CN116790472A - In-vitro blood brain barrier model with tight connection structure and application thereof - Google Patents
In-vitro blood brain barrier model with tight connection structure and application thereof Download PDFInfo
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Abstract
The invention provides an in-vitro blood brain barrier model with a tight connection structure and application thereof. Wherein the in vitro blood brain barrier model comprises: a tubular orifice plate carrier; a mesh substrate support for capping the lower end of the orifice plate support; a cell membrane supported on the substrate support and having a tight connection structure; and encapsulating the cell membrane, substrate scaffold and well plate scaffold as a unitary hydrogel structure. The blood brain barrier model disclosed by the invention is close to the blood brain barrier of physiological conditions in the aspects of tissue structure, transmembrane resistance value, permeability, toxic drug reaction and the like, provides an important research foundation for drug development and disease diagnosis of nervous system diseases, and improves the accuracy of drug development of the blood brain barrier. The blood brain barrier model has the characteristics of simple and convenient operation and easy popularization on the premise of ensuring the effectiveness and the accuracy of the blood brain barrier model, and has great commercial value.
Description
Technical Field
The invention belongs to the technical field of tissue engineering and biological manufacturing under biomedical engineering, and particularly relates to an in-vitro blood brain barrier model with a tight connection structure and application thereof.
Background
The blood brain barrier refers to the existence of a "barrier" between the cerebral blood vessels and the brain that selectively blocks the passage of certain specific substances from the blood through the cerebral blood vessels into the brain. The essence of the blood brain barrier is a complex cellular or tissue structure that exists between peripheral blood and brain tissue that controls the passage and exchange of substances between blood and cerebrospinal fluid, regulating and ensuring homeostasis of the brain's internal environment. The cells constituting the blood brain barrier are mainly endothelial cells, and the endothelial cells form a tight connection structure under the action of various tight connection proteins and interact with cells such as glial cells and pericytes to form a special barrier system of the blood brain barrier.
The blood brain barrier has low permeability, which makes it a natural obstacle to delivering drugs into the brain, preventing the drugs from being effectively transported into the brain. Therefore, research and development of delivering drugs into the brain must be considered to be effective in penetrating the blood brain barrier. In addition, dysplasia and loss of function of the blood brain barrier can disrupt the homeostasis of the brain microenvironment, leading to neurological dysfunction that can lead to numerous neurological disorders such as stroke, alzheimer's disease, parkinson's disease, etc. Therefore, development and in-depth research on the blood brain barrier in-vitro model will provide an important theoretical basis for drug development and disease diagnosis and treatment of nervous system diseases.
The existing blood brain barrier model mainly comprises: an in vivo animal model, an in vitro microfluidic chip technology-based model, and a Transwell cell-based model. In many animal models, the blood brain barrier of animals such as mice, rats and rabbits is studied, and the accuracy of the blood brain barrier model is difficult to guarantee due to the species difference between small animals and human bodies. The micro-environment of the human blood brain barrier can be better reproduced by the in-vitro model based on the micro-fluidic chip. However, the model operation difficulty based on the microfluidic chip is high, and the wide application of the model is hindered by high technical threshold. The in vitro Transwell-based model has the characteristic of convenient operation, and the use of human cells can reduce the species difference as much as possible. However, the model of the common Transwell chamber has difficulty in well promoting the formation of a tight connection structure between cells, so that the effectiveness of the blood brain barrier is difficult to ensure.
Therefore, it would be desirable to provide an effective blood brain barrier model that is simple to operate, has a low technical threshold, and is capable of promoting the formation of tight junctions between cells.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides an in-vitro blood brain barrier model with a tight connection structure, which can effectively simulate the in-vivo blood brain barrier function, has the characteristics of simple operation and low technical threshold, and has great commercial value.
The invention also provides application of the in-vitro blood brain barrier model with the tight connection structure in research and development of blood brain barrier related medicines.
An in vitro blood brain barrier model having a tight junction structure, comprising:
a tubular orifice plate carrier;
a mesh substrate support for capping the bottom of the orifice plate support;
a cell membrane supported on the substrate support and having a tight connection structure;
and encapsulating the cell membrane, substrate scaffold and well plate scaffold as a unitary hydrogel structure.
