In-vitro skin stretching model construction method

Abstract

The invention discloses an in-vitro skin stretching model construction method. The method comprises the following steps: acquiring sheet-shaped in-vitro skin; the flaky in-vitro skin is subjected to pretreatment operation for standby application; the skin tissue is installed on a fixed part and a movable part which are arranged on a distraction support and are adjustable in relative position, a culture solution is injected into a culture dish, the distraction support is placed in the culture dish, and the skin tissue is soaked in the culture solution; the relative positions of the fixed part and the movable part are adjusted, and the traction force borne by the skin tissue is changed; the skin tissue is taken down from the distraction support; skin tissue parameters are collected for in-vitro skin stretching model evaluation. According to the invention, the construction of the experimental model completely relying on the human skin for mechanical research can be realized, and the accuracy of data is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of first aid testing equipment, and in particular to a method for constructing an in vitro skin stretch model. Background Art

[0002] Current clinical research on human skin primarily involves creating an internal expansion model using water bladders inserted into mice and rats for mechanical studies. This approach presents a problem: the skin structure of experimental mice and rats differs from that of human skin, making experimental models based on mouse and rat skin incapable of fully replacing those based on human skin and mechanical studies conducted on rat and rat skin. The resulting experimental data fail to fully capture the true mechanism by which human skin expands under mechanical stress. Therefore, developing a new device for constructing human skin models that overcomes these limitations of existing technologies is a topic for further research by those skilled in the art. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for constructing an in vitro skin stretch model, which can realize the construction of an experimental model for mechanical mechanics research based on human skin and improve the accuracy of the data.

[0004] The present invention discloses a method for constructing an in vitro skin stretch model, which comprises the following steps:

[0005] Step 010: Obtaining ex vivo skin in the form of a sheet, wherein the ex vivo skin in the form of a sheet refers to human skin that is separated from the human body;

[0006] Step 020: Pre-treating the ex vivo skin sheet and obtaining skin tissue from the ex vivo skin sheet for later use;

[0007] Step 030: Mounting the skin tissue on a fixed portion and a movable portion of a stretching support, wherein the relative positions of the fixed portion and the movable portion are adjustable;

[0008] Step 040: injecting culture fluid into a culture dish and placing the stretching scaffold in the culture dish so that the skin tissue is immersed in the culture fluid;

[0009] Step 050: Adjusting the relative positions of the fixed portion and the movable portion to change the tension applied to the skin tissue;

[0010] Step 060: removing the skin tissue from the distraction support;

[0011] Step 070: Collect the parameters of the skin tissue to perform in vitro skin stretch model evaluation.

[0012] This technical solution utilizes discarded human skin obtained from clinical settings for in vitro culture. The two ends of the human skin sample are secured to a fixed portion and a movable portion, respectively. The stretching stent is then placed in a culture dish, allowing the human skin to be immersed in a culture medium. During the construction of the model for mechanical research, the movable portion is gradually moved away from the sutured side of the fixed portion, thereby maintaining a certain tensile force on the human skin in the culture medium. This allows the construction of an in vitro experimental model for mechanical research using human skin.

[0013] Preferably, in the above-mentioned in vitro skin stretch model construction method,

[0014] In step 100,

[0015] The ex vivo skin flakes are obtained by obtaining human skin discarded during clinical surgery, or more specifically, by obtaining clinically discarded human skin after obtaining informed consent from the patient.

[0016] Preferably, in the above-mentioned in vitro skin stretch model construction method,

[0017] In step 200, the pre-processing operation includes:

[0018] Soak the excised skin slices in pre-cooled PBS for later use;

[0019] Wash the excised skin sheet with pre-cooled PBS and chlorhexidine;

[0020] The soaked ex vivo skin sheet was transferred to a clean bench and washed several times with sterile PBS and chlorhexidine;

[0021] Using sterile surgical scissors to cut subcutaneous tissue from the ex vivo skin sheet to obtain the skin tissue;

[0022] By adopting this technical solution, it is ensured that the obtained skin graft can be kept in a relatively sterile state and the activity of the skin graft can be maintained for a period of time.

[0023] Preferably, in the above-mentioned in vitro skin stretch model construction method,

[0024] In step 030,

[0025] The distraction bracket is made of medical stainless steel and includes a fixed part and a movable part.

