Three-dimensional substrate for microorganism culture

Abstract

The present invention relates to a three-dimensional substrate with a structural and compositional gradient for microbial culture and co-culture and a method for preparing the same, wherein the three-dimensional substrate comprises: - a diffusion system (1) comprising a first compartment (2) and a second compartment (3), wherein the first compartment (2) is placed above the second compartment (3), and the first and second compartments (2, 3) are separated by a semipermeable membrane (4); - a substrate solution comprising a polysaccharide, a protein and a salt or a preformed hydrogel comprising a polysaccharide, a protein and a salt; - Crosslinking medium containing salts, culture medium and distilled water Includes:

Description

Technical Field

[0001] The present invention relates to a three-dimensional gradient substrate for microbial culture and co-culture, and a method for simultaneously preparing a gradient with the substrate and the same substrate.

[0002] In certain embodiments, the present invention relates to a three-dimensional substrate for microbial culture and co-culture and a method for preparing the same, wherein the three-dimensional substrate is - A diffusion system (1) including a first compartment (2) and a second compartment (3), wherein the first compartment (2) is placed on the second compartment (3), and the first and second compartments (2, 3) are separated by a semipermeable membrane (4); - A substrate solution containing a preformed hydrogel containing a polysaccharide, a protein, and a salt or a polysaccharide, a protein, and a salt; - A cross-linking medium containing a salt, a medium, and distilled water and includes.

Background Art

[0003] Background Art Currently, bacterial cultures are generally obtained on agar, with its attendant limitations. Among others, it is noteworthy that soft agar gels allow for a certain degree of bacterial motility in addition to the diffusion of substances therein. However, since the gel is necessarily placed on a hard agar substrate (1.5 - 2.0% w / v) and is limited by the substrate, three-dimensional movement is partially restricted.

[0004] Furthermore, the addition of soft agar gels to high-strength supports such as cell culture plates or multi-well plates for high-throughput screening and the interpretation of the results require some skills and complex structures, thus limiting the screening ability and increasing the risk of contamination.

Summary of the Invention

Problems to be Solved by the Invention

[0005] There is a strong need for another method for microbial culture and co - culture that can mimic both the conditions seen in mucus and physiopathological culture conditions.

Means for Solving the Problem

[0006] Object of the Invention The first object of the present invention is to form a three - dimensional substrate adapted for the study of bacterial and / or microbial culture and co - culture.

[0007] In a second object, the present invention describes a method for simultaneously preparing a three - dimensional substrate and a gradient therein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0019] Detailed Description of the Invention For a first object, the present invention describes a three-dimensional substrate.

[0020] In the following description, for the term "substrate", examples of the substrate are described.

[0021] The three-dimensional substrate of the present invention contains a mixture of multiple components that can form a gel using various crosslinking agents by a diffusion process. The diffusion process controls the process of forming a gradient.

[0022] The present inventors surprisingly showed that the rheological properties of this substrate can be accurately modified by changing the crosslinking parameters.

[0023] The "matrix" includes a diffusion system that is a two-compartment system (formed by the distance between two cross-linking points) that forms a cross-linking gradient to mimic and reproduce the typical diffusion of nutrients and molecules in the microbial microenvironment.

[0024] As shown in Figure 1, the diffusion system 1 includes a first compartment 2 (also referred to as the apical side) and a second compartment 3 (also referred to as the basal side). In a preferred embodiment of the present invention, the first compartment 2 is placed on top of the second compartment 3. The first and second compartments are separated by a semi-permeable membrane 4. For the purposes of the present invention, the semi-permeable membrane 4 allows salts to pass through but does not allow polysaccharides and proteins to pass through. For example, this membrane does not allow an alginate and / or mucin solution to pass through. In a preferred embodiment, the semi-permeable membrane 4 is formed of a material such as polycarbonate, polystyrene, poly(diallyldimethylammonium chloride), polyethylene terephthalate, or polyamide (nylon).

[0025] In a preferred embodiment, the diffusion system 1 is designed by design software and manufactured by three-dimensional printing, or is processed by rapid prototyping, subtractive manufacturing, and manufactured by injection molding.

[0026] The solution used as the substrate solution is deposited in the first apical compartment 2. For the purposes of the present invention, the substrate solution contains polysaccharides, proteins, and salts. In certain embodiments, the substrate solution has a viscosity of 0.05 to 100 Pa·s, preferably 0.2 to 10 Pa·s.

[0027] In another embodiment, a preformed gel containing polysaccharides, proteins, and salts can be introduced into the first apical compartment 2 instead of the solution.

[0028] In a preferred embodiment, the polysaccharide of the substrate solution is selected from sodium alginates of various molecular weights, pectins of various molecular weights and various degrees of esterification and amidation, hyaluronic acids of various molecular weights, gellans of various molecular weights, and dextrans of various molecular weights.

[0029] In a particularly preferred embodiment, the substrate solution contains alginate as the polysaccharide.

[0030] Alginate is present in an amount of about 0.2 to 8%, preferably about 5% (w / v).

[0031] In a preferred embodiment, the protein of the substrate solution is selected from mucin, serum albumin, fibrinogen, fibronectin, collagen, elastin, insulin, and transferrin.

[0032] In a particularly preferred embodiment, the substrate solution contains mucin.

[0033] Even more preferably, the mucin has a concentration of about 25 mg / mL.

