3D mammary gland printing method based on HAMA fat cell microspheres and mammary gland organs

Abstract

The invention provides a 3D mammary gland printing method based on HAMA fat cell microspheres and mammary gland organs, relates to the technical field of biomedicine, and aims to solve the problems of incomplete structural function simulation, low cell survival rate, uneven distribution and the like in the prior art. According to the core technical scheme, HAMA fat cell microspheres serve as fat layer function units, mammary gland organs serve as gland layer units, and precise positioning of a double-layer structure is achieved through a double-nozzle 3D printer; the survival rate of adipose cells is improved by regulating and controlling the crosslinking degree of HAMA and the porosity of the microspheres; special bio-ink and optimized printing parameters are adopted, so that the agglomeration rate of a functional unit is smaller than or equal to 5%, and a cross-linking transition layer is arranged on an interface to promote structural connection; by adding gelatin and adopting a dual-crosslinking process, the compression modulus of the model reaches 10-15kPa, the degradation period is 3-6 months, and the model has good mechanical properties and biocompatibility. The application effectively simulates the physiological microenvironment of the mammary gland, and has important application value in the fields of tissue engineering reconstruction, drug screening, disease research and the like.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for 3D printing breast tissue based on HAMA adipocyte microspheres and breast organoids. Background Technology

[0002] In the field of 3D printing breast technology, existing technologies mainly focus on simulating the structure and function of breast tissue, and can be divided into two key approaches. The first approach is based on HAMA (methacryloyl hyaluronic acid) and single breast organoids for 3D printing. This technology uses HAMA as a bio-ink matrix, combined with in vitro cultured breast organoids (functional units containing alveolar structures), and constructs the model through extrusion-based photopolymerization 3D printing. It aims to simulate the breast glandular layer using the natural functional characteristics of organoids, but completely lacks fat support. The second approach is based on synthetic materials (such as PCL, PLGA) or simple natural materials (such as gelatin, sodium alginate) and dispersed fat cells for 3D printing breast scaffolds. This technology prepares porous scaffolds through melt deposition or extrusion molding and inoculates dispersed fat cells to simulate the breast fat layer. It is mainly used in breast reconstruction scenarios, but neglects the integration of glandular functional units. Both technologies attempt to address some structural or functional issues in breast models, but neither achieves the synergistic simulation of fat and glandular tissue.

[0003] Existing technologies have significant drawbacks that limit the realism and practicality of breast tissue models. For technologies based on HAMA and single breast organoids, the main deficiency is the lack of adipose tissue support, leading to a mismatch between the model structure and the physiological microenvironment. Experimental data shows that the alveolar maturation rate is only 35%, far lower than in vivo levels, and the organoids are unevenly distributed (aggregation rate approximately 25%), failing to form a continuous ductal network and exhibiting poor functional synergy. For technologies based on synthetic materials / simple natural materials and dispersed adipocytes, the drawbacks are even more pronounced: low adipocyte survival rate (only 40% after 7 days post-inoculation), poor colonization efficiency, and inability to form a functional fat layer; insufficient material compatibility, such as the long degradation cycle of synthetic materials which easily triggers foreign body reactions, and the poor mechanical strength of simple natural materials leading to scaffold collapse; furthermore, this technology lacks glandular functional units, failing to simulate core functions such as breast secretion or hormone response, and the structural stratification is inaccurate, with a similarity to real breast tissue differing by more than 50%, making it difficult to meet the needs of precision medicine. Summary of the Invention

[0004] The purpose of this invention is to solve the problem that existing technologies have failed to achieve the synergistic simulation of fat and glands.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids includes the following steps:

[0007] S1: HAMA adipocyte microspheres were prepared using the "emulsification-ultraviolet crosslinking method";

[0008] S2: Extraction and culture of mammary gland organoids;

[0009] S3: Preparation of bio-ink: The bio-ink includes mammary gland organoid and HAMA adipocyte microsphere ink;

[0010] S4: 3D printing of breast structure: Printing is performed using a dual-channel syringe. 10% HAMA hydrogel and adipocyte suspension are loaded into the syringe, respectively. HAMA adipocyte microsphere ink is then loaded into the syringe. The arrow of the dual-channel syringe points to the inlet of the microfluidic chip. A coaxial flow channel structure is marked inside the microfluidic chip, with the adipocyte suspension in the middle and the HAMA hydrogel on the outer layer. The outlet of the microfluidic chip is connected to a UV curing device. A collection bottle is located below the UV curing device to collect the microspheres. The printed fat layer and the extracted glandular layer are mixed in a matrix gel to allow the fat layer and glandular layer to bond.

[0011] S5: In vitro culture and performance testing of 3D printed breast models.

[0012] Preferably, the steps in S1 are as follows:

[0013] S1-1: Preparation of HAMA solution: Dissolve HAMA powder in sterile PBS, stir magnetically, prepare a 3% (w / v) HAMA solution and filter;

[0014] S1-2: Preparation of adipocyte suspension: After resuscitating human primary adipocytes (HADC), they were cultured in adipocyte culture medium, digested with trypsin, and then centrifuged.

[0015] S1-3: Emulsification and Mixing: Mix the HAMA solution of S1-1 with the adipocyte suspension of S1-2, add LAP, and stir magnetically until homogeneous; slowly drip the mixture into liquid paraffin containing Span 80, and ultrasonically emulsify to form a water-in-oil emulsion;

[0016] S1-4: Place the water-in-oil emulsion under a UV lamp to crosslink and solidify HAMA to form microspheres;

[0017] S1-5: Microsphere purification: Collect microspheres by centrifugation, wash with anhydrous ethanol, and finally resuspend in adipocyte culture medium.

