Patent Publication Number: US-2023149930-A1

Title: Toxicity evaluation system and method for toxicological evaluation using the same

Description:
STATEMENT REGARDING PRIOR DISCLOSURES BY THE INVENTORS 
     The inventors of the present application authored and disclosed the subject matter of the present application on Sep. 10, 2021. This prior disclosures has been submitted in an Information Disclosure Statement in the present application as “NAM, Sung-Wook et al. “Xenopus chip for single-egg trapping, in vitro fertilization, development, and tadpole escape.” Biochemical and Biophysical Research Communications 569 (2021) 29-34. Sep. 10, 2021. English Language. 6 pages.” 
    
    
     CROSS-REFERENCE TO RELATED APPLICATIONS 
     A claim for priority under 35 U.S.C. § 119 is made to Korean Patent Application No. 10-2021-0157039 filed on Nov. 15, 2021, in the Korean Intellectual Property Office, the entire contents of which are hereby incorporated by reference. 
     BACKGROUND 
     Embodiments of the inventive concept described herein relate to a toxicity evaluation system, and a method for a toxicological evaluation using the same. In more detail, the inventive concept relates to an invention regarding a system including a chip that allows in vitro fertilization, development of embryos, and toxicological evaluation of spawns of a South African Xenopus laevis having claws, which is one of vertebrates. 
     A Xenopus laevis is an animal that does in vitro fertilization, and it is easy to observe a generation process outside the body. 678 studies supported by National Institutes of Health (NIH) in 2010 use the Xenopus laevis as an experiment animal, and about 217,000,000 dollars (about 2 trillion Won) are supported. South African Xenopus laevis having claws is widely used as an animal model for studying development of vertebrates. In particular, because a development process that continues after fertilization of oocytes and sperms of the Xenopus laevis may be observed outside the body, it corresponds to an important experiment animal in studying an initial development step of the vertebrates. 
     SUMMARY 
     Embodiments of the inventive concept provide an evaluation system, by which an initial development step of vertebrates may be easily observed. 
     The problems that are to be solved by the inventive concept are not limited to the above-mentioned problems, and the unmentioned problems will be clearly understood by those skilled in the art to which the inventive concept pertains from the specification and the accompanying drawings. 
     A toxicity evaluation system according to an embodiment of the inventive concept is disclosed. 
     The toxicity evaluation system includes a chip including a passage, in which a sample flows, and a plurality of recesses provided on the passage, and that traps objects included in the sample, and a pump that injects the sample into the chip. 
     According to an example, each of the plurality of recesses may have a size, by which one spawn included in the sample is trapped. 
     According to an example, an inlet port and an outlet port may be formed at one end and an opposite end of the passage. 
     According to an example, the passage may be a combination of a first passage having a first width and a second passage having a second width, the first width may have a size, by which the objects included in the sample flow, and the second width may have a size, by which the objects included in the sample is prevented from flowing. 
     According to an example, the recesses may be provided in a direction that faces the outlet port from the inlet port. 
     According to an example, the pump may cause the sample to flow in any one of the first direction or the second direction. 
     According to an example, the sample may be any one of a solution containing the spawns, a solution containing sperms that fertilize the spawns, or a pharmaceutical sample, by which a toxicity is evaluated. 
     A method for performing a toxicological evaluation by using the toxicity evaluation system according to another embodiment of the inventive concept is disclosed. 
     The method includes trapping the spawns in the plurality of recesses by injecting the solution containing the spawns into the chip, and inducing in vitro fertilization by injecting the solution containing the sperms that fertilize the spawns into the chip. 
     According to an example, the method may further include injecting a sample for evaluating a toxicity of an organism, on which the in vitro fertilization has been completed, into the passage. 
     According to an example, the method may further include trapping the organism, on which the in vitro fertilization has been completed, by the recesses, by adjusting the pump such that the sample flows in a first direction. 
     According to an example, the method may further include causing the organism, on which the in vitro fertilization has been completed, to escape, by adjusting the pump such that the sample flows in a second direction. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
       The above and other objects and features will become apparent from the following description with reference to the following figures, wherein like reference numerals refer to like parts throughout the various figures unless otherwise specified, and wherein: 
         FIG.  1    is a view illustrating an example of a toxicity evaluation system according to an embodiment of the inventive concept; 
         FIG.  2    is a view illustrating a chip according to an embodiment of the inventive concept; 
         FIGS.  3 A- 3 D  is a view illustrating a process of manufacturing a chip according to  FIG.  2   ; 
         FIGS.  4 A- 4 D  is a view illustrating that spawns are trapped by using a chip according to  FIG.  3   ; 
         FIGS.  5 A- 5 E  is a view illustrating that an embryo is sequentially grown by using a chip according to the inventive concept; 
         FIGS.  6 A- 6 H  is a view illustrating that trapping or escaping of a tadpole may be adjusted by adjusting a direction of a pump; and 
         FIG.  7    is a flowchart illustrating a method for a toxicological evaluation according to an example of the inventive concept. 