[0034] In an even more preferred embodiment, in the substrate solution, mucin and alginate are contained in the following ratio (weight / weight). [Table 1]

[0035] In a preferred embodiment, the salt of the substrate solution is selected from sodium chloride, ammonium phosphate, potassium chloride, sodium hydrogen phosphate, sodium bicarbonate, potassium chloride, dipotassium hydrogen phosphate trihydrate, magnesium chloride hexahydrate, sodium sulfate, tris(hydroxymethyl)aminomethane, sodium nitrate, sodium nitrite, potassium nitrate, silver nitrate, ammonium nitrate, calcium nitrite, potassium bisulfate, potassium sulfate, sodium bisulfate, sodium sulfate and / or copper(I) sulfate.

[0036] In a preferred embodiment, the salt of the substrate solution is typically NaCl, which is present in an amount of about 0.007 to 9 mg / mL, preferably about 7 mg / mL, in an even more preferred embodiment.

[0037] For the second substrate side compartment 3, a crosslinking medium is deposited thereon. For the purposes of the present invention, such a crosslinking medium comprises salts, a culture medium and distilled water.

[0038] In certain embodiments, the salts included in the crosslinking medium are selected from calcium acetate, calcium citrate, calcium chloride, calcium gluceptate, calcium gluconate, calcium nitrate, calcium phosphate, calcium hydrogen phosphate, hydroxyapatite, hydroxycarbonate apatite, tricalcium phosphate, octacalcium phosphate, potassium nitrate, zinc nitrate, magnesium nitrate, barium nitrate, titanium nitrate, iron(III) nitrate, copper nitrate, gallium nitrate, calcium nitrite, aluminum sulfate, aluminum sulfacetate, zinc sulfate, tetraamminecopper(II) sulfate, copper sulfate, iron(III) sulfate hydrate, iron sulfate, calcium sulfate and / or magnesium sulfate.

[0039] In a preferred embodiment, the salt is typically calcium gluconate, which is present in an amount of about 0.05 - 1.2% (w / v), preferably about 0.16% (w / v) in an even more preferred embodiment.

[0040] In a preferred embodiment, the culture medium is typically Mueller - Hinton and Luria - Bertani culture media.

[0041] In certain embodiments, the components of the "substrate" are dissolved in a bacterial culture medium or aqueous solution suitable for this purpose.

[0042] One skilled in the art knows how to adjust the thickness of the "substrate" by varying the volume of the solution introduced and the diameter of the first compartment 2.

[0043] As described above, in other embodiments, a pre - formed hydrogel containing the same components as described for the solution is introduced into the first apical side compartment (2).

[0044] For a second object, in fact, a method for preparing the "substrate" of the present invention is described.

[0045] In particular, such a method comprises - a diffusion system (1) comprising a first compartment (2) and a second compartment (3), wherein the first compartment (2) is placed above the second compartment (3), and the first and second compartments (2, 3) are separated by a semipermeable membrane (4) that allows salts to pass through but does not allow polysaccharides and proteins to pass through; - filling the first compartment (2) with a substrate solution or a preformed hydrogel containing a polysaccharide, a protein, and a salt; - filling the second compartment (3) with a crosslinking medium containing a salt, a medium, and distilled water, - incubating steps.

[0046] In particular, the substrate solution and the crosslinking medium are as described above.

[0047] In particular, the incubation continues for a time sufficient to allow salts to diffuse from the second basal compartment (3) through the semipermeable membrane (4) into the first apical compartment and to allow crosslinking. The time also controls the simultaneous formation of the gradient.

[0048] For example, the incubation can continue for a time between 2 minutes and 20 hours at a temperature of 4 to 25 °C.

[0049] In a preferred embodiment, the incubation continues for 20 hours at a temperature of 4 °C.

[0050] In a preferred embodiment, the "substrate" is prepared using a 5% (w / v) alginate solution prepared in a 7.07 mg / mL sodium chloride solution and 0.16% (w / v) calcium gluconate prepared in Mueller-Hinton culture medium acting as a crosslinking agent. Once the solutions are prepared, 500 μL of the alginate solution is introduced into the upper chamber of the diffusion device, while 6 mL of 0.16% (w / v) calcium gluconate is introduced into the lower chamber. Finally, the two-compartment diffusion chamber is left standing at a temperature of about 4 °C for 20 hours.

[0051] Advantageously, the preparation process does not expose the "substrate" to high temperatures or reagents incompatible therewith by filling with eukaryotic or prokaryotic cells, proteins or heat-labile substances.

[0052] Also advantageously, the present three-dimensional substrate obtained for the growth of microorganisms in pure culture or co-culture Adjustable is characterized by crosslinking, gas concentration and composition gradients.

[0053] The inventors have surprisingly shown that the addition of human serum albumin to the "substrate" increases its viscoelasticity. This observation indicates not only that the protein is denatured by the "substrate", but also the fact that the "substrate" can reproduce the physiological interactions of albumin in mucus.

[0054] This observation makes the "substrate" particularly advantageous for the mimicry or reproduction of mucus. In fact, currently available mucins cannot be crosslinked because they lose crosslinking sites during the extraction and purification process. Gelation of mucin is only possible under strongly acidic conditions that are incompatible with microbial culture. The "substrate" makes it possible to prepare hydrogels from commercially available mucins and is defined as "mucin-substrate".

