CN115851575A - Culture medium for culturing stomach organoid and application thereof - Google Patents

Abstract

The present disclosure relates to the technical field of organoid culture, in particular to a medium for culturing gastric organoids and the application of the medium for culturing mouse gastric organoids. The culture medium is a basic medium and specific addition factors; the specific addition factors are SB505124, artificial gastric juice and amphotericin B; wherein, the addition amount of the specific addition factors accounts for 0.02-0.2% of the total amount of the base medium. The culture method shown in the present invention helps to solve the problem of bacterial and fungal contamination during the culture of mouse gastric organoids, and provides a more optimized way for the culture of mouse gastric organoids.

Description

A kind of medium for gastric organoid culture and application thereof

technical field

The present disclosure relates to the technical field of organoid culture, in particular to a medium for culturing gastric organoids and the application of the medium for culturing mouse gastric organoids.

Background technique

Gastric epithelium is the main site of gastric diseases, so gastric epithelial cells are an important research object of gastric diseases. For a long time, research on gastric inflammation and diseases has been mainly based on cell or animal models, most of which are gastric epithelial cells and mouse models. Gastric epithelial cell GES-1 has been successfully constructed so far, which can be subcultured in vitro for a long time, but its epithelial cell type is single. Because the structure of mice is similar to that of humans, mouse models are also widely used in the study of stomach-related diseases.

The gastric structure is divided into mucosal layer, submucosa layer, muscular layer, and serosa layer. The mucous layer contains glands in which stem cells reside and have the ability to self-renew. Organoid is a collection of epithelial cells developed from stem cells. It is a three-dimensional structure that simulates epithelial cells in vitro. Compared with traditional two-dimensional structures, organoids have spatial extension and cell polarity has also changed. The cell composition and function of organoids are similar to those of epithelium, and are closer to the physiological state of cells in vivo. Compared with a single primary cell culture, organoids contain more types of epithelial cells, which can better simulate the physiological conditions of the internal structure of the human body.

Mouse gastric organoids are obtained by extracting adult stem cells from gastric gland cells, that is, Lgr5+ stem cells, adding certain growth factors, and culturing them into a 3D model. The cultured gastric organoids can contain a variety of epithelial cell types, such as E-cardherin, PCNA, Lgr5, etc., which can well simulate the physiological state of mouse stomach in vitro. Gastric organoids are richer in epithelial cell types than traditional epithelial cell cultures. Therefore, gastric organoids can provide a more reliable model for studying the physiological and pathological mechanisms of the stomach. With the continuous development of organoid technology, researchers have successfully established methods for culturing mouse liver, lung, and intestinal organoids, but there are few reports on 3D organoid models of the stomach.

Contents of the invention

The purpose of the present disclosure is to provide a medium for organ culture and the application of the medium for culturing mouse gastric organoids.

In order to achieve the above object, the technical solution adopted by the present invention is:

A medium for culturing gastric organoids, the medium is basal medium and specific added factors; the specific added factors are SB505124, artificial gastric juice and amphotericin B; wherein, the added amount of specific added factors accounts for the base 0.02-0.2% of the total medium.

The final concentration of SB505124 in the medium is 1-10 μM; the final concentration of amphotericin B is 5 μg; 2-20 μl of artificial gastric juice is added to every 10 ml of medium.

The artificial gastric juice, also known as gastric juice simulated liquid, is composed of saline and hydrochloric acid, etc., does not contain enzymes, and can be used to check the time limit of drug disintegration. The sterile solution can simulate the gastric juice environment of the human body.

The SB 505124, SB 505124 is a selective inhibitor of TGF-β type I receptors ALK4, ALK5, ALK7, and the transforming growth factor β signaling pathway is a signal transmission process mediated by transforming factors. It plays an important regulatory role in the growth, differentiation, apoptosis, and organ formation of cells and tissues. At the same time, SB505124 can effectively block the growth inhibition induced by transforming growth factor β, and at the same time inhibit the phosphorylation of Smad, maintain the stemness of stem cells, and inhibit cell apoptosis and senescence.

