TWI817291B - Device and method for culturing tissue - Google Patents

Abstract

A device and a method for culturing tissue is used to solve the problem that the fish skin is difficult to be cultured in vitro. The device for culturing tissue comprises a base having a first lateral wall and a pressing member having a second lateral wall. A chamber is defined by the first lateral wall, and an observing space is defined by the second lateral wall. A first opening and a second opening are provided at a top end and a bottom end of the base, respectively, and the first opening intercommunicates with the second opening via the chamber. A third opening and a fourth opening are provided at a top end and a bottom end of the pressing member, respectively, and the third opening intercommunicates with the fourth opening via the observing space. The pressing member detachably couples to the base, and the observing space intercommunicates with the chamber.

Description

Tissue culture device and tissue culture method

The present invention relates to a tissue culture device and a tissue culture method, in particular to a tissue culture device and a tissue culture method that only make the endothelial surface of the fish skin tissue close to the fish body cavity come into contact with the culture fluid.

Due to the abundant fish resources in the waters around Taiwan and the popularity of domestic fisheries in recent years, all walks of life have actively invested in fish research, such as virology, physiology or genetics, in order to maintain precious fish resources and promote the vigorous development of related industries. Study etc. Among them, because it is distributed in complex water bodies, fish skin often interacts with microorganisms in the environment, and is closely related to the occurrence of many fish diseases. In view of this, fish skin tissue has become one of the key research directions.

At present, because fish skin tissue is difficult to culture in vitro, fish cells cultured in vitro or living fish are the most important research resources. However, cell experiments cannot fully reflect the function of the tissue, causing the credibility of the experimental results to remain uncertain. Doubts; direct use of live fish for animal experiments often requires the sacrifice of a large number of fish bodies. In addition to consuming feeding costs and breeding space, it also leads to a waste of fish resources. On the other hand, the experimental results are also easily caused by individual differences among fish bodies. There is a large error, so there is a need to improve it.

In order to solve the above problems, the object of the present invention is to provide a tissue culture device that can culture a fish skin tissue in vitro and make the fish skin tissue have normal tissue morphology and integrity to exert physiological functions.

A secondary object of the present invention is to provide a tissue culture device that can separate the inner and outer skin surfaces of the fish skin tissue and enable the inner and outer skin surfaces of the fish skin tissue to be in contact with different solutions respectively.

Another object of the present invention is to provide a tissue culture method, which uses the above-mentioned tissue culture device to culture the fish skin tissue in vitro.

Directionality or similar terms are used throughout the present invention, such as "front", "back", "left", "right", "upper (top)", "lower (bottom)", "inner", "outer" , "side", etc. mainly refer to the directions of the attached drawings. Each directionality or its approximate terms are only used to assist in explaining and understanding the various embodiments of the present invention, and are not intended to limit the present invention.

The use of the quantifier "a" or "an" in the elements and components described throughout the present invention is only for convenience of use and to provide a common sense of the scope of the present invention; in the present invention, it should be interpreted as including one or at least one, and single The concept of also includes the plural unless it is obvious that something else is meant.

Approximate terms such as "combination", "combination" or "assembly" used throughout the present invention mainly include forms that can be separated without damaging the components after being connected, or components that are inseparable after being connected, and are based on common knowledge in this field. You can choose according to the material of the components to be connected or the assembly requirements.

The tissue culture device of the present invention includes: a base with a first ring wall. The first ring wall surrounds a chamber. The upper and lower ends of the base each have a first opening and a second opening. , the first opening and the second opening communicate with the chamber; a pressing member has a second ring wall, the second ring wall surrounds an observation space, and the upper and lower ends of the pressing member each have a The third opening and A fourth opening, the third opening and the fourth opening communicate with the observation space, the pressing member is detachably connected to the base, the observation space communicates with the chamber, and the outer periphery of the second ring wall of the pressing member The surface can have an annular groove; and a fixing ring, the fixing ring is an elastic O-ring, the fixing ring can be aligned with the annular groove, and the fixing ring can abut against the first ring wall.

The tissue culture method of the present invention includes the following steps: using the above-mentioned tissue culture device, clamping a fish skin tissue between the base and the pressing member; and placing the tissue culture device in a culture medium, so that the The inner skin surface of the fish skin tissue located in the second opening and close to the fish body cavity contacts the culture medium.