[0055] Therefore, the "mucin-substrate" was used for the evaluation of the diffusion of active ingredients through the mucus barrier. The "substrate" has been shown to act as a barrier to the diffusion of active ingredients both in the presence and absence of mucin. The "substrate" also acts as a barrier to the diffusion of nanoparticles.

[0056] The technology for manufacturing the "substrate" makes it possible to apply the process to a preformed hydrogel, thus resulting in gradients that provide the viscoelasticity of interest for a particular application.

[0057] The use of "substrate" in an antibiogram enables obtaining more reliable results than those achieved by current methods for calculating the minimum inhibitory concentration (MIC) for both single-strain cultures and complex cultures (microbial cultures containing various genera, species, or strains). In fact, the "substrate" is a material that makes it possible to provide bacteria in a situation suitable for mimicking a three-dimensional matrix such as mucus to form aggregates or biofilms or to reproduce phenomena related to the spatial inhomogeneity of the medium. This is made possible by the three-dimensional structure of the "substrate" in addition to gradients of nutrients, oxygen, and water, which are factors generally not adjustable in the media found in the background art.

[0058] In certain embodiments, the "substrate" is separated from the diffusion system in which it is produced and placed within a high-strength support such as a cell culture plate or multi-well plate for high-throughput screening, enabling obtaining meaningful data with short-time analysis and simple operation while avoiding cross-contamination.

[0059] In certain embodiments, the "substrate" can be divided into multi-well plates of a desired size.

[0060] The following examples are to be construed as illustrative of the invention and not limiting thereof, the scope of which is defined by the appended claims.

Example

[0061] Example 1: "Substrate" with Added Human Serum Albumin Human serum albumin (HSA) was added to the alginate solution and mucin-alginate solution before crosslinking using a double-syringe method. 10 μL of a 56.2 mg / mL HSA solution was mixed with the alginate or mucin / alginate solution to give a final concentration of HSA of 1.182 mg / mL. The viscoelasticity of the hydrogel thus obtained is shown in Figure 2.

[0062] Example 2: Production and Characterization of "Mucin-Substrate" Add mucin to the "substrate" in the amounts shown in Table 1, where the mucin concentration is shown relative to the final product.

Table 2

[0063] Example 3: "Substrate" and "Mucin - Substrate 2" (Mus 3 SUB) The "substrate" having the properties described in Example 2 and Mus 3 SUB were tested for their ability to mimic a mucus barrier. In addition to gold nanoparticles, the active ingredients cephalexin and epirubicin were tested. The gold nanoparticles have a steric hindrance of approximately 25 nm, cephalexin has a steric hindrance of approximately 3 nm, and epirubicin has a steric hindrance of approximately 4 nm. The chart in Figure 4 shows the ability of the molecules to pass through a barrier consisting of a permeable support, the "substrate" or Mus 3 SUB. The results indicate that the translocation of the drug is decelerated when the drug has a strong interaction with mucin, as in the case of epirubicin (pKa = 8.01, logD7.4 = 0.03). In contrast, when the interaction with mucin is low, as in the case of cephalexin (pKa = 3.26 and 7.23, logD7.4 = -2.5), the translocation is extremely fast. This shows the ability to act as an interaction barrier and indicates that the mucin in the "substrate" maintains the ability to interact with the molecule. If the interactions are equal, the steric hindrance effect can be shown by comparing between gold nanoparticles (25 nm) and cephalexin (3 nm), and it is observed that the larger gold nanoparticles are decelerated by the presence of the "substrate".

[0064] Example 4: Production of a "substrate" having viscoelasticity and a differential gradient The "substrate" produced from a pre-crosslinked hydrogel is denoted with "double". Some of the "substrates" were manufactured in a two-compartment diffusion system using various cross-linking times to form a double cross-linked structure. Sodium alginate was dissolved at 2.8% (w / v) in an NaCl solution (16.33 mg / mL) over 12 hours with slow magnetic stirring. The alginate solution and distilled water were mixed at a 1:4 ratio using a double syringe method (Step 1). A suspension of calcium carbonate (7 mg / mL) in an NaCl solution (16.33 mg / mL) was sonicated for 5 minutes (UP200S, sonicator, Hielscher, Ultrasound Technology) and centrifuged at 3500 rpm for 1 minute (Vortex IKA MS3 100 - 240 V orbital stirrer) and further mixed with the solution prepared in Step 1 at a 1:5 ratio (Step 2). Finally, a GDL solution (10 mg / mL) was prepared in NaCl (16.33 mg / mL) and mixed with the solution prepared in Step 2 at a 1:6 ratio. Approximately 706 μL of the final mixture was inserted into the first apical side compartment and the mixture was allowed to react overnight ("substrate"-single). After overnight cross-linking, 0.2% calcium gluconate or calcium chloride was dissolved in 40 mM alizarin red and 6 mL of this solution was introduced into the second basal side compartment and reacted for various times, e.g., 5 minutes, 30 minutes, and 60 minutes as well as 20 hours to enable the formation of a gradient ("substrate"-double). Alizarin red is an organic red stain used for the identification of calcium deposition in cell cultures with a well-defined staining protocol. As seen in Figure 5A, the diffusion of calcium gluconate resulted in the formation of a calcium gradient with a deep red or mixed region (lower and upper parts of the sample respectively) within the "substrate", while the "substrate"-single was transparent and the "substrate"-double after 20 hours of the second cross-linking showed a uniform red (black in the figure). The viscoelastic changes induced by the second cross-linking of the "substrate" were also analyzed (Figure 5B). Both the storage and loss modules increased with the time of the second cross-linking and were more prominent between 60 minutes and 20 hours. In fact, the maximum value of the storage module was detected in the sample exposed to 20 hours of diffusion and no significant difference was seen between 30 minutes and 60 minutes of diffusion.

