CN115354016A - Method for constructing in-vitro blood brain barrier model - Google Patents
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Abstract
The invention provides a method for constructing an in vitro blood brain barrier model, which comprises the following steps: 1) Selecting a cell line; 2) Coating; 3) And (4) inoculating the cells. The invention belongs to the technical field of tissue engineering, comprehensively screens a cell line for constructing an in vitro blood brain barrier model, adopts a humanized immortalized brain tissue cell line, namely a brain microvascular endothelial cell hCMEC/D3 cell, a pericyte HBVP cell and an astrocyte SVG p12 cell, utilizes a Transwell cell culture device to construct an in vitro three-dimensional blood brain barrier model, and preferably selects an optimal arrangement mode of the three cells, which is closer to the real blood brain barrier environment of human beings; in addition, the construction process of the model is simple, complex equipment is not needed, the time required by the model to mature is short, the long maturation time can be stably maintained, and the method is convenient to popularize and apply in a large range.
Description
Technical Field
The invention belongs to the technical field of tissue engineering, and particularly relates to a method for constructing an in-vitro blood brain barrier model.
Background
The nervous system is one of the most complex and functionally important life systems of the human body. Scientific research and exploration carried out around the nervous system, such as neurotoxicity risk assessment of chemicals, drug research and development of a targeted nervous system and the like, constantly face various challenges such as ethical problems, difficulty in obtaining materials of relevant tissues, difference of intergeneric reactions and the like. The blood-brain barrier (BBB) is a dynamic interface between the blood circulation system and the central nervous system of the brain, maintaining homeostasis in the brain and achieving normal nervous system function by strictly regulating the exchange of substances between the blood and the brain. A number of brain diseases are associated with a breakdown in the defense function of the blood brain barrier. Researchers hope urgently to construct an in vitro cell model capable of simulating the blood-brain barrier (BBB) in vivo to develop related scientific exploration, and the method has important significance for further researching the functional principle of the blood-brain barrier and treating brain injury caused by barrier function failure. Brain microvascular endothelial cells, pericytes and astrocytes are the main cellular components constituting the BBB. Wherein, the tight connection and adhesion connexin expressed by the brain microvascular endothelial cells enables the cells to have low paracellular and intercellular permeability, and a dense and closed barrier is formed; pericytes and astrocytes maintain barrier integrity.
At present, different in vitro BBB models, including a brain microvascular endothelial cell monolayer culture model, a multi-cell model in which brain microvascular endothelial cells are co-cultured with pericytes or/and astrocytes, a 3D organoid model, and the like, have advantages and disadvantages, and are specifically shown in table 1.
TABLE 1 Excellent and Defect of different in vitro BBB models
The parameters used by existing in vitro BBB models vary, and endothelial cells that construct barrier properties also come from a variety of sources, including human, murine, porcine and bovine, and primary cell cultures. However, the use of cells of animal origin has the disadvantage of a high paracellular permeability and a differential reaction between species which is not negligible. The high paracellular permeability makes the model far from the actual physiological properties of the blood brain barrier; inter-species reaction differences make extrapolation of experimental results to human reactions uncertain. Due to inter-species response variability, more than 90% of neuro-candidate drugs with promise in animal experiments failed in human clinical trials. On the other hand, the separation of primary endothelial cells is time-consuming and labor-consuming, and the experimental result is often influenced by the cell separation purity and has low repeatability. Therefore, in order to be closer to the real blood brain barrier environment of human, the cell lines selected in the construction of in vitro blood brain barrier model need to be considered comprehensively. In addition, recently developed methods for preparing blood brain barrier endothelial cells by utilizing pluripotent induction stem cells or embryonic stem cells can also establish a good blood brain barrier environment, but the requirements of cell culture, induced differentiation and other related technologies are high, the price of reagent consumables is high, the method is not beneficial to large-scale popularization and use, and meanwhile, the reproducibility of the method is yet to be verified.