[0026] The fixing portion includes a first transverse plate, and a plurality of first puncture holes are distributed on the plate body of the first transverse plate and respectively penetrate the upper and lower sides of the plate body of the first transverse plate;

[0027] The movable portion includes a movable beam parallel to the first transverse plate, and the movable beam is provided with a plurality of second puncture holes respectively penetrating the upper and lower sides of the plate body of the movable beam;

[0028] Mounting the skin tissue on the fixed portion and the movable portion of a stretching bracket refers to suturing and fixing the two ends of the skin tissue to the first puncture holes on the first horizontal plate and the second puncture holes on the movable beam by surgical suturing.

[0029] By adopting this technical solution: the two ends of the sheet of ex vivo skin are fixed to the first horizontal plate and the movable beam respectively by surgical suturing. Fixing the skin sheet by surgical suturing can ensure that it remains sterile during in vitro culture and ensure the stability of fixation.

[0030] Preferably, in the above-mentioned in vitro skin stretch model construction method, in step 040,

[0031] The culture dish is a circular culture dish with an inner diameter of 6 cm;

[0032] The immersing of the skin tissue in the culture solution refers to allowing the dermis layer of the skin tissue to be immersed in the culture solution and the epidermis layer of the skin tissue to be exposed to air.

[0033] By adopting this technical solution, it is ensured that the skin graft remains relatively sterile and maintains its activity over a period of time.

[0034] Preferably, in the above-mentioned in vitro skin stretch model construction method,

[0035] In step 030,

[0036] The stretching bracket is configured as follows: the fixing portion further includes a first longitudinal straight rod, a second longitudinal straight rod and a second transverse plate; one end of the rod body of the first longitudinal straight rod and one end of the rod body of the second longitudinal straight rod are respectively fixedly connected to the two ends of the plate body of the first transverse plate as a whole; the other end of the rod body of the first longitudinal straight rod and the other end of the rod body of the second longitudinal straight rod are respectively fixedly connected to the two ends of the plate body of the second transverse plate as a whole; a plurality of positioning pins are symmetrically provided on the rod body of the first longitudinal straight rod and the rod body of the second longitudinal straight rod; positioning sockets matching the positioning pins are respectively provided at both ends of the movable crossbeam;

[0037] In step 050,

[0038] Adjusting the relative positions of the fixed part and the movable part refers to using medical tweezers to clamp the movable beam and move the movable beam so that the positioning sockets at both ends are respectively inserted into a positioning column on the first longitudinal straight rod and a positioning column on the second longitudinal straight rod.

[0039] By adopting this technical solution: the distance between the fixed part and the movable part is adjusted in a detachable manner, the device structure is simplified, and it is ensured that when the fixed part and the movable part are relatively fixed, the distance between them is stable, so as to facilitate the acquisition of experimental samples.

[0040] Preferably, in the above-mentioned in vitro skin stretch model construction method,

[0041] In step 060,

[0042] Removing the skin tissue from the stretching support means using medical tweezers to clamp the movable beam, moving the movable beam until the positioning holes at both ends are separated from the positioning pins, and then using surgical scissors to remove the skin tissue from the culture dish.

[0043] Preferably, in the above-mentioned in vitro skin stretch model construction method,

[0044] In the step 060, it further includes:

[0045] After the skin tissue is removed from the culture dish, at least one operation of mechanical-related functional experiments, primary cell extraction, bioinformatics testing, gene editing and regulation, etc. is performed on the skin tissue.

[0046] Preferably, in the above-mentioned in vitro skin stretch model construction method,

[0047] In step 070, the in vitro skin stretch model assessment includes at least one of the following:

[0048] The morphological changes of the model were described by HE and Masson staining;

[0049] Skin activity was evaluated at different culture time points using CCK8 and LDH assays;

[0050] Epidermal cell apoptosis and proliferation were evaluated by TUNEL staining and Ki67 immunofluorescence staining;

[0051] Skin activity was evaluated at different culture time points through bioinformatics quality control;

[0052] The skin barrier function was evaluated by transepidermal water loss and immunofluorescence staining;

[0053] Transmission electron microscopy was used to observe the cell connections of the skin at different culture time points and to evaluate the effect of tension on cell connections.

[0054] Compared with the prior art, the present invention has the following technical advantages:

[0055] First, the present invention realizes the construction of an experimental model for mechanical mechanics research based on human skin, thereby improving the accuracy of experimental data.

