CN214937363U - Device for establishing in-vitro model for three-dimensional culture of biological membrane - Google Patents

Abstract

The utility model relates to an immunochromatography equipment technical field, concretely relates to an equipment for establishing external model of biomembrane three-dimensional cultivation, including cultivateing the container, be equipped with soft solid layer in the bottom of cultivateing the container, the sample has been inserted in the soft solid layer. The technical problem that the operation of the three-dimensional culture of the bacterial plaque biomembrane in the prior art is complex or high in cost is solved, the scheme has no special requirement on the material or the shape of the sample, and the equipment is used as long as the ordinary aseptic operation principle is followed, pollution is not easily generated in the experimental process, a single sample can be taken out to be tested without influencing the test process of other samples, and the equipment is particularly suitable for large-scale test and verification processes.

Description

Device for establishing in-vitro model for three-dimensional culture of biological membrane

Technical Field

The utility model relates to an immunochromatography equipment technical field, concretely relates to a device for establishing the vitro model of biomembrane three-dimensional culture.

Background

Plaque biofilm is a complex three-dimensional structure composed of a multitude of microbial cells and an extracellular polymeric matrix that adheres and colonizes the surface of a solid support (e.g., the surface of a tooth). Bacteria on plaque biofilms have greater resistance, virulence and ability to fight host immune defenses than bacteria of the same species in a planktonic state. Therefore, in the study of antibacterial substances, it is necessary to study not only the inhibitory action of these antibacterial substances on bacteria in a planktonic state but also the inhibitory action of the antibacterial substances on bacteria on a plaque biofilm. The establishment of an in vitro model of the bacterial plaque biofilm is a conventional way for researching the inhibition effect of the antibacterial substance on bacteria on the bacterial plaque biofilm and is also a necessary process for the preclinical or pre-market research of the antibacterial substance.

There are a wide variety of devices on the market for creating in vitro models of plaque biofilm, such as: commercial 96-well-plate MBEC for biomembrane determination by Innovotech corporation

This equipment has set up a plurality of activity stud on cultivating and has covered, scribbles the coating (hydroxyapatite, cellulose and titanium dioxide etc.) on the stud, forms the plaque biomembrane on this coating, can test the antibacterial material of multiple different concentrations simultaneously, is an effectual bacteriostat screening tool (Innovotech, MBEC)

). However, the tool is not suitable forThe material cannot be made into a coating, and the tool is expensive. For some substances that are not suitable for making coatings, such as dental films, Sande et al have devised a method of fixing dental film samples using orthodontic iron wire and then placing the fixed dental film into a petri dish perpendicular to the bottom of the dish to form a plaque biofilm on the three-dimensional dental film (F.H. van de Sande, M.S. Azevedo, R.G. Lund, M.C.D.N.J.M.Huysmans, and M.S.Cenci, "An in vitro biological model for architectural removal and antimicrobial biological site-stress testes," biofoulding, vol.27, No.9, pp.1057-1063,2011, doi: 10.1080/08927014.2011.625473.). The method has low cost, can simultaneously test various samples, but has quite complicated preparation process and is easy to cause pollution in the operation process. Exterkate et al use a method of directly fixing a dental plaque or the like vertically on a petri dish lid, thereby forming a plaque biofilm on the three-dimensional dental plaque (r.a.m. Exterkate, w.crislaard, and j.m.ten cat, "differential stress to amine fluoride by microorganism bacteria and microbial bio-in a novel high-throughput active attachment model," bones res, vol.44, No.4, pp.372-379,2010, doi: 10.1159/000316541.). However, with this method, the angle of the dental plaque cannot be adjusted and the environment of plaque biofilm formation cannot be adequately simulated. And the method has complicated manufacturing process, is easy to cause pollution in the operation process and influences the test result.

In summary, because the methods and apparatuses for creating in vitro models of three-dimensional culture of biofilm in the prior art have various defects, it is necessary to develop an apparatus that is easy and convenient to operate, avoids pollution, and can perform three-dimensional culture of plaque biofilm on substances unsuitable for coating.

SUMMERY OF THE UTILITY MODEL

The utility model aims at providing an equipment for establishing the external model of biomembrane three-dimensional cultivation, solved prior art's plaque biomembrane three-dimensional cultivation complex operation's technical problem.

