Method for screening for therapeutic agent for preterm premature rupture of membrane

Abstract

The present invention provides a method for screening for a therapeutic agent for preterm premature rupture of membrane, the method comprising the steps of: (1) treating amniotic tissue including incision damage or perforation damage with a candidate material; (2) fastening the amniotic tissue treated with the candidate material to a container containing a solution, so as to cover an opening of the container; and (3) measuring leakage of the solution through the amniotic membrane.

Description

Premature rupture of membranes treatment screening method

[0001] The present invention relates to a method for rapidly and simply screening therapeutic agents for premature rupture of membranes.

[0002] The amniotic membrane is a thin membrane, 0.02 to 0.5 mm thick, located at the innermost part of the placenta. It consists of five layers: the epithelial layer, basal layer, compact layer, fibroblast layer, and spongy layer. As an avascular placental membrane lacking nerves, lymphatic vessels, or blood vessels, it surrounds the fetus and acts as a protective barrier against various infections and immune responses from the mother. In humans, the amniotic membrane initially contacts the fetus when it first forms, but after 4 to 5 weeks of pregnancy, amniotic fluid begins to fill the membrane. As the fluid accumulates, the membrane gradually expands and eventually comes into contact with the chorionic membrane located on the outside. The amniotic fluid allows the fetus inside the membrane to move freely and acts to cushion external shocks.

[0003] Preterm premature rupture of membranes (PRP) is a condition in which amniotic fluid leaks out of the membrane due to an abnormality in the amniotic membrane surrounding the fetus during pregnancy. Preterm premature rupture of membranes can be broadly divided into two types depending on the cause: spontaneous pPRP, which occurs due to the mother's health condition or inflammatory response, and iatrogenic pPRP, which occurs due to amniocentesis performed in obstetrics and gynecology.

[0004] Premature rupture of membranes (PROM) is a major cause of perinatal complications and neonatal morbidity and mortality; it is known to account for 50% of neonatal deaths and 30–40% of preterm births, with 30–50% occurring before 37 weeks of gestation. Generally, if PROM occurs before 24–25 weeks of gestation, fetal survival is difficult, leading to a high likelihood of miscarriage. This is because PROM concomitantly causes a severe increase in the risk of intrauterine infection and impaired development of the fetal lung system. Furthermore, as society becomes increasingly older, the frequency of preterm births is rising, and consequently, the frequency of PROM is also rapidly increasing.

[0005] As such, despite the high frequency of perinatal complications and neonatal morbidity / mortality associated with premature rupture of membranes, methods for prediction and prevention, as well as therapeutic approaches, are not yet clearly defined, and the clinical protocols implemented are very limited.

[0006] Traditional treatment focuses on conservative management based on the evidence that gestational age is the most critical factor in neonatal morbidity and mortality, emphasizing the maintenance of the pregnancy; however, this approach is highly limited in preventing premature rupture of membranes due to the potential for numerous obstetric complications. Furthermore, regarding diagnostic approaches, the utility of ultrasound examination has not been proven, and cervical examinations remain challenging due to the increased risk of infection and the potential for shortened pregnancy duration. Finally, regarding current therapeutic approaches, the administration of antibiotics and corticosteroids is not appropriate for long-term treatment as it can have adverse effects on fetal growth, adrenal function, neurological development, and survival rates.

[0007] Under the circumstances described above, some attempts to develop treatments for premature rupture of membranes have been identified. For example, experimental methods involving artificially damaging the amniotic membrane of pregnant mammals (e.g., rats) and then administering a treatment to assess efficacy are known; however, these methods are not ethically sound and present problems such as the significant time and cost required to establish animal models and verify therapeutic efficacy.

[0008] Against this backdrop, the inventors have completed the present invention by developing a novel screening system for selecting therapeutic substances in vitro in a rapid and simple manner for the development of a treatment for premature rupture of membranes.

[0009] One objective of the present invention is to provide a screening method for a treatment for preterm premature rupture of membrane.

[0010] Another objective of the present invention is to provide a screening vessel for a treatment for premature rupture of membranes.

[0011] Another objective of the present invention is to provide a device for evaluating the airtightness of a container for screening early amniotic rupture of membranes therapeutic agents based on changes in internal pressure.

[0012] Each description and embodiment disclosed in this invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention should not be considered limited by the specific descriptions provided below.

[0013] In the following, redundant details will be omitted to prevent clutter. In other words, the content of the invention is not limited solely to the following, and should be interpreted in accordance with the overall context of the invention.

[0014] Furthermore, terms used in this specification are for illustrative purposes only and should not be interpreted as intended to be limiting. Singular expressions include plural expressions unless the context clearly indicates otherwise. Terms such as “comprising” or “having” in this specification are intended to specify the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0015] In addition, terms such as first, second, A, B, (a), (b), (1), (2), etc. may be used when describing the components of the present invention. These terms are intended only to distinguish the components from other components, and the essence, order, or sequence of the components is not limited by the terms. Where it is stated that a component is "connected," "combined," or "connected" to another component, it should be understood that the component may be directly connected or connected to the other component, but that other components may also be "connected," "combined," or "connected" between each component.