Accordingly, the tissue culture device and tissue culture method of the present invention can culture the fish skin tissue in vitro by clamping the fish skin tissue between the base and the pressing member. Only the endothelial surface close to the fish body cavity is in contact with the culture solution. Compared with the fresh fish skin tissue, the fish skin tissue can be composed of the same type of cells and has relatively high tissue integrity, and the fish skin tissue In addition to having a certain number and size of goblet cells in the skin tissue, they can also function to express specific genes. In this way, since the fish skin tissue can have physiological functions, compared with fish cells, using the fish skin tissue to conduct experiments (such as bacterial infection experiments, etc.) can improve the credibility of the experimental results. On the other hand, Because a large amount of fish skin tissue can be obtained from one fish body for in vitro culture, it not only reduces experimental errors caused by individual differences between fish bodies, but also reduces the number of fish bodies required during the experiment, which not only saves The cost and space required for breeding can also significantly reduce the waste of fish resources. In addition, a simple structure can be used to fix the fish skin tissue on the pressing member, which has the effect of improving operation convenience and reducing manufacturing costs. Moreover, the fish skin tissue can be separated from the endothelial surface close to the fish body cavity and from the external environment. The outer skin surface in contact has the effect of separating the inner and outer skin surfaces of the fish skin tissue.

Wherein, the bottom surface of the first ring wall may have at least one fixed foot. In this way, the base can be stood firmly on a flat surface, which has the effect of improving operation convenience.

Wherein, the first ring wall may have a receiving part, and the receiving part may be located in the chamber and abut against the pressing member. In this way, the pressing member can be prevented from sliding downward from the chamber after being connected to the base, which has the effect of improving the stability of the combination of the components.

Wherein, a predetermined reactant can be added to the observation space, so that the outer skin surface of the fish skin tissue located in the fourth opening and in contact with the external environment contacts the predetermined reactant. In this way, a possible reaction can occur between the predetermined reactant and the fish skin tissue, which has the effect of understanding the relationship between the predetermined reactant and the fish skin tissue.

Wherein, the predetermined reactant may be a bacterium. In this way, a possible interaction between the bacteria and the fish skin tissue can be generated, which has the effect of understanding the interaction between the bacteria and the fish skin tissue.

Wherein, the predetermined reactant may be a toxin. In this way, the toxin can have a possible effect on the fish skin tissue, and the effect of the toxin on the fish skin tissue can be understood.

Wherein, the predetermined reactant may be water from a predetermined water source. In this way, the water from the predetermined water source can have a possible effect on the fish skin tissue, and has the effect of understanding the impact of the water from the predetermined water source on the fish skin tissue.

[Invention]

1: Base

1a: First opening

1b: Second opening

11:The first ring wall

12: Undertaking department

13: Fixed feet

2: Press firmware

2a: The third opening

2b: The fourth opening

21:Second ring wall

22: Ring groove

3: Fixed ring

4: Fish skin tissue

M: observation space

S: chamber

[Figure 1] An exploded perspective view of a preferred embodiment of the present invention.

[Figure 2] A schematic diagram of the combination of a preferred embodiment of the present invention and a fish skin tissue.

[Figure 3] A front view of a combination of a preferred embodiment of the present invention and a fish skin tissue.

[Picture 4] Histological section of the dorsal fin skin tissue of Pangasianodon hypophthalmus .

[Picture 5] A tissue section diagram of the dorsal fin skin tissue of Pangasianodon hypophthalmus after it was soaked in culture medium and cultured for five days.

[Picture 6] A tissue section diagram of the endothelial surface of the dorsal fin skin tissue of Pangasianodon hypophthalmus after it was in contact with the culture medium and cultured for five days.

[Figure 7] Histological section of the pelvic fin skin tissue of Pangasianodon hypophthalmus .

[Picture 8] A tissue section of the pelvic fin skin tissue of Pangasianodon hypophthalmus soaked in culture medium and cultured for five days.

[Picture 9] Tissue section diagram of the endothelial surface of the pelvic fin skin tissue of Pangasianodon hypophthalmus , which is close to the body cavity of the fish, in contact with the culture medium and cultured for five days.

[Picture 10] The thickness of the skin tissue of the dorsal and pelvic fins of cultured Pangasianodon hypophthalmus .

