KR20230106074A - Manufacturing method of cancer organoid using 3d printing - Google Patents

Abstract

The present invention is a cancer organoid using 3D printing comprising extruding matrix ink containing a collagen solution and an alginate-hyaluronic acid solution, and extruding bioink containing cells, hyaluronic acid, and gelatin on the matrix ink. It's about manufacturing methods.

Description

Cancer organoid manufacturing method using 3D printing {MANUFACTURING METHOD OF CANCER ORGANOID USING 3D PRINTING}

The present invention is a cancer organoid using 3D printing comprising extruding matrix ink containing a collagen solution and an alginate-hyaluronic acid solution, and extruding bioink containing cells, hyaluronic acid, and gelatin on the matrix ink. It's about manufacturing methods.

Breast cancer has the highest incidence among Western women, and various standard treatments such as breast-conserving surgery, radiation therapy, and chemotherapy are applied to breast cancer patients. However, these treatments do not reflect the specific variability of each patient, limiting their effectiveness. In this respect, personalized medicine is emerging as a new paradigm for breast cancer patient treatment.

Personalized medicine predicts the patient's treatment response and provides customized drugs for effective treatment results. In personalized medicine, a patient-specific cancer model is an essential tool for simulating drug response, and the most widely used breast cancer model is patient-derived xenotransplantation (PDX).

The PDX model is produced by implanting patient-derived breast cancer tissue into an animal that has the advantage of reproducing genetic and morphological characteristics similar to natural cancer. However, this model requires a long preparation time of about 3-18 months and is expensive. Also, its grafting success rate (approximately 18% success rate) and reproducibility are low.

To overcome the limitations of the PDX model, many researchers are working to develop patient-specific in vitro breast cancer models. Various organoid technologies are being developed to generate biomimetic microstructures with patient-derived breast cancer cells. These organoids show a strong correlation between morphological microstructure and drug response.

However, since organoids are produced by spontaneous morphogenesis of cancer cells, it is difficult to control the microstructure and size, resulting in low reproducibility. In vitro breast cancer models based on microfluidics and cell aggregate technology are being introduced.

Microfluidic technology can reproduce various tissue microstructures resembling native breast cancer, such as ductal and solid morphologies. Breast cancer tissue exhibits heterogeneous morphological characteristics ranging from less aggressive ducts to highly invasive solid tissue microstructures. These tissue types show different responses to drugs. For example, in vitro breast cancer models with solid tissue microstructures show higher sensitivity to hypoxia-activated prodrugs than models with ductal microstructures.

In a study using a cell aggregate-based breast cancer model, researchers observed that genotypic and phenotypic characteristics of cancer cells were transformed into advanced stage cancer-like characteristics as the size of cell aggregates increased. The large aggregates helped activate the migratory behavior of non-migrating breast cancer cells and helped increase drug resistance. Therefore, various types of in vitro breast cancer models are being developed. Many researchers report that the ultrastructure of these models' cells has a significant impact on drug response.

However, only a single type of microstructure (ductal or solid tissue microstructure) has been used in in vitro model studies of breast cancer. Because many heterogeneous cancers have different morphological structures that respond differently to a given drug, using a single tissue microstructure as a model to develop patient-specific therapies may not work. Although numerous techniques for breast cancer are being developed, none are capable of reproducing this morphological heterogeneity in vitro.

Republic of Korea Patent No. 10-2272551

In this study, a 3D bioprinting technique was used to develop an in vitro breast cancer model that reproduces the morphological heterogeneity of cancer tissue. A printing process for cell aggregates (organoids) with conduits and solid microstructures was established using human breast cancer cells (MCF7). Genotypic and phenotypic characteristics of breast cancer organoids were evaluated. In addition, the present inventors investigated the effect of morphological heterogeneity on drug response using a heterogeneous cancer model generated through co-printing of ductal and solid breast cancer organoids. Creating in vitro models of xenogeneic breast cancer tissue that recapitulates in vivo cellular morphology will enable precise development of effective therapies for breast cancer patients.

According to one embodiment of the present invention, extruding a matrix ink containing a collagen solution and an alginate-hyaluronic acid solution, and extruding bioink containing cells, hyaluronic acid and gelatin on the matrix ink, A method for manufacturing cancer organoids using 3D printing is provided.

The bio-ink may further include collagen, but is not limited thereto.

After the bio-ink extrusion step, a step of crosslinking by treatment with a calcium ion solution may be further included, but is not limited thereto.

The collagen solution may be prepared by dissolving pepsin and collagen in a hydrochloric acid solution, but is not limited thereto.

