KR20220153306A - Method for pretreating tissue for decellularization using electroporation and sonication - Google Patents

Abstract

The present invention relates to a pretreatment method which increases the efficiency of decellularization from a tissue by improving tissue permeability, and a method for decellularization of a tissue using the same. The present invention provides a pretreatment method of a skin tissue for decellularization, which includes the following steps: treating a separated skin tissue with sodium dodecyl sulfate (SDS); and treating the tissue treated with sodium dodecyl sulfate (SDS) with Triton X-100.

Description

Method for pretreating tissue for decellularization using electroporation and sonication {Method for pretreating tissue for decellularization using electroporation and sonication}

The present invention relates to a tissue pre-treatment method for increasing tissue decellularization efficiency through electroporation and ultrasonic treatment, and a method for decellularization of the pre-treated tissue through electroporation and ultrasonic treatment.

Decellularized living tissue is a technology that can be applied to various tissue engineering fields that require extracellular matrix (ECM) proteins, such as investigation of cell reactivity to its three-dimensional structure, tissue transplantation, and tissue regeneration. Be in the spotlight.

Tissue decellularization is a technology for using the remaining extracellular matrix after selectively removing cell components from tissues obtained from living organisms. It is used extensively.

Previously reported decellularization methods mainly use a combination of physical, chemical, and enzymatic treatments. A general decellularization method is to destroy the cell membrane using a physical treatment or an ionic solution, separate cellular components from the extracellular matrix through an enzymatic treatment, and then use a washing solution to separate the cells. It consists of the process of removing endogenous material, cell nuclear material, and cellular debris.

Tissues rich in extracellular matrix, such as the skin, maintain a relatively undamaged original form even after decellularization, and thus tissues having a three-dimensional structure maintained after decellularization can be used as they are. On the other hand, in the case of tissues with many cellular components, a post-processing process is essential, such as dissolving the extracellular matrix obtained after decellularization and using it as bioink for 3D printing. In particular, in the case of using limited resources due to practical and ethical problems, such as human tissue, there is a limit in that only extremely limited tissues can be used after decellularization.

When decellularization is performed using existing physical, chemical, and enzymatic methods, the structure inherent in biological tissues is destroyed or extracellular matrix proteins are lost. In addition, while going through such a treatment process, a problem in that biological tissues are physically weakened may occur. In particular, long-term decellularization of large animals requires a long treatment process, which inevitably leads to loss of extracellular matrix proteins and degradation of structural properties. Additional measures are being taken to shorten the decellularization time, such as using strong washing solutions or increasing physical force, but these also entail loss of extracellular matrix proteins and disruption of structural properties.

Therefore, in performing decellularization from tissue, there is a demand for a method for increasing decellularization efficiency and minimizing cell damage by shortening the decellularization time.

One object of the present invention relates to a pretreatment method that increases the efficiency of tissue decellularization by improving tissue permeability.

Another object of the present invention relates to a method for decellularizing tissues with high efficiency by improving tissue permeability through the above pretreatment method.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

According to one embodiment of the present invention, it relates to a method for pretreatment of tissue for decellularization.

In the present invention, the "decellularization" is to separate the extracellular matrix (ECM) of the tissue from the cells present in the tissue, leaving an ECM scaffold of the original tissue that can be used for artificial organs and tissue regeneration This is the process used to

In the present invention, the tissue to be subjected to the decellularization is not particularly limited as long as it is a biological tissue derived from a vertebrate, and a biological tissue derived from livestock such as mammals, livestock such as birds, or humans is more preferable. As mammalian livestock, cattle, horses, camels, llamas, donkeys, yaks, sheep, pigs, goats, deer, alpacas, dogs, raccoons, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, mice, squirrels, and Americas raccoons, etc. In addition, as livestock of birds, parakeets, parrots, chickens, ducks, turkeys, geese, guinea fowls (helmeted guinea fowl), pheasants, ostriches, quails, emu, etc. may be mentioned, but preferably mouse cows, pigs, It may be rabbit or human tissue.

