BIO-INK COMPOSITION HAVING IMPROVED PHYSICAL AND BIOLOGICAL PROPERTIES

Abstract

The present invention relates to a bio-link composition having improved physical and biological properties and, more specifically to a bio-link composition, which exhibits, through a combination of specific contents of components, high viscosity, strong shear-thinning tendencies, formations of fast cross linkages, and appropriate mechanical properties after printing. The bio-ink composition of the present invention is capable of being used very usefully in the preparation of three-dimensional bio-printed tissue-like organs and internal transplantable tissue structures.

Claims

1. A bio-ink composition comprising 0.05-6010.sup.6/mL of cell, 0.1 to 10 w/v % of a cell carrier material, 0.01 to 1 w/v % of a viscosity enhancer, 1 to 30 v/v % of a lubricant and 0.1 to 10 w/v % of a structural material.

2. The bio-ink composition of claim 1, wherein the bio-ink composition further comprises a tissue-derived component material.

3. The bio-ink composition of claim 1, wherein the bio-ink composition further comprises a regulator material of differentiation.

4. The bio-ink composition of any of claims 1 to 3, wherein the bio-ink composition is one or more selected from the group consisting of a stem cell, an osteoblast, a myoblast, a tenocyte, a neuroblast, a fibroblast, a glioblast, a germ cell, a hepatocyte, a renal cell, a sertoli cell, a chondrocyte, an epithelial cell, a cardiovascular cell, a keratinocyte, a smooth muscle cell, a cardiomyocyte, a glial cell, a endothelial cell, a hormone-secreting cell, an immune cell, a pancreatic islet cell and a neuron.

5. The bio-ink composition of any of claims 1 to 3, wherein the cell carrier material is gelatin or collagen, the viscosity enhancer is hyaluronic acid or dextran, the lubricant is glycerol, and the structural material is fibrinogen or gelatin methacrylate.

6. The bio-ink composition of any of claims 1 to 3, wherein the bio-ink composition further comprises a thrombin solution or photoinitiator.

7. A method for preparing a tissue-like organ, the method comprising: (a) charging the bio-ink composition of any of claims 1 to 3 into a three dimensional bio-printer; (b) three-dimensionally printing a desired tissue-like organ; and (c) cross-linking the three-dimensionally printed bio-ink composition.

8. The method of claim 7, wherein the method further comprises (d) culturing the tissue-like organ in a culture medium.

9. A tissue-like organ (organoid) prepared according to the method of claim 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 is a schematic view showing a process of preparing a three-dimensional hydrogel tissue structure using the bio-ink of the present invention.

[0062] FIG. 2 shows the result of evaluating the structural stability of the bio-ink of the present invention after 21 days of culture in the cell culture conditions (A: appearance change of tissue structure after 21 days of culture, B: measurement of tissue structure size change for 21 days after bio-printing).

[0063] FIG. 3 shows the change in the stability of the bio-printed structure depending on the content of the viscous enhancer contained in the bio-ink composition.

[0064] FIG. 4 is a diagram for evaluating the resolution according to the printing performance and feed rate of the bio ink of the present invention, showing that the pattern resolution is improved as the feed rate is increased and that the uniformity is maintained under the used feed rate.

[0065] FIG. 5 shows the result of evaluating the viability of cells for 7 days via fluorescent staining after three-dimensional printing with bio-inks of various compositions containing 3T3 fibroblasts (Gel-MA 50:50% methacrylated gelatin).

[0066] FIG. 6 shows the result of evaluating the viability of cells for 7 days via fluorescent staining after three-dimensional printing with bio-inks of various compositions containing a C2Cl2 myoblast cell line (Gel-MA; gelatin methacrylate).

[0067] FIG. 7 shows that it was confirmed that the proliferation and differentiation of muscle cells were further improved as compared with a control group containing no tissue-derived components after printing muscle cells using a bio-ink composition containing muscle tissue-derived components (Control: Tissue-derived components-untreated control group, muscle ECM: composition group containing muscle-derived extracellular matrix).

[0068] FIG. 8 is a schematic diagram showing a method of incorporating a peptide into the bio-ink composition via chemical bonding, wherein the peptide mimics a growth factor involved in the regulation of cell differentiation.

[0069] FIG. 9 is photographic results showing a tissue-like organ prepared using the bio-ink composition of the present invention (A: liver tissue organoid, B: cardiac tissue organoid, C: muscle tissue organoid).