Therefore, the development of an in vitro blood brain barrier model which is rapid, reliable and easy to popularize in a large range is of great significance.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a method for constructing an in vitro blood brain barrier model, which comprehensively screens a cell line for constructing the in vitro blood brain barrier model, adopts a humanized immortalized brain tissue cell line, namely a brain microvascular endothelial cell hCMEC/D3 cell, a pericyte HBVP cell and an astrocyte SVG p12 cell, utilizes a Transwell cell culture device to construct an in vitro three-dimensional blood brain barrier model, and preferably selects an optimal arrangement mode of the three cells, so that the model is closer to the real blood brain barrier environment of a human; in addition, the construction method of the model is simple, complex equipment is not needed, the time required by the model to mature is short, the long maturation time can be stably maintained, and the method is convenient to popularize and apply in a large range.
The objects of the invention will be further illustrated by the following detailed description.
The invention provides a method for constructing an in vitro blood brain barrier model, which comprises the following steps:
1) Cell line selection: selecting hCMEC/D3 cells as brain microvascular endothelial cells, selecting HBVP cells as pericytes and selecting SVG p12 cells as astrocytes;
2) Coating: coating a Transwell membrane layer of the Transwell cell culture device by adopting a coating solution; the Transwell cell culture device comprises a Transwell chamber;
3) Cell inoculation: selecting corresponding cells in logarithmic growth phase, and inoculating the cells in sequence: (1) inoculating SVG p12 cells in a first pore plate for culturing; (2) reversely buckling the Transwell chamber in a second pore plate, inoculating HBVP cells in the lower membrane layer of the Transwell chamber for culture, and culturing the Transwell chamber in a third pore plate in a positive mode after adherent growth; (3) hCMEC/D3 cells were seeded on the upper membrane layer of the Transwell chamber for culture, the Transwell chamber seeded with hCMEC/D3 and HBVP cells was combined with the first well plate seeded with SVG p12 cells, and the HBVP cells indirectly contacted with hCMEC/D3 cells through the Transwell membrane layer, thereby constructing an in vitro blood brain barrier model.
By adopting the technical scheme, complex equipment is not needed, the invention inoculates three human-derived cells hCMEC/D3, HBVP and SVGP12 respectively according to the sequence of the upper layer, the middle layer and the lower layer, the upper layer cavity simulates the intravascular environment, the lower layer cavity simulates the brain tissue internal environment, the integrity and functionality verification is carried out, and the model has better tight barrier property and transport and discharge functions and is closer to the real blood brain barrier environment of human beings; the constructed in-vitro blood brain barrier model begins to mature after being cultured in a cell culture box for 48 hours (namely the second day after construction), the TEER value, the compactness and the like begin to be rapidly increased until the third day to reach the maximum value, the in-vitro blood brain barrier model is completely developed, the time required by the model maturation is short, and the longer maturation time can be stably maintained (can be continued until the sixth day).
Preferably, the coating solution is a rat tail collagen I type solution, the concentration of the coating solution is 10-20 mug/mL, and the coating solution is prepared by adopting a precooled acetic acid solution.
More preferably, the concentration of the rat tail collagen type I solution is 12-18 mug/mL, and the concentration of the acetic acid solution is 0.004-0.008M.
More preferably, the hCMEC/D3 is fineThe inoculation density of the cells was 1.8X 10 5 -2.2×10 5 Cells/cm 2 。
More preferably, the SVG p12 cells are seeded at a density of 1.2X 10 4 -1.8×10 4 Cells/cm 2 。
More preferably, the seeding density of the HBVP cells is 0.8X 10 6 -1.2×10 6 Cells/cm 2 。
More preferably, the method for constructing the in vitro blood brain barrier model provided by the invention further comprises the following step 4): and (5) detecting the barrier property and the transport function of the constructed model.
More preferably, the first orifice plate is a 12-orifice plate, the second orifice plate is a 6-orifice plate, and the third orifice plate is a 12-orifice plate.
More preferably, the Transwell cell culture apparatus is purchased from corning, usa and employs a 0.4 μm pore size polycarbonate filter.
In addition, the invention also provides an in vitro blood brain barrier model constructed by the construction method of the in vitro blood brain barrier model.