After the construction of the above-described in vitro blood brain barrier model with a tight junction structure is completed, the lower end (cell membrane) needs to be placed in a cell culture medium to maintain cell activity. The cross section of the orifice plate support is round, square, rectangular, triangular or special-shaped.
Cell membranes, which are composed of one or more blood brain barrier-associated cells, can be used to mimic the tight junctions between blood brain barrier cells.
Preferably, the orifice plate support is of a circular tube structure. In order to facilitate processing and installation and improve the sealing effect of the blood brain barrier model, the substrate support is of a circular sheet structure matched with the circular tube structure of the orifice plate support.
Preferably, a positioning part for positioning the orifice plate support is arranged at the upper end of the orifice plate support. The positioning part is used for positioning the blood brain barrier model, so that the lower end of the pore plate support with the cell membrane can be suspended in the cell culture medium, and the cell membrane can divide the cell culture medium into an inner part and an outer part of the blood brain barrier model and is used for simulating the inner part and the outer part of the blood brain barrier, thereby realizing the physiological function of the blood brain barrier in vitro. Wherein the inner part (cell culture medium) of the blood brain barrier model is used as an upper chamber, and the outer part (cell culture medium) is used as a lower chamber.
As a further preferred aspect, the positioning part is a positioning rod perpendicular to the central axis of the orifice plate support and fixedly connected to the upper end of the orifice plate support, and when the orifice plate support is placed in a container containing a cell culture medium, the outer end (the end not connected to the orifice plate support) of the positioning rod is placed on the top of the container. The size of the orifice plate carrier is set according to the size of the container.
As still further preferable, the three positioning rods are uniformly distributed along the circumferential direction of the upper end of the orifice plate support.
In order to improve the stability of the positioning part structure, as a further preferred aspect, the positioning part further comprises a positioning ring sleeved outside the orifice plate support, the positioning rod is connected with the upper end of the orifice plate support and the positioning ring respectively, and the radius of the positioning ring is smaller than the sum of the radius of the orifice plate support and the length of the positioning rod.
Preferably, a support part for mounting the substrate support is provided at the lower end of the orifice plate support. The supporting part is an annular mounting table arranged along the inner wall of the lower end of the orifice plate support, and the inner diameter of the mounting table is larger than the outer diameter of the substrate support.
Preferably, the substrate support is a near-field direct-writing high-precision 3D printed Polycaprolactone (PCL) support, wherein the distance between two adjacent filaments in the same direction is 0.1-10 mm. More preferably 0.8 to 1.2mm.
Preferably, the hydrogel structural material is a substance that changes from a liquid state to a gel state after being stimulated by external conditions (such as temperature, light, etc.).
As a further preferred aspect, the hydrogel structural material is one or more of gelatin, gelatin derivatives, hyaluronic acid derivatives, alginate compounds, pluronic F-127, fibrinogen, collagen, silk fibroin, chitosan, agarose, polyethylene glycol, polyethylene oxide. More preferably, methacrylic acid-modified gelatin (GelMA).
Preferably, the culture method of the cell membrane having a tight junction structure comprises:
inoculating the cells subjected to culture medium resuspension on bottom supporting liquid in a culture container, and forming the cell membrane with the tight connection structure after the culture is completed on the surface of the bottom supporting liquid;
the density of the bottom support liquid is greater than the density of the culture medium and is not miscible with the culture medium.
In the culture method of the cell membrane, the bottom supporting liquid is used as the growth support of cells, and when the liquid matrix is cultured, the extracellular matrix secreted by the cells cannot adhere to the liquid matrix and can only adhere to other adjacent cells, so that the cells secrete more extracellular matrix, and the closely connected cell membrane with high cell density is formed.
As a further preference, the bottom support liquid includes, but is not limited to, one or more of fluorinated oils, fluorinated alkanes, siloxanes (e.g., silicone oils, uncured polydimethylsiloxanes), esters (e.g., dimethyl carbonate, dimethyl sulfate).
The main factor in choosing a hydrophobic liquid, represented by fluorinated oil, as the bottom support liquid is that the hydrophobic effect of the fluorinated oil of the liquid substrate will provide the cell with a radially inward surface tension from various aspects. The shape of the cells is governed by the young-laplace equation; that is, potential energy is minimized under the constraint of each cell volume conservation, so that cells become stereo spheroids, which spontaneously self-assemble to form a hexagonal tight junction structure.