In order to achieve the above purpose, the utility model adopts the following technical scheme:

an apparatus for creating an in vitro model for the three-dimensional cultivation of a biofilm comprises a culture container, at the bottom of which a soft solid layer is arranged, in which a sample is inserted.

The application method, the principle and the advantages of the scheme are as follows: an agar solution is first prepared, and then the sterilized agar solution is injected into a culture vessel before coagulation. After the agar has cooled and solidified to form a soft solid, the sample is inserted into the soft solid layer at an angle, and then a culture solution (e.g., a liquid medium in which bacteria are dispersed) for culturing a plaque biofilm is added to the culture vessel. And covering the culture container cover, and culturing for a certain time to form a bacterial plaque biological film on the surface of the sample. By adopting the scheme, the sample is fixed in the culture container very simply and conveniently, and unlike the method in the prior art, the sample is not required to be fixed on the culture container cover through structures such as screws, and the structure for specially fixing the sample by using orthodontic iron wires is not required. The scheme not only brings convenience to the fixation of the samples, but also simplifies the taking-out process of the samples, and each sample can be taken out independently. And by adopting the mode of the scheme, the sample can be inserted into the soft solid layer at any angle. As the angle of inclination of the sample can affect the rate of bacterial deposition and attachment, as well as the morphology and degree of enrichment of plaque biofilm, etc. The scheme can conveniently research the relation between the spatial position of the sample and the formation of the bacterial plaque biological film.

In conclusion, the device for establishing the in vitro model for three-dimensional culture of the biological membrane simplifies the fixing process of the sample in the culture container, and particularly can conveniently fix the sample at different spatial positions (can realize different inclination angles), realize three-dimensional culture of the biological membrane in vitro and simulate a bacterial plaque growth structure. And the three-dimensional culture of the bacterial plaque biological film can avoid the influence of the floating bacteria which are not adhered on the test result compared with the plane culture (for example, a layer of bacterial plaque biological film is cultured at the bottom of the culture container). The scheme has wide applicability, the sample to be tested can be in various shapes such as a rod shape, a sheet shape and a tubular shape, and can also be made of various materials such as metal, hydroxyapatite, glass and dental tissue slices, and the test can be carried out as long as the sample can be inserted into the soft solid layer for fixing. If no special requirement exists, the material is not required to be made into a special shape. Due to the simplification of the operation flow, the pollution phenomenon is not easy to occur in the correct sterile operation by using the scheme, and a single sample can be taken out for testing without influencing the test process of other samples, so that the method is particularly suitable for the large-scale test verification process.

Preferably, as a refinement, the sample is a hard sheet.

By adopting the technical scheme, the flaky substance has a smooth surface, and is more favorable for observing the bacterial plaque biofilm after the bacterial plaque biofilm is formed.

Preferably, as a refinement, the thickness of the soft solid layer is from 0.25 to 0.3 mm; the soft solid layer is made of agar gel.

By adopting the technical scheme, the thickness can expose the sample to be tested to the maximum extent to participate in the test on the premise of stabilizing the sample. The agar gel has certain hardness and can be used for fixing a sample, and the agar gel does not contain nutrients which can be absorbed and utilized by bacteria, has no influence on the growth of the bacteria and does not influence the process of forming a bacterial plaque biofilm on the sample.

Preferably, as a modification, the number of the culture vessels is several.

By adopting the technical scheme, the action effects of different bacteriostats, or the effects of different action concentrations of the same bacteriostat (the concentration-effect curve is obtained by research), or the effects of the same bacteriostat under the condition of the same concentration and different action time (the time-effect curve is obtained by research) can be simultaneously researched.

Preferably, as a refinement, the angle between the hard platelets and the soft solid layer is 30 °, 60 ° or 90 °.

By adopting the technical scheme, the condition is the inclination angle of the hard sheet-shaped object which is often adopted in the experiment. In fact, the hard sheet-shaped object and the soft solid layer can form any included angle, and the different inclination angles of the hard sheet-shaped object can influence the speed of deposition and attachment of bacteria on the hard sheet-shaped object, the shape and enrichment degree of a bacterial plaque biological film and the like. The device of the scheme can realize the culture of the dental plaque biological film in multiple directions and multiple angles, and creates conditions for fully researching the formation rule of the dental plaque biological film and related bacteriostatic agents.

