WO2019245110A1 - Hybrid bio-ink, method of preparing same, and method of preparing artificial tissue using same - Google Patents

Abstract

The present invention relates to: a hybrid bio-ink which comprises a mixture of a first bio-ink obtained by liquefying extracellular matrices of decellularized tissues and a second bio-ink containing alginate or fibrinogen, provides advantages in terms of the survival, proliferation, and differentiation of cells due to excellent biocompatibility, and due to excellent physical strength, does not undergo deformation due to cells even after ionic crosslinking; a method of preparing the hybrid bio-ink; and a method of preparing artificial tissues using same, the present invention comprising: a first bio-ink obtained by liquefying the extracellular matrices of the decellularized tissues; and a second bio-ink containing alginate or fibrinogen, wherein the first bio-ink and the second bio-ink are mixed preferably in a volume ratio (v/v) of 1.5-9:1.

Description

 2019/245110 （1/10 公 018/012996

【Specification】

[Name of invention]

 Hybrid bio ink and its manufacturing method and artificial tissue manufacturing method using the same

TECHNICAL FIELD The present invention mixes a first bio ink liquefied with an extracellular matrix of decellularized tissue and a second bio ink containing alginate or fibrinogen, and thus has excellent biocompatibility, thereby allowing cell survival, proliferation and The present invention relates to a hybrid bio ink, which is advantageous for differentiation and has excellent physical strength and does not change shape by cells even after ion crosslinking, and a method of manufacturing the same, and a method of manufacturing artificial tissue using the bio 30 printing process using the same.

 [Project No. 1711064779 [Department name] Ministry of Science and Technology Information and Communication [Research Management Specialized Institution] Korea Research Foundation [Name of Research Project] Cell Regeneration Technology Development Project

[Project name] Development of microvascular internal type liver tissue mimic printing technology [Organizer] Ulsan Institute of Science and Technology

 [Research organization] 2017.04.01. -2021.12.31.

[Project 1, 201, her 1 show 284010353 [Department name] Basic research project of science and engineering field

1 Alternative paper (Article 26) 2019/245110 1 »（： 1/10 公 018/012996

[Research Management Specialized Institution] Ministry of Science and Technology Information and Communication

 [Name of research project] Medium-sized researcher support project

 [Project name] Production of Heterogeneous Liver Cancer Model Using 30-Bioprinting

 [Organizer] Korea University of Technology

 [Research Period] 2017.03.01 ~ 2019.02.29

 Background Art

 Bio 30 printing technology refers to a technology capable of manufacturing a specific shape by forming and stacking a shape desired by a user based on a bio printer, a bio ink, a cell, a growth factor, and the like.

 This bio 30 printing technology can be used to help heal diseases such as organoids (0 8! 101 (1), organ-like chips (0 311-011-3-()), and tissue and organ analogs for animal replacements. Many researches are available to give.

 In particular, bio inks include collagen, fibrin gel, matrigel, alginate, gelatin, etc., because intrinsic cells can survive, proliferate, and differentiate, and must maintain a specific shape or structure for a long time like human tissue. Material may be utilized.

 However, various growth factors are required to differentiate various cells as human tissues, and thus, a culture technology for decellularizing and liquefying specific tissues of animals and re-culturing the cells and restoring the original tissues in a similar form has been studied. .

 However, the above-mentioned conventional bio inks are advantageous for survival, proliferation and differentiation of specific cells, but are easy to use during long-term culturing due to their weak physical strength.

2 Alternative paper (Article 26) 2019/245110 （1/10 公 018/012996 It can be shrunk or disassembled, and it is difficult to maintain a specific shape or structure for a long time.

 [Prior art document]

 US Patent No. 9442105

 Detailed Description of the Invention

 [Technical problem]

 The present invention is a mixture of the first bio ink, which liquefied the extracellular matrix of the decellularized tissue and the second bio ink containing alginate or fibrinogen, has excellent biocompatibility and is advantageous for survival, proliferation and differentiation of cells. It is to provide a hybrid bio ink, a method of manufacturing the same, and a method of manufacturing artificial tissue using the bio 30 printing process using the hybrid bio ink, in which the physical strength is excellent and the shape is not changed by the cells even after ionic crosslinking.

 Technical Solution

 One embodiment of the present invention for achieving the object as described above, relates to a hybrid bio ink, low 11 bio ink liquefied extracellular matrix of decellularized tissue; And a second bio ink comprising alginate and / or fibrinogen, wherein the first bio ink and the second bio ink are preferably mixed in a volume ratio of 1.5-9: 1 (V8).