As still further preferred, the bottom support liquid is one or more of 3M Novec HFE series fluorinated oils (e.g., HFE 7500), 3M Fluorinert FC series fluorinated oils, TECCEM fluorinox series fluorinated oils, silicone oils, uncured polydimethyl siloxane, dimethyl carbonate, dimethyl sulfate.
As a further preferable aspect, the addition amount of the bottom supporting liquid in the culture vessel is more than 0.08mL/cm 2 . More preferably, the addition amount of the bottom supporting liquid is 0.3 to 0.7mL/cm 2 。
As a further preference, the cells are cryopreserved cells after resuscitation or cells after passaging digestion.
As a further preference, the cells are blood brain barrier model-related cells including, but not limited to, one or more of endothelial cells, glial cells, and pericytes. Even more preferably endothelial cells.
More preferably, the cell culture is performed at an seeding concentration of 2X 10 cells 4 ～2×10 8 Individual/cm 2 . More preferably 1X 10 6 Individual/cm 2 ～2×10 6 Individual/cm 2 。
More preferably, the cell culture is carried out at a temperature of 35 to 39℃for a period of 1 to 28 days. More preferably, the culture temperature is 37℃and the culture time is 1 to 14 days, and the cells after the culture form a membranous cell sheet having a tightly-connected structure.
More preferably, the cells are cultured in a carbon dioxide incubator at a concentration of 5% after inoculation.
More preferably, the medium is replaced every 10 to 15 hours during the cell culture, and the volume of the replaced cell culture medium is 70 to 90% of the volume of the original cell culture medium. Still more preferably, the medium is replaced every 12 hours during the cultivation.
More preferably, when the medium is replaced, the new medium is preheated and then replaced.
As a further preferred aspect, the bottom support liquid is sterilized and then added to the culture vessel.
As a further preferred aspect, the bottom support liquid is sterilized by one or more of chemical sterilization, radiation sterilization, dry heat sterilization, wet heat sterilization, and filter sterilization. Ultraviolet sterilization is even more preferred.
The culture vessel may be a culture plate or a culture dish, or any vessel suitable for ordinary cell culture. The culture container can also be a container with self-defined materials, shapes and structures. Preferably, the culture vessel is a commercial multi-well culture plate.
As a further preferred option, the substrate support is first buried in the bottom supporting liquid, then cell inoculation is carried out, after cell culture is completed to form cell membrane, the substrate support is lifted upwards and taken out, and the cell membrane is attached to the substrate support during the period, so as to obtain the substrate support carrying the cell membrane. Because the formed cell membrane is fragile, the cell membrane is difficult to completely take out without external force. According to the technical scheme, the reticular substrate support is used as a supporting structure of the cell membrane, the cell membrane is completely taken out under the support of the substrate support, and the substrate support and the cell membrane are integrally applied to a blood brain barrier model.
In order to facilitate the taking out of the substrate support, as a further preferable mode, a collecting support can be added as an auxiliary taking and placing tool to take and place the substrate support, the collecting support comprises an annular structure at the bottom and a lifting rod connected with the annular structure, and an annular boss for installing the substrate support is arranged on the inner side of the annular structure;
the lifting rod is parallel to the central axis of the annular structure, the lower end of the lifting rod is connected with the annular structure, and the upper end of the lifting rod is provided with a bent lifting handle. When the collecting bracket is placed in the culture container, the handle can be hung on the top of the culture container. The size of the collection support is set according to the size of the culture container.
When the cell membrane collecting device is used, the substrate support is firstly arranged on the collecting support and then placed in the bottom support liquid, the collecting support is directly taken out when the cell membrane is taken out after the cell membrane is formed, and then the substrate support and the cell membrane are taken down from the collecting support.
As a further preference, the collection scaffold is a 3D printed polylactic acid (PLA) scaffold.
Preferably, the substrate holder and the collection holder are sterilized and then added to the culture vessel. Further preferably, the sterilization is performed by one or more sterilization methods selected from the group consisting of chemical agent sterilization, radiation sterilization, dry heat sterilization, wet heat sterilization, and filter sterilization. Ultraviolet sterilization is even more preferred.
