Method for constructing vascular tissue structure and vascular tissue structure constructed using same

Abstract

The present invention relates to a method for constructing a vascular tissue structure by using growth media containing hyaluronic acid and polyethylene glycol, respectively, without a basement membrane extract (BME), and a vascular tissue structure constructed by using the same. The constructing method of the present invention can construct a vascular tissue structure having a vascular wall-like structure and a lumen by culturing vascular endothelial cells without BME. Since the vascular tissue structure of the present invention does not use BME, the vascular tissue structure has high bio-similarity, can form a vascular tissue provided with functionality, and can be variously utilized as a vascular tissue suitable for a regenerative medical purpose by increasing the possibility of expanding the size of an organoid to the scale of a tissue for transplantation.

Description

Method for manufacturing a vascular tissue structure and a vascular tissue structure manufactured using the same

[0001] The present invention relates to a method for producing a vascular tissue construct using a growth medium containing hyaluronic acid and polyethylene glycol, respectively, without BME (Basement membrane extract), and to a vascular tissue construct produced using the same.

[0002]

[0003] Organoids are tissues cultured in vitro to replicate structures similar to living tissues in terms of cell-cell connections and three-dimensional morphogenesis. Because they can exhibit tissue-specific functionality, organoids have been recognized as valuable alternatives to animal testing and for elucidating organ morphogenesis and disease mechanisms. Organoids are expected to be useful in elucidating disease mechanisms and developing new drugs by providing morphological / functional interactions and disease models of 3D cultured tissues that transcend the limitations of 2D culture.

[0004] However, since organoids are structures formed based on active cells, they have limitations in reproducing the complex activities exhibited by living tissues.

[0005] First, organoids have a problem of lacking consistency in terms of structural regularity because it is difficult to secure consistency in organizational units during the process of structural formation through spontaneous cell-cell or cell-ECM interactions.

[0006] In particular, to induce tubule formation similar to blood vessels using vascular endothelial cells in vitro, cell-ECM interactions and structural maintenance must be induced through modulation of the surrounding microenvironment. To this end, culture methods that add Matrigel, a bio-derived matrix containing various growth factors necessary for angiogenesis, are essential. However, Matrigel has critical shortcomings, such as being a mixture of animal-derived matrix proteins and a lack of reproducibility due to the random composition of the matrix mixture components, making it unsuitable for the development of advanced formulations aimed at clinical application. Furthermore, limitations in genetic and imaging analysis have been reported due to the excessive presence of matrix.

[0007] In addition, in order to form tissue for transplantation, it is essential to maintain function even during long-term culture, but culture additives such as Matrigel have characteristics that make them unsuitable for long-term culture due to the difficulty in controlling the decomposition behavior of the substrate by the decomposition enzymes produced by the cells.

[0008] Therefore, these bio-derived matrix molecule mixtures pose concerns about component consistency and the risk of immune rejection, making them difficult to apply to organoids developed for regenerative medicine purposes. Furthermore, technical limitations in inducing blood vessel formation complicate the process of creating organoids with complex microstructures and complex cellular compositions, making it difficult to achieve biosimilarity.

[0009] Therefore, there is a need for methods for manufacturing vascular tissues that can be used for regenerative medicine purposes, thereby overcoming technological limitations by forming regenerative medicine organoids with biosimilarity and functionality.

[0010]

[0011] Accordingly, the inventors of the present invention studied a method for producing a vascular tissue structure without BME (Basement membrane extract), and confirmed that a stable vascular structure can be formed in a long-term culture environment without BME by using SlabON technology, a culture method using a growth medium containing hyaluronic acid and a growth medium containing polyethylene glycol, and completed the present invention.

[0012] Accordingly, the present invention aims to provide a method for producing a vascular tissue construct, including the steps of 1) culturing vascular endothelial cells by sedimenting them in a growth medium containing hyaluronic acid; and 2) culturing them by adding a growth medium containing polyethylene glycol; and a vascular tissue construct produced using the same.

[0013]

[0014] To achieve the above purpose, the present invention provides a method for producing a vascular tissue structure, comprising: 1) a step of culturing vascular endothelial cells by sedimenting them in a growth medium containing hyaluronic acid; 2) a step of culturing them by adding a growth medium containing polyethylene glycol.