[0016] Furthermore, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as generally understood by those skilled in the art to which the embodiments pertain. Terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an ideal or overly formal sense unless explicitly defined in this specification.

[0017] The present invention will be described in detail below.

[0018] The present invention provides a screening method for a treatment for preterm premature rupture of membrane, comprising: (1) a step of treating amniotic tissue including incision damage or punch damage with a candidate substance; (2) a step of attaching the amniotic tissue treated with the candidate substance to a container so that the opening of the container containing the solution is covered; and (3) a step of measuring leakage of the solution through the amniotic membrane.

[0019] The term 'amniotic membrane or amniotic tissue' refers to a membrane observed during pregnancy in amniotic organisms such as birds, mammals, and reptiles, consisting of an avascular matrix that surrounds the fetus and serves to protect it from the external environment. More specifically, it may refer to a thin avascular membrane that forms a sac that binds to the fetus and constitutes the environment, consisting of a single layer that forms the placenta in a triple-layered structure together with the chorionic membrane and decidua. In the present invention, it is preferable to use a material of human origin.

[0020] The above amniotic membrane or amniotic tissue may be separated from placental tissue obtained through premature birth, cesarean section, natural childbirth, etc. For example, the amniotic membrane may be separated from placental tissue through blunt dissection and cut into appropriate sizes. In the case of such amniotic membrane, for example, after undergoing a washing process using a buffer solution such as DPBS to remove blood clots from the amniotic membrane, it may be stored in a medium capable of maintaining or culturing the amniotic membrane or amniotic tissue.

[0021] These media may include, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM / F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax complete medium, AminoMax ± complete medium, EBM (Endothelial Basal Medium) medium, Chang's Medium, MesenCult-XF, DMEM / HG (Dulbecco's Modified Eagle's Medium high glucose) medium, or MCDB+DMEM / LG (MCDB+Dulbecco's Modified Eagle's Medium low glucose), but are not particularly limited thereto.

[0022] If necessary, the separated amniotic membrane or amniotic tissue may be frozen and stored under conditions further containing polyhydroxy compounds, such as sugars (monosaccharides, disaccharides, and polysaccharides), polyalcohols, and derivatives thereof, and may be thawed for use. Cryoprotective agents that can be used as such freezing agents include, for example, sucrose, trehalose, mannitol, sorbitol, glucose, raffinose, maltose, glycerol, lactose, fructose, galactose, and combinations thereof.

[0023] A screening method for a therapeutic agent for premature rupture of membranes according to the present invention includes the step of treating amniotic tissue containing an incision injury or perforation with a candidate substance.

[0024] In the present invention, an incision injury refers to a separation of continuity in a part of the amniotic tissue, and the incision injury can be filled back into tissue according to the recovery process of the amniotic membrane.

[0025] The above incision may be an injury with a length of 0.1 mm to 20 mm, preferably 0.5 to 18 mm, more preferably 1 mm to 16 mm, even more preferably 2 mm to 14 mm, and most preferably 3 to 10 mm. The above incision may produce injuries of various lengths depending on the tool used. The above incision may be made with a medical scalpel.

[0026] In the present invention, a perforation refers to a hole penetrating the amniotic membrane, and means a state in which no tissue or cells exist within the perforation when it is created. The perforation may be filled with tissue again as the amniotic membrane recovers. The shape of the perforation may be circular, elliptical, or polygonal depending on the tool used; while circular is preferred, it is not limited thereto, and irregular perforations are also possible. The perforation may be created using a syringe needle. The diameter of the perforation may be 0.01 mm to 10 mm, preferably 0.05 to 9 mm, more preferably 0.1 mm to 8 mm, even more preferably 0.2 mm to 7 mm, and most preferably 0.3 to 5 mm.

[0027] In one embodiment, the cross-sectional area of ​​the amniotic membrane tissue may be 30 mm to 70 mm, preferably 40 mm to 60 mm, but is not limited thereto and can be appropriately adjusted according to the diameter of the opening of the container used for screening and the size of the damage.

[0028] The amniotic tissue is preferably separated amniotic tissue. More specifically, it is amniotic tissue separated from placental tissue obtained through premature birth, cesarean section, or natural childbirth.

[0029] The above candidate substances include, but are not limited to, cells, synthetic compounds, natural compounds, low molecular weight compounds, high molecular weight compounds, nucleic acid molecules (e.g., DNA, RNA, PNA, and aptamers), proteins, sugars, or lipids. The above candidate substances may include substances that have an effect on treating premature rupture of membranes or are expected to have a therapeutic effect. The treatment may involve the re-filling of an incision or perforation of the amniotic membrane with tissue or the regeneration of amniotic membrane tissue.