     
    
    
     DETAILED DESCRIPTION 
     The terms used in the specification and the accompanying drawings are provided to easily describe the inventive concept, and the inventive concept is not limited by the terms and the drawings. 
     A detailed description of, among the technologies used in the inventive concept, known technologies that are not closely relevant to the spirit of the inventive concept will be omitted. 
     Hereinafter, exemplary embodiments of the inventive concept will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the inventive concept pertains may easily carry out the inventive concept. However, the inventive concept may be implemented in various different forms, and is not limited to the embodiments. Furthermore, in a detailed description of the preferred embodiment of the inventive concept, a detailed description of related known functions or configurations will be omitted when they may make the essence of the inventive concept unclear. In addition, the same reference numerals are used for parts that perform similar functions and operations throughout the drawings. 
     The expression of ‘including’ some elements may mean that another element may be further included without being excluded unless there is a particularly contradictory description. In detail, the terms “including” and “having” are used to designate that the features, the numbers, the steps, the operations, the elements, the parts, or combination thereof described in the specification are present, and may be understood that one or more other features, numbers, step, operations, elements, parts, or combinations thereof may be added. 
     The terms such as first and second may be used to describe various elements, but the elements are not limited to the terms. The terms may be used only for distinguishing one component from other components. For example, without departing the scope of the inventive concept, a first element may be referred to as a second element, and similarly, a second element may be referred to as a first element. 
     The terms of a singular form may include plural forms unless otherwise specified. Furthermore, in the drawings, the shapes and sizes of the elements may be exaggerated for clearer description. 
     The term ‘unit’ or ‘module’ used in the entire specification is a unit for processing at least one function or operation, and for example, may refer to a hardware element such as an FPGA or an ASIC. However, the ‘unit’ or the ‘module’ is not limited to software or hardware. The ‘unit’ and “module” may be constituted in a storage medium that may perform addressing, and may be configured to reproduce one or more processors. 
     Accordingly, as an example, the ‘unit’ and ‘the module’ may include elements such as software elements, object-oriented software elements, class elements, and task elements, processes, functions, attributes, procedures, sub-routines, segments of a program code, drivers, firmware, micro-codes, circuits, data, a database, data structures, tables, arrays, and parameters. The functions provided by the elements and the ‘units’ and the ‘modules’ may be separately performed by a plurality of elements and ‘units’, and may be integrated with other additional elements, and ‘the unit’ and ‘the module’. 
     The inventive concept discloses a Xenopus laevis chip  10  for evaluating external fertilization of a vertebrate, development of an embryo, and a development toxicity. The chip  10  according to the inventive concept may induce fertilization and embryogenesis after trapping an embryo of a single entity level in a 3D chip, and may evaluate a toxicity of a medicine. The chip  10  of the inventive concept may function as an incubator that significantly increases a probability of fertilization in a process of performing external fertilization of eyes or embryos. Furthermore, the inventive concept may supply sperms, therapeutic substances, or biomolecules at a uniform velocity through a fluid circulation system using a pump  20 , and may cause continuous flows and circulations. Furthermore, after an embryo is grown up to a tadpole, the tadpole may be extracted out of a 3D chip reversely. 
     Hereinafter, a toxicity evaluation system  1  according to the inventive concept will be described in detail with reference to the accompanying drawings. 
       FIG.  1    is a view illustrating an example of the toxicity evaluation system  1  according to an embodiment of the inventive concept. 
     The toxicity evaluation system  1  according to the inventive concept may include the chip  10  including a passage  11 , in which the sample may flow, and a plurality of recesses  12  provided on the passage  11 , and by which objects included in the sample may be trapped. The toxicity evaluation system  1  according to the inventive concept may include the pump  20  that may inject the sample into the chip  10 . Hereinafter, the chip  10  and the Xenopus laevis chip  10  may have the same meaning. 
     Referring to  FIG.  1   , in vitro fertilization may be induced by circulating a solution  30 , in which sperms are mixed with Xenopus laevis spawns located in the passage  11  in the chip  10  in the Xenopus laevis chip  10  through a peristaltic pump  20 . The Xenopus laevis chip  10  includes the plurality of recesses  12 , and may function to trap organisms, on which spawning and in vitro fertilization of Xenopus laevis have been completed. A detailed structure of the Xenopus laevis chip  10  will be described in detail in  FIG.  2   . The organisms, on which the in vitro fertilization has been completed, may be tadpoles. 