[0018] Preferably, the preparation method of the HAMA adipocyte microsphere ink is as follows:

[0019] After mixing HAMA solution with LAP, add HAMA adipocyte microspheres and hyaluronidase inhibitor, gently pipetting (to avoid breaking the microspheres) until homogeneous, and store at 4°C for later use; the formulation ratio is 4% (w / v) HAMA solution + HAMA adipocyte microspheres (5×10⁻⁶). 4 The HAMA adipocyte microsphere ink has a viscosity of 3500-4000 cP (25℃), with microspheres uniformly dispersed in the ink and an aggregation rate of only 4.8±1.2%.

[0020] Preferably, step S2 is as follows:

[0021] Autologous breast tissue was extracted, and the tissue block was transferred to a sterile culture dish and rinsed three times with sterile PBS (containing double antibodies).

[0022] Tissue dissociation and acquisition of single-cell suspension;

[0023] Cell counting and cell viability detection;

[0024] Organoid culture is performed, followed by organoid purification and identification to obtain qualified functional units.

[0025] Preferably, the following culture medium is used for organoid culture:

[0026] Element Final concentration DMEM / F12 medium basal culture medium Fetal bovine serum (FBS) 2% Epidermal growth factor (EGF) 20 ng / mL insulin 10 μg / mL R-spondin 1 500 ng / mL Noggin 100 ng / mL Double antibiotics (penicillin-streptomycin) 1%

[0027] .

[0028] Preferably, the 3D printing parameters are as follows:

[0029] Printing layer Extrusion pressure Printing speed Layer thickness Ultraviolet light irradiation time fat layer 7-9 kPa 6 mm / s 150 μm 20 s

[0030] .

[0031] Preferably, the specific steps in S5 are as follows:

[0032] S5-1: In vitro culture: The culture environment is 37℃, 5% CO2, and 95% humidity. The culture medium is changed every 2 days, and the culture cycle is 21 days.

[0033] S5-2: Key performance testing.

[0034] Preferably, the culture medium used for in vitro culture in S5-1 is a mixture of "mammary organoid culture medium + adipocyte culture medium", wherein the volume ratio of "mammary organoid culture medium + adipocyte culture medium" is 1:1, and 1% penicillin-streptomycin is added.

[0035] This application also provides a 3D-printed mammary gland based on HAMA adipocyte microspheres and mammary organoids, which is prepared using the preparation method described above.

[0036] Compared with the prior art, this application has at least the following beneficial effects:

[0037] 1. This application combines HAMA adipocyte microspheres (as a functional unit of the adipose layer) with mammary gland organoids (as a functional unit of the glandular layer) to form a "fat-gland" dual-functional unit system. HAMA adipocyte microspheres provide a three-dimensional growth microenvironment for adipocytes, while mammary gland organoids retain the specific functions of mammary tissue. The two achieve cross-unit synergistic regulation through cytokine signals (such as leptin and VEGF).

[0038] 2. In this application, microspheres encapsulating adipocytes are prepared by using HAMA as a carrier and through the "emulsification-crosslinking method". This solves the pain points of the prior art, namely "poor survival of adipocytes and inability to form a functional fat layer". Specifically, by adjusting the ultrasonic power and crosslinking time, the porosity of the microspheres (50%-60%) is matched with the growth requirements of adipocytes, avoiding the problems of "excessive pore size leading to cell loss and insufficient pore size affecting nutrient exchange" in the prior art.

[0039] 3. This application also employs a dual-nozzle 3D printer, loading "HAMA-specific ink for mammary organoids" and "HAMA-specific ink for fat cell microspheres" respectively, and printing layer by layer according to a preset three-dimensional mammary model (including the anatomical structure of the fat layer and glandular layer). The extrusion pressure of the glandular layer nozzle (diameter 200-250μm) is 6-8kPa, and the extrusion pressure of the fat layer nozzle (diameter 250-300μm) is 7-9kPa. The interface between the two layers is connected by a HAMA transition layer (with 0.5% collagen added), ultimately forming an integrated 3D printed mammary model. This solves the pain points of "uneven distribution of functional units and inaccurate structural layering" in existing technologies. Furthermore, the special ink formula and printing process are highly synergistic, ensuring the uniform distribution of functional units and structural integrity during the printing process. This is the core of achieving precise "fat-glandular" layering and should be considered a key protection point at the application level.

[0040] 4. In this application, the performance of the HAMA matrix is ​​also controlled by a dual process of ultraviolet light crosslinking and chemical crosslinking, so that the compression modulus of the 3D printed breast model reaches 10-15 kPa and the degradation cycle is controlled within 3-6 months, matching the mechanical properties and tissue repair cycle of normal breast tissue; specifically, the above-mentioned method of this application solves the pain points of "poor mechanical properties and mismatched degradation cycle" in the prior art. Attached Figure Description

[0041] Figure 1 Bright field image of HAMA microspheres;

[0042] Figure 2 To track the life and death of HAMA microspheres loaded with HUVEC cells;

[0043] Figure 3 For data statistics on whether a device is alive or dead;

[0044] Figure 4 It is a differentiated human-shaped mammary gland organoid;

[0045] Figure 5 CD31 markers for mammary organoids;

[0046] Figure 6 SMA markers for mammary organoids;

[0047] Figure 7 This is a diagram illustrating the printing of a simulation operation in one embodiment of this application;

[0048] Figure 8 Bright field image of a human autologous breast organoid combined with HAMA microspheres transplanted three months later;

[0049] Figure 9 Bright-field plot of differentiation of mouse mammary gland organoids;

[0050] Figure 10 For the detection of protein secretion in human mammary gland organoids;

[0051] Figure 11 Bright-field diagram of lipid droplet formation in human primary fat HAMA microspheres. Detailed Implementation

[0052] The present invention will be further described in detail below with reference to specific embodiments.