[0030] Additionally, in step (1), the treatment of the candidate substance may involve directly applying or coating the therapeutic candidate substance to the site of incision or perforation damage of the amniotic membrane, or adding it to a culture medium containing the amniotic membrane.

[0031] Specifically, adding to the culture medium of the amniotic membrane may involve directly adding a candidate substance to the culture medium containing the amniotic membrane. Additionally, a method of co-culturing using a transwell system may be included, and secretion factors can be further identified by co-culturing using the transwell.

[0032] More specifically, a method of co-culturing using the above-described transwell system may include placing amniotic tissue at the bottom of the transwell and inoculating a candidate substance (preferably, cells) at the top of the transwell for co-culturing. Through such co-culturing, therapeutic efficacy, such as paracrine effects, may also be confirmed.

[0033] In step (1) of the present invention, the step of treating the amniotic membrane with a candidate substance and then culturing the amniotic membrane treated with the candidate substance may be further included.

[0034] In addition, step (1) of the present invention may further include a step of treating the amniotic membrane with a candidate substance and then restoring the amniotic membrane treated with the candidate substance.

[0035] The step of culturing or restoring the amniotic membrane treated with the above candidate substance may involve maintaining the amniotic membrane containing the incision damage or perforation for a certain period of time after treating the amniotic membrane containing the candidate substance so that the incision damage or perforation of the amniotic membrane is filled with tissue again, regenerated, or healed.

[0036] The culture medium used in the step of culturing or restoring the amniotic membrane containing the above-mentioned amniotic membrane or the amniotic membrane treated with the candidate substance is, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, DMEM / F12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax complete medium, AminoMax ± complete medium, EBM (Endothelial Basal Medium) medium, Chang's Medium, MesenCult-XF, DMEM / HG (Dulbecco's Modified Eagle's Medium high glucose) medium, or MCDB+DMEM / LG (MCDB+Dulbecco's Modified Eagle's Medium low glucose) medium under a certain It may be maintained for a period of time or longer. Additionally, the medium may contain antibiotics, antifungal agents, and / or substances commonly used in the industry to prevent the growth of Mycoplasma in order to prevent infection by bacteria, fungi, etc. Additionally, the medium may include serum isolated from animals such as fetuses of cattle, calves, horses, sheep, pigs, dogs, and goats, if necessary.

[0037] This culture period varies depending on the candidate substance being treated, but may be, for example, 12 hours, 24 hours, 36 hours, 48 ​​hours, 3 days, 4 days, 5 days, 6 days or longer.

[0038] A screening method for a therapeutic agent for premature rupture of membranes according to the present invention includes the step of attaching an amniotic tissue treated with a candidate substance to a container so that the opening of the container containing the solution is covered.

[0039] In the present invention, the solution is a solution containing a dye so that the degree of recovery and / or treatment status of an amniotic membrane damaged by incision or perforation can be visually confirmed after treatment with a candidate substance, and the degree of leakage of the solution from the amniotic membrane can be easily confirmed visually.

[0040] The above solution may include a dye and distilled water, and the dye included in the solution may be Trypan Blue, Hematoxylin, Eosin, or Naphthol Yellow S, but is not limited thereto. Preferably, the dye may be Trypan Blue.

[0041] In the present invention, the container may be made of materials such as glass, PETG (Polyethylene Terephthalate Glycol), PETE (polyethylene terephthalate), OPET (oriented polyethylene terephthalate), PCTG (Poly Cyclohexylenedimethylene Terephthalate), PCTC (Polycyclodimethylterephthalate glycol), polyester, polypropylene (PP), polyethylene (PE), or polystyrene (PS), but is not limited thereto.

[0042] In the present invention, the container may include a bottle shape, a cylinder shape, or a polygonal shape, but is not limited thereto. Any container having the shape of a conventional container capable of holding a liquid, including an opening on one side that allows for the injection or leakage of liquid, may be used. In one embodiment, the container may be used in which the opening of the container has a narrower diameter than the bottom of the container, but is not limited thereto.

[0043] In addition, the container may have a size capable of holding a sufficient amount of solution to allow the therapeutic effect to be confirmed by leaking the solution for a certain period of time, specifically 20 to 200 mL, more specifically 30 to 100 mL of solution. In one embodiment, the container may use a Square PETG Media Bottle from Nalgene™, and the container may hold 60 mL of solution.

[0044] In step (2) above, the fastening may be achieved by securing a portion of the membrane in contact with the upper outer surface including the opening of the container by tightening it with an elastic band.

[0045] The opening of the above-mentioned container refers to an open inlet through which a solution within the container can be injected or a solution contained within the container can leak out, and the diameter and / or cross-sectional area of ​​the opening may be smaller than the diameter and / or cross-sectional area of ​​the amniotic membrane.

[0046] In one embodiment, the diameter of the opening of the container may be 16 to 20 mm, preferably about 18 mm, and the cross-sectional area of ​​the membrane may have a size that can surround the opening and the outer surface of the top of the container.