[0065] Example 5: The "substrate" mimics a physiological or pathological situation A bacterial starter culture was inoculated into an appropriate medium and cultured overnight. For inoculating the starter culture, in this case Pseudomonas aeruginosa, a small amount of frozen bacterial culture was mechanically removed from the original frozen vial stored at -80 °C, suspended in 10 mL of Mueller-Hinton (MH) medium, and then maintained at 37 °C overnight with stirring at 200 rpm. The next day, the estimation of the bacterial count was performed spectrophotometrically at λ = 600 nm (optical density 600; OD600). To prepare samples for the absorbance test, the suspension was diluted 1:10 with fresh MH medium. The same medium was used as a blank. Once the absorbance of the diluted sample was measured, the bacterial count was estimated using a calibration curve showing the bacterial count at the OD600 value. This concentration was then converted to the non-diluted medium concentration. To test the bacterial viability within the "substrate", each "substrate" was infected with 500, 1000, and 5000 bacteria with 24-hour and 48-hour incubations. For this purpose, 1 mL of the suspension was taken from the initial starter culture and diluted to the required concentration. The "substrate" was then infected by introducing 100 μL of the bacterial suspension and incubated at 37 °C under static conditions. CFU counting was used to test for the presence of bacteria within the "substrate", i.e., bacteria that migrated into and effectively proliferated within the "substrate", as compared to bacteria cultured under planktonic conditions. In this case, sterile cultures were performed both under planktonic conditions and in the "substrate". To estimate the number of bacteria that migrated and proliferated inside the "substrate", the residual bacteria seen on the top of the "substrate" and the well walls were removed by means of two washes with fresh MH medium. The "substrate" was then dissolved using 50 mM sodium citrate solution at pH 7.4. After dissolution, the suspension was diluted and seeded after CFU counting. Briefly, the medium was introduced into a 1.5 mL centrifuge tube and then diluted with an aqueous 0.9% sodium chloride solution, pH 7.4, at [10 -6 ~10 -10 fold dilutions. Then 10 μl of the diluted suspension was evenly distributed onto MH agar plates. The MH agar plate was left standing in an overnight incubator. The actual colonies in the infected "substrate" were calculated by multiplying by the dilution factor. For the "substrate", 150 μL of lysing agent was considered for calculating CFU / mL. The results are shown in Figure 6A. In further experiments, the "substrate" was inoculated with 10 8 bacteria necessary to obtain 4 bacteria / mL. After this time, the supernatant of the "substrate" was removed and the "substrate" was washed twice with fresh culture medium. Then, 100 μL of antibiotics at 3 concentrations of 0.1 MIC, 1 MIC and 10 MIC were added. The antibiotics were allowed to act for 24 hours under static incubation at 37 °C. After the effective antibiotic action time, viable CFU counts were performed. Various controls were carried out: (I) planktonic bacteria with antibiotics at each concentration; (II) planktonic bacteria without any treatment; (III) untreated infected "substrate"; and (IV) solution for "substrate" formation. For the infected "substrate" tested with antibiotics, even in the latter case, only the CFUs of the lysed "substrate" were taken into account. The data obtained are shown in Figure 6B.

[0066] Example 6: Adding Escherichia coli to the "substrate" prepared in Example 5 ("substrate"-single) The bacterial starter culture was inoculated into Luria-Bertani (LB) broth and cultured overnight. For inoculating the starter culture, in this case Escherichia coli, a small amount of frozen bacterial culture was mechanically removed from the original cryovial stored at -80 °C, suspended in 10 mL of Luria-Bertani (LB) broth medium, and then maintained at 37 °C overnight with stirring at 200 rpm. The next day, the estimation of the number of bacteria was carried out spectrophotometrically at λ = 600 nm (optical density 600; OD600). To prepare samples for the absorbance test, the suspension was flushed with fresh medium at a 1:10 ratio. The same medium was used as a blank. Once the absorbance of the diluted sample was measured, the number of bacteria was estimated using a calibration curve showing the number of bacteria at the OD600 value. This concentration was then converted to the non-diluted medium concentration. To test the bacterial viability in the "substrate", each "substrate" was infected with 500, 1000, and 5000 bacteria with a 24-hour incubation. For this purpose, 1 mL of the suspension was taken from the initial starter culture and diluted to the required concentration. Then, the "substrate" was infected by introducing 100 μL of the bacterial suspension and incubated at 37 °C under static conditions. Using the CFU count, the presence of bacteria in the "substrate", i.e., the bacteria that moved into and effectively proliferated in the "substrate", was tested in comparison with the bacteria cultured under planktonic conditions. In this case, sterile cultures were performed both under planktonic conditions and in the "substrate". To estimate the number of bacteria that moved into and grew inside the "substrate", the residual bacteria found on the top of the "substrate" and the well walls were removed by means of two washes with fresh medium. Then, the "substrate" was dissolved using a 50 mM sodium citrate solution at pH 7.4. After dissolution, the suspension was streaked and seeded after CFU counting. Briefly, the medium was introduced into a 1.5 mL centrifuge tube and then diluted with an aqueous 0.9% sodium chloride solution, pH 7.4, at a -6 ~10 -10 -fold dilution. Then, 10 μl of the diluted suspension was evenly distributed onto an LB agar plate. The LB agar plate was left in an incubator overnight. The actual colonies in the infected "substrate" were calculated by multiplying the dilution factor. In the case of the "substrate", 150 μL of the lysing agent was considered for calculating CFU / mL. The results are shown in Figure 7. Various controls were performed: (I) plankton-like bacteria with antibiotics at each concentration; (II) the "substrate" without infection; and (III) the solution for "substrate" formation.