조직을포함하며, 상기 제 1 바이 The tissue is derived from a pig

Tissue, wherein the first bi

액상화（ （1116£3（：11011）될 수 있다. 1 to 3 ， Pep at a temperature of about 15 to 25 ^° 0

Can be liquefied (1116 £ 3 (11011)).

 3

Alternative Paper (Article 26) 2019/245110 1 »（： 1 ^ 1 {2018/012996 Specifically, the first bio ink or the second bio ink is one or more selected from embryonic fibroblasts, stem cells derived from embryonic stem cells, and human stem cells. It may include, and the concentration of 1.5 ~ 4.0% (wt ₂ 0 is preferred, respectively. Meanwhile, another embodiment of the present invention relates to a method for producing a hybrid bio ink, a decellular step of removing cells from the tissue Liquefying the extracellular matrix of the decellularized tissue to prepare a first bio ink; preparing a second bio ink comprising alginate or fibrinogen; and 1.5 preparing the first bio ink and the second bio ink. And a volume ratio of 9 to 1 (mixing with a furnace).

 At this time, the tissue may include liver (l iver) tissue derived from the pig, the decellular phase step, it is possible to remove the cells of the tissue using a decellular solution containing a surfactant and hypertonic solution.

 Specifically, the breakdown solution is preferably a solution of 0.03 to 1.0 M including any one or more selected from the group containing NaCl, KC1, CaCh, MgCh, BaCh, and NaHCOs, and the surfactant is Triton X-100 (Triton X-100), Tween 80, sodium dodecyl sul fate (SDS), sodium deoxycholate and triton X-200 (Tri ton X-200) is preferably any one or more selected from the group containing.

In the preparing of the sieve 1 bio ink, the extracellular matrix of the decellularized tissue may be liquefied by pessin at a temperature of pH 1 to 3 and about 15 to 25 ^° C. It is preferable that the 1st bio-ing a and the 2nd bio-inks are 1.5-4.0% (wt eight r), respectively.

 On the other hand, another embodiment of the present invention relates to a method for manufacturing artificial tissue,

4 Alternative paper (Article 26) 2019/245110 1 »（： 1/10 公 018/012996 printing artificial tissue using the hybrid bioinks; And a sol idi f icat ion step of gelling and cross-inking the artificial tissue using an inorganic salt.

 At this time, the inorganic salt is preferably at least one selected from the group consisting of calcium, manganese, barium, cobalt, zinc, copper.

 In addition, the solidification step, after gelling the collagen by heat (gelat ion), can be cross-linked kross-1 inking alginate or fibrinogen using a solution containing an inorganic salt.

 【Effects of the Invention】

 The present invention, by mixing the first bio ink, which liquefied the extracellular matrix of the decellularized tissue and the second bio ink containing alginate or fibrinogen, it is excellent in biocompatibility, which is advantageous for survival, proliferation and differentiation of cells. ， Excellent physical strength has the effect that the shape does not change by cells even after ion crosslinking.

 In addition, the crosslinking time required to prepare the artificial tissue according to the present invention is shortened, and the artificial tissue prepared according to the present invention includes a three-dimensional attachment space to which the cells can be attached and at the same time the cells can be attached. Aggregation between cells is possible, including numerous voids, which have beneficial effects on cell survival, proliferation and differentiation.

 【Brief Description of Drawings】

1 is a graph showing a result of measuring the viscosity according to the temperature change of the first bio-ink according to an embodiment of the present invention over time. Alternative Paper (Article 26) 2 and 3 are graphs showing the results of the viscosity measurement according to the change of the content of the hybrid bio ink according to an embodiment of the present invention over time.

 4 is a view showing a change in shape of the hybrid bio ink according to an embodiment of the present invention.

 5 and 6 are graphs showing the results of measuring the compressive strength according to the change in the content of the hybrid bio ink according to an embodiment of the present invention over time.

 Figure 7 is a view showing the cells cultured in artificial tissue according to an embodiment of the present invention.

 8 is a graph showing the results of measuring the cell viability cultured in artificial tissue according to an embodiment of the present invention over time.

 9 is a graph showing the results of measuring the cell proliferation rate cultured in artificial tissue according to an embodiment of the present invention over time.

 10 is a view showing a void of the artificial tissue according to an embodiment of the present invention. [Form for implementation of invention]

 Before describing in detail through the preferred embodiment of the present invention, the terms or words used in the present specification and claims should not be construed as being limited to the common or dictionary meanings, and are consistent with the technical spirit of the present invention. Be interpreted as meaning and concept.