[0070] FIG. 10 is photographic results showing the preparation of a three-dimensional bio-printed and manipulated tissue structure using the bio-ink composition of the present invention (A: Ear tissue structure, B: nose tissue structure, and C: blood vessel-like tissue structure).

[0071] FIG. 11 is photographic results showing that the linkage among tissues was able to be reproduced by simultaneously printing various cells (A: hepatocyte-vascular cell printing, B: osteocyte-chondrocyte printing, C: muscle-tendon cell printing)

[0072] FIG. 12 is photographic results showing that a muscle tissue structure having an improved function was formed by simultaneously printing nerve cells and muscle cells using the composition of the present invention (red: muscle, green: nerve cell)

MODE FOR CARRYING OUT INVENTION

[0073] Hereinafter, the present invention will be described in detail.

[0074] However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1: Cell Culture

[0075] In order to evaluate the bio-ink composition of the present invention, various cells were utilized as follows: 3T3 fibroblast cell line (ATCC, Manassas Va., USA) was cultured in high glucose DMEM culture medium (Life Technologies) containing 10% fetal bovine serum (FBS, Life Technologies, Carsbad, Calif., USA) and 1% penicillin/streptomycin at 37 C. in a 5% C02 cell incubator.

[0076] Chondrocytes separated from the cartilage of a rabbit by enzymes were cultured in DMEM/F-12 culture medium (Life Techologies) containing 10% FBS and 1% penicillin/streptomycin at 37 C. in a 5% C02 cell incubator.

[0077] The C2Cl2 muscle cell line (ATCC) was cultured in high glucose DMEM culture medium containing 10% FBS and 1% penicillin/streptomycin, and then cultured in DMEM/F-12 medium and 1% horse serum (HS, Life Technologies) for muscle fiberization.

Example 2: Preparation of Bio-Ink Composition

[0078] The bio-ink composition of the present invention comprises a temperature-sensitive gelatin (35 mg/mL, Sigma-Aldrich, St. Louis, Mo., USA) which has a sufficient viscosity so that printing can be performed in a uniformly mixed form of cells, hyaluronic acid (3 mg/mL, Sigma-Aldrich) which can enhance the viscosity of the bio-ink during printing and improve the uniformity of the pattern, glycerol (10 v/v %, Sigma-Aldrich) as a lubricant which reduces nozzle clogging, and a chemically modified gelatin (20 mg/mL methacrylated gelatin) which provides structural stability by cross-linking after printing. The bio-ink composition in which such components were fully mixed and dissolved was sterilized using a 0.45 um injector-type filter.

Example 3: Evaluation of Dispensing Rate and Structural Stability of Bio-Ink Composition

[0079] The uniformity of the pattern and the stability of the printed structure were evaluated upon printing with the bio-ink of the present invention. These are indices indicating the superiority of the bio-ink of the present invention. For evaluation, the bio-ink of the present invention was filled into a bio-printer and printed through a nozzle having a diameter of 300 um at a pressure of 50-80 kPa. The bio-printer used in this experiment is composed of a three-axis moving stage, a dispensing module capable of pneumatic injection, and an injector-type reservoir and a nozzle for charging bio-ink.

[0080] In order to evaluate the change of dispensing rate according to the concentration of gelatin or hyaluronic acid, the content of the remaining components except gelatin or hyaluronic acid was kept the same as that of Example 2. By varying the content of gelatin or hyaluronic acid, the volume of the printed bio-ink was measured over time. Using the measured values, the coefficient of variation (COV) was calculated to evaluate the printing performance according to the concentration, and the optimal content was determined based on the evaluation.

[0081] The results are shown in Table 1 below.

TABLE-US-00001 TABLE 1 Conc. Dispensing rate (mg/mL) (L/sec) COV (%) Gelatin 30 2.73 0.82 30.07 35 0.39 0.14 35.70 40 0.28 0.09 31.60 45 0.12 0.04 30.65 HA 0 0.40 0.14 35.70 3 0.52 0.05 9.81 6 0.45 0.02 4.42

[0082] As shown in Table 1, the dispensing rate was varied depending on the gelatin concentration. Dispensing rate is the volume of bio-ink printed per hour. The lower the dispensing rate is, the higher the resolution of the pattern becomes.

[0083] In the case of gelatin, the resolution of the pattern rapidly increased at 35 mg/mL or higher. Coefficient of variation (COV) is an index representing the uniformity of the printed pattern. The higher the value of this index is, the more dispensing rate becomes uneven. As a result, the resolution of the pattern was improved at 35 mg/mL or higher of gelatin, while there was no change in uniformity.