[0067] Example 7: The "substrate" of the present invention was produced using a 5% (w / v) alginate solution prepared in a sodium chloride (7.07 mg / mL) solution and 0.16% (w / v) calcium gluconate prepared in Mueller-Hinton culture medium as a crosslinking agent. Once the solutions were prepared, 500 μL of the alginate solution was placed in the upper chamber of the diffusion device, while 6 mL of 0.16% (w / v) calcium gluconate was introduced into the lower chamber. Finally, the two-compartment diffusion chamber was stored at 4 °C for 20 hours overnight. In parallel, bacterial cultures were inoculated and cultured overnight. To inoculate the cultures, a small amount of frozen bacterial culture was mechanically taken from the original cryovial (-80 °C storage) suspended in 10 mL of Mueller-Hinton culture medium and maintained at 37 °C and 200 rpm. After overnight inoculation, the bacterial count was spectro-optically quantified at λ = 600 nm (OD600). Staphylococcus aureus ATCC 25923 and Pseudomonas aeruginosa PAO1 ATCC 15692 were cultured at a 1:1 ratio under substrate and plankton conditions and incubated at 37 °C for the evaluation of bacterial growth within the substrate. The "substrate" was infected with 100 μL of 103 Staphylococcus aureus and Pseudomonas aeruginosa at a 1:1 ratio. After 24 hours of culture, 150 μL of 50 mM trisodium citrate dihydrate, pH 7.4 was used to dissolve the substrate. After a 2-minute reaction, the new suspension was flushed out with a [10 -6 ~10 -10 - fold dilution with 0.9% NaCl solution, pH 7.4. Then, 10 μL of the diluted suspension was evenly dispensed onto Mueller-Hinton medium agar plates and incubated at 37 °C overnight. Then, colony-forming units (CFUs) were calculated taking into account the dilution count. The results are shown in Figure 8. In a suspension of the culture medium (plankton-like conditions), Staphylococcus aureus could not be cultured in the co-presence of Pseudomonas aeruginosa. Only Pseudomonas aeruginosa was counted (black bars), while Staphylococcus aureus was undetectable (ND in Figure 8). If the co-culture was carried out on the substrate of the present invention (grey bars), both bacteria were viable and could form colonies. This result represents the co-existence state of the infections of the two bacteria in chronic infections only when the substrate of the present invention is used, rather than under standard culture conditions, indicating the possibility of testing the competitive effects.

[0068] Example 8: The "substrate" of the present invention was prepared using a 5% (w / v) alginate solution prepared in a 7.07 mg / mL sodium chloride solution and 0.16% (w / v) calcium gluconate prepared in Mueller-Hinton culture medium acting as a cross-linking agent. Once the solution was prepared, 250 or 500 μL of the alginate solution was introduced into the upper chamber of the diffusion device, while 6 mL of 0.16% (w / v) calcium gluconate was introduced into the lower chamber. The "substrate" prepared using 250 μL of the alginate solution was designated as "thin", while that prepared using 500 μL was termed "thick". Finally, the two-compartment diffusion chamber was stored at 4 °C overnight for 20 hours. The O2 tension within the "substrate" was measured using a Clark-type oxygen sensor (OX-25; Unisense, Aarhus N, Denmark) connected to a highly sensitive picoampere 4-channel amplifier designated as Microsensor Multimeter S / N 8678 (Unisense). O2 diffuses into the environment through a silicone membrane with the sensor chip, is reduced at the gold cathode surface, and thus generates an electric current. The picoammeter converts the obtained reduction current into a signal. The signal from the oxygen sensor is prepared in picoamperes. Therefore, the oxygen sensor needs to be connected to the picoampere amplifier during measurement. Subsequently, the Unisense SensorTrace software automatically converts the signal from the partial pressure (oxygen partial pressure) to an equivalent oxygen concentration (μmol / L). Before measurement, the electrodes, i.e., the reference anode and the guard cathode, were polarized overnight and further calibrated with air-saturated water (positive control) and 2% w / v sodium dithionite (negative control). Before measurement, low melting point agarose consisting of 2% (w / v) agarose in 7.07 mg / mL NaCl was placed in a Petri dish. The newly prepared "substrate" was placed on the agarose layer. The microelectrode was placed using a motorized micromanipulator (MXU2; PyroScience, Aquisgrana, Germany). Measurements were carried out at the center of the "substrate" from its surface (0 mm) through its thickness until the tip was completely penetrated into the structure. To avoid oxygen diffusing from the side of the "substrate", an O-ring with an inner diameter corresponding to the "substrate" diameter was used. The maximum depth reached by the tip, called the final depth, was approximately 2200 μm and 3200 μm for the "thin" and "thick" "substrates", respectively. Successive points were taken 100 μm apart. Each point was carried out 3 times. The results are shown in Figure 9. A continuous decrease in oxygen partial pressure was observed through the thickness of both types of "substrates", with variations of 83.0 μmol / L and 70.3 μmol / L for the "thin" and "thick" "substrates", respectively. A large decrease in oxygen partial pressure was seen in the thin "substrate". This result indicates the possibility of obtaining an oxygen gradient within the same substrate.