Herein ¾ sieve, assuming that no part comprises a certain component """, a particularly meant the opposite, not excluding the other elements do not have a base material that can more ¾ ^· Four other components do.

6 Alternative paper (Article 26) 2019/245110 ^ 1/100018/012996 Hereinafter will be described in more detail with respect to the hybrid bio ink of the present invention, a method for producing the same, and a method for manufacturing artificial tissue using the same.

 First, the hybrid bio ink, the first bio ink liquefied the extracellular matrix of the decellularized tissue; And a second bio ink including alginate or fibrinogen; wherein the first bio ink and the second bio ink are mixed in a volume ratio of 1.5 to 9: 1.

 Decellularized tissue refers to tissue from which the rest of cellular components are removed except for the extracellular matrix of the tissue. The tissue used at this time may be tissues derived from mammals such as humans, pigs, cows, rabbits, dogs, goats, sheep, chickens and horses (eg, liver tissues, heart tissues, muscle tissues), The tissue used in the present invention is most preferably including liver (:]! ·) Tissue derived from pigs to favor the survival, proliferation and differentiation of hepatocytes.

미만인 경우에는， 펠신 ? !!）의 활성도가 감소하여 원활 한 소화（（1 63 010가 이루어질 수 없고, 251：를 초과하는 경우에는 펩신 6?3比） 의 활성이 지나치게 증가되어， 점도가 낮아져서 바이오 잉크로 사용하기에 부적합 하다. Specifically, the first bio ink is liquefied by pepsin 6-3 at a temperature of 1 to 3, about 15 to 25 ° (degrees) of decellularized tissue, and is suitable for use as a bioprinting material. It is desirable to have a viscosity (for example, 100 to 1000 to 3), where pepsin has different reactivity properties depending on 1) 11 and temperature conditions, which leads to temperature

If less than Pelsin? !!) activity is reduced, so that smooth digestion ((1 63 010 cannot be achieved, and when it exceeds 251 :), the activity of pepsin 6-3 is excessively increased, and the viscosity is low, so that it can be used as a bio ink. Not suitable

 As described above, the first bio ink is liquefied to an appropriate viscosity, so that the bioprinting aptitude is excellent, and biocompatibility is excellent, including extracellular matrix of decellularized tissue.

등을 활용하여 특정 세포의 생존， 증식 및 분화에 유 리하다. 7 Alternative Paper (Article 26) 2019/245110 ^ 1/1012018/012996 Nago, a variety of extracellular matrix components, glycoproteins and glycosaminoglyco

It is useful for survival, proliferation and differentiation of specific cells.

 The 12 bio inks may include, but are not limited to, natural polymers such as alginate, fibrinogen, carboxymethyl cellulose, heunsulfuric acid, hyaluronic acid, collagen, dextran, and most preferably, alginate or fibri Hydrogels such as nogen.

 Hydrogels including alginate or fibrinogen have high moisture content, excellent biocompatibility, excellent mechanical properties for crosslinking by ionic bonding by inorganic salts, and thus have no change in shape by cells even after crosslinking. Suitable for use It also provides pores that cells can't adhere to, leading to aggregation between cells.

 The present invention is a hybrid in which the first bio ink and the second bio ink are mixed at a volume ratio of 1.5 to 9: 1, so that biocompatibility is excellent, physical strength is excellent, and the shape does not change by cells even after ion crosslinking. Bio ink can be provided.

 In addition, the present invention provides a three-dimensional attachment space in which the first bio ink can adhere to cells by mixing the first bio ink and the second bio ink in a volume ratio of 1.5 to 9: 1 (V8 0). At the same time, the second bio ink may provide a hybrid bio ink that provides a void to which cells cannot adhere, thereby inducing aggregation between cells. In contrast, the mixing ratio of the first bio ink and the second bio ink is in the aforementioned range.

8 Alternative Paper (Article 26) 0 2019/245110 1 »（： 1/10 公 018/012996 If the first bio ink has a high content, the physical strength is weak and the crosslinking time is long, so that it is easily contracted and differentiated by cells If the second bio ink has a high content, the cell affinity is low, thereby limiting the survival, proliferation and differentiation of cells.

 For example, the low U bio ink or the second bio ink may further include, but is not limited to, one or more selected from embryonic fibroblasts, stem cells derived from embryonic stem cells, and human stem cells.

 In addition, the first bio-ink and the second bio-ink each preferably have a concentration of 1.5 to 4.0% (wt O 0, and most preferably may be 3% (wt O).