Add 5ml of Wnt conditioned medium to each 10ml of basal medium; 1 μg of recombinant mouse Noggin; 500 ng of recombinant mouse epidermal growth factor; 1 μg of recombinant mouse fibroblast growth factor-10; 10 μg of recombinant mouse R-Spondin 1; Glutamine 10nM; Niacinamide 10mM; N-acetylcysteine 1mM; Penicillin/Streptomycin 100μl; B27 200μl; the balance was filled with Advanced DMEM F12.

Preparation of Wnt conditional medium in the basal medium:

a: Take a portion of L-Wnt-3A cells preserved in liquid nitrogen and inoculate them in Wnt basal medium (Wnt basal medium is 5ml DMEM+10%FBS+G418 (0.4mg/ml)) %CO ₂ incubator, cultivate for 1-2 days until the cells cover the entire bottom;

b: After the cells are congested, passage to the T75 cell culture flask according to 1:2, add 15ml DMEM+10% FBS, and culture for 2-3 days until the cells cover the entire bottom;

c: After the cells are congested, passage to T175 cell culture flask according to 1:3, add 25ml Wnt final medium (by volume, 10% FBS + 1% HEPES + 1% GlutaMAX + 1% P/S, the rest Filled with Advanced DMEM F12), cultured continuously for 3 days in a 5% CO ₂ incubator at 37°C, collected the medium by filtration, then added fresh Wnt final medium to the culture bottle, cultured for two days according to the above culture conditions, and collected by filtration Culture medium;

d: Mix and centrifuge the two culture media collected above, collect the supernatant and filter, aliquot according to the experimental amount and store at -80°C to avoid repeated freezing and thawing.

An application of the medium, the application of the medium in culturing mouse gastric 3D organoids.

Use of a medium to inhibit the use of fungi in culturing gastric organoids.

The fungus is Aureobasidium pullulans.

A method for culturing mouse gastric organoids. The cells to be cultured are resuspended in Advanced DMEM F12 to obtain a single cell suspension (concentration of 1×10 ⁵ /L), and then the single cell suspension is mixed with Matrigel Mix according to the volume ratio of 1:1, solidify after mixing, then add the medium described in claim 1, cultivate it at 37°C and 5% CO ₂ environment, change the medium once every two days, and obtain it in 5-7 days Mouse gastric organoids.

The mouse gastric tissue pieces were washed with DPBS, and the precipitate was collected by natural sedimentation. Digestion solution was added to the precipitate, and it was placed in a 5% CO ₂ incubator at 37°C for 1 hour of digestion, and the precipitate was collected for use.

100 mg of type IV collagenase, 50 mg of hyaluronidase, 2 ml of FBS, and 100 μl of penicillin/streptomycin in each 10 ml of digestive juice.

Further said:

1. Take the mouse stomach, shave off the surrounding mesentery, cut it along the greater curvature of the stomach, and clean the contents. Remove the muscle layer under a stereomicroscope, then cut the mouse stomach tissue into 5mm ² fragments, and collect them in a centrifuge tube; wash with ice DPBS, let the tissue settle naturally, discard the supernatant, repeat the washing until the supernatant is clear, and then separate the tissue The fragments were transferred to a sterile petri dish, cut into 2mm ² tissue fragments, and continued to wash with ice DPBS until the supernatant was clear.

2. The mouse stomach was digested by the combination of glass slide and digestive juice. That is, the digestion solution was first added to a 37°C CO ₂ incubator for digestion for 1 hour, the supernatant was discarded after standing still, and the digestion was terminated with DPBS containing 0.1% BSA. Collect the tissue fragments at the bottom of the centrifuge tube and put them into a 10mm ² sterile petri dish, press them with a sterile glass slide, and you can see the release of cloudy glands.

3. Collect all the pressed glands and tissue fragments into a centrifuge tube, resuspend them with Advanced DMEM F12, filter them with a 200-mesh cell mesh, and after 3-4 times, blow the cells evenly with a pipette. Make a single cell suspension.

4. Centrifuge the single cell suspension at 1000rpm at 4°C for 10min, discard the supernatant, add an appropriate amount of Advanced DMEM F12 to resuspend it, then add Matrigel at a ratio of 1:1, mix well and inoculate with 50μl per well until the preheated 24 in the orifice plate. When inoculating, be careful not to touch the wall or bottom of the well, and inoculate to the center of the 24-well plate in a round drop shape.

