Biofilm culture simulation device and method for simulating and cultivating wound biofilm

Abstract

The present invention discloses a biofilm culture simulation device and a method for simulating wound biofilm cultivation. By assembling a heat source, an exudate container, a biofilm reactor, and a sealing cover, and using an exudate drainage tube to control flow, the device simulates the actual biofilm growth environment. The device can simulate the growth process of biofilms in difficult-to-heal wounds, as well as the growth process of biofilms in wounds with exudate (both low and high exudate conditions), providing favorable conditions for medical researchers to conduct in vitro research on wound biofilms.

Description

Technical Field

[0001] The invention relates to the field of microbial experiments or wound simulation experiments, in particular to a biofilm culture simulation device and a method for simulating and cultivating wound biofilm. Background Art

[0002] Wound biofilms are formed when bacteria adhere to the wound surface and fuse with their own extracellular matrix components. They are composed of bacteria and their products, extracellular matrix, and necrotic tissue. They are commonly seen in chronic wounds such as pressure ulcers, diabetic foot ulcers, and venous leg ulcers. Biofilms complicate clinical treatment and can affect healing outcomes.

[0003] In order to evaluate the impact of biofilms on existing medical dressings, or to evaluate their effectiveness in removing biofilms, it is often necessary to simulate biofilm growth in the laboratory. At present, the main methods for artificial biofilm generation include test tube method, microplate method, sheet method, flat membrane method, pipeline method, flow chamber method and spinning disc method. The most widespread are the microplate method and sheet method, in which a microplate refers to a plate with several micropores opened along a rectangular array, and bacteria are cultured by adding them into the micropores. However, the existing microplates lack corresponding auxiliary equipment when in use, causing the microplates to be completely exposed to the external environment, which can easily cause the microplates to be contaminated. In recent years, there have been more and more patents for biofilm reactors. For example, China's utility model patent CN212199223U (announcement date 2020-12-22) discloses a flow-type biofilm culture device that uses flowing water for biofilm culture, but its application in natural water bodies cannot meet the growth and evaluation of wound biofilms. Chinese utility model patent CN 212770674 U (announcement date 2021-03-23) discloses a biofilm culture device that solves the problem of biofilm peeling, but the product still cannot simulate and evaluate the growth of biofilm on the wound surface.

[0004] Since wound biofilms often appear on chronic wounds, they are difficult to detect using traditional culture methods. In addition, wound biofilms are more difficult to remove than those in other environments, have strong regenerative abilities, and can change with the physiological and biochemical conditions of the wound and tissue fluid. Currently, there are no reports publicly available on any simulation devices for simulating the growth of biofilms in the physiological and biochemical environments of wounds, nor are there any public reports on biofilm simulation culture methods that truly simulate the wound environment. Summary of the Invention

[0005] Purpose of the invention: The purpose of the present invention is to provide a biofilm culture device that simulates the growth environment of wound biofilm; another purpose of the present invention is to provide a method for simulating the culture of wound biofilm based on the device.

[0006] Technical solution: In order to achieve the above-mentioned purpose of the invention, a biofilm culture simulation device of the present invention includes an exudate container, a heat source, and a biofilm reactor in contact with the heat source. A sealing cover is provided on the top of the biofilm reactor to form a culture space for accommodating simulated wound components and carriers; the sealing cover is provided with no less than two through holes, and the exudate container is provided with an exudate drainage tube corresponding one-to-one to the no less than two through holes, and the liquid outlet end of the drainage tube is located in the culture space; each exudate drainage tube is provided with a flow control mechanism for adjusting the flow rate.

[0007] The biofilm culture simulation device provided by the present invention is used to simulate the growth of biofilm on the wound surface. The heat source provides the temperature required for the growth of microorganisms. The simulated wound components in the biofilm reactor provide carbon sources, nitrogen sources and water for the microorganisms. The carriers selected include but are not limited to medical care products such as wound care, negative pressure drainage, bandage and hemostasis, such as dressings, patches, bandages, non-woven fabrics, etc. The shape of the biofilm reactor can be the same as the container of traditional laboratory instruments, such as beakers, flasks, conical flasks, test tubes, wide-mouth bottles, etc.; it can also be designed as a combination of any one or more shapes of hexahedron, columnar, spherical, and terraced. The through hole design of the sealing cover allows the exudate drainage tube to extend into the culture space, but still leaves a certain gap, so that the assembled biofilm culture simulation device has a basically sealed culture space and provides oxygen for the microorganisms.

[0008] Because wound exudate is often not concentrated at a single site, a multi-site exudate design is crucial for realistically simulating wound exudate scenarios. Furthermore, flow control can be used to simulate varying degrees of wound exudate. This allows researchers to conduct experiments based on wound size, exudate volume, and wound type, for example, assessing biofilm growth under specific dressings. This invention is particularly suitable for simulating biofilm growth in high-exudate wounds, such as those caused by burns, scalds, chronic venous ulcers, and postoperative dehiscence.