Preferably, the substrate support carrying the cell membrane is mounted on the orifice plate support after being removed from the collecting support, and the substrate support carrying the cell membrane is suspended in a container containing a cell culture medium through a positioning part of the orifice plate support, so that the in-vitro blood brain barrier model is obtained.
Taking cell culture in a 12-well culture plate as an example, the above-mentioned in-vitro blood brain barrier model with a tight junction structure is constructed as follows:
(1) Preparation of cell membranes with tight junctions
A clean sterilized 12-well culture plate was prepared, into which a clean sterilized custom-sized collection support and a substrate support (substrate support mounted on collection support) were sequentially placed.
The outer diameter of the collecting bracket with the customized size is slightly smaller than the inner diameter of the 12-hole culture plate; the height is slightly higher than the depth of the plate hole of the 12-hole culture plate.
Sequentially adding bottom supporting liquid (the liquid level at least exceeds the substrate bracket) and cell culture medium containing cells into a 12-hole culture plate for culturing, and forming a cell membrane with a tight connection structure on the surface of the bottom supporting liquid after culturing;
wherein the density of the bottom support liquid is greater than the density of the culture medium and is immiscible with the culture medium.
(2) Establishing an in vitro blood brain barrier model
Preparing another clean sterilized 6-hole culture plate, slowly taking out the collecting bracket from the 12-hole culture plate, and attaching the cell membrane to the substrate bracket during the period; separating the substrate support carrying the cell membrane from the collection support and placing the substrate support in a clean sterilized orifice plate support with a customized size;
the orifice plate support needs to be suspended on the orifice plate, and the distance between the bottom of the orifice plate support (namely the cell membrane) and the orifice plate is 1-10 mm.
And (3) encapsulating the substrate bracket carrying the cell membrane and the orifice plate bracket by using hydrogel, and placing the encapsulated orifice plate bracket into a 6-hole culture plate added with a cell culture medium for culturing so as to keep the cell activity.
Preferably, the collection scaffold is a 3D printed polylactic acid (PLA) scaffold with an outer diameter of 21.2mm and a height of 19mm.
Preferably, the substrate support is a circular net structure with a diameter of 15-21mm and a wire spacing of 0.1-10 mm.
Preferably, the substrate support is a near field direct write high precision 3D printed Polycaprolactone (PCL) support with a diameter of 19mm and a wire spacing of 1mm.
Preferably, the orifice plate support is a 3D printed polylactic acid (PLA) support, the outer diameter of the orifice plate support is 35mm, the height of the orifice plate support is 17mm, and the distance between the bottom of the orifice plate support and the orifice plate is 5mm.
Use of the in vitro blood brain barrier model with tight junctions according to any of the preceding claims for development of blood brain barrier related drugs.
Preferably, the bottom (cell membrane) of the blood brain barrier model is suspended in a cell culture medium, a blood brain barrier-related drug is added to the cell culture medium (upper chamber) inside the blood brain barrier model, and the permeation effect of the drug in the blood brain barrier model is determined by detecting the concentration of the drug in the cell culture medium (lower chamber) outside the blood brain barrier model.
According to the method for establishing the in-vitro blood brain barrier model with the tight connection structure, provided by the invention, endothelial cells related to the blood brain barrier are prepared into the cell membrane with the tight connection structure, the cell membrane, the pore plate support and the substrate support with the customized sizes are packaged into a whole through hydrogel, the pore plate support is divided into an upper part and a lower part, the two parts are used for simulating the inner part and the outer part of the blood brain barrier, the physiological function of the blood brain barrier can be reproduced in vitro, and the permeability of a medicine in the blood brain barrier is observed.
The method for establishing the external blood brain barrier model with the tight connection structure constructs a platform for simulating the internal blood brain barrier, and the platform can be applied to life science and clinical medicine research and provides an important theoretical basis for drug development and disease diagnosis of nervous system diseases.
The invention provides a method for establishing an in-vitro blood brain barrier model with a tight connection structure, which comprises the following steps: (1) Culturing a cell patch by a culture method of adherent cells producing a tight junction structure; (2) printing a cell patch substrate support; (3) printing a custom-sized orifice plate carrier; (4) And packaging the cell membrane, the substrate support and the pore plate support through hydrogel.
The cell membrane consists of one or more blood brain barrier related cells, and can be used for simulating a tight connection structure between blood brain barrier cells; the cell membrane substrate support is used for scooping up the cell membrane and providing mechanical support for the cell membrane; the orifice plate support can be suspended in orifice plates of different types to divide the orifice plate into an upper chamber and a lower chamber. The invention integrates the functions of constructing, characterizing, barrier function, evaluating drugs and the like of an in-vitro blood brain barrier model, and can be used for in-vitro simulation of the blood brain barrier model and testing application of drugs related to the blood brain barrier. Compared with the existing blood brain barrier model, the method solves the contradiction between the accuracy and the simplicity of the in-vitro blood brain barrier model construction, is closer to the in-vivo real environment, improves the experimental efficiency, and has good application prospect in the research and the drug development of blood brain barrier related diseases.
Compared with the prior art, the invention has the beneficial effects that:
(1) Compared with an in-vivo blood brain barrier animal model, the blood brain barrier model eliminates the species difference, and experimental data obtained through the platform (blood brain barrier model) better accords with the real situation of a human body, so that the accuracy of research and development of blood brain barrier medicines is improved.
(2) Compared with an in-vitro blood brain barrier model based on a microfluidic chip, the blood brain barrier model reduces the operation difficulty and the technical threshold. On the premise of ensuring the effectiveness and accuracy of the blood brain barrier model, the method has the characteristics of simple and convenient operation and easy popularization, and has great commercial value.
(3) Compared with the blood brain barrier model of an in-vitro common Transwell cell, the blood brain barrier model of the invention promotes the formation of a tight connection structure between cells, has a tissue structure similar to the real situation of human tissues, and forms an effective blood brain barrier model.
(4) The invention constructs the blood brain barrier close to physiological conditions in the aspects of tissue structure, transmembrane resistance value, permeability, toxic drug reaction and the like for the first time in vitro, and provides an important research platform for drug development and disease diagnosis of nervous system diseases.
Drawings
FIG. 1 is a schematic diagram of a process for preparing a cell membrane with a tight junction structure and establishing an in vitro blood brain barrier model according to an embodiment of the present invention; wherein, 1 is a clean and sterilized 12-hole culture plate, 2 is a collecting bracket, 3 is a substrate bracket, 4 is a bottom supporting liquid, 5 is a cell, 6 is a cell culture medium, 7 is a clean and sterilized 6-hole culture plate, 8 is a pore plate bracket, and 9 is hydrogel;
FIG. 2 is a schematic diagram of an embodiment of an in vitro blood brain barrier model;
FIG. 3 is a schematic view of the structure of a collection support and an orifice plate support according to an embodiment of the present invention; wherein, a in fig. 3 is an oblique view, a top view and a side view of the collecting bracket; fig. 3 b is an oblique view, a top view and a side view of the orifice plate carrier; wherein 10 is an annular structure, 11 is a lifting rod, 12 is a handle, and 13 is an annular boss; 80 is a circular tube structure, 81 is a positioning rod, 82 is a positioning ring, and 83 is an annular mounting table;
FIG. 4 is a pictorial representation of a substrate support and an enlarged microscopic view of the edge and center portions of an embodiment of the present invention;
FIG. 5 is a process diagram of an embodiment of the present invention; wherein, a in FIG. 5 is a physical diagram for preparing a cell membrane having a tight junction structure; FIG. 5 b is a physical view (top view) of a cell membrane with a tight junction structure; FIG. 5 c is a diagram showing the cell membrane with tight junction structure after being collected by the collection support; FIG. 5 d is a physical view of a cell patch on a substrate holder after preparation of a cell patch with a tight junction structure (substrate holder with cell patch); FIG. 5 e is a physical diagram of an in vitro blood brain barrier model;
FIG. 6 is a micrograph and an electron micrograph of a cell membrane with a tight junction structure according to an embodiment of the present invention; wherein, a is a 4-fold micrograph of a cell patch on a substrate support after the cell patch has a tightly-connected structure in FIG. 6; FIG. 6 b is a 10-fold micrograph of a cell patch on a substrate support after the cell patch has a tight junction structure; FIG. 6 c is a 300-fold electron microscope image of a cell patch on a substrate support after a cell patch having a tight junction structure; FIG. 6 d is a 4000 Xelectron microscope image of a cell patch on a substrate holder after a cell patch having a tight junction structure;
FIG. 7 is an electron microscope image of a hydrogel in an embodiment of the invention; wherein, a is a 100-time electron microscope image of the surface of the hydrogel in fig. 7; FIG. 7 b is a 300-fold electron microscope image of the hydrogel surface; FIG. 7 c is a 50-fold electron micrograph of the interior of the hydrogel; FIG. 7 d is a 200-fold electron microscope image of the interior of the hydrogel;
FIG. 8 is a test result for verifying the sealability of an orifice plate carrier (blood brain barrier model) in an embodiment of the present invention;
FIG. 9 is a graph showing the results of the blood brain barrier transmembrane resistance test under different blood brain barrier construction conditions;
FIG. 10 is a graph showing the results of a test of the passage of fluorescent substances of different molecular weights through the blood brain barrier under different conditions of blood brain barrier construction;
FIG. 11 shows the results of blood brain barrier permeability test before and after treatment with a blood brain barrier toxic drug (Bingpin) according to an embodiment of the present invention;
FIG. 12 is a graph showing the results of a blood-brain barrier test of a blood-brain barrier related agent (dopamine, L-dopamine) according to an embodiment of the present invention; wherein, fig. 12 a is a schematic diagram of the blood brain barrier related drug passing through the blood brain barrier; fig. 12 b shows the result of detecting the passage of the blood-brain barrier-related drug through the blood-brain barrier.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in further detail with reference to examples, and the apparatus and reagents used in each example and test example are commercially available unless otherwise specified. The specific embodiments described herein are to be considered in an illustrative sense only and are not intended to limit the invention.
As shown in fig. 1, a method for establishing an in vitro blood brain barrier model with a tight junction structure includes the following steps:
a. a clean sterilized 12-well culture plate was prepared, into which a clean sterilized custom-sized collection support and a substrate support (substrate support mounted on collection support) were sequentially placed.
b. A bottom support liquid (liquid surface is over the substrate support) and a cell culture medium containing endothelial cells are sequentially added to the 12-well plate for culturing.
c. After 24h of culture, the collection support is slowly taken out from the 12-hole culture plate, and in the taking out process, the cell membrane is attached to the substrate support, and the substrate support carrying the cell membrane is separated from the collection support.
d. Another clean sterilized 6-well plate was prepared, the cell membrane loaded substrate holder was placed into a clean sterilized custom-sized well plate holder, and the cell membrane loaded substrate holder was encapsulated with the well plate holder using hydrogel.
e. The packaged pore plate bracket is placed into a 6-hole culture plate added with a cell culture medium for culture, wherein the cell membrane is suspended in the cell culture medium, namely, the upper side and the lower side of the cell membrane are immersed in the cell culture medium.
f. The constructed in vitro blood brain barrier model with tight junction structure can add blood brain barrier related drugs from the upper chamber (cell culture medium on the upper side of cell membrane, i.e. cell culture medium in blood brain barrier model) and detect the effect of drugs penetrating the blood brain barrier in the lower chamber (cell culture medium in 6-well culture plate).
Product (blood brain barrier model) appearance structure:
an in vitro blood brain barrier model with a tight junction structure can be constructed through the above process, and the structure is mainly shown in fig. 2. A6-hole culture plate is suspended with a hole plate bracket, and the distance between the bottom of the hole plate bracket and the 6-hole culture plate is 5mm. The bottom of the pore plate bracket bears a cell membrane formed by the substrate bracket and endothelial cells, and the substrate bracket and the cell membrane are encapsulated through hydrogel.
The collecting bracket structure is shown in fig. 3 a, the collecting bracket comprises an annular structure 10 at the bottom and a lifting rod 11 connected with the annular structure 10, and an annular boss 13 for installing a substrate bracket is arranged on the inner side of the annular structure 10; the lifting rod 11 is parallel to the central axis of the annular structure 10, the lower end of the lifting rod is connected with the annular structure 10, and the upper end of the lifting rod is provided with a bent lifting handle 12. When the collecting bracket is placed in the culture container, the handle can be hung on the top of the culture container.
The pore plate support structure is shown in fig. 3 b, the pore plate support is a circular tube structure 80, the upper end of the circular tube structure 80 is sleeved with a positioning ring 82 and three positioning rods 81 which fixedly connect the upper end of the circular tube structure 80 with the positioning ring 82, and the positioning rods 81 are arranged perpendicular to the central axis of the circular tube structure 80; the three positioning rods 81 are uniformly arranged along the circumferential direction of the circular tube structure 80, and the radius of the positioning ring 81 is smaller than the sum of the radius of the circular tube structure 80 and the length of the positioning rods 81; the lower inner wall of the tubular structure 80 is provided with an annular mounting table 83 for mounting the substrate support. The substrate holder carrying the cell membrane mounted on the annular mounting table 83 is suspended in the cell culture medium by a positioning portion composed of a positioning ring 82 and three positioning rods 81.
The structure and microscopic view of the substrate holder is shown in fig. 4, and the substrate holder has a circular net structure.
In fig. 5 a and b are shown a front view and a top view of an object of the process of preparing a cell membrane with a tight junction structure. FIG. 5 c shows a physical image after the preparation of cell membranes with tight junctions and collection from the collection scaffold. In fig. 5 d is shown a physical image of the cell membrane on the substrate holder after the cell membrane having the tight junction structure is prepared, and as can be seen in fig. 5 d, the membrane-like cell membrane forms a macroscopic tight junction and is integrally attached to the substrate holder. Fig. 5 e is a physical diagram of an in vitro blood brain barrier model.
Product characterization:
the tight junction structure of endothelial cell membranes in this example was verified by microscopy and electron microscopy. As can be seen from fig. 6, the membranous cell sheets form a tight connection and are integrally attached to the substrate holder on a microscopic scale.
It was verified by electron microscopy that the hydrogels used for encapsulation in this example did not produce a barrier function, i.e. the barrier function was produced by the constructed cell membrane. As can be seen from fig. 7, the hydrogel for encapsulation is a loose porous hollow structure on a microscopic scale, and can ensure the passage of substances.
The edge sealability of the hydrogels used for encapsulation in this example was verified using fluorescence permeation experiments.
The specific experimental process is as follows: the well plate support and the substrate support carrying the cell membrane were encapsulated with hydrogel with/without a barrier, respectively, and then fluorescent dyes were added to the upper chamber, respectively, and after a period of time, the fluorescence intensity in the lower chamber was measured to reflect the degree of fluorescence leakage, and the measurement results are shown in fig. 8.
As can be seen from the results of fig. 8, the edges encapsulated by the hydrogel do not significantly penetrate after 12 hours, and have good sealability.
And (3) testing the functions of the product:
the difference between the blood brain barrier model constructed in this example and the hydrogel control group, hydrogel+monolayer cell control group, and the transmembrane resistance of the blood brain barrier in vivo under the same conditions was measured by immersing the electrodes of the Millicell-ERS volt-ohm meter transmembrane resistance measuring apparatus in the liquid (cell culture medium) in the upper and lower chambers, respectively, and the results are shown in FIG. 9. The results in FIG. 9 show that the blood brain barrier model constructed in this example (shown as hydrogel+cell membrane in the figure) has a transmembrane resistance of up to about 1900 Ω/cm 2 Is similar to the blood brain barrier data of human body (in vivo), and is significantly different from the control group.
The difference in permeability (barrier function) between the blood brain barrier model constructed in this example and the hydrogel control group and the hydrogel+monolayer cell control group under the same conditions was measured by the permeation test of substances of different molecular weights, and the results are shown in fig. 10. The results in fig. 10 show that the blood brain barrier model constructed in this example (shown as hydrogel+cell membrane in the figure) has significantly different permeability from the control group, i.e., has good barrier function.
The response of the blood brain barrier model constructed in this example to the blood brain barrier toxic drug was measured by the blood brain barrier toxic drug test, and the results are shown in fig. 11. The results in fig. 11 show that the blood brain barrier model constructed in this example (shown as blood brain barrier) has a significantly reduced barrier function after treatment with the blood brain barrier toxic drug (icetoxin), and has a significantly improved barrier function after 24 hours of self-recovery, which is consistent with the response of the human blood brain barrier to the blood brain barrier toxic drug (icetoxin).
The permeability (permselective function) of the blood-brain barrier model constructed in this example to the blood-brain barrier-related drugs was measured by the blood-brain barrier-related drug permeation test, and the results are shown in fig. 12. The results in fig. 12 show that after dopamine and L-dopamine are added to the upper chamber, the L-dopamine content detected in the lower chamber is significantly higher than the dopamine content, which indicates that the blood brain barrier model constructed in this example has a selective permeation function for the blood brain barrier-related drugs, which is consistent with the selective permeation result of the human blood brain barrier for the blood brain barrier-related drugs.
In conclusion, the in-vitro blood brain barrier model with the tight connection structure constructed by the invention can well simulate the function of the human blood brain barrier, improves the accuracy and the effectiveness of research and development of blood brain barrier drugs, has simple operation and easy popularization, and provides an important research foundation for development of nervous system disease drugs and diagnosis and treatment of diseases.
Claims (10)
1. An in vitro blood brain barrier model with tight junction structures, comprising:
a tubular orifice plate carrier;
a mesh substrate support for capping the bottom of the orifice plate support;
a cell membrane supported on the substrate support and having a tight connection structure;
and encapsulating the cell membrane, substrate scaffold and well plate scaffold as a unitary hydrogel structure.
2. The model of claim 1, wherein the upper end of the orifice plate carrier is provided with a positioning part for positioning the orifice plate carrier.
3. The in vitro blood brain barrier model with tight connection structure according to claim 1, wherein the lower end of the orifice plate carrier is provided with a support portion for mounting the substrate carrier.
4. The in vitro blood brain barrier model with tight junctions according to claim 1, wherein the hydrogel structural material is a substance that changes from a liquid state to a gel state upon stimulation by external conditions.
5. The in vitro blood brain barrier model with tight junction structure according to any one of claims 1 to 4, wherein the culture method of cell membrane with tight junction structure comprises:
inoculating the cells subjected to culture medium resuspension on bottom supporting liquid in a culture container, and forming the cell membrane with the tight connection structure after the culture is completed on the surface of the bottom supporting liquid;
the density of the bottom support liquid is greater than the density of the culture medium and is not miscible with the culture medium.
6. The model of an in vitro blood brain barrier with tight junctions of claim 5, wherein the bottom support liquid is a mixture of one or more of fluorinated oils, fluoroalkanes, siloxanes, esters;
the cells are cryopreserved cells after resuscitation or cells after subculture and digestion.
7. The model of an in vitro blood brain barrier with tight junctions of claim 5, wherein the cells are seeded at a concentration of 2 x 10 when the cells are cultured 4 ～2×10 8 Individual/cm 2 。
8. The model of claim 5, wherein the substrate support is embedded in a bottom supporting liquid, and then the cell is inoculated, and after the cell culture is completed to form a cell membrane, the substrate support is lifted upwards and taken out, and the cell membrane is attached to the substrate support during the period of time, so as to obtain the substrate support carrying the cell membrane.
9. Use of the in vitro blood brain barrier model with tight junction structure according to any one of claims 1 to 4 in the development of blood brain barrier related drugs.
10. The use according to claim 9, wherein the bottom of the blood brain barrier model is suspended in a cell culture medium, a blood brain barrier-related drug is added to the cell culture medium inside the blood brain barrier model, and the permeation effect of the drug in the blood brain barrier model is determined by detecting the concentration of the drug in the cell culture medium outside the blood brain barrier model.
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| CN202210245504.4A CN116790472A (en) | 2022-03-14 | 2022-03-14 | In-vitro blood brain barrier model with tight connection structure and application thereof |
| PCT/CN2022/128137 WO2023093454A1 (en) | 2021-11-29 | 2022-10-28 | Method for culturing adherent cells producing tight junction structures and product application thereof |
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| CN117398519A (en) * | 2023-12-15 | 2024-01-16 | 中国医学科学院生物医学工程研究所 | Quick-forming artificial blood vessel and preparation method thereof |
| CN117398519B (en) * | 2023-12-15 | 2024-03-22 | 中国医学科学院生物医学工程研究所 | Quick-forming artificial blood vessel and preparation method thereof |
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