Bioink for printing adipose tissue-mimicking structure

Abstract

The bioink for printing a human adipose tissue-mimicking structure of the present invention comprises a porcine adipose-derived decellularized extracellular matrix, and provides a human inflammatory disease model and a human diabetes model, both comprising a human adipose tissue-mimicking structure produced by printing with the bioink for printing a human adipose tissue-mimicking structure.

Description

Bioink for printing adipose tissue-mimicking structures

[0001] The present invention relates to a bioink for printing adipose tissue-mimicking structures. Specifically, the present invention relates to a bioink for printing human adipose tissue-mimicking structures that can construct an adipose cell culture environment similar to actual human adipose tissue using tissue not derived from humans and can implement cell behavior similar to that of actual humans, and to the use thereof.

[0002] Three-dimensional culture methods involving encapsulation in hydrogels such as collagen and alginate have been widely used to culture cells derived from body tissues. However, since the aforementioned hydrogels cannot completely mimic the microenvironment and composition of actual human tissues, cells cultured in these hydrogels exhibit differences in behavior and growth patterns compared to the cells that constitute the actual tissue. In particular, adipose tissue is deeply related to inflammatory responses, possesses a tissue composition different from other organs, and is known to have significantly different compositions and roles between visceral and subcutaneous adipose tissues. As an example, while subcutaneous fat has health benefits, such as inhibiting the formation of visceral fat, in pathological conditions like diabetes, adipocytes attract and activate macrophages. Consequently, these activated macrophages produce inflammatory cytokines that exacerbate the chronic inflammatory state of adipose tissue. This characteristic is known to be more pronounced in visceral fat than in subcutaneous fat. Therefore, in order to realize a cell culture environment and behavioral mimicking similar to human tissue in vitro, the characteristics of the hydrogel used to encapsulate and grow cells need to mimic a microenvironment specific to human tissue.

[0003] Accordingly, the inventors intended to solve these problems by developing a hydrogel that can establish a cell culture environment similar to actual human adipose tissue in vitro and implement behaviors similar to reality, particularly a hydrogel bioink for using bioprinting techniques such as 3D printing.

[0004] Therefore, the main objective of the present invention is to provide a bioink for printing human adipose tissue mimicking structures that can establish a cell culture environment similar to actual human adipose tissue in vitro and implement behavioral mimicry similar to that of the real.

[0005] Another objective of the present invention is to provide a human adipose tissue mimic structure manufactured using the bioink described above, which has a cell culture environment similar in form to actual human adipose tissue and implements behavioral mimicry similar to that of the real.

[0006] According to one aspect of the present invention, the present invention provides a bioink for printing human adipose tissue mimicking structures comprising a porcine fat-derived decellularized extracellular matrix.

[0007] In the bioink for printing human adipose tissue mimicking structures of the present invention, the porcine fat-derived decellularized extracellular matrix is ​​preferably derived from porcine visceral fat or subcutaneous fat.

[0008] In the bioink for printing human adipose tissue mimicking structures of the present invention, the porcine fat-derived decellularized extracellular matrix is ​​preferably derived from porcine visceral fat.

[0009] In the bioink for printing human adipose tissue mimicking structures of the present invention, it is preferable that the porcine adipose-derived decellularized extracellular matrix be obtained by treating porcine adipose tissue with isopropanol, sodium dodecyl sulfate, and Triton X-100.

[0010] In the bioink for printing a human adipose tissue mimicking structure of the present invention, it is preferable that the human adipose tissue mimicking structure is a structure of an inflammatory disease model or a diabetes model.

[0011] In the bioink for printing human adipose tissue mimicking structures of the present invention, the porcine fat-derived decellularized extracellular matrix is ​​derived from porcine visceral fat and contains 5 to 15 weight percent of the porcine fat-derived decellularized extracellular matrix, and the human adipose tissue mimicking structure is preferably a structure of an inflammatory disease model.

[0012] According to another aspect of the present invention, the present invention provides a human adipose tissue mimic structure produced by printing with the bioink for printing human adipose tissue mimic structures of the present invention.