[0047] The above top refers to a part of the container including an opening, and the outer surface of the top refers to the surface of the top.

[0048] The amniotic membrane and the container can be secured by covering the opening of the container with the amniotic membrane to block the opening, bringing the membrane into contact with the upper outer surface including the opening, and tightening it with an elastic band. At this time, an incision and / or perforation damage portion of the amniotic membrane may be located over the opening.

[0049] The above tightening can be performed by wrapping an elastic band once or multiple times, and can be tightly secured so that the solution does not leak out from the part tightened by the elastic band.

[0050] The above elastic band may be made of an elastic material such as rubber or synthetic resin having excellent elasticity, and may be a composite fiber containing an elastic material, but is not limited thereto.

[0051] The leakage of the above solution can be performed for 30 seconds to 30 minutes, preferably for 30 seconds to 15 minutes, more preferably for 30 seconds to 10 minutes, even more preferably for 30 seconds to 5 minutes, and most preferably for 30 seconds to 2 minutes. In one embodiment, the leakage of the solution can be performed for approximately 1 minute.

[0052] The absorbent pad described above may be composed of a material such as a sheet-shaped fiber, paper, or non-woven fabric, using a Super Absorbent Polymer (SAP) that has the property of absorbing a solution. The absorbent pad possesses absorbency in order to contain the solution leaked from the amniotic membrane and to evaluate the amount of leakage visually or quantitatively.

[0053] The above step (3) may specifically involve inverting a container covered with amniotic tissue so that the amniotic membrane comes into contact with the absorbent pad, thereby leaking the solution contained within the container and measuring the amount of leakage.

[0054] In step (3) above, leakage of the solution can be performed under conditions where the pressure acting on the opening covered by amniotic tissue is 30 to 50 mmHg. Since the pressure acting on the amniotic membrane is generally in the range of 20 to 30 mmHg, the pressure acting on the amniotic membrane at the time of preterm labor may be approximately 40 mmHg or higher. Therefore, in the present invention, the amount of leakage of the solution can be confirmed by setting the pressure applied to the opening covered by amniotic tissue to 30 to 45 mmHg, preferably around 40 mmHg.

[0055] The pressure acting on the opening covered by the amniotic tissue is the pressure acting on the amniotic membrane, specifically, the pressure acting on the amniotic tissue by the solution contained within the container inverted with the container covered by the amniotic tissue.

[0056] In one embodiment, the diameter of the opening is 18 mm, and the volume of the solution contained in the container may be about 60 mL so that the pressure acting on the amniotic membrane covering the opening is 30 to 50 mmHg. However, the volume of the solution contained in the container is not limited thereto, and the amount of the solution can be adjusted so that the pressure acting on the amniotic membrane is 30 to 50 mmHg, preferably about 40 mmHg.

[0057] Alternatively, the above step (3) may involve quantitatively measuring the airtightness of the container by causing leakage of the solution contained in the container under a reduced pressure or pressurized environment.

[0058] In step (3) above, the airtightness may be a measurement of the leakage pressure of the solution or the pressure at which leakage begins.

[0059] For example, the above step (3) may involve placing a container inside a device including a depressurizing or pressurizing pump and applying additional pressure inside to cause leakage of the solution contained in the container, and the lower the pressure at which the solution leaks from the container, or the lower the device pressure at which the solution begins to leak from the container, the more quantitatively the airtightness of the container is measured.

[0060] In step (3) above, the depressurization or pressurization environment can be controlled using a device including a depressurization or pressurization pump, and can be controlled in the range of -3000 mbar to 8000 mbar.

[0061] In the present invention, after step (3), (4) a step of selecting a candidate substance that reduces the leakage amount or increases the airtightness by comparing the leakage amount or airtightness of the solution leaked from the amniotic membrane treated with the candidate substance with the leakage amount or airtightness of the solution leaked from a control group not treated with the candidate substance.

[0062] In the step of selecting candidate substances, if the amount of solution leakage from the amniotic membrane treated with the candidate substance decreases compared to the amount of solution leakage of the control group, or if the airtightness of the container containing the amniotic membrane treated with the candidate substance increases compared to the airtightness of the control group container, this indicates that the tissue at the site of the incision or perforation injury where the solution may leak has regenerated and / or healed, thereby signifying that the incision or perforation injury of the amniotic membrane is recovered or treated by treatment with the candidate substance. In other words, the candidate substance can be used as a treatment for premature rupture of membranes.

[0063] When a container with the attached amniotic membrane tissue is inverted onto an absorbent pad and maintained for a certain period of time so that the opening covered by the amniotic membrane contacts the absorbent pad, if the incision or perforation injury is treated by the candidate substance, it can be confirmed that there is almost no solution absorbed by the absorbent pad, or that it is significantly reduced compared to the amniotic membrane not treated with the candidate substance.