[0053] In this application, HAMA adipocyte microspheres (rather than dispersed adipocytes) and mammary organoids are used as the core functional units of the adipocyte and glandular layers, respectively. 3D printing technology is used to achieve precise spatial positioning of the two-layer structure, simulating the anatomical structure of the real mammary gland. At the same time, relying on the cytokines (such as leptin and VEGF) secreted by the adipocyte microspheres, physiological microenvironmental signals are provided to the mammary organoids, solving the problem of mismatch between the structure and microenvironment in the prior art, and improving the alveolar maturation rate and functional responsiveness of the mammary organoids.

[0054] Furthermore, adipocyte microspheres (200-300 μm in diameter) were constructed using HAMA as a carrier to provide a three-dimensional growth microenvironment for adipocytes, reducing mechanical damage and nutrient deprivation. By regulating the degree of cross-linking of HAMA (methacrylylation 60%-80%) and the porosity of the microspheres (50%-60%), the adhesion, proliferation, and maturation conditions of adipocytes were optimized, ensuring that the adipocyte survival rate was ≥70% and the maturation rate was ≥60% within 21 days after printing, forming a continuous and functional fat layer, thus solving the problems of poor adipocyte survival and inability to form a functional fat layer in existing technologies.

[0055] Furthermore, specialized bio-inks were designed for mammary organoids and HAMA adipocyte microspheres respectively (organoid ink: HAMA + mammary organoids + growth factors; adipocyte microsphere ink: HAMA adipocyte microspheres + nutrient slow-release particles). The printing parameters were precisely controlled using a dual-nozzle 3D printer (nozzle diameter 300 μm, extrusion pressure 6-9 kPa, layer thickness 150 μm) to ensure that the two types of functional units are evenly distributed within their respective layers (aggregation rate ≤5%). At the same time, a HAMA cross-linking transition layer was introduced at the interface between the two layers to promote signal transmission and structural connection between adipocyte microspheres and mammary organoids, forming an overall synergistic functional network and solving the problems of uneven unit distribution and poor connectivity in existing technologies.

[0056] This application also adjusts the composition of HAMA ink (such as adding 5%-10% gelatin to improve mechanical properties) and the cross-linking process (ultraviolet light cross-linking + chemical cross-linking dual curing) to achieve a compressive modulus of 10-15 kPa for the printed model (matching the mechanical properties of normal breast tissue), and controls the degradation cycle to 3-6 months (synchronized with the tissue repair or model function maintenance cycle). At the same time, relying on the excellent biocompatibility and degradability of HAMA, foreign body reactions are avoided, meeting the comprehensive needs of "structure-function-safety" for models in multiple scenarios such as personalized breast reconstruction, drug screening, and basic medical research.

[0057] The above content is elaborated below with specific verification experiments. A method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids, the steps of which are as follows:

[0058] I. Experimental materials, specifications, and sources:

[0059] Raw material name Specifications source Methacrylamide hyaluronic acid (HAMA) Molecular weight 800-1000 kDa, degree of methacrylation 60%-80% Shanghai Aladdin Biochemical Technology Co., Ltd. Human primary mammary epithelial cells (HBEC) Purity ≥ 95%, Viability ≥ 90% Wuhan Pronosei Life Science Technology Co., Ltd. Human primary adipocytes (HADC) Purity ≥ 90%, Viability ≥ 85% Shenzhen Cyagen Biotechnology Co., Ltd. Photoinitiator (LAP) Purity ≥ 98% Sigma-Aldrich, Inc. Mammary organoid culture medium Contains EGF (20 ng / mL) and insulin (10 μg / mL) STEMCELL Technologies, Inc. Adipocyte culture medium Contains FBS (10%) and dexamethasone (1 μmol / L) Gibco, USA

[0060] II. Experimental Procedure:

[0061] S1: Preparation of HAMA adipocyte microspheres: In this application, the "emulsification-ultraviolet crosslinking method" is used to encapsulate adipocytes with HAMA as a carrier. By adjusting the emulsification parameters and crosslinking time, microspheres with porous structures are constructed to provide a three-dimensional growth microenvironment for adipocytes.

[0062] In one embodiment, step S1 is as follows:

[0063] S1-1: Preparation of HAMA solution

[0064] Dissolve HAMA powder in sterile PBS (pH=7.4), stir magnetically (300 rpm, 25℃) for 24 h to prepare a 3% (w / v) HAMA solution, and filter it through a 0.22 μm filter membrane for sterilization.

[0065] S1-2: Preparation of adipocyte suspension

[0066] Human primary adipocytes (HADCs) were revived, cultured in adipocyte culture medium until the logarithmic growth phase, digested with trypsin, and centrifuged (1000 rpm, 5 min) to adjust the cell concentration to 1×10⁻⁶. 6 cells / mL;

[0067] S1-3: Emulsion Mixing

[0068] The HAMA solution prepared in S1-1 was mixed with the adipocyte suspension in S1-2 at a volume ratio of 3:1, and 0.1% (w / v) LAP was added. The mixture was magnetically stirred (200 rpm, 10 min) until homogeneous. The mixture was then slowly dripped into liquid paraffin containing 5% Span 80 (oil phase: water phase = 5:1) and ultrasonically emulsified (power 300 W, time 30 s, amplitude 50%) to form a water-in-oil emulsion.

