, the spinal cord microvascular endothelial cells show better vascularization phenotype compared with the brain microvascular endothelial cells of the central nervous system, which indicates that the spinal cord microvascular endothelial cells have specificity compared with other endothelial cells, and the cells are worthy of being separated and cultured and further studied.
Test example 3
The influence of the combination of the glass tube and the plunger in the tissue grinder used in the step (1) on the obtained microvascular fragments and the number of cells was investigated
The microvascular segments obtained in examples 1-3 and the number of spinal cord microvascular endothelial cells were characterized by the following specific method: inoculating 1mL of suspension containing spinal cord microvascular fragments into a 6-well plate, and observing the number of suspended blood vessels under a phase contrast microscope; culturing spinal cord microvascular endothelial cells, observing the culture time required when 80% -90% of cells in each 6-well plate are fused under a phase contrast microscope, and recording. The results are shown in Table 1.
TABLE 1
The results show that: the use of a glass tube of the same specification in combination with a plunger directly results in the disruption of tissue cells during the grinding process, which is not feasible because living cells or dissociated microvascular fragments cannot be obtained. When the glass tubes with different specifications are combined with the plunger, when the difference value between the inner diameter of the glass tube and the outer diameter of the plunger is smaller than 1 mm, microvascular fragments and spinal cord microvascular endothelial cells can be obtained, and when the difference value between the inner diameter of the glass tube and the outer diameter of the plunger is larger than 1 mm, insufficient grinding or even incapability of grinding tissues can be caused, and the number of the obtained microvascular fragments and the spinal cord microvascular endothelial cells is small.
Test example 4
Investigating the influence of the treatment mode of the cell culture plate in the step (5) on the cell adhesion
The growth of the spinal cord microvascular endothelial cells obtained on day 3 of the culture of the primary spinal cord microvascular endothelial cells obtained in example 1, examples 4 to 7 and comparative example 1 (control) was observed by a phase contrast microscope and photographed. The specific experimental method is described in test example 1.
The results are shown in fig. 4, and the spinal cord microvascular endothelial cells obtained in each example are high in number and purity. Compared with a control group, the protein-treated cell culture plate can remarkably promote the adhesion of the spinal cord microvascular endothelial cells, wherein the effect of promoting the adhesion of the spinal cord microvascular endothelial cells by adopting a collagen 2+ fibronectin 2 treatment mode is more remarkable.
Test example 5
Investigating the effect of puromycin concentration on cell purity and yield in the ECSM formulation of step (5)
The purity and yield of spinal cord microvascular endothelial cells obtained on day 5 of primary spinal cord microvascular endothelial cell culture obtained in example 1 and examples 8-11 were characterized using the spinal cord microvascular endothelial cell marker CD31, the astrocyte marker GFAP and the pericyte marker Desmin.
The specific experimental method comprises the following steps: 4% PFA fixed cells; washing with PBS for three times, 5min each time; treating with 0.5% triton ice for 15 min; washing with PBS for three times, 5min each time; blocking with 5% BSA for 1 h; adding primary antibody (CD 31, GFAP and Desmin from different species), and standing at 4 deg.C overnight; washing with PBS for three times, 5min each time; add secondary antibody and DAPI and incubate for 1h at room temperature and take pictures.
The results are shown in figure 5 (scale 100 microns) and as shown, spinal cord microvascular endothelial cells obtained are of high purity (fewer heterocytes) and high yield when the concentration of puromycin in the ECSM formulation is 400 ng/mL compared to other concentration options.
The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention is implemented only by relying on the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
Claims (10)
1. A primary spinal cord microvascular endothelial cell isolation and culture method is characterized by comprising the following steps:
(1) mixing spinal cord tissue with balanced salt solution, grinding, centrifuging, and collecting precipitate;
(2) mixing the precipitate obtained in the step (1) with an enrichment reagent, centrifuging, and discarding the supernatant to obtain enriched spinal cord microvascular fragments;
(3) mixing the obtained spinal cord microvascular fragments with enzyme, and digesting to obtain spinal cord microvascular endothelial cells;
(4) mixing spinal cord microvascular endothelial cells with a proliferation culture medium, inoculating the mixture on a cell culture plate coated with protein and/or polypeptide, carrying out primary culture, and then carrying out secondary culture by using a selective culture medium to obtain primary spinal cord microvascular endothelial cells;
the polypeptide comprises polylysine and the protein comprises any one of laminin, gelatin, collagen or fibronectin, or a combination of at least two of these.
2. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein said grinding in step (1) is performed using a tissue grinder, said tissue grinder comprising a glass tube and a plunger, said glass tube having a gauge larger than that of the plunger, and said plunger having an inner diameter that differs from an outer diameter of the plunger by less than 1 mm.
3. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein the enrichment reagent of step (2) comprises any one of bovine serum albumin, dextran, colloidal silica cell isolate or ficoll cell isolate or a combination of at least two thereof.
4. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein said discarding of supernatant in step (2) further comprises mixing with balanced salt solution, washing, centrifuging, discarding supernatant to obtain washed spinal cord microvascular fragments.
5. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein said enzyme of step (3) comprises any one or a combination of at least two of deoxyribonuclease, collagenase or neutral protease.
6. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein said protein of step (4) comprises collagen and/or fibronectin.
7. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein the time of the primary culture in step (4) is 30-40 h.
8. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein said proliferation medium and selection medium of step (4) each independently comprises any one of fetal bovine serum, penicillin, streptomycin, or endothelial cell growth supplements or a combination of at least two thereof.
9. The method for isolated culture of primary spinal cord microvascular endothelial cells according to claim 1, wherein said selection medium of step (4) further comprises puromycin 100-800 ng/mL.
10. A spinal cord microvascular endothelial cell prepared by the isolated culture method of primary spinal cord microvascular endothelial cells according to any one of claims 1-9.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210231390.8A CN114292806A (en) | 2022-03-10 | 2022-03-10 | Isolation culture method of primary spinal cord microvascular endothelial cells and spinal cord microvascular endothelial cells obtained by same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210231390.8A CN114292806A (en) | 2022-03-10 | 2022-03-10 | Isolation culture method of primary spinal cord microvascular endothelial cells and spinal cord microvascular endothelial cells obtained by same |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN114292806A true CN114292806A (en) | 2022-04-08 |
Family
ID=80978522
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210231390.8A Pending CN114292806A (en) | 2022-03-10 | 2022-03-10 | Isolation culture method of primary spinal cord microvascular endothelial cells and spinal cord microvascular endothelial cells obtained by same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114292806A (en) |
-
2022
- 2022-03-10 CN CN202210231390.8A patent/CN114292806A/en active Pending
Non-Patent Citations (7)
| Title |
|---|
| P MARC D WATSON等: "Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier", 《BMC NEUROSCIENCE》 * |
| SHUJUN GE等: "Isolation and culture of microvascular endothelial cells from murine spinal cord", 《JOURNAL OF NEUROIMMUNOLOGY》 * |
| 李琼等: "人视网膜血管内皮细胞的分离与培养", 《海峡科学》 * |
| 苏震等: "人肾小球微血管内皮细胞体外培养方法和鉴定的实验研究", 《浙江医学》 * |
| 苑晓晨等: "大鼠脊髓微血管内皮细胞的分离培养与鉴定", 《基础医学与临床》 * |
| 苑晓晨等: "小鼠脊髓微血管周细胞的体外培养及鉴定", 《基础医学与临床》 * |
| 荆瀛黎: "C57BL/6小鼠脊髓损伤后的微循环功能调控", 《中国博士学位论文全文数据库》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101818127B (en) | Method for separating and culturing mouse primitive spermatogonia | |
| TW200521234A (en) | Method of single cell culture of undifferentiated human embryonic stem cells | |
| CN107142242B (en) | Isolated culture method of naked mole rat skeletal muscle myoblasts | |
| CN114591905B (en) | Method for preparing apoptotic vesicles from human erythrocytes and application of apoptotic vesicles | |
| CN107557329A (en) | A kind of Brain Microvascular Endothelial separation and cultural method | |
| WO2023016029A1 (en) | Method for separating fibroblasts derived from human induced pluripotent stem cells, and use thereof | |
| CN113583952B (en) | Culture solution for increasing yield of exosomes of stem cells | |
| CN114874979A (en) | Separation and purification method of donkey skeletal muscle satellite cells | |
| CN116836934B (en) | Osteosarcoma organoid culture solution, culture reagent combination and culture method | |
| CN112175909A (en) | VSX2 green fluorescent reporter gene human induced pluripotent stem cell line and construction method thereof | |
| CN114292806A (en) | Isolation culture method of primary spinal cord microvascular endothelial cells and spinal cord microvascular endothelial cells obtained by same | |
| CN109609453B (en) | Tree shrew microglial cell culture medium and in-vitro isolated culture and purification method thereof | |
| JP5564679B2 (en) | Cardiomyocyte differentiation induction promoter and method of use thereof | |
| CN114958746B (en) | Method and kit for inducing pluripotent stem cells to generate 3D brain-like bodies | |
| CN111004776A (en) | Method for separating and culturing equine skeletal muscle satellite cells | |
| CN115369073A (en) | Preparation method of epidermal stem cell exosome | |
| CN113684174B (en) | Preparation method of human kidney foot cells | |
| CN114276986A (en) | Method for separating and purifying buffalo primary myoblasts and application thereof | |
| CN113151153A (en) | Purification method and identification method of mouse peritoneal perivascular cells | |
| CN112501115A (en) | Extraction, separation and purification method of rabbit muscle stem cells | |
| CN107475198B (en) | Tree shrew dopaminergic neuron cell isolation and culture method | |
| WO1997001628A1 (en) | Liquid medium for nerve cells, process for producing the same and method for incubating nerve cells with the use of the same | |
| CN113652394B (en) | Method for in vitro separation of primary sperm cells of rats | |
| CN112175995B (en) | VSX2 green fluorescence reporter gene vector system and construction method thereof | |
| CN115181722B (en) | In-vitro separation and culture method of goose skeletal muscle satellite cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20220408 |