[0013] According to another aspect of the present invention, the present invention provides a human inflammatory disease model comprising a human adipose tissue mimicking structure of the present invention.

[0014] According to another aspect of the present invention, the present invention provides a human diabetes model comprising a human adipose tissue mimicking structure of the present invention.

[0015] According to another aspect of the present invention, the present invention provides a method for manufacturing a human inflammatory disease model comprising: a step of preparing a bioink containing 5 to 15 weight percent of porcine visceral fat-derived decellularized extracellular matrix; and a step of printing an adipose tissue mimic structure using the bioink.

[0016] According to the present invention, a bioink for printing human adipose tissue-mimicking structures is provided, which can construct an adipose cell culture environment similar in form to actual human adipose tissue using tissue not derived from humans and can implement cell behavior similar to that of actual humans. In particular, the bioink of the present invention has significant advantages in that it has characteristics suitable for application in 3D printing technology.

[0017] FIG. 1 illustrates the process of preparing a porcine fat-derived decellularized extracellular matrix according to one embodiment of the present invention.

[0018] FIGS. 2 to 5 show the results of proteomic analysis for comparing porcine visceral fat-derived decellularized extracellular matrix (vadECM) and porcine subcutaneous fat-derived decellularized extracellular matrix (sadECM) according to an embodiment of the present invention. (Fig. 2 (A)) Comparison of protein types having an effective abundance of 0.01% or more; (Fig. 2 (B)) Matrixome profile; (Fig. 2 (C)) Analysis of the Gene Ontology-Biological Process (GO-BP); (Fig. 3) Results of the GO-BP overrepresentation test; (Fig. 4) Results of the Gene Ontology-pathway (GO-P) overrepresentation test; (Fig. 5) Heatmap displaying protein type content by category.

[0019] FIGS. 6 to 11 show the results of characteristic evaluation of vadECM and sadECM bioinks according to an embodiment of the present invention. (Fig. 6) Results of sol-gel transition experiment; (Fig. 7) Results of confirmation of collagen, GAG (glycosaminoglycan), and DNA content; (Fig. 8) Survival / death images of human adipose-derived stem cells (green: surviving cells; red: dead cells); (Fig. 9) Results of confirmation of cell proliferation rate; (Figs. 10 and 11) Results of confirmation of flow characteristics, 1: viscosity of sadECM, 2: gelation kinetics of sadECM, 3: complex modulus of sadECM, 4: viscosity of vadECM, 5: gelation kinetics of vadECM, 6: complex modulus of vadECM.

[0020] FIGS. 12 to 21 show the results of comparing the effects of vadECM and sadECM on hASC differentiation ability, cytokine secretion, and macrophage activity according to one embodiment of the present invention. (Fig. 12) Oil Red O staining results; (Fig. 13) PPARG and FABP4 expression levels confirmed by qRT-PCR; (Fig. 14) Adipocyte size measurement results; (Fig. 15) Degree of macrophage activation confirmed using IL-6 and TNF-alpha ELISA; (Fig. 16) Cell proliferation rate on day 14; (Fig. 17) Schematic diagram of a platform for evaluating macrophage migration; (Fig. 18) Degree of macrophage migration through migration channels at 6, 12, and 24 hours; (Fig. 19) Survival / death analysis results of the macrophage chamber portion at 24 hours; (Fig. 20) TNF-alpha and IL-6 ELISA results; (Fig. 21) Number of macrophages remaining in the macrophage chamber at 24 hours.

[0021] Figure 22 shows the results of an analysis of the amount of inflammatory molecules released from adipocytes according to the concentration of vadECM according to one embodiment of the present invention.

[0022] The bioink for printing human adipose tissue mimicking structures according to the present invention is characterized by comprising a porcine adipose-derived decellularized extracellular matrix.

[0023] Due to the characteristics of the porcine fat-derived decellularized extracellular matrix, which is almost identical to the composition of proteins constituting human adipose tissue, the bioink of the present invention can mimic human adipose tissue to a very high degree.

