CN103396946B - Biological reaction apparatus and its preparation method and application - Google Patents

Abstract

The present invention proposes a bioreactor and its preparation method and application, wherein the bioreactor comprises: a first plate, the first plate has a first through hole; a second plate, the second plate has a second through hole; the first plate It is integrally connected with the second board, and the first through hole corresponds to the second through hole one by one. The bioreactor can be used for culturing cells and screening drugs. In particular, the bioreactor can realize high-throughput parallel automatic loading of cells and drugs, which is more time-saving and labor-saving than traditional single-sample complex operations. It is simple and convenient, and can quickly screen drugs.

Description

Biological reaction device and its preparation method and application

technical field

The invention relates to the field of biology, in particular, the invention relates to a biological reaction device and a preparation method and application thereof.

Background technique

In vitro research on the interaction between cells and small molecule chemical drugs, extracellular matrix (ECM) protein molecules, and other cells has gradually become more and more important in the fields of molecular cell biology, clinical diagnosis, and drug development. The two-dimensional culture environment of traditional culture dishes or multi-well plates cannot well simulate and reproduce the three-dimensional environment of cell growth in vivo. Therefore, constructing a three-dimensional microenvironment in vitro to realize the interaction between cells and other biochemical factors or cells at the three-dimensional level is of great significance for accelerating the further development of biomedicine and drug research and development. And with the introduction and development of the concept of individualized medicine, people have realized the importance of individualized differences in drug development. The therapeutic effect of a single drug class and its dosage varies greatly among different individuals. The combination of three-dimensional cell culture and personalized medicine is bound to develop a new model of biological and drug research and development, and promote the vigorous development of the field of biomedicine.

Modern biomedical research needs to obtain and analyze a large amount of information, and high-throughput (high-throughput) technology has emerged as the times require, which greatly improves the efficiency of obtaining biological information and reduces the required reagents (such as antibodies, drugs), materials (such as extracellular matrix proteins) and cells (e.g. limited number of primary cells), etc.

The currently commonly used high-throughput platform technology is based on the form of microwell plates (such as 96- and 384-well plates) or chips (such as genes, proteins, materials, and cell chips), with automated operating systems (manipulators, row guns, chip spotting systems, personal Spotting instrument, etc.) to execute the test process and realize the study of micro samples. However, expensive automated operating systems have not effectively reduced the total cost of research, and more efficient and easier loading methods need to be developed

method to achieve high-throughput 3D microscale arrays of drugs, materials, and cells.

The micron-scale processing (micro-processing) technology born in the semiconductor industry is more and more widely used in biomedical research to achieve precise control and high-throughput arrangement of molecules, materials and cells in space. The field of high-throughput 3D microenvironments is powerful. For example, spatially precisely controlled 3D microenvironments can be used to reconstruct in vitro biomimetic models (such as the multilayered physiological structure of blood vessels, and the fine physiological structure of liver lobules); high-throughput arrayed 3D microenvironments can be used to Build 3D arrays of drugs, materials, and cells on a chip. Commonly used micromachining methods to achieve three-dimensional patterned arrangement include: photolithography, micro-molding, stencil patterning, imprint lithography, fluid lithography Technology (flow lithography) and so on. The design of a three-dimensional microenvironment needs to involve many factors, such as the source of biological materials (natural or synthetic), the physicochemical properties of materials (chemical properties, hardness, degradability, structure), and the biological activities of materials (adhesion sites). Presence or absence, presence or absence of inducer molecules) and the way drugs, materials and cells are encapsulated (encapsulated during the scaffolding process, encapsulated on the formed scaffold).

Today, the application of micromachining technology in the construction of three-dimensional microenvironments is mostly limited to laboratories with engineering manufacturing backgrounds. There are still technical bottlenecks in its wide application in traditional biology, pharmacy and medical laboratories, especially in the field of Construction of three-dimensional microenvironments for living cells and bioactive materials and drugs. For example, the extracellular matrix materials commonly used in laboratories, matrigel and gelatin, are commercialized gel proteins with good biological activity, biocompatibility, and degradability, and are recognized as excellent materials for culturing cells. Materials, especially matrigel, are the standard base materials for culturing embryonic stem cells. The way of gelation is generally temperature transition (4°C-37°C), and it is difficult to achieve three-dimensional patterned arrangement with conventional methods. The mold is prepared with the help of microfabrication technology, and the three-dimensional microscale arrangement of gel proteins and cells can be realized by relying on the microscale space limitation of the mold. But to a certain extent, it increases the cost of research or the difficulty of operating skills.

As mentioned above, in order to meet the increasing demand for precisely controllable and high-throughput 3D microenvironments in different research fields such as biology, pharmacy, and medicine, there is an urgent need for a simple and widely applicable platform technology to quickly, non-destructively and High-throughput realization of 3D patterned microscale arrangements of drugs, materials, and cells, as well as their mixtures. An ideal microfabricated three-dimensional microenvironment high-throughput screening platform should be easy to operate for those who are familiar with traditional two-dimensional drug, cell and material research, without special expertise and means (such as microfabrication technology and special synthetic materials) and expensive equipment (e.g. automation, microfabrication equipment) to achieve a wide range of applications in traditional biology, pharmacy and medicine.

Contents of the invention

The present invention aims at solving one of the above technical problems at least to a certain extent or at least providing a useful commercial choice. Therefore, an object of the present invention is to provide a bioreactor and its preparation method and application.

In one aspect of the present invention, the present invention provides a biological reaction device, according to an embodiment of the present invention, the biological reaction device includes: a first plate, the first plate has a first through hole; a second plate, the The second board has a second through hole; the first board and the second board are connected by double-sided adhesive tape, and the first through hole corresponds to the second through hole one by one. The organelle culture device has simple structure and comprehensive functions, and can automatically load cells, culture medium and drugs in parallel with high throughput.

In addition, the bioreactor according to the above-mentioned embodiments of the present invention may also have the following additional technical features:

According to an embodiment of the present invention, the first through hole includes a plurality of through holes, and the second through hole includes a plurality of through holes.

According to an embodiment of the present invention, the diameter of the first through hole is smaller than the diameter of the second through hole.

According to an embodiment of the present invention, the first board and the second board are formed of polymethyl methacrylate, and the first board and the second board are connected as a whole by biocompatible glue.

In the second aspect of the present invention, the present invention proposes a method for preparing the above-mentioned bioreactor, the method comprising: providing a first plate, preparing a first through hole on the first plate; providing a second plate, preparing second through holes on the second board; connecting the first board and the second board together, wherein the first through holes correspond to the second through holes one by one.

In the third aspect of the present invention, the present invention proposes a method for culturing cells using the above-mentioned bioreactor device, the method comprising: subjecting the first through hole and the second through hole to hydrophilic treatment; gelatin is loaded in parallel in the first through hole; cells are continuously planted in parallel in the first through hole loaded with gelatin; culture solution is loaded in parallel in the second through hole.

In the fourth aspect of the present invention, the present invention proposes the use of the above-mentioned bioreactor in culturing cells.

In the fifth aspect of the present invention, the present invention proposes the use of the above-mentioned bioreactor in screening drugs.

Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of drawings

The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:

Fig. 1 is a longitudinal sectional view of a bioreactor according to an embodiment of the present invention.

Fig. 2 is a top view of a bioreactor according to an embodiment of the present invention.

Fig. 3 is a schematic structural view of a drug-loaded chip according to an embodiment of the present invention.

Fig. 4 is an image of the drug concentration distribution before and after the diffusion of the drug in the screened drug by the bioreactor.

Fig. 5 is the detection result of culturing human fibrosarcoma cell lines using the biological reaction device of the present invention and a conventional two-dimensional porous plate.

detailed description

Embodiments of the present invention are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals designate the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary and are intended to explain the present invention and should not be construed as limiting the present invention.

In describing the present invention, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", " Orientation or position indicated by "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc. The relationship is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, therefore It should not be construed as a limitation of the present invention.

In addition, the terms "first" and "second" are used for descriptive purposes only, and cannot be interpreted as indicating or implying relative importance or implicitly specifying the quantity of indicated technical features. Thus, a feature defined as "first" and "second" may explicitly or implicitly include one or more of these features. In the description of the present invention, "plurality" means two or more, unless otherwise specifically defined.

In the present invention, unless otherwise clearly specified and limited, terms such as "installation", "connection", "connection" and "fixation" should be understood in a broad sense, for example, it can be a fixed connection or a detachable connection , or integrally connected; it may be mechanically connected or electrically connected; it may be directly connected or indirectly connected through an intermediary, and it may be the internal communication of two components. Those of ordinary skill in the art can understand the specific meanings of the above terms in the present invention according to specific situations.

The bioreactor of the embodiment of the present invention is described in detail below with reference to Fig. 1, specifically, this bioreactor comprises: the first plate 10, and the first plate 10 has the first through hole 11; There is a second through hole 21; the first plate 10 and the second plate 20 are connected as a whole, and the first through hole 11 corresponds to the second through hole 21 one by one. According to a specific embodiment of the present invention, one-to-one correspondence means that the center line of the first through hole coincides with the center line of the second through hole. According to a specific embodiment of the present invention, the first through hole is suitable for loading a solid cell culture support, such as gelatin, and the cells are further planted in the through hole filled with gelatin. The second through hole 21 is suitable for loading nutrient solution, and the first through hole corresponds to the second through hole one by one, so that the nutrient solution in each second through hole 21 can supply the cells planted in the corresponding first through hole 11 .

According to a specific embodiment of the present invention, the first through hole 11 on the first board 10 includes a plurality of through holes, and the second through hole 21 on the second board 20 also includes a plurality of through holes. According to a specific embodiment of the present invention, the diameter of the first through hole 11 is smaller than the diameter of the second through hole 21 . Therefore, when the cells are planted in the first through hole and the nutrient solution is loaded in the second through hole, sufficient nutrient solution support can be ensured. In a micron-scale system, the rapid evaporation of the liquid is likely to occur due to the small amount of liquid, so that the nutrients obtained by the cells are quickly lost. Therefore, the diameter of the second through hole 11 is designed to be larger than the first through hole 21, so that the culture medium can Sufficient to support the growth of cells in the first through-hole for a short time.

According to a specific embodiment of the present invention, the first board and the second board are formed of polymethyl methacrylate, and the first board 10 and the second board 20 are connected as one by biocompatible glue, wherein the first board 10 is Ice glue carrier. Thus, the survival rate of cells can be improved when cells are cultured using the bioreactor. The biocompatible adhesive involved in the present invention is a substrate-free transparent double-sided adhesive, which is commonly used in optical research and biochemical research, and is odorless and non-toxic. The first plate and the second plate are bonded and connected as a whole through biocompatible glue, so that each pair of corresponding through holes can be regarded as a separate reflection unit without affecting each other, so that it can be realized in the same device Simultaneously set different experimental conditions.

In another aspect of the present invention, the present invention proposes a method for preparing the above-mentioned bioreactor, the method comprising: providing a first plate, preparing a first through hole on the first plate; providing a second plate, preparing second through holes on the second board; connecting the first board and the second board as a whole, wherein the first through holes correspond to the second through holes one by one. According to a specific embodiment of the present invention, the method for preparing a bioreactor of the present invention can be used to prepare a bioreactor as shown in FIGS. 1 and 2 .

According to an embodiment of the present invention, the first through hole includes a plurality of through holes, and the second through hole includes a plurality of through holes. According to a specific embodiment of the present invention, each of the through holes can be used as a separate bioreaction device, so that the bioreaction device can have multiple bioreaction spaces at the same time, and high-throughput parallelism can be achieved by using the bioreaction device. Automatic loading of cells and drugs, experimental three-dimensional cell culture, and drug screening.

According to a specific embodiment of the present invention, the method of preparing the first through hole and the second through hole is not particularly limited. According to a specific embodiment of the present invention, methods such as laser engraving technology and rapid prototyping machining can be used To prepare through-holes, you only need to use computer design software to design the pattern, which eliminates the complicated mold making process, realizes rapid processing of the device, and further improves the pass rate of through-holes.

According to an embodiment of the present invention, the diameter of the first through hole is smaller than the diameter of the second through hole. According to a specific embodiment of the present invention, the radii of the specific first through hole and the second through hole can be 0.5mm and 0.8mm respectively, and through size design, the center distance between the holes on the device and the size of the single hole can be It matches the size of the traditional commercial 384 multi-well plate, which greatly increases the wide applicability of the device, and can realize seamless connection with the existing multi-well plate testing equipment, making it more convenient for the use and testing of laboratories.

According to an embodiment of the present invention, the first plate and the second plate are formed of polymethyl methacrylate, and the first plate and the second plate are connected as a whole by biocompatible glue, wherein the first plate is made of ice Adhesive carrier board. According to a specific embodiment of the present invention, the size specifications of the first plate and the second plate can be designed according to specific needs, for example, it can be 76×26cm ² (consistent with the specifications of commercially available glass slides), which can facilitate subsequent processing detection. According to a specific embodiment of the present invention, the thickness of the first plate and the second plate is not particularly limited. According to a specific embodiment of the present invention, the thickness of the first plate can be the same as that of the second plate, and the specific thickness can be 0.5 ~1mm, which makes it more convenient to microscopically observe the cell samples in the device.

According to a specific embodiment of the present invention, the number of through holes on each plate is not particularly limited. According to a specific example of the present invention, it can be 3×8 or 6×16, which can improve its use for culturing Improve the efficiency of cells or improve the efficiency of using it to screen drugs.

In yet another aspect of the present invention, the present invention proposes a method for cultivating cells using the above-mentioned bioreactor device. According to a specific embodiment of the present invention, the method may include: conducting affinity between the first through hole and the second through hole Water treatment; parallel loading of gelatin in the first through hole; parallel planting of cells in the first through hole loaded with gelatin; parallel loading of culture solution in the second through hole. Therefore, the method can be used to effectively realize the unitized three-dimensional culture of cells, and realize the independent processing of each unit by means of the bonding of two plates.

In yet another aspect of the present invention, the present invention proposes the use of the above-mentioned bioreactor in culturing cells. Therefore, using the bioreactor can significantly improve the efficiency of culturing cells, and realize high-throughput parallel automatic loading of cells and effective culturing thereof. Using this bioreactor to culture cells is more time-saving, labor-saving, simple, convenient, and faster than traditional single-sample complex operations. At the same time, using the bioreactor to cultivate cells can also achieve fast, efficient and accurate sample addition, and realize three-dimensional cell culture. At the same time, it can reduce the use of cells, cell culture medium, and culture consumables, which is of great significance for saving costs. For example, when using the device for 96-throughput drug screening, it takes only half a minute because cells and other materials are loaded in parallel, but it takes at least 5 to 10 minutes when using a 96-well plate for manual operation.

According to a specific embodiment of the present invention, a three-dimensional cell culture array chip based on gelatin micro-ice gel formed in a bioreactor can promote cell aggregation to form a three-dimensional micro-scale tissue (referred to as micro-tissue) in a simulated body. This kind of micro-tissue of three-dimensional cells can not only avoid the common problem of uneven oxygen and nutrient transfer in three-dimensional tissues, but also better simulate the three-dimensional environment of cells in the body, ensuring that the cultured cells have a structure similar to that of cells in tissues and organs in vivo. Skeleton morphology, interaction between cells and signal transduction mechanism, etc. Due to the microscale nature of the three-dimensional scaffold, the device can save a large amount of cells, drugs, and cell culture fluid. For example, if cells are planted on the device at a cell planting density of 5×10 ⁶ /ml, only 100 μl, or 5×10 ⁵ cells, can be used to seed 96 scaffolds. Compared with traditional three-dimensional cell seeding, the dosage is only is the inoculum amount for one scaffold. In addition, in order to satisfy the cell culture of 96 wells, only 200 μl of culture medium is needed, which is 48 times less than the minimum amount of 100 μl per well of a 96-well plate. However, only a maximum of 1 μl of liquid is required for drug loading in each microscale culture unit, thereby significantly saving precious drug compounds.

In the fifth aspect of the present invention, the present invention proposes the use of the above-mentioned bioreactor in screening drugs. The bioreactor can be used to screen drugs effectively. Since the above-mentioned bioreactor can simulate the three-dimensional environment of cells in the body, it is ensured that the cultured cells have a skeleton shape similar to that of cells in tissues and organs in the body. The mutual function and signal transduction mechanism between the cells make the use of the cells for drug screening have a more accurate response, making the use of the bioreactor to cultivate cells for drug screening with better predictability. For example, when cancer cells grow in a three-dimensional porous scaffold, partial differentiation will occur, that is, epithelial-mesenchymal transition. Cancer cells will gradually differentiate into a malignant state, such as changes in cell shape, cell membrane surface The decrease of E-cadherin expression, the increase of N-cadherin expression, and the activation of other gene expression in the nucleus result in decreased sensitivity and increased drug resistance of cancer cells to chemotherapeutic drugs.

The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and do not limit the present invention in any way.

The preparation method of embodiment 1 bioreactor:

Select a 0.5mm thick PMMA plate (with protective film on both sides) as the ice gel carrier, and design the sketch of the device in the drawing software according to the dimensions mentioned in the implementation mode (the size of the device is 76×26mm, and the ice gel carrier has a total of holes 96, the array arrangement is 6×16, and the hole diameter is designed to be 1mm). Then transfer the graphics file to the control program of the laser engraving machine for mold cutting, and rinse the cut PMMA ice-gel carrier board with deionized water and dry it in an oven at 60°C. During this process, the protective film on both sides of the PMMA is not peeled off to prevent contamination of the hydrophobic PMMA surface when the pre-polymerization solution of the subsequent loading of the biomaterial scaffold is used. The prepared mold can be stored in a clean sealed bag at room temperature for later use.

When preparing the biomaterial scaffold for three-dimensional culture, the above-mentioned mold is pre-treated with a plasma cleaning machine for hydrophilization for 1 min. There is a wide choice of biomaterials, commonly used polyethylene glycol and gelatin, etc. can be used to prepare the scaffold in this device. Taking gelatin scaffold as an example, prepare 4% gelatin aqueous solution, place it on ice to pre-cool, then add glutaraldehyde solution to make the final concentration of glutaraldehyde reach 0.1%, then take 200 μl and drop it on one end of the mold, and use a thin plate (such as Thin glass slide) evenly spread the droplet on the surface of the mold, and the solution can naturally fill in the array holes on the mold. After the required number of molds are loaded with the material solution, place them in a low-temperature refrigerator (-12 to -20°C) as soon as possible for 12 hours to complete the reaction. Afterwards, the mold with the preformed stent was taken out, immersed in a 0.1M sodium borohydride solution for 10 minutes to neutralize the unreacted aldehyde groups and reduce the autofluorescence of the stent, and then rinse the mold with deionized water for two to three minutes. three times. After the cleaning process is over, immerse the mold in water to freeze it into ice, and place it in a freeze dryer for 12 hours. The ice crystals in the bracket will sublimate, and interconnected channels of tens to 100 microns will be generated in the bracket. At this time, the mold can be taken out , placed in a dry environment for storage, so far, the preparation of the biological reaction device is completed.

The specific structure of the bioreactor prepared by the above method is as follows:

The device is divided into upper and lower layers, both of which are designed with standardized dimensions. The appearance is consistent with standard microscope slides, with a length of 76mm and a width of 26mm. The array well design is consistent with the commercially available 384-well plate, which is a 6×16 array. The upper layer is a culture medium storage plate with a hole diameter of 1.6mm, and the lower layer is an ice gel carrier plate with a hole diameter of 1mm. The center-to-center distance of the holes on both plates is 4.5mm, and they are firmly bonded by biocompatible double-sided adhesive.

Embodiment 2 utilizes the operating method of bioreactor to cultivate cells

Firstly, the device is sterilized by ultraviolet light to prevent contamination of cell samples in the experiment. The cells pre-used for the experiment were prepared into a cell suspension at the required concentration, 1-5×10 ⁶ cells per milliliter. Pipette 100-120 μl of cell suspension dropwise onto one end of the ice gel carrier plate, and evenly spread the cell suspension on the surface of the plate with a clean and sterile thin glass slide, the ice gel on the plate can automatically absorb the cells, so far , the cell loading is complete.

Then take 200 μl of cell culture solution and add it dropwise to the other end of the device, that is, the end of the culture solution storage plate, and also use clean and sterile thin glass slides to scrape on the surface of the plate to complete the loading of the culture solution. Finally, place the loaded reaction device in a container with sufficient humidity and perform routine cultivation at 37°C.

The purposes of embodiment 3 bioreactors in screening medicine

If the screening experiment of cell drug toxicity is carried out, since each culture system in the device is independent of each other, it is convenient to add different concentrations and different types of drug molecules.

If the experiment is equipped with a high-throughput operation platform, the corresponding type and concentration of drugs can be directly added to each reaction unit in the device.

For general laboratory testing, the drug solution can be dripped onto the simple drug-loaded chip prepared by the present invention, and then the chip loaded with the drug (Figure 3) is buckled on the side of the culture solution storage plate of the device, and the drug will slowly Diffusion into the cell culture system.

Preparation method of drug-loaded chip: The drug-loaded chip in this example is made by traditional photoetching method, and the material is made of photo-crosslinkable polyethylene glycol material with a molecular weight of 4000 and a concentration of 10% w/t, accompanied by 0.5% w/t photoinitiator I2959, irradiated with UV light for 35 seconds under the cover of a photomask. After that, wash the irradiated polyethylene glycol gel chip with clean water, freeze at -20°C for 6-7 hours, and vacuum freeze-dry for about 12 hours.

A brief description of the structure of the drug-loaded chip: the polyethylene glycol scaffold on the chip also adopts a cylindrical array pattern, with a diameter of 1mm and a distance of 4.5mm between the columns, which is consistent with the size of the cell-loaded chip in this invention, which is convenient for experiments.

Test results:

Figure 4 shows the drug concentration distribution images before and after the drug diffusion process. The simulation results show that the drug can reach equilibrium in about 4.8 hours, and it can also be seen from the right figure of Figure 4 that the drug still maintains a concentration gradient after undergoing diffusion. Table 1 lists the loading concentration before drug diffusion, the theoretically calculated concentration after diffusion, and the actual measured concentration. After comparison, it can be clearly seen that the actual diffusion process of the drug has a very good correspondence with the theoretical prediction.

Table 1

Drug loading concentration (μg/ml) 97.66 195.3 390.6 781.3 1562.5 3125 6250 Theoretical predicted concentration after diffusion (μg/ml) 16.76 33.52 67.03 134.1 268.1 534.6 1069 Actual measured concentration after diffusion (μg/ml) 16.80 33.48 66.90 133.83 267.6 535.8 1071

After the drug has acted for a period of time (usually 24 hours or more), put the device directly into a 50ml centrifuge tube (if using a drug-loaded chip, just remove it), centrifuge at 1000rpm for 2 minutes, remove the liquid in the device, and then add In the same way as the cell culture solution, evenly smear the reagent for detecting cell activity (such as Almaran solution) on the device, and react at 37°C for 1 to 2 hours, then take out the device and place it on the detection rack. The detection value of each unit is used to calculate the relative activity of cells under different drug conditions.

Fig. 5 shows the comparison results of the detection results of the device of the present invention and the conventional two-dimensional multi-well plate in culturing human fibrosarcoma cell lines. It can be clearly seen from Fig. 5 that in the device of the present invention, the drug resistance of the cells is significantly improved. In the three-dimensional culture state, the half-inhibitory concentration of the broad-spectrum antineoplastic drug doxorubicin to human fibrosarcoma cells is 130.02 μM, while in In the traditional two-dimensional multi-well plate culture mode, the half inhibitory concentration of the drug is only 9.55 μM, and the lower half inhibitory concentration measured in the two-dimensional culture mode cannot truly reflect the anti-drug response of the cells in the original three-dimensional growth environment. It is basically consistent with the literature reports that the three-dimensional culture mode improves the drug resistance of tumor cells in recent years, so it also proves the accuracy and reliability of the device.

In the description of this specification, descriptions with reference to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be construed as limitations to the present invention. Variations, modifications, substitutions, and modifications to the above-described embodiments are possible within the scope of the present invention.

Claims (7)

1. a kind of biological reaction apparatus, it is characterised in that be formed by following structures：

First plate, first plate has first through hole；

Second plate, second plate has the second through hole；

First plate and the second plate are connected as one, and the first through hole is corresponded with second through hole,

Wherein, the first through hole includes multiple through holes, and second through hole includes multiple through holes,

Solid cell culture support is mounted with each first through hole, the solid cell culture support is ice glue,

Nutrient solution is loaded in each second through hole,

The size of the biological reaction apparatus is 76 × 26cm²,

Multiple through holes that the first through hole and the second through hole include are in 3 × 8 or 6 × 16 branches simultaneously respectively,

The radius of the first through hole and the second through hole is respectively 0.5mm and 0.8mm.

2. biological reaction apparatus according to claim 1, it is characterised in that first plate and the second plate are by poly- methyl-prop E pioic acid methyl ester is formed, and first plate and the second plate are connected as one by biocompatible glue.

3. a kind of method of the biological reaction apparatus prepared described in claim 1 or 2, it is characterised in that including：

First plate is provided, first through hole is prepared on first plate；

Second plate is provided, the second through hole is prepared on second plate；

First plate and second plate are connected as one,

Wherein, the first through hole is corresponded with second through hole,

The first through hole includes multiple through holes, and second through hole includes multiple through holes,

The diameter of the first through hole is less than the diameter of the second through hole.

4. method according to claim 3, it is characterised in that first plate and the second plate are by polymethyl methacrylate Formed, first plate and the second plate are connected as one by biocompatible glue.

5. a kind of method of the biological reaction apparatus culture cell described in utilization claim 1 or 2, it is characterised in that including：

The first through hole and the second through hole are subjected to hydrophilic treated；

The loaded in parallel ice glue in the first through hole；

Continue parallel repopulating cell in the first through hole for being mounted with ice glue；And

The loaded in parallel nutrient solution in second through hole.

6. purposes of the biological reaction apparatus in culture cell described in claim 1 or 2.

7. purposes of the biological reaction apparatus in screening medicine described in claim 1 or 2.