[Figure 11] Transepithelial resistance values of cultured dorsal and pelvic fin skin tissues of Pangasianodon hypophthalmus .

[Figure 12] Distribution of goblet cells (indicated by black arrows) in the dorsal fin skin tissue of Pangasianodon hypophthalmus .

[Picture 13] After the endothelial surface of the dorsal fin skin tissue of Pangasianodon hypophthalmus , which is close to the fish body cavity, was in contact with the culture medium and cultured for five days, the goblet cells (indicated by black arrows) were distributed.

[Picture 14] After the entire dorsal fin skin tissue of Pangasianodon hypophthalmus was immersed in culture medium and cultured for five days, the goblet cells (indicated by black arrows) were distributed.

[Figure 15] Distribution of goblet cells (indicated by black arrows) in the pelvic fin skin tissue of Pangasianodon hypophthalmus .

[Figure 16] After the endothelial surface of the pelvic fin skin tissue of Pangasianodon hypophthalmus , which is close to the fish body cavity, was in contact with the culture medium and cultured for five days, the goblet cells (indicated by black arrows) were distributed.

[Picture 17] After the entire pelvic fin skin tissue of Pangasianodon hypophthalmus was immersed in culture medium and cultured for five days, the goblet cells (indicated by black arrows) were distributed.

[Figure 18] Number of goblet cells in the cultured dorsal and ventral fin skin tissues of Pangasianodon hypophthalmus .

[Figure 19] The size of goblet cells in the dorsal and ventral fin skin tissue of cultured pangasianodon hypophthalmus .

[Figure 20] Relative expression of the MUC5AC gene in the dorsal and ventral fin skin tissues of cultured pangasianodon hypophthalmus .

In order to make the above and other objects, features and advantages of the present invention more obvious and understandable, preferred embodiments of the present invention are illustrated below and described in detail with reference to the accompanying drawings; in addition, the same symbols are used in different drawings. are considered to be the same and their description will be omitted.

Please refer to Figures 1 and 2, which is a preferred embodiment of the tissue culture device of the present invention. It includes a base 1 and a holding member 2. The holding member 2 is detachably connected to the base 1.

The base 1 has a first ring wall 11, which surrounds a chamber S. In this embodiment, the chamber S can be selected to be a cylindrical cavity; The lower two ends each have a first opening 1a and a second opening 1b, and the first opening 1a and the second opening 1b communicate with the chamber S. The inner surface of the first ring wall 11 has a receiving portion 12. In this embodiment, the receiving portion 12 can be made into a circular ring. For example, but not limited to, the bottom surface of the first ring wall 11 may also have at least one fixing foot 13. The at least one fixing foot 13 is preferably columnar, so that the base 1 can be stably stood on a flat surface. superior.

The holding member 2 has a second ring wall 21, and the second ring wall 21 surrounds an observation space M. In this embodiment, the observation space M can be selected to be a cylindrical cavity; The upper and lower ends each have a third opening 2a and a fourth opening 2b. The third opening 2a and the fourth opening 2b communicate with the observation space M; the outer diameter of the holding member 2 is preferably slightly larger than the receiving portion. The inner diameter 12 is slightly smaller than the outer diameter of the receiving portion 12 , so that when the pressing member 2 extends into the chamber S, it can contact the receiving portion 12 and allow the observation space M to communicate with the chamber S. The outer side surface of the second ring wall 21 can also have an annular groove 22. The ring groove 22 can be opened along the outer circumferential surface of the second ring wall 21. The ring groove 22 can be used for another fixing ring 3 to align. The ring 3 is elastic and can be an O-ring, but is not limited thereto. The fixing ring 3 can be used to fix a fish skin tissue 4 on the pressing member 2. When the pressing member 2 extends into the chamber S, the fixing ring 3 can be located between the pressing member 2 and the first ring wall 11. Thereby, the inner skin surface of the fish skin tissue 4 close to the fish body cavity and the outer skin surface in contact with the external environment can be separated.