[0015] The present invention also provides a vascular tissue structure manufactured by the above method.

[0016] The present invention also provides a kit for producing a vascular tissue construct, comprising: one compartment containing a growth medium containing hyaluronic acid, two compartments containing a growth medium containing polyethylene glycol, and three compartments containing vascular endothelial cells.

[0017]

[0018] The manufacturing method of the present invention can produce a vascular tissue construct having a structure and lumen similar to a vascular wall by culturing vascular endothelial cells without the use of BME. The vascular tissue construct of the present invention has high biosimilarity because it does not use BME, can form functional vascular tissue, and increases the possibility of expanding the size of the organoid to the size of transplantable tissue, making it suitable for various uses as a vascular tissue suitable for regenerative medicine purposes.

[0019]

[0020] Figure 1 is a diagram showing the results of confirming the cell sheet morphology and the aggregation phenomenon of the plate-shaped cell sheet over time of vascular endothelial cells cultured using only general culture medium (Comparative Example 1, control) and vascular endothelial cells cultured using the SlabON culture method using hyaluronic acid (HA) and polyethylene glycol (PEG) (SlabON-EPC).

[0021] Figure 2 is a diagram showing the shape and size of a culture frame with a branched shape used to confirm whether an organizational structure of the intended size was formed.

[0022] Figure 3 is a diagram showing the results of confirming the change in shape of the tissue structure formed in Comparative Example 1 (control) and SlabON-EPC of the present invention over time.

[0023] Figure 4 is a diagram showing the results of observing changes in the arrangement of the cytoskeleton at the edge of the cell sheet formed depending on whether the SlabON culture method was applied and changes in the localization characteristics of the YAP protein.

[0024] Figure 5 is a diagram showing the results of histological staining to confirm the microstructure of the internal cross-section of a cell sheet of vascular endothelial cells cultured using only a general culture medium (Comparative Example 1, control) and a cell sheet of vascular endothelial cells cultured using the SlabON culture method (SlabON-EPC).

[0025] Figure 6 is a diagram showing the results of culturing vascular endothelial cells (SlabON-EPC) without a separate BME (Basement membrane extract) using SlabON medium in culture frames with different width and height ratios, and confirming the formation of a tube-shaped tissue structure.

[0026] Figure 7 is a diagram showing the results of confirming the expression of ZO-1 protein to confirm cell-cell bonds formed between vascular endothelial cells in the cultured tissue obtained through the experiment of Figure 6 and the expression of CD34, a vascular endothelial cell marker, in the cells used.

[0027]

[0028] The present invention relates to a method for producing a vascular tissue construct, comprising the steps of: 1) culturing vascular endothelial cells by sedimenting them in a growth medium containing hyaluronic acid; and 2) culturing them by adding a growth medium containing polyethylene glycol. The present invention also relates to a method for producing a vascular tissue construct, and a vascular tissue construct produced by the method.

[0029] The vascular tissue structure according to the present invention has high biosimilarity because it does not use BME, an external factor for shape formation, and macro-scale blood vessel formation of a desired structural shape is possible through a simple process.

[0030] The present invention is described in detail below.

[0031]

[0032] The present invention relates to a method for producing a vascular tissue structure using a “SlabON culture method.” In the present invention, a growth medium containing hyaluronic acid and a growth medium containing polyethylene glycol may be referred to as SlabON medium, and vascular endothelial cells cultured using the SlabON medium as described above may be referred to as SlabON-EPC.

[0033] More specifically, the method for manufacturing a vascular tissue structure using the SlabON culture method may include the following two culture steps.

[0034] First, step 1) is the step of culturing vascular endothelial cells by sedimenting them in a growth medium containing hyaluronic acid.

[0035] In the above step 1), the vascular endothelial cells used for culture can be cells of a passage that can form tight junctions between cells on the outside and an inner lumen structure on the inside through the SlabON culture method of the present invention, and cells of passages 2 to 10, preferably cells of passages 3 to 5, can be used.

[0036] In addition, the culture in step 1) may be characterized by culturing for 1 to 3 hours, preferably 1 to 2 hours, so that vascular endothelial cells can exhibit changes in cytoskeletal arrangement and form vascular cell wall morphology.