[0056] Secondly, the present invention can ensure that both ends of the human skin cultured in vitro are uniformly stretched during the process of constructing the model.

[0057] Thirdly, the present invention can withstand high-temperature sterilization and at the same time ensure that it has no toxic effects on in vitro cultured skin that it contacts.

[0058] Finally, the present invention has a simple structure and is easy to prepare and operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 Schematic diagram of the process of Example 1.

[0060] Figure 2 Schematic diagram of the structure of the culture dish and culture support in Example 1.

[0061] Figure 3 Schematic diagram of the evaluation of skin sheet activity of the in vitro culture model at different time points by CCK8 detection in Example 1.

[0062] Figure 4 Schematic diagram of the evaluation of skin sheet activity of the in vitro culture model at different time points by LDH detection in Example 1.

[0063] Figure 5 for Figure 3-4 Schematic diagram of skin graft activity assessment for the control group.

[0064] In the figure, the corresponding components of the reference numerals are as follows:

[0065] 100, culture dish; 200, culture rack; 210, first transverse plate; 220, first vertical rod; 230, second vertical rod; 240, second transverse plate; 250, movable beam; 260, positioning pin; 211, first puncture hole; 251, second puncture hole; 252, positioning hole. DETAILED DESCRIPTION

[0066] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0067] Example 1, please refer to Figure 1-5 :

[0068] A method for constructing an in vitro skin stretch model comprises the following steps:

[0069] Step 010: Obtaining ex vivo skin in the form of a sheet, wherein the ex vivo skin in the form of a sheet refers to human skin that is separated from the human body;

[0070] In this example, the ex vivo skin obtained refers to human skin discarded during clinical surgery. Furthermore, it refers to obtaining clinically discarded human skin after obtaining the patient's informed consent.

[0071] Step 020: Pre-treating the ex vivo skin sheet for later use;

[0072] In this example, the pretreatment operation specifically includes: soaking the excised skin sheet in pre-cooled PBS for later use; rinsing the skin sheet with pre-cooled PBS and chlorhexidine; transferring the skin sheet to a clean bench, and washing the skin tissue several times with sterile PBS and chlorhexidine; trimming the skin tissue into a size of 1 mm-1.5 mm (length) * 1 mm-1.5 mm (width) and immersing it in complete culture medium for later use.

[0073] Step 030: Mounting the skin tissue on a fixed portion and a movable portion of a stretching support, wherein the relative positions of the fixed portion and the movable portion are adjustable;

[0074] In this example, the culture support 200 is entirely made of medical stainless steel, and the size of the culture support 200 is configured so that the culture support 200 can be placed in the culture dish 100. Here, the culture dish 100 is a conventional laboratory culture dish 100, a round transparent culture dish 100 with an inner diameter of 6 cm.

[0075] The distraction stent is made of medical stainless steel and includes a fixed portion and a movable portion. The fixed portion includes a first transverse plate 210, which is a long rectangular plate with a plurality of first puncture holes 211 vertically extending through the upper and lower sides of the first transverse plate 210. The first puncture holes 211 are evenly distributed along the extension direction of the first transverse plate 210. The movable portion includes a movable beam 250 parallel to the first transverse plate 210, which is a long rectangular plate with a length comparable to that of the first transverse plate 210. The movable beam 250 includes a plurality of second puncture holes 251 vertically extending through the upper and lower sides of the movable beam 250. The second puncture holes 251 are evenly distributed along the extension direction of the movable beam 250. The step of mounting the ex vivo skin sheet on the fixed portion and the movable portion of a stretching support refers to suturing and fixing the two ends of the ex vivo skin sheet to the first puncture holes 211 on the first horizontal plate 210 and the second puncture holes 251 on the movable beam 250 by surgical suturing. The tensioning bracket is configured as follows: the fixed part also includes a first longitudinal straight rod 220, a second longitudinal straight rod 230 and a second horizontal plate 240; one end of the rod body of the first longitudinal straight rod 220 and one end of the rod body of the second longitudinal straight rod 230 are respectively fixedly connected to the two ends of the plate body of the first horizontal plate 210 as a whole; the other end of the rod body of the first longitudinal straight rod 220 and the other end of the rod body of the second longitudinal straight rod 230 are respectively fixedly connected to the two ends of the plate body of the second horizontal plate 240 as a whole; there are several symmetrical positioning columns 260 on the rod body of the first longitudinal straight rod 220 and the rod body of the second longitudinal straight rod 230; the two ends of the movable beam 250 are respectively provided with positioning sockets 252 matching the positioning columns 260. (That is, the first transverse plate 210, the first vertical rod 220, the second vertical rod 230, and the second transverse plate 240 together form a hollow rectangular frame, and the length and width of the rectangular hollow frame are both less than 6 cm to facilitate its overall placement within the culture dish 100.) Furthermore, before suturing and fixation, the direction of the dermatoglyphics must be confirmed. The direction of stretching and dermatoglyphics can be determined based on the research content.