 On the other hand, another embodiment of the present invention relates to a method for producing a hybrid bio-ink, Decelled step of removing cells from the tissue; Liquefying the extracellular matrix of the decellularized tissue to prepare a first bio ink; Preparing a second bio ink comprising alginate or fibrinogen; And mixing the first bio ink and the second bio ink at a volume ratio (v 八 r) of 1.5 to 9: 1.

Tissues used in the decellularization stage are tissues derived from mammals such as humans, pigs, cows, rabbits, dogs, goats, sheep, chickens and horses as described above (for example, liver tissues and heart tissues). , be in muscle tissue, etc.), but the tissue used in the present invention is preferred to include a cross-section is derived from pig (li _ver) tissue in favor of survival, proliferation and differentiation of the liver cells.

For example, liver tissue derived from pigs is agitated ^and washed with distilled water at room temperature for about 2 hours and then washed with liver tissue using a decellularized solution.

9 Alternative Paper (Article 26) 2019/245110 1 »(1 ^ 1 {2018/012996 can remove cellular components of tissues.

 In this case, the surfactant contained in the decellularized solution may be an ionic surfactant such as sodium dodecyl sulfate (SDS), sod deoxycholate, Triton X-200, or Triton X-100, Tween. Nonionic surfactants such as 80 may be used, but Triton X-W0 is preferably used. Therefore, various proteins and glycoproteins of the extracellular matrix and ¾ glycosaminoglycans (glycosaminoglycan, GAG) are preferred. Damage can be minimized and the DNA contained in the cells can be destroyed,

 And the hypertonic solution contained in the decellularization solution is used together with the surfactant to increase the decellularization efficiency, can use the hypertonic solution more than three times the normal saline concentration, and increase the remaining amount of the extracellular matrix of the tissue Preference is given to using a solution of 0.03 to 1.0 M comprising any one or more selected from the group comprising NaCl, KC1, CaCh, MgCls, BaCh and NaHCOs.

 Decellularized tissue obtained through the decellularization step of the present invention, by breaking the DNA contained in the cell significantly lowered the DNA content contained in the tissue can not only significantly lower the immune rejection reaction when introduced into the body, In addition, various proteins, glycoproteins, and glycosaminoglycans (GAG), which are components of extracellular matrix, can be utilized, which is advantageous for survival, proliferation, and differentiation of specific cells.

 Meanwhile, in the step of preparing the first bio ink, the extracellular matrix of the decellularized tissue obtained through the decellularization step is liquefied under a specific temperature and pH conditions to prepare a first bio ink having an appropriate viscosity.

 Specifically, the step of preparing the first bio ink is a cell of decellularized tissue

액상화되어, 바 이오 프린팅 소재로 쓰이기 위한 적절한 점도（예를 들어, 100 내지 1000 ? 3）를 갖는 것이 바람직하다. 여기서 펩신은如 및 온도조건에 따라 반응성이 다른 특성 을 가지고 있으며, 이로 인해 온도가 15^°0 미만인 경우에는

활성 도가 감소하여 원활한 소화（ 은63 011）가 이루어질 수 없고, 25껀를 초과하는 경우 에는

활성이 지나치게 증가되어， 점도가 낮아져서 바이오 잉크로 사용하기에 부적합하다. 10 Alternative Paper (Article 26) 2019/245110 1 »(1 ^ 1 {2018/012996 outside air temperature is 1 to 3, at a temperature of about 15 to 25

Liquefied and suitable viscosity for bioprinting materials (eg 100-1000? It is preferable to have 3). Here, pepsin has different reactivity according to 如 and temperature condition, so if the temperature is less than 15 ^° 0

If the activity decreases, smooth extinguishing (silver 63 011) cannot be achieved,

The activity is excessively increased and the viscosity is low, making it unsuitable for use as a bio ink.

 The preparing of the second bio ink may include preparing a second bio ink including natural polymer such as alginate, fibrinogen, carboxymethyl cellulose, heparan sulfate, hyaluronic acid, collagen, dextran, etc. It is not limited to this, and most preferably may include a hydrogel including alginate or fibrinogen.

 The second bio ink may be prepared by adding a natural polymer material to distilled water and then stirring it at room temperature.

 Thereafter, the first bio ink and the second bio ink are mixed at a volume ratio of 1.5-9: 1 (V 八 0), so that the biocompatibility is excellent, the physical strength is excellent, and the hybrid does not change its shape even after ion crosslinking. Bio inks can be prepared.

 In addition, the first bio ink provides a three-dimensional attachment space for the cells to attach, while the second bio ink provides a void that the cells can not attach to produce a hybrid bio ink that induces aggregation between cells can do.