5. Put the inoculated 24-well plate into the CO ₂ incubator stably and incubate for 30 minutes to make the Matrigel completely solidify.

6. After the Matrigel is solidified, add 500 μl of the organoid culture medium to each well of the 24-well plate, culture at 37° C. and 5% CO ₂ , and completely change the medium every two days. After 3-5 days of culture, budding of organoids can be seen. Mouse gastric organoids with a diameter of about 50 μm can be obtained in 5-7 days.

The beneficial effects of the present invention are:

① The culture medium of the present invention contains the minimum components required for the culture of mouse gastric organoids, and can cultivate organoids derived from normal gastric tissues of mice.

② The medium of the present invention uses Wnt conditioned medium, which is derived from mouse L-Wnt 3A cells and has high homology. Secondly, the conditioned medium is obtained by culturing Wnt cells, which is more economical than directly purchasing recombinant cytokines.

③ The culture medium of the present invention is added with artificial gastric juice to promote the growth of organoids and help maintain a good growth state.

④ SB505124 is added to the culture medium of the present invention, which can inhibit the growth of fungi and promote the germination of organoids.

⑤ Amphotericin B is added to the medium of the present invention, which has the effect of preventing fungal contamination, and can effectively solve the problem of bacterial and fungal contamination in the organoid culture process.

⑥ When the medium of the present invention cooperates with the culture method, when the number of single cell suspension obtained after digestion is 1×10 ³ , mouse gastric organoids can still be cultured.

Description of drawings

Figure 1 is a diagram of the gland obtained after digesting and pressing the slide;

Figure 2 is a picture under an optical microscope for 7-day culture of mouse gastric organoids using the culture medium of the embodiment of the present invention;

Fig. 3 is a picture under an optical microscope of mouse gastric organoids cultured for 7 days using the medium of the present invention in Example 3;

Fig. 4 is a picture under an optical microscope of mouse gastric organoids cultured in Example 4 of the embodiment medium of the present invention for 7 days;

Fig. 5 is a picture under an optical microscope of mouse gastric organoids cultured in the culture medium of Comparative Example 1 for 7 days;

Fig. 6 is a picture under an optical microscope of mouse gastric organoids cultured in the medium of Comparative Example 2 for 7 days.

Detailed ways

The specific embodiments of the present invention will be further described below in conjunction with examples. It should be noted that the specific embodiments described here are only for illustrating and explaining the present invention, and are not intended to limit the present invention.

Example 1

Organoid culture medium is basal medium and specific additive factors; 10 μl of artificial gastric juice is added to every 10 ml of basal medium; SB505124 2 μM; amphotericin B 5 μg.

5ml of Wnt conditioned medium in every 10ml of basal medium in the basal medium; 1 μg of recombinant mouse Noggin; 500 ng of recombinant mouse epidermal growth factor; 101 μg of recombinant mouse fibroblast growth factor; 10 μg of recombinant mouse R-Spondin 1; Gastrin 10nM; glutamine 10nM; nicotinamide 10mM; N-acetylcysteine 1mM penicillin/streptomycin 100μl; B27 200μl; the balance was filled with Advanced DMEM F12.

The preparation process of the Wnt conditioned medium is as follows:

a: Take a portion of L-Wnt-3A cells stored in liquid nitrogen, resuscitate them in a T25 cell culture flask, and then add 5ml of Wnt basal medium (Wnt basal medium is 5ml DMEM+10%FBS+G418 (0.4mg /ml)), in a 5% CO ₂ incubator at 37°C, cultivate for 1-2 days until the cells cover the entire bottom;

b: After the cells are confluent, passage to the T75 cell culture flask at a ratio of 1:2, add 15ml DMEM+10%FBS, and culture for 2-3 days until the cells cover the entire bottom;

c: After the cells are congested, passage to T175 cell culture flask according to 1:3, add 25ml Wnt final medium (by volume, 10% FBS + 1% HEPES + 1% GlutaMAX + 1% P/S, the rest Filled with Advanced DMEM F12), cultured continuously for 3 days in a 5% CO ₂ incubator at 37°C, collected the medium by filtration, then added fresh Wnt final medium to the culture bottle, cultured for two days according to the above culture conditions, and collected by filtration Culture medium;

d: Mix and centrifuge the two culture media collected above, collect the supernatant and filter, aliquot according to the experimental amount and store at -80°C to avoid repeated freezing and thawing.