[0076] Step 040: injecting culture fluid into the culture dish 100 and placing the stretching scaffold in the culture dish 100 so that the skin tissue is immersed in the culture fluid;

[0077] In this example, the culture dish 100 is a circular culture dish 100 with an inner diameter of 6 cm; immersing the ex vivo skin in the culture solution means that the dermis layer of the skin tissue is immersed in the culture solution and the epidermis layer of the skin tissue is exposed to the air.

[0078] Step 050: Adjusting the relative positions of the fixed portion and the movable portion to change the tension applied to the skin tissue;

[0079] In this example, adjusting the relative positions of the fixed part and the movable part refers to using medical tweezers to clamp the movable beam 250 and move the movable beam 250 so that the positioning holes 252 at both ends are respectively inserted into a positioning column 260 on the first longitudinal straight rod 220 and a positioning column 260 on the second longitudinal straight rod 230.

[0080] Step 060: removing the skin tissue from the distraction support;

[0081] In this example, removing the skin tissue from the stretching frame involves using medical forceps to grasp the movable crossbeam 250, moving the movable crossbeam 250 until the positioning sockets 252 at both ends are separated from the positioning pins 260, and then using surgical scissors to remove the skin tissue from the culture dish 100. Furthermore, the skin tissue may be subjected to at least one of mechanical function experiments, primary cell extraction, bioinformatics testing, gene editing, and regulation.

[0082] Step 070: Collect the parameters of the skin tissue to perform in vitro skin stretch model evaluation. The in vitro skin stretch model evaluation includes at least one of the following:

[0083] The morphological changes of the model were described by HE and Masson staining; CCK8 and LDH detection were used to evaluate skin activity at different culture time points; TUNEL staining and Ki67 immunofluorescence staining were used to evaluate epidermal cell apoptosis and proliferation; single-cell sequencing quality inspection was used to evaluate skin activity at different culture time points; transepidermal water loss and Claudin immunofluorescence staining were used to evaluate skin barrier function; and transmission electron microscopy was used to observe cell connections at different culture time points and evaluate the effect of tension on cell connections.

[0084] In this case, CCK8 and LDH assays were used to evaluate the activity of the skin grafts in the in vitro culture model at different time points. It was found that the skin grafts maintained good skin activity and a relatively intact skin structure within 7 days of in vitro culture compared to before culture. During the culture process, the epidermal thickness of the skin grafts in the stretching group gradually increased, exceeding that of the control group on the third day.

[0085] The embodiments of the present invention are described in detail above with reference to the accompanying drawings, but the present invention is not limited to the above embodiments. Even if various changes are made to the present invention, if these changes fall within the scope of the claims of the present invention and their equivalents, they still fall within the scope of protection of the present invention.

Claims

1. A method for constructing an in vitro skin stretch model, characterized in that: The steps include: Step 010: Obtaining ex vivo skin in the form of a sheet, wherein the ex vivo skin in the form of a sheet refers to human skin that is separated from the human body; Step 020: Pre-treating the ex vivo skin sheet and obtaining skin tissue from the ex vivo skin sheet for later use; Step 030: Mounting the skin tissue on a fixed portion and a movable portion of a stretching support, wherein the relative positions of the fixed portion and the movable portion are adjustable; Step 040: injecting culture fluid into a culture dish and placing the stretching scaffold in the culture dish so that the skin tissue is immersed in the culture fluid; Step 050: Adjusting the relative positions of the fixed portion and the movable portion to change the tension applied to the skin tissue; Step 060: removing the skin tissue from the distraction support; Step 070: Collect the parameters of the skin tissue to perform in vitro skin stretch model evaluation.