[0024] In the present invention, the porcine adipose tissue-derived decellularized extracellular matrix (adECM) may be derived from various porcine tissues, but preferably is derived from porcine visceral adipose tissue-derived decellularized extracellular matrix (vadECM) or subcutaneous adipose tissue-derived decellularized extracellular matrix (sadECM). Accordingly, it is possible to produce structures similar to human adipose tissue to a higher degree, particularly in terms of protein composition.

[0025] In particular, the porcine adECM in the present invention is preferably vadECM. According to the present invention, porcine vadECM is rich in proteins associated with signaling pathways such as inflammation mediated by chemokine and cytokine signaling pathways, histamine H2 receptor-mediated signaling pathways, and tyrosine biosynthesis; therefore, using vadECM as the porcine adECM of the present invention allows for more effective realization of inflammatory responses and pathological environments. Furthermore, porcine vadECM can induce human adipose-derived stem cells (hASCs) to form larger and synthesize more lipids, and can more effectively support the survival of hASCs, thereby enabling the creation of more effective human adipose tissue-mimicking structures.

[0026] In the present invention, porcine adECM may be obtained from porcine adipose tissue through a conventional tissue decellularization method. Preferably, it may be obtained by separating and collecting white fat attached to the subcutaneous tissue or pancreas, liver and / or intestines of a porcine, chopping it finely, and then treating it with isopropanol, SDS (sodium dodecyl sulfate), and Triton X-100, followed by freeze-drying. At this time, the isopropanol treatment is preferably performed by immersing the cut fat in isopropanol for at least 1 day, preferably at least 2 days, and more preferably at least 3 days; the SDS treatment is performed by immersing the isopropanol-treated fat in a 0.1 to 1% (w / v) SDS solution for at least 1 day, preferably at least 2 days, and more preferably at least 3 days; and the Triton X-100 treatment is performed by immersing the SDS-treated fat in a 0.5 to 2% (w / v) Triton X-100 solution for at least 1 day, preferably at least 2 days. At this time, it is preferable to wash the fat with water between each treatment. Preferably, after all treatments, the fat is freeze-dried to powder.

[0027] The bioink of the present invention is preferably prepared by adding the porcine adECM of the present invention to an acetic acid-containing solution. In this case, the acetic acid-containing solution is preferably a 0.25 to 1 M acetic acid solution, more preferably a 0.3 to 0.8 M acetic acid solution, and more preferably a 0.4 to 0.6 M acetic acid solution. The bioink prepared accordingly may have properties useful for 3D printing.

[0028] The bioink of the present invention may contain porcine adECM at a concentration of, for example, 0.5% by weight or more, preferably 1% by weight or more, more preferably 1.5% by weight or more, and more preferably 2% by weight or more. The upper limit of the adECM content in the bioink of the present invention is not particularly limited, but is preferably 20% by weight or less, more preferably 15% by weight or less. The bioink according to this may have properties useful for 3D printing.

[0029] The bioink of the present invention can very effectively mimic an inflammatory disease environment or a diabetes environment by including porcine adECM. Therefore, the bioink of the present invention may be a bioink for printing structures of an inflammatory disease model or a diabetes model.

[0030] As mentioned above, using vadECM as the porcine adECM of the present invention allows for more effective implementation of inflammatory responses and pathological environments. Furthermore, according to the research results of the present invention, using vadECM allows for the release of inflammation-related molecules from human adipocytes to be simulated at levels similar to those observed in actual patients with inflammatory diseases, compared to using other adECMs, such as sadECM; in particular, it has been shown that using a high dose of vadECM can enhance this effect. Therefore, the bioink of the present invention preferably contains porcine vadECM as the porcine adECM, and includes this vadECM in a high dose, for example, 2% by weight or more, preferably 3% by weight or more, more preferably 4% by weight or more, and more preferably 5% by weight or more, and the human adipose tissue mimic structure is a structure of an inflammatory disease model. In this regard, the upper limit of the vadECM content in the bioink of the present invention is not specifically limited, but is preferably 20% by weight or less, and more preferably 15% by weight or less.