In the present invention, as the site of the tissue, a site having an extracellular matrix structure is used, and examples of the site include liver, kidney, ureter, bladder, urethra, tongue, tonsil, esophagus, stomach, small intestine, large intestine, and anus. , pancreas, heart, blood vessel, spleen, lung, brain, bone, spinal cord, cartilage, testis, uterus, fallopian tube, ovary, placenta, cornea, skeletal muscle, tendon, kidney, skin, etc., but for the purpose of the present invention, skin organization is preferred.

The decellularization pretreatment method of the present invention is a sodium chloride (NaCl) aqueous solution for the separated tissue; trypsin-EDTA solution; sodium dodecyl sulfate (SDS); And at least one selected from the group consisting of Triton X-100, preferably an aqueous solution of sodium chloride (NaCl); trypsin-EDTA solution; sodium dodecyl sulfate (SDS); and processing Triton X-100.

The decellularization pretreatment method of the present invention may include a first washing step of removing residual blood or impurities from the separated tissue.

In the present invention, the first washing may be performed by washing the tissue with a washing solution containing distilled water.

In the present invention, the time for performing the first washing is not particularly limited, but may be, for example, greater than 0 and less than 24 hours, preferably greater than 8 and less than 20 hours.

The pretreatment method for decellularization of the present invention may include treating the firstly washed tissue with an aqueous solution of sodium chloride (NaCl).

In the present invention, the concentration of the sodium chloride aqueous solution is not particularly limited, but may be, for example, 0.1 to 10 M, preferably 1 to 3 M.

In the present invention, the treatment time of the aqueous sodium chloride solution is not particularly limited, but may be, for example, greater than 0 and less than 24 hours, preferably 12 to 18 hours.

The decellularization pretreatment method of the present invention may include treating a trypsin-EDTA solution to a tissue treated with an aqueous sodium chloride solution.

In the present invention, the concentration of the trypsin-EDTA (trypsin-EDTA) solution is not particularly limited, but may be, for example, 0.01 to 1% by weight / volume, preferably 0.05% by weight / volume.

In the present invention, the treatment time of the trypsin-EDTA solution is not particularly limited, but may be, for example, greater than 0 and less than 24 hours, preferably 12 to 18 hours.

The decellularization pretreatment method of the present invention may include treating the tissue treated with a trypsin-EDTA solution with sodium dodecyl sulfate (SDS).

In the present invention, the "sodium dodecyl sulfate (SDS)" is a kind of anionic surfactant, the molecular formula is NaC ₁₂ H ₂₅ SO ₄ , the molecular weight is 288.38, the density is 1.01 g/cm ³ , the melting point is Silver has a temperature of 206 ℃ and is a white or pale yellow crystal with a slightly peculiar odor. SLS is made by the esterification reaction of dodecanol (lauryl alcohol, C ₁₂ H ₂₅ OH) with sulfuric acid.

In the present invention, the concentration of sodium dodecyl sulfate (SDS) is not particularly limited, but may be, for example, 0.1 to 10% by weight/volume, preferably 1 to 5% by weight/volume. And, more preferably, it may be 2% by weight / volume.

In the present invention, the treatment time of the sodium dodecyl sulfate (SDS) is not particularly limited, but may be, for example, 1 to 10 days, preferably 3 days.

The decellularization pretreatment method of the present invention may include treating the tissue treated with sodium dodecyl sulfate (SDS) with Triton X-100.

In the present invention, the "Triton X-100 (Triton X-100, Triton X-100, C ₁₄ H 22 _O (C ₂ H 4 _O ) n)" is a hydrophilic polyethylene oxide chain (having an average of 9.5 ethylene oxide units) and an aromatic hydrocarbon It is a nonionic surfactant with a lipophilic or hydrophobic group.

In the present invention, the concentration of Triton X-100 is not particularly limited, but may be, for example, 0.1 to 10% by weight/volume, preferably 1% by weight/volume.