[0069] S1-4: Ultraviolet crosslinking

[0070] The emulsion obtained in S1-3 was irradiated under a 405 nm UV lamp (10 cm away) for 20 s to allow HAMA to crosslink and solidify into microspheres.

[0071] S1-5: Microsphere purification

[0072] Collect the microspheres by centrifugation (1500 rpm, 10 min), wash three times with anhydrous ethanol (5 min each time) to remove residual liquid paraffin, and finally resuspend in adipocyte culture medium for later use.

[0073] Please see Figure 1 Observation under an optical microscope (Olympus CX43) revealed that the microspheres were spherical with smooth surfaces, a particle size distribution of 200-300 μm, an average particle size of 252.6±18.3 μm, and a particle size uniformity of 92%.

[0074] Using the Beyo3D™ 7-AAD cell viability assay kit, the endothelial cell viability within the microspheres was 87% at 1 day, 98% at 5 days, 99% at 15 days, and 99% at 25 days. Figure 2 The result was significantly higher than 40% of the existing technology (p<0.01).

[0075] S2: Extraction and culture of mammary gland organoids:

[0076] In one embodiment, breast organoids are extracted from the patient's own breast tissue, strictly following medical ethics and aseptic operation principles, and the entire process is completed in a GMP-level cell laboratory. The core objective is to efficiently obtain breast organoids with tissue-specific functions from a small amount of autologous tissue, providing functional units for subsequent 3D printing of breast tissue.

[0077] In one embodiment, the specific steps of S2 are as follows:

[0078] S2-1: Preoperative preparation stage

[0079] 1. Ethical approval and patient informed consent

[0080] (1) Submit an application to the hospital ethics committee for “autologous breast tissue for organoid culture”, specifying the purpose of the tissue (only for the patient’s own 3D printed breast construction), the amount extracted (≤5 g) and the risks (minor tissue damage, infection probability <0.5%), and obtain the ethics approval document (the approval document number needs to be archived).

[0081] (2) Inform the patient of the operation process, tissue extraction method, organoid culture cycle (7-10 days) and potential risks, and sign the "Autologous Breast Tissue Donation Informed Consent Form" to clarify the ownership and usage restrictions of the tissue (prohibited from being used for other research or by others).

[0082] 2. Material and Equipment Preparation

[0083] All sterile consumables must be autoclaved (121°C, 30 min) to avoid tissue contamination. In addition, the equipment used must be disinfected in advance (wiped with 75% ethanol and irradiated with ultraviolet light for 30 min).

[0084] S2-2: Autologous breast tissue extraction stage

[0085] 1. Organizational Extraction Scenarios and Methods

[0086] Depending on the patient's condition, tissue extraction is mainly divided into two scenarios, with slight differences in the operation methods:

[0087] (1) When performing breast surgery, such as breast-conserving surgery for breast cancer or resection of benign breast tumors, during the operation, the surgeon selects a non-lesion area from the removed breast tissue (confirmed by pathological frozen section to be free of cancer cells or abnormal cells) and cuts a tissue block with a volume of about 0.5 cm × 0.5 cm × 0.3 cm (weighing about 0.075 g) to avoid damaging the glandular structure;

[0088] (2) When there is no need for surgery, a minimally invasive extraction is performed. Specifically, under local anesthesia (2% lidocaine subcutaneous injection), a breast puncture needle (14 G) is used to puncture the upper outer quadrant of the breast and extract about 0.1-0.2 g of breast tissue (containing glandular components). After puncture, pressure is applied to stop bleeding for 5-10 minutes, and antibiotic ointment (such as mupirocin ointment) is applied to prevent infection.

[0089] 2. Organization of transfer and preliminary treatment

[0090] (1) Immediately place the extracted tissue block into pre-cooled sterile tissue preservation solution (the liquid level should cover the tissue), mark the patient information (name, ID number, extraction date, tissue location), and transport it to the laboratory with an ice pack (4°C). The transport time should be ≤2 hours to avoid tissue ischemia and necrosis.

[0091] The sterile tissue preservation solution contains penicillin (100 U / mL), streptomycin (100 μg / mL), and amphotericin B (2.5 μg / mL), and is pre-cooled at 4°C for tissue transport.

[0092] (2) In the biosafety cabinet, transfer the tissue block to a sterile culture dish and rinse it three times with sterile PBS (containing double antibiotics). Gently shake the culture dish for 1 minute each time to remove residual blood and tissue fluid until the rinsing solution is clear.

[0093] III. Tissue dissociation to obtain single-cell suspension

[0094] 1. Tissue mincing and enzymatic digestion

[0095] (1) Use sterile tissue scissors to cut the rinsed tissue block into 1 mm pieces. 3 The following fine powder should be used to avoid tangling of tissue fibers;

[0096] (2) Transfer the tissue fragments to a 50 mL centrifuge tube, add 5 mL of enzymatic hydrolysate, the formulation of which is: DMEM / F12 medium + 0.1% collagenase type I + 0.05% neutral protease + 2% FBS, and gently invert to mix; wherein the collagenase activity is ≥150 U / mg, and the FBS needs to be heat-inactivated (56℃, 30 min).

[0097] (3) Place the enzymatically digested tissue fragments obtained in the above steps in a 37°C, 5% CO2 incubator and shake to digest at a shaking speed of 50 rpm for 60-90 min. During this period, take out the centrifuge tube every 30 min and observe the dissociation under an inverted microscope. Stop digestion when most of the tissue fragments are dispersed into cell clusters or single cells.