The alginate-hyaluronic acid solution may be prepared by dissolving alginate and hyaluronic acid in an animal cell culture medium, but is not limited thereto.

The animal cell culture medium is MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI 1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE), Keratinocyte-SFN (with BPE), KnockOut It may be selected from D-MEM, AmnioMAX-II Complete Medium and AmnioMAX-C100 Complete Medium, but is not limited thereto.

The matrix ink may be a mixture of a collagen solution and an alginate-hyaluronic acid solution in a weight ratio of 1 to 5:1, but is not limited thereto.

The cells may be mixed at a concentration of 1×10 ⁷ to 20×10 ⁷ cells/mL, but is not limited thereto.

In the bioink, hyaluronic acid may be mixed at a concentration of 1 to 5 mg/mL, but is not limited thereto.

In the bioink, gelatin may be mixed at a concentration of 15 to 25% (w/v), but is not limited thereto.

The matrix ink may be extruded at a speed of 0.5 to 0.9 μL/s and a printing speed of 100 to 300 mm/min, but is not limited thereto.

The bioink may be extruded at a rate of 0.005 to 0.02 μL/s and printed in a dot form, but is not limited thereto.

The cells are cancer cells, preferably breast cancer cells.

According to another embodiment of the present invention, a cancer organoid prepared by the above production method is provided.

The ductal and solid organoids bioprinted with human breast cancer cells (MCF7) of the present invention showed similar genotypic and phenotypic characteristics of early and advanced cancers, respectively. Bioprinted solid organoids showed significantly higher hypoxic (>8-fold) and mesenchymal (>2-4-fold) marker expression, invasive activity (>15-fold), and drug resistance than bioprinted ductal organoguides. seemed Co-printing of conduit and solid organoids produced heterogeneous breast cancer tissue models that reproduced different stages of breast cancer histomorphology. Bioprinted cancer tissue organoids, representing advanced cancer stages, were more resistant to doxorubicin (an anthracycline antibiotic) and more resistant to human-like tirapazamine (a hypoxia-activating prodrug). tolerance was lower. Our results show that the spatial structure of cancer organoids has a significant effect on drug response, even though the model reproduces the same cancer stage. This study demonstrates that it is possible to bioprint cancer organoids that mimic the morphological pathology of human breast cancer to generate in vitro patient-specific breast cancer tissue representative of the patient's clinical cancer stage.