Preferably, as a modification, an angle assisting unit for assisting the insertion of the hard sheet into the soft solid layer at a prescribed angle is further included.

By adopting the technical scheme, the angle auxiliary unit can be used for more accurately confirming the inclination angle of the hard sheet-shaped object instead of inserting the hard sheet-shaped object into the soft solid layer at a certain angle by experience, so that the research process is more accurate.

Preferably, as a refinement, the angle auxiliary unit is triangular prism-shaped.

By adopting the technical scheme, the angle auxiliary unit is simple in structure and easy to manufacture.

Preferably, as a refinement, the angles of the three apex angles of the cross section of the angle assisting unit are 30 °, 60 ° and 90 °, respectively.

By adopting the technical scheme, the angle of 30 degrees, 60 degrees or 90 degrees is the inclination angle of the hard sheet-shaped object which is often adopted, so the angles of three top angles are respectively set as 30 degrees, 60 degrees and 90 degrees

Preferably, as a refinement, a thickness auxiliary unit for defining the thickness of the soft solid layer is further included.

By adopting the technical scheme, the thickness of the soft solid layer needs to be maintained at 0.25-0.3mm, and operators can add the soft solid layer according to experience when the agar solution is injected into the culture container, so that the situation that the soft solid layer is too thick or too thin is easy to occur, and the thickness auxiliary unit is particularly arranged in the scheme.

Preferably, as an improvement, the thickness auxiliary unit comprises a positioning sheet, a middle hole is formed in the middle of the positioning sheet, the distance between the positioning sheet and the bottom of the culture container is 0.25-0.3mm, and the positioning sheet is hung at the upper end of the culture container.

With the above technical solution, if the culture container is from a commercial well plate or culture dish, there is usually no structure for identifying the thickness of the soft solid layer on these products, and the thickness auxiliary unit of the present solution is applicable to various commercial well plates or culture dishes.

Drawings

FIG. 1 is a schematic perspective view of an apparatus for creating an in vitro model of the three-dimensional culture of biofilms according to example 1.

FIG. 2 is a front view of the culture vessel of example 1 (containing a hard sheet, the angle between the hard sheet and the soft solid layer being 60 ℃).

FIG. 3 is a front view of the culture vessel of example 1 (containing a hard sheet, the hard sheet having an angle of 90 ℃ to a soft solid layer).

FIG. 4 is a front view of the apparatus for creating an in vitro model of the three-dimensional culture of a biofilm according to example 2

Fig. 5 is a perspective view of the angle assist unit of embodiment 2.

FIG. 6 is a plan view of the culture vessel set of example 3.

FIG. 7 is a longitudinal sectional view of an apparatus for in vitro modeling of a three-dimensional culture of a biofilm according to example 4.

Detailed Description

The following is further detailed by way of specific embodiments:

reference numerals in the drawings of the specification include: the culture container comprises a culture container 1, a soft solid layer 2, a hard sheet 3, an angle auxiliary unit 4, a first plane 5, a second plane 6, a third plane 7, a horizontal rod 8, a connecting rod 9, a positioning piece 10, a connecting cylinder 11, a middle hole 12, a culture container group 13 and a culture container cover 14.

Example 1

As shown in FIG. 1, an apparatus for creating an in vitro model of three-dimensional culture of a biofilm comprises a culture vessel 1 and a soft solid layer 2, the soft solid layer 2 being located at the bottom of the culture vessel 1. The culture container 1 may be a conventional culture dish of various types (i.e., a cylindrical container having an upper opening), or may be a container having an upper opening of another shape (e.g., a rectangular parallelepiped shape or a triangular prism shape). A culture container lid 14 is placed on the culture container 1, and a sample is inserted into the soft solid layer 2. The thickness of the soft solid layer 2 is 0.25-0.3mm, and the component is agar gel, and the thickness can maximally expose the sample to be tested to participate in the test on the premise of stabilizing the sample. The sample is a substrate for the adhesion of the bacterial plaque biological film, can be a solid substance with any shape which can be inserted into the soft solid layer 2, and can be a rod, a sheet, a tube and the like, and can also be an irregular shape. The material of the sample can be metal, hydroxyapatite, glass, dental tissue slices and the like, and different detection purposes can be realized by selecting samples of different materials. For example, a section of dental tissue is selected as a sample, and the device using the scheme can study the formation rule of the bacterial plaque biological film in the dental tissue, the inhibition effect of the antibacterial substance on the bacterial plaque biological film on the dental tissue and the like. For example, by selecting metal as a sample, the corrosion rule of the plaque biofilm on metal materials (such as metal materials to be used in water environment) can be researched, or the inhibition effect of the antibacterial and anticorrosive agent on the plaque biofilm attached on the metal materials can be tested. The flaky sample, namely the hard flaky object 3, is selected to be used in the scheme, because the flaky object has a flat surface, the plaque biofilm can be observed (for example, observation under a laser confocal microscope) after being formed, and the failure of establishing an experimental model caused by the culture failure of the plaque biofilm is avoided. The hard sheet 3 may be a metal sheet, a rock sheet, a glass sheet, a hydroxyapatite sheet, a dental sheet, or the like.

