COMPOSITION FOR TRANSPLANTATION OF ORGANOID

Abstract

The present invention relates to a composition for transplantation comprising an organoid, and a use of same. According to one example, using collagen, gelatin or fibrin glue as a scaffold for organoid transplantation results in a high transplantation rate and a high survival rate of organoid as well as desirable stability.

Claims

1. A composition for biotransplantation comprising organoid; and gelatin, collagen, fibrin glue, or a combination thereof.

2. The composition for biotransplantation according to claim 1, wherein the organoid is selected from the group consisting of intestinal organoid, retinal organoid, kidney organoid, liver organoid, gastric organoid, prostate organoid, breast organoid, inner ear organoid, cardiac muscle fiber organoid, hepatic endothelial organoid, pancreatic organoids, fallopian tube organoids, and cerebral organoids.

3. The composition for biotransplantation according to claim 1, wherein the gelatin is comprised in an amount of 2.5 to 10% (w/v) based on the total weight of the composition.

4. The composition for biotransplantation according to claim 1, wherein the collagen is comprised in an amount of 10 to 20% (w/v) based on the total weight of the composition.

5. The composition for biotransplantation according to claim 1, wherein the fibrin glue is comprised in an amount of 10 to 15% (w/v) based on the total weight of the composition.

6. A method for transplanting an organoid comprising mixing gelatin, collagen, fibrin glue, or a combination thereof with the organoid; and administering the mixture to a subject.

Description

BRIEF DESCRIPTION OF FIGURES

[0037] FIG. 1 is a photograph of measuring the GFP signal emitted from an organoid transplanted into the colon tissue on the 7th day after transplanting the colon organoid for each scaffold.

[0038] FIG. 2 is a graph showing the area of an organoid transplanted into the colon tissue on the 7th day after transplanting the colon organoid.

[0039] FIG. 3 is a photograph showing the result of observing a colon tissue section on the 7th day after transplanting colon organoids.

[0040] FIG. 4 is a photograph showing the morphology of a colon transplanted with an organoid on the 7th day after transplanting the colon organoid.

[0041] FIG. 5 is a graph showing the result of calculating the weight/length of a colon transplanted with an organoid on the 7th day after transplanting the colon organoid.

[0042] FIG. 6 is a photograph of the result of confirming the GFP signal after forming a secondary organoid after transplanting the colon organoid.

[0043] FIG. 7 is a graph showing the results of measuring a correlation with a transplanted area by measuring a number of GFP-expressing organoids after forming a secondary organoid after transplanting a colonic organoid.

[0044] FIG. 8a is a schematic diagram showing the manufacturing process of the acute colon injury model induced by EDTA.

[0045] FIG. 8b is a schematic diagram illustrating a transplantation process of an organoid according to one embodiment.

[0046] FIG. 8c is a result showing the tissue analysis result after transplantation of the organoid according to one embodiment.

EXAMPLES

[0047] Hereinafter, it will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Reference 1. Preparation and Culture of Colon Organoids

[0048] Colon tissue was separated from EGFP mice, and colonic crypt was isolated using an enzyme. It was inoculated into an uncoated 48-well plate wherein matrigel and medium for colon organoids were mixed in a 1:1 ratio. After checking that the matrigel was hardened 20 minutes after placed in an incubator, a medium for colon organoids was added and cultured for 5 days to prepare colon organoids for biotransplantation to be used below.

Example 1. Preparation and Transplantation of Organoids for Biotransplantation

1.1. Colon Tissue Damage Model Construction

[0049] A colon tissue damage model to produce organoids was prepared as follows. Wild-type mice to be transplanted with colon organoids were exposed to 0.5M EDTA for 5 minutes, and physical damage was applied to remove the crypts of the colonic lining for 2 minutes with an electric toothbrush.

1.2. Preparation and Transplantation of Organoids for Biotransplantation

[0050] A colon organoid was prepared so that the colon organoid prepared above expresses GFP. All colon organoids were treated with 10 μM of Y-27632 in the organoid medium one day before transplantation to maximize the survival rate. In addition, the matrix used during culture was completely removed by treatment with a cell recovery solution.