     The pump  20  according to the inventive concept may perform a control such that the sample flows in any one of a first direction or a second direction. The pump  20  may adjust a direction and a velocity of the flows of the sample. According to an example, the pump  20  is used to induce the flows of the solution  30 , and according to an example, the pump  20  may be a peristaltic pump or a syringe pump. According to an embodiment, when the peristaltic pump  20  is used, a fluid may be made to circulate by causing sperms, therapeutic substances, or biomolecules to continuously flow at a constant velocity for several hours or several days. 
     According to an embodiment, the sample supplied to the chip  10  by using the pump  20  may be any one of the solution  30  containing spawns, the solution  30  containing sperms that may sterilize the spawns, or a pharmaceutical sample that may evaluate toxicity. According to an example, the sample may be a sample that is related to development of a Xenopus laevis frog. 
     According to an embodiment, the Xenopus laevis chip  10  included in the toxicity evaluation system  1  according to  FIG.  1    may control physical movements of eggs and embryos in a single entity level. 
     According to the inventive concept, an effect and a toxicological operation of a material may be evaluated by trapping an egg at one trapping site, that is, in the recess  12 , inducing egg fertilization by circulating sperms through a fluid circulating system, and circulating therapeutic substances or biomolecules. According to an example, operations, expressive types of toxics, and mechanism of various medicines, such as therapeutic substances or biomolecules, in particular, NSAIDs may be known. Furthermore, a direction of the flows of the fluid may be adjusted by using the pump  20 . According to an example, when the sample flows in a forward direction, a single entity may be trapped, and when the sample flows in a reverse direction, the trapped entities may be extracted out of the 3D chip  10 . 
       FIG.  2    is a view illustrating the chip  10  according to an embodiment of the inventive concept. 
     Referring to  FIG.  2   , the chip  10  may be configured to include the passage  11  and the recesses  12 . According to an example, each of the plurality of recesses  12  may have a size, by which one spawn included in the sample is trapped. The spawn may be an egg. According to an example, an inlet port  13  and an outlet port  14  may be formed at one end and an opposite end of the passage  11 . The inlet port  13  and the outlet port  14  may be connected to the pump  20  to function as an introduction port  13  or a discharge port  14  of the sample. 
     Referring to  FIG.  2   , the passage  11  may be a combination of a first passage  11   a  having a first width and a second passage  11   b  having a second width. The first width may be larger than the second width. The first passage  11   a  may be a passage, in which the sample mainly flows. The second passage  11   b  may connect the first passage  11   a  and the recess  12 . The second passage  11   b  may connect the first passage  11   a  and the recess  12 , and may function to add a pressure such that the sample flows better as it has the second width that is smaller than the first width. 
     According to an example, the second passage  11   b  may be omitted. 
     According to an example, the first width may have a size, by which the object included in the sample may flow, and the second width may have a size, by which the object included in the sample is prevented from flowing. The recess  12  may be provided in a direction that faces the outlet port  14  from the inlet port  13 . 
     Referring to  FIG.  2   , because the recess  12  is provided at a front part in the direction that faces the outlet port  14  from the inlet port  13 , it may be a site, at which the spawn included in the sample is trapped. Because the recess  12  has a size, by which one spawn may be trapped, the spawns may be trapped in the recesses to be easily observed individually when the sample continuously flows through the plurality of recesses  12  disposed on the passage  11 . Conventionally, because the chip itself is not used, the spawns gather together so that it is difficult to directly observe a development process. The inventive concept suggests a chip structure that may solve the problem. 
     According to an example, the Xenopus laevis chip  10  according to the inventive concept may have four functions. A single spawn may be trapped (single egg trapping), external fertilization (in vitro fertilization) may be possible, and development and escaping of a tadpole may be performed. 
     The drawing of the Xenopus laevis chip  10  disclosed in  FIG.  2    may be merely an example, and another shape may be provided as long as it has a shape that satisfies the described condition. 
       FIG.  3    is a view illustrating a process of manufacturing the chip  10  according to  FIG.  2   . 
     According to  FIG.  3 A , implementation of a 3D design is illustrated. According to  FIG.  3 B , an example of outputting a mold by using a 3D printer is disclosed. According to  FIG.  3 C , manufacturing of a PDMS chamber using the mold is illustrated. According to  FIG.  3 D , it may be identified that the Xenopus laevis chip  10  is manufactured. 