In the present invention, the treatment time of the Triton X-100 is not particularly limited, but may be, for example, greater than 0 and less than 24 hours, preferably 12 to 18 hours.

The decellularization pretreatment method of the present invention may include a secondary washing step to remove by-products of destroyed cells or residual impurities from the tissue treated with Triton X-100.

In the present invention, the secondary washing may be performed using distilled water or an aqueous sodium chloride solution. Here, the concentration of the sodium chloride aqueous solution is not particularly limited, but may be, for example, 0.1 to 10 M, preferably 1 M.

In addition, in the present invention, the solution used for the secondary washing may contain antibiotics for sterilization and disinfection. Although the type of the antibiotic is not particularly limited, for example, penicillin/streptomycin may be used.

In general, among living tissues, skin tissue in particular has lower decellularization efficiency than other tissues, so the decellularization process requires a lot of time along with the treatment of high-concentration chemical solutions, resulting in loss of extracellular matrix proteins and collapse of structural characteristics. There is a problem being. By the way, when a series of decellularization pretreatment methods according to the present invention are performed, the permeability of skin tissue increases, and as a subsequent decellularization process, the degree of influence of electrical stimulation applied to skin tissue during electroporation and/or ultrasonic treatment It is possible to significantly increase the decellularization efficiency by increasing the decellularization process, and furthermore, it is possible to solve problems such as loss of extracellular matrix proteins and collapse of structural characteristics by increasing the efficiency of outflow of dissolved cells from the tissue during the decellularization process.

According to another embodiment of the present invention, it relates to a method for decellularizing tissues pretreated by the pretreatment method of the present invention.

The decellularization method of the present invention may perform a step of performing electroporation on the pretreated tissue.

In the present invention, the electroporation may be performed by adding an aqueous solution of sodium chloride to the pretreated tissue and applying an electric current.

In the present invention, the concentration of the aqueous sodium chloride solution used during the electroporation may be preferably 0.1 to 10 M, more preferably 1 M.

In the present invention, the intensity of the pulse current applied during the electroporation may be preferably 0.1 to 5 A, more preferably 1 to 3 A, and most preferably 2 A.

In the present invention, the duration of the pulse current applied during the electroporation is 1 second to 5 seconds, preferably 1 second to 3 seconds, and the rest time, that is, the interval between pulses is 3 seconds to 10 seconds, preferably 3 seconds to 5 seconds.

Also, in the present invention, the application time of the total pulse current may be 5 minutes to 60 minutes, preferably 10 minutes to 30 minutes.

In the present invention, the electroporation may be performed while maintaining a temperature of 37 °C or less.

The decellularization method of the present invention may further include ultrasonicating the pretreated tissue one or more times before, after, or before and after performing the electroporation.

In the present invention, the ultrasonic treatment may be preferably performed for 30 minutes to 6 hours, and more preferably for 1 to 3 hours.

In the present invention, the ultrasonic treatment may be performed within a frequency range of 20 to 40 kHz, preferably 30 to 40 kHz.

In the process of decellularization by conventional chemical methods, there is a problem in that it must be treated for a long time along with the treatment of a high-concentration chemical solution, and there is a problem of wastewater due to organic solvents that are inevitably generated after the decellularization process. In the present invention, all of the above problems could be solved by using the electroporation method and ultrasonic treatment in combination during the decellularization process and performing the tissue pretreatment process prior to this.

In the case of the decellularization pretreatment method of the present invention, by increasing the permeability of skin tissue with very low decellularization efficiency, the degree of influence of electrical stimulation applied to skin tissue during electroporation and/or ultrasonic treatment in the subsequent decellularization process is reduced. The efficiency of decellularization increases according to the increase, and moreover, the efficiency of dissolving cells from the tissue in the above decellularization process is increased or decellularization can be performed under mild conditions, so that extracellular matrix protein (ECM) It is possible to solve the problems such as the loss of the structure and the collapse of the structural characteristics.