[0037] The vascular endothelial cells of the present invention can be dispensed into a medium at a high cell density. When vascular endothelial cells are cultured using the SlabON culture method of the present invention, unlike the existing culture method where cells form a structure divided into multiple spheroids and show aggregation from the edge of the cell sheet, the cells dispensed at a high density can form a vascular tissue structure in which the cells are cohesively connected as a whole. In the present invention, the high cell density is 1x10 3 Inside 1X10 5Cell density of / ㎕, 5x10 4 Inside 1X10 5 / ㎕ cell density, and in a preferred embodiment of the present invention, 1×10 7 Cells were seeded at a cell density of 200㎕ to form vascular tissue structures.

[0038] In addition, the hyaluronic acid of step 1) of the present invention may be included in the growth medium at 0.5 to 5 mg / ml, 0.5 to 2.5 mg / ml, and preferably 0.5 to 1 mg / ml.

[0039]

[0040] In the present invention, the culturing step 2) is a step of culturing by adding a growth medium containing polyethylene glycol.

[0041] After the cell layer is uniformly settled through the culture in step 1 above, a growth medium containing polyethylene glycol is added to perform culture, thereby forming a vascular tissue structure of the desired shape. At this time, the polyethylene glycol may be included in the growth medium at 1 to 10% (w / v), and preferably at 3 to 7% (w / v). In a preferred embodiment of the present invention, a growth medium containing 5% polyethylene glycol was used.

[0042] The culture in step 2) above can be cultured for 16 to 72 hours after adding a growth medium containing polyethylene glycol (PEG).

[0043] Meanwhile, when this culture step is performed, vascular endothelial cells can form a plate-shaped cell sheet, and the formed vascular tissue structure can be characterized by forming a cortical actin layer on the outside and a lumen on the inside.

[0044] More specifically, the vascular tissue construct of the present invention has multiple layers, for example, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 cell layers, on the outside of the construct, and most of the actin of each cell layer forms a cortical actin layer. In particular, by using the manufacturing method of the present invention, the YAP protein co-localizes with actin, thereby causing YAP-mediated cellular mechanotransduction, so that the shape of the manufactured vascular tissue construct can be maintained for a long period of time.

[0045] In contrast, a lumen is formed inside the vascular tissue structure of the present invention. That is, the cells inside do not aggregate, but rather exist individually in a state of isolation, such as in the form of cell debris. These isolated internal cells can be easily removed using media perfusion methods known in the art.

[0046] Therefore, the manufacturing method of the present invention may further include, after step 2), a step of removing free cells present inside the vascular tissue structure manufactured through medium perfusion.

[0047] Through the above method, a vascular tissue structure in the form of a blood vessel can be manufactured by forming a tube with a vascular wall formed on the outside through tight junctions between cells and forming a lumen on the inside.

[0048] In the present invention, the culturing steps 1) and 2) may be performed in a patterned culture frame. By performing culturing in a patterned culture frame, various desired patterned cell sheets can be harvested, and cell arrangements suitable for directional tissues and tissues with unique cell patterns among human tissues can be induced. The patterned culture frame can be used in the same sense as a patterned support layer.

[0049] The above culture frame can include various culture frames with different width-to-height ratios, thereby producing a vascular tissue structure of the desired shape. For example, the culture frame can take on a branched or rectangular shape, and its width, length, etc. can be appropriately adjusted depending on the intended purpose.

[0050] In steps 1) and 2) of the present invention, various growth media for MSC culture and growth media for EPC culture including DMEM and / or alpha-MEM-based culture solutions known in the art suitable for culturing vascular endothelial cells can be used, and endothelial cell basal medium can be used.

[0051] Meanwhile, the manufacturing method of the present invention may be characterized by not including a BME (Basement Membrane Extract) treatment step. BME is a bio-derived matrix containing various growth factors used to induce tubule formation similar to blood vessels in vitro, and refers to Matrigel or ECM gel. According to the manufacturing method of the present invention, a vascular tissue structure having a vascular shape can be formed without the addition of a separate BME material, and therefore, the vascular tissue structure of the present invention has superior biocompatibility and safety, and can be more widely used as a regenerative therapeutic agent.