Compared with the prior art, the invention has the beneficial effects that:
(1) The invention comprehensively screens a cell line for constructing an in vitro blood brain barrier model, and adopts a humanized immortalized brain tissue cell line, namely a brain microvascular endothelial cell hCMEC/D3 cell, a pericyte HBVP cell and an astrocyte SVG p12 cell to construct the model, thereby avoiding the defects of high paracellular permeability and species reaction difference of animal-derived cells and avoiding the instability caused by using primary cells.
(2) The invention utilizes a Transwell cell culture device to construct an in-vitro three-dimensional blood brain barrier model, optimizes the optimal arrangement mode of the three cells, respectively inoculates three human-derived cells of hCMEC/D3, HBVP and SVG p12 according to the sequence of an upper layer, a middle layer and a lower layer, an upper layer cavity simulates the internal environment of a blood vessel, a lower layer cavity simulates the internal environment of brain tissues, and the integrity and functionality verification is carried out, so that the model has better tight barrier property and transport and discharge functions and is closer to the real blood brain barrier environment of human beings.
(3) The method optimizes the inoculation density of the brain microvascular endothelial cells hCMEC/D3 cells at the core part of the model, determines the optimal cell inoculation density, and ensures that the transmembrane resistance value (TEER value) of the model is higher and relatively stable.
(4) The model construction method provided by the invention is simple, does not need complex equipment, has short time for model maturation (the model begins to mature on the second day after construction and is completely developed on the third day), can stably maintain longer maturation time (can be continued to the sixth day), and is convenient for large-scale popularization and application.
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FIG. 1 is a schematic diagram of model construction according to a third embodiment of the present invention.
FIG. 2 results of BBB model transmembrane resistance measurements of hCMEC/D3 cell monolayer cultures at different seeding densities.
FIG. 3 shows the results of measurements of transmembrane resistance values of three cells in different arrangement patterns; wherein, a refers to hCMEC/D3-HBVP-SVG p12, b refers to hCMEC/D3-SVG p12-HBVP, c refers to hCMEC/D3- (HBVP-SVG p 12) -up, D refers to hCMEC/D3- (HBVP-SVG p 12) -down,1 refers to hCMEC/D3-HBVP-SVG p12,2 refers to (HBVP-hCMEC/D3) -SVG p12, and 3 refers to (hCMEC/D3-HBVP) -SVG p12.
FIG. 4 shows the results of measurements of transmembrane resistance values of different cell arrangement patterns.
FIG. 5 maximum TEER values of the BBB model constructed in the present invention compared to the hCMEC/D3 monolayer culture model.
FIG. 6 shows the results of the detection of mRNA and protein expression of the BBB models ZO-1 and Claudin-5 constructed according to the present invention; wherein A and B respectively refer to the mRNA expression detection results of ZO-1 and Claudin-5; c means the protein expression detection result of ZO-1 and Claudin-5.
FIG. 7 shows the results of the detection of permeability coefficient and recovery rate of sodium fluorescein and Huang Er lithium salt in the BBB model constructed by the present invention; wherein A refers to the detection result of 7-day permeability coefficient of fluorescein sodium; b refers to the detection result of 7-day recovery rate of fluorescein sodium; c refers to a detection result of 7-day permeability coefficient of the fluorescein dilithium salt; d refers to the detection result of the recovery rate of the fluorescein dilithium salt in 7 days.
FIG. 8 is a standard curve for rhodamine 123.
FIG. 9 shows the results of the transport efflux function validation of the BBB model constructed according to the present invention; wherein, A refers to the transport capacity of rhodamine 123 in an upper chamber and a lower chamber of a BBB model respectively; b refers to the transport efflux capacity of the BBB model after P-gp inhibitor cyclosporine A pretreatment.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples.
In the present invention, the cells, reagents and materials are all conventional commercially available products, or can be obtained by means of conventional techniques in the art. For example: hCMEC/D3 cells were purchased from Shanghai Yubo Biotech, inc., and HBVP and SVGP12 cells were purchased from Guangzhou Huatuo Biotech, inc. In the present invention, the percentages are understood in accordance with the customary practice in the art. For example: 5% fetal bovine serum means 5% volume percent fetal bovine serum.