     Referring to  FIG.  3   , a 3D structure is drawn by using software for a 3D design, and a 3D mold may be output by using 3D printer equipment. A polydimethlysiloxane (PDMS) mixture may be poured into the output 3D mold, may be solidified at a temperature of about 70° C. to 80° C., and may be separated. Thereafter, the Xenopus laevis chip  10  may be manufactured by attaching another flat PDMS layer or a slide after the inlet port  13  and the outlet port  14 , through which the solution  30  may be injected into the PDMS layer, are punched with a biopsy punch. That is, according to the inventive concept, the mold maybe manufactured through the 3D printer, and the Xenopus laevis chip  10  may be manufactured of polydimethlysiloxane (PDMS) through soft lithography. 
       FIG.  4    is a view illustrating that spawns are trapped by using the chip  10  according to  FIG.  3   . 
     According to an embodiment, when the solution  30  having eggs is injected in the forward direction, one egg may be trapped at a trapping site, that is, in the recess  12 . When one trapping site, that is, the recess  12  is filled, the remaining entities may move to the next trapping site, that is, another recess  12  through a bypass, that is, the first passage  11   a . Furthermore, when the fluid flows in the reverse direction, the entities trapped in the recesses  12  may be extracted out of the chip  10 . 
     In more detail, the Xenopus laevis chip  10  may have the inlet port  13 , through which the solution  30  may be injected, and the outlet port  14 , through which the solution  30  may be extracted, and may include the plurality of recesses  12  that may be trapping sites, the first passage  11   a,  and the second passage  11   b . When the Xenopus laevis spawns are collected in a pipette tip and are injected through the inlet port  13 , the spawns move along the first passage  11   a . When one spawn is trapped in the recess  12  while the sample containing the spawns flow along the first passage  11   a,  the remaining spawns return through the first passage  11   a . By repeating the process, the spawns are trapped in the plurality of recesses  12  sequentially one by one, and after the spawns are trapped in all of the plurality of recesses  12 , the remaining spawns are extracted toward the outlet port  14 . 
       FIG.  5    is a view illustrating that an embryo is sequentially grown by using the chip  10  according to the inventive concept. In more detail, it is a view illustrating a development process of a spawn, on which in vitro fertilization has been performed in the Xenopus laevis chip  10 . 
       FIG.  5 A  illustrates a step, in which cell cleavages are observed in a spawn, on which the in vitro fertilization has been performed in the Xenopus laevis chip  10 . A two cell state of  FIG.  5 A  corresponds to stage 2 of the development of a Xenopus laevis. A four cell state of  FIG.  5 B  corresponds to stage 3 of the development of the Xenopus laevis. A blastula state of  FIG.  5 C  corresponds to stage 7 of the development of a Xenopus laevis.  FIG.  5 D  illustrates a picture corresponding to stage 25. According to  FIG.  5 E , a tadpole is observed when development of about 50 hours or more is progressed. 
     When movement of the tadpole that was in vitro fertilized in the Xenopus laevis chip  10  and was grown up is observed, it may be observed heat beats, tailing, and swimming. 
       FIG.  6    is a view illustrating that trapping or escaping of the tadpole may be adjusted by adjusting a direction of the pump  20 . 
     A structure of the Xenopus laevis chip  10  has an asymmetrical shape. The flows of the solution  30  may be adjusted to a forward or reverse direction through the peristaltic pump  20 , and thus has different functions.  FIGS.  6 A to  6 D  illustrate that a trapping process of the tadpole may be induced when the sample flows in the forward direction.  FIGS.  6 E to  6 H  illustrate that an escaping process of the tadpole may be induced when water flows in the reverse direction. 
     Referring to  FIG.  6 A , three tadpoles are in the Xenopus laevis chip  10 , and two of them are at the trapping sites, that is, in the recesses  12  and one of them is in the first passage  11   a . The solution  30  may flow to the recess  12  as in  FIG.  6 B  by causing the solution  30  to flow in the forward direction. Next, as in  FIG.  6 B , when the solution  30  flows in the reverse direction, one of the three tadpoles may be extracted from the recess  12 . As in  FIG.  6 C , it may be observed that the tadpole extracted from the recess  12  may be located in the first passage  11   a , and in the process, the remaining two tadpoles may be resistant to the reverse flows. To move all the tadpoles to the recesses  12 , as in  FIGS.  6 C and  6 D , the solution  30  may be made to flow in the forward direction. 
