Preparation method and application of blood-brain barrier penetrating biological in-vitro model using rBMECs

Abstract

The invention discloses a preparation method and application of a blood-brain barrier penetrating biological in-vitro model using rBMECs, and relates to the technical field of drug screening. The method comprises the following steps: step 1, cell culture; step 2, preparing a model: inoculating rBMEC and MDCK-MDR1 cells into a 24-pore plate filter, and standing for four days; the monolayer cells and preheated HBSS are pre-incubated together for 15 minutes; after the pre-incubation period, the cross-epithelium / endothelium resistance of each single layer is measured by using a chopstick electrode. According to the method disclosed by the invention, the rBMECs cells are easy to obtain, and compared with a traditional MDCK-MDR1 cell blood brain barrier model, the rBMECs cells are more in line with brain barrier physiological characteristics, the modeling is easy, the modeling cost is low, and the modeling is rapid and more reliable; according to the method, the rBMECs and the MDCK-MDR1 cells are combined for the first time, it is found that the rBMECs and the MDCK-MDR1 cells are high in compatibility, and the permeable membrane of the blood-brain barrier can be formed, so that the problem that the in-vitro blood-brain barrier is difficult to form through the single rBMECs cells can be solved, and the problem that the permeable membrane formed through the single MDCK-MDR1 cells lacks brain specificity can also be solved.

Description

Technical Field

[0001] The present invention relates to the technical field of drug screening, and specifically provides a preparation method and application of an in vitro model for blood-brain barrier penetration using rBMECs. Background Art

[0002] The MDCK-MDR1 cell static culture model is the most common traditional brain endothelial permeability screening model in many laboratories at home and abroad. By using various cell membrane transporters of MDCK-MDR1 cells, the permeability of tens of millions of various small molecule drugs to brain tissues is evaluated, and it is widely used to identify the drug delivery and its permeation kinetics of P-glycoprotein drug screening molecules in the early stage of new drug discovery.

[0003] Although the MDCK-MDR1 cell model can predict CNS drug penetration, MDCK-MDR1 cells are non-brain cells, and there are significant differences in cell physiological metabolism compared with brain cells. As a result, the in vitro model of blood-brain barrier penetration of MDCK-MDR1 cells lacks brain-specific characteristics and cannot fully meet the key physiological characteristics of the blood-brain barrier, such as tight junction integrity, transporter protein activity, and receptor-mediated transport, etc., resulting in a low success rate in identifying the drug delivery and its permeation kinetics of P-glycoprotein drug screening molecules in the early stage of new drug discovery.

[0004] Therefore, it is necessary to replace MDCK-MDR1 non-brain cells with a solution that better conforms to the physiological metabolism characteristics of the brain. Due to medical ethics reasons, it is difficult to obtain real human brain cells for preparation and application to in vitro models. Therefore, the present invention proposes a preparation method and application of an in vitro model for blood-brain barrier penetration using rBMECs, preparing rat brain capillary endothelial cells using rat brain capillary endothelial cells (rBMECs) to solve the defects of the blood-brain barrier of MDCK-MDR1 cells and improve the success rate of identifying the drug delivery and its permeation kinetics of P-glycoprotein drug screening molecules in the early stage of new drug discovery.

[0005] The above information disclosed in this background art is only used to increase the understanding of the background art of the present invention. Therefore, it may include prior art that is not known to those of ordinary skill in the art. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method and application of an in vitro model for blood-brain barrier penetration using rBMECs to solve the defects of the blood-brain barrier of MDCK-MDR1 cells and improve the success rate of identifying the drug delivery and its permeation kinetics of P-glycoprotein drug screening molecules in the early stage of new drug discovery.

[0007] To achieve the above object, the present invention provides the following technical solution: A method for preparing an in vitro model of blood-brain barrier penetration using rBMECs, comprising the following steps:

[0008] Step 1, cell culture:

[0009] Rat rBMECs and astrocytes are maintained in a basal medium supplemented with 10% FBS; the astrocytes are added to the bottom of the Trans well and placed for one day, and then the insert is placed in a 24-well plate and placed for one day;

[0010] The MDCK-MDR1 cell line is maintained in a basal medium containing 0.1 mM sodium pyruvate, 2 mM L-glutamine, 10% FBS, and 0.01 mM MEM non-essential amino acids;

[0011] Step 2, model preparation:

[0012] The rBMECs and MDCK-MDR1 cells are inoculated in a 24-well plate filter and allowed to stand for four days; the monolayer cells are pre-incubated with pre-warmed HBSS for 15 minutes; after the pre-incubation period, the trans-epithelial / endothelial resistance of each monolayer is measured using a chopstick electrode.