[0031] The bioink of the present invention may further comprise adipocytes and / or macrophages. In this case, the adipocytes and macrophages may be of human origin, for example, cultured through isolation and tissue culture from humans. Furthermore, the concentrations of adipocytes and macrophages are, for example, 5 × 10⁶ each separately based on the total volume of the bioink. 5 Up to 5×10 6 It can be cells / ml.

[0032] The human adipose tissue mimic structure of the present invention is characterized by being produced by printing with the bioink of the present invention. The structure of the present invention can realize human adipose tissue to a very high degree according to the effects of the bioink of the present invention as mentioned above. At this time, printing can be performed through a printing method commonly used to produce bio-tissue mimic structures using bioink, for example, a 3D printing method.

[0033] The human inflammatory disease model of the present invention is characterized by including the human adipose tissue mimicking structure of the present invention. The inflammatory disease model of the present invention can realize a human inflammatory disease environment to a very high degree based on the effects of the bioink of the present invention as mentioned above and the effects of the structure of the present invention based thereon. In this case, an inflammatory disease refers to a disease in which inflammation is the primary lesion, and may be, for example, a disease selected from the group consisting of gastritis, enteritis, hepatitis, pneumonia, nephritis, cystitis, arthritis, dermatitis, allergy, atopy, conjunctivitis, periodontitis, rhinitis, otitis media, and pharyngitis, but is not limited thereto.

[0034] Furthermore, the human diabetes model of the present invention is characterized by including the human adipose tissue mimicking structure of the present invention. Based on the effects of the bioink of the present invention as mentioned above and the effects of the structure of the present invention based thereon, the diabetes model of the present invention can realize a human diabetes environment at a very high level.

[0035] The model of the present invention may further include other tissue-mimicking structures associated with other related diseases, such as blood vessel-mimicking structures, in addition to the human adipose tissue-mimicking structure of the present invention.

[0036] The method for manufacturing a human inflammatory disease model according to the present invention is characterized by comprising the steps of: preparing a bioink containing 5 to 15 weight percent of porcine vadECM; and printing an adipose tissue mimic structure using the bioink.

[0037] The step of preparing the bioink may be, for example, a step of adding vadECM to a suitable solution. The suitable solution may be, for example, an acetic acid-containing solution, preferably a 0.25 to 1 M acetic acid solution, more preferably a 0.3 to 0.8 M acetic acid solution, and more preferably a 0.4 to 0.6 M acetic acid solution.

[0038] Preferably, a bioink containing 6 to 14 weight% of porcine vadECM is prepared, more preferably, a bioink containing 7 to 13 weight% of porcine vadECM is prepared, more preferably, a bioink containing 8 to 12 weight% of porcine vadECM is prepared, and more preferably, a bioink containing 9 to 11 weight% of porcine vadECM is prepared.

[0039] The step of preparing the bioink may be a step of preparing a bioink that further includes adipocytes and / or macrophages. In this case, the adipocytes and macrophages may be of human origin, for example, cultured through isolation and tissue culture from humans. Additionally, the concentrations of adipocytes and macrophages here are, for example, 5 × 10⁶ each separately based on the total volume of the bioink. 5 Up to 5×10 6 It can be cells / ml.

[0040] Printing can be performed using printing methods commonly used to create biomimetic structures using bioink, such as 3D printing methods.

[0041] The present invention will be described in more detail below through examples. These examples are merely illustrative of the present invention, and therefore the scope of the present invention should not be interpreted as being limited by these examples.

[0042]

[0043] [Example]

[0044] Example 1. Preparation of porcine fat-derived decellularized extracellular matrix

[0045] Using visceral fat and subcutaneous fat of pigs, visceral fat-derived decellularized extracellular matrix (vadECM) and subcutaneous fat-derived decellularized extracellular matrix (sadECM) were prepared, respectively, through the process shown in Fig. 1.