[0064] Alternatively, when a container with an amniotic membrane attached is placed in a device including a depressurization or pressurization pump and additional pressure is applied inside the device to induce leakage of the solution contained within the container, if the incision or perforation injury is treated by the candidate substance, it can be confirmed that the airtightness of the container is significantly higher (lower leakage pressure of the solution or higher device pressure at which leakage begins) compared to a container containing an amniotic membrane not treated with the candidate substance.

[0065] The present invention is a container (1),

[0066] A main body (2) having an opening (22) at the top (21) and an internal space (23);

[0067] Amniotic membrane (3) covering the opening (22) above;

[0068] An elastic band (4) for fixing the above amniotic membrane; and

[0069] It includes a solution (5) contained in an internal space (23),

[0070] A portion (31) of the above membrane covers a portion of the upper outer surface (24), and

[0071] A portion (31) of the above membrane is fastened by tightening a portion of the upper outer surface (24) with an elastic band (4), and

[0072] The above amniotic membrane provides a container that is amniotic tissue containing incision damage or perforation damage.

[0073] The above container can preferably be used for screening purposes for the treatment of premature rupture of membranes.

[0074] More specifically, the amniotic membrane may be an amniotic tissue treated with a candidate substance, including incision damage or perforation damage.

[0075] In the container of the present invention, the definitions of “amniotic membrane,” “elastic band,” “solution,” “incision injury,” and “perforation” are as described above.

[0076] The above opening (22) refers to an open inlet through which a solution contained in a container can be injected or leaked, and the diameter and / or cross-sectional area of ​​the opening may be smaller than the diameter and / or cross-sectional area of ​​the membrane.

[0077] The upper part (21) refers to a part of the container including an opening in the container, and the outer surface (24) of the upper part refers to the surface.

[0078] The portion (31) of the above-mentioned amniotic membrane refers to the remaining portion that does not cover the opening, which is in contact with the outer surface of the top of the main body so that the amniotic membrane covering the opening is fixed to the main body of the container, and the portion (31) of the amniotic membrane in contact with the outer surface (24) can be fixed to the main body by an elastic band (4).

[0079] The above-mentioned tightening method may involve covering the opening of the container with the membrane to block the opening, bringing the upper outer surface containing the opening into contact with a portion of the membrane not covering the opening, and securing the membrane and the container by tightening with an elastic band. Specifically, this can be performed by wrapping the elastic band once or multiple times, and the connection can be made tight so that the solution does not leak out from the part tightened by the elastic band.

[0080] The above internal space (23) may include a space having a volume of 20 to 200 mL capable of holding a solution.

[0081] The diameter of the opening (22) of the container may be narrower than the diameter of the container body (2), but is not limited thereto.

[0082] The pressure acting on the amniotic membrane (3) may be 30 to 50 mmHg, preferably around 40 mmHg. The pressure acting on the amniotic membrane (3) may be the pressure exerted on the amniotic membrane tissue by the solution contained in the internal space of the container when the container is inverted.

[0083] The present invention is a device (6) for evaluating the airtightness of the container based on changes in internal pressure,

[0084] A vacuum chamber (61) having a storage space (62) formed inside for storing a container and a sealing part (63) that can be opened or sealed to the outside; and

[0085] A device is provided that includes a control unit (65) connected to the chamber and tube (64) and controlling pressure changes in the internal space.

[0086] Specifically, evaluating the airtightness of the above-mentioned container may involve evaluating the airtightness of the amniotic membrane attached to the container.

[0087] Water (66) can be filled into a part of the storage space above.

[0088] The above device may additionally include a display unit that graphs and displays the results of the airtightness measurement.

[0089] The above device can adjust the internal pressure to a range of -3000 mbar to 8000 mbar.

[0090] The screening method for a therapeutic agent for premature rupture of membranes according to the present invention has the effect of enabling rapid and convenient screening of the therapeutic agent ex vivo. In particular, it has the advantage of allowing rapid visual evaluation without the need for separate devices such as a microscope, as well as accurate analysis through quantification.

[0091] Figure 1 is a schematic diagram showing a normal control amniotic membrane without incision damage (a), an amniotic membrane with incision damage (b), and a candidate substance treatment (c) on an amniotic membrane with incision damage.

[0092] Figure 2 is a figure showing a container capable of screening an early rupture of membranes therapeutic agent prepared according to Example 1.

[0093] Figure 3 is a diagram showing the leakage of a solution through the amniotic membrane using a container.

[0094] Figure 4 is an image (a) confirming the amount of solution leaked through the amniotic membrane using a container, and a graph (b) summarizing it.

[0095] Figure 5 is a cross-sectional view illustrating a container capable of screening for a treatment for premature rupture of membranes.

[0096] Figure 6 is a figure briefly showing the conditions of the normal control group (normal amniotic membrane), the disease control group (damaged amniotic membrane), and the experimental group (treatment of the damaged amniotic membrane with a therapeutic candidate substance).