[0098] 2. Termination of digestion and filtration purification

[0099] (1) Add 5 mL of DMEM / F12 medium containing 10% FBS to the centrifuge tube to terminate the enzymatic digestion reaction;

[0100] (2) Filter the digestion solution with a 70 μm sterile filter and collect the filtrate into a new 50 mL centrifuge tube to remove undigested tissue fragments; then filter the filtrate a second time with a 40 μm sterile filter to further remove cell clumps and retain a single mammary epithelial cell and a small number of basal cells.

[0101] (3) Centrifuge the cell suspension after secondary filtration (1000 rpm, 5 min, room temperature), discard the supernatant, add 2 mL of DMEM / F12 medium to resuspend the cells, and obtain a single cell suspension.

[0102] IV. Cell Counting and Viability Assay Stage

[0103] 1. Cell counting

[0104] (1) Take 10 μL of cell suspension and mix it with 10 μL of trypan blue staining solution (0.4%). Let it stand at room temperature for 3 min and then stain the dead cells with trypan blue staining solution.

[0105] (2) Add the mixture to a hemocytometer and count the cells under an inverted microscope. The formula for calculation is: Cell concentration (cells / mL) = (Total number of cells in 4 large squares / 4) × 10 4 ×Dilution factor (the dilution factor for this step is 2);

[0106] (3) Cell concentration ≥ 1 × 10 6 If the concentration is insufficient, it can be concentrated by centrifugation (1000 rpm, 5 min) to adjust to the target concentration.

[0107] 2. Cell viability assay

[0108] Procedure: Differentiate between live cells (clear, unstained) and dead cells (blue) on a hemocytometer, and calculate cell viability: Cell viability (%) = (number of live cells / total number of cells) × 100%;

[0109] Acceptable criteria: Cell viability ≥ 85%. If viability is < 85%, the cause needs to be investigated (such as excessive tissue digestion or excessive transport time), and the tissue should be re-extracted if necessary.

[0110] In one embodiment, flow cytometry is also required to further identify cell purity. Specifically, the markers CK18 (positive rate ≥90%) and CK14 (positive rate ≥5%) of mammary epithelial cells are detected to ensure that there is no contamination from other cells (such as fibroblasts and immune cells) (the proportion of contaminating cells <5%).

[0111] V. Organoid Culture Stage

[0112] 1. Matrix adhesive embedding

[0113] (1) Take the Matrigel matrix out of the -80℃ freezer and place it in the 4℃ freezer to thaw overnight to avoid repeated freeze-thaw cycles that could damage its activity. The entire operation should be carried out on ice to prevent the matrix from solidifying.

[0114] (2) Take 1×10 5 One qualified mammary gland cell was resuspended in DMEM / F12 medium to 100 μL, mixed with 100 μL of Matrigel matrix gel (concentration 10 mg / mL), and gently pipetted 3 times (avoiding the generation of air bubbles) to obtain a cell-matrix mixture.

[0115] (3) Drop the mixture into each well of a 24-well plate (200 μL per well) to form a "droplet" with a diameter of about 5 mm. Place the 24-well plate in a 37°C, 5% CO2 incubator and incubate for 30 min to allow Matrigel to solidify completely, thereby forming a solid scaffold to simulate the extracellular matrix in vivo.

[0116] 2. Addition and culture of organoid-specific culture medium

[0117] (1) Prepare a special culture medium for mammary gland organoids, filter it to remove bacteria (0.22 μm filter membrane) and set it aside for use;

[0118] The formula for the special culture medium is as follows:

[0119] Table 1: Formulation of Culture Medium Specific for Mammary Organoids

[0120] Element Final concentration DMEM / F12 medium basal culture medium Fetal bovine serum (FBS) 2% Epidermal growth factor (EGF) 20 ng / mL insulin 10 μg / mL R-spondin 1 500 ng / mL Noggin 100 ng / mL Double antibiotics (penicillin-streptomycin) 1%

[0121] (2) After Matrigel solidifies, slowly add 500 μL of special culture medium to each well and incubate in a CO2 incubator;

[0122] (3) During the culture process, the culture medium is changed every 2 days. When removing the old culture medium, avoid touching the gel droplets. On the 3rd day, observe the organoid formation under an inverted microscope. Cells can be seen to aggregate into small clusters and begin to form hollow structures. On the 7th-10th day, they reach maturity and form organoids with acinar-like structures.

[0123] VI. Organoid purification and identification stage, obtaining qualified functional units.

[0124] 1. Organoid purification

[0125] (1) After culturing for 7-10 days, rinse the wells with sterile PBS twice, add 200 μL of cell recovery solution (containing 0.5 mg / mL dispase enzyme), and incubate at 37°C for 15 min to degrade Matrigel;

[0126] (2) Gently pipette the droplets to release the organoids from the matrix gel, transfer them to a 15 mL centrifuge tube, add 3 mL of DMEM / F12 medium to dilute, and centrifuge (800 rpm, 5 min, room temperature).

[0127] (3) Discard the supernatant, add 2 mL of DMEM / F12 medium to resuspend, filter with a 40 μm sterile filter to remove residual matrix gel fragments and single cells, collect the organoids on the filter, wash twice with PBS to obtain purified mammary gland organoids.

[0128] 2. Organoid quality assessment

[0129] Quality assessment includes morphological assessment and assessment of functional markers.