[0009] As a preferred embodiment, the exudate drainage tubes are provided with 4-12 tubes, which are distributed in an array or in a centrally symmetrical distribution. Those skilled in the art can make adaptive adjustments according to the desired simulated wound size.

[0010] Considering the differences in exudate flow rates caused by wounds at different locations on the patient, the present invention also necessarily includes a pressurizing device to simulate biofilm growth in high-exudate wounds. This pressurizing device is used to regulate the hydraulic pressure in the exudate drainage tube. It can be a valve located in the exudate container, an air pump that continuously pressurizes the sealed exudate container, or a plunger that applies pressure to the simulated wound exudate (SBF simulated body fluid) within the exudate container.

[0011] The present invention also provides dynamic regulation of exudate volume and distribution based on the different stages of wound healing. To regulate exudate volume, a rotating valve core can be installed on the sidewall of each exudate drainage tube. This rotating valve core has a fluid channel, and the angle of the rotating valve core is used to control the flow rate of exudate from the simulated wound. The present invention also provides an alternative solution, which is achieved by adjusting the cross-section of the exudate drainage tube's outlet end. Generally speaking, the flow rate of an outlet with a flat end is lower than that of a pointed end. A higher slope of the pointed end results in a faster exudate flow rate. The present invention can also combine the rotating valve core with the cross-sectional design of the exudate drainage tube's outlet end to control exudate flow rate. To simulate biofilm growth during wound healing, the flow rate of each exudate drainage tube can be gradually reduced by controlling the rotating valve core. Alternatively, the outlet ends of the peripheral exudate drainage tubes can be designed with a flat end, with the slope of the pointed end increasing as it approaches midday. To simulate wound deterioration, the flow rate of each exudate drainage tube can be gradually increased by controlling the rotating valve core.

[0012] The heat source described in the present invention is selected from, but not limited to, an electric heating device or a water bath heating device, and the contact surface between the biofilm reactor and the heat source includes, but not limited to, the bottom surface, the side surface, or a combination of the bottom surface and the side surface.

[0013] Taking into account factors such as biofilm growth being observable and easy to sterilize, the exudate container, exudate drainage tube, sealing cover and biofilm reactor are preferably made of transparent glass material.

[0014] Regarding the biofilm culture simulation device described above, the present invention also provides a method for simulating and culturing wound biofilm based on the device. The method comprises the following steps:

[0015] (1) sterilizing the biofilm culture simulation device at high temperature;

[0016] (2) preparing simulated wound exudate, steam sterilizing, adding agar and sodium chloride according to the ratio, heating in a water bath, adding an equal volume of bovine serum solution to obtain simulated wound components, pouring into the biofilm reactor, steam sterilizing, and standing to cool until solidified; the remaining simulated wound exudate is poured into the exudate container for subsequent steps;

[0017] (3) The carrier is placed in the biofilm reactor, and the mixed bacterial solution is dripped onto the carrier. The sealing cover is put on and the exudate container is assembled. The heat source is adjusted to 35-42°C to start the culture;

[0018] (4) Maintain a constant flow rate or gradually increase the flow rate of the exudate drainage tube during the culture process. Remove the tube after 5-15 days and stain and examine under a microscope.

[0019] The volume ratio of the mixed bacterial solution to the simulated wound surface components is 0.1-10:50, preferably 1:50. The flow rate of the exudate drainage tube is 0.01-1 mL / day. When simulating low-volume exudation, the flow rate of the exudate drainage tube is 0.01-0.1 mL / day; when simulating high-volume exudation, the flow rate of the exudate drainage tube is 0.1-1 mL / day. The flow rate can be gradually increased or decreased over time depending on the experimental purpose.

[0020] Furthermore, the amount of agar added is 0.6-1v% of the simulated wound exudate in the biofilm reactor, and the amount of sodium chloride added is 0.7-1v% of the simulated wound exudate in the biofilm reactor. The solution can be processed to form a semi-solidified state, which is closer to the wound morphology.

[0021] Preferably, there are 4-12 exudate drainage tubes, which are distributed in an array or in a centrally symmetrical distribution; the flow rate of the exudate drainage tubes located at the periphery is 1 / 4-1 / 2 of the flow rate of the exudate drainage tubes located in the middle.