Example 2

Organoid culture medium is basal medium and specific additive factors; 2 μl of artificial gastric juice is added to every 10 ml of basal medium; 1 μM of SB505124; 5 μg of amphotericin B.

5ml of Wnt conditioned medium in every 10ml of basal medium in the basal medium; 1 μg of recombinant mouse Noggin; 500 ng of recombinant mouse epidermal growth factor; 101 μg of recombinant mouse fibroblast growth factor; 10 μg of recombinant mouse R-Spondin 1; Gastrin 10nM; glutamine 10nM; nicotinamide 10mM; N-acetylcysteine 1mM; penicillin/streptomycin 100μl; B27 200μl; the balance was filled with Advanced DMEM F12.

The preparation process of the Wnt conditioned medium is as follows:

a: Take a portion of L-Wnt-3A cells stored in liquid nitrogen, resuscitate them in a T25 cell culture flask, and then add 5ml of Wnt basal medium (Wnt basal medium is 5ml DMEM+10%FBS+G418 (0.4mg /ml)), in a 5% CO ₂ incubator at 37°C, cultivate for 1-2 days until the cells cover the entire bottom;

b: After the cells are confluent, passage to the T75 cell culture flask at a ratio of 1:2, add 15ml DMEM+10%FBS, and culture for 2-3 days until the cells cover the entire bottom;

c: After the cells are congested, passage to T175 cell culture flask according to 1:3, add 25ml Wnt final medium (by volume, 10% FBS + 1% HEPES + 1% GlutaMAX + 1% P/S, the rest Filled with Advanced DMEM F12), cultured continuously for 3 days in a 5% CO ₂ incubator at 37°C, collected the medium by filtration, then added fresh Wnt final medium to the culture bottle, cultured for two days according to the above culture conditions, and collected by filtration Culture medium;

d: Mix and centrifuge the two culture media collected above, collect the supernatant and filter, aliquot according to the experimental amount and store at -80°C to avoid repeated freezing and thawing.

Example 3

Organoid culture medium is basal medium and specific additive factors; 20 μl of artificial gastric juice is added to every 10 ml of basal medium; SB505124 10 μM; amphotericin B 5 μg.

5ml of Wnt conditioned medium in every 10ml of basal medium in the basal medium; 1 μg of recombinant mouse Noggin; 500 ng of recombinant mouse epidermal growth factor; 101 μg of recombinant mouse fibroblast growth factor; 10 μg of recombinant mouse R-Spondin 1; Gastrin 10nM; glutamine 10nM; nicotinamide 10mM; N-acetylcysteine 1mM; penicillin/streptomycin 100μl; B27 200μl; the balance was filled with Advanced DMEM F12.

The preparation process of the Wnt conditioned medium is as follows:

a: Take a portion of L-Wnt-3A cells stored in liquid nitrogen, resuscitate them in a T25 cell culture flask, and then add 5ml of Wnt basal medium (Wnt basal medium is 5ml DMEM+10%FBS+G418 (0.4mg /ml)), in a 5% CO ₂ incubator at 37°C, cultivate for 1-2 days until the cells cover the entire bottom;

b: After the cells are confluent, passage to the T75 cell culture flask at a ratio of 1:2, add 15ml DMEM+10%FBS, and culture for 2-3 days until the cells cover the entire bottom;

c: After the cells are congested, passage to T175 cell culture flask according to 1:3, add 25ml Wnt final medium (by volume, 10% FBS + 1% HEPES + 1% GlutaMAX + 1% P/S, the rest Filled with Advanced DMEM F12), cultured continuously for 3 days in a 5% CO ₂ incubator at 37°C, collected the medium by filtration, then added fresh Wnt final medium to the culture bottle, cultured for two days according to the above culture conditions, and collected by filtration Culture medium;

d: Mix and centrifuge the two culture media collected above, collect the supernatant and filter, aliquot according to the experimental amount and store at -80°C to avoid repeated freezing and thawing.

Example 4

Validation of the effect of SB505124:

Organoid culture medium is basal medium and specific additive factors; 20 μl of artificial gastric juice is added to every 10 ml of basal medium; different amounts of SB505124 (2 μM and 10 μM) are added. At the same time, no SB505124 was added as a control.