In the present invention, the first and second liquid crystal display panels, * the expression P is less than 0.05, ** the expression P is less than 0.01, *** the expression P is less than 0.001, **** represents P < 0.0001.
Cell culture: brain microvascular endothelial cells hCMEC/D3 cells were cultured in ECM medium (ScienCell, USA) containing 5% fetal bovine serum, 1% endothelial growth factor and 1% diabody; pericytes HBVP and astrocyte SVGP12 were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum and 1% double antibody. All three cells were cultured at 37 ℃ and 5% CO 2 The constant temperature cell culture box.
Example selection of cell lines
For three cell components forming the blood brain barrier, human immortalized cell lines are mainly considered, and particularly the brain microvascular endothelial cells forming the most main blood brain barrier are considered. On the basis of the research of literature search, the physiological characteristics of each cell are relatively known, and the cell line which meets the requirements most is selected. As for the brain microvascular endothelial cells, there are 8 human immortalized brain microvascular endothelial cell lines, including BB19, HCEC, HBEC-5i, HBMEC-3, TY08, HBMEC/Ci β, NKIM-6 and hCMC/D3 cell lines, as shown in Table 2, for each immortalization mode and the order of merits and demerits.
TABLE 2 immortalized human brain microvascular endothelial cells
The hCMEC/D3 cell line is formed by immortalizing primary human brain microvascular endothelial cells by co-expression of hTERT and SV40 large T antigen via a lentiviral vector system, and the hCMEC/D3 cells express typical endothelial markers (such as CD31, VE-cadherin and von Willebrand factor), have stable normal karyotype, maintain a contact inhibitory monolayer in culture, and form capillaries in the matrix. In addition, the cell lines express chemokine receptors and upregulate cell adhesion molecules in response to inflammatory cytokines. Finally, it was experimentally found that hCMEC/D3 cells exhibit blood-brain barrier characteristics such as tight junction protein formation and the ability to restrict efflux of drugs. In combination with the above factors, the present invention selects hCMEC/D3 cells as model endothelial cells. Pericytes are a class of parietal cells attached to the basement membrane of vascular endothelial cells, and one of their functions is to regulate endothelial cell permeability. Since there are no immortalized pericytes at present, the present invention selects the second layer of cells using commercially available primary human cerebrovascular pericytes as a model. Astrocytes are the most abundant glial cells in the brain, and the final foot of the astrocytes covers up to 99% of the surface of blood vessels, and plays an important role in maintaining the specific function of brain microvascular endothelial cells and the normal function of neurons, promoting proteoglycan synthesis and maintaining the integrity of the blood brain barrier. The immortalized astrocyte cell line SVG p12 is derived from primary fetal brain glial cells transfected by SV40 virus, and still retains the characteristics of primary astrocytes after multiple passages (about 80 generations), so that the instability caused by using primary cells can be reduced. In conclusion, the invention selects the brain microvascular endothelial cells hCMEC/D3, the pericytes HBVP and the astrocytes SVG p12 to construct an in vitro BBB model.