[0052]

[0053] Accordingly, the present invention provides a vascular tissue structure manufactured by the above method.

[0054] Through the above method, vascular endothelial cells dispersed at a high concentration can form a single tissue structure without forming a structure divided into spheroids, and a structure similar to a blood vessel with a formed lumen is formed.

[0055] The vascular tissue construct of the present invention may be used interchangeably with terms such as artificial vascular construct, vascular tissue scaffold, and vascularized cell construct. The vascular tissue construct of the present invention is a tubular construct.

[0056] The above vascular tissue structure may be formed with a cortical actin layer on the outside and a lumen on the inside, and may be characterized by suppression of cell aggregation and contraction compared to a non-cultured comparative control group including hyaluronic acid and polyethylene glycol.

[0057] In addition, the vascular tissue construct of the present invention does not include BME, and forms vasculogenesis by inducing spontaneous organization through cell-cell connection and activation of cell matrix formation without the external factor BME for maintaining and supporting the shape of cells. Since the vascular tissue construct manufactured by the method of the present invention exhibits a cell arrangement form according to a patterned culture frame rather than a random cell arrangement, a macro-scale vascular tissue construct with a desired structural form is provided through a simple process.

[0058] That is, organoids formed through conventional techniques are formed with a limited size in the range of 100 to 250 μm in order to take into account the exchange of nutrients and waste products and to prevent necrosis of cells inside the tissue, and therefore do not sufficiently exhibit the characteristics of living tissue in vitro. However, the vascular tissue structure according to the present invention enables the formation of macro-scale blood vessels in a desired structural shape through the application of a vascular structure in a simple manner, and thus a more scaled-up organoid can be formed.

[0059]

[0060] The present invention also provides a kit for producing a vascular tissue construct, comprising: one compartment containing a growth medium containing hyaluronic acid, two compartments containing a growth medium containing polyethylene glycol, and three compartments containing vascular endothelial cells.

[0061] Using the components included in the above individual compartments, 1) a step of culturing vascular endothelial cells by sedimenting them in a growth medium containing hyaluronic acid; 2) a step of culturing them by adding a growth medium containing polyethylene glycol; and thereby, a vascular tissue structure can be effectively manufactured.

[0062]

[0063] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0064]

[0065] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0066]

[0067] Example 1. Method for manufacturing a cell sheet using hyaluronic acid and polyethylene glycol

[0068] A plate-shaped cell sheet was prepared using hyaluronic acid (HA) and polyethylene glycol (PEG). Hereinafter, the vascular cell sheet prepared using hyaluronic acid and polyethylene glycol is referred to as “SlabON-EPC.” Specifically, rabbit bone marrow-derived vascular endothelial cells at passage number 3 to 5 were seeded at a density of 1 × 10 in growth medium (EBM basal medium (Lonza), EGM supplement (Lonza)) containing 1 mg / mL hyaluronic acid (HA) in a square silicone mold with a silicone sheet on the bottom to prevent cell aggregation. 7 / 200㎕ of the cell layer was seeded and cultured for 1.5 hours to induce uniform settlement of the cell layer. Afterwards, a growth medium containing 5% (w / v) polyethylene glycol (PEG) was added and cultured for 16 to 72 hours to prepare a cell sheet.

[0069] As a comparative control, a cell sheet (Comparative Example 1) was manufactured by culturing the cells under the same conditions without adding hyaluronic acid and polyethylene glycol.

[0070]

[0071] Example 2. Analysis of SlabON-EPC cell sheet morphology

[0072] The morphology of the cell sheets manufactured by the methods of Example 1 and Comparative Example 1 was confirmed over time. Specifically, the degree of cell aggregation was confirmed after 13 hours, 22 hours, and long-term 6 days while maintaining the culture state for SlabON-EPC and the cell sheets of Comparative Example 1, and the results are shown in Fig. 1.