     That is, the tadpoles may be moved to desired recesses  12  by adjusting the flows of the solution in the forward direction and the reverse direction. In particular, the heads of the tadpoles are positioned toward the recesses  12  and the tails of the tadpoles are positioned toward the passage  11  by inducing the repeated forward and reverse flows, and thus the directions, in which the tadpoles are positioned, may be adjusted. Through this, a toxicity evaluation may be possible in the same condition. 
     According to  FIGS.  6 E to  6 H , a process of causing the tadpole to escape out of the chip  10  to obtain the tadpole that has been externally fertilized, developed, and grown up in the chip  10  is illustrated. 
     As in  FIG.  6 E , a tube connected to the inlet port  13  of the chip  10  is extracted and stronger reverse flows are induced. As in  FIG.  6 F , it may be observed that the first tadpole is extracted. It may be observed in  FIG.  6 G  that the second tadpole is extracted. As in  FIG.  6 H , it may be observed that all of the three tadpoles are extracted. The extracted tadpoles may be contained in a Petri dish. Through this, it is possible to secure the three tadpoles that have been in vitro-fertilized, developed, and grown up in the chip  10 . 
     The tadpoles may be located at desired trapping sites, that is, in the recesses  12  by carefully adjusting the flows of the solution  30  in the forward or reverse direction through the peristaltic pump  20  to control movements of the tadpoles. 
       FIG.  7    is a flowchart illustrating a method for a toxicological evaluation according to an example of the inventive concept. 
     Referring to  FIG.  7   , a method for performing a toxicity evaluation by using the toxicity evaluation system  1  is disclosed. First, the 3D chip  10  may be designed and manufactured. This is illustrated in  FIGS.  2  and  3   . The method may include an operation of trapping the spawns in the plurality of recesses  12  by injecting the solution  30  containing the spawns into the chip  10 , and an operation of inducing in vitro fertilization by injecting the solution  30  containing the sperms that fertilize the spawns into the chip  10 . In more detail, ovulation may be induced through injection of HCG into the Xenopus laevis chip  10 , and it may be injected into the 3D chip  10  after the spawns are squeezed. Thereafter, in a process of injecting the spawns into the 3D chip  10 , single entities may be physically fixed to the recesses  12 . Then, based on the peristaltic pump  20 , the fluid circulating system  1  that may inject sperms into the 3D chip  10  may be constituted. Thereafter, in vitro fertilization may be induced by circulating the sperms for several hours or several days. Growth of cleavages or tadpoles may be observed through this, or growth of cleavages or tadpoles may be observed while medicines are injected. 
     Thereafter, an operation of injecting a sample for evaluating a toxicity of a living body, on which the in vitro fertilization has been completed, into the passage  11  may be included. According to an example, in addition to the toxicity evaluation of an organism, on which the in vitro fertilization has been completed, the toxicity evaluation may become possible by injecting the sample for the toxicity evaluation even in the development process. 
     Thereafter, when the growth of the tadpoles are finished, the entities may be extracted out of the 3D chip  10 . Thereafter, embryogenesis may be studied through a molecular-biological analysis including an analysis of genes. 
     According to an example, by causing the solution to flow in a first direction by adjusting the pump  20 , the organism, on which the in vitro fertilization has been completed, may be trapped in the recess  12 . According to another example, by causing the solution to flow in a second direction by adjusting the pump  20 , the organisms, on which the in vitro fertilization has been completed, may be caused to escape. 
     That is, according to the inventive concept, external fertilization may be observed, and may be utilized for pharmaceutical toxicology. Furthermore, side effects that may be caused as drugs are mixed with the solution  30  and are caused to flow in the external fertilization, development, and growth process in the chip  10 . 
     Furthermore, the inventive concept may circulate the solution  30 , in which the sperms are mixed for external fertilization, by using the pump  20 . Alternatively, the solution for cultivating Xenopus laevis may be circulated, or drugs may be mixed therewith to be circulated. Furthermore, the chip  10  may be utilized for the study of embryogenesis. 
     According to the inventive concept, after external fertilization of embryos, a toxicity, by which therapeutic substances or biomolecules influence development of a vertebrate, may be evaluated. 
     The effects of the inventive concept are not limited to the above-mentioned effects, and the unmentioned effects can be clearly understood by those skilled in the art to which the inventive concept pertains from the specification and the accompanying drawings. 
     It is noted that the above embodiments are suggested for understanding of the inventive concept and do not limit the scope of the inventive concept, and various modifiable embodiments also fall within the scope of the inventive concept. It should be understood that the technical protection range of the inventive concept has to be determined by the technical spirit of the claims, and the technical protection range of the inventive concept is not limited to the lexical meaning of the claims but reaches even to the equivalent inventions.