[0097] FIG. 7 is a simplified illustration of a quantitative functional evaluation system for a premature rupture of membranes disease model, showing an image (a) of amniotic tissue attached to a test container according to each condition and an image (b) showing the step of installing the test container in a water tank to measure airtightness due to changes in external pressure.

[0098] Figure 8 is a figure showing the pressure (a) when the test container maintains airtightness and the pressure (b) when leakage occurs in a quantitative functional evaluation method of a premature rupture of membranes disease model.

[0099] Figure 9 is a graph showing the results of the airtightness evaluation in a pressurized environment for each experimental group of an in vitro model of premature rupture of membranes. In the graph, the disease control group used amniotic membranes damaged by 16G, 19G, and 20G needles, respectively, while the experimental group used amniotic membranes damaged by 16G, 19G, and 20G needles treated with MSCs.

[0100] FIG. 10 is a cross-sectional view illustrating a sealing evaluation system including a container and a depressurization or pressurization pump capable of screening for a treatment for premature rupture of membranes.

[0101] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0102] In this invention, MEM Alpha (Minimum Essential Medium, Gibco), FBS (Fetal Bovine Serum, Gibco), and Gentamicin Reagent Solution (Gibco) were used for the cell culture of mesenchymal stem cells (WJ-MSC). For the in vitro model of premature rupture of membranes, Nalgene™ Square PETG Media Bottles with Septum Closure: Sterile, Shrink-Wrapped Trays were purchased from Thermo Scientific™.

[0103] Example 1. Preparation of an in vitro model of premature rupture of membranes and a method for screening therapeutic agents using the same

[0104] (1) Mesenchymal stem cell culture (candidate substance)

[0105] Wharton jelly-derived mesenchymal stem cells (WJ-MSC) were cultured in a growth medium containing 90% MEM Alpha, 10% fetal bovine FBS, and 0.5% gentamicin at 37°C under 5% CO2 conditions.

[0106] (2) Preparation of amniotic membrane with incision damage

[0107] With the mother's consent, amniotic membrane tissue was received from the placenta discarded during childbirth (Department of Obstetrics and Gynecology, Samsung Seoul Hospital). Specifically, the placenta was placed in a sterile field with the umbilical cord facing upward. Subsequently, the umbilical cord was trimmed close to the placenta, and an X-shaped incision was made to wash away any remaining blood in the amniotic membrane before placing it in a sterile bottle for collection. To remove blood clots from the collected amniotic membrane, it was washed 3 to 4 times using DPBS (Dulbecco's phosphate-buffered salin).

[0108] The cross-sectional area of ​​the amniotic membrane was prepared so that it did not exceed 60 mm (Fig. 1(a)), and an incision damage of a certain depth was created using a No. 11 medical knife (PARAGON) (Fig. 1(b)). The amniotic membrane containing the incision damage was stored in DPBS to prevent it from drying out.

[0109] (3) Treatment of the candidate substance on the amniotic membrane where the incision damage occurred

[0110] After transferring the amniotic tissue containing the incision wound to a 60pi dish, cell culture medium was filled. Subsequently, 5×10⁶ cultured WJ-MSCs (candidate substance) were added. 5 After direct seeding at 100 µl per cell / 100 µl at the site of the amniotic membrane incision, the cells were cultured for 24 hours in a 37°C, 5% CO2 incubator (Fig. 1(c)).

[0111] (4) Manufacture of a container for screening premature rupture of membranes

[0112] A square PETG media bottle (60 mL, Nalgene™) was filled with a solution mixed with 0.01% Trypan blue solution and distillation (60 mL), and the opening of the bottle was covered with the amniotic membrane cultured for 24 hours and secured tightly using a rubber band to fix the membrane. At this time, the incision site was positioned over the opening (Fig. 2).

[0113] The overall cross-sectional view of the screening container for the treatment of premature rupture of membranes is shown in Fig. 5.

[0114] (5) Functional evaluation (solution leakage and leakage amount measurement)

[0115] The prepared screening container was inverted onto an absorbent pad (diaper) ([CNL] DELUXE DOG PADS 80 g / 600x760 mm) so that the amniotic membrane portion was in contact with the absorbent pad and maintained for 1 minute (Fig. 3). Afterward, the amount of solution absorbed by the absorbent pad was visually checked or quantified to see if the solution leaked out through the incision damage of the amniotic membrane.

[0116] Example 2. Screening of therapeutic agents for premature rupture of membranes

[0117] In Example 1, the container was inverted onto an absorbent pad ([CNL] DELUXE DOG PADS 80 g / 600x760 mm) so that the amniotic membrane portion was in contact with the absorbent pad and maintained for 1 minute, after which the absorbent pad was photographed with a camera, and the area of ​​solution leaking from the amniotic membrane was quantified using ImageJ. The results are shown in Figure 4.