[0073] As shown in Fig. 1, the control group that was not treated with hyaluronic acid and polyethylene glycol showed a shrinkage pattern after 13 hours of formation, but the SlabON-EPC of the present invention did not show shrinkage even after 6 days of formation, and it was confirmed that the plate-like shape with the outline formed by the culture frame was maintained even after 6 days of formation. Specifically, Comparative Example 1, which did not contain either hyaluronic acid or polyethylene glycol, aggregated to about 60% of the initial area after 13 hours of culture, to about 42% after 22 hours of culture, and to about 13% on the 6th day of culture. On the other hand, the SlabON-EPC cell sheet of the present invention maintained the same initial area after 13 hours of culture, and aggregated to about 82% of the initial area after 22 hours of culture, but even after 6 days of culture, the degree of aggregation of about 82% was maintained and no further aggregation was observed. These results demonstrate that the differential actions of hyaluronic acid and polyethylene glycol components present in the culture medium can effectively regulate the behavior of vascular endothelial cells, and indicate that SlabON-EPC can effectively maintain a plate-like morphology without cell aggregation.

[0074]

[0075] Example 3. Morphological and structural analysis of SlabON-EPC using a branched culture frame

[0076] 3.1 Checking the branched culture frame culture and shrinkage phenomenon

[0077] In order to confirm whether the SlabON-EPC cell sheet of the present invention can maintain the shape of a plate-shaped tissue structure in culture frames of various shapes and sizes, culture was performed using a branched culture frame. The structure of the branched culture frame used in the experiment is shown in Fig. 2. The SlabON-EPC manufacturing method of Example 1 and the manufacturing method of Comparative Example 1 were performed in the branched culture frame of Fig. 2, and the results of confirming the shape of the cell sheet over time are shown in Fig. 3.

[0078] As shown in Fig. 3, in Comparative Example 1, cultured without hyaluronic acid and polyethylene glycol, shrinkage was observed 13 hours after formation, with the edges of the tissue structure beginning to aggregate, and severe shrinkage occurred after 27 hours. Continued aggregation was observed on days 3, 6, and 9, and the shape of the cell sheet was not maintained.

[0079] On the other hand, in the SlabON-EPC experimental group, slight aggregation at the edge was observed after 27 hours, but no additional cell aggregation was observed thereafter, and it was confirmed that the shape of the tissue structure was maintained until the 9th day.

[0080]

[0081] 3.2 Tissue ultrastructural analysis using immunofluorescence

[0082] In order to analyze the ultrastructure of the tissue construct formed in 3.1 above, on the 9th day of tissue construct formation, the tissue construct was fixed in 4% paraformaldehyde in a refrigerator for 16 hours, washed twice with 1X Phosphate-Buffered Saline (PBS), and then freeze-embedded in OCT compound embedding medium to perform cryosectioning. For cell membrane permeability of the tissue sections cut into 5 μm thick, they were washed with 0.1% Triton X-100 (Siamg-Aldrich), and a blocking reaction was induced in a 10% goat serum (VectorLabs) solution for 1 hour to prevent nonspecific antibody reactions. After incubation in a refrigerator for 16 hours for primary antibody binding, secondary antibodies conjugated with Alexa Fluor 488 or Alexa Fluor 647 (abcam) were reacted for 1 hour at room temperature for staining. Actin filaments were stained using phalloidin conjugated with Alexa-Fluor 594 (abcam). Fluorescence images were captured using a confocal laser scanning microscope (Zeiss). Tissue sections cut to 5 μm thickness were dried, stained with hematoxylin and eosin (H&E), and observed under a high-resolution optical microscope. The results of tissue ultrastructural analysis using immunofluorescence are shown in Figure 4, and the results of confirming the structure of the tissue structure sections through staining and high-resolution optical microscopy are shown in Figure 5.

[0083] As shown in Fig. 4, in the SlabON-EPC of the present invention, most of the actin was observed in the form of cortical actin in the outer 1-2 layers, and in particular, YAP protein was observed to co-localize with actin. This result shows that the addition of hyaluronic acid and polyethylene glycol led to co-localization of YAP and actin in vascular endothelial cells, and through this, YAP-mediated cellular mechanotransduction occurred, thereby maintaining the shape. On the other hand, in the control group, Comparative Example 1, it was confirmed that contraction occurred as a high expression of contractile actin stress fibers was observed in the outer 6 layers.