2. The method for constructing an in vitro skin stretch model according to claim 1, characterized in that: In step 100, The method of obtaining ex vivo skin in sheet form refers to obtaining human skin discarded during clinical surgery.

3. The method for constructing an in vitro skin stretch model according to claim 2, wherein: In step 200, the pre-processing operation includes: Soak the excised skin slices in pre-cooled PBS for later use; Wash the excised skin sheet with pre-cooled PBS and chlorhexidine; The soaked ex vivo skin sheet was transferred to a clean bench and washed several times with sterile PBS and chlorhexidine; Using sterile surgical scissors to cut subcutaneous tissue from the ex vivo skin sheet to obtain the skin tissue; The skin tissue was trimmed into a size of 1-1.5 mm*1-1.5 mm and immersed in complete culture medium for later use.

4. The method for constructing an in vitro skin stretch model according to claim 3, wherein: In step 030, The distraction bracket is made of medical stainless steel and includes a fixed part and a movable part. The fixing portion includes a first transverse plate, and a plurality of first puncture holes are distributed on the plate body of the first transverse plate and respectively penetrate the upper and lower sides of the plate body of the first transverse plate; The movable portion includes a movable beam parallel to the first transverse plate, and the movable beam is provided with a plurality of second puncture holes respectively penetrating the upper and lower sides of the plate body of the movable beam; Mounting the skin tissue on the fixed portion and the movable portion of a stretching bracket refers to suturing and fixing the two ends of the skin tissue to the first puncture holes on the first horizontal plate and the second puncture holes on the movable beam by surgical suturing.

5. The method for constructing an in vitro skin stretch model according to claim 4, characterized in that: In step 040, The culture dish is a circular culture dish with an inner diameter of 6 cm; The immersing of the skin tissue in the culture solution refers to allowing the dermis layer of the skin tissue to be immersed in the culture solution and the epidermis layer of the skin tissue to be exposed to air.

6. The method for constructing an in vitro skin stretch model according to claim 5, characterized in that: In step 030, The stretching bracket is configured as follows: the fixing portion further includes a first longitudinal straight rod, a second longitudinal straight rod and a second transverse plate; one end of the rod body of the first longitudinal straight rod and one end of the rod body of the second longitudinal straight rod are respectively fixedly connected to the two ends of the plate body of the first transverse plate as a whole; the other end of the rod body of the first longitudinal straight rod and the other end of the rod body of the second longitudinal straight rod are respectively fixedly connected to the two ends of the plate body of the second transverse plate as a whole; a plurality of positioning pins are symmetrically provided on the rod body of the first longitudinal straight rod and the rod body of the second longitudinal straight rod; positioning sockets matching the positioning pins are respectively provided at both ends of the movable crossbeam; In step 050, Adjusting the relative positions of the fixed part and the movable part refers to using medical tweezers to clamp the movable beam and move the movable beam so that the positioning sockets at both ends are respectively inserted into a positioning column on the first longitudinal straight rod and a positioning column on the second longitudinal straight rod.

7. The method for constructing an in vitro skin stretch model according to claim 6, wherein: In step 060, Removing the skin tissue from the stretching support means using medical tweezers to clamp the movable beam, moving the movable beam until the positioning holes at both ends are separated from the positioning pins, and then using surgical scissors to remove the skin tissue from the culture dish.

8. The method for constructing an in vitro skin stretch model according to claim 7, characterized in that: In the step 060, it further includes: After the skin tissue is removed from the culture dish, at least one operation of mechanical-related functional experiments, primary cell extraction, bioinformatics testing, gene editing and regulation, etc. is performed on the skin tissue.

9. The method for constructing an in vitro skin stretch model according to claim 8, characterized in that: In step 070, the in vitro skin stretch model assessment includes at least one of the following: The morphological changes of the model were described by HE and Masson staining; Skin activity was evaluated at different culture time points using CCK8 and LDH assays; Epidermal cell apoptosis and proliferation were evaluated by TUNEL staining and Ki67 immunofluorescence staining; Skin activity was evaluated at different culture time points through bioinformatics quality control; The skin barrier function was evaluated by transepidermal water loss and immunofluorescence staining; Transmission electron microscopy was used to observe the cell connections of the skin at different culture time points and to evaluate the effect of tension on cell connections.

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