[0084] In contrast, when the hyaluronic acid was added to the gelatin hydrogel, the dispensing rate did not change significantly (HA: hyaluronic acid). However, there was markedly improved in the uniformity of the printed pattern as shown by the low COV values. Thus, it was found that although the inclusion of HA to the bio-ink composition insignificantly contributes to the resolution of the pattern, while exerting a great influence on the uniformity of the pattern.

[0085] Therefore, the above results suggest that the inclusion of gelatin and HA in the bio-ink composition of the present invention is essential.

[0086] In addition, in order to evaluate the structural stability of the bio-ink of the present invention, the tessellated-like structure was printed three-dimensionally using the bio-ink of the present invention. The bio-printed structure was exposed to UV for 1 to 1,000 seconds for crosslinking, followed by incubation at 37 C. in a cell culture medium. The change in size was measured over time. The evaluation was carried out for 21 days.

[0087] The results are shown in FIG. 2.

[0088] As shown in FIG. 2, after the bio-printed structure was cultured for 21 days, the structure stability was excellent enough to maintain about 70% of the initial size (Day 0).

[0089] Then, a change in the stability of the bio-printed structure depending on the content of the viscous enhancer contained in the bio-ink composition was determined. The bio-ink composition was prepared in the same manner as in Example 2 except that only the concentration of hyaluronic acid, which is a viscosity increasing agent, was changed to 0, 1, 2 and 3 mg/ml to evaluate the structural stability.

[0090] The results are shown in FIG. 3.

[0091] As shown in FIG. 3, the stability of the bio-printed structure was found to improve with the increasing content of hyaluronic acid.

Example 4: Performance Evaluation of Bio-Ink Composition: Feed Rate and Pattern Resolution

[0092] In order to evaluate the printing performance, i.e., the resolution according to the feed rate of the bio-ink of the present invention, the bio-ink of the present invention was printed through a nozzle having a diameter of 200 um under adjustment of a feed rate of 100 to 700 mm/min.

[0093] The results are shown in FIG. 4.

[0094] As shown in FIG. 4, the bio-ink of the present invention showed an improved pattern resolution as the feed rate increased, while maintaining uniformity under the feed rates as used.

Example 5: Evaluation of Cell Survival Rate in Bio-Ink Composition

[0095] In order to evaluate the cell suitability of the bio-ink composition of the present invention, the bio-ink of the present invention was mixed with a 3T3 fibroblast cell line, filled in a bio printer, passed through a nozzle having a diameter of 300 um at a pressure of 50-80 kPa, completing a three-dimensional structure. As for three-dimensional structures containing bio-printed cells, cell viability was evaluated using Live/Dead assay kit (Life Technologies). The bio-ink composition used in this experiment was prepared according to the method of Example 2. By changing the molecular weight and degree of crosslinking (20, 50 and 80%) of gelatin in gelatin methacrylate, the physical rigidity of the bio-ink was changed.

[0096] Phosphate buffer (PBS, Life Technologies) in which 0.5 uL/ml Calcein-AM and 2 uL/mL Eth-D were dissolved was exposed to the printed bio-ink structure with the cells. Two hours later, the cells were observed under a fluorescence microscope to identify cells with green fluorescence (calcein), that is, living cells. The optimization of the bio-ink was determined under conditions of cell viability of 80% or more.

[0097] The results are shown in FIG. 5. In FIG. 5, Gel-MA 50 refers to 50% methacrylated gelatin.

[0098] As shown in FIG. 5, green fluorescence, which indicates living cells, was observed in most of the cells in the bio-printed three-dimensional structure. When quantified, the cell survival rate showed 90% or more (see the graph) under all the composition conditions used in the experiment. Thus, it was verified that the bio-ink composition of the present invention minimizes cell damage at a high shear pressure generated during printing.

[0099] The bio-ink of the same composition as the above experiment was used for printing except that the concentration of Gel-MA 50 was changed and the C2Cl2 myoblast cell line was contained. 50% methacrylated gelatin was used for the experiments with two concentrations of 20 mg/ml and 30 mg/ml, respectively. In addition, the printing conditions were as follows: feed rate of 120 mm/min and the nozzle size of 300 Teflon nozzle.

[0100] The results are shown in FIG. 6.

[0101] As shown in FIG. 6, in all the compositions, most of the cells in the bio-printed three-dimensional structure exhibited green fluorescence which indicates living cells. While the gelatin concentration is usually increased to improve the strength of the gel, such an increased gelatin concentration may reduce the diffusion rate within the gel and decrease the cell survival rate. In the case of the bio-ink composition according to the present invention, it was confirmed that Gel-MA 50 did not affect cell viability even at a concentration of 30 mg/ml.