Please refer to Figures 2 and 3. In this embodiment, a tissue culture method can be performed through the above-mentioned tissue culture device, including the following steps: first, place the fish skin tissue 4 on the fourth part of the presser 2 On the opening 2b, the part of the fish skin tissue 4 exposed outside the fourth opening 2b is recessed in the direction of the third opening 2a. In addition, the fixing ring 3 can be fitted on the fish skin tissue 4 and positioned at the opening 2b. The ring groove 22 allows the fish skin tissue 4 to be fixed on the pressing member 2 by the fixing ring 3; then, extend the pressing member 2 into the chamber S of the base 1, and push the pressing member 2 Until the fish skin tissue 4 is in contact with the receiving part 12, the fish skin tissue 4 is clamped between the base 1 and the holding member 2; then, the tissue culture device is placed in a culture medium. The inner surface of the fish skin tissue 4 located in the second opening 1b and close to the fish body cavity contacts the culture medium to maintain the normal growth of the fish skin tissue 4 . In addition, the inner edge of the fixing ring 3 can be tightly bound to the fish skin tissue 4, and the outer edge of the fixing ring 3 can be tightly pressed against the first ring wall 11, so that the first opening 1a of the base 1 can be connected with the first ring wall 11. The second opening 1b may be formed between It is liquid-tightly disconnected. This ensures that the culture solution only contacts the inner surface of the fish skin tissue 4 and prevents the culture solution from contacting the outer surface of the fish skin tissue 4 . In another embodiment, a predetermined reactant can be added to the observation space M, so that the outer surface of the fish skin tissue 4 located in the fourth opening 2b that is in contact with the external environment contacts the predetermined reactant. After the culture time, the morphological changes and physiological function changes of the fish skin tissue 4 after contact with the predetermined reactant can be observed.

In order to confirm that the tissue culture device and its method can maintain the shape, tissue integrity and physiological functions of fish skin tissue, the following tests were conducted:

(A) The tissue culture device of the present invention maintains the morphology and integrity of fish skin tissue.

After removing approximately 10×10 square millimeters of skin tissue 4 from the dorsal fin and pelvic fin of Pangasianodon hypophthalmus , the fish skin tissue 4 was cultured. The fish skin tissue 4 is clamped between the base 1 and the pressing member 2, and then the tissue culture device is placed in the culture solution, so that the endothelial surface of the fish skin tissue 4 close to the fish body cavity is in contact with the culture medium. The fish skin tissue 4 is in contact with the culture liquid; and, the entire fish skin tissue 4 is immersed in the culture liquid, so that the inner and outer skin surfaces of the fish skin tissue 4 are in contact with the culture liquid, and then the fish skin tissue 4 is placed at 25°C without containing The culture was carried out in a carbon dioxide incubator for five days, and new culture medium needed to be replaced every two days during the culture period. After the culture is completed, the dorsal and pelvic fin fish skin tissues cultured in different ways are compared with the fresh uncultured dorsal and pelvic fin fish skin tissues to observe the morphology and integrity of the fish skin tissue.

Please refer to Figures 4, 5, and 6, which show that superficial epithelial cells (SC), basal epithelial cells (BC), and melanophores (ME) can be observed in fresh dorsal fin fish skin tissue. ) and dense collagenous tissue (DCT) (Figure 4); when the entire dorsal fin fish skin tissue was immersed in the culture medium, the surface epithelial cells (SC) and basal epithelial cells (BC) were almost completely lost (Figure 4 5); and the surface epithelial cells of dorsal fin fish skin tissue using this tissue culture device (SC), basal epithelial cells (BC) and melanocytes (ME) are still almost completely distributed in the dorsal fin fish skin tissue (Figure 6), showing the various types of cells that make up the dorsal fin fish skin tissue when using this tissue culture device It can still be roughly distributed in the dorsal fin fish skin tissue.

Please refer to Figures 7, 8, and 9, which show that superficial epithelial cells (SC), rod cells (CC), basal epithelial cells (BC), and melanocytes (ME) can be observed in fresh pelvic fin fish skin tissue. ) and dense colloid tissue (DCT) (Picture 7); when the entire pelvic fin fish skin tissue is immersed in the culture medium, almost only a small amount of melanocytes (ME) remain distributed in it (Picture 8); and using this tissue for culture The surface epithelial cells (SC), rod cells (CC), basal epithelial cells (BC), and melanocytes (ME) of the pelvic fin skin tissue of the device are still almost completely distributed in the pelvic fin skin tissue (Figure 9). It shows that when using this tissue culture device, various types of cells that make up the pelvic fin fish skin tissue can still be roughly distributed in the pelvic fin fish skin tissue.