In addition, the decellularization method of the present invention not only shortens the time required for decellularization by reducing the processing time and amount of chemical solvents and enzymes used in the existing general decellularization process by performing electroporation and ultrasonic treatment, but also decellularization It is preferable because it can reduce the amount of wastewater due to the organic solvent inevitably generated after saturation.

1 shows a photograph of a skin tissue derived from a human body, which is a subject of the following experiment in Preparation Example 1.
Figure 2 shows a photograph observed under a microscope after treating the skin tissue with a 1 M aqueous solution of sodium chloride in Preparation Example 1 and staining with hematoxylin & eosin (H&E).
Figure 3 is electroporation for the skin tissue treated with sodium chloride aqueous solution in Experimental Example 1; and/or ultrasound; The result of measuring the double-stranded DNA content in the tissue after treatment is shown in a graph.
4 is a flowchart of a pretreatment and decellularization process for human-derived skin tissue according to an example of the present invention.
5 shows a photograph of the skin tissue pretreated in Experimental Example 2 observed under an electron microscope.
Figure 6 shows a photograph observed under a microscope after performing hematoxylin & eosin (H&E) staining on the skin tissue pretreated in Experimental Example 2.
Figure 7 shows a photograph observed under a microscope after performing hematoxylin & eosin (H&E) staining on the decellularized skin tissue in Experimental Example 2.
8 is a graph showing the result of measuring the temperature change of sodium chloride added to the chamber after applying the pulse current in performing electroporation on the skin tissue pretreated in Experimental Example 3.
9 is a graph showing the results by measuring the double-stranded DNA content in the skin tissue after performing the decellularization process of ultrasonic treatment-electroporation-ultrasound treatment under various conditions for the skin tissue pretreated in Experimental Example 3 .
10 is a flowchart of a pretreatment and decellularization process for human-derived skin tissue according to an example of the present invention.
11 is a graph showing the results of measuring the double-stranded DNA content in the decellularized skin tissue in Experimental Example 4.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for explaining the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

[Preparation Example 1]

Human-derived skin tissue to be used in the experiment was taken and a photograph thereof is shown in FIG. 1 . These skin tissues were treated with a 1 M aqueous solution of sodium chloride, stained with hematoxylin & eosin (H&E), and the results of observing changes in the skin tissues under a microscope are shown in FIG. 2 .

As shown in Figure 2, it was confirmed that the separation of the skin dermal layer and the epidermal layer occurs by treatment with a 1 M sodium chloride aqueous solution.

[Experimental Example 1]

Electroporation under the conditions shown in Table 1 for the skin tissue treated with the sodium chloride aqueous solution in Preparation Example 1; and/or ultrasound; After the treatment, the double-stranded DNA content in the tissues after each treatment was measured, and the results are shown in FIG. 3 .

division processing conditions electroporation Pulse current strength: 2 A Pulse current duration/rest time: 2s/4s Total pulse current application time: 1 hour sonication Processing time: 2 hours Frequency: 40 kHz

As shown in FIG. 3, compared to the case where electroporation or ultrasonic treatment is performed alone on the skin tissue immersed in sodium chloride solution, when all of these treatments are performed, in particular, ultrasonic treatment-electroporation-ultrasonic treatment is performed in the order In one case, it was confirmed that the DNA content decreased the most. However, it was found that the decellularization of skin tissue was difficult due to the insignificant effect of reducing the DNA content by such treatment alone. Accordingly, the inventors of the present invention developed a pretreatment process for skin tissue decellularization and conducted the following experiments.