Example construction of an extrabody blood brain barrier model
A method for constructing an in vitro blood brain barrier model comprises the following steps:
1) Cell line selection: selecting hCMEC/D3 cells as brain microvascular endothelial cells, selecting HBVP cells as pericytes and selecting SVG p12 cells as astrocytes;
2) Coating: coating a Transwell membrane layer of the Transwell cell culture device by adopting a coating solution, adding 200 mu L of the coating solution into the upper layer of the Transwell cell culture device, adding 800 mu L of the coating solution into the lower layer of the Transwell cell culture device, and placing the Transwell cell culture device in a refrigerator at 4 ℃ for overnight standby; the coating solution is a rat tail collagen I type solution, the concentration of the coating solution is 15 mug/mL, and the coating solution is prepared by precooled 0.006M acetic acid solution; the Transwell cell culture device comprises a Transwell chamber;
3) Cell inoculation: selecting corresponding cells in logarithmic growth phase, and sequentially inoculating SVG p12, HBVP and hCMEC/D3 cells: (1) SVG p12 cells were seeded in a first well plate (12 well plate) and cultured at 1.5X 10 4 Cells/cm 2 The inoculation density of (2) is that the resuspended SVG p12 cells are uniformly added with the culture medium amount of 1 mL/hole, and then the cells are put into a cell culture box for culture; (2) the coated Transwell chamber was inverted in a second well plate (6 well plate), and HBVP cells were seeded in the lower layer of the Transwell chamber and cultured at 1.0X 10 6 Cells/cm 2 The inoculation density of (2) is that the resuspended HBVP cells are uniformly added according to the culture medium amount of 100 mul/hole, a plate cover is covered, the HBVP cells are placed into a cell culture box for culture for 4 hours after adherent growth, a second hole plate is taken out, a Transwell chamber is washed by PBS solution, the Transwell chamber is placed in a third hole plate (12 hole plates) for culture, 600 mul of culture medium is added, and the cell culture box is placed for culture; (3) after 12h incubation with the Transwell chamber facing the third well plate (overnight), hCMEC/D3 cells were plated at 2X 10 5 Cells/cm 2 The density of (2) was inoculated on the membrane upper layer of the Transwell cell for culture, 500. Mu.L of the culture medium was added to the upper Transwell chamber, the Transwell cell inoculated with hCMEC/D3 and HBVP cells was combined with the first well plate inoculated with SVG p12 cells, 1.5mL of the culture medium was added to the lower chamber to keep the liquid level of the upper chamber level, and the HBVP cells indirectly contacted with hCMEC/D3 cells through the Transwell membrane layer to construct an in vitro blood brain barrier model.
Example construction of a three-in-vitro blood brain Barrier model
A method for constructing an in vitro blood brain barrier model, wherein a schematic diagram of model construction is shown in fig. 1, and the method comprises the steps 1) to 3) of the second embodiment, and further comprises the step 4): and (5) detecting the barrier property and the transport function of the constructed model.
EXAMPLE four inoculation Density Studies of hCMEC/D3 cells
The inventors experimentally found that hCMEC/D3 cells play a crucial role in maintaining the barrier properties of BBB, and that their cell seeding density influences the maturation time and compactness of the BBB model. The invention sets BBB model cultured by hCMEC/D3 cell monolayer with different inoculation density, then detects transmembrane resistance value (TEER value) of the model, and the result is shown in figure 2, and is 5 multiplied by 10 5 Cells/cm 2 Density seeded hCMEC/D3 cells with maximum TEER value of 46.92 + -1.97 Ω. Cm on day 2 2 But then the TEER value drops rapidly; to be at 2x 10 5 Cells/cm 2 The TEER value on day 2 of the density-seeded hCMEC/D3 cells of 41.96. + -. 1.98. Omega. Cm 2 The second largest TEER in all the seeded cells and the TEER value decreased more slowly, with the TEER value being greater than 5X 10 on day 3 5 Cells/cm 2 TEER values of hCMEC/D3 cells seeded at a density of 2X 10 5 Cells/cm 2 The density of the inoculated hCMEC/D3 cells can maintain the TEER of the cells relatively stable. TEER values were smaller for both remaining cell seeding densities. In summary, the present invention finally determined that hCMEC/D3 cell seeding density for constructing in vitro BBB model is 2 × 10 5 Cells/cm 2 。
Example examination of different arrangement patterns of five BBB model cells
The inventor finds out through experiments that different arrangement patterns (contact among cells) of in vitro BBB model cells can influence the barrier property of the model, and the optimal arrangement pattern is selected by setting different arrangement patterns of three cells, namely pericyte HBVP and astrocyte SVG p12 and pericyte HBVP and brain microvascular endothelial cell hCMEC/D3 cell, and then detecting the TEER value of the model, and the result is shown in figure 3. hCMEC/D3 cells seeded on Transwell platesThe best model was constructed by constructing an in vitro BBB model with the largest TEER value of 61.600 + -3.677 Ω. Cm in such a cell arrangement pattern that HBVP cells were seeded on the upper layer of the cell membrane in the lower layer of the Transwell chamber membrane and indirectly contacted with hCMEC/D3 cells through the membrane, and SVGP12 cells were seeded in a 12-well plate 2 Indicating that the tight barrier properties of the model are optimal. In addition, the characteristics begin to mature the next day after modeling, so that the maturation time of the model is effectively shortened, and the model can be continued to the sixth day, so that the corresponding functions can be effectively maintained for a longer time.