Example 6: Evaluation of Cell Proliferation and Differentiation Ability of Bio-Ink Composition Containing Tissue-Derived Components

[0102] It is virtually impossible for any artificially generated composition to reproduce all of the properties possessed by natural tissue-derived components such as an extracellular matrix. Thus, by adding the de-cellularized tissue-derived components to the bio-ink compositions, it was sought to determine whether the cells could normally grow and differentiate within the bio-printed structures.

[0103] The composition contained 12% of the muscle tissue-derived component in the bio-ink composition (20 mg/ml Gel-MA 50 (bloom 300), 30 mg/ml gelatin (bloom 90-100), 3 mg/ml HA, 10% glycerol) tested above (Cell: C2Cl2 myoblast cell line (110.sup.6 cells/ml), feed rate: 120 mm/min, nozzle size: 300 Teflon nozzle). The muscle tissue-derived components were obtained as follows: the muscle tissues of a pig were de-cellularized, followed by the separation of the remaining extracellular matrix proteins by dissolving in weak acidic solution and pepsin. Those components were neutralized and then contained in the bio-ink composition. The main components of the muscle tissue-derived components were collagen, glycosaminoglycan, growth factor and cytokine.

[0104] The results are shown in FIG. 7.

[0105] As shown in FIG. 7, when the muscle cells were printed using the bio-ink composition containing the components derived from muscle tissue, it was confirmed that the proliferation and differentiation of the cells were further improved as compared with the control group containing no tissue-derived components.

Example 7: Preparation of Bio-Ink Composition Containing a Regulator Material of Differentiation

[0106] The present inventors have prepared a bio-ink composition containing a regulator material of differentiation in order to induce the differentiation of stem cells (including embryonic stem cells, inducible pluripotent stem cells, and adult stem cells) contained in the bio ink composition into specific cells.

[0107] Specifically, in this Example, the bio-ink composition contained, via chemical bonding, peptides that mimic bone-forming protein-2 (BMP-2, SEQ ID NO: 1) used to induce differentiation of the bio-printed stem cells into osteocytes, growth factors such as TGF-beta3 (SEQ ID NO: 2, SEQ ID NO: 3) used to induce their differentiation into chondrocytes, or a vascular endothelial growth factor (SEQ ID NO: 4) used to induce their differentiation into vascular endothelial cells, respectively:

TABLE-US-00002 SEQIDNO:1: KIPKASSVPTELSAISTLYL SEQIDNO:2: ANVAENA SEQIDNO:3: LIANAK SEQIDNO:4: KLTWQELYQLKYKGI

[0108] A schematic diagram of a method for preparing a bio-ink composition containing growth factors involved in the regulation of differentiation is shown in FIG. 8.

Example 8: Preparation of Tissue-Like Organs and Implantable Tissue Structures Using Bio-Ink Compositions

[0109] Various tissue-like organs and implantable tissue structures were prepared using the bio-ink of the present invention. For this purpose, in order to obtain a structure that is characteristic of a specific organ, histologically stained images or bio-images (obtained by bio-imaging techniques such as CT or MRI) were converted into a 3D CAD model and then again converted into a printable motion file. The converted motion file was linked with the bio-printer to print the specific structure.

[0110] The results are shown in FIGS. 9 to 12.

[0111] As shown in FIG. 9, through observing that the liver tissue (FIG. 9A), cardiac tissue (FIG. 9B) and muscle tissue (FIG. 9C) organoids were favorably formed using the bio-ink composition of the present invention, it was confirmed that it is possible to print an internal structure with a sophisticated orientation associated with the functionality of a tissue.

[0112] As shown in FIG. 10, it was observed that the structures of the three-dimensional bio-printed manipulated ear (FIG. 10A), nose (FIG. 10B) and tubular tissue (FIG. 10C) could also be prepared favorably, verifying that several tissues and organs that can be transplanted could be printed using the bio-ink composition developed in the present invention.

[0113] As shown in FIG. 11 and FIG. 12, it was confirmed that the interaction among tissues could be reproduced by simultaneously printing various cells using the composition of the present invention. That is, by confirming a linkage between hepatocytes and blood vessel cells (FIG. 11A), a linkage between osteocytes and chondrocytes (FIG. 11B), and a linkage between muscle cells and tendon cells (FIG. 11C), it was verified that a tissue structure improved in its functionality could be prepared using the composition of the present invention.