[0046] Specifically, visceral or subcutaneous fat of pigs was washed with PBS (phosphate buffered saline), then washed again with water, treated with isopropanol, then treated with 0.5% SDS, then treated with 1% Triton X-100, then washed with water, and the decellularized tissue was collected. Subsequently, the collected decellularized tissue was subjected to freeze-thaw treatment, freeze-dried, and ground to produce vadECM and sadECM in powder form.

[0047] The decellularization protocol for visceral fat tissue is as follows:

[0048] 1. After receiving pig pancreases, livers, intestines, etc. from a slaughterhouse, the white visceral fat attached to each organ is separated, gathered into a single mass, and then finely chopped into small pieces;

[0049] - The following treatment processes are carried out at a cool temperature of 18℃ to prevent spoilage;

[0050] 2. Finely chopped visceral fat was washed with 1× PBS for 2 hours, then washed three times for 30 minutes each in ultrapure distilled water (DW);

[0051] 3. Treatment in 100% isopropanol for 3 days;

[0052] 4. Wash twice for 30 minutes each in ultrapure DW;

[0053] 5. Treated for 3 days using 0.5% SDS detergent;

[0054] 6. Wash twice for 30 minutes each in ultrapure DW;

[0055] 7. Treated with 1% Triton X-100 for 2 days until the visceral fat tissue turned completely white;

[0056] 8. Wash three times for 30 minutes each in ultrapure DW;

[0057] The produced vadECM is freeze-dried and stored in powder form at -20℃.

[0058] The subcutaneous fat tissue decellularization protocol is as follows:

[0059] 1. Purchase pork subcutaneous fat (for lard production) from a slaughterhouse and cut it into small pieces;

[0060] - The subsequent protocol is identical to the above visceral fat tissue decellularization protocol.

[0061]

[0062] Experimental Example 1. Comparison of vadECM and sadECM

[0063] In order to specifically determine what differences exist between vadECM and sadECM, proteomics was performed on each dECM prepared in Example 1 above.

[0064] Figure 2 (A) compares the types of proteins with a valid abundance of 0.01% or more, with 469 types detected in sadECM and 563 types in vadECM, and 322 types of proteins found to be identical in both. Through this, it was confirmed that sadECM and vadECM are composed of significantly different proteins and are very similar to the composition of proteins that make up human adipose tissue.

[0065] Figure 2 (B) shows the results of examining the matrisome profiles of sadECM and vadECM, confirming that sadECM is composed of more collagen than vadECM, while vadECM is composed of more glycoproteins than sadECM. In particular, by confirming that some secreted factors, including inflammatory cytokines, were detected in vadECM, it can be inferred that vadECM is more suitable than sadECM for inflammatory responses and the realization of pathological environments.

[0066] Figure 2 (C) shows the analysis of the Gene Ontology-Biological Process (GO-BP) for sadECM and vadECM. When verifying the functions of sadECM and vadECM, it was confirmed that while there were quantitative differences, the functional differences between sadECM and vadECM in terms of GO-BP were not significant.

[0067] Figure 3 shows the results of the GO-BP overrepresentation test conducted on sadECM and vadECM. The GO-BP overrepresentation test is a method to statistically determine which biological function a given protein composition is most associated with. As shown in Figure 3, the test results confirmed that both sadECM and vadECM are directly involved in the metabolism of various organic compounds and exhibit mostly identical biological functions; however, it was found that sadECM is associated with the alcohol metabolism process, while vadECM is more associated with the fatty acid metabolism process.