1 shows schematic (a) and image (b) of the bioprinter for preparing cancer organoids of the present invention.
Figure 2 is a schematic diagram of the production of ductal cancer and solid cancer organoids for breast cancer models; (a) Printed images of cancer organoids, (b) Representative tissue images of human breast invasive ductal epithelial carcinoma (H&E staining), (c) Illustration depicting production of ductal carcinoma and solid carcinoma organoids.
Figure 3 shows fluorescence microscopy images (scale bar, 200 μm) of bioprinted cancer organoids with various MCF7 concentrations in day 4 bio-ink.
Figure 4 is a diagram showing the effect of collagen concentration on the induction of solid tumor organoids; ( a ) Immuno and H&E staining results of bioprinted solid organoids with various collagen type 1 concentrations in day 4 bio-ink (scale bar, 200 μm). Immunotherapy was performed with COL1 and Hoechst to identify collagen and nuclei, respectively, in the organoids. ( b ) Corresponding cell density in printed solid organoids (n = 4; *P < 0.05).
5 is a graph showing the relative mRNA expression level of the SNAIL gene for cell concentration and presence (+)/absence (-) collagen type I (Col I) in bio-ink (n = 3; *P < 0.05, * *P < 0.005, and ***P < 0.001).
Figure 6 depicts bioprinted ductal carcinoma/solid carcinoma organoids; (a) Bright-field and fluorescence microscopy images of bioprinted and stained ductal carcinoma/solid carcinoma organoids at day 4 (live/cadaver stained (green, live; red, cadaveric cells) and immunosuppressed (green, HIF) -1a, hypoxic marker; blue, DAPI) (scale bar, 100 μm (b)) (b) Cell viability at day 4 after printing (n = 5) (c) Relative mRNA expression of HIF1A and HIF2A genes (n = 3) (df) Measured cell area (d), nuclear area (e), and nuclear-cytoplasmic ratio of ductal/solid organoids (n = 20).(*P < 0.05, ***P < 0.001 )
7 is a graph showing the relative mRNA expression (epithelial tissue to mesenchymal transition) amount of EMT genes (E-CAD, SNAIL, VIM, N-CAD, and SLUG) of ductal cancer/solid cancer organoids (n = 3; *P < 0.05, **P < 0.005, ***P < 0.001).
Figure 8 shows the results of three-dimensional invasion analysis using breast cancer organoids; (a) Bright-field microscopy images of ductal/solid breast cancer organoids in collagen/alginate hydrogel matrices at day 1 and day 10 after printing (scale bar, 200 μm). White arrowheads point to collective cell invasion. (b) Invaded area quantified at day 10 (n = 4; *P < 0.05).
Figure 9 shows the results of drug resistance measurement of breast cancer organoids; (A) Relative cell proliferation of ductal/solid breast cancer organoids after treatment with doxorubicin (n = 4; ***P < 0.001). Data are normalized across DMSO-treated controls. (b) Relative mRNA expression of specific ABC (ATP binding cassette) transporter genes (ABCB1, ABCC1, ABCC2, and ABCG2) (n = 3; *P <0.05; ***P < 0.001).
10 is an experimental result showing drug resistance of a breast cancer model having a change in the ratio of ductal carcinoma organoids to solid organoids; (a,b) Bright-field microscopy images (a) and corresponding relative cell proliferation of a breast cancer model consisting of ductal carcinoma and solid carcinoma organoids 7 days after treatment with doxorubicin (n = 4) (b). The proportion of ductal and solid organoids in the model was varied through the bioprinting process. Proliferation data are normalized to DMSO-treated control. Black and white arrowheads indicate ductal organoids and solid organoids, respectively. (**P <0.005; ***P < 0.001)
Figure 11 is a breast cancer model with morphological heterogeneity and bioprinting of their drug response. (a) Bright-field microscopic images of bioprinted breast cancer models (scale bar, 1 mm). Groups 1, 2b and 3 reproduced the morphology of cancer with grades 1, 2 and 3, respectively. Group 2a is a model with the same ductal/solid organoid ratio as group 2b, but with a different pattern. Black and white arrowheads indicate ductal and solid organoids, respectively. (b,c) Relative cell proliferation in bioprinted cancer models treated with doxorubicin (b) or tirapazamine (c) (n = 5, *P < 0.05, **P < 0.005, * **P < 0.001). Proliferation data are normalized to DMSO-treated control. (d) Procedures for designing and printing breast cancer models with heterogeneous patient cancer morphologies: (i) histology of human breast (H&E staining) invasive ductal epithelial carcinoma, (ii) localization results of ductal and solid organoids, (iii) bright-image of the bioprinted breast cancer model. In image (ii), circles in red and blue lines represent solid and ductal organoids, respectively. In image (iii), red and blue dotted lines represent bioprinted solid and ductal organoids, respectively.

In this specification, when a member is said to be located “on” another member, this includes not only the case where a member is in contact with another member, but also the case where another member exists between the two members.

In this specification, when a part is said to "include" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

According to one embodiment of the present invention, extruding a matrix ink containing a collagen solution and an alginate-hyaluronic acid solution, and extruding bioink containing cells, hyaluronic acid and gelatin on the matrix ink, A method for manufacturing cancer organoids using 3D printing is provided.

Cancer organoids prepared by the above method can precisely mimic the cell microstructure of heterogeneous cancer tissue of a patient, which produces a drug response similar to that of a human. Therefore, a bioprinted process for generating cancer aggregates (organoids) with ductal and solid tissue microstructures reflecting the morphological reproduction of breast cancer tissue was established and applied to develop breast cancer models.

The bio-ink may further include collagen, but is not limited thereto. Collagen concentration of the bio-ink may be added in the range of 0 to 1% v/v. As the collagen concentration of the bioink increased, the organoid cell density increased, peaked at 0.6% v/v (3,600 cells/mm ² ), and then decreased. In addition, it was confirmed that the expression of intracellular SNAIL increased as the concentration of collagen in the bioink increased.

After the bio-ink extrusion step, a step of crosslinking by treatment with a calcium ion solution may be further included, but is not limited thereto. The calcium ion is calcium hypochlorite, calcium perchlorate, calcium bromide, calcium iodide, calcium nitrate, calcium chloride acetic acid It may be selected from the group consisting of calcium acetate and mixtures thereof, but is not limited thereto.

The collagen solution may be prepared by dissolving pepsin and collagen in a hydrochloric acid solution, but is not limited thereto.

The alginate-hyaluronic acid solution may be prepared by dissolving alginate and hyaluronic acid in an animal cell culture medium, but is not limited thereto.