[Experimental Example 2]

4 is a flow chart of a pretreatment and decellularization process for human-derived skin tissue according to the present invention. Specifically, the skin tissue is first washed with distilled water to remove blood or impurities remaining in the tissue for 16 hours, then treated with 1 M aqueous sodium chloride solution for 16 hours, and 0.05% by weight/volume of trypsin- EDTA (trypsin-EDTA) solution was treated for 16 hours. Then, it was treated with 2 wt/vol% SDS for 3 days and then treated with 1 wt/vol% Triton X-100 for 16 hours. Thereafter, a second washing was performed to remove impurities or cell by-products with distilled water. Thereafter, ultrasonication-electroporation-ultrasound treatment was sequentially performed under the conditions shown in Table 1 above. A photograph of the pretreated skin tissue observed under an electron microscope after performing the secondary washing is shown in FIG. 5, and a photograph observed under a microscope after performing hematoxylin & eosin (H&E) staining on the pretreated skin tissue is shown in Figure 6. In addition, after performing hematoxylin & eosin (H&E) staining on the decellularized skin tissue that has been subjected to electroporation and sonication, a photograph observed under a microscope is shown in FIG. 7 .

As shown in FIGS. 5 and 6, in the case of the pretreated skin tissue, the fiber bundles constituting the extracellular matrix are released compared to the skin tissue that has not been pretreated, and the epidermal layer is separated and the dermis layer has a microspace. You can see it formed.

In addition, as shown in FIG. 7 , while cells were observed in untreated skin tissue, these cells were not observed in skin tissue subjected to decellularization after pretreatment. Therefore, it was confirmed that decellularization was effectively performed.

Through this, it was found that the microspace in the skin tissue formed through the above pretreatment process effectively transmits electrical stimulation to the skin tissue during electroporation and helps the penetration of ultrasound at the same time, ultimately increasing the decellularization efficiency.

[Experimental Example 3]

In the same manner as in Experimental Example 1, the pretreatment process was performed on the human skin tissue, and then the decellularization process of ultrasonic treatment-electroporation-ultrasound treatment was performed, but the electroporation conditions were performed under the conditions shown in Table 2 below. . At this time, the temperature change over time in the chamber was measured, and the results are shown in FIG. 8, and the double-stranded DNA content in the skin tissue treated with the second ultrasonic wave after electroporation for each condition was measured and the results are shown in FIG. was

division processing conditions electroporation 1 condition Pulse current strength: 2 A Pulse current duration/rest time: 2s/4s Total pulse current application time: 20 minutes Total electroporation time: 1 hour 2 conditions Pulse current strength: 2 A Pulse current duration/rest time: 5min/5min Total pulse current application time: 20 minutes Total electroporation time: 40 minutes 3 conditions Pulse current strength: 2 A Pulse current duration/rest time: 5min/10min Total pulse current application time: 20 minutes Total electroporation time: 1 hour 4 conditions Pulse current strength: 2 A Pulse current duration/rest time: 5min/25min Total pulse current application time: 20 minutes Total electroporation time: 2 hours sonication Processing time: 2 hours Frequency: 40 kHz

As shown in FIG. 8, when a pulse current of 2 A is applied under conditions of 2 seconds/4 seconds and 5 minutes/25 minutes for the duration/pause time (interval between pulses), the temperature in the chamber ranges from 35 to 37 It was found that it was possible to apply the maximum without exceeding ℃. As shown in FIG. 9, among the above two conditions, in particular, when a pulse current of 2 A is applied under a condition of 2 seconds/4 seconds for a duration/pause time (interval between pulses), respectively, it is applied under a condition of 5 minutes/25 minutes. It was found that the decellularization efficiency was more excellent by significantly lowering the DNA content in the skin tissue than in one case.

[Experimental Example 4]

In order to prove that an excellent decellularization efficiency improvement effect can be obtained only by a series of pretreatment processes shown in FIG. 4, the following experiment was performed. Specifically, according to the flow chart shown in FIG. 10, pretreatment and decellularization processes were performed on human-derived skin tissue, and in detail, 1 step of removing blood or impurities remaining in the tissue with distilled water for 16 hours. After performing the car washing, 1 M sodium chloride aqueous solution was treated for 16 hours, and 0.05 weight/vol % trypsin-EDTA (trypsin-EDTA) solution was treated for 16 hours. Thereafter, a second washing was performed to remove impurities or cell by-products with distilled water. Thereafter, ultrasonic treatment-electroporation-ultrasonic treatment was sequentially performed under the conditions shown in Table 3 below. After the second ultrasonic treatment, the double-stranded DNA content in the skin tissue was measured and the results are shown in FIG. 11 .