[0068] Figure 4 shows the results of the Gene Ontology-Pathway (GO-P) overrepresentation test performed on sadECM and vadECM. The GO-P overrepresentation test is a method to statistically determine which signaling pathways the proteins are directly associated with. It was confirmed that sadECM is associated with signaling pathways such as glycolysis, the TCA cycle, and pyruvate metabolism. This implies that sadECM is directly associated with signaling pathways related to cell survival and metabolic activity, and suggests that it can mimic the function of subcutaneous fat, which plays a role in energy storage. On the other hand, it was confirmed that vadECM is associated with signaling pathways such as inflammation mediated by chemokine and cytokine signaling pathways, histamine H2 receptor-mediated signaling pathways, and tyrosine biosynthesis. Numerous studies have reported that inflammation mediated by chemokine and cytokine signaling pathways is associated with the release of various inflammatory cytokines, histamine H2 receptor-mediated signaling pathways are linked to pathological processes that lead to sepsis upon the onset of diabetes, and that tyrosine biosynthesis, which generates tyrosine through glycolysis, causes the overproduction of tyrosine as a cause of diabetes. In other words, vadECM is deeply involved in signaling pathways directly linked to diabetes, suggesting that it can significantly aid in mimicking the pathological environment of diabetes.

[0069] Additionally, to support the content of Figure 4, Figure 5 presents a heatmap of proteins with high content that overlap with the proteins constituting sadECM and vadECM among the proteins constituting each signaling pathway. The upper heatmap of Figure 5 shows the types and content of proteins with high content within sadECM, categorized by glycolysis, the TCA cycle, and pyruvate metabolism. The lower heatmap of Figure 5 shows the types and content of proteins with high content within vadECM, categorized by inflammation mediated by chemokine and cytokine signaling pathways, histamine H2 receptor-mediated signaling pathways, and tyrosine biosynthesis. In particular, it can be confirmed that a significant portion of the proteins constituting vadECM are deeply involved in signaling pathways directly associated with diabetes.

[0070]

[0071] Example 2. Preparation of bioink using vadECM and sadECM

[0072] The freeze-dried vadECM and sadECM powders prepared in Example 1 above were each dissolved in a 0.5M acetic acid solution, and a 10N NaOH solution was added to finally prepare a bioink in solution form adjusted to pH 7.

[0073]

[0074] Comparative Example 1. Preparation of bioink using collagen

[0075] Collagen (Dalim Tisen, Seoul, Korea) was dissolved in pepsin-acetic acid for 3 days and neutralized to pH 7 with 0.1 N NaOH and 10× DMEM to finally produce collagen bioink.

[0076]

[0077] Experimental Example 2. Characterization of vadECM and sadECM Bioinks

[0078] The basic characteristics of vadECM and sadECM bioinks were verified.

[0079] First, the sol-gel transition of each vadECM and sadECM bioink of Example 2 was tested, and as shown in Fig. 6, each bioink remained in a fluid state at 4°C and exhibited thermal-crosslinking behavior when exposed to 37°C. Through this, it was confirmed that vadECM and sadECM are capable of gelation, and thus it was confirmed that they can be used for fabricating structures.

[0080] As a result of comparing vadECM and sadECM with natural tissues for biochemical evaluation, as shown in Figure 7, almost no DNA (sadECM: 3.2% ± 0.5%; vadECM: 2.8% ± 0.5%) remained during the decellularization process, but other extracellular matrix (ECM) components, such as collagen (sadECM: 128.07% ± 15.08%; vadECM: 134.81% ± 19.52%) and glycosaminoglycans (GAG) (sadECM: 65.08% ± 7.02%; vadECM: 62.79% ± 11.13%), were maintained at high levels. Therefore, it was confirmed that the bioink produced through the decellularization process had cellular components capable of causing a heterologous immune response sufficiently removed and retained sufficient ECM components necessary for cell growth.

[0081] To confirm biocompatibility, human adipose-derived stem cells (hASCs) were encapsulated in the 2% (w / w) vadECM bioink and 2% (w / w) sadECM bioink of Example 2 and the 2% (w / w) collagen bioink of Comparative Example 1, respectively, and cultured in three dimensions. Subsequently, a live / dead assay was performed on each using a cell counting kit (CCK). As shown in Fig. 8, all bioinks were found to possess excellent biocompatibility. Furthermore, the encapsulated hASCs maintained high viability for 14 days. In particular, the hASCs exhibited the highest proliferation rate in the vadECM bioink.