The animal cell culture medium is MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI 1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE), Keratinocyte-SFN (with BPE), KnockOut It may be selected from D-MEM, AmnioMAX-II Complete Medium and AmnioMAX-C100 Complete Medium, but is not limited thereto.

The matrix ink may be a mixture of a collagen solution and an alginate-hyaluronic acid solution in a weight ratio of 1 to 5:1, but is not limited thereto.

The cells may be mixed at a concentration of 1×10 ⁷ to 20×10 ⁷ cells/mL, but is not limited thereto. As the concentration of the cells decreased, ductal organoids were formed, and as the concentration of cells increased, solid organoids were formed. In addition, it was confirmed that intracellular SNAIL expression increased as the cell concentration increased.

In the bioink, hyaluronic acid may be mixed at a concentration of 1 to 5 mg/mL, but is not limited thereto. The hyaluronic acid serves to increase viscosity in order to homogeneously distribute and maintain cells during printing.

In the bioink, gelatin may be mixed at a concentration of 15 to 25% (w/v), but is not limited thereto. Gelatin is included in both the matrix ink and the bioink, and the gelatin commonly included in the ink is used to temporarily thermally crosslink the printed structure until irreversible crosslinking immediately after printing to maintain the structure. Alginate in the matrix ink is added to permanent cross-linking through calcium in the temporary cross-linking of gelatin. Ultimately both gelatin and alginate are added to maintain the print structure. Hyaluronic acid and gelatin, which are not cross-linked by calcium, have the property of dissolving in a culture medium at 37° C., so they are naturally removed.

The matrix ink may be extruded at a speed of 0.5 to 0.9 μL/s and a printing speed of 100 to 300 mm/min, but is not limited thereto.

The bioink may be extruded at a rate of 0.005 to 0.02 μL/s and printed in a dot form, but is not limited thereto.

The cells are cancer cells, preferably breast cancer cells, specifically, the breast cancer cells are MCF7 cells.

According to another embodiment of the present invention, a cancer organoid prepared by the above production method is provided. Bioprinted cancer tissue organoids, representing advanced cancer stages, were more resistant to doxorubicin (an anthracycline antibiotic) and more resistant to human-like tirapazamine (a hypoxia-activating prodrug). tolerance was lower.

Hereinafter, the present invention will be described in detail through examples. Since the embodiments described in this specification and the configurations shown in the drawings are only one of the most preferred embodiments of the present invention and do not represent all of the technical spirit of the present invention, various equivalents that can replace them at the time of this application It should be understood that there may be variations and examples.

<Example>

1. Cell culture

MCF7 cells (ATCC, Manassas, VA, USA) were grown in an incubator maintained at 37° C. and 5% CO ₂ with fetal bovine serum (10% v/v, Henne, Germany, Capricorn) and penicillin-streptomycin ( 1% v/v, P/S, Capricorn, Henne, Germany) supplemented with RPMI 1640 (LM 0131-03, Welgene, Gyeongsan, Korea). Cultures were exchanged every 2-3 days. MCF7 cells were subcultured to 70% confluency by dissociation with 0.25% trypsin EDTA solution (Thermo Fisher Scientific, Waltham, MA, USA).

2. Ink preparation for bioprinting

A matrix ink and two bio-inks for ductal and solid organoids were prepared for printing an in vitro breast cancer model. The matrix ink consisted of collagen (Dalim Tissen, Seoul, Korea), alginate (Sigma, St. Louis, PA, USA) and hyaluronic acid (HA, Sigma).

Collagen solution (2% w/v) was incubated in 0.01 N hydrochloric acid solution (diluted in phosphate-buffered saline (PBS), Sigma) supplemented with pepsin (2.27 mg/mL, Sigma) for 72 h at 18°C. prepared by dissolving collagen.

Alginate-HA solution dissolved alginate (4% w/v) and HA (6 mg/mL) overnight at a temperature of 37°C in modified Eagle's minimum essential broth (MEM, Gibco, Wiltham, MA, USA). made by making

The matrix ink was prepared by mixing collagen and alginate-HA solution in a weight ratio of 3:1.

Bio-ink for ductal cancer and solid cancer organoids were composed of gelatin, HA, MCF7 cells or collagen. HA (3 mg/mL, Sigma) was dissolved in MEM overnight at 37°C. Gelatin (22.5 mg/mL for ductal organoids, 27.5 mg/mL for solid organoids, Sigma) was then dissolved in the HA-MEM solution at 37°C for 2 hours. These materials were sterile-filtered (0.45 μm filter, Millipore, Danvers, MA, USA).