11 Alternative Paper (Article 26) 2019/245110 1 »(1 ^ 112018/012996 On the other hand, the mixing ratio of the first bio ink and the second bio ink is outside the above-mentioned range, so that the physical strength is weak when the first bio ink has a high content, The cross-linking time is long, can be easily contracted and differentiated by the cell, when the second bio ink has a high content, the cell affinity is low, the survival, proliferation and differentiation of the cell is limited.

미만의 온도 범위가유지되도록 적절히 온도를 제어하면서 혼합하는 것이 바람직하며， 적절한 점성을가질 수 있도록교반속도를제어하면서 혼합하는 것이 적절하다. Specifically, the first bio ink and the second bio ink are each preferably at a concentration of 1.5 to 4.0% (/ V), and most preferably at a concentration of 3¾ (桃 八). At this time, the first bio ink is vulnerable to heat because it contains a variety of protein components, since the formulation may be modified by deformation or aggregation at the above temperature, the fibrosis reaction proceeds,

It is preferable to mix with proper temperature control so that the temperature range below is maintained, and it is appropriate to mix while controlling the stirring speed so as to have an appropriate viscosity.

 As another embodiment of the present invention, an artificial tissue manufacturing method using a hybrid bio ink manufactured by the aforementioned manufacturing method may be used. The artificial tissue manufacturing method according to the present invention may be performed using a hybrid bio ink. Printing artificial tissue; And solidifying (301 compared with 11) a step of gelling (for 6101 1) and crosslinking (033-1 1) 1 artificial tissue using an inorganic salt.

 The step of printing artificial tissue means a bio 30 printing step of printing artificial tissue, which is a three-dimensional structure, using a mixed hybrid bio ink. Artificial tissues may be, for example, organoids (0 ^ 81101 (1), organ-like chips (0 ^ 811-011-011-3-11)), liver tissues, heart tissues, bone tissues, and the like. Can be done using

12 Alternative Paper (Article 26) 2019/245110 1 »(1 ^ 1 {2018/012996

 Next, the printed artificial tissue is subjected to the solidification (sol idi f icat ion) step of gelling and cross-1 inking the inorganic tissue using inorganic salts. In this case, the inorganic salt may be any one or more selected from the group consisting of calcium, manganese, barium, cobalt, zinc, and copper, but is not limited thereto. Most preferably, artificial tissue is crosslinked using calcium. To form.

 Specifically, the solidification step is to gel the collagen by heat and then cross- lking alginate or fibrinogen using a solution containing an inorganic salt, thereby printing the printed artificial tissue by the cells. It allows the construction of a rigid structure so that the shape does not change. In this case, the cells may be variously changed according to the type of artificial tissue, and cells may be cultured in the artificial tissue using a cell culture device.

 The solidification step may crosslink the alginate or fibrinogen using a solution containing an inorganic salt and then gelate the collagen by heat, but as mentioned above, by heat After gelling the collagen, it is most preferable to cross-bilize alginate or fibrinogen using a solution containing an inorganic salt.

 The artificial tissue preferably includes a three-dimensional attachment space to which cells can attach due to the first bio ink, and at the same time includes voids to which cells cannot attach due to the second bio ink. It induces aggregation between multiple cells, thereby promoting cell survival, proliferation and differentiation.

13 Alternative Paper (Article 26) 2019/245110 1 »[" ^ 0 技 018/012996 The hybrid bio ink prepared according to the present invention comprises a first bio ink in which an extracellular matrix of decellularized tissue is liquefied and a second bio ink including alginate or fibrinogen By mixing with a volume ratio of 1.5 to 9: 1, the biocompatibility is excellent, which is advantageous for the survival, proliferation and differentiation of cells, and the physical strength is excellent, and the shape is not changed by the cells even after ion crosslinking.

 In addition, the crosslinking time required to prepare the artificial tissue according to the present invention is shortened, and the artificial tissue prepared according to the present invention includes a three-dimensional attachment space to which the cells can be attached and at the same time the cells can be attached. Aggregation between cells is possible, including numerous pores, which have beneficial effects on cell survival, proliferation and differentiation.

 Specifically, in order to maximize the effect of the present invention, it is most preferable to mix the first bio ink and the second bio ink in a volume ratio of 4: 1 (V 八 0).

 In the following, an embodiment of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art to which the invention belongs, various modifications of the contents described herein within the scope of the present invention Can be done.