The soft solid layer 2 of the present embodiment is prepared by the following method: dispersing agar in deionized water with the mass fraction of agar being 1.5-2% to obtain agar dispersion, and sterilizing the agar dispersion by conventional wet heat sterilization method (usually wet heat sterilization at 121 deg.C for 15min) to obtain sterile agar solution. Similarly, the culture vessel 1 and the culture vessel lid 14 are sterilized by moist heat sterilization, or commercially available sterilized dishes may be used. Then, in an aseptic station, the sterilized agar solution is poured into the culture container 1, and after the agar solution is cooled, the agar solution can be solidified to form agar gel, thereby forming the soft solid layer 2 of the present embodiment. In addition, the mass fraction of agar is a key factor for determining the hardness of the agar gel, and the larger the mass fraction is, the harder the agar gel becomes, and the more viscous the resulting agar solution becomes. The device of the scheme is different from the conventional culture medium flat plate in the prior art (namely, a solid culture medium layer is arranged at the bottom of the culture dish). The prior art media plates have a solid media layer much thicker than 0.3mm and the components of the solid media layer contain nutrients in addition to agar. Because the solid media layer provides support and nutrients for bacterial growth, it is not practical to provide the necessary conditions for bacterial growth with too little thickness and no nutrients.

The detailed description of the specific implementation process is carried out by taking the example of studying plaque biofilm on hydroxyapatite pressure plates:

the hard sheet 3 is a hydroxyapatite pressed sheet, which is a common plaque biofilm attachment matrix (also called as a film forming medium), and can enable microorganisms to gather and densely attach to the surface of the hydroxyapatite pressed sheet to form a stable biofilm due to the loose and porous characteristic of the hydroxyapatite pressed sheet. Before the cultivation of the bacterial plaque biomembrane, the hard sheet-shaped object 3 needs to be subjected to the conventional aseptic treatment in the prior art, so as to avoid the influence of mixed bacteria on the formation of the bacterial plaque biomembrane. According to the above method, the culture vessel 1 having the soft solid layer 2 attached to the bottom thereof is prepared, and then the hard sheet 3 is inserted into the soft solid layer 2 at an angle such that the hard sheet 3 and the soft solid layer 2 form an angle of 60 ° and an angle of 90 ° respectively, as shown in FIGS. 2 and 3. In this scheme, stereoplasm flake 3 can be arbitrary contained angle with soft solid layer 2, and the difference of the inclination of stereoplasm flake 3 can influence the speed that the bacterium deposits and adheres to on stereoplasm flake 3 to and influence the form and the enrichment degree etc. of plaque biomembrane. The device of the scheme can realize the culture of the dental plaque biological film in multiple directions and multiple angles, and creates conditions for fully researching the formation rule of the dental plaque biological film and related bacteriostatic agents. After the hard sheet 3 is inserted, bacterial suspension is added into the culture container 1 (the process of adding the bacterial suspension is also called inoculation), the culture container cover 14 is covered, and the culture container 1 is placed under specific conditions for culturing for a period of time, so that a bacterial plaque biofilm can be formed on the surface of the hard sheet 3. The bacterial suspension is formed by dispersing specific oral common bacteria into a specific liquid culture medium according to a certain concentration. Common oral bacteria include, but are not limited to, Streptococcus sanguis, Streptococcus mutans, Actinomyces viscosus, Lactobacillus rhamnosus, Porphyromonas gingivalis, and the like. In addition, in order to study the inhibition effect of different bacteriostatic agents on the bacterial plaque biofilm, the bacteriostatic agent can be used for carrying out surface pretreatment on the hard sheet 3 (for example, the bacteriostatic agent is coated on the surface of the hard sheet 3), and the effect of the bacteriostatic agent is evaluated by observing the formation condition of the bacterial plaque biofilm.