[0130] Please see Figure 3 Under an inverted microscope, qualified organoids must meet the following criteria: diameter 50-100 μm, spherical or elliptical shape, and internal formation of hollow acini-like structures. Figure 3 Organoids ≥ 1×10 4 One (obtained from 0.1 g of tissue);

[0131] In addition, please see Figures 4-6 Immunofluorescence staining was used to detect the breast epithelial cell marker CK8 (positive rate ≥90%, demonstrating organoid epithelial cell characteristics). Figure 4 ); CD31 in vascular endothelial cells of human breast organoids (positive rate ≥80%, proving that the organoids have vascularization ability) Figure 5 ); Functional marker SMA of myoepithelial / mesenchymal cells in human breast organoids (positive rate ≥80%, ensuring the organoids have contractile function) Figure 6 )

[0132] S3: Preparation of specialized bio-ink.

[0133] The bio-ink comprises mammary gland organoids (glandular layer) and HAMA adipocyte microspheres (adipocyte layer), wherein the ink for the mammary gland organoids is referenced in S2, and the steps for producing the HAMA adipocyte microsphere ink are as follows:

[0134] After mixing HAMA solution with LAP, add HAMA adipocyte microspheres and hyaluronidase inhibitor, gently pipetting (to avoid breaking the microspheres) until homogeneous, and store at 4°C for later use; the formulation ratio is 4% (w / v) HAMA solution + HAMA adipocyte microspheres (5×10⁻⁶). 4 (pcs / mL) + 1% (w / v) hyaluronidase inhibitor + 0.1% (w / v) LAP.

[0135] Specifically, in one embodiment, the following ink was prepared:

[0136] Preparation of HAMA-RGD:

[0137] Dissolve 200 mg HAMA in 4 mL PBS solution and stir. After complete dissolution, add 58 mg NHS and 78 mg EDC and stir for 30 min. Then add 232 mg RGD (75% grafting rate) and stir overnight. Measure the pH and adjust it to 7-7.5 with NaOH (EDC is acidic). Pour the solution into a dialysis bag (ensuring that small molecular weight NHS and EDC molecules are dialyzed out; the molecular weight cutoff should be >1 kDa). Dialyze in pure water for 2-3 days, changing the pure water every half day. Place the solution in a plastic lunchbox or petri dish (the liquid level should not be too high) and pre-freeze at -20℃ overnight; freeze-dry for 2-4 days.

[0138] Preparation of HAMA-FN:

[0139] Take 10 μL of fibronectin stock solution (FN stock solution concentration is 10 mg / mL), add 990 μL of PBS, filter through a 0.22 μm filter membrane, and in an ice bath, mix the FN solution and HAMA solution at a volume ratio of 1:9 and gently vortex; add LAP solution to make the final concentration 0.05-0.2%; treat under vacuum for 2-3 times, 3-5 minutes each time, at room temperature; pour the mixture into a culture dish or a custom mold (thickness < 5 mm); irradiate under a 405 nm UV lamp for 5-10 minutes at a distance of 5-10 cm; soak the gel in PBS, shake at 4℃ for 24 h, and replace the PBS 3 times; store in PBS at 4℃, or freeze-dry and store at -20℃.

[0140] Preparation of HAMA / gelatin / nano-hydroxyapatite composite ink

[0141] Weigh 1.0-2.0 g of HAMA powder and dissolve it in 10 mL of PBS buffer (pH 7.4). Stir magnetically at 37°C for 2-4 h until completely dissolved to prepare a 10%-20% HAMA stock solution. Add 0.5-2.0 g of gelatin powder to the HAMA stock solution and continue stirring magnetically at 37°C for 1 h to allow the gelatin to fully swell and dissolve, forming a HAMA / gelatin mixed solution (final gelatin concentration 5%-20%). Weigh out n-HA powder at a final concentration of 5%-15% and slowly add it to the mixed solution. First, stir magnetically for 30 min to initially mix, then sonicate in an ice bath for 15-30 min (300 W power, 5 s working / 5 s intermittent) to prevent n-HA aggregation and form a homogeneous suspension. Add Irgacure 2959 (final concentration 0.5%-1%) and stir magnetically at 37°C for 30 minutes. Let it dissolve completely; transfer the mixed ink to a vacuum degassing chamber and evacuate for 10-15 minutes to remove air bubbles from the solution (to avoid pore defects after printing or curing), and you will get HAMA / gelatin / n-HA composite ink. Store at 4°C away from light for later use.

[0142] The ink prepared in this application has a viscosity of 3500-4000 cP (25℃), and the microspheres are uniformly dispersed in the ink with an aggregation rate of only 4.8±1.2%.

[0143] S4: 3D printing of breast structures.

[0144] The equipment used for printing in this application includes a Lange constant flow pump, an ultraviolet irradiator, and a high-voltage DC power supply.

[0145] Set the printing parameters: nozzle diameter 200-400 μm, printing speed 5-10 mm / s, air pressure 0.1-0.3 MPa, platform temperature 37℃ (to prevent gelatin from solidifying); after printing the preset structure, immediately irradiate it with a 365nm UV curing instrument (light intensity 200-300 mW / cm²) for 30 s to rapidly crosslink HAMA, achieve structural fixation, and prevent scaffold collapse; prepare a 0.1% genipin PBS solution, completely immerse the UV pre-crosslinked scaffold in the solution, and incubate it at 37℃ in a constant temperature shaker (speed 50-100 rpm) for 2 h to complete the chemical crosslinking of gelatin and genipin.

[0146] In one implementation, please refer to Figure 6 By loading 10% HAMA hydrogel (containing 0.5% photoinitiator I2959) and autologous fat cell suspension (concentration 5×10⁻⁶) into a dual-channel syringe, respectively... 6 (each cell / mL), and then the HAMA adipocyte microsphere ink is loaded into the device syringe.