[Production Example 1]

 Preparation of the first biotick

 After cutting the pork liver to 1ä size to prepare a number of sections, the sections were put in distilled water and washed for 2 hours at room temperature using a stirrer. After award

14 Alternative Paper (Article 26) 2019/245110 1 »(The 1 ^ 1 {2018/012996 section fragments were removed at 10 ^° C using decellular solution containing 0.5% Triton X-100, 0.5M NaCl solution. Decellularized for time. At this time, the decellular solution was changed every 3 hours. Next, the decellularized sections were stirred with distilled water and washed at room temperature for 2 hours, and then sterilized decellularized sections by stirring for 1 hour at room temperature with a disinfectant solution containing 0.1% peracetic acid in PBS. It was. Finally, decellularized tissue was prepared by stirring with distilled water and washing at room temperature for 1 hour.

After the prepared decellularized tissue and pepsin (P 印 sin) was put in the container, the temperature was 201 :, was added to the inside of the chamber to be kept constant at 沈^° C. to prepare a first bio-ink.

 Preparation of Second Bio Ink

 Alginate * was added to distilled water and stirred at room temperature for 12 hours to prepare a second bio ink.

 Preparation of Hybrid Bio-Iq

 The first bio ink prepared in the 25 ° C chamber and the second bio ink were mixed in the volume ratio (v 八 ，) as shown in Table 1 below, followed by mixing for 15 minutes using a paste mixer, and hybrid bio. Ink was prepared.

 Table II

15

Alternative Paper (Article 26) 2019/245110 1 »（1 ^ 1 {2018/012996

Manufacture example

 Manufacturing organization

 The hybrid bio ink prepared according to Preparation Example 1 was printed into a three-dimensional structure having a hexahedron shape using a 3D printing apparatus, and then heat was applied to gel the hybrid bio ink. Then, artificial tissues were prepared by stirring and crosslinking with 20 (M concentration of calcium chloride solution for 30 minutes).

[Experimental example 1] Viscosity measurement experiment of the first bio ink

바이오 잉크의 점도를측정하였다. 도 1의 결과를 살펴보면, 25 ^°C 챔버에서 제조된 3%농도의 제 1 바이오 잉크 의 경우가장높은 점도를나타내는 것으로 확인되었다. In order to check the viscosity according to the temperature change of the first bio ink prepared in Preparation Example 1, using a Brookfield viscometer (Brookfield RVDV-3, Brookf ield, US)

The viscosity of the bio ink was measured. Looking at the results of Figure 1, it was confirmed that the highest viscosity of the first bio ink of 3% concentration prepared in a 25 ^° C chamber.

 In addition, for the same concentration, the first bio ink manufactured in a 25 ° C chamber, 20 ° C

16

Alternative Paper (Article 26) \ 20 2019/245110 It is believed that all of them exhibit higher values than the first bio inks produced in the chamber, and thus exhibit excellent viscosity characteristics in the chamber at 25 ° C.

Experimental Example 2 Viscosity Measurement Experiment of Hybrid Bio-Ink

 In order to check the viscosity according to the change in the content of the hybrid bio ink prepared in Preparation Example 1, Comparative Examples 1 to 4 and Examples using a Brookfield viscometer (Brookfield RVDV-3, Brookf ield, US) at 4 ° C The viscosity of 1 to 4 was measured, and the results are shown in FIGS. 2 and 3.

 Referring to the results of FIG. 2, it was confirmed that the Comparative Example 1 including only the first bio ink showed a significantly lower viscosity than Examples 1 to 4 in which the first bio ink and the second bio ink were mixed.

 However, in Examples 1 to 4 and Comparative Examples 2 to 4 in which the first bio ink and the second bio ink are mixed as shown in FIGS. 2 and 3, the second bio ink is compared with the first bio ink. The higher the content of the higher the viscosity was confirmed that.

Experimental Example 3 Shape Change Experiment of Hybrid Bio Ink

 Changes in the shapes of Comparative Examples 1 to 4 and Example 2 prepared by Preparation Example 2 were observed and measured, and the results are shown in FIG. 4. Referring to FIG. 4, Comparative Example 1 comprising only the first bio ink, the first bio ink

17 Alternative Paper (Article 26) 2019/245110 1 »(： 1 ^ 1 {2018/012996 and 2: 2 volume ratio of 8: 2 Example 2: The first and second bio ink and the second bio ink is 5: 5 by volume ratio 八) In Comparative Example 2 mixed with, the shape change was slight, whereas in Comparative Example 3 and Comparative Example 4 in which the ratio of the second bio ink was higher than that of the first bio ink, the change in shape was large, resulting in moldability after crosslinking. There was a problem that was not guaranteed.