[0084] As shown in Fig. 5, Comparative Example 1 did not exhibit a morphology similar to vascular tissue and showed an aggregated morphology in which no cavity structure was formed inside the blood vessel. On the other hand, it was confirmed that the cells inside SlabON-EPC were not aggregated and the cells existed in an individually isolated state. That is, the cross-section of the SlabON-EPC structure shows a morphology in which the inside is filled with loosely distributed cell debris and the outside maintains cell-cell connections. This structure is a result showing that the lumen can be formed and the blood vessel structure can be completed by easily removing the cell debris inside through media perfusion.

[0085]

[0086] Example 4. Confirmation of angiogenesis induction

[0087] Since the possibility of forming blood vessel structures through the SlabON-EPC method was confirmed through the above Examples 1 to 3, an experiment was conducted to confirm whether blood vessel formation was induced with only SlabON-EPC without a separate BME (Basement membrane extract) such as Matrigel or ECM gel. Culture frames with various width x length ratios such as 4x10 mm and 1x10 mm were manufactured as shown in Fig. 6, and SlabON-EPC was formed in each culture frame. Afterwards, blood vessel formation was confirmed through the expression of ZO-1, a marker of tight junction expressed in vascular tissue, and CD34 expressed in vascular endothelial cells.

[0088] As a result, as shown in Fig. 6, a tubular tissue structure was observed in the SlabON-EPC experimental group, and in particular, in the case of SlabON-EPC formed in a 1 X 10 mm culture frame, endothelial cells formed by 1 to 2 layers of tight junctions on the outside were observed, while the inside was clearly composed of cells forming relatively loose junctions.

[0089] In addition, as shown in Figure 7, ZO-1 and CD34 were positively expressed in all SlabON-EPC experimental groups, confirming that tight junctions were effectively induced and blood vessels were formed.

[0090] These results indicate that the SlabON culture method using hyaluronic acid and polyethylene glycol can induce tube formation and vascular tissue microstructure similar to that induced when vascular endothelial cell differentiation was induced using BME without using BME.

Claims

1. 1) A step of culturing vascular endothelial cells by sedimenting them in a growth medium containing hyaluronic acid; 2) A method for producing a vascular tissue structure, comprising: a step of culturing by adding a growth medium containing polyethylene glycol; 2. A method for producing a vascular tissue structure in claim 1, wherein the vascular endothelial cells are cells of passages 2 to 10.

3. A method for producing a vascular tissue structure, wherein the culture in step 1) is performed for 1 to 3 hours.

4. In the first paragraph, the vascular endothelial cells are 1x10 3 Inside 1X10 5 A method for producing a vascular tissue structure, wherein the vascular tissue structure is dispensed into a medium at a cell density of / ㎕.

5. A method for producing a vascular tissue structure, wherein the hyaluronic acid of step 1) is contained in an amount of 0.5 to 5 mg / ml.

6. A method for producing a vascular tissue structure, wherein in the first paragraph, the polyethylene glycol of step 2) is included at 1 to 10% (w / v).

7. A method for manufacturing a vascular tissue structure in accordance with claim 1, wherein the vascular tissue structure has a cortical actin layer formed on the outside and a lumen formed on the inside.

8. A method for producing a vascular tissue structure, wherein the culturing steps 1) and 2) in the first paragraph are performed in a patterned culture frame.

9. A method for manufacturing a vascular tissue structure, wherein the manufacturing method in claim 1 does not include a BME (Basement membrane extract) processing step.

10. A vascular tissue structure manufactured by any one of the methods of claims 1 to 9.

11. In the 10th paragraph, the vascular tissue structure is a vascular tissue structure having a cortical actin layer formed on the outside and a lumen formed on the inside.

12. In claim 10, the vascular tissue structure is a vascular tissue structure in which cell aggregation and contraction are suppressed.

13. A vascular tissue structure in claim 10, wherein the vascular tissue structure does not contain BME (Basement membrane extract).

14. 1 compartment containing growth medium containing hyaluronic acid; 2 compartments containing a growth medium containing polyethylene glycol, and A kit for manufacturing a vascular tissue construct comprising three compartments containing vascular endothelial cells.

Images