[0069] Example 9: The "substrate" of the present invention was prepared using a 5% (w / v) alginate solution prepared in a 7.07 mg / mL sodium chloride solution and 0.16% (w / v) calcium gluconate prepared in Mueller-Hinton culture medium acting as a cross-linking agent. Once the solutions were prepared, 500 μL of the alginate solution was introduced into the upper chamber of the diffusion device, while 6 mL of 0.16% (w / v) calcium gluconate was introduced into the lower chamber. Finally, the two-compartment diffusion chamber was stored at 4 °C overnight for 20 hours. In parallel, bacterial cultures were inoculated and cultured overnight. To inoculate the cultures, a small amount of frozen bacterial culture was mechanically taken from the original frozen vial (-80 °C storage) suspended in 10 mL of Mueller-Hinton culture medium and maintained at 37 °C and 200 rpm. After overnight inoculation, the bacterial count was spectro-optimically quantified at λ = 600 nm (OD600). 100 μL of Pseudomonas aeruginosa PAO1 ATCC 15692 was cultured in the "substrate" at a bacterial count equal to 103, and the oxygen partial pressure was measured as described in Example 2 after 12 hours, 24 hours, and 48 hours of infection. The results are shown in Figure 10. The oxygen partial pressure profile inside the "substrate" continued to decrease with the incubation time, and after 48 hours, an anoxic condition was achieved at a depth of about 300 μm. As the culture time increased, for example, from 12 hours to 24 hours, the oxygen partial pressure difference between the shallowest layer and the innermost layer became gradually more significant. This result shows the synergy of the bacteria and the "substrate" matrix in reconstructing the culture conditions suitable for a specific type of bacteria.

[0070] Example 10: The "substrate" of the present invention was prepared using a 5% (w / v) alginate solution prepared in a 7.07 mg / mL sodium chloride solution and 0.16% (w / v) calcium gluconate prepared in Mueller-Hinton culture medium acting as a cross-linking agent. Once the solutions were prepared, 500 μL of the alginate solution was introduced into the upper chamber of the diffusion device, while 6 mL of 0.16% (w / v) calcium gluconate was introduced into the lower chamber. Finally, the two-compartment diffusion chamber was stored at 4 °C overnight for 20 hours. To express the fluorescent protein, Pseudomonas aeruginosa PA01 was transformed with a plasmid by the process suggested by Cadoret et al. (2014). Briefly, electroporation of electrocompetent Pseudomonas aeruginosa and the fluorescent plasmid pMF440. Pseudomonas aeruginosa was subjected to electroporation by means of a series of dilution steps in electroporation buffer (300 mM sucrose solution) from an overnight culture in Luria-Bertani (LB). The overnight culture was pelleted and resuspended in electroporation buffer. The suspension was then recentrifuged and resuspended in fresh electroporation buffer at half the volume of the initial culture. This process was repeated until the volume was reduced to 0.01 of the initial volume. These steps aim to provide the bacteria with a high-nutrient environment for growth even in the absence of ions that can create an electric arc during the electroporation process. Plasmid pMF440 was obtained from the database Addgene (ID code: 62550) as a bacterial transformation of Escherichia coli DH5α. For plasmid DNA extraction, Escherichia coli was cultured in LB containing 100 μg / mL ampicillin. Plasmid DNA was extracted using a QIAprep Spin Miniprep (Qiagen) kit according to the manufacturer's instructions. After extraction, the DNA was eluted with MilliQ water and stored at -20 °C until needed. The DNA was extracted with deionized water to achieve a concentration of 0.1 - 1 μg / mL. 80 mL of electrocompetent Pseudomonas aeruginosa cell suspension was mixed with 10 mL of DNA suspension, and this suspension was placed on ice for 30 minutes before electroporation. The electroporation process was carried out using a gene pulser electroporation system with a conductive cuvette. An electronic impulse was applied at 2.5 kV, 200 Ω, 25 μF for 5 milliseconds. After this shock, the cuvette contents were transferred to a Falcon tube containing 2 mL of optimal glucose-enriched broth and incubated at 37 °C for 2 hours under stirring conditions. After 2 hours of recovery, the suspension was plated on selective agar plates containing 300 μg / mL carbenicillin. Further culturing and expansion of the transformed bacteria were carried out in carbenicillin-containing medium. Bacterial cultures were inoculated and cultured overnight. To inoculate the cultures, a small amount of frozen bacterial material was mechanically taken from the original cryovial (-80 °C storage) suspended in 10 mL of Mueller-Hinton culture medium and maintained at 37 °C and 200 rpm. After overnight inoculation, the bacterial count was spectrophotometrically quantified at λ = 600 nm (OD600). 100 μL out of 103 Pseudomonas aeruginosa PACC 1 ATCC 15692 expressing green was cultured in the "substrate" and incubated at 37 °C. The bacterial tissue was evaluated by confocal microscopy. After 24 hours of incubation, the samples were observed under a confocal laser microscope. Scanning was performed using excitation with a wavelength of 587 nm and a maximum depth of approximately 50 μm. The augmentation was analyzed using the LasX software provided by Leica. The results are shown in Figure 11. The "substrate" enables the formation of bacterial aggregates similar to those observed in human infections, while these aggregates cannot be obtained under suspension culture conditions. Bacteria respond to gradients by organizing themselves into multicellular aggregates with a morphology and size similar to those previously observed in cystic fibrosis sputum and chronic wounds. These bacterial aggregates correlate with the oxygen gradient and then result in the production of alginate by Pseudomonas aeruginosa, the latter being considered the root cause of chronic infections by this pathogen.