[0022] The above method can be used to analyze the effectiveness of different clinical techniques for biofilm removal. For example, the effectiveness of different liquid dressings on biofilm removal can be analyzed by evaluating low-volume exudate biofilms, or the biofilm growth pattern of infected wounds can be analyzed by evaluating high-volume exudate biofilms, which can help develop corresponding dressing products. The biofilm culture simulation device and its method for simulating wound biofilm culture can help medical researchers simulate in vitro wound sampling and analysis, evaluate biofilm growth in different scenarios, and develop corresponding dressing products. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is a structural diagram of embodiment 1 of the present invention;

[0024] Figure 2 This is a biofilm staining microscopy image from Example 2 of the present invention;

[0025] Figure 3 This is a microscopic examination diagram of biofilm staining in Example 3 of the present invention. DETAILED DESCRIPTION

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0027] Example 1

[0028] A biofilm culture simulation device includes a constant-temperature electric heater 1, a biofilm reactor 2, a sealing cover 3, and an exudate container 4. The biofilm reactor 2, the sealing cover 3, and the exudate container 4 are all made of glass. The biofilm reactor 2 contains simulated wound components 8 for cultivating microorganisms to form a biofilm, while the exudate container 4 contains simulated wound exudate 9 for penetration of the simulated skin wound. The constant-temperature electric heater 1 controls the temperature and heating state via a control panel 10 and adjustment buttons 11.

[0029] The sealing cover 3 is open on one side and has seven through-holes 5 on the other. It is inverted and placed above the biofilm reactor 2. Seven exudate drainage tubes 6 extend from the lower end of the exudate container 4 and extend through the seven through-holes 5 into the inner cavity of the biofilm reactor 2. Each exudate drainage tube 6 has an inner diameter of 0.5 cm and a length of 5-10 cm. The liquid outlet end is flat and has a flow valve 7 installed on the tube wall.

[0030] Example 2

[0031] This example simulates the biofilm culture on a hypotonic wound based on the biofilm culture simulation device provided in Example 1. The specific steps are as follows:

[0032] (1) All components of the biofilm culture simulation device were sterilized by pressure steam at a pressure of 100 kPa, a sterilization temperature of 121°C, and a sterilization time of 20 minutes;

[0033] (2) Prepare simulated wound exudate (SBF simulated body fluid, purchased from Huizhi Taikang), steam sterilize at a pressure of 100kpa and 121℃ for 20 minutes. Then add agar and sodium chloride according to the ratio, heat in a water bath for 1 hour to dissolve until clear, take 10ml, add an equal volume of bovine serum solution and mix evenly to obtain simulated wound components. Pour the simulated wound components into the biofilm reactor, steam sterilize at a pressure of 100kpa, a sterilization temperature of 121℃, and a sterilization time of 20 minutes, then place it in a biosafety cabinet and let it stand for 2 hours to cool to room temperature, at which time it is solidified;

[0034] (3) A carrier (such as qualitative filter paper, microporous filter membrane, etc.) is placed in the biofilm reactor, and 200 μL of mixed bacterial solution (Escherichia coli, sterile Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans mixed at a ratio of 1:1:1:1) is added dropwise to the carrier. The reactor is sealed with a lid, the exudate container is assembled, and the heat source is adjusted to 37°C to start culturing.

[0035] (4) The flow rate of each exudate drainage tube was controlled by a flow valve, with an initial flow rate of 10 μL / day.

[0036] (5) Observe the growth of the biofilm daily. After 10-15 days of culture, remove the biofilm carriers and analyze them by staining microscopy.

[0037] Crystal violet staining is used. If the biofilm carriers are lightly stained and unevenly distributed, showing a patchy pattern, it indicates an immature biofilm. If the biofilm carriers are darkly stained and evenly and densely distributed, it indicates a more mature biofilm.

[0038] like Figure 2 As shown, microscopic observation of the biofilm carriers revealed darker coloration, uniform and dense distribution, indicating a mature biofilm. This low-exudate wound biofilm can be used for in vitro testing to analyze the effectiveness of different clinical techniques on biofilm removal, such as the effectiveness of different liquid dressings on biofilm removal.

[0039] Example 3

[0040] This example simulates the biofilm culture on a hypertonic wound based on the biofilm culture simulation device provided in Example 1. The specific steps are as follows:

[0041] 1) All components of the biofilm culture simulation device were sterilized by pressure steam at a pressure of 100 kPa, a sterilization temperature of 121°C, and a sterilization time of 20 minutes;

[0042] (2) Prepare simulated wound exudate (SBF simulated body fluid, purchased from Huizhi Taikang), steam sterilize at a pressure of 100kpa and 121℃ for 20 minutes. Then add agar and sodium chloride according to the ratio, heat in a water bath for 1 hour to dissolve until clear, take 10ml, add an equal volume of bovine serum solution and mix evenly to obtain simulated wound components. Pour the simulated wound components into the biofilm reactor, steam sterilize at a pressure of 100kpa, a sterilization temperature of 121℃, and a sterilization time of 20 minutes, then place it in a biosafety cabinet and let it stand for 2 hours to cool to room temperature, at which time it is solidified;

[0043] (3) A carrier (such as qualitative filter paper, microporous filter membrane, etc.) is placed in the biofilm reactor, and 200 μL of mixed bacterial solution (Escherichia coli, sterile Staphylococcus aureus, Pseudomonas aeruginosa, and Candida albicans mixed at a ratio of 1:1:1:1) is added dropwise to the carrier. The reactor is sealed with a lid, the exudate container is assembled, and the heat source is adjusted to 37°C to start culturing.