To prepare bio-inks for conduit and solid organoids, prior to bioprinting, each gelatin-HA mixture was mixed homogeneously with cell and/or collagen solutions to the final concentrations specified in Table 1.

category MCF7 cells Collagen type I HA gelatin ductal cancer 3Х10 ⁷ cells/mL 0% w/v 3 mg/mL 22.5% w/v solid cancer 15Х10 ⁷ cells/mL 0.6% w/v 2.1 mg/mL 19.3% w/v

The cell-laden bio-ink was filled into a 1 mL syringe (Henke Sas Wolf, Nürten-Hartenberg, Germany) and cooled in a 4° C. refrigerator for 8 minutes for gelation. Then, the syringe was connected to a micronozzle with an inner diameter of 300 μm (Mitsumi Electric Co., Ltd., Tokyo, Japan) for matrix ink and a micronozzle with an inner diameter of 120 μm for bio-ink. The prepared bioink was installed in a bioprinting system (FIG. 1).

3. 3D bioprinting system and process for breast cancer model production

A custom-designed bioprinting system (Kang et al.) was utilized to create a breast cancer model. The printing system consists of a three-axis stage system, a multi-cartridge dispensing module, and an enclosure that helps maintain temperature, humidity and cleanliness.

To print cancer organoids, the previously reported spheroid printing process (Jeon et al.) was applied. A 3D hydrogel matrix with a size of 5 Х 5 Х 0.6 mm ³ was printed using matrix ink at 18 °C (Fig. 2).

The matrix bio-ink was extruded at a speed of 0.69 μL/s and a printing speed of 200 mm/min. Thereafter, the bio-ink for ducts and solid organoids was extruded onto the matrix ink in a predefined pattern. The organoids were extruded at a rate of 0.01 μL/s for 5 seconds and printed in a dot form. After the printing process, the structures were first cross-linked in a 37° C. incubator for 30 minutes and then cross-linked with a 40 mM CaCl ₂ solution (dissolved in 0.9% w/v saline, Sigma) for 15 minutes.

The interconnected structures were incubated in a culture medium at 37° C. and 5% CO ₂ to remove sacrificial HA and gelatin from the printed bio-ink.

4. Cell viability assay

To measure the cell viability of the printed cancer organoids, live and dead staining (L3224, Thermo Fisher Scientific) was performed on day 4. Models were incubated in staining solution (calcein AM and ethidium homodimer-1 (2 μM and 4 μM, Therm Fisher Scientific) in PBS) for 40 minutes at room temperature. After washing with PBS, the samples were photographed using an FV-1000SPD confocal laser scanning microscope (Olympus; Tokyo, Japan). After counting live and dead cells, the cell viability was calculated by dividing the number of live cells by the total number of cells.

5. Histological analysis

To analyze the histology of the printed breast cancer models, the models were fixed overnight in 4% v/v formaldehyde solution (diluted in PBS; Junsei, Tokyo, Japan) on day 4. After tissue processing and paraffin embedding, the models were cut to 7 μm thickness using a microtome. For each cut slide, hematoxylin and eosin (H&E, Sigma) staining was performed according to the manufacturer's manual for histological analysis of tissue. Slides were incubated for 7 minutes in hematoxylin solution and 2 minutes in eosin solution for staining and then covered with coverslips (DURAN Group GmbH, Wertheim, Germany). Stained specimens were observed through a microscope (Leica Microsystems AG). Nuclear and cytoplasmic areas of MCF7 cells in ductal carcinoma and solid carcinoma organoids were measured using ImageJ software (NIH). The nuclear-cytoplasmic ratio (N:C ratio) was calculated by dividing the nuclear area by the total cell area.

6. Immunostaining

The cut slides were immunostained to observe the expression of hypoxia-inducible factor 1 alpha (HIF1A) in MCF7 cells. Briefly, the cut slides were washed twice in xylene (Samchun chemical, Seoul, Korea) and then rehydrated stepwise in ethanol with PBS at concentrations of 100, 90, 80, and 70% v/v. ). The rehydrated specimens were blocked with 5% w/v bovine serum albumin (Sigma) in PBS for 1 hour, followed by HIF1A antibody (1:50 dilution ratio, Novus Biologicals, Litten, CO, USA) in PBS overnight at 4°C. ) was cultured. After washing, the samples were incubated in Goat anti-mouse lgG(H+L) Alexa Floor 488 (1:500 dilution, Thermo Fisher Scientific) in PBS. After further washing, the samples are stained with Hoechst 33258 (1:1000 dilution, Sigma) in PBS. Stained specimens were observed using a fluorescence microscope (Leica DM2500, Leica Microsystems AG).