Experimental Example 4 Compressive Strength Measurement Experiment of Hybrid Bio-Ink

 The compressive strength of the comparative examples 1 to 4 and Examples 1 to 4 prepared by Preparation Example 2 were measured using a compressive strength device (1e01011) based on Shoyoä 0 790, which is an international standard. Is the same as FIG. 5 and FIG. Referring to the results of FIGS. 5 and 6, as the ratio of the second bio ink is increased, the compressive strength increases, and it is confirmed that the physical strength is excellent.

미만의 압축 강도로 측 정되었으며， 제 2 바이오 잉크만을포함한 비교예 4의 압축 강도는 72.98 3로, 상 대적으로높은 압축 강도를 나타낸 것을 확인할수 있었다. Specifically, Examples 1 to 4 of

It was measured with a compressive strength of less than, and the compressive strength of Comparative Example 4 including only the second bio ink was 72.98 3, it was confirmed that the relatively high compressive strength.

Experimental Example 5 Cell survival rate and proliferation rate of artificial tissue

시켰다. 이후, 2001 농도의 염화칼슘 용액과 함께 30분동안 교 Mouse embryo-derived fibroblasts 111- 況 3) in Comparative Examples 1 and 2 and Example 2 were mixed at a ratio of 1 × 1 (广 7 _/ ½1) and printed using a 30 printing apparatus, and then heated.

I was. After 30 minutes of incubation with calcium chloride solution at 2001 concentration

 18

Alternative Paper (Article 26) 2019/245110 1 »（： 1 ^ 1 {2018/012996 and crosslinked. Thereafter, the cells were cultured in a cell culture apparatus at 37 ^° C. for 4 days, and cell viability from day 1 to day 4 of the cell culture was evaluated by using Live / dead cel l viabi lity kit (Li fe Technologies), and Proliferation rates were assessed using Cel counting kit (CCK-8, Doj indo) and are shown in FIGS. 7 to 9. 7 to 8, the cell viability of Comparative Example 1 containing only the first bio ink and the second bio ink in which the ratio of the first bio ink is higher than the ratio of the second bio ink are each on the first day of culture. 90% and 83% showed relatively high values, and the 4th day of cultivation showed 92% and 90%, respectively, and the cell survival rate increased as the ratio of the first bio ink increased or as the culture period increased. I could confirm.

 On the other hand, the ratio of the first bio ink and the second bio ink (Comparative Example 2 in which v8 0 is 5: 5 is about 67% at the first day of culture, but it is 62% at the fourth day of culture. As the culture period increased, cell viability decreased.

 Referring to FIG. 9, the cell proliferation rate also increased as the ratio of the first bio ink increased. In particular, the value of Comparative Example 1 was 1.6 and the value of Example 2 was 1.2 when the culture was 4 days old. , The value of Comparative Example 2 was measured to 0.8, it was confirmed that the value of the relatively larger difference than 0.3, 0.4, 0.5 that is the value of the first day of culture.

Experimental Example 6 Pore Observation Experiment of Artificial Tissue

 Tissues of Comparative Examples 1 and 2 and Example 2 of the culture day 4 prepared in Experimental Example 5

염색한 후， 현미경을사용하여.공극을관찰하였다. 19 Alternative Paper (Article 26) 2019/245110 1 »（： 1 ^ 1 {2018/012996 Hemalock to confirm academic structure

After staining, the pores were observed using a microscope.

 Referring to FIG. 10, in the case of Comparative Example 2 in which the first bio ink and the second bio ink are in a volume ratio of 1: 1 (including V8 0, the first bio ink is included at a higher ratio than the second bio ink. Relatively more voids were observed compared to Comparative Examples 1 and 2. These pores serve to allow cells to aggregate with each other, but relatively small three-dimensional attachment spaces to which cells can attach are judged to be small.

 On the other hand, in Comparative Example 1 containing only the first bio ink, very small amount of pores were observed, which provided a three-dimensional attachment space for the cells to attach to, but the effect of coagulation between the cells was expected to be small. .

 In the case of Example 2, in which the low bio ink and the second bio ink were mixed in a volume ratio of 8: 2, it was observed to contain appropriate pores, which had the effect of coagulating with each other and at the same time the three-dimensional cell attachment. It is also possible to provide an attachment space.

 The present invention is not limited to the above specific embodiments and descriptions, and various modifications can be made by those skilled in the art without departing from the spirit of the invention as claimed in the claims. Yes, such modifications are within the protection scope of the present invention.

 Industrial Applicability

 The present invention provides a biocompatible method by mixing a first bio ink that liquefies extracellular matrix of decellularized tissue and a second bio ink including alginate or fibrinogen.