[0013] Preferably, in step 1, 100 μL of astrocytes, 1×10 4 / mL.

[0014] Preferably, in step 2, the rBMECs are 4 - 6×10 4 cells / cm 2 and the MDCK-MDR1 cells are 4 - 6×10 4 cells / cm 2 .

[0015] Preferably, in step 2, the inoculation of rBMECs and MDCK-MDR1 cells is to dissociate the cells using 0.25% trypsin and 0.02% EDTA in PBS.

[0016] Preferably, in step 2, the TEER value is between 450 - 800 Ω / cm 2 .

[0017] The present invention also provides the application of an in vitro model of blood-brain barrier penetration prepared by the method for preparing an in vitro model of blood-brain barrier penetration using rBMECs as described above in drug screening.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] (1) The method of the present invention can easily obtain rBMECs cells, which is more in line with the physiological characteristics of the blood-brain barrier than the traditional MDCK-MDR1 cell blood-brain barrier model. It is easy to establish the model, and the modeling cost is cheap, fast and more reliable. The method of the present invention takes the lead in combining rBMECs cells and MDCK-MDR1 cells, and finds that the compatibility of rBMECs cells and MDCK-MDR1 cells is relatively high, and they can form a permeable membrane of the blood-brain barrier more easily. This can solve the problem that it is difficult for single rBMECs cells to form an in vitro blood-brain barrier, and can also solve the problem that the permeable membrane formed by single MDCK-MDR1 cells lacks brain-specific characteristics.

[0020] (2) Comparing the results of in vivo animal studies, the method of the present invention is more accurate compared with the MDCK-MDR1 cell blood-brain barrier model.

[0021] (3) In terms of application, it can replace the MDCK-MDR1 cell blood-brain barrier model and improve the success rate of drug screening involving P-glycoprotein in the early stage of new drug development.

[0022] The above summary is only for the purpose of the specification and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments and features described above, further aspects, embodiments and features of the present invention will be readily apparent by reference to the drawings and the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 : Schematic diagram of the comparison results between the in vitro blood-brain barrier penetration model of rBMECs and MDCK (traditional);

[0024] Figure 2 : Schematic diagram of the comparison results between animal experiments and the in vitro blood-brain barrier penetration model of rBMECs;

[0025] Figure 3 : Table of comparison results of rBMECs and MDCK-MDR1 of known CNS drugs. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0027] Example 1

[0028] A preparation method of an in vitro model for blood-brain barrier penetration using rBMECs includes the following steps:

[0029] Step 1: Cell culture:

[0030] Rat rBMEC (rat brain microvascular endothelial cells) and astrocytes were maintained in basal medium (Cell Application, Inc.) supplemented with 10% FBS (Fetal Bovine Serum); astrocytes (100 μL, 1×10 4 / mL) were added to the bottom of a Trans well (membrane filter) and placed for one day, and then the insert was placed in a 24-well plate and left for one day;

[0031] The MDCK-MDR1 cell line was maintained in basal medium containing 0.1 mM sodium pyruvate, 2 mM L-glutamine, 10% FBS, and 0.01 mM MEM non-essential amino acids;

[0032] Step 2: Model preparation:

[0033] rBMEC (4 - 6×10 4 cells / cm 2 ) and MDCK-MDR1 cells (4 - 6×10 4 cells / cm 2 ) were seeded in a 24-well plate filter and left standing for four days; the two-cell seeding was separated using 0.25% trypsin and 0.02% EDTA in PBS;

[0034] The monolayer cells were pre-incubated with pre-warmed HBSS (Hanks' balanced salt solution) for 15 minutes; after the pre-incubation period, the trans-epithelial / endothelial resistance (TEER) of each monolayer was measured using a chopstick electrode (Millicell ERS, Millipore Co., Bedford, MA, USA).