[0082] To predict the usability of the bioinks for 3D bioprinting and the stability of structures fabricated using the bioinks, the rheological properties of the 1% (w / w), 2% (w / w), and 3% (w / w) sadECM and vadECM bioinks from Example 2 were measured. As a result, as shown in Figures 10 and 11, shear-thinning behavior was confirmed in all experimental groups, in which the viscosity of the bioink decreased as the shear rate increased (see Figure 10, 1 and Figure 11, 4). This shear-thinning behavior is essential for reducing shear stress generated during bioprinting and enhancing cell viability. Additionally, gelation occurred in all experimental groups when the temperature increased from 4°C to 37°C (see Figure 10, 2 and Figure 11, 5). In addition, it was confirmed that the 2% (w / w) bioink has properties suitable for use in bioprinting compared to other groups (see Fig. 10, 3 and Fig. 11, 6).

[0083] These results indicate that the fabricated vadECM and sadECM bioinks can be used as bioinks for 3D bioprinting, are suitable for fabricating tissue mimics containing cells, and that these bioinks have sufficient physical properties to support structures.

[0084]

[0085] Experimental Example 3. Comparison of the effects of vadECM and sadECM on hASC differentiation ability, cytokine secretion, and macrophage activity

[0086] By comparing the adipocyte differentiation ability and macrophage-induced chemokine secretion ability of collagen (negative control; not AT-derived dECM), vadECM, and sadECM, we determined which hydrogel was most suitable for mimicking inflammatory conditions and the diabetic environment.

[0087] Based on previous research results, it is known that lipid synthesis is more efficient when hASCs differentiate well into adipocytes and there are more functionally superior adipocytes. Therefore, equal concentrations of hASCs were inoculated into culture dishes coated with vadECM, sadECM, and collagen, respectively, differentiation into adipocytes was induced, and Oil Red O staining was performed to measure the amount of lipids. In addition, the expression levels of adipogenesis markers PPARG and FABP4 in each group were confirmed using qRT-PCR. As a result, as shown in Figures 12 and 13, it was confirmed that the vadECM bioink produced a larger adipocyte-like phenotype and synthesized more lipids compared to sadECM and collagen. Furthermore, as shown in Figure 14, the vadECM bioink group showed the largest cell size because the adipocytes contained the highest amount of lipids. Additionally, as shown in Figure 16, the vadECM bioink was found to be most beneficial for the survival of adipocytes. Additionally, adipocytes and macrophages derived from THP-1 (human monocytic cell line) were co-cultured on culture substrates coated with vadECM, sadECM, and collagen, respectively. The culture media used for each were then collected and subjected to ELISA. As a result, as shown in Figure 15, it was confirmed that IL-6 and TNF-alpha, representative inflammatory cytokines, were detected at the highest levels in the vadECM bioink group. Therefore, it was proven that vadECM is superior to sadECM and collagen in mimicking the inflammatory state induced by cytokine secretion.

[0088] A cell migration assay was performed to confirm the correlation between each bioink and macrophage activity. For this purpose, a platform was constructed using 3D bioprinting technology to create AT chambers containing adipocytes and macrophage chambers containing macrophages within each bioink, connected by migration channels. Three types of cell migration platforms were created, each containing vadECM, sadECM, and collagen, respectively, within the AT chambers (Fig. 17). Macrophage migration pattern images were then analyzed at time points of 6, 12, and 24 hours. Additionally, macrophages remaining in the macrophage chambers were identified and counted through a survival / death analysis. As a result, as shown in Figs. 18, 19, and 21, macrophages migrated most actively in the group containing vadECM in the AT chamber. In addition, ELISA was performed on the medium collected from the chamber, and as shown in Figure 20, the production of IL-6 and TNF-alpha in adipocytes encapsulated in vadECM was higher than in other groups. Although there are various factors controlling macrophage migration, such as the microstructure of migration channels and the chemoactivation of external chemokines, in this experiment we focused on the chemokine effects of dECM on macrophage migration under the same microstructure of migration channels for each group. Therefore, it can be inferred that adipocytes encapsulated in vadECM in the AT chamber produced more IL-6 and TNF-alpha. The cytokines generated in the AT chamber further activated the macrophages in the macrophage chamber and increased their motility.