The invention also examines the arrangement mode of different cells, and the TEER value detection result is shown in figure 4. As can be seen from fig. 4. Model maturation times for other permutation patterns of different cells took around 4-7 days, more time was spent waiting for model maturation, and TEER values for some models were significantly lower. Therefore, the in vitro BBB model provided by the invention can effectively save time and cost and improve research efficiency.
EXAMPLE six integrity verification of the in vitro BBB model constructed
And (3) carrying out integrity identification on the in vitro BBB model constructed in the second embodiment, wherein the integrity identification comprises the TEER value, the expression condition of the tight junction protein and the permeability coefficients of sodium fluorescein and Huang Er lithium salt of the model.
Teer value monitoring: the transmembrane resistance of the model was measured using a MERS00002 Millicell-ERS cell resistance meter. Soaking and sterilizing a detection electrode of a cell resistance instrument for 30min by using 75% ethanol, washing the electrode by using HBSS (acetyl beta-cyclodextrin) and drying in the air; next, the Transwell chamber was gently washed with HBSS preheated at 37 ℃ and fresh medium was added, the detection electrodes of the cell resistance meter were placed inside and outside the Transwell chamber to detect the model resistance value and the TEER value was calculated according to equation (1).
TEER(Ω·cm 2 ) = (model determination resistance-blank cell resistance) × cell area (1)
The result is shown in figure 5, the maximum value of the TEER of the model appears at the beginning of the third day, and the maximum TEER value of the model group is obviously higher than that of the hCMEC/D3 monolayer culture group, which indicates that the blood brain barrier model of the invention has a complete dense barrier and the model modeling is successfully judged preliminarily.
b. And (3) detecting the expression of the zonulin ZO-1 and Claudin-5: the compactness of the blood-brain barrier is mainly influenced by the tight junction proteins expressed by brain microvascular endothelial cells. The invention adopts real-time fluorescent quantitative PCR and Western-blot method to detect the expression condition of the brain microvascular endothelial cell tight junction protein ZO-1 and Claudin-5 in the model, and the result is shown in figure 6. The expression levels of Claudin-5 and ZO-1 increased with time, reached the maximum expression level on the third day, and then decreased until the sixth day. The results indicate that the model developed fully mature at day three, consistent with TEER results.
c. Sodium fluorescein and fluorescent Huang Er lithium salt permeability coefficients: firstly, preparing fluorescein sodium and fluorescein standard curves, wherein the concentrations of the standard curves are respectively as follows: 0. 0.01, 0.025, 0.05, 0.1, 0.25,0.5,1,2,4,8 microgram/mL, detecting by a multifunctional microplate reader, measuring each sample for 3 times, and drawing a standard curve of the two by taking an average value. Wherein the wavelength of the fluorescein sodium excitation light is 485nm, and the emission wavelength is 535nm; the fluorescence Huang Jifa has a light wavelength of 428nm and an emission wavelength of 536nm. Next, 1.5mL of warm Hanks solution was added to the lower Transwell chamber (receiving well) of the model, 0.5mL of Hanks solution containing 10. Mu.g/mL of sodium fluorescein or fluorescein was added to the upper chamber (supply well), 100. Mu.L of the solution was taken from the receiving well to a black 96-well plate after 30min and 1h, respectively, the fluorescence intensity of both was measured at the above wavelength using a multi-function microplate reader, and the amount of sodium fluorescein and fluorescein penetrating through the BBB model and the cell-free control group, respectively, was calculated from the respective standard curves. The clearance volume is then calculated according to equation (2).