[0071] From the above, the advantages of the present invention will be immediately apparent to those skilled in the art.

[0072] The "substrate" of the present invention has properties that are beneficial for applications in the fields of food, environment, and health. The "substrate" also includes, as examples, cellulose, polyester, insulin, antibacterial, antifungal, antiviral, antithrombotic agents, immunomodulatory substances, anticancer agents, enzyme inhibitors, insecticides, herbicides, fungicides (such as alkaloids and flavonoids), and substances that promote the growth of animals and plants, and is also used in the field of products related to bacteria. As an example, the "substrate" is beneficially used for analysis aimed at monitoring bacterial growth, interactions between microbial communities, and / or the antibacterial ability of drugs and micro / nanoparticles, pollutants, probiotics, and probiotic release. Further, the three-dimensional substrate is suitable for atmospheric research to understand microbial adaptation to space environments such as microgravity and high radiation levels, in addition to screening methods for selecting effective antibacterial treatment agents and initial steps in the search for new drugs. For example, understanding how a certain diet therapy and / or probiotics and a probiotic carrier as a dietary supplement affect bacterial behavior and microbial communities in the food industry, and environmental science for evaluating pollutants or biocides in detergents related to microbial communities, products for increasing agricultural production, or environmental pollution remediation are further applications. Other examples include the cosmetics field, such as cosmetics that release skin-affecting microorganisms. The "substrate" can also be used as a method for reproducing the microenvironment of microorganisms in a microbial pad for treating water and reducing pollution. The co-culture of different microorganisms in a three-dimensional substrate can also be used in the production of biofuels and bioenergy conversion. This is because these applications require the physical and spatial configuration of microorganisms. Finally, the three-dimensional substrate can also be used as a microbial fuel cell.

[0073] The use of the three-dimensional substrate described herein is immediately understandable and does not require any technical experience or special equipment. Furthermore, the three-dimensional substrate of the present invention is compatible with commonly used platforms such as 6-well, 12-well, 24-well, 48-well, 96-well or 384-well multi-well plates, Transwell® systems and microfluidic devices. The three-dimensional substrate is an absolute competitive cost, and its production can be easily modified according to the research purpose in terms of viscoelasticity and gradient, can be scaled up, and the modules can be exchanged.

[0074] Experiments using the three-dimensional substrate of the present invention have given realistic results with high reproducibility.

[0075] The "substrate" has further advantages summarized below compared to synthetic or agar-based materials. - The components are carbon sources for bacteria and in some cases are produced by the bacteria themselves. For example, Pseudomonas aeruginosa secretes alginate. - The "substrate" cultures bacteria in a three-dimensional gradient structure that is not achievable by current methods. - Microorganisms cultured on the "substrate" are supported on a high-intensity support (e.g., a high-throughput screening plate), and meaningful data can be obtained with short analysis time and minimal manual work while avoiding cross-contamination. - In the preparation process, the use of high temperatures or reagents that are incompatible with the encapsulation of microorganisms (e.g., bacterial cells) or the formation of a single cell layer is avoided. - The preparation process avoids the use of high temperatures or reagents that are incompatible with the addition of eukaryotic or prokaryotic cells, proteins or thermally unstable substances. - The reaction rate control of the "substrate" enables the effective division of the "substrate" into multi-well plates with 6 - 96 or 384 wells of different sizes later.

[0076] Agar gels available in the prior art, especially 0.2 - 0.8% w / v agar gels, have the potential for three-dimensional microorganism culture but have some limitations. - Without showing a gradient, it remains in the arrangement on a two-dimensional hard agar plate; - In soft agar gels, the motility of bacteria and the possibility of diffusion of substances into the gel are shown. However, they are placed on a hard agar substrate (1.5 - 2.0% w / v). As a result, cells can move partially in three dimensions because they are blocked by the basal level. - The addition of soft agar gels to a high-strength support (high-throughput screening plate) and the interpretation of results require manual work and complex configurations, increasing the possibility of contamination, and the screening ability related to cost efficiency is limited compared to the "substrate" of the present invention. - Agar needs to be boiled to completely dissolve and maintained at 50 - 55 °C to maintain a liquid form until inoculation and plating. These temperatures are disadvantageous to many microorganisms, affect the heat-labile components of the medium, and may require enriching the culture composition (such as proteins and vitamins). The "substrate" was considered in terms of synthesis and polymerization under conditions that do not affect the encapsulation of heat-labile components or microorganisms (such as bacterial cells) or the layering on cell monolayers.