[0051] Thereafter, the following three scaffolds were mixed under the conditions described below and transplanted in a volume of 50 μl into the anus of the colon tissue damage mouse model prepared in 1.1. [0052] 1) Gelatin: GFP+ colon organoids were mixed with PBS in which 5% gelatin was dissolved in a ratio of gelatin:organoid containg medium=1:2, and the resultant was transplanted. Herein, the organoid containg medium comprises 10 μM of Y-27632. [0053] 2) Collagen: GFP+ colon organoids were mixed with 100% collagen stock solution in a ratio of collagen:organoid containg medium=1:9, and the resultant was transplanted. Herein, the organoid containg medium comprises 10 μM of Y-27632. [0054] 3) Fibrin Glue: a colonoid solution was prepared by mixing GFP+ colon organoids with 45 μl of colonoid culture medium containing 10 μM of Y-27632. Fibrin was mixed with a ratio of 1:1 in a solution in which thrombin is diluted 1:100 in PBS to make 5 μl of the solution. And then, it was mixed with the prepared 45 μl of colonoid solution and the resultant was transplanted.

[0055] Matrigel was used as a control group.

[0056] Matrigel was transplanted after mixing it with a colonoid culture medium containing organoids at a concentration of 10%. At this time, the medium containing the organoids contains 10 μM of Y-27632.

[0057] After transplantation, the anus was closed using 10 μl of 3M Vetbond, and the suture was released 14 hours later to induce normal bowel activity.

Example 2. Evaluation of Transplantation Rate of Organoids

[0058] In order to evaluate the transplantation rate according to the scaffold of the colon organoid transplanted in Example 1, it was carried out as follows.

[0059] On the 7th day after transplantation of colon organoids, the colon tissue was autopsied to measure GFP signals emitted from the transplanted organoids. Specifically, the organoid-implanted colon tissue was vertically incised to create a planar structure, and this tissue was spread thinly on a slide glass and a glass cover was covered thereon. At this time, the crypt was directed downward. The prepared slide glass was placed on the stage of a fluorescence microscope to observe the region where GFP fluorescence was expressed.

[0060] FIG. 1 is a photograph of measuring the GFP signal emitted from the organoid transplanted into the colon tissue on the 7th day after transplanting the colon organoid for each scaffold.

[0061] FIG. 2 is a graph showing the area of the organoid transplanted into the colon tissue on the 7th day after transplanting the colon organoid.

[0062] As shown in FIGS. 1 and 2, in the absence of a scaffold, it was confirmed that the GFP signal was the weakest and the transplantation rate was low. On the other hand, it was confirmed that when gelatin, collagen, and fibrin glue were used as scaffolds, the transplantation rate and transplanted area were similar to those when Matrigel, a positive control, was used as a scaffold.

[0063] Therefore, when gelatin, collagen, or fibrin glue is used as a scaffold for organoid transplantation, the transplantation rate of organoids is significantly increased.

Example 3. Evaluation of Survival Rate and Engraftment Ratio of Organoids

[0064] In order to evaluate the survival rate and engraftment ratio according to the scaffold of the colon organoid transplanted in Example 1, it was carried out as follows.

[0065] 10 to 13 animals were used for each scaffold experimental group, and tissue autopsy was performed one week after transplantation of GFP organoids into the colon. Those expressing GFP in the colon were classified as successfully transplanted animals, and those not expressing GFP were classified as unsuccessfully transplanted animals. The final engraftment ratio was determined by calculating the percentage of the number of successfully transplanted animals out of the total number. And the final survival rate was calculated by counting the animals who died within 7 days after transplantation.