For some bacteria, the soft solid layer 2 is an agar gel, contains no nutrients, bacteria do not tend to adhere to the soft solid layer 2, and bacteria adhere to the hard sheet 3 in large numbers (e.g., when using hydroxyapatite and dental plates). Sometimes. In order to allow the bacteria to attach more efficiently to the hard plate 3 rather than to the agar gel, some modification of the surface of the hard plate 3 may be made, for example, by modifying the surface of the hard plate 3 with proteins by conventional means of the prior art. In particular, when a cover slip is used as the hard sheet 3 for bacteria such as Streptococcus mutans, Streptococcus mutans tends to grow in a liquid as a lump precipitate (i.e., suspension growth). In this case, it is critical that the surface of the hard sheet 3 be modified to form a protein layer on the outer surface thereof.

Example 2

This embodiment is an improvement of embodiment 1, in which an angle assisting unit 4 is additionally provided (in use, the angle assisting unit 4 subjected to sterile processing is used). As shown in fig. 4 and 5, the angle assisting unit 4 has a triangular prism-like structure. The cross section of the angle auxiliary unit 4 is triangular, and the three vertex angles are respectively 90 degrees, 30 degrees and 60 degrees, and the angles of the three vertex angles can also be designed according to actual requirements. The angle assisting unit 4 includes three planes as shown in fig. 4: a first plane 5, a second plane 6 and a third plane 7. The length of the angle assisting unit 4 is larger than the caliber of the upper opening of the culture vessel 1 so that the angle assisting unit 4 can be placed on the upper end of the culture vessel 1.

The following description will be given taking as an example the vertical insertion of the hard sheet 3 into the soft solid layer 2 using the angle assisting unit 4: the angle assisting unit 4 is first placed on the upper end of the culture container 1, the third flat surface 7 is in contact with the culture container 1, the vertical first flat surface 5 serves as a guide surface, the left side surface of the hard sheet 3 is brought close to the first flat surface 5, then the hard sheet 3 is slid downward along the first flat surface 5, and finally the lower end of the hard sheet 3 is inserted into the soft solid layer 2 in a vertical state. The angle assisting unit 4 plays a role of prompting the insertion angle of the hard sheet 3, and the insertion angle of the hard sheet 3 is more controllable than that without the angle assisting unit 4.

Example 3

This embodiment is a modification of embodiment 1 or embodiment 2, and as shown in fig. 6, the number of culture vessels 1 is eight, and eight culture vessels 1 are located in a culture vessel group 13, and a rectangular culture vessel lid 14 (not shown) is provided above the culture vessel group 13. In fact, the number of the culture vessels 1 may be several, and the culture vessel group 13 may be various types of cell culture plates such as a commercially available 24-well plate. For example, when the culture container group 13 with eight culture containers 1 of this embodiment is used, different bacteriostatic agents may be added to each culture container 1, or the same bacteriostatic agent may be added at different concentrations, so that the effects of different bacteriostatic agents or the effects of different concentrations of the same bacteriostatic agent may be studied simultaneously (the concentration-effect curves are obtained). The culture container 1 may also be a single culture dish in the prior art, and multiple culture dishes are taken for plaque biofilm research, so that the effect of the same bacteriostatic agent at the same concentration and different action times can be researched (namely, the culture time of each culture dish is different, and a time-effect curve is obtained through research).