Please refer to Figure 10, which shows that when the dorsal fin or pelvic fin skin tissue is used in this tissue culture device, the thickness of the fish skin tissue is roughly the same as that of fresh fish skin tissue; however, when the dorsal fin or pelvic fin skin tissue is soaked as a whole in When the culture medium was used, the thickness of the fish skin tissue decreased significantly compared with the fresh fish skin tissue, indicating that the tissue culture device can maintain the structural integrity of the dorsal and ventral fin fish skin tissue.

Please refer to Figure 11, which was used to detect the resistance value of the dorsal and ventral fin fish skin tissue during 21 days of culture. The resistance value can reflect the tight connections between cells in the fish skin tissue. The tissue barrier function exerted by the fish skin tissue is greater. The greater the resistance value per unit area of the fish skin tissue, the higher the degree of tight connections between cells in the fish skin tissue. The results showed that the resistance value per unit area of the dorsal and pelvic fin fish skin tissues gradually increased during the first to fifth days of culture using the tissue culture device; during the fifth to eighteenth days During the culture period, the resistance value per unit area of the dorsal and pelvic fin fish skin tissue can be maintained in a range similar to that on the fifth day. This experiment shows that the tissue culture device can maintain the dorsal and pelvic fin fish skin tissue. tissue health.

(B) The tissue culture device of the present invention maintains the distribution and function of goblet cells in fish skin tissue.

Please refer to Figures 12 to 14. In fresh dorsal fin fish skin tissue (Figure 12) and dorsal fin fish skin tissue using this tissue culture device (Figure 13), goblet cells are distributed in the outermost epithelial cells. on the surface and in the epithelial tissue; however, when the entire dorsal fin fish skin tissue was immersed in the culture medium, the goblet cells were only distributed on the surface of a single layer of epithelial tissue (Figure 14). This experiment shows that the tissue culture device can maintain the dorsal fin Goblet cell distribution in fish skin tissue.

Please refer to Figures 15 to 17. In fresh pelvic fin fish skin tissue (Figure 15) and pelvic fin fish skin tissue using this tissue culture device (Figure 16), goblet cells are distributed in the outermost epithelial cells. on the surface and in the epithelial tissue; however, when the entire pelvic fin fish skin tissue was immersed in the culture medium, the goblet cells were only distributed on the surface of a single layer of epithelial tissue (Figure 17). This experiment shows that the tissue culture device can maintain the pelvic fin Goblet cell distribution in fish skin tissue.

Please refer to Figure 18. When using this tissue culture device, the number of goblet cells in the dorsal and pelvic fin fish skin tissues is not significantly different from the fresh dorsal and pelvic fin fish skin tissues respectively; however, when the dorsal and pelvic fin fish skin tissues are in total When soaked in culture medium, the number of goblet cells in the fish skin tissue was significantly reduced compared with fresh dorsal and pelvic fin skin tissue. This experiment shows that the tissue culture device can maintain the number of goblet cells in the dorsal and pelvic fin skin tissue. .

Please refer to Figure 19. When using this tissue culture device, the size of goblet cells in the dorsal and pelvic fin fish skin tissues is not significantly different from that of fresh dorsal and pelvic fin fish skin tissues respectively; however, when the dorsal and pelvic fin fish skin tissues are whole When soaked in culture medium, the size of goblet cells in the fish skin tissue decreased significantly compared with fresh dorsal and pelvic fin skin tissue. This experiment shows that the tissue culture device can maintain the size of goblet cells in the dorsal and pelvic fin skin tissue. .

Please refer to Figure 20, which shows the function of mucus secretion in epithelial cells. The MUC5AC gene is targeted and its gene expression level is detected. The results showed that in the dorsal fin skin tissue using this tissue culture device, the relative gene expression of MUC5AC decreased by about 55% compared with the fresh dorsal fin skin tissue. When the dorsal fin skin tissue was soaked in the culture medium, the relative gene expression of MUC5AC decreased by about 55%. Compared with the fresh dorsal fin fish skin tissue, the expression amount decreased by about 88%; in the pelvic fin fish skin tissue using this tissue culture device, there was no significant difference in the relative gene expression amount of MUC5AC compared with the fresh pelvic fin fish skin tissue. When the pelvic fin fish skin tissue When the whole tissue was immersed in the culture medium, the relative gene expression of MUC5AC decreased by about 60% compared with fresh pelvic fin fish skin tissue. This experiment shows that the dorsal and pelvic fin fish skin tissues using this tissue culture device are better at maintaining goblet cells. Feature.