7. 3D invasion assay

To evaluate the invasion of MCF7 cells in the 3D matrix, ductal and solid organoids printed on days 1 and 10 were observed using a microscope (Leica Microsystems AG). To quantitatively assess cell invasion, the total area of organoids on day 1 and day 10 was measured using ImageJ software (NIH). The invasion area (%) was calculated using Equation 1:

[Equation 1]

8. Measurement of cancer drug response

To evaluate the drug response of the breast cancer model, the printed model was cultured in culture medium for 1 day in 24 well-plates (Corning, Inc., Corning, USA). For drug treatment, doxorubicin (HY-15142, NJ, USA, MedChemExpress, Monmouth Junction) and tirapazamine (Sigma) were dissolved in dimethyl sulfoxide (DMSO, Sigma). On day 1, cultures were removed from the wells. Metabolic activity of the specimen was measured by adding 500 μL of alamarBlue solution (10% v/v, diluted in growth medium, Thermo Fisher Scientific). Cultures containing alamarBlue dye (10% v/v) were used as blank samples. After incubation in the alamarBlue solution for 3 hours, 100 μL of the assay solution was taken from each container. Their fluorescence intensities were measured using a microplate reader (Synergy NEO2 hybrid multi-mode reader; Winooski, Bio-Tech, VT, USA). After removing the residual alamarBlue solution from each vessel, culture medium containing 1 μM doxorubicin was added. A control group was added together with the cultures containing the same amount of DMSO. Six days after drug treatment, the metabolic activity of the cancer model was measured again by the method described above. Relative cell proliferation was calculated using Equation 2:

[Equation 2]

Similar to doxorubicin treatment, the relative cell proliferation of each model was calculated in the same manner for 3 days after treatment with 100 μM tirapazamine.

9. RT-qPCR

In each cancer organoid, messenger ribonucleic acid (mRNA) expression associated with hypoxia, epithelial-to-mesenchymal transition (EMT) and drug efflux was measured. Each sample was separated using a bioMasher-II (Optima, Tokyo, Japan). The samples were incubated in TriSure solution (Bioline, London, UK) for 10 minutes, and RNA was isolated by vigorous vortexing. Helixcript?? to synthesize cDNA corresponding to each RNA sample. Thermo Reversed Transcriptase (Nanohelix, Daejeon, Korea) was used. cDNA was synthesized using a Biometra Professional TRIO Thermo-cycler (Analytik Jena AG, Jena, Germany). cDNA mixed with primers (Table S2) was amplified in a light cycler 480 ± (Roche Diagnostics Gmbh) to 480 SYBR Green ° (Roche Diagnostics Gmbh, Mannheim, Germany) with a Roche SYBR light cycler. Each mRNA expression level was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the ΔΔCt method.

10. Statistical analysis studies

Results are presented as mean ± standard error of the mean. Statistical analysis was performed using Origin (2020 version, OriginLab Corp., MA, USA). Multiple comparisons between test groups were performed using one-way analysis of changes followed by Tukey's multiple comparison test. For all analyses, p < 0.05 indicates statistical significance.

<Experiment result>

3.1. Bioprinting of ductal and solid tumor organoids

The effect of cancer cell concentration on conduit/solid microstructure printing was investigated. At a cell concentration of 3×10 ⁷ cells/mL, MCF7 cells adhered to the interface between the bio-ink and the surrounding matrix to form conduit microstructures (FIG. 3). As the concentration of MCF7 cells in the bio-ink increased, the ductal size of cancer organoids decreased, and solid organoids were formed at a cell concentration of 15Х10 ⁷ cells/mL.

Cancer cells were densely packed with native solid tumors. In this regard, the effect of collagen concentration on the cell density of printed solid tumor organoids was evaluated. Collagen fibers were evenly distributed in the printed solid organoids (Fig. 4(a)). As the collagen concentration of the bio-ink increased, the organoid cell density increased, peaked, and then decreased (Fig. 4(b)). The highest cell density (about 3,600 cells/mm ² ) was observed at a collagen concentration of 0.6% w/v, which was about 20% higher than that of the 0% w/v collagen group. In addition, the present inventors investigated the effect of changes in cell and collagen concentration on the expression of SNAIL, one of the representative indicators of ongoing cancer, through RT-qPCR. As a result, it was shown that the increase in cell and collagen concentrations of bio-ink increased the expression level of SNAIL (FIG. 5). SNAIL expression in the group with 0.6% v/v collagen concentration was 1.3 to 1.4 times higher than that in the group without collagen, regardless of cell concentration. The group with a cell concentration of 15Х10 ⁷ cells/mL showed 1.4-1.5 times higher SNAIL expression than the 3Х10 ⁷ cells/mL group. Based on these results, the composition of bio-ink for printing ductal cancer and solid organoids was determined (FIG. 1).