[0147] The arrow of the dual-channel syringe points to the inlet of the microfluidic chip. The coaxial flow channel structure is marked inside the microfluidic chip. The middle layer is a mixture of fat cell suspension and HAMA hydrogel (inner phase), and the outer layer is paraffin oil containing Span 80 (outer phase). The flow rate ratio is marked as 1:3 (inner phase 0.5 mL / h, outer phase 1.5 mL / h).

[0148] The microfluidic chip outlet is connected to the UV curing device, and a collection bottle is located below the UV curing device to collect the microspheres.

[0149] Set microfluidic parameters, print the fat layer, and cure it by irradiation with 405 nm ultraviolet light during printing;

[0150] The printed fat layer and the extracted gland layer are mixed in a matrix gel to allow the fat layer and gland layer to bind together.

[0151] S5: In vitro culture and performance testing of 3D-printed breast models

[0152] S5-1: In vitro culture: The culture medium used for in vitro culture is a mixture of "mammary gland organoid culture medium + adipocyte culture medium" (volume ratio 1:1), with 1% double antibiotics (penicillin-streptomycin). The culture environment is 37℃, 5% CO2, and 95% humidity. The culture medium is changed every 2 days, and the culture cycle is 21 days.

[0153] S5-2: Key Performance Test Data:

[0154] Please see Figure 8 The model was transplanted into nude mice, and the integrity rate was 90% after 90 days, with a degradation period of 24±0.5 months, which was synchronized with the tissue repair cycle.

[0155] After 21 days of culture, the maturation rate of glandular organoids in the glandular layer reached 82.3 ± 4.5%. Figure 9 The protein concentration was 100 μg / mL (ELISA detection). Figure 10 The lipid droplets within the lipid layer microspheres are abundant ( Figure 11 This demonstrates that the model possesses the core functions of breast tissue.

[0156] In this application, dual-nozzle 3D printing technology is used to print "HAMA adipocyte microsphere ink" and "mammary organoid-HAMA ink" as the fat layer and glandular layer, respectively. The interface between the two layers is connected by a HAMA cross-linking transition layer, which accurately restores the anatomical structure of the real mammary gland "fat support-glandular function". At the same time, cytokines such as leptin and VEGF secreted by adipocyte microspheres can penetrate into the glandular layer through the transition layer, providing physiological microenvironment signals for the mammary organoid.

[0157] Furthermore, due to the synergistic effect of fat and glands, experimental verification (n=3, number of samples per group=5) showed that the alveolar maturation rate of mammary organoids reached 82.3%±4.5% after 14 days of culture, which is close to the normal level in vivo (about 80%); the sensitivity to progesterone response was increased to 1.8 times that of the prior art (the progesterone-induced whey protein expression level was 2.1 times that of the prior art).

[0158] Secondly, through layered printing and transition layer design, the structural layering accuracy in this application reaches over 95% (the deviation between the two layers is ≤50 μm), and the continuity and integrity of the fat layer reaches 90%. Micro-CT scans show that the model structure matches human breast tissue by 85%, far exceeding the 30%-40% of existing technologies.

[0159] Furthermore, in this application, adipocytes are encapsulated within HAMA microspheres (200-300 μm in diameter, with a porosity of 50%-60%). The microspheres provide a three-dimensional growth space for the cells, reducing mechanical damage during the printing process. Simultaneously, the degree of methacrylylation of HAMA is controlled to 60%-80%, balancing scaffold stability and nutrient permeability. Additionally, the fatty acid slow-release particles added to the ink continuously provide energy to the adipocytes, promoting their maturation. Experimental testing (using the CCK-8 assay to detect survival rate and Western blotting to detect PPARγ expression) showed that the survival rate of the adipocyte microspheres reached 91.2%±3.8% after 7 days and remained at 73.5%±5.2% after 21 days. The PPARγ expression level was 68.3%±6.1% of that in normal adipose tissue, 2.3 times that of existing technologies. Oil Red O staining revealed that the accumulation of fat droplets within the microspheres was 3.1 times that of dispersed cells, demonstrating the formation of a functional adipose layer.

[0160] Furthermore, the adipocyte microspheres of the present invention not only achieve structural filling, but also regulate the proliferation rate of mammary organoids by secreting adiponectin. Experiments show that under the condition of adding adipocyte microspheres, the proliferation rate of mammary organoids is increased by 45% compared with the single organoid model, and the cell apoptosis rate is reduced by 32%.

[0161] Finally, this application designs specialized bio-inks for two types of functional units: the mammary gland organoid ink contains 0.1% RGD peptide (to promote organoid adhesion) and 0.5% fibronectin (to guide cell connection), while the adipocyte microsphere ink contains 1% hyaluronidase inhibitor (to maintain microsphere structural stability). Simultaneously, the extrusion pressure of the dual-nozzle printer is precisely controlled, and the nozzle movement speed is synchronously adjusted to 5-10 mm / s to avoid unit aggregation caused by ink flow. Compared to existing technologies, this application, through ink optimization and parameter control, reduces the organoid aggregation rate to 4.8%±1.2%, and achieves a 92% uniformity in the distribution of adipocyte microspheres. After 21 days of culture, immunofluorescence staining shows that continuous duct-like structures are formed within the glandular layer (ducts ≥500 μm in length account for 65%), and the duct ends are connected to the acinar structure. The microspheres in the adipose layer are physically connected to the basement membrane of the glandular layer through collagen fibers in the transition layer, achieving functional synergy between "fat" and "gland". Furthermore, the adipocyte microspheres of the present invention and the mammary gland organoids achieve chemical signal transmission through signaling molecules (such as EGF) in the transition layer. The experiment detected the cytokine concentration gradient at the interface between the two layers (the leptin concentration decay rate from the adipocyte layer to the glandular layer is only 15%), which ensures the effective transmission of signals. In contrast, the cytokine decay rate of the prior art exceeds 60%, which makes it impossible to achieve cross-layer signal regulation.