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Alternative Paper (Article 26) 0 2019/245110 1 »（： 1 ^ 112018/012996 It is excellent for cell survival, proliferation and differentiation, and its physical strength is excellent so that its shape is not changed by cells even after ionic crosslinking and its manufacturing method , And a method for manufacturing artificial tissue using the Baqiao 30 printing process using the same, the crosslinking time required when manufacturing the artificial tissue is short, and in the case of the artificial tissue prepared according to the present invention, cells It is possible to aggregate the cells, including the three-dimensional attachment space attachable at the same time, including the pores that the cells can not attach, there is an advantageous effect on the survival, proliferation and differentiation of the cells, there is industrial availability.

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Claims

2019/245110 1 »（： 1 ^ 1 {2018/012996 【Scope of request】  [Claim 1] A first bio ink liquefying extracellular matrix of decellularized tissue; And A second bio ink comprising alginate or fibrinogen; A hybrid bio ink, wherein the first bio ink and the second bio ink are mixed at a volume ratio of 1.5 to 9: 1. [Claim 2] The method of claim 1, And said tissue comprises liver (1 ^ 61 ·) tissue derived from pig.  [Claim 3] The method of claim 1, The first bio ink, the decellularized tissue

At a temperature of about 15 to 25 ° （：

Hybrid bio-ink, characterized in that the liquefied by liquefying. [Claim 4] The method of claim 1, The first bio ink or sieve 2 bio ink further comprises one or more selected from embryonic-derived fibroblasts, stem cells derived from embryonic stem cells, and human-derived stem cells.  [Claim 5] 22 Alternative Paper (Article 26) 0 2019/245110 1 »（： 1 ^ 1 {2018/012996 According to claim 1, A hybrid bio ink, wherein the first bio ink and the second bio ink are 1.5 to 4.0% (concentration of 18, respectively).  [Claim 6] A decellularization step of removing cells from the tissue; Preparing a first bio-anch by liquefying extracellular matrix of decellularized tissue; Preparing a low 2 bio ink comprising alginate or fibrinogen; And mixing the first bio ink and the second bio ink with a volume ratio of 1.5 to 9: 1 (V 8).  [Claim 7] The method of claim 6, The tissue is a method for producing a hybrid bio-ink, comprising liver (110) tissue derived from pigs,  [Claim 8] The method of claim 6, The decellularization step, characterized in that to remove the cells of the tissue using a decellularization solution containing a surfactant and the hypertonic solution, a method for producing a hybrid bio ink.  [Claim 9] The method of claim 8, The fault solution is selected from the group consisting of 池, ^ 1, 0 & 0 \ 2, ¾1 (： 12, 63012 and 附 敗 0 ₃₎ ^.23 Alternative Paper (Article 26) 2019/245110 1 »(： 1 ^ 1 {2018/012996) A method for producing a hybrid bioink, characterized in that a solution of 0.03 to 1.0 containing at least one selected.  [Claim 10] The method of claim 8, The surfactant is triton) (-1000 1; 011 Table-100),

80, 3 ^ (30 ^ 11111 X-ray), a method for producing a hybrid bio ink, characterized in that any one or more selected from the group containing Sodium deoxycholate and Triton Table-200 (1 ^ 1011 Table-200) .  [Claim 11] The method of claim 6, In the preparing of the first bio ink, the extracellular matrix of the decellularized tissue is liquefied by pepsin 6 to 3 at a temperature of 1 to 3 and about 15 to 25 ^° (hybrid). Method of producing bio ink.  [Claim 12] The method of claim 6, The first bio ink and the second bio ink are 1.5-4.0% (八), respectively, characterized in that the method for producing a hybrid bio ink.  [Claim 13] A hybrid bio ink according to any one of claims 1 to 5; Or printing artificial tissue using a hybrid bio ink prepared by the method according to any one of claims 6 to 12; And 24 Alternative Paper (Article 26) 2019/245110 1 »（Solid 1 ^ 1 {2018/012996 Inorganic salts to solidify and crosslink (033-111 止 ^ 1 增) the artificial tissues (301 to 比比 比 11) Step; artificial tissue manufacturing method comprising a.  [Claim 14] The method of claim 13, The inorganic salt is artificial tissue manufacturing method of any one or more selected from the group containing calcium, manganese, barium, cobalt, zinc, copper.  [Claim 15] The method of claim 13, The solidifying step is characterized by gelling collagen by heat (Yo 61! 011), and then crosslinking ( ₀₃₃ -11 _{11 1} thumb) with alginate or fibrinogen using a solution containing an inorganic salt. Manufacturing method. 25  Alternative Paper (Article 26)