Conduit/solid organoids were bioprinted with engineered bio-ink. In the printed conduit organoids, conduits surrounded by one to three layers of MCF7 cells were observed, and dense cores appeared in the solid organoids (Fig. 6(a)). The viability of MCF7 cells in the printed organoids was over 95% (Fig. 6(b)). Expression of the hypoxic marker (HIF1A) was investigated via immunosuppression. In ductal carcinoma organoids, the HIF1A marker was hardly expressed, whereas in solid oronoids, strong expression was observed throughout the cell structure (Fig. 6(a)). This trend was more evident in the RT-qPCR results. The expression of HIF1A and HIF2A mRNA in solid organoids was about 10-fold and 8-fold higher than that of ductal organoids, respectively, and statistical significance was also observed (Fig. 6(c)).

To quantitatively evaluate the cellular morphology of cancer organoids, cell and nuclear areas were measured. Conduit organoids had approximately 64% higher average cell area and approximately 17% lower average nuclear area than solid organoids (Fig. 6(d),(e)). Statistical significance was only observed for cell area data. In the N:C ratio calculated based on the above measured results, solid organoids were about 2 times higher than ductal organoids (Fig. 6(f)). Interestingly, the measured N:C ratio of bioprinted cancer organoids (approximately 0.31/0.65 for ductal/solid organoids) was similar to that of native human breast cancer (approximately 0.4-0.8). As a result, it was confirmed that Oroguided printing of solid tumors induced a hypoxic environment and increased the N:C ratio of MCF7 cells.

2. EMT-related gene expression and 3D invasion assay

EMT-related gene expression of bioprinted cancer organoids was investigated via RT-qPCR (FIG. 7). Although the cells had the same morphology, E-CAD gene expression (an epithelial marker) was more than doubled in ductal organoids compared to solid organoids. On the other hand, the expression of the mesenchymal markers SNAIL, VIM, N-CAD, and SLUG genes was about 2-4 times higher in solid organoids than in ductal organoids. Statistical significance was also observed for all differences.

EMT of cancer cells weakens cell-cell adhesion and promotes invasion and metastasis. We performed a 3D invasion assay on cancer organoids printed on a 3D collagen/alginate hydrogel matrix. Ten days after printing, little invasion occurred in ductal organoids, whereas active collective cell invasion was observed in solid organoids (Fig. 8(b)). The invasion area of solid organoids was about 15 times higher than that of ductal organoids (Fig. 8(b)). These results show that solid cancer organoids showed higher EMT-related gene expression and invasion characteristics similar to advanced stage cancers than ductal cancer organoids.

3. Drug Resistance of Cancer Organoids

The resistance of ductal cancer/solid cancer organoids to doxorubicin, a representative breast cancer chemotherapy agent, was investigated. To this end, relative cell proliferation of bioprinted organoids was measured after treatment with doxorubicin for 6 days. Solid organoids showed about 92% relative cell proliferation, whereas ductal organoids only about 10% (Fig. 9(a)). These results indicate that there is little growth inhibition of solid tumor organoids by doxorubicin. Similar results were observed for drug-efflux related gene expression levels measured via RT-qPCR. The mRNA expression of ATP-binding cassette (ABC) transporter genes (ABCB1, ABCC1, ABCC2 and ABCG2; drug-efflux related genes) in solid organoids was about 6-60 times greater than that in ductal organoids. times higher (FIG. 9(b))). These results showed that solid organoids were more resistant to doxorubicin than ductal organoids.

4. Effect of simultaneous printing of ductal and solid organoids on drug response

As native breast cancer progresses, the proportion of ductal microstructures decreases, while the proportion of solid microstructures increases. In this study, considering the morphological characteristics of native breast cancer, breast cancer models with various ratios between ductal cancer and solid organoids were prepared (FIG. 10(a)). Then, resistance to doxorubicin was evaluated. As the proportion of solid organoids increased from 0% to 100%, the relative cell proliferation of the cancer model also increased from 9% to 93%. There were statistically significant differences between each group. This result confirmed that doxorubicin resistance increased as the proportion of solid organoids increased in cancer models.