[0162] Furthermore, this application also incorporates gelatin (to enhance elasticity) and nano-hydroxyapatite (to strengthen strength) into the HAMA ink, employing a dual curing process of "UV crosslinking for 30 s + 0.1% genipin chemical crosslinking for 2 h." By controlling the degree of crosslinking, the model's compressive modulus is stabilized at 12.5 ± 1.8 kPa, perfectly matching the mechanical properties of normal breast tissue (10-15 kPa). The degradation cycle is adjusted by the degree of methacrylylation of HAMA, ultimately controlled at 4.5 ± 0.5 months. Specifically, the mechanical properties of this application achieve a 98% match with human tissue. In animal subcutaneous implantation experiments (SD rat model), there was no significant inflammatory response within 3 months (the number of macrophage infiltrations was only 1 / 5 of that of the PCL scaffold), and the degradation rate was synchronized with the rate of new tissue growth (after 4 months, the residual amount in the model was ≤20%, and the new tissue filling rate was ≥75%). Furthermore, the model of the present invention maintains 88% morphological integrity after 30 days of culture, with a degradation rate of only 22%. In the breast reconstruction simulation experiment, the model can maintain a stable morphology for up to 3 months, while the model of the prior art undergoes significant deformation within 1 month and cannot meet the reconstruction requirements.

Claims

1. A method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids, characterized in that: Includes the following steps: S1: HAMA adipocyte microspheres were prepared using the "emulsification-ultraviolet crosslinking method"; S2: Extraction and culture of mammary gland organoids; S3: Preparation of bio-ink: The bio-ink includes mammary gland organoid and HAMA adipocyte microsphere ink; S4: 3D printing of breast structure: Printing is performed using a dual-channel syringe. 10% HAMA hydrogel and adipocyte suspension are loaded into the syringe respectively. Then, HAMA adipocyte microsphere ink is loaded into the syringe. The arrow of the dual-channel syringe points to the inlet of the microfluidic chip. A coaxial flow channel structure is marked inside the microfluidic chip, with the adipocyte suspension in the middle and the HAMA hydrogel on the outer layer. The outlet of the microfluidic chip is connected to a UV curing device. A collection bottle is placed below the UV curing device to collect the microspheres. The printed fat layer and the extracted glandular layer are mixed in a matrix gel to allow the fat layer and glandular layer to bond. S5: In vitro culture and performance testing of 3D printed breast models.

2. The method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids according to claim 1, characterized in that: The steps in S1 are as follows: S1-1: Preparation of HAMA solution: Dissolve HAMA powder in sterile PBS, stir magnetically, prepare a 3% (w / v) HAMA solution and filter; S1-2: Preparation of adipocyte suspension: After resuscitating human primary adipocytes (HADC), they were cultured in adipocyte culture medium, digested with trypsin, and then centrifuged. S1-3: Emulsification and Mixing: Mix the HAMA solution of S1-1 with the adipocyte suspension of S1-2, add LAP, and stir magnetically until homogeneous; slowly drip the mixture into liquid paraffin containing Span 80, and ultrasonically emulsify to form a water-in-oil emulsion; S1-4: Place the water-in-oil emulsion under a UV lamp to crosslink and solidify HAMA to form microspheres; S1-5: Microsphere purification: Collect microspheres by centrifugation, wash with anhydrous ethanol, and finally resuspend in adipocyte culture medium.

3. The method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids according to claim 2, characterized in that: The preparation method of the HAMA adipocyte microsphere ink is as follows: After mixing HAMA solution with LAP, add HAMA adipocyte microspheres and hyaluronidase inhibitor, gently pipetting (to avoid breaking the microspheres) until homogeneous, and store at 4°C for later use; the formulation ratio is 4% (w / v) HAMA solution + HAMA adipocyte microspheres (5×10⁻⁶). 4 The HAMA adipocyte microsphere ink has a viscosity of 3500-4000 cP (25℃), with microspheres uniformly dispersed in the ink and an aggregation rate of only 4.8±1.2%.

4. The method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids according to claim 1, characterized in that: The S2 step is as follows: Autologous breast tissue was extracted, and the tissue block was transferred to a sterile culture dish and rinsed three times with sterile PBS (containing double antibodies). Tissue dissociation and acquisition of single-cell suspension; Cell counting and cell viability detection; Organoid culture is performed, followed by organoid purification and identification to obtain qualified functional units.

5. The method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids according to claim 1, characterized in that: The following culture media are used for organoid culture: 。 6. The method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids according to claim 2, characterized in that: The 3D printing parameters are as follows: 。 7. The method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids according to claim 1, characterized in that: The specific steps in S5 are as follows: S5-1: In vitro culture: The culture environment is 37℃, 5% CO2, and 95% humidity. The culture medium is changed every 2 days, and the culture cycle is 21 days. S5-2: Key performance testing.

8. A method for 3D printing a breast based on HAMA adipocyte microspheres and breast organoids according to claim 7, characterized in that: The culture medium used in S5-1 for in vitro culture is a mixture of "mammary organoid culture medium + adipocyte culture medium", wherein the volume ratio of "mammary organoid culture medium + adipocyte culture medium" is 1:1, and 1% penicillin-streptomycin is added.

9. A 3D-printed mammary gland based on HAMA adipocyte microspheres and mammary gland organoids, characterized in that: It is prepared using the preparation method according to any one of claims 1-8.

Images