[0077] Finally, the three-dimensional substrate of the present invention, which consists of a water-soluble polymer of natural origin and is prepared without using toxic solvents, is an environmentally friendly device. Furthermore, the present invention includes the following aspects. 1. A diffusion system (1) for preparing a microbial growth substrate having a modular cross-linking gradient, comprising a first top-side compartment (2) and a second bottom-side compartment (3), wherein the first compartment (2) is placed on top of the second compartment (3), a substrate solution containing a polysaccharide, a protein and a salt is contained in the first top-side compartment (2), a cross-linking medium containing a salt, a medium and distilled water is contained in the second bottom-side compartment (3), and the first and second compartments (2, 3) are separated by a semi-permeable membrane (4) that allows salts to permeate but does not allow polysaccharides and proteins to permeate, the diffusion system (1). 2. The diffusion system (1) according to item 1, wherein the semi-permeable membrane is made of polycarbonate, polystyrene, poly(diallyldimethylammonium chloride), polyethylene terephthalate or polyamide (nylon). 3. The diffusion system (1) according to item 1 or 2, wherein the polysaccharide in the substrate solution is selected from sodium alginate of various molecular weights, pectin of various molecular weights and various degrees of esterification and amidation, hyaluronic acid of various molecular weights, gellan of various molecular weights, and dextran of various molecular weights. 4. The diffusion system (1) according to any one of items 1 to 3, wherein the solution has a viscosity of 0.05 to 100 Pa·s, or 0.2 to 10 Pa·s. 5. The diffusion system (1) according to any one of items 1 to 4, wherein in the substrate solution, the protein is selected from mucin, serum albumin, fibrinogen, fibronectin, collagen, elastin, insulin, and transferrin. 6. The diffusion system (1) according to any one of items 1 to 5, wherein in the substrate solution, the salt is selected from sodium chloride, ammonium phosphate, potassium chloride, sodium hydrogen phosphate, sodium bicarbonate, potassium chloride, dipotassium hydrogen phosphate trihydrate, magnesium chloride hexahydrate, sodium sulfate, tris(hydroxymethyl)aminomethane, sodium nitrate, sodium nitrite, potassium nitrate, silver nitrate, ammonium nitrate, calcium nitrite, potassium bisulfate, potassium sulfate, sodium bisulfate, sodium sulfate and / or copper(I) sulfate. 7. In the base material solution, the salt is selected from calcium acetate, calcium citrate, calcium chloride, calcium gluconate, calcium glyceptate, calcium gluceptate, calcium nitrate, calcium phosphate, calcium hydrogen phosphate, hydroxyapatite, hydroxycarbonate apatite, tricalcium phosphate, octacalcium phosphate, potassium nitrate, zinc nitrate, magnesium nitrate, barium nitrate, titanium nitrate, iron(III) nitrate, copper nitrate, gallium nitrate, calcium nitrite, aluminum sulfate, aluminum sulfacetate, zinc sulfate, tetraamminecopper(II) sulfate, copper sulfate, iron(III) sulfate hydrate, iron sulfate, calcium sulfate and / or magnesium sulfate, and is any of the diffusion systems (1) of Items 1 to 6. 8. A preformed hydrogel containing a polysaccharide, a protein, and a salt is inserted into the first apical side compartment (2) instead of the base material solution, and is any of the diffusion systems (1) of Items 1 to 7. 9. A method for producing a three-dimensional substrate for microorganism culture, - providing a diffusion system (1) including a first compartment (2) and a second compartment (3), wherein the first compartment (2) is placed above the second compartment (3), and the first and second compartments (2, 3) are separated by a semipermeable membrane (4) that allows salts to permeate but does not allow polysaccharides and proteins to permeate; - filling the first compartment (2) with a base material solution or a preformed hydrogel containing a polysaccharide, a protein, and a salt; - filling the second compartment (3) with a crosslinking medium containing a salt, a culture medium, and distilled water; - incubating for a time sufficient to allow the salt to diffuse from the second base side compartment (3) through the semipermeable membrane (4) into the first apical side compartment and to crosslink. The method includes the above steps. 10. The method of Item 9, wherein the base material solution contains a polysaccharide represented by alginate and a protein represented by mucin. 11. The method of Item 9 or 10, wherein the crosslinking medium contains a salt represented by calcium gluconate. 12. The method of any of Items 9 to 11, wherein the crosslinking medium contains a culture medium selected from Mueller-Hinton and Luria-Bertani. 13. The method of any of Items 9 to 12, wherein the incubation is continued for a time of 2 minutes to 20 hours at a temperature of 4 to 25 °C, preferably 20 hours at 4 °C. 14. A three-dimensional substrate for growing microorganisms in pure culture or co-culture, characterized by module crosslinking, gas concentration, and compositional gradients, obtained by the method of item 13. 15. A method for culturing a microbial culture, comprising the use of a three-dimensional substrate obtained by any of the methods of items 9 to 14. 16. The method of item 15, wherein the microbial culture is a bacterial culture that may contain various strains.

Claims

**Claim 1**: A three-dimensional substrate having a structural gradient and a compositional gradient for the growth of microorganisms, comprising a polysaccharide, a protein, and a salt that form a gel having a cross-linking gradient, wherein - the polysaccharide is selected from sodium alginate and pectin; the protein is mucin; the salt is selected from sodium chloride and calcium chloride or is calcium gluconate, and the culture medium is selected from Mueller-Hinton and Luria-Bertani; - the culture medium and the salt are in the range of a high concentration at the bottom to a low concentration at the surface of the three-dimensional substrate; - the oxygen partial pressure is from 290 μmol / L at the surface to 195 μmol / L at the bottom, - the substrate has a storage modulus G' of 10 Pa to 3,000 Pa at a shear strain frequency of 0.01 Hz to 100 Hz, a three-dimensional substrate.

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