[0089] Therefore, these results confirm that vadECM bioink supports the differentiation and survival of encapsulated cells into the adipocyte lineage, and that it induces the production of cytokines that promote the migration or proliferation of immune cells more effectively compared to sadECM. In conclusion, the use of vadECM bioink strongly upregulates inflammatory responses, enabling the stable simulation of inflammation-inducing models that embody pathological environments such as diabetes.

[0090]

[0091] Experimental Example 4. Analysis of the amount of inflammatory molecule release from adipocytes according to the concentration of vadECM

[0092] 2% (w / w) sadECM hydrogel, 2% (w / w) vadECM hydrogel, or 10% (w / w) vadECM hydrogel were prepared in Transwell, human adipocytes and macrophages were encapsulated in them, and then cultured for 7 days in a medium at a high blood glucose concentration (25 mmol / L). Afterward, the amount of released inflammatory molecules was analyzed using ELISA. The inflammatory molecules analyzed were TNF-alpha, pro-inflammatory cytokines IL-6, IL-1 beta, CXCL8 / IL-8, adipose tissue-specific secretory factor resistin, adiponectin involved in glucose regulation, and factor D / adipsin, adipokines associated with insulin secretion and fat accumulation. In an inflammatory environment, it is generally known that the levels of TNF-alpha, IL-6, IL-1 beta, CXCL8 / IL-8, and resistin increase, while the levels of adiponectin and factor D / adipsin decrease.

[0093] As a result of the analysis, as shown in Figure 22, it was confirmed that a clearer trend was observed in vadECM than in sadECM, and in 10% vadECM than in 2% vadECM. It was also confirmed that inflammatory molecules were released at 10% vadECM at levels observed in actual patients with inflammatory diseases. These results demonstrate that using a high concentration of vadECM of 10% or more is suitable for simulating an inflammatory environment in vitro.

Claims

1. Bioink for printing human adipose tissue mimic structures containing porcine fat-derived decellularized extracellular matrix.

2. In Paragraph 1, The above-mentioned porcine fat-derived decellularized extracellular matrix is ​​a bioink for printing human adipose tissue mimicking structures derived from porcine visceral fat or subcutaneous fat.

3. In Paragraph 2, The above-mentioned porcine fat-derived decellularized extracellular matrix is ​​a bioink for printing human adipose tissue mimicking structures derived from porcine visceral fat.

4. In Paragraph 1, The above-mentioned porcine fat-derived decellularized extracellular matrix is ​​a bioink for printing human adipose tissue mimicking structures, obtained by treating porcine adipose tissue with isopropanol, sodium dodecyl sulfate, and Triton X-100.

5. In Paragraph 1, The above human adipose tissue mimicking structure is a bioink for printing human adipose tissue mimicking structures, which is a structure of an inflammatory disease model or a diabetes model.

6. In Paragraph 1, A bioink for printing a human adipose tissue mimic structure, wherein the above-mentioned porcine fat-derived decellularized extracellular matrix is ​​derived from porcine visceral fat and contains the above-mentioned porcine fat-derived decellularized extracellular matrix in an amount of 5 to 15 weight%, and the above-mentioned human adipose tissue mimic structure is a structure of an inflammatory disease model.

7. A human adipose tissue mimic structure produced by printing with a bioink for printing a human adipose tissue mimic structure according to any one of paragraphs 1 to 6.

8. A human inflammatory disease model comprising the human adipose tissue mimicking structure of claim 7.

9. A human diabetes model comprising the human adipose tissue mimicking structure of claim 7.

10. A step of preparing a bioink comprising 5 to 15 weight% of porcine visceral fat-derived decellularized extracellular matrix; and A step of printing an adipose tissue mimicking structure using the above bioink; including Method for manufacturing a human inflammatory disease model.

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