[0044] (4) The flow rate of each exudate drainage tube was controlled by a flow valve. The initial flow rate was 200 μL / day, and the flow rate was increased to 600 μL / day on the 3rd to 5th day.

[0045] (5) Observe the growth of the biofilm daily. After culturing for 5-7 days, remove the biofilm carriers and analyze them by staining microscopy.

[0046] (6) Maintain a constant flow rate during the culture process or remove the cells after 10-15 days of culture and stain and examine under a microscope.

[0047] Crystal violet staining is used. If the biofilm carriers are lightly stained and unevenly distributed, showing a patchy pattern, it indicates an immature biofilm. If the biofilm carriers are darkly stained and evenly and densely distributed, it indicates a more mature biofilm.

[0048] like Figure 3 As shown in the figure, microscopic observation shows that the biofilm carrier is darker in color, evenly distributed and dense, indicating that the biofilm is mature. The evaluation of high-exudate biofilm and the analysis of biofilm growth patterns in infected wounds will help to prepare corresponding dressing products.

[0049] Example 4

[0050] This embodiment is basically the same as embodiment 1, with the only difference being that the outlet ends of the six peripheral exudate drainage tubes are flat-end designed, while the outlet end of the central exudate drainage tube is pointed-end designed with an inclination angle of 45°.

[0051] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A biofilm culture simulation device, characterized in that: It includes an exudate container, a heat source, and a biofilm reactor in contact with the heat source. A sealing cover is provided on the top of the biofilm reactor to form a culture space for accommodating simulated wound components and a carrier, and the carrier is a qualitative filter paper or a microporous filter membrane; the sealing cover is provided with no less than two through holes, and the exudate container is provided with an exudate drainage tube corresponding to the no less than two through holes, the exudate drainage tube extends into the culture space and forms a gap with the through holes; the liquid outlet end of the drainage tube is located in the culture space; each exudate drainage tube is provided with a flow control mechanism for adjusting the flow rate; the exudate container is provided with a pressurizing device; 4-12 exudate drainage tubes are provided, distributed in an array or in a centrally symmetrical distribution; a rotating valve core is provided on the side wall of each exudate drainage tube, and the rotating valve core is provided with a fluid channel; the liquid outlet cross-section of the exudate drainage tube located at the periphery is smaller than that of the exudate drainage tube located in the middle.

2. A biofilm culture simulation device according to claim 1, characterized in that: The heat source is an electric heating device or a water bath heating device, and the contact surface between the biofilm reactor and the heat source includes a bottom surface or a combination of a bottom surface and a side surface.

3. A biofilm culture simulation device according to claim 2, characterized in that: The exudate container, the exudate drainage tube, the sealing cover and the biofilm reactor are all made of transparent glass materials.

4. A method for simulating the cultivation of wound biofilm using the device as claimed in claim 1, characterized in that The steps include: (1) sterilizing the biofilm culture simulation device at high temperature; (2) preparing simulated wound exudate, steam sterilizing, adding agar and sodium chloride according to the ratio, heating in a water bath, adding an equal volume of bovine serum solution to obtain simulated wound components, pouring into the biofilm reactor, steam sterilizing, and then standing to cool until solidified; (3) Place the carrier in the biofilm reactor, drip the mixed bacterial solution onto the carrier, cover with a sealing cover, assemble the exudate container, and adjust the heat source to 35-42°C to start cultivation; (4) During the culture process, maintain a constant flow rate or gradually increase the flow rate of the exudate drainage tube. Remove the tube after 5-15 days and stain and examine under a microscope.

5. The method for simulating and culturing a wound biofilm using the device according to claim 1 according to claim 4, characterized in that: The volume ratio of the mixed bacterial solution to the simulated wound surface components is 0.1-10:50; the flow rate of the exudate drainage tube is 0.01-1 mL / day.

6. The method for simulating and culturing a wound biofilm using the device according to claim 1 according to claim 5, characterized in that: There are 4 to 12 exudate drainage tubes, which are distributed in an array or in a centrally symmetrical distribution; the flow rate of the exudate drainage tubes located at the periphery is 1 / 4 to 1 / 2 of the flow rate of the exudate drainage tubes located in the middle.

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