In addition, the tissue culture device can also be used to explore the interaction between fish skin tissue and certain substances. First, the endothelial surface of the fish skin tissue close to the fish body cavity is contacted with the culture medium to maintain the normal state of the fish skin tissue. To grow, a predetermined reactant can be mixed with the culture solution to form a reaction solution, and then added to the observation space of the tissue culture device, so that the outer surface of the fish skin tissue that is in contact with the external environment is in contact with the predetermined reactant. The reactant can be a bacterium, a toxin or water from a predetermined water source. After a predetermined incubation time, the changes in the fish skin tissue can be observed. In this way, methods such as histopathological sections or molecular techniques can be used to understand the interaction between specific bacteria and fish skin tissue, as well as the effects of specific toxins or water quality on fish skin tissue.

To sum up, the tissue culture device and tissue culture method of the present invention can culture the fish skin tissue in vitro by clamping the fish skin tissue between the base and the pressing member, so that the fish skin tissue can be cultured in vitro. Only the endothelial surface of the tissue close to the fish body cavity is in contact with the culture solution. Compared with fresh fish skin tissue, the fish skin tissue can be composed of the same type of cells and has a fairly high tissue integrity, and In addition to having a certain number and size of goblet cells in the fish skin tissue, it can also function to express specific genes. In this way, since the fish skin tissue can have physiological functions, compared with fish cells, using the fish skin tissue to conduct experiments (such as bacterial infection experiments, etc.) can improve the credibility of the experimental results. On the other hand, Because you can get a lot of it from one fish body The use of fish skin tissue for in vitro culture not only reduces experimental errors caused by individual differences between fish, but also reduces the number of fish required during the experiment. This not only saves the cost and space required for breeding, but also Significantly reduce the waste of fish resources.

Although the present invention has been disclosed using the above-mentioned preferred embodiments, they are not intended to limit the invention. Anyone skilled in the art can make various changes and modifications to the above-described embodiments without departing from the spirit and scope of the invention. The technical scope protected by the invention, therefore, the protection scope of the invention shall include all changes within the literal and equivalent scope described in the appended patent application scope.

1: Base

1a: First opening

1b: Second opening

11:The first ring wall

12: Undertaking department

13: Fixed feet

2: Press firmware

2a: The third opening

2b: The fourth opening

21:Second ring wall

22: Ring groove

3: Fixed ring

M: observation space

S: Chamber

Claims (8)

A tissue culture device, including: a base with a first ring wall, the first ring wall surrounds a chamber, the upper and lower ends of the base each have a first opening and a second opening, the The first opening and the second opening communicate with the chamber; a pressing member has a second ring wall, the second ring wall surrounds an observation space, and the upper and lower ends of the pressing member each have a third opening and a fourth opening, the third opening and the fourth opening communicate with the observation space, the pressing member is detachably connected to the base, the observation space communicates with the chamber, and the second surrounding wall of the pressing member There is an annular groove on the outer peripheral surface; and a fixing ring, the fixing ring is an elastic O-ring, the fixing ring is aligned with the annular groove, and the fixing ring is in contact with the first ring wall. The tissue culture device of claim 1, wherein the bottom surface of the first ring wall has at least one fixed foot. The tissue culture device of claim 1, wherein the first ring wall has a receiving portion, the receiving portion is located in the chamber and contacts the pressing member. A tissue culture method, including the following steps: using the tissue culture device according to any one of claims 1 to 3, clamping a fish skin tissue between the base and the holding member; and placing the tissue culture device on In a culture medium, the endothelial surface of the fish skin tissue located in the second opening and close to the fish body cavity is in contact with the culture medium. The tissue culture method of claim 4, wherein a predetermined reactant is added to the observation space so that the outer skin surface of the fish skin tissue located in the fourth opening that is in contact with the external environment contacts the predetermined reactant. The tissue culture method of claim 5, wherein the predetermined reactant is a bacterium. The tissue culture method of claim 5, wherein the predetermined reactant is a toxin. The tissue culture method of claim 5, wherein the predetermined reactant is water from a predetermined water source.

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