5. Bioprinting of a breast cancer model that mimics the morphology of a patient's cancer tissue

The present inventors prepared four breast cancer models by co-printing ductal cancer and solid organoids (FIG. 11(a)). Groups 1, 2b, and 3 were created considering the median proportion of ductal microstructure in grade 1, 2, and 3 breast cancer tissues. The solid microstructure proportions of groups 1, 2b, and 3 were 8%, 45%, and 88%, respectively. Group 2a had the same number of ductal/spherical organoids as group 2b, but was designed to have a different pattern. As a result of doxorubicin treatment for 6 days, the relative cell proliferation decreased as the ratio of ductal organoids increased, similar to FIG. 10 (groups 1, 2b, and 3 in FIG. 11(b)). Interestingly, group 2b with a mixed pattern showed a higher relative cell proliferation than group 2a with a separate pattern (groups 2a and 2b in FIG. 11(b)). Statistical significance was also observed between each group. On the other hand, the opposite trend was observed in the tirapazamine experiment (FIG. 11(c)). Relative cell proliferation decreased in the order of groups 1, 2a, 2b, and 3. These results indicated that the ratio between ductal and solid microstructure and morphology has a significant impact on the drug response of cancer models.

Finally, the present inventors investigated whether a cancer model having a heterogeneous patient's cancer type could be created using the developed printing technology. In this study, the location and size of ducts/solid microstructures in breast cancer tissue were measured using ImageJ software based on histological data of human breast cancer (Fig. 11(d)-(i)). Based on the measurement results, a biomimetic cancer model was designed and printed (FIG. 11(d)-(ii),(iii)). As a result, it was confirmed that the developed technology could produce a patient-specific in vitro breast cancer model. Although design was performed manually in this study, an automated design process is planned as part of future work.

Claims (14)

extruding a matrix ink comprising a collagen solution and an alginate-hyaluronic acid solution; and
A method for producing cancer organoids using 3D printing, comprising extruding bioink containing cells, hyaluronic acid, and gelatin on the matrix ink.
According to claim 1,
The method of manufacturing cancer organoids using 3D printing, wherein the bioink further comprises collagen. According to claim 1,
A method for producing cancer organoids using 3D printing, further comprising crosslinking by treating a calcium ion solution after the bioink extrusion step.
According to claim 1,
The method for producing cancer organoids using 3D printing, characterized in that the collagen solution is prepared by dissolving pepsin and collagen in a hydrochloric acid solution.
According to claim 1,
The alginate-hyaluronic acid solution is characterized in that prepared by dissolving alginate and hyaluronic acid in an animal cell culture medium, cancer organoid production method using 3D printing.
According to claim 5,
The animal cell culture medium is MEM (Minimum Essential Media), DMEM (Dulbecco's Modified Eagle Media), RPMI 1640, IMDM (Iscove's Modified Dulbecco's Media), Defined Keratinocyte-SFM (without BPE), Keratinocyte-SFN (with BPE), KnockOut A method for manufacturing cancer organoids using 3D printing, characterized in that selected from D-MEM, AmnioMAX-II Complete Medium and AmnioMAX-C100 Complete Medium.
According to claim 1,
The matrix ink is a cancer organoid manufacturing method using 3D printing, characterized in that the collagen solution and the alginate-hyaluronic acid solution are mixed in a weight ratio of 1 to 5: 1.
According to claim 1,
The method of manufacturing cancer organoids using 3D printing, characterized in that the cells are mixed at a concentration of 1×10 ⁷ to 20×10 ⁷ cells/mL.
According to claim 1,
In the bioink, hyaluronic acid is mixed at a concentration of 1 to 5 mg / mL, characterized in that, cancer organoid manufacturing method using 3D printing.
According to claim 1,
In the bioink, gelatin is mixed at a concentration of 15 to 25% (w / v), characterized in that, cancer organoid manufacturing method using 3D printing.
According to claim 1,
The method for producing cancer organoids using 3D printing, characterized in that the matrix ink is extruded at a speed of 0.5 to 0.9 μL / s and a printing speed of 100 to 300 mm / min.
According to claim 1,
The bioink is extruded at a speed of 0.005 ~ 0.02 μL / s and printed in a dot form, characterized in that, cancer organoid manufacturing method using 3D printing.
According to claim 1,
The method of manufacturing cancer organoids using 3D printing, characterized in that the cells are cancer cells.
A cancer organoid prepared by the method of any one of claims 1 to 13.

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