[0035] Establishment of in vivo blood-brain barrier test evaluation

[0036] Polyethylene glycol 400 dissolved in water (40:55, v / v, containing 5% dimethyl sulfoxide) was used as a carrier for the test compound and intraperitoneally injected into rats at a dose of 5 mg / kg; blood and brain tissue samples were collected 30 minutes and 3 hours after intraperitoneal injection, respectively. The blood samples were centrifuged at 13,000 rpm for 5 minutes at 4°C, and the supernatant plasma samples were collected and stored at -80°C before LC-MS / MS analysis;

[0037] LC-MS / MS analysis

[0038] The analytes were separated and analyzed using an aqueous solution of 0.1% formic acid - acetonitrile (10:90, v / v) at a flow rate of 0.3 mL / min;

[0039] To prepare plasma samples, an aliquot (10 μL) was mixed with 390 μL of PBS containing 20 ng / mL of gliclazide as an internal standard;

[0040] To prepare brain samples, an equal portion of homogenate (10 μL) was added to 390 μL of PBS containing 20 ng / mL of gliclazide as an internal standard;

[0041] Using HLB μElution 96-well plates (Waters, Milford, MA, USA), solid-phase extraction was performed to extract the analytes for the preparation of plasma and brain homogenate aliquot samples;

[0042] Two calibration standards for all compounds were used: 0.025, 0.05, 0.1, 0.5, 1, and 2 μM for in vitro BBB permeability studies based on cells, and 1, 3, 5, 10, 50, 100, and 500 ng / mL for in vivo BBB permeability studies;

[0043] Only 3 μL of the sample was directly injected into the LC-MS column, and the separated eluate was transferred to the mass spectrometer; the temperatures of the column and the autosampler tray were maintained at 40 °C and 4 °C, respectively.

[0044] 1. The comparison of the screening results of 45 drug molecules related to the CNS showed that Figure 1 The Permibility in Figure (A) and the Efflux ratio parameter in Figure (B) had a high degree of consistency. Therefore, it was considered that the results of the rBMECs in vitro blood-brain barrier penetration model were basically consistent with those of the MDCK (traditional) protocol.

[0045] 2. The comparison of the screening results of 45 drug molecules related to the CNS showed that Figure 2 The Permibility in Figure (A) and the Efflux ratio parameter in Figure (B) had a high degree of consistency. Therefore, it was considered that the rBMECs in vitro blood-brain barrier penetration model, using rBMECs cells, was more likely to mimic the physiological environment of the brain, and thus could replace the MDCK (traditional) protocol in the initial stage of new drug screening.

[0046] 3. The screening results of known CNS drugs showed that, compared with the MDCK-MDR1 non-brain cell model, rBMECs were more in line with the real data of the drugs. Therefore, it was considered that the rBMECs cell in vitro blood-brain barrier penetration model protocol, compared with the MDCK-MDR1 cell model, could improve the success rate of CNS drug permeability screening in the initial stage of new drug research and development.

[0047] In the description of this specification, the description referring to terms such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0048] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing an in vitro blood-brain barrier penetration model using rBMECs, characterized in that: The steps include: Step 1: Cell culture: Rat rBMECs and astrocytes were maintained in basal medium supplemented with 10% FBS; astrocytes were added to the bottom of a Trans well and left for one day, and then the chamber was placed in a 24-well plate and left for one day; The MDCK-MDR1 cell line was maintained in basal medium containing 0.1 mM sodium pyruvate, 2 mM L-glutamine, 10% FBS, and 0.01 mM MEM non-essential amino acids; Step 2: Model preparation: rBMEC and MDCK-MDR1 cells were seeded in 24-well plate filters and left to rest for four days; the monolayers were preincubated with prewarmed HBSS for 15 minutes; after the preincubation period, the transepithelial / endothelial resistance of each monolayer was measured using chopstick electrodes.

2. The method for preparing an in vitro blood-brain barrier penetration model using rBMECs according to claim 1, characterized in that: In step 1, 100 μL of astrocytes, 1×10 4 / mL.

3. The method for preparing an in vitro blood-brain barrier penetration model using rBMECs according to claim 1, characterized in that: In step 2, the number of rBMECs is 4 to 6 × 10 4 cells / cm 2 MDCK-MDR1 cells are 4 to 6×10 4 cells / cm 2 .

4. The method for preparing an in vitro model of blood-brain barrier penetration using rBMECs according to claim 1, characterized in that: In step 2, the rBMEC and MDCK-MDR1 cells are inoculated by using 0.25% trypsin and 0.02% EDTA in PBS to separate the cells.

5. The method for preparing an in vitro model of blood-brain barrier penetration using rBMECs according to claim 1, characterized in that: In step 2, the TEER value is between 450 and 800 Ω / cm 2 between.

6. Use of the blood-brain barrier penetration in vitro model prepared by the method for preparing the blood-brain barrier penetration in vitro model using rBMECs as described in any one of claims 1 to 5 in drug screening.

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