division processing conditions electroporation Pulse current strength: 2 A Pulse current duration/rest time: 2s/4s Total pulse current application time: 20 minutes Total electroporation time: 1 hour sonication Processing time: 2 hours Frequency: 40 kHz

As shown in FIG. 11, although the experiment was performed while adjusting the concentration of the aqueous sodium chloride solution supported during electroporation, the DNA content was still high in the skin tissue after the decellularization process, so it is predicted that a large number of cells remain in the skin tissue. Bar, it was confirmed that the decellularization efficiency was low.

Having described specific parts of the present invention in detail above, it is clear that these specific techniques are merely preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (22)

treating the separated skin tissue with sodium dodecyl sulfate (SDS); and
A method for pre-treatment of skin tissue for decellularization comprising the step of treating the tissue treated with sodium dodecyl sulfate (SDS) with Triton X-100. According to claim 1,
The concentration of the sodium dodecyl sulfate (SDS) is 0.1 to 10% by weight / volume, the pretreatment method. According to claim 1,
The treatment time of the sodium dodecyl sulfate (SDS) is 1 to 10 days, the pretreatment method. According to claim 1,
The concentration of the Triton X-100 is 0.1 to 10% by weight / volume, the pretreatment method. According to claim 1,
The treatment time of Triton X-100 is more than 0 and less than 24 hours, pretreatment method. According to claim 1,
The pretreatment method further comprises the step of treating the separated skin tissue with an aqueous sodium chloride (NaCl) solution prior to the step of treating the sodium dodecyl sulfate (SDS). According to claim 6,
The concentration of the sodium chloride aqueous solution is 0.1 to 10 M, pretreatment method. According to claim 6,
The pretreatment method, wherein the treatment time of the aqueous sodium chloride solution is greater than 0 and less than or equal to 24 hours. According to claim 1,
The pretreatment method further comprises the step of treating the separated skin tissue with a trypsin-EDTA solution prior to the step of treating the sodium dodecyl sulfate (SDS). According to claim 9,
The concentration of the trypsin-EDTA (trypsin-EDTA) solution is 0.01 to 1% by weight / volume, pretreatment method. According to claim 9,
The treatment time of the trypsin-EDTA (trypsin-EDTA) solution is greater than 0 and less than 24 hours, pretreatment method. According to claim 1,
The pretreatment method further comprises the step of firstly washing the separated skin tissue prior to the step of treating the sodium dodecyl sulfate (SDS). According to claim 1,
The pretreatment method further comprises the step of secondarily washing the skin tissue after the step of treating Triton X-100. A method for decellularizing skin tissue comprising performing electroporation on the skin tissue pretreated by the method of any one of claims 1 to 13. According to claim 14,
The decellularization method further comprises the step of ultrasonicating the pretreated tissue one or more times before, after, or before and after performing the electroporation, the method for decellularizing skin tissue. According to claim 14,
The electroporation is performed by adding an aqueous solution of sodium chloride at a concentration of 0.1 to 10 M to the pretreated tissue and applying an electric current, a method for decellularizing skin tissue. According to claim 14,
The intensity of the pulse current applied during the electroporation is 0.1 to 5 A, the decellularization method of skin tissue. According to claim 14,
The duration of the pulse current applied during the electroporation is 1 second to 5 seconds, the rest time is 3 seconds to 10 seconds, the decellularization method of skin tissue. According to claim 14,
The application time of the total pulse current during the electroporation is 5 minutes to 60 minutes, the decellularization method of skin tissue. According to claim 14,
Wherein the electroporation is performed while maintaining a temperature of 37 ° C. or less, a method of decellularizing skin tissue. According to claim 15,
The ultrasonic treatment is performed for 30 minutes to 6 hours, a method of decellularizing skin tissue. According to claim 15,
The ultrasonic treatment is performed within a frequency range of 20 to 40 kHz, a method for decellularizing skin tissue.

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