5ml of Wnt conditioned medium in every 10ml of basal medium in the basal medium; 1 μg of recombinant mouse Noggin; 500 ng of recombinant mouse epidermal growth factor; 101 μg of recombinant mouse fibroblast growth factor; 10 μg of recombinant mouse R-Spondin 1; Gastrin 10nM; glutamine 10nM; nicotinamide 10mM; N-acetylcysteine 1mM; penicillin/streptomycin 100μl; B27 200μl; the balance was filled with Advanced DMEM F12.

Then, the culture medium prepared in the above comparative example was used to culture mouse gastric organoids in vitro according to the culture method described in the above Example 5 (see FIG. 4 ).

It can be seen from Figure 4 that, compared with the blank control, SB505124 can inhibit fungi at 2 μM, and the inhibitory effect is enhanced at a concentration of 10 μM. Therefore, it can be concluded that SB505124 has a certain inhibitory effect on fungi, and this effect is enhanced with the increase of its concentration. Adding SB505124 in the medium can play a certain role in preventing fungal contamination, but its effect is weaker than that of amphotericin B.

Example 5

Using the organoid medium obtained in Example 1 above to culture mouse gastric organoids in vitro, specifically:

1. Take normal mouse stomach tissue, cut into ^2mm2 tissue fragments, wash with DPBS until the supernatant is clear, discard the supernatant,

2. Add 10ml of the preheated digestion solution to the precipitate and digest it in a CO ₂ incubator at 37°C for 1 hour. After the end, let it settle naturally, discard the supernatant, and add DPBS containing 0.1% BSA to stop the digestion.

100mg of type IV collagenase, 50mg of hyaluronidase, 2ml of FBS, and 100μl of penicillin/streptomycin in every 10ml of digestive juice.

3. Transfer the precipitate to a 10mm ² sterile Petri dish, press it with a sterile glass slide to release the gland, collect the gland and tissue fragments into a centrifuge tube, resuspend with Advanced DMEM F12, and pass through 70μm cells Filter through a strainer to remove tissue debris.

4. Centrifuge the filtered cell suspension at 500×g at 4°C for 5 minutes, discard the supernatant, and take an appropriate amount of Advanced DMEM F12 to resuspend the pellet (concentration is 1×10 ⁶ /L).

5. Mix the resuspended cell suspension with the pre-packaged Matrigel at a ratio of 1:1, inoculate it into a preheated 24-well plate in the form of water droplets, and put it in a 37°C incubator to cure for 30 minutes.

6. After the Matrigel was solidified, 500 μl of the medium of Example 1 was added to each well, and cultured for 5-7 days to obtain normal gastric organoids in mice.

It can be seen from Figures 2-4 that the specific additive factors artificial gastric juice, SB505124, and amphotericin B added to the culture medium can promote the growth of organoids, and the concentration should be set to artificial gastric juice 5 μl/5ml; SB505124 2 μM; amphotericin Factor B 5μg.

Comparative example 1:

The organoid medium is the basal medium and specific addition factors; 5 μg of amphotericin B is added to every 10 ml of the basal medium.

5ml of Wnt conditioned medium in every 10ml of basal medium in the basal medium; 1 μg of recombinant mouse Noggin; 500 ng of recombinant mouse epidermal growth factor; 101 μg of recombinant mouse fibroblast growth factor; 10 μg of recombinant mouse R-Spondin 1; Gastrin 10nM; glutamine 10nM; nicotinamide 10mM; N-acetylcysteine 1mM; penicillin/streptomycin 100μl; B27 200μl; the balance was filled with Advanced DMEM F12.

Then, the culture medium prepared in the above ratio was used to culture mouse gastric organoids in vitro according to the culture method described in the above-mentioned Example 5 (see FIG. 5 ).

It can be seen from Figure 5 that when artificial gastric juice and SB505124 were not added, the organoids grew slowly, had small diameters, and sprouted late. However, compared with the above-mentioned examples, the organoids obtained by adding SB505124 and artificial gastric juice in the culture medium grew slightly faster and were in good condition.

Comparative example 2

Organoid medium is the base medium and specific addition factors; 10 μl of artificial gastric juice is added to every 10 ml of base medium; SB505124 2 μM.