[0118] As can be seen in Fig. 4, it was visually confirmed that there was almost no leakage of the solution on the absorbent pad in the experimental group treated with the candidate substance WJ-MSC (#2+WJ-MSC) compared to the control group (#2 Ctrl) that was not treated with the candidate substance (Fig. 4(a)). In addition, it was quantitatively confirmed through a quantified graph that there was almost no leakage of the solution on the absorbent pad in the experimental group treated with the candidate substance WJ-MSC (Fig. 4(b)).

[0119] Example 3. Preparation of an in vitro model of premature rupture of membranes simulating a gestational environment and a screening method for premature rupture of membranes therapeutic agents using the same

[0120] (1) Mesenchymal stem cell culture (candidate substance)

[0121] The mesenchymal stem cell (candidate substance) culture step was prepared in the same manner as in Example 1 above.

[0122] (2) Preparation of amniotic membrane with perforation damage

[0123] In the case of the amniotic membrane manufacturing step in which perforation damage is generated, it was manufactured in a manner similar to Example 1 above, except for the method of amniotic membrane damage.

[0124] Premature rupture of membranes is divided into iatrogenic pPROM and spontaneous pPROM depending on the cause of occurrence, and in this embodiment, we intended to construct an in vitro disease model of premature rupture of membranes suitable for the cause of each disease.

[0125] The iatrogenic premature rupture of membranes in vitro disease model induced perforation injury using 16G, 19G, and 20G needles, with the perforation diameter being largest with 16G and smallest with 20G. The spontaneous premature rupture of membranes in vitro disease model used a surgical mass.

[0126] (3) Treatment of candidate substances on the amniotic membrane where perforation damage occurred

[0127] The conditions of the normal control group, disease control group, and experimental group in this embodiment are briefly shown in FIG. 6.

[0128] The experimental group may include experimental group A, in which stem cells were treated on the damaged amniotic membrane, and experimental group B, in which a therapeutic candidate substance was treated, and the stem cell treatment was performed in the same manner as in Example 1 above.

[0129] (4) Manufacture of a container for screening premature rupture of membranes

[0130] The step of manufacturing the screening container was performed in the same manner as in Example 1 above.

[0131] (5) Functional evaluation (airtightness measurement)

[0132] We established an ex vivo functional evaluation method to simulate the pressure conditions exerted on the amniotic membrane in the gestational environment and to quantify the degree of amniotic membrane damage and recovery in normal control groups, disease control groups, and experimental groups.

[0133] First, as shown in FIG. 7, the test containers of the normal control group, disease control group, and experimental group prepared in (4) of Example 3 (Fig. 7(a)) were placed in a water tank equipped with a depressurization or pressurization pump (Fig. 7(b)). Then, the airtightness of the amniotic membrane damage site was compared while depressurizing or pressurizing the pressure of the water tank.

[0134] For example, in the pressurized environment experiment, the set pressure (device pressure) of the water tank was gradually increased while simultaneously measuring the internal pressure of the tank to determine the set pressure (device pressure) at which leakage from the amniotic membrane begins, and through this, the airtightness of the normal control group, disease control group, and experimental group was compared.

[0135] Specifically, at a specific pressure, if the red line (pressure inside the tank) is maintained parallel to the green line (set pressure) as in Fig. 8(a), it is determined that the amniotic membrane maintains airtightness, and if the red line rises as in Fig. 8(b), it is determined that the amniotic membrane loses airtightness and leakage occurs.

[0136] A full cross-sectional view of a leak evaluation device including a container for screening premature rupture of membranes therapeutic agents and a depressurization or pressurization pump is shown in FIG. 10.

[0137] Example 4. Screening of therapeutic agents for premature rupture of membranes in an in vitro model simulating a gestational environment

[0138] Functional evaluation (airtightness measurement) in a pressurized environment was performed on the normal control group, disease control group, and experimental group prepared through the method of Example 3, and the results are shown in Fig. 9.

[0139] As a result of pressurized environment experiments, all disease control groups showed significantly lower set pressures (device pressure) at which leakage begins, regardless of needle diameter, and it was confirmed that the stem cell-treated experimental groups maintained airtightness even under relatively high pressure conditions compared to the disease control groups. In particular, the stem cell-treated 19G damaged amniotic membrane maintained airtightness up to an average pressure of about 2000 mbar, confirming that stem cells have the effect of partially regenerating the damaged amniotic membrane and restoring its function.

[0140] Through these results, it was confirmed that the screening model for therapeutic agents for premature rupture of membranes according to the present invention can be usefully employed for the discovery of therapeutic agents in vitro in a simple and rapid manner, and can also quantitatively measure the degree of amniotic membrane damage and the amniotic membrane regenerative capacity of therapeutic candidate substances.