[0066] The results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Number Engraft- Engraft- Dead Survival of trans- ment ment rate ratio Engraft- plantation successes failed (%) (%) ment No 13 8 1 2 84.62 61.54 scaffold Matrigel 10 9 0 1 90.00 90.00 Gelatin 13 9 1 3 76.92 69.23 Collagen 10 9 1 0 100.00 90.00 Fibrin 10 9 0 1 90.00 90.00 glue

[0067] As shown in Table 1, when gelatin, collagen, and fibrin glue were used as scaffolds, the survival rates were 76.92, 100, and 90%, respectively, and the engraftment ratio was confirmed as high as 69.23, 90, and 90%, respectively. Therefore, it was confirmed that the survival rate and engraftment ratio were excellent. This was at a level similar to that in the case of using Matrigel, a positive control. FIG. 3 is a photograph showing the results of observing the colon tissue section on the 7th day after transplantation of colon organoids.

[0068] As shown in FIG. 3, when gelatin, collagen, and fibrin glue were used as scaffolds, it was confirmed that the organoids were successfully settled to form colonic crypts.

Example 4. Stability Evaluation

[0069] In order to evaluate the stability of the colon organoid transplanted in Example 1, an autopsy was performed on the 7th day after transplantation, and the morphology of the autopsied tissue and the occurrence of lesions were checked.

[0070] FIG. 4 is a photograph showing the morphology of the colon transplanted with the organoid on the 7th day after transplantation of the colon organoid.

[0071] As shown in FIG. 4, when gelatin, collagen, or fibrin glue was used as a scaffold, an edema or bloody stool that may appear in the colon transplanted with organoid was not observed in the autopsy tissue.

[0072] FIG. 5 is a graph showing the result of calculating the weight/length of the colon transplanted with the organoid on the 7th day after transplanting the colon organoid.

[0073] As shown in FIG. 5, the volume of edema that may appear when inflammation occurred was checked by calculating the weight/length of the colon transplanted with organoid. As a result, there was no significant difference compared with the control group using Matrigel.

[0074] Therefore, it was confirmed that when the organoid was transplanted using gelatin, collagen, or fibrin glue as a scaffold, the stability was excellent.

Example 5. Evaluation of Normal Organoid Formation from Transplanted Tissue

[0075] In order to evaluate whether normal organoids are formed from the transplanted colonic organoids in Example 1, a second colonic organoid (secondary organoid) was formed from the transplanted colonic tissue.

[0076] Specifically, after checking the GFP of the transplanted colon tissue with a fluorescence microscope, it was cut with surgical scissors, put in a tube containing crypt chelating buffer, and reacted in a shaking incubator at 37° C. for 20 minutes. And then, it was put in a 10 ml syringe equipped with an 18 gage needle, and crypt is separated by grinding 20 times. The separated crypt was centrifuged, collected, filtered through a 70 μm filter, mixed with Y-27632-added medium and matrigel at a 1:1 ratio, and inoculated at a concentration of 20 μl/well in a 48-well plate. After hardening the matrigel by putting it in an incubator at 37° C. for 30 minutes, a Y-27632-added medium was added and cultured for 5 days. On the 5th day of culture, colonic organoids expressing GFP were followed up.

[0077] FIG. 6 is a photograph of the result of confirming the GFP signal after forming a secondary organoid after transplanting the colon organoid.

[0078] As shown in FIG. 6, when gelatin, collagen, or fibrin glue was used as a scaffold, secondary organoids expressing GFP were effectively formed. This was confirmed to be at a level similar to that of the positive control using matrigel.

[0079] FIG. 7 is a graph showing the results of measuring the correlation with the transplanted area by measuring the number of GFP-expressing organoids after forming a secondary organoid after transplanting colonic organoids.

[0080] As shown in FIG. 7, it was confirmed that the number and area of secondary organoids increased in proportion.

[0081] Therefore, it indicates that gelatin, collagen, and fibrin glue do not significantly affect normal organoid formation.

[0082] According to one aspect of the present invention, when collagen, gelatin, or fibrin glue is used as a scaffold for organoid transplantation, the transplantation rate and survival rate of the organoid is high and the stability is also excellent, therefore the composition comprising the same can be used usefully for biotransplantation in vivo.