5ml of Wnt conditioned medium in every 10ml of basal medium in the basal medium; 1 μg of recombinant mouse Noggin; 500 ng of recombinant mouse epidermal growth factor; 101 μg of recombinant mouse fibroblast growth factor; 10 μg of recombinant mouse R-Spondin 1; Gastrin 10nM; glutamine 10nM; nicotinamide 10mM; N-acetylcysteine 1mM; penicillin/streptomycin 100μl; B27 200μl; the balance was filled with Advanced DMEM F12.

Then, the culture medium prepared in the above ratio was used to culture mouse gastric organoids in vitro according to the culture method described in the above-mentioned Example 5 (see Figure 6)

It can be seen from Figure 6 that when amphotericin B is not added, the organoids are seriously polluted, but when amphotericin B is added according to the above examples, the culture medium is clear and pollution-free, and the organoids grow well.

Comparative example 3:

The culture method provided in this comparative example is the same as that in Example 5, but no glass slide is used after the digestion treatment, and the total number of cells digested is quite different from that in Example 5, which shows that the way of adding digestive fluid to the glass slide can improve the utilization rate of tissue cells, so that It becomes possible to grow organoids from small pieces of tissue.

Embodiment 5 (use glass slide) Comparative example 3 (without slides) cell number 2.0×10⁵/ml 1.5×10⁵/ml

To sum up, the culture medium of mouse normal gastric organoids in vitro can improve the success rate of mouse gastric organoid culture, adding artificial gastric juice to simulate the acidic environment in the human body, making the culture medium environment closer to the body to promote the growth of gastric organoids Growth; adding SB505124 can promote organoid budding; adding amphotericin B to the medium can solve the problem of fungal contamination in the early stage of culture without affecting the growth of organoids. The step of adding slides to press after the digestion of the digestive juice can increase the extraction rate of glandular cells and make the culture efficiency of mouse gastric organoids higher.

The above-mentioned embodiments only express several implementation modes of the present invention, and the description thereof is relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be pointed out that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (9)

1. A medium for gastric organoid culture, characterized in that: the medium is a base medium and a specific addition factor; the specific addition factor is SB505124, artificial gastric juice and amphotericin B; wherein, the specific addition The added amount of the factor accounts for 0.02-0.2% of the total amount of the basal medium. 2. The medium according to claim 1, characterized in that: the final concentration of SB505124 in the medium is 1-10 μM; the final concentration of amphotericin B is 5 μg; 2-20 μl is added in the medium of every 10 ml of artificial gastric juice. 3. by the medium described in claim 1, it is characterized in that: add Wnt condition medium 5ml in the described every 10ml basal medium; Recombinant mouse Noggin 1 μ g; Recombinant mouse epidermal growth factor 500ng; Recombinant mouse fibroblast growth Factor-10 1μg; recombinant mouse R-Spondin 1 10μg; human gastrin 10nM; glutamine 10nM; nicotinamide 10mM; N-acetylcysteine 1mM; penicillin/streptomycin 100μl; Advanced DMEM F12 completed. 4. The application of the medium according to claim 1, characterized in that: the application of the medium in culturing mouse gastric organoids. 5. The application of the medium according to claim 1, characterized in that: the application of the medium to inhibit fungi in culturing gastric organoids. 6. The application of the medium according to claim 5, characterized in that: the fungus is Aureobasidium pullulans. 7. A culture method for mouse gastric organoids, characterized in that: cells to be cultured are resuspended through DMEM F12, and single cell suspension is obtained after resuspension, then single cell suspension and matrigel are mixed at a ratio of 1:1 Mix the volume ratio, solidify after mixing, and then add the medium described in claim 1, culture at 37°C and 5% CO ₂ environment, change the medium once every two days, and obtain mouse gastric organoids in 5-7 days . 8. The culture method according to claim 7, characterized in that: the mouse gastric tissue block is washed with DPBS and then collected by natural sedimentation, adding digestive juice to the precipitate, and placing it in a 5% _CO2 incubator at 37°C Digest for 1 h, collect the precipitate, and set aside. 9. The culture method according to claim 8, characterized in that: 100 mg of type IV collagenase, 50 mg of hyaluronidase, 2 ml of FBS, and 100 μl of penicillin/streptomycin in each 10 ml of digestive juice.

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