[0141] [Explanation of the symbol]

[0142] 1: Courage

[0143] 2: Main body

[0144] 21: Top of the main body

[0145] 22: Opening

[0146] 23: Internal space

[0147] 24: Outsourcing

[0148] 3: Amniotic membrane

[0149] 31: Part of the amniotic membrane

[0150] 4: Elastic band

[0151] 5: Solution

[0152] 6: Confidentiality evaluation device

[0153] 61: Vacuum chamber

[0154] 62: Storage space

[0155] 63: Sealing part

[0156] 64: Tube

[0157] 65: Control unit

[0158] 66: Water

Claims

1. (1) A step of treating amniotic tissue including incision damage or punch damage with a candidate substance; (2) A step of attaching the amniotic tissue treated with the candidate material to the container so that the opening of the container containing the solution is covered; and (3) A screening method for a treatment for preterm premature rupture of membrane, comprising the step of measuring leakage of the solution through the amniotic membrane.

2. A screening method for a treatment for premature rupture of membranes according to claim 1, wherein the length of the incision injury is 0.1 mm to 20 mm.

3. A screening method for an early rupture of membranes treatment agent according to claim 1, further comprising the step of treating the amniotic membrane with a candidate substance in step (1) and then culturing the amniotic membrane treated with the candidate substance.

4. A screening method for a therapeutic agent for premature rupture of membranes according to claim 1, wherein the candidate substance comprises one or more selected from the group consisting of cells, synthetic compounds, natural compounds, low molecular weight compounds, high molecular weight compounds, nucleic acid molecules, proteins, sugars, and lipids.

5. A screening method for a therapeutic agent for premature rupture of membranes, wherein in step (1), the treatment of the candidate substance involves directly applying or coating the therapeutic agent candidate substance to the site of incision or perforation damage of the amniotic membrane, or adding it to a culture medium containing the amniotic membrane.

6. A screening method for a treatment for premature rupture of membranes, wherein, in claim 1, the solution comprises a dye and distilled water.

7. A screening method for a treatment for premature rupture of membranes according to claim 1, wherein the solution comprises one or more selected from the group consisting of Trypan Blue, Hematoxylin, Eosin, and Naphthol Yellow S.

8. A screening method for a treatment for premature rupture of membranes according to claim 1, wherein the container is made of one or more materials selected from the group consisting of glass, PETG (Polyethylene Terephthalate Glycol), PETE (polyethylene terephthalate), OPET (oriented polyethylene terephthalate), PCTG (Poly Cyclohexylenedimethylene Terephthalate), PCTC (Polycyclodimethylterephthalate glycol), polyester, polypropylene (PP), polyethylene (PE), and polystyrene (PS).

9. A screening method for an early rupture of membranes treatment, wherein in step (2), the fastening is secured by tightening a portion of the amniotic membrane and the upper outer surface including the opening of the container with an elastic band.

10. A screening method for a treatment for premature rupture of membranes, wherein, in claim 1, the leakage of the solution is performed for 30 seconds to 30 minutes.

11. A screening method for an early rupture of membranes treatment, wherein, in paragraph 1, step (3) is to invert a container covered with amniotic tissue so that the amniotic membrane contacts the absorbent pad, thereby leaking the solution contained in the container and measuring the amount of leakage.

12. A screening method for a treatment for premature rupture of membranes, wherein in step (3) of claim 11, the leakage of the solution is performed under conditions where the pressure acting on the opening covered by amniotic tissue is 30 to 50 mmHg.

13. A screening method for a treatment for premature rupture of membranes, wherein, in paragraph 1, step (3) is to quantitatively measure the airtightness of the container by causing leakage of the solution contained in the container under a reduced pressure or pressurized environment.

14. A screening method for a treatment for premature rupture of membranes, wherein, in paragraph 13, the above-mentioned airtightness measures the leakage pressure of the solution or the pressure at which leakage begins.

15. A screening method for an early rupture of membranes treatment, comprising, in addition to step (3) step (4), a step of comparing the leakage amount or airtightness of the solution leaked from the amniotic membrane treated with the candidate substance with the leakage amount or airtightness of the solution leaked from a control group not treated with the candidate substance, and selecting a candidate substance that reduces the leakage amount or increases the airtightness.

16. A main body (2) having an opening (22) at the top (21) and an internal space (23); Amniotic membrane (3) covering the opening (22) above; An elastic band (4) for fixing the above amniotic membrane; and It includes a solution (5) contained in an internal space (23), A portion (31) of the above membrane covers a portion of the upper outer surface (24), and A portion (31) of the above membrane is fastened by tightening a portion of the upper outer surface (24) with an elastic band (4), and The above amniotic membrane is a vessel that is amniotic tissue containing incision damage or perforation damage.

17. In paragraph 16, the amniotic membrane comprises a container in which a candidate substance is treated on amniotic tissue including incision damage or perforation damage.

18. A device (6) for evaluating the airtightness of the container of claim 13 based on changes in internal pressure, A vacuum chamber (61) having a storage space (62) formed inside for storing a container and a sealing part (63) that can be opened or sealed to the outside; and A device comprising: a control unit (65) connected to the chamber and tube (64) to regulate pressure changes in the internal space.

19. A device according to claim 18, wherein water (66) is filled in a part of the storage space.

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