Clearance volume (μ L) = (C) A ×V A )/C L (2)
Wherein, C A Is the receiving pond concentration; v A Is the receiving well volume; c L Is the initial concentration of the feed tank. The slope of the clearance rate (. Mu.L/min) is recorded as the corresponding permeability (P). Times.surface area (S) by plotting the clearance volume against time. Endothelial cell permeability x surface area (PS) in sodium fluorescein in vitro permeation model e ) Can be calculated by the following formula: 1/PSe =1/PS t -1/PS f In which PS is t Permeability of the representation modelProduct of PS f The permeability of the cell-free culture chamber control group is represented by the surface area, S is the membrane area of the cell culture chamber, and S in the Transwell chamber used in the present invention is 1.12cm 2 。
The results are shown in FIG. 7, which shows the mean P of fluorescein sodium and fluorescein in the in vitro BBB model of the invention e Less than 6 x 10 from the first day to the sixth day after model construction -4 cm/min (there is research showing that when P is e ＞6×10 -4 cm/min, indicating that the model is leaky or open), mean P starting on day seven e ＞6×10 -4 cm/min, and minimum P of the model e Appearing on the third day after modeling, are respectively: 3.317 ± 0.514 × 10 -4 cm/min and 2.510 +/-0.376 multiplied by 10 -4 cm/min. In addition, the recovery rate of fluorescein sodium and fluorescein is more than 80% in 7 days, and the absorption metabolism condition of the fluorescein sodium and fluorescein yellow by a model can be eliminated. The permeability coefficient results of fluorescein sodium and fluorescein show that the tight barrier property of the in vitro BBB model of the invention starts to appear on the first day, reaches maturity on the third day, and can be continued to the sixth day, and the results are consistent with the TEER results; in addition, the tight barrier properties may prevent the permeation of polar small molecule species.
EXAMPLE seven functional characterization of the in vitro BBB model constructed
The blood brain barrier has a powerful restricted efflux function to ensure homeostasis in the brain. Among them, P glycoprotein (P-gp) expressed by brain microvascular endothelial cells plays an important role. According to the results, on the third day of model construction, the efflux function of the model is verified by using a P-gp substrate rhodamine 123, and a P-gp function inhibitor cyclosporine A is used as a control.
First, the model medium was discarded and the model was gently washed with warm HBSS solution. Then, 0.5mL of HBSS solution containing 1. Mu.M rhodamine 123 was added to the upper chamber of the model, and 1.5mL of LHBSS solution (AP-BL) was added to the lower chamber; meanwhile, in another model, 0.5mL of a solution of rhodamine 123 was added to the upper chamber and 1.5mL of a HBSS solution (BL-AP) containing 1. Mu.M of rhodamine 123 was added to the lower chamber. Placing the model back into an incubator for incubation for 1h, respectively sucking 100 mu LHBSS solution from the receiving pool into a special 96-well fluorescent plate, and detecting the solution by adopting a fluorescent microplate readerFluorescence intensity, set conditions were: the excitation wavelength is 485nm, and the emission wavelength is 535nm. Each model was assayed in 3 replicates. Meanwhile, 0,0.125,0.25,0.5,1,2,4,8. Mu.M rhodamine 123 solution was set separately for standard curve measurement. The results are shown in FIG. 8. The rhodamine 123 standard curve obtained by the invention is as follows: y =6500.2x-982.61 2 ＝9985。
Then, the apparent permeability coefficient P of rhodamine 123 in the model is calculated according to the formula (3) app 。
Wherein V is the volume of the receiving pool; ca is the concentration of the stock solution in the supply tank; cr is the concentration of the detection liquid of the receiving pool; t is rhodamine 123 incubation time; s is the bottom area of the Transwell chamber of the model.
When the apparent permeability coefficient P of rhodamine 123 on different sides of the model is calculated app And then, calculating the efflux rate ER of the model according to the formula (4). By ER>2 is the standard, and the judgment is made.
As a result, as shown in FIG. 9, the apparent permeability coefficient of rhodamine 123 on the model BL side was significantly higher than that on the model AP side, and ER was 2.231. + -. 0.323, which was greater than 2, indicating that rhodamine 123 was largely discharged from the outside of the model substrate toward the top side. Meanwhile, after cyclosporin A pretreatment, the amount of rhodamine 123 transported from the model BL side to the AP side was reduced, indicating that the efflux activity of P-gp was inhibited. Research results show that the in vitro BBB model has a perfect transport efflux function.
In conclusion, the following three human-derived cell lines (hCMEC/D3, HBVP and SVG p12 cells) are selected for the first time, a Transwell cell culture device is used for modeling the blood brain barrier in vitro, and then the barrier property and the functionality of the model are verified by measuring the transmembrane resistance of the model, the permeability coefficient of tight junction protein and small molecular substances and the restrictive transport efflux function, and the result shows that the BBB model in vitro has the complete barrier property and the functionality of the model, and the blood brain barrier in vivo can be well reproduced to a certain extent.
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions can be made without departing from the spirit of the invention, and all shall be considered as belonging to the protection scope of the invention.
Claims (10)
1. A method for constructing an in vitro blood brain barrier model is characterized in that: the method comprises the following steps:
1) Cell line selection: selecting hCMEC/D3 cells as brain microvascular endothelial cells, selecting HBVP cells as pericytes and selecting SVG p12 cells as astrocytes;
2) Coating: coating a Transwell membrane layer of the Transwell cell culture device by adopting a coating solution; the Transwell cell culture device comprises a Transwell chamber;
3) Cell inoculation: selecting corresponding cells in logarithmic growth phase, and inoculating the cells in sequence: (1) inoculating SVG p12 cells in a first pore plate for culturing; (2) reversely buckling the Transwell chamber in a second pore plate, inoculating HBVP cells in the lower membrane layer of the Transwell chamber for culture, and after adherent growth, rightly arranging the Transwell chamber in a third pore plate for culture; (3) hCMEC/D3 cells are inoculated on the upper membrane layer of the Transwell chamber for culture, the Transwell chamber inoculated with hCMEC/D3 and HBVP cells is combined with the first pore plate inoculated with SVG p12 cells, and the HBVP cells indirectly contact with the hCMEC/D3 cells through the Transwell membrane layer to construct an in vitro blood brain barrier model.
2. The method of constructing an in vitro blood brain barrier model according to claim 1, characterized in that: the coating solution is a rat tail collagen I type solution, the concentration of the coating solution is 10-20 mug/mL, and the coating solution is prepared by precooled acetic acid solution.
3. The method of constructing an in vitro blood brain barrier model according to claim 2, characterized in that: the concentration of the rat tail collagen I type solution is 12-18 mu g/mL, and the concentration of the acetic acid solution is 0.004-0.008M.
4. The method of constructing an in vitro blood brain barrier model according to claim 1, characterized in that: the inoculation density of the hCMEC/D3 cells is 1.8 multiplied by 10 5 -2.2×10 5 Cells/cm 2 。
5. Method of construction of an in vitro blood brain barrier model according to any one of claims 1 to 4, characterized in that: the inoculation density of the SVG p12 cells is 1.2 multiplied by 10 4 -1.8×10 4 Cells/cm 2 。
6. Method of construction of an in vitro blood brain barrier model according to any one of claims 1 to 4, characterized in that: the seeding density of the HBVP cells is 0.8 multiplied by 10 6 -1.2×10 6 Cells/cm 2 。
7. The method of constructing an in vitro blood brain barrier model according to any one of claims 1 to 4, characterized in that: further comprising step 4): and (5) detecting the barrier property and the transport function of the constructed model.
8. The method of constructing an in vitro blood brain barrier model according to any one of claims 1 to 4, characterized in that: the first orifice plate is a 12-orifice plate, the second orifice plate is a 6-orifice plate, and the third orifice plate is a 12-orifice plate.
9. The method of constructing an in vitro blood brain barrier model according to any one of claims 1 to 4, characterized in that: the Transwell cell culture apparatus was purchased from corning, usa and used a polycarbonate filter with a pore size of 0.4 μm.
10. The in vitro blood brain barrier model constructed according to the method of constructing an in vitro blood brain barrier model of any one of claims 1 to 9.
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