Example 4

This embodiment is an improvement over embodiment 3, in that the thickness of the soft solid layer 2 needs to be maintained at 0.25 to 0.3mm, and the operator is added empirically when pouring the agar solution into the culture vessel 1, and the soft solid layer 2 is likely to be too thick or too thin, so that the present embodiment is provided with a thickness auxiliary means (in use, a thickness auxiliary means subjected to aseptic processing needs to be used). As shown in FIG. 6, the thickness auxiliary unit comprises a positioning sheet 10, the outer diameter of the positioning sheet 10 is slightly smaller than the inner diameter of the culture container 1, a middle hole 12 is arranged at the center of the positioning sheet 10, and the distance from the lower surface of the positioning sheet 10 to the bottom wall of the culture container 1 is 0.25-0.3 mm. The upper surface integrated into one piece of spacer 10 has connecting cylinder 11, and the quantity of connecting cylinder 11 is two, and two connecting cylinders 11 use the axis of spacer 10 to be the symmetry axis and be axisymmetric distribution. The connecting cylinder 11 is inserted with a connecting rod 9, and the connecting rod 9 is detachably fixed with the connecting cylinder 11 by means of friction force between the connecting rod 9 and the inside of the connecting cylinder 11. The upper end of the connecting rod 9 is bonded with a horizontal rod 8, and the horizontal rod 8 is hung at the upper end of the culture container 1. When the agar positioning plate is used, agar solution is injected into the middle hole 12 until the liquid level of the agar solution reaches the lower surface of the positioning plate 10, then the whole thickness auxiliary unit is taken out, and after the agar solution is cooled, a soft solid layer 2 with the thickness of 0.25-0.3mm can be formed. In addition, the connecting rod 9 is detachable from the connecting cylinder 11, and in the case where the depth of the culture vessel 1 is changed, the connecting rod 9 having a different length can be replaced, thereby controlling the distance between the spacer 10 and the bottom of the culture vessel 1. If the culture vessel 1 is from a commercial well plate or culture dish, there is usually no structure for indicating the thickness of the soft solid layer 2 on any of these products, so this embodiment has been devised particularly for a thickness auxiliary unit applicable to a variety of commercial well plates or culture dishes. The connecting rod 9 can be made of silica gel, the length of the connecting rod 9 can be designed to be longer in the initial state, and whether the distance between the positioning piece 10 and the bottom of the culture container 1 needs to be adjusted by cutting one connecting rod 9 according to the depth of the culture container 1.

While the embodiments of the present invention have been described in detail with reference to the accompanying drawings, it is to be understood that the present invention is not limited to the embodiments and that various changes and modifications may be made therein by those skilled in the art without departing from the spirit and scope of the invention. These should also be considered as the scope of protection of the present invention, and these do not affect the effect of the implementation of the present invention and the utility of the patent. The techniques, shapes, and structural parts, which are omitted from the description of the present invention, are all known techniques.

Claims (10)

1. An apparatus for creating an in vitro model for the three-dimensional cultivation of biofilms, comprising a culture vessel provided at its bottom with a soft solid layer in which a sample is inserted.

2. An apparatus for creating an in vitro model for the stereoscopic cultivation of biological membranes according to claim 1, characterized in that the sample is a rigid sheet.

3. The apparatus according to claim 2, wherein the thickness of the soft solid layer is 0.25-0.3mm, and the material of the soft solid layer is agar gel.

4. An apparatus for creating an in vitro model of the stereoscopic cultivation of a biofilm according to claim 3, wherein the number of said culture vessels is several.

5. An apparatus for creating an in vitro model for stereoscopic cultivation of biofilms according to claim 4, wherein the angle between the hard sheet and the soft solid layer is 30 °, 60 ° or 90 °.

6. The apparatus for creating an in vitro model for stereoscopic culture of biofilm according to any of claims 1 to 5, further comprising an angle assisting unit to assist insertion of the hard sheet into the soft solid layer at a designated angle.

7. An apparatus for creating an in vitro model of stereoscopic cultivation of biological films according to claim 6, characterized in that said angular auxiliary unit is triangular prism-shaped.

8. An apparatus for creating an in vitro model for the stereoscopic cultivation of biological membranes according to claim 7, characterized in that the angles of the three top corners of the cross section of the angle assisting unit are respectively 30 °, 60 ° or 90 °.

9. An apparatus for creating an in vitro model for the stereoscopic cultivation of biological membranes according to claim 6, further comprising a thickness auxiliary unit for defining the thickness of the soft solid layer.

10. The apparatus according to claim 9, wherein the thickness auxiliary unit comprises a spacer, the spacer is provided with a middle hole, the distance between the spacer and the bottom of the culture container is 0.25-0.3mm, and the